EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell l...

MISSING IMAGE

Material Information

Title:
EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer
Physical Description:
Mixed Material
Language:
English
Creator:
Singh, Sandeep
Trevino, Jose
Bora-Singhal, Namrata
Haura, Eric
Altiok, Soner
Chellappan, Srikumar P.
Coppola, Domenico
Publisher:
BioMed Central
Publication Date:

Notes

Abstract:
Background: Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling. Results: SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples. Conclusions: Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs. Keywords: Cancer stem-like cells, Side-population cells, Self-renewal, EGFR, Sox2
General Note:
Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository at the University of Florida community, with research, news, outreach, and educational materials.
General Note:
doi:10.1186/1476-4598-11-73 Cite this article as: Singh et al.: EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer. Molecular Cancer 2012 11:73.
General Note:
Singh et al. Molecular Cancer 2012, 11:73 http://www.molecular-cancer.com/content/11/1/73; Pgs. 1-15

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All rights reserved by the source institution.
System ID:
AA00013233:00001

Full Text


Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


o MOLECULAR
CANCER


EGFR/Src/Akt signaling modulates Sox2

expression and self-renewal of stem-like

side-population cells in non-small cell lung cancer

Sandeep Singh '4, Jose Trevino ', Namrata Bora-Singhall, Domenico Coppola2, Eric Haura3, Soner Altiok2
and Srikumar P Chellappan*


Abstract
Background: Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In
non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have
stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self renew;
however the mechanistic correlation between oncogenic pathways and self renewal of cancer stem-like cells has
remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate
tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self renewal of SP cells
is dependent on EGFR mediated signaling.
Results: SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary
human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to
self renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic
implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers
like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP
cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic
inhibitors suppresses the self renewal growth and expansion of SP-cells and resulted in specific downregulation of
Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its
self renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in
regulating self renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples.
Conclusions: Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates
self renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted
therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.
Keywords: Cancer stem-like cells, Side-population cells, Self renewal, EGFR, Sox2


Introduction
Despite significant therapeutic advances, lung cancer
causes the maximum number of cancer related deaths
worldwide [1]. In the United States, -85% of the patients
diagnosed with NSCLCs, die within five years [2-4], thus,
highlight a need for better understanding of the cellular
and molecular events underlying the genesis of this dis-
ease. Cancer stem cell model has emerged as a viable

Correspondence' I
Department of Tumor Biology, H Lee Moffitt Cancer Center and Research
Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA
Full list of author information is available at the end of the article


explanation for the initiation and progression of the ag-
gressive cancers like NSCLCs.
Cancer stem cell model suggests that cancer stem-like
cells (CSCs) are a subpopulation of cells within the
tumor that have the deregulated properties of normal
stem cells with sustained self-renewal, and can generate
secondary tumors that recapitulate the heterogeneity
and diversity of original tumor [5-8]. CSCs are consid-
ered to be responsible for tumor initiation, propagation,
recurrence and resistance to therapy [9,10]. Hoechst
33342 dye excluding cells, termed side-population (SP)
cells, have been described as CSCs in a variety of tumor


S 2012 Singh et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
l ed Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted ue, distribution, and
reproduction in any medium, provided the original work is properly cited.






Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


types, including NSCLCs [11], where they have been
shown to display increased tumorigenicity when trans-
planted into immunocompromised mice [12,13] as com-
pared to major population (MP) cells. SP phenotype is
dependent on the differential ability of cells to efflux the
Hoechst 33342 dye via the ATP-binding cassette (ABC)
family of transporter protein, mainly ABCG2 (breast
cancer resistance protein, BRCP1) which is .:... r;.11
expressed on the cell membrane of stem cell populations
[14]. Earlier studies have demonstrated the existence of
SP cells in various established human NSCLC cell lines
[11] but their ability to generate tumors in lung micro-
environment as well as the signaling pathways governing
their stem-like properties remain to be elucidated.
The transcription factors Oct4, Sox2 and Nanog have
been identified as core regulators that maintain the self-
renewal of embryonic stem cells [15]. These factors are
overexpressed in various cancers and are associated with
malignant progression and poor prognosis i.:i!!,,
NSCLCs [16,17], suggesting that the core regulators that
govern normal stem cell self-renewal may also maintain
the stem-like properties of CSCs in cancers. However,
the influence of NSCLC specific oncogenic pathways on
the expression of these factors remains relatively un-
known. Alterations in ECFP-L- L. like copy number
gains and/or mutant allele-specific ..iiJ. .*.n are
associated with NSCLC pathogenesis. In addition, activa-
tion of EGFR -;g ....:.;. increases the self-renewal cap-
acity of neural precursor cells and brain tumor stem
cells [18-20]. In this study, we provide biochemical and
biological evidence that SP cells isolated from estab-
lished human NSCLC cell lines and tumors are highly
enriched in NSCLC-CSCs and EGFR-Src-Akt ..
axis contributes ....i,. ... i to the self-renewal of SP
cells. Interestingly, Sox2 transcription factor is the pre-
dominant downstream target of EGFR signaling in these
cells and plays a major role in self-renewal growth and
expansion of SP cells, independent of Oct4 and Nanog.

Results
SP cells are enriched with tumorigenic cells and produce
highly invasive tumors
In an attempt to identify NSCLC stem-like cells, SP ana-
lysis was conducted on four primary human NSCLC
explants grown in athymic nude mice. SP cells appeared
as a well separated population as .1 .1i..I previously
[21]. As shown in Figure 1A, a *-.... ,. inhibitor of
ABCG2 [22], F..,,-;. _..-, ..gI0., C (FTC) could block the ap-
pearance of SP phenotype. All the four tumor samples dis-
played the presence of SP cells with varying frequency
ranging from 0.6-3%, and could be .; .i; .. I.! blocked by
FTC (Figure 1B).
Self-renewing normal or cancer-stem-like cells can be
expanded as ), -..i..: i. .i spheres when cultured at low


density in serum free, stem cell selective medium; differ-
entiated cells do not grow or form spheres under these
conditions [6,23,24]. The self-renewal property of SP
cells was examined by performing sphere formation
assay on sorted SP and MP cells isolated from human
tumor xenografts. While sorted SP cells were able to
grow as spheres, MP cells had .. ,. I ii. less capacity to
grow under identical i.l ihii. (Figure 1C).
Attempts were then made to assess the presence of
SP cells in human NSCLC cell lines. As shown in later
sections, A549 (K-Ras mutant; wild type EGFR amplifi-
cation), H1650 (EGFR mutant; Exon 20 deletion:
delE746-A750) and H1975 (EGFR mutant; L858R and
T790M mutations), contained SP-cells with varying fre-
quency. Appearance of SP cells was completely blocked
by FTC. Sorted SP cells were able to grow as spheres
whereas MP cells showed :-:!i .1.:!; reduced capability
(Figure ID) suggesting that NSCLC-SP cells are
enriched with CSCs.
The stem cell like property of NSCLC-SP cells was
verified by evaluating its ability to form tumors in the
lung microenvironment. Sorted SP and MP cells from
A549 cells stably expressing the luciferase gene were
orthotopically implanted into the left lung of SCID mice
and tumor growth was monitored for 12 weeks. As
shown in Figure IE, SP cells generated primary tumors
in the lung more efficiently than MP cells. At the end of
the experiment, lungs, liver, kidney and brain were
excised from each mouse and ex-vivo images were
examined for the presence of metastasized luciferase
positive cells. Mice injected with SP cells demonstrated
substantial tumor burden throughout the lungs and
showed luminescent metastatic loci in liver, kidney
adrenalss) and brain (Figure 1F). In contrast, MP cells
formed only one luminescent focus in the lung of one
mouse (out of five) injected with 50 000 MP cells and
there was no metastasis (Figure 1F). These results were
.. .n.. I by H&E staining; further, tumors formed
within the lung from SP cells, i. 'iq iii a. the histo-
pathology of adenocarcinoma as confirmed by positive
staining with pan-keratin antibody as well as mucicar-
mine dye (Figure 1G). These data .. ,. .1 that SP cells
are enriched with tumorigenic cells and can develop
metastatic tumors in vivo.

SP cells exhibit molecular markers of stem-like cells
Recent reports suggest that epithelial cells acquire can-
cer stem cell properties upon induction of epithelial to
mesenchymal transition iF ..T) [25]. To evaluate
whether SP cells show features of EMT, SP and MP cells
from A549, H1650 and H1975 were examined for the
levels of EMT markers like E-cadherin, Vimentin and
Fibronectin. As shown in Figure 2A, ABCG2 expression
was significantly higher in the SP fraction in all the three


Page 2 of 15







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


B
Control ECon
.FMC

1.05% 2

a-
C,)


0 I000 200 3000 000
Fumitremorgin C

00 00 0

0.04%
" /




0 100 2000 3000 4000



trol


5 10 20


Patient ID
- SP Cells 5






S oI
-----~P Cells-------5


Cells Mice with
Type Injected tumor Metastasis
SP 50,000 3/5 Yes
10,000 1/3 Yes

MP 50,000 1/5 No
10.000 0/3 No


F ----Sfp MP
Lung Liver Lung Liver 2000


1600




Adrenals Brain Adrenals Brain
and Kidney and Kidney counts


H&E


Muclcarmlne





ilK WOW


Figure 1 SP cells exhibit stem-like properties. (A) FACS analysis on single cell suspension of human NSCLC xenograft stained with Hoechst
33342 dye showing SP cells. Fumitremorgin C (FTC) inhibited the efflux of the dye and caused the disappearance of SP cells. (B) SP cell frequency
in presence or absence of FTC in four different human tumor xenografts. (C) Sphere formation assay on SP or MP cells grown in stem cel
selective media for 10 days. The pictures of representative spheres are presented. The bar diagram show the average (+SD) number of spheres
formed from 2000 cells. (D) SP or MP cells from HI650 and HI975 cell lines were plated in serum free medium supplemented with EGF and
bFGF for 10 days under self-renewal assay conditions. The pictures of representative spheres are depicted (E) Indicated number of SP and MP
cells from A549-Luc cell line were implanted into the right lung tissue of SCID mice. The tumor incidence and metastasis was monitored for
12 weeks by bioluminescence imaging. (F) Ex-vivo images of the lung, liver, kidney/adrenals and brain captured at the end of experiment.
Metastasis was prevalent in SP cells implanted mice. (G) H&E, pan-keratin (brown staining) and mucicarmine (pink staining, indicated by arrow
head) staining of whole lungs from mice implanted with SP or MP cells.


cell lines. The levels of E-cadherin was lower in H1650-
SP cells as compared to MP cells, however, it was un-
detectable in A549 and unchanged in H1975 cells. Fibro-
nectin was detected at higher levels in A549 and H1975-
SP cells, but undetectable in H1650 cells. Vimentin level


was higher in A549-SP cells, but low in H1975 and
H1650 SP cells. Although the levels vary in a cell type
dependent manner, these results suggest that, SP cells
express proteins indicative of EMT without any external
stimuli to the cells (Figure 2A). The molecular basis for


3000-


Page 3 of 15


U H1975


v, tars


ia


I


(4


H1650







mi m







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


H1650
SP MP
em



I m


B
A549 H1975
SP MP SP MP I3 *As49MP
S -m ABCG2 DA4e SP
-- ECadherin 2
- m .- Fibronectin
S-- % VImentin t
dm -- aa -ActIn E


C
m- *gr6 MP







Twist Slug Snail
E
-101
II A54 MP
2 A4. OAS4SP P


4J


ABCG2 Oct4 Sox2 Nanog
G


Twist Slug Snail

D





s1S n


Twist Slug Snail


Figure 2 SP cells express stem cell-like markers. (A) Western blot analysis on SP and MP cells from A549, H1650 and H1975 cell lines for the
indicated proteins. 3-Actin was used as the loading control. (B, C, D) SP and MP cells were analyzed by real time qRT-PCR method for the
expression of Twist, Slug and Snail genes. Average (+SD) fold change between MP and SP cells were plotted. (E, F, G) SP and MP cells were
analyzed by real time qPCR method for the expression of ABCG2, Oct4, Sox2 and Nanog genes. Average (+SD) fold change between MP and SP
cells were plotted. (H) The spheres formed in self-renewal assays of H1650 SP cells were stained for the expression of ABCG2, Oct4, Sox2 and
Nanog. Confocal image of one of the z-stacks for a sphere is presented. DAPI was used to stain the nucleus.


the differential expression of the EMT markers was then
examined. Transcription factors like Twist, Slug and
Snail have been demonstrated to be capable of coordin-
ating the EMT program during embryonic development
and in cancers [26,27]. Therefore, we next assessed the
expression of these transcription factors in SP and MP
cells. Real-time PCR analysis revealed that Twist, Slug
and Snail transcription factors are expressed at higher
levels in SP cells in all the three NSCLC cell lines
(Figure 2B, C, D).


The expression of Oct4, Sox2 and Nanog transcrip-
tion factors was next examined in SP cells. Real-time
PCR analysis showed elevated levels of ABCG2, Oct4,
Sox2, and Nanog in the SP fraction in all the three
cell lines. (Figure 2E, F, G). Further, SP cells from
H1650 cells growing as spheres showed expression of
ABCG2, Oct4, Sox2 and Nanog proteins by fluores-
cence microscopy (Figure 2H), indicating the undiffer-
entiated growth of self-renewing SP cells within the
spheres.


Page 4 of 15






Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


EGFR tyrosine kinase inhibitors downregulate self-renewal
and SP phenotype
Experiments were conducted to explore the molecular
mechanisms involved in the self-renewal of SP cells. Since
aberrant EGFR signaling is implicated with the initiation
and progression of lung cancer, we first assessed SP fre-
quency and expression of ABCG2 in the presence of an
antagonistic ,ooi;..,., against EGFR. Cells were mixed
with 10 pg/ml anti-EGFR antibody or an isotype control
and plated in 2% FBS containing media for 5 days. Block-
ing EGF-receptors resulted in a significant decrease in SP
frequency in both A549 and H1650 cells (Figure 3A, B),
along with decreased EGFR phosphorylation as well as
ABCG2 expression in both the cell lines (Figure 3C). Con-
firming these results, depletion of EGFR expression by a
siRNA resulted in decreased SP frequency and ABCG2 ex-
pression in A549, H1650 and H1975 cells (Figure 3D, E).
To further evaluate whether EGFR signaling contribu-
ted to the self-renewal property of H1650 SP cells, sphere
formation assay was conducted in the presence or ab-
sence of EGFR inhibitors Gefitinib or Erlotinib. As shown
in Figure 3F, inhibition of EGFR-kinase activity by
500 nM of Gefitinib or Erlotinib, demonstrated a 5-7
fold (p < 0.001) decrease in the number of spheres; further
the size of the spheres was also significantly reduced.
A secondary point mutation in exon 20 of EGFR
(T790M) is associated with acquired resistance to gefiti-
nib or Erlotinib, but this can be overcome by the irre-
versible EGFR-tyrosine kinase inhibitor BIBW2992
(BIBW). We tested the effect of 500 nM of gefitinib and
200 nM of BIBW on EGFR phosphorylation and self-
renewal growth of SP cells from H1975 cell line, which
harbors gefitinib-resistant-T790M mutation along with
Gefitinib responsive-L858R mutation in exon 21. West-
ern blot analysis showed that tyrosine phosphorylation
of EGFR was insensitive to 500 nM concentration of
*-. Ii1 .. I. whereas ;,i ,,:; :..t l....... L! .t: ..." occurred
after treatment with 200 nM of BIBW in H1975 cells
,.i.iti.. I1 file 1: Figure Sl). Consistent with this, BIBW
could significantly inhibit the self-renewal of SP cells
from H1975 cells (Figure 3G).

Adherent cultures of SP cells maintain stem-like
properties
To conduct further molecular studies on SP cells, we
attempted to establish adherent cell cultures of isolated
SP cells from A549, H1975 and H1650 cell lines, as sug-
gested for --..!',: stem cells [28]. Isolated SP cells were
plated on uncoated or Poly-D Lysine + Laminin coated
culture plates in serum free, stem cell media. While
A549-SP and H1975-SP cells detached from the surface,
H1650-SP cells grew as an adherent culture. As shown
in Figure 3A, H1650-SP cells cultured on uncoated sur-
face failed to maintain SP phenotype with high


frequency (Figure 4A, (i)), but 80% of the cells main-
tained as SP cells even after 5 passages when plated on
PDL + laminin coated surface (Figure 4A, (ii); H1650-
SI-'\.lh cells). H1650-SPAdh cells cultured back in 5%
FBS containing medium for 10 days could recapitulate
the proportion of SP and MP cells found in parental
H1650 cells (F:.. ..- 4A, (iii)), with a concomitant reduc-
tion in expression of ABCG2 (FTi.... I': as well as Oct4,
Sox2 and Nanog mRNA as seen by R-PCR (Figure 4C).
Cell cycle analysis showed that H1650-SPAdh cells were
slow cycling compared to parental cells (Figure 4D), hav-
ing approximately 20% higher number of cells in Go/G1
phase; but upon serum-induced differentiation, H1650-
SPAdh cells acquired cell-cycle phase distribution com-
parable to H1650-parental cells (F; ni:- 4D).
Treatment of H1650-SPAdh cells with 200 nM BIBW
c.;.. I:-'l suppressed the number as well as the size of
spheres (Figure 4E); at the same time, treatment with
30 VM cisplatin did not affect the number or the size of
the spheres formed by H1650-SP cells, suggesting
enhanced chemoresistance of these cells. Further, the
sphere-formation ability of SP was not altered by the
ABCG2 inhibitor, FTC, suggesting that self-renewal of
SP cells was independent of ABCG2 activity (Figure 4E).

Inhibition of EGFR-Src-Akt signaling downregulates Sox2
expression
Experiments were conducted to examine the down-
stream ;*, .';,_ events from EGFR that modulates self-
renewal of SP cells and whether these pathways impinge
transcription factors associated with stemness. Role of c-
Src in the process was first examined since Src is altered
in NSCLC [29,30]. H1650-SPAdh cells were treated with
EGFR or Src TKIs and the levels of Oct4 and Sox2 was
assessed by western blotting T ..., .. 5A). EGFR inhib-
ition by 500 nM gefitinib or 200 nM BIBW as well as in-
hibition of Src activity by 200 nM i 1..1. I. or 1 IM
PP2 markedly reduced Sox2 expression; Oct4 level was
not ill .'.i (Figure 5A). These results were verified by
immunoflorescence experiments. Similar to Oct4, there
was no i;..:i. :..i difference in Nanog expression; how-
ever, the number Sox2 positive cells were significantly
decreased in response to the treatment of EGFR- and
Src-TKIs (Additional file 1: Figure S2).
Inhibition of EGFR as well as Src signaling resulted in
decreased phosphorylation of EGFR, Src, ERK and Akt
(Figure 5A). Contribution of ERK and Akt pathways to
EGFR mediated induction of Sox2 was next examined
in H1650SPAdh cells. Phosphorylation of ERK was
suppressed by MEK inhibitor PD98059 and AKT-
phosphorylation was suppressed by the PI3-kinase in-
hibitor, LY294002. However, PI3-Kinase inhibited
H1650SPAdh cells also resulted in 'lii, inhibition in
ERK phosphorylation IF-. ....- 5B). A similar observation


Page 5 of 15






Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


A isotype igu


o*" 1 .
1W8.62%
j *FMC
o -
i ,







o o5K 10~0 10K 200K 20KO


7

I3!


o
0
o5.5-
u.
I



F
20"


15-


10.
0.


d 5
5-
C
a.


1- .

S"13.2..% ..



6 5K tooK 1501 2K 250K


SEGFR SiRNA




L*


7 **


1 2


o u. a u.
00 00
0 uj ui
O- ON" ABCG2
1 0.4 1 0.7
-* -4 w4 Phos-EGFR
1 0.5 1 0.6
W1I PTTotal EGFR
mll i p-Actin
A549 H1650


z< z < z <

SL. C L. toC .


i,- I m I ABCG2

"m- rn EGFR

mam m -7 l-Actin
A549 H1975 H1650


Control Gefitinib




BIBW

60 pm
Al !.
^^H ..r'''a* 'I^


_1 = = = 01 Mmm
Control Gefitinib Erlotinib Control Gefitinib BIBW
Figure 3 Inhibition of EGFR suppresses SP frequency and self-renewal. (A) H1650 and A549 cells revealed a decrease in SP
frequency after treatment with 10 pg/ml EGFR-neutralizing antibodies for 5 days. (B) Frequency of SP cells in control antibody (1) and EGFR-
neutralizing antibodies (2) treated cells. (C) Western blot analysis for ABCG2 and phospho-EGFR after EGFR-neutralizing antibody treatment. (D) SP
frequency in NSCLC cells transfected with siRNA against EGFR or a control siRNA. (E) Expression of ABCG2 and EGFR in antibody treated cells.
(F and G) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres
generated per well from 1000 cells is plotted (mean + SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are
presented. represent the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.


Page 6 of 15


I Control SiRNA


H1650 Control Gefitinib Erlotinib

1#''


60 pm







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


SP on normal surface
(I)

46.3%


0 1000 2000 3000 4000
Hoechst Red-A

C
1.2
1; ]
S -g :2
i 1.0
u.
o E
0 0.8.
Z a

I ABCG2 E 0.6
S0.4
-g O 3-Actin Z
W 0.2
E


0J--


GO/GI= 54%
S= 34%


GO/GI= 74%
S= 19%
G2/M= 7%


50 100
H1650-SPAdh
(N21EGFIFGF)


4000-


3000-


92000-

1


SP on PDL-Laminin
(ii)
82.7%


1000 2000
Hoechst Red-


* N2/EGFIFGF
1 Serum (5%)


G2


GOIGI= 48%


50 100
H1650-SPAdh
(Serum 5%)


Figure 4 Establishment and characterization of H1650-SPAdh cells. (A) SP cells from H1650 cell line was plated on normal tissue culture
plate (i) or poly-D-lysine-laminin (PDL-Laminin) coated surface (ii) in serum free medium containing N2-supplement, EGF and bFGF. H1650-SPAdl
cells growing in self-renewing condition was cultured in serum for 5 days to induce differentiation, and reanalyzed for SP frequency (iii). (B)
Serum induces differentiation of SPAdh cells as seen by ABCG2 expression and (C) real time qPCR analysis for stem cell markers, ABCG2, Oc4,
Sox2, Nanog. (D) Cell cycle analysis for parental-H1650 or H1650-SPAdh and serum differentiated H1650-SPAdh cells grown on PDL-Laminin
coated surface. Histograms were plotted using ModFit program. (E) The average number of spheres generated from 1000 H1650-SPAdh cells is
plotted (mean + SD). Phase contrast microscopy images of the spheres in presence or absence of indicated drugs.


Page 7 of 15


0.1%
do=-i


3000 000
3000 40W0


0 1000 2000 3000



4000


Sox2


inog






Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


-


Sox2


-!11 m Oct4
Phos AKT

I ~ a -. Phos ERK

II -- ^ ^| Phos-EGFR

ll lTotal oEGFR

Phos-Src

S me rlTotal Src


o 0
o a
0m


Sox2


m- -- Phos-AKT
S- -- Phos-ERK

a P-Actin


A549 H1975


S- ABCG2

4 nnm. P -Actin
a m- r a ABCG2
Actin
x: i-Actin


*Control SiRNA ESrc SiRNA DAKT SiRNA






mIE


F I I I
g 4 <4 <
I Z Z Z Z OZ

O a O, 0 < O t W
O-f -m Om0 m ABCG2

m mm -ma Pam -Actin


A549


H1650


H1975


H1650 Control Dasatinib



PP2


4s 60 pmP



Dasatinib PP2


LY294002 -15
E



PD98059


5 .



)8059 LY294002 0.


H1975 Control Dasatinib



PP2




Control Dasatinib PP2

Control Dasatlnlb PP2


PD98059



LY294002


LY294002


98059
Figure 5 Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors
for 4 days and western blot analysis was performed for the indicated proteins. (B) H1650-SPAdh cells were treated with MEK or P 3K
inhibitor for 4 days and levels of Sox2 and activation ofAkt or ERK was evaluated by western blot. (C) A549 and H975 cells were treated with
MEK or P13K inhibitor for 5 days and the frequency of SP cells were examined by SP analysis. Data represents the fold change in mean (+ SD) of
SP frequency. (D) ABCG2 expression upon inhibitor treatment as detected by western blotting. (E) SP cell frequency in NSCLC cell lines
transfected with specific siRNAs against Src or AKT. (F) ABCG2 expression in siRNA transfected cells as detected by western blotting. (G and H) SP
cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated from
1000 cells is plotted (mean SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. represent
the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.


Page 8 of 15


- 1,
,

3.
0

0.5,


u.


20'
E






d 5l
I-.


0


I







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


has been reported in earlier studies where PI3-Kinase sig-
naling was demonstrated to regulate the ERK phosphor-
ylation in T-cell-receptor (TCR) signaling [31] and
PDGFR mediated signaling [32]. However, as shown in
Figure 5B, inhibition of MEK activity did not affect the
levels of Sox2 while the PI3-kinase inhibition, markedly
reduced its levels with corresponding reduction in SP fre-
quency (Figure 5C) and ABCG2 expression (Figure 5D).
These results were confirmed using siRNAs to Src and
Akt. As shown in Figure 5E, SP frequency was signifi-
cantly downregulated in both Akt and Src siRNA trans-
fected A549, H1650 and H1975 cells as compared to the
control siRNA transfected cells, with a corresponding re-
duction in ABCG2 expression (Figure 5F). Similar inhibi-
tory effects were observed upon silencing of two other
Src family members, Fyn and Yes (data not shown).
To determine whether Src or Akt signaling facilitates
self-renewal of SP cells, sphere formation assay was con-
ducted on SP cells in presence or absence of Src inhibi-
tors Dasatinib or PP2, MEK inhibitor PD98059 as well
as Akt inhibitor LY294002. As shown in Figures 5G and


di
A I 5 I B



W40.












S25 -Control SiRNA o Sox2 S
Q- 0Ct4 30
I W Nanog 20'

C 10.






1.25- EOControl SiRNA ISox2 S

1

o 0.75-



i0.25,


5H, Src-kinase inhibitors dasatinib or PP2, as well as
PI3K/Akt inhibitor LY294002 showed a significant (5-7
fold; p <0.001) decrease in sphere formation; MEK in-
hibition by PD98059 did not have any significant effect
on self-renewal. The average size of the spheres formed
was found to be 7-10 folds smaller than the untreated
cells. Collectively, these data indicated that inhibition of
EGFR/Src/Akt signaling results in depletion of Sox2 ex-
pression and decreased self-renewal of SP cells.

Suppression of Sox2 expression is sufficient to inhibit the
self-renewal of SP cells
Since inhibition of EGFR/Src/Akt signaling specifically
downregulated the expression of Sox2, we examined the
contribution of Sox2 to the self-renewal of H165SP-Adh
cells. Transient transfection of EGFR and Src siRNA in
H1650-SPadh cells reduced EGFR expression by 60%
and Src expression by 50%. Reduction in EGFR or Src
expression decreased the levels of Sox2 by 50% and 40%
respectively; the expression of Oct4 and Nanog was not
altered (Figure 6A). In addition, depletion of EGFR or


Control EGFR Src Sox2
SIRNA SIRNA SIRNA SIRNA



60.im


Control EGFR Src Sox2
SIRNA SIRNA SIRNA SIRNA

D < < <

IA








A 0 0H
A549 H1650 H1975


Figure 6 Sox2 is necessary for maintaining the self-renewal of SP cells. (A) H1650-SPAdh cells were transfected with non-targeting control
siRNA, or siRNA against EGFR, Src, or Sox2 and western blot analysis was performed for Sox2, Oct4, EGFR and Src expression. (B) siRNA transfected
cells were plated for self-renewal assay and analyzed after 5 days of plating. Average number of spheres generated per well from 1000 cells is
plotted (mean + SD). (C) NSCLC cell lines were transfected with control or Sox2 siRNAs and SP frequency was evaluated. Average fold change in
SP-frequency (+ SD) is plotted. (D) Decrease in ABCG2 expression in Sox2 siRNA transfected cells was detected by western blotting. The Sox2
expression was evaluated by qRT-PCR and results are presented as bar diagram.


Page 9 of 15






Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


Src by siRNA suppressed the sphere formation by 2-3
folds (rF,:. 6B). To further explore the function of
Sox2 in self-renewal of SP cells, we depleted Sox2 ex-
pression in H1650-SPadh cells. Transient transfection of
Sox2 siRNA reduced the expression of Sox2 by 60%
(Frli:. 6A). Depletion of Sox2 expression did not sig-
nificantly alter the expression of Oct4 or Nanog expres-
sion in H1650-SPadh cells (FTi ... 6AB), and reduced
the sphere formation by approximately 2.5 folds (Fig-
ure 6) with a corresponding reduction in the average size
(p = 0.003). Depletion of Sox2 expression resulted in a
pronounced decrease in the frequency of SP cells
(Figure 6C) as well as ABCG2 expression (Figure 6D) in
A549, H1650 and H1975 cells compared to control
siRNA transfected cells. -. Ini., results were obtained
when a .: irr...-.. siRNA to Sox2 was used (. ::.:i:t..:!
file 1: Figure S3). Collectively, these results suggest that
Sox2 gene has a direct role in maintaining cancer stem
cell characteristics and self-renewal of SP cells from
NSCLC.

Sox2 is expressed in NSCLC and is associated with
metastatic progression
Our data showing that depletion of Sox2 affects the self-
renewal properties of stem-like cells, we next examined
Sox2 expression in a panel of NSCLC tumor samples
obtained from stage I/II or stage IV patients on tissue
microarrays (TMAs) by immunohistochemistry. Samples
from 193 patients with NSCLC-stage I/II disease includ-
ing 73 with adenocarcinoma were on one I I ', samples
from 103 stage IV-NSCLC patients including 45 with
adenocarcinoma from primary site and 17 adenocarcin-
oma samples from the metastatic sites were on the sec-
ond TMA. In accordance with earlier reports, Sox2 was
i...l... expressed in squamous cell carcinoma (SCC)
samples for both stage I/II and IV patients (Figure 7A (i
and ii)). In contrast to SCCs, adenocarcinoma samples
had ', 1 ..'. '.i; lower expression of Sox2. Sox2 positive
cells were 1l t I ... ii. ... 1: distributed in adenocarcin-
oma samples for both stage I/II and IV patients
(Figure 7A (iii and iv). While there was no significant
ii, ., ....- in Sox2 expression between ,i::; .. i grades
of tumors, elevated expression of Sox2 was positively
associated with metastatic progression. Representative
images for adenocarcinoma-metastases are shown in
Figure 7A (v-viii). Approximately 67% of stage I/II
(n=73) and 73% of stage IV (primary site, n=45)
tumors were detected as positive for Sox2 expression
(score> 1) using a semi-quantitative scoring system.
Compared to the primary site tumor for stage IV
patients, l,:1. :I numbers of metastasized tumors were
positive for Sox2 (Figure 7B). The median score for Sox2
expression is represented as !p: l..- : ... "- .....- 7C). The
average score for Sox2 expression was found to be


S'-d._;;..:u higher (p=0.01) in metastasized tumors as
compared to the primary site or lower stage tumors.
Overall, Sox2 was expressed in all stages of adenocarcin-
oma and its levels were 1..i. ,:i, higher in metastatic
lesions.

Discussion
In the current study, we used the SP phenotype to iden-
tify and enrich a subpopulation of NSCLCs with the
properties ascribed to CSCs. The studies presented here
demonstrates a specific and ii;:. iif role for EGFR
signaling cascade in facilitating the self-renewal growth
and expansion of the side population cells from
NSCLCs.
Our study, in accordance with earlier studies [11],
[33], confirmed the presence of SP cells in established
human NSCLC cell lines and in human tumor xeno-
grafts with the properties of CSCs. Comparing the self-
renewal ability of SP and MP cells isolated from human
tumor .. I ... ,.~: we found that approximately 0.2% SP
cells were able to self-renew and form spheres, whereas
MP cells were unable to self-renew. Comparing the per-
centage of sphere forming cells in SP cells, we estimate
that approximately 1-2% of SP cells from established cell
lines may have stem-like properties; therefore, SP pheno-
type may not be the exclusive marker for CSCs, but can
be used to enrich stem-like l. from NSCLCs.
SP cells were found to be more tumorigenic in vivo,
confirming the enrichment of tumor initiating cells in
SP compartment. These cells were able to produce
highly invasive disease upon implantation into the lungs.
Also, the direct association of stem-like cells with gener-
ation of metastatic disease may be supported by our ob-
servation where a I 1,.' ..'. correlation was observed
between high Sox2 expressions in the metastatic tumors
of lung adenocarcinoma patients. Recent reports indicate
that the normal epithelial cells acquire the CSCs proper-
ties upon induction of EMT governed by various cyto-
kines and growth factors from stromal cells [10,25]. Our
results demonstrate that SP-cells intrinsically exhibit loss
of epithelial markers and/or the gain of mesenchymal
markers as compared to MP cells and could be due to
the 1.:.l1 i expression of transcription factors Twist, '.hIn.-
and Snail, which are known to be involved in maintain-
ing the mesenchymal phenotype. Together with the ex-
pression of embryonic stem cell transcription factors like
Oct4, Sox2, and Nanog along with the exhibition of
EMT like features and orthotopic tumor-forming ability,
,II. ... i; suggest that SP cells isolated from NSCLC
cell lines and tumors have stem-like properties. The ob-
servation that EGFR .--., i i- affects stern-like functions
of SP cells is intriguing, given that several EGFR
tyrosine-kinase inhibitors have efficacy L I;.. i NSCLCs
[34,35]. IJi. :*...1 EGFR appears to regulate Sox2


Page 10 of 15







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


levels, through the Src-Akt pathway; Sox2 has been
shown to be regulated by Akt in ES cells, through the in-
hibition of proteasomal degradation [36]. Consistent
with these results, our observation suggest that inhib-
ition of EGFR-Src-Akt signaling downregulates Sox2
levels along with a reduction in ABCG2 levels. This de-
crease in ABCG2 expression upon EGFR inhibition is
probably a causal effect of Sox2 depletion-mediated dif-
ferentiation of SP into MP cells.
The fact that EGFR-pathway inhibition resulted in spe-
cific depletion of Sox2 without any significant effect on
Oct4 or Nanog expression suggests that their expression
may be regulated through independent mechanisms in
NSCLC SP cells. Our results as well as an earlier report
[37] suggest that Sox2 is expressed in both low as well
as high stage adenocarcinomas irrespective of their
grades. However, Oct4 or Nanog expression was found
to be associated only with the high grade lung adenocar-
cinoma and not expressed in low grade tumors [17,37].
Therefore, we predict that the EGFR pathway inhibition
may exert its favorable effects only for those tumors
where Sox2 is the major determinant in controlling the
self-renewal of CSCs. Interestingly, a recent study


showed that the ectopic overexpression of Oct4 and
Nanog increases the tumor initiating property of A549
cells [17]. In agreement with these reports, we find that
specific and independent depletion of Oct4 or Nanog
also resulted in decrease in SP phenotype but in a cell
type dependent fashion (Data not shown). Two recent
reports demonstrate that ectopic expression of Sox2
increased the frequency of side population cells and
tumor formation in mouse and human NSCLC cell lines
[33,38]. These reports strongly suggest that Sox2 expres-
sing cells harbor the stem cell-like properties. Our ob-
servation further strengthens this postulation where we
demonstrate that Sox2 depletion was sufficient to inhibit
the self-renewing property SP cells in all the three
NSCLC cell lines.
In addition to the mutation in EGFR signaling, per-
turbation of p53 activity is another important event
occurs in initiation and progression of NSCLCs [39]. Re-
cently, p53 is shown to have certain roles in promoting
the differentiation of human embryonic stem cell
through repression of factors like Oct4, Klf4, Lin28A,
and Sox2 [40]. However, there is not much information
available on the direct role of p53 transcriptional


Page 11 of 15


A Squamous cell carcinoma Adenocarcinoma
SStaget Sge IV Stage 1ll stage iv

i'-s,




Metastatic Adenocarcinoma






MV ..A>." l. _Y I-.,
Lymph Node Bone Adrenal Brain

B C p=0.01
Lung Adenocarcinoma Sox2 Staining 2.5 ----1
Stage Negaive Posllve 20 (3.3+0.2)
I and II (n=73) 329% 67 1i%
IV Primary site (n-45) 26.7% 73.3% 1.5
Metastatic (n17) 17.6% 82.4% 1.0.
o (1.5+0.2) (1.60.2)
1.0 -
X0.5
0
Stage Primary Metastatic
Stage IV
Figure 7 Sox2 expression correlates with metastatic progression of adenocarcinoma. (A) Sox2 expression in Lung squamous cells
carcinoma, adenocarcinoma and metastatic adenocarcinoma was analyzed from stage 1/11 or stage IV patients samples by immunohistochemistry.
(B) Semi-quantitative scoring was performed for adenocarcinoma samples and the tumors with score of one or more was considered positive for
Sox2 expression and listed. (C) Median score for Sox2 expression was calculated and plotted for different stages of NSCLC progression. The mean
(+ SD) of score is mentioned in parenthesis. Metastatic tumors showed I (p=0.01) higher expression of Sox2.







Singh et al. Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


activities in regulating Sox2 expression in stem-like cells
in cancer, and would be interesting to explore in future.


Conclusions
Figure 8 summarizes the role of Sox2 in SP cell biology
and tumor growth. While certain frequency of isolated
SP cells from NSCLC exhibit stem cell-like properties
and can form metastatic tumors, more differentiated MP
cells are greatly impaired in their ability to generate
tumors. Further, inhibition of EGFR pathway including
Src and PI3-kinase could strongly inhibit the expression
of Sox2, suppressing the self-renewal properties of SP
cells. Therefore, relative Sox2 expression and functions
within the tumor-CSCs may be a major determinant in
EGFR-targeted therapy against NSCLCs. This informa-
tion might also be potentially useful to overcome the
acquired resistance to EGFR therapies, by targeting
downstream targets of EGFR signaling, including Sox2.
Additional investigations in this direction might lead to
the development of more effective therapeutic agents to
combat NSCLC, especially those harboring EGFR
mutations.


Materials and methods
Cell lines and tumor samples
H1650, and H1975 cell lines were obtained from
ATCC and maintained in RPMI or DMEM contain-
ingl0% fetal bovine serum (FBS; Mediatech) in 5%
CO2 at 37C. Human tumor xenografts were obtained
from SA laboratory.


Dye efflux
assay


00

MP cells




SP cells


Inhibitors, siRNAs and antibodies
Gefitinib, Erlotinib, BIBW2992 and Dasatinib were pur-
chased from LC laboratories. PP2 and Fumitremorgin C
(FTC) were purchased from Sigma Inc. In the present
study, Gefitinib or erlotinib is used at 500 nM, dasatinib
or BIBW2992 is used at 200 nM and PP2 is used at
1 tM dose. siRNA against EGFR, Src family kinases, Akt
and Sox2, Oct4 and Nanog was purchased from Santa
Cruz Biotechnology or OriGene Technology Inc. Pri-
mary antibodies against Sox2 (#3579), Oct4 (#2750),
Nanog (#4903), Phos-Src-pY416 (#2101), pERKI/2
(#4376) and phospho-AKT-pS473 (#4058) were pur-
chased from Cell Signaling Technology; Phos-EGFR-
pY168 (#44788G) from Invitrogen; EGFR neutralizing
antibody (#05-101) from Milipore and isotype matched
mouse IgG were purchased from Biolegend.

RNA preparation and qRT PCR analysis
RNA preparation and RT-PCR analysis was performed
as described earlier [41]. Fold inductions were calculated
using the formula 2-(ddCt) using GAPDH as internal con-
trol gene. The gene-specific primer pairs were as follows.
ABCG2 (F) 5'-CAC AAG GAA ACA CCA ATG GCT-3;
ABCG2 (R) 5'-ACA GCT CCT TCA GTA AAT GCC
TTC-3'; Oct4 (F) 5'-ACA TCA AAG CTC TGC AGA
AAG AAC-3; Oct4 (R) 5'-CTG AAT ACC TTC CCA
AAT AGA ACC C-3; Sox2 (F) 5'-GGG AAA TGG GAG
GGG TGC AAA AGA-3; Sox2 (R) 5'-TTG CGT GAG
TGT GGA TGG GAT TGG-3; Nanog (F) 5'-AGA AGG
CCT CAG CAC CTA-3; Nanog (R) 5'-GGC CTG ATT
GTT CCA GGA TT-3'; Twist (F) 5'-CTC GGA CAA


SNon-
tumorigenic
SNon-
metastatic


Highly
tumorigenic
Highly
metastatic


_ Loss of


0


S Self-renewal Sox2

T Loss of SP phenotype
PI3K Loss of self-renewal
inhibition
Figure 8 A schematic depiction of the signaling events that regulate the biology of SP cells. Isolated SP cells form metastatic tumors but
MP cells do not. Inhibiting EGFR, Src or P13-kinase I impairs the self-renewal properties of SP cells.


Orthotopic
implantation
into the lungs


Page 12 of 15






Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


GCT GAG CAA GAT TCA GA-3; Twist (R) 5'-CGT
GAG CCA CAT AGC TGC AGC-3; Slug (F) 5'- ACA
CAT TAC CTT GTG TTT GCA AGA TCT-3; Slug (R)
5'- TGT CTG CAA ATG CTC TGT TGC AGT G-3;
Snail (F) 5'- CCT CAA GAT GCA CAT CCG AAG
CCA C-3; Snail (R) 5'- CCG GAC ATG GCC TTG TAG
CAG C-3; GAPDH (F) 5'-GGT GGT CTC CTC TGA
CTT CAA CA-3; GAPDH (R) 5'-GTT GCT GTA GCC
AAA TTC GTT GT-3'.


Hoechst 33342 dye efflux assay for SP analysis and cell
sorting
Adherent cells were harvested using accutase reagent
m Inc). Human Tumor tissue grown in athymic
nude mice was minced, enzymatically digested with
0.2% ..*..i :, ,-._ IV (Worthington Biochemical Corpor-
ation) prepared in 10% FBS containing medium for
60 min at 37C. The digest was further disaggregated
by passing through 10 ml pipette several times and fil-
tered through 100/70-pm cell strainer to obtain a sin-
gle cell suspension. Cells were washed and resuspended
in HBSS at 1X106 cells/ml density and incubated with
4 pg/ml of Hoechst 33342 dye (Invitrogen) for 90 min
at 37C in presence or absence of 1 pM FTC, as
described by Goodell et al. [21]. Cells were incubated
with 2 gg/ml Propidium iodide (PI;; :-.. m. Inc) before
analysis to visualize and exclude the non-viable cells.
The Hoechst 33342 dye was excited at 350 nm using
UV laser and its fluorescence was analyzed using 400-
500 nm BP filter for blue emission and 640-680 nm
BP filter in combination with 655 nm LP-filter for red
emission. Flow cytometers from BD Biosciences were
used for data acquisition. Data were acquired using
LSRII or FACS Vantage ii i,,. I and sorted using FACS
Vantage (DiVa) cell sorter. Data analyses were done
using FlowJo software (Tree Star). Cell cycle analyses
for fixed cells were performed for PI stained cells using
\ ..l. i.I.- method with similar protocol as .I ..1i.1
earlier [41].


Sphere formation or Self renewal assay
Sorted SP or MP cells were plated in 96 well plates at
the density of 10,000 cells/ml (1000 ....11 .- .II in 100 pl
medium) in serum free stem cell selective media
(DMEM/F12K (1:1) 'Tl. I..-; ,,. supplemented with IX-
N2 supplement i'i... 1n.. ...1. .10 .., ',,1 EGF and 10 ng/
ml bFGF (Sigma)) and allowed to grow as spheres for
10 days. Images of the spheres were taken using phase
contrast microscope (Nikon) and total numbers were
counted. To study the effect of drugs on the self-renewal
of SP cells, drugs were added to the respective wells on
day 1 and 5 and size and number of the spheres were
analyzed on day 10.


Immunofluorescence
For immunostaining, spheres were transferred to poly
D-lysine/Laminin coated glass surface for 18 h. For
monolayer cultures, cells were directly plated over the
poly D-lysin/Laminin coated glass surface and cultured
or treated in stem cell selective media as indicated. Im-
munofluorescence staining was performed as described
previously [42]. Cells were observed using a Leica TCS
SP5 confocal microscope (Leica Microsystems) at x 630
magnification.

Immunohistochemistry
Human lung cancer tissue microarray (TMA) slides with
stage I/II or stage IV NSCLC patients were obtained
through Lung Cancer Specialized Program of Research
Excellence (SPORE). TMA slide with stage I/II tumor
samples contained usable cores from 193 patients, and
TMA slide with stage IV tumor samples contained usable
cores from 103 patients including 17 adenocarcinoma
samples from the metastatic sites. The Immunohisto-
chemical i ;,,:..i* was performed as 1I ..I. .i [42]. The
samples were scored by a pathologist (D. Coppola). The
....i.:, .i...: .!i... score was reached by taking into consid-
eration both cellularity and intensity of expression (semi-
i: .**'.; .1r- score = cellularity x intensity). Cellularity was
scored as follows: a score of 3 equals to greater than 66%
cellularity, a score of 2 equals to 34%-65% cellularity, and
a score of 1 equals to less than 33% cellularity. Intensity
was scored as follows: a score of 3 equals to strong inten-
sity, a score of 2 equals to moderate intensity, and a score
of 1 equals to weak intensity I The score of 1 or above
was considered as positive expression of Sox2. The images
were captured at x 200 magnification.

In vivo tumor formation assay and bioluminescence
imaging
5-weeks-old female SCID-beige mice were used for these
experiments under an IACUC approved protocol. For
orthotopic implantation of tumor cells, sorted SP or MP
cells from A549 cell line stably expressing luciferase
gene (A549-Luc) were washed with serum-free DMEM-
F12K medium and resuspended at indicated numbers in
HBSS containing 500 '.1,,1 growth factor reduced
Matrigel. Surgical procedure for orthotopic lung implant-
ation was followed as .'!-_. i. 1 earlier for intrapulmon-
ary implantation of tumor cells with some modifications
[43]. C:.. if; i11.. cells were inoculated with 1 ml syringes
with 30-gauge hypodermic in._._.:l. in an open technique
under direct visualization into the right lung tissue of
SCID mice anesthetized by gas anesthesia (3% isoflur-
ane). Tumor growth/metastases were imaged weekly
using bioluminescence by IVIS-200 imaging system from
Caliper Corporation. Mice were anesthetized and 30 mg/
Kg of D-luciferin in PBS was administered by


Page 13 of 15







Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


intraperitoneal (i.p.) injection. Ten minutes after injec-
tion, bioluminescence was imaged with a charge-coupled
device camera (Caliper) with an imaging time of 2 min.
At the end of the experiment, or when mice become
moribund, all of the mice were euthanized and individual
organs harvested for evaluation of tumor size; distant
metastases was determined by bioluminescence of luci-
ferase expressing cells.


Statistical methods
Data were presented as the mean + standard deviation
(SD). To assess the statistical significance of i'11' ,.
student's t test was performed. The data were consid-
ered statistically significant when the P value was less
than 0.05.


Additional file


Additional file 1: Figure S1. BIBW2992 Inhibits EGFR phophorylation
H1975 cells were treated wth 500 nM geftiinib or 200 nM BIBW2992 for
5 days EGFR phosphorylation and total EGFR expression was detected in
presence or absence of drug treatment Figure 52 Downregulation of
Sox2 expression after EGFR and Sc inlh bt:on H1650-SPAdh cells were
treated plated over PD [ amtnin coated gcass surface and treated with
indicated drugs for 4 days (A) Expression of Sox2 was monitored by
mmunofiuorescence confocal imaging Isotype antibody was used to
show the specify c staining of Sox2 (B) Number of Sox2 positive cels for
each treatment condition were converted into percentage and plotted P
values were calculated from three different experiments and suggested a
significant decrease in Sox2 positive ceils after EGFR and Src inhibition
(C) Under similar treatment conditions cel s were stained with Nanog
specific antibodies Drug tieatmeni did not a ter Ihe expes sion of Nanog
in H1650-SPAdh cells Figure 53 Depetion of Sox2 expression
suppresses SP frequency (A) A549, 111650 and 11975 cells were
Iransiently transfected with second set siRNA (purchased form Origene)c
48 h after tiansfection, cells were analyzed for SP frequency Similar to
first set of siRNA (purchased from SantaCruz), depletion of Sox2 rested
in significant decrease in SP frequency in NSCLCs (B) NSCLC cells were
transfected w3th Sox2 SIRNA and ABCG2 expression was detected by
western blotting P Actin was used as ,nterna contro for equal loading *
p<005


Competing interest
We do not have any conflict of interest

Authors' contributions
SS conducted the experiments and wrote the initial version of the
manuscript; JT and NBS conducted certain experiments; DC did pathological
analysis of Ihe samples; EH proved intel lecua inputl; SA provided human
tumor xenografts and input; SC directed the project and finalized the
manuscript A: authors read and approved the final manuscript

Acknowledgments
We thank Jennifer Gemmer for technical support These studies were
supported by the grant CA127725 from the NC to SPC Support of the Lung
Cancer SPORE, National Functional Genomrics Center and the Core Facilities
at the Moff:tt Cancer Center is greatly appreciated

Author details
Department of Tumor Biology, H Lee Moffitt Cancer Center and Research
Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA 2Department of
Anatomic .1 1 H Lee Moffitt Cancer Center and Research Institute,
12902 Magnolia Drive, Tampa, FL 33612, USA Department of Thoracic
Oncology, H Lee Moffitt Cancer Center and Research Institute, 12902
Magnolia Drive, Tampa, FL 33612, USA 4Current address National Institute of


Biomedical Genomics, 2nd floor, Netaji Subash Sanatorium, Kalyani 741251,
India Current address Department of Surgery, University of Florida, 1600
SW Archer Rd, Rm 6175, Gainesville, FL 32610-0109, USA

Received: 14 June 2012 Accepted: 18 September 2012
Published: 25 September 2012


References
1 Parkin DM, Bray F, Ferlay J, Pisani P Global cancer statistics, 2002. CA
Cance J lin 2005, 55:74-108
2 emal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ Cancer statistics,
2008. CA Cance' J Cin 2008, 58:71-96
3 ernal A, Thun M1, Ries I A, Howe eir Hr K, Center MM, Ward E, Wu XC,
Eheman C, Anderson R, et a\' Annual report to the nation on the status of
cancer, 1975-2005, featuring trends in lung cancer, tobacco use, and
tobacco control. J Nat/Cancer nst 2008, 100:1672-1694
4 Siegd R, Naishadham D, Jernal A Cancer statistics, 2012. CA Cancer Clin
2012, 62:10-29
5 Bonnet D, Dick JE Human acute myeloid leukemia is organized as a
hierarchy that originates from a primitive hematopoietic cell. Nat Med
1997, 3:730-737
6 Clarke M, Dick i DFirks PB, Eaves CJ, Jamieson CH, Jones DI, Visvader ,
Weissman IL, Wahl GMi Cancer stem cells-perspectives on current status
and future directions: AACR Workshop on cancer stem cells. Cancer Res
2006, 66:9339-9344
7 Clevers H Stem cells, asymmetric division and cancer. Nat Genet 2005
37:1027-1028
8 Morrison J, Kimble J Asymmetric and symmetric stem-cell divisions in
development and cancer. Nature 2006, 441:1068-1074
9 Salnikov AV Gladkich J, Moldnhauer G, Volii M, Mattern J, Heir I' CD133 is
indicative for a resistance phenotype but does not represent a
prognostic marker for survival of non-small cell lung cancer patients.
lni J Cancer 2010. 126:950-958
10 Singh A, Settleman J' EMT, cancer stem cells and drug resistance:
an emerging axis of evil in the war on cancer. Onrogene 2010
29:4741-4751
I1 io MM, Ng AV, Lam S, I lung JY Side population in human lung cancer
cell lines and tumors is enriched with stem-like cancer cells. Cancer Res
2007, 67:4827-4833
12 Golebiewska A, Brons NI, Bjerkvig R, Niclou SP' Critical appraisal of the
side population assay in stem cell and cancer stem cell research. Cell
Stem Cel 2011,8:136-147
13 Wu C. Alrnan BA Side population cells in human cancers. Cancer iett
2008, 268:1-9
14 Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath Morris JJ
Lagutina 1, Gicsvld GC, Osawa M, Nakau(hi H, Sorrentino BP The ABC
transporter Bcrpl/ABCG2 is expressed in a wide variety of stem cells and
is a molecular determinant of the side-population phenotype. Nat Med
2001,7:1028-1034
15 Kirn J, Chu i, Shen X, Wang J, Orkin SH' An extended transcriptional
network for pluripotency of embryonic stem cells. Cell 2008,
132:1049-1061
16 Schoenhals M, Kassambara A, De Vos I, Hose D, Moreaux J, Klein B
Embryonic stem cell markers expression in cancers. Biochem Biophys Res
Cmmrnrnu 2009, 383:157-162
17 Chiou Si1, Wang ML, Chou YT, Chen Ci, I ong CF, ilsieh WJ, Chang iT,
Chen YS, Lin NT, T su IS, Wu CW' Coexpression of Oct4 and Nanog
enhances malignancy in lung adenocarcinoma by inducing cancer stem
cell-like properties and epithelial-mesenchymal transdifferentiation.
Cancer Res 2010, 70:10433-10444
18 Jin X, Yin Kim SH, Sohn YW, Beck S, Lim YC, Nam DH, Choi Y, Kim Ht
EGFR-AKT-Smad signaling promotes formation of glioma stem-like cells
and tumor angiogenesis by ID3-driven cytokine induction. Cancer Res
2011, 71:7125-7134
19 Soeda A, Inagaki A, Oka N, Ikegame Y, Aoki H, Yoshimura S, Nakashima S,
Kunisada i, Iwarr!a Epidermal growth factor plays a crucial role in
mitogenic regulation of human brain tumor stem cells, j Bi' Chem 2008,
283:10958-10966
20 ilu Q, Zhang L, Wen J, Wang S, Li M, Feng R, Yang X, Li L The EGF
receptor-sox2-EGF receptor feedback loop positively regulates the
self-renewal of neural precursor cells. Stem Cels 2010, 28:279-286


Page 14 of 15








Singh et al Molecular Cancer 2012, 11:73
http://www.molecular-cancer.com/content/11/1/73


21 Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC Isolation and
functional properties of murine hematopoietic stem cells that are
replicating in vivo. Exp Med 1996, 183:1797-1806
22 Robey RW, Steadman K, Polgar O, Morisaki K, Blayney M, Mistry P, Bates SE
Pheophorbide a is a specific probe for ABCG2 function and inhibition.
Cancer Res 2004, 64:1242-1246
23 Pastrana E, Silva Vargas V, Doetsch F Eyes wide open: a critical review of
sphere-formation as an assay for stem cells. Cell Stem Cel 2011,
8:486-498
24 Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM,
Cusimano MD, Dirks PB Identification of human brain tumour initiating
cells. Nature 2004, 432:396-401
25 Mani SA, Guo W, Liao MJ, Eaton EN, Ayanan A, Zhou AY, Brooks M,
Reinhard F, Zhang CC, Shipitsin M, et al The epithelial-mesenchymal
transition generates cells with properties of stem cells. Cell 2008,
133:704-715
26 Moreno-Bueno G, Portillo F, Cano A Transcriptional regulation of cell
polarity in EMT and cancer. Oncogene 2008, 27:6958-6969
27 Polyak K, Weinberg RA Transitions between epithelial and mesenchymal
states: acquisition of malignant and stem cell traits. Not Rev Cancer 2009,
9:265-273
28 Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J,
Head R, Lee M, Bernstein M, et al Glioma stem cell lines expanded in
adherent culture have tumor-specific phenotypes and are suitable for
chemical and genetic screens. Cell tem Cell 2009, 4:568-580
29 Zhang J, Kalyankrishna S, Wislez M, Thilaganathan N, Saigal B, Wei W, Ma L,
Wistuba II, Johnson FM, Kurie JM SRC-family kinases are activated in non-
small cell lung cancer and promote the survival of epidermal growth
factor receptor-dependent cell lines. Am J Patho 2007, 170:366-376
30 Kim LC, Song L, Haura EB Src kinases as therapeutic targets for cancer.
Not Rev Clin Oncol 2009, 6:587-595
31 Robertson LK Mireau LR, Ostergaard HL A role for phosphatidylinositol
3-kinase in TCR-stimulated ERK activation leading to paxillin
phosphorylation and CTL degranulation. J mmuno/ 2005, 175 8138-8145
32 Wang CC, Cirit M, Haugh JM P13K-dependent cross-talk interactions
converge with Ras as quantifiable inputs integrated by Erk. Mol Syst Biol
2009, 5:246
33 Nakatsugawa M, Takahashi A, Hirohashi Y, Torigoe T, Inoda S, Murase M,
Asanuma H, Tamura Y, Morita R, Michifuri Y, et al SOX2 is overexpressed in
stem-like cells of human lung adenocarcinoma and augments the
tumorigenicity. Lab invest 2011, 91:1796-1804
34 Gazdar AF, Minna JD Deregulated EGFR signaling during lung cancer
progression: mutations, amplicons, and autocrine loops. Cancer Prev Res
2008, 1:156-160
35 Amann J, Kalyankrishna S, Massion PP, Ohm JE, Girard L, Shigematsu H,
Peyton M, Juroske D, Huang Y, Stuart Salmon J, et al Aberrant epidermal
growth factor receptor signaling and enhanced sensitivity to EGFR
inhibitors in lung cancer. Cancer Res 2005, 65:226-235
36 Jeong CH, Cho YY, Kim MO, Kim SH, Cho EJ, Lee SY, Jeon YJ, Lee KY, Yao K
Keum YS, et al Phosphorylation of Sox2 cooperates in reprogramming to
pluripotent stem cells. Stem Cells 2010, 28:2141-2150
37 Sholl LM, Barletta JA, Yeap BY, Chirieac LR, Hornick JL Sox2 protein
expression is an independent poor prognostic indicator in stage I lung
adenocarcinoma. Am J Surg Pathol 2010, 34:1193-1198
38 Xiang R, Liao D, Cheng T, Zhou H, Shi Q, Chuang TS, Markowitz D, Reisfeld
RA, Luo Y Downregulation of transcription factor SOX2 in cancer stem
cells suppresses growth and metastasis of lung cancer. Br Cancer 2011,
104:1410-1417
39 Mogi A, Kuwano H TP53 mutations in nonsmall cell lung cancer.
J Biomed Biotechnol 2011, 2011:583929
40 Jain AK Allton K, lacovino M, Mahen E, Milczarek RJ, Zwaka TP, Kyba M,
Barton MC p53 regulates cell cycle and microRNAs to promote
differentiation of human embryonic stem cells. PLoS Biol 2012,
10:el001268
41 Singh S, Davis R, Alamanda V, Pireddu R, Pernaza D, Sebti S, Lawrence N,
Chellappan S Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis
in metastatic melanoma cells and synergizes with dacarbazine. Mol
Cancer Ther 2010, 9:3330-3341
42 Dasgupta P, Rizwani W, Pillai S, Davis R, Banerjee S, Hug K, Lloyd M, Coppola
D, Haura E, Chellappan SP ARRB1-mediated regulation of E2F target


genes in nicotine-induced growth of lung tumors. J Nao Cancer Inst 2011,
103:317-333
43 Doki Y, Murakami K, Yamaura T, Sugiyama S, Misaki T, Saiki I Mediastinal
lymph node metastasis model by orthotopic intrapulmonary
implantation of Lewis lung carcinoma cells in mice. Br Cancer 1999,
79:1121-1126

doi:10.1186/1476-4598-11-73
Cite this article as: Singh et al EGFR/Src/Akt signaling modulates Sox2
expression and self-renewal of stem-like side-population cells in
non-small cell lung cancer. Molecular Cancer 2012 11 73


Page 15 of 15


Submit your next manuscript to BioMed Central
and take full advantage of:

* Convenient online submission
* Thorough peer review
* No space constraints or color figure charges
* Immediate publication on acceptance
* Inclusion in PubMed, CAS, Scopus and Google Scholar
* Research which is freely available for redistribution


Submit your manuscript at
www.biomedcentral.com/submit 0 1 BiIIoMl Central







Supplementary Figures:


.0

C
U 0

l w S pY1068-EGFR


r Total EGFR

H1975

Supplemetary Figure S1

Supplementary Figure S1: BIBW2992 inhibits EGFR phophorylation. H1975 cells were
treated with 500 nM gefitinib or 200 nM BIBW2992 for 5 days. EGFR phosphorylation and total
EGFR expression was detected in presence or absence of drug treatment.


















C


Sox2 positive cells (%)
0 25 50 75 100

p-0.03


p40.003
Sp 0.1
mmooo


Supplementary Figure S2

Supplementary Figure S2: Downregulation of Sox2 expression after EGFR and Src
inhibition. H1650-SPAdh cells were treated plated over PDL-Laminin coated glass surface and
treated with indicated drugs for 4 days. (A) Expression of Sox2 was monitored by
immunofluorescence confocal imaging. Isotype antibody was used to show the specific staining
of Sox2. (B) Number of Sox2 positive cells for each treatment condition were converted into
percentage and plotted. P values were calculated from three different experiments and


BIB




Istp oto





BlV




Istp oto









suggested a significant decrease in Sox2 positive cells after EGFR and Src inhibition. (C) Under

similar treatment conditions cells were stained with Nanog specific antibodies. Drug treatment

did not alter the expression of Nanog in H1650-SPAdh cells.











' 1.25 OControl SiRNA *Sox2 SiRNA z Z Z


0.75 oo ) o o
-0 I U ) OT U (0
S0.25 0 0 Actin


S0.25


A549 H1650 H1975
Supplementary Figure S3


Supplementary Figure S3: Depletion of Sox2 expression suppresses SP frequency. (A)

A549, H1650 and H1975 cells were transiently transfected with second set siRNA (purchased

from Origene). 48 hr after transfection, cells were analyzed for SP frequency. Similar to first set

of siRNA (purchased from SantaCruz), depletion of Sox2 resulted in significant decrease in SP

frequency in NSCLCs. (B) NSCLC cells were transfected with Sox2 SIRNA and ABCG2

expression was detected by western blotting. P-Actin was used as internal control for equal

loading. p<0.05.




Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E1IHQGEVD_FKAF2T INGEST_TIME 2013-01-22T15:47:13Z PACKAGE AA00013233_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES



PAGE 1

1 Supplementary Figure s: Supplementary Figure S 1: BIBW 2992 inhibits EGFR phophorylation H1975 cells were treated with 500 nM gefitinib or 200 nM BIBW2992 for 5 days. EGFR phosphorylation and total EGFR expression was detected in presence or absence of drug treatment.

PAGE 2

2 Supplementary Figure S2 : Downregulation of Sox2 expression after EGFR and Src inhibition. H1 650 SPAdh cells were treated plated over PDL Laminin coated glass surface and treated with indicated drugs for 4 days. (A) Expression of Sox2 was monitored by immuno fluorescence confocal imaging. Isotype antibody was used to show the specific staining of Sox2. (B) Number of Sox2 posi tive cells for each treatment condition were converted into percentage and plotted. P values were calculated from three different experiments and

PAGE 3

3 suggested a significant decrease in Sox2 positive cells after EGFR and Src inhibition. (C) Under similar treat m,ent conditions cells were stained with Nanog specific antibodies. Drug treatment did not alter the expression of Nanog in H1650 SPAdh cells.

PAGE 4

4 Supplementary Figure S3 : Depletion of Sox2 expression suppresses SP frequency (A) A549, H1650 and H1975 ce lls were transiently transfected with second set siRNA (purchased from Origene). 48 hr after transfection, cells were analyzed for SP frequency. Similar to first set of siRNA (purchased from SantaCruz), depletion of Sox2 resulted in significant decrease in SP frequency in NSCLCs. (B) NSCLC cells were transfected with Sox2 SIRNA and ABCG2 expression was detected by western blotting. Actin was used as internal control for equal loading. p<0.05.



PAGE 1

RESEARCHOpenAccessEGFR/Src/AktsignalingmodulatesSox2 expressionandself-renewalofstem-like side-populationcellsinnon-smallcelllungcancerSandeepSingh1,4,JoseTrevino1,5,NamrataBora-Singhal1,DomenicoCoppola2,EricHaura3,SonerAltiok2andSrikumarPChellappan1*AbstractBackground: Cancerstemcellsarethoughttoberesponsiblefortheinitiationandprogressionofcancers.In non-smallcelllungcancers(NSCLCs),Hoechst33342dyeeffluxingsidepopulation(SP)cellsareshowntohave stemcelllikeproperties.Theoncogeniccapacityofcancerstem-likecellsisinpartduetotheirabilitytoself-renew; howeverthemechanisticcorrelationbetweenoncogenicpathwaysandself-renewalofcancerstem-likecellshas remainedelusive.HerewecharacterizedtheSPcellsatthemolecularlevelandevaluateditsabilitytogenerate tumorsattheorthotopicsiteinthelungmicroenvironment.Further,weinvestigatediftheself-renewalofSPcells isdependentonEGFRmediatedsignaling. Results: SPcellsweredetectedandisolatedfrommultipleNSCLCcelllines(H1650,H1975,A549),aswellasprimary humantumorexplantsgrowninnudemice.SPcellsdemonstratedstem-likepropertiesincludingabilityto self-renewandgrowasspheres;theywereabletogenerateprimaryandmetastatictumorsuponorthotopic implantationintothelungofSCIDmice.Invitrostudyrevealedelevatedexpressionofstemcellassociatedmarkers likeOct4,Sox2andNanogaswellasdemonstratedintrinsicepithelialtomesenchymaltransitionfeaturesinSP cells.Further,weshowthatabrogationofEGFR,SrcandAktsignalingthroughpharmacologicalorgenetic inhibitorssuppressestheself-renewalgrowthandexpansionofSP-cellsandresultedinspecificdownregulationof Sox2proteinexpression.siRNAmediateddepletionofSox2significantlyblockedtheSPphenotypeaswellasits self-renewalcapacity;whereasothertranscriptionfactorslikeOct4andNanogplayedarelativelylesserrolein regulatingself-renewal.Interestingly,Sox2waselevatedinmetastaticfociofhumanNSCLCsamples. Conclusions: OurfindingssuggestthatSox2isanoveltargetofEGFR-Src-AktsignalinginNSCLCsthatmodulates self-renewalandexpansionofstem-likecellsfromNSCLC.Therefore,theoutcomeoftheEGFR-Src-Akttargeted therapymayrelyupontheexpressionandfunctionofSox2withintheNSCLC-CSCs. Keywords: Cancerstem-likecells,Side-populationcells,Self-renewal,EGFR,Sox2IntroductionDespitesignificanttherapeuticadvances,lungcancer causesthemaximumnumberofcancerrelateddeaths worldwide[1].IntheUnitedStates,~85%ofthepatients diagnosedwithNSCLCs,diewithinfiveyears[2-4],thus, highlightaneedforbetterunderstandingofthecellular andmoleculareventsunderlyingthegenesisofthisdisease.Cancerstemcellmodelhasemergedasaviable explanationfortheinitiationandprogressionoftheaggressivecancerslikeNSCLCs. Cancerstemcellmodelsuggeststhatcancerstem-like cells(CSCs)areasubpopulationofcellswithinthe tumorthathavethederegulatedpropertiesofnormal stemcellswithsustainedself-renewal,andcangenerate secondarytumorsthatrecapitulatetheheterogeneity anddiversityoforiginaltumor[5-8].CSCsareconsideredtoberesponsiblefortumorinitiation,propagation, recurrenceandresistancetotherapy[9,10].Hoechst 33342dyeexcludingcells,termedside-population(SP) cells,havebeendescribedasCSCsinavarietyoftumor *Correspondence: Srikumar.Chellappan@moffitt.org1DepartmentofTumorBiology,H.LeeMoffittCancerCenterandResearch Institute,12902MagnoliaDrive,Tampa,FL33612,USA Fulllistofauthorinformationisavailableattheendofthearticle 2012Singhetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Singh etal.MolecularCancer 2012, 11 :73 http://www.molecular-cancer.com/content/11/1/73

PAGE 2

types,includingNSCLCs[11],wheretheyhavebeen showntodisplayincreasedtumorigenicitywhentransplantedintoimmunocompromisedmice[12,13]ascomparedtomajorpopulation(MP)cells.SPphenotypeis dependentonthedifferentialabilityofcellstoeffluxthe Hoechst33342dyeviatheATP-bindingcassette(ABC) familyoftransporterprotein,mainlyABCG2(breast cancerresistanceprotein,BRCP1)whichisspecifically expressedonthecellmembraneofstemcellpopulations [14].Earlierstudieshavedemonstratedtheexistenceof SPcellsinvariousestablishedhumanNSCLCcelllines [11]buttheirabilitytogeneratetumorsinlungmicroenvironmentaswellasthesignalingpathwaysgoverning theirstem-likepropertiesremaintobeelucidated. ThetranscriptionfactorsOct4,Sox2andNanoghave beenidentifiedascoreregulatorsthatmaintaintheselfrenewalofembryonicstemcells[15].Thesefactorsare overexpressedinvariouscancersandareassociatedwith malignantprogressionandpoorprognosisincluding NSCLCs[16,17],suggestingthatthecoreregulatorsthat governnormalstemcellself-renewalmayalsomaintain thestem-likepropertiesofCSCsincancers.However, theinfluenceofNSCLCspecificoncogenicpathwayson theexpressionofthesefactorsremainsrelativelyunknown.AlterationsinEGFR-genelikecopynumber gainsand/ormutantallele-specificamplificationsare associatedwithNSCLCpathogenesis.Inaddition,activationofEGFRsignalingincreasestheself-renewalcapacityofneuralprecursorcellsandbraintumorstem cells[18-20] Inthisstudy,weprovidebiochemicaland biologicalevidencethatSPcellsisolatedfromestablishedhumanNSCLCcelllinesandtumorsarehighly enrichedinNSCLC-CSCsandEGFR-Src-Aktsignaling axiscontributessignificantlytotheself-renewalofSP cells.Interestingly,Sox2transcriptionfactoristhepredominantdownstreamtargetofEGFRsignalinginthese cellsandplaysamajorroleinself-renewalgrowthand expansionofSPcells,independentofOct4andNanog.ResultsSPcellsareenrichedwithtumorigeniccellsandproduce highlyinvasivetumorsInanattempttoidentifyNSCLCstem-likecells,SPanalysiswasconductedonfourprimaryhumanNSCLC explantsgrowninathymicnudemice.SPcellsappeared asawellseparatedpopulationasdescribedpreviously [21].AsshowninFigure1A,aspecificinhibitorof ABCG2[22],FumitremorginC(FTC)couldblocktheappearanceofSPphenotype.AllthefourtumorsamplesdisplayedthepresenceofSPcellswithvaryingfrequency rangingfrom0.6-3%,andcouldbesignificantlyblockedby FTC(Figure1B). Self-renewingnormalorcancer-stem-likecellscanbe expandedasnon-adherentsphereswhenculturedatlow densityinserumfree,stemcellselectivemedium;differentiatedcellsdonotgroworformspheresunderthese conditions[6,23,24].Theself-renewalpropertyofSP cellswasexaminedbyperformingsphereformation assayonsortedSPandMPcellsisolatedfromhuman tumorxenografts.WhilesortedSPcellswereableto growasspheres,MPcellshadmarkedlylesscapacityto growunderidenticalconditions(Figure1C). Attemptswerethenmadetoassessthepresenceof SPcellsinhumanNSCLCcelllines.Asshowninlater sections,A549(K-Rasmutant;wildtypeEGFRamplification),H1650(EGFRmutant;Exon20deletion: delE746-A750)andH1975(EGFRmutant;L858Rand T790Mmutations),containedSP-cellswithvaryingfrequency.AppearanceofSPcellswascompletelyblocked byFTC.SortedSPcellswereabletogrowasspheres whereasMPcellsshowedmarkedlyreducedcapability (Figure1D)suggestingthatNSCLC-SPcellsare enrichedwithCSCs. ThestemcelllikepropertyofNSCLC-SPcellswas verifiedbyevaluatingitsabilitytoformtumorsinthe lungmicroenvironment.SortedSPandMPcellsfrom A549cellsstablyexpressingtheluciferasegenewere orthotopicallyimplantedintotheleftlungofSCIDmice andtumorgrowthwasmonitoredfor12weeks.As showninFigure1E,SPcellsgeneratedprimarytumors inthelungmoreefficientlythanMPcells.Attheendof theexperiment,lungs,liver,kidneyandbrainwere excisedfromeachmouseandex-vivoimageswere examinedforthepresenceofmetastasizedluciferase positivecells.MiceinjectedwithSPcellsdemonstrated substantialtumorburdenthroughoutthelungsand showedluminescentmetastaticlociinliver,kidney (adrenals)andbrain(Figure1F).Incontrast,MPcells formedonlyoneluminescentfocusinthelungofone mouse(outoffive)injectedwith50000MPcellsand therewasnometastasis(Figure1F).Theseresultswere confirmedbyH&Estaining;further,tumorsformed withinthelungfromSPcells,recapitulatedthehistopathologyofadenocarcinomaasconfirmedbypositive stainingwithpan-keratinantibodyaswellasmucicarminedye(Figure1G).ThesedatasuggestedthatSPcells areenrichedwithtumorigeniccellsandcandevelop metastatictumors invivo .SPcellsexhibitmolecularmarkersofstem-likecellsRecentreportssuggestthatepithelialcellsacquirecancerstemcellpropertiesuponinductionofepithelialto mesenchymaltransition(EMT)[25].Toevaluate whetherSPcellsshowfeaturesofEMT,SPandMPcells fromA549,H1650andH1975wereexaminedforthe levelsofEMTmarkerslikeE-cadherin,Vimentinand Fibronectin.AsshowninFigure2A,ABCG2expression wassignificantlyhigherintheSPfractioninallthethreeSingh etal.MolecularCancer 2012, 11 :73 Page2of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 3

celllines.ThelevelsofE-cadherinwaslowerinH1650SPcellsascomparedtoMPcells,however,itwasundetectableinA549andunchangedinH1975cells.FibronectinwasdetectedathigherlevelsinA549andH1975SPcells,butundetectableinH1650cells.Vimentinlevel washigherinA549-SPcells,butlowinH1975and H1650SPcells.Althoughthelevelsvaryinacelltype dependentmanner,theseresultssuggestthat,SPcells expressproteinsindicativeofEMTwithoutanyexternal stimulitothecells(Figure2A).Themolecularbasisfor Figure1 SPcellsexhibitstem-likeproperties. ( A )FACSanalysisonsinglecellsuspensionofhumanNSCLCxenograftstainedwithHoechst 33342dyeshowingSPcells.FumitremorginC(FTC)inhibitedtheeffluxofthedyeandcausedthedisappearanceofSPcells.( B )SPcellfrequency inpresenceorabsenceofFTCinfourdifferenthumantumorxenografts.( C )SphereformationassayonSPorMPcellsgrowninstemcell selectivemediafor10days.Thepicturesofrepresentativespheresarepresented.Thebardiagramshowtheaverage(SD)numberofspheres formedfrom2000cells.( D )SPorMPcellsfromH1650andH1975celllineswereplatedinserumfreemediumsupplementedwithEGFand bFGFfor10daysunderself-renewalassayconditions.Thepicturesofrepresentativespheresaredepicted( E )IndicatednumberofSPandMP cellsfromA549-LuccelllinewereimplantedintotherightlungtissueofSCIDmice.Thetumorincidenceandmetastasiswasmonitoredfor 12weeksbybioluminescenceimaging.( F )Ex-vivoimagesofthelung,liver,kidney/adrenalsandbraincapturedattheendofexperiment. MetastasiswasprevalentinSPcellsimplantedmice.( G )H&E,pan-keratin(brownstaining)andmucicarmine(pinkstaining,indicatedbyarrow head)stainingofwholelungsfrommiceimplantedwithSPorMPcells. Singh etal.MolecularCancer 2012, 11 :73 Page3of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 4

thedifferentialexpressionoftheEMTmarkerswasthen examined.TranscriptionfactorslikeTwist,Slugand SnailhavebeendemonstratedtobecapableofcoordinatingtheEMTprogramduringembryonicdevelopment andincancers[26,27].Therefore,wenextassessedthe expressionofthesetranscriptionfactorsinSPandMP cells.Real-timePCRanalysisrevealedthatTwist,Slug andSnailtranscriptionfactorsareexpressedathigher levelsinSPcellsinallthethreeNSCLCcelllines (Figure2B,C,D). TheexpressionofOct4,Sox2andNanogtranscriptionfactorswasnextexaminedinSPcells.Real-time PCRanalysisshowedelevatedlevelsofABCG2,Oct4, Sox2,andNanogintheSPfractioninallthethree celllines.(Figure2E,F,G).Further,SPcellsfrom H1650cellsgrowingasspheresshowedexpressionof ABCG2,Oct4,Sox2andNanogproteinsbyfluorescencemicroscopy(Figure2H),indicatingtheundifferentiatedgrowthofself-renewingSPcellswithinthe spheres. Figure2 SPcellsexpressstemcell-likemarkers. ( A )WesternblotanalysisonSPandMPcellsfromA549,H1650andH1975celllinesforthe indicatedproteins. -Actinwasusedastheloadingcontrol.( B C D )SPandMPcellswereanalyzedbyrealtimeqRT-PCRmethodforthe expressionofTwist,SlugandSnailgenes.Average(SD)foldchangebetweenMPandSPcellswereplotted.( E F G )SPandMPcellswere analyzedbyrealtimeqPCRmethodfortheexpressionofABCG2,Oct4,Sox2andNanoggenes.Average(SD)foldchangebetweenMPandSP cellswereplotted.( H )Thespheresformedinself-renewalassaysofH1650SPcellswerestainedfortheexpressionofABCG2,Oct4,Sox2and Nanog.Confocalimageofoneofthez-stacksforasphereispresented.DAPIwasusedtostainthenucleus. Singh etal.MolecularCancer 2012, 11 :73 Page4of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 5

EGFRtyrosinekinaseinhibitorsdownregulateself-renewal andSPphenotypeExperimentswereconductedtoexplorethemolecular mechanismsinvolvedintheself-renewalofSPcells.Since aberrantEGFRsignalingisimplicatedwiththeinitiation andprogressionoflungcancer,wefirstassessedSPfrequencyandexpressionofABCG2inthepresenceofan antagonisticantibodyagainstEGFR.Cellsweremixed with10 g/mlanti-EGFRantibodyoranisotypecontrol andplatedin2%FBScontainingmediafor5days.BlockingEGF-receptorsresultedinasignificantdecreaseinSP frequencyinbothA549andH1650cells(Figure3A,B), alongwithdecreasedEGFRphosphorylationaswellas ABCG2expressioninboththecelllines(Figure3C).Confirmingtheseresults,depletionofEGFRexpressionbya siRNAresultedindecreasedSPfrequencyandABCG2expressioninA549,H1650andH1975cells(Figure3D,E). TofurtherevaluatewhetherEGFRsignalingcontributedtotheself-renewalpropertyofH1650SPcells,sphere formationassaywasconductedinthepresenceorabsenceofEGFRinhibitorsGefitiniborErlotinib.Asshown inFigure3F,inhibitionofEGFR-kinaseactivityby 500nMofGefitiniborErlotinib,demonstrateda5 – 7 fold(p<0.001)decreaseinthenumberofspheres;further thesizeofthesphereswasalsosignificantlyreduced. Asecondarypointmutationinexon20ofEGFR (T790M)isassociatedwithacquiredresistancetogefitiniborErlotinib,butthiscanbeovercomebytheirreversibleEGFR-tyrosinekinaseinhibitorBIBW2992 (BIBW).Wetestedtheeffectof500nMofgefitiniband 200nMofBIBWonEGFRphosphorylationandselfrenewalgrowthofSPcellsfromH1975cellline,which harborsgefitinib-resistant-T790Mmutationalongwith Gefitinibresponsive-L858Rmutationinexon21.Westernblotanalysisshowedthattyrosinephosphorylation ofEGFRwasinsensitiveto500nMconcentrationof gefitinib,whereassignificantdownregulationoccurred aftertreatmentwith200nMofBIBWinH1975cells (Additionalfile1:FigureS1).Consistentwiththis,BIBW couldsignificantlyinhibittheself-renewalofSPcells fromH1975cells(Figure3G).AdherentculturesofSPcellsmaintainstem-like propertiesToconductfurthermolecularstudiesonSPcells,we attemptedtoestablishadherentcellculturesofisolated SPcellsfromA549,H1975andH1650celllines,assuggestedforgliomastemcells[28].IsolatedSPcellswere platedonuncoatedorPoly-DLysine+Laminincoated cultureplatesinserumfree,stemcellmedia.While A549-SPandH1975-SPcellsdetachedfromthesurface, H1650-SPcellsgrewasanadherentculture.Asshown inFigure3A,H1650-SPcellsculturedonuncoatedsurfacefailedtomaintainSPphenotypewithhigh frequency(Figure4A,(i)),but80%ofthecellsmaintainedasSPcellsevenafter5passageswhenplatedon PDL+laminincoatedsurface(Figure4A,(ii);H1650SPAdhcells).H1650-SPAdhcellsculturedbackin5% FBScontainingmediumfor10dayscouldrecapitulate theproportionofSPandMPcellsfoundinparental H1650cells(Figure4A,(iii)),withaconcomitantreductioninexpressionofABCG2(Figure4B),aswellasOct4, Sox2andNanogmRNAasseenbyR-PCR(Figure4C). CellcycleanalysisshowedthatH1650-SPAdhcellswere slowcyclingcomparedtoparentalcells(Figure4D),havingapproximately20%highernumberofcellsinG0/G1phase;butuponserum-induceddifferentiation,H1650SPAdhcellsacquiredcell-cyclephasedistributioncomparabletoH1650-parentalcells(Figure4D). TreatmentofH1650-SPAdhcellswith200nMBIBW significantlysuppressedthenumberaswellasthesizeof spheres(Figure4E);atthesametime,treatmentwith 30 Mcisplatindidnotaffectthenumberorthesizeof thespheresformedbyH1650-SPcells,suggesting enhancedchemoresistanceofthesecells.Further,the sphere-formationabilityofSPwasnotalteredbythe ABCG2inhibitor,FTC,suggestingthatself-renewalof SPcellswasindependentofABCG2activity(Figure4E).InhibitionofEGFR-Src-AktsignalingdownregulatesSox2 expressionExperimentswereconductedtoexaminethedownstreamsignalingeventsfromEGFRthatmodulatesselfrenewalofSPcellsandwhetherthesepathwaysimpinge transcriptionfactorsassociatedwithstemness.RoleofcSrcintheprocesswasfirstexaminedsinceSrcisaltered inNSCLC[29,30].H1650-SPAdhcellsweretreatedwith EGFRorSrcTKIsandthelevelsofOct4andSox2was assessedbywesternblotting(Figure5A).EGFRinhibitionby500nMgefitinibor200nMBIBWaswellasinhibitionofSrcactivityby200nMdasatinibor1 M PP2markedlyreducedSox2expression;Oct4levelwas notaffected(Figure5A).Theseresultswereverifiedby immunoflorescenceexperiments.SimilartoOct4,there wasnosignificantdifferenceinNanogexpression;however,thenumberSox2positivecellsweresignificantly decreasedinresponsetothetreatmentofEGFR-and Src-TKIs(Additionalfile1:FigureS2). InhibitionofEGFRaswellasSrcsignalingresultedin decreasedphosphorylationofEGFR,Src,ERKandAkt (Figure5A).ContributionofERKandAktpathwaysto EGFRmediatedinductionofSox2wasnextexamined inH1650SPAdhcells.PhosphorylationofERKwas suppressedbyMEKinhibitorPD98059andAKTphosphorylationwassuppressedbythePI3-kinaseinhibitor,LY294002.However,PI3-Kinaseinhibited H1650SPAdhcellsalsoresultedinslightinhibitionin ERKphosphorylation(Figure5B).AsimilarobservationSingh etal.MolecularCancer 2012, 11 :73 Page5of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 6

Figure3 InhibitionofEGFRsuppressesSPfrequencyandself-renewal. ( A )H1650andA549cellsrevealedasignificantdecreaseinSP frequencyaftertreatmentwith10 g/mlEGFR-neutralizingantibodiesfor5days.( B )FrequencyofSPcellsincontrolantibody(1)andEGFRneutralizingantibodies(2)treatedcells.( C )WesternblotanalysisforABCG2andphospho-EGFRafterEGFR-neutralizingantibodytreatment.( D )SP frequencyinNSCLCcellstransfectedwithsiRNAagainstEGFRoracontrolsiRNA.( E )ExpressionofABCG2andEGFRinantibodytreatedcells. ( F and G )SPcellsweresortedandplatedforself-renewalassayinthepresenceorabsenceofindicateddrugs.Averagenumberofspheres generatedperwellfrom1000cellsisplotted(meanSD).Phasecontrastmicroscopyimagesofthespheresinpresenceorabsenceofdrugsare presented.*representthepvalueof<0.05;**representthepvalueof<0.01;***representthepvalueof<0.001. Singh etal.MolecularCancer 2012, 11 :73 Page6of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 7

Figure4 EstablishmentandcharacterizationofH1650-SPAdhcells. ( A )SPcellsfromH1650celllinewasplatedonnormaltissueculture plate(i)orpoly-D-lysine-laminin(PDL-Laminin)coatedsurface(ii)inserumfreemediumcontainingN2-supplement,EGFandbFGF.H1650-SPAdh cellsgrowinginself-renewingconditionwasculturedinserumfor5daystoinducedifferentiation,andreanalyzedforSPfrequency(iii).( B ) SeruminducesdifferentiationofSPAdhcellsasseenbyABCG2expressionand( C )realtimeqPCRanalysisforstemcellmarkers, ABCG2,Oct4, Sox2,Nanog .( D )Cellcycleanalysisforparental-H1650orH1650-SPAdhandserumdifferentiatedH1650-SPAdhcellsgrownonPDL-Laminin coatedsurface.HistogramswereplottedusingModFitprogram.( E )Theaveragenumberofspheresgeneratedfrom1000H1650-SPAdhcellsis plotted(meanSD).Phasecontrastmicroscopyimagesofthespheresinpresenceorabsenceofindicateddrugs. Singh etal.MolecularCancer 2012, 11 :73 Page7of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 8

Figure5 InhibitionofEGFRsignalingdownregulatesSox2expression(A)H1650-SPAdhcellsweretreatedwithEGFRorSrcinhibitors for4daysandwesternblotanalysiswasperformedfortheindicatedproteins. ( B )H1650-SPAdhcellsweretreatedwithMEKorPI3K inhibitorfor4daysandlevelsofSox2andactivationofAktorERKwasevaluatedbywesternblot.( C )A549andH1975cellsweretreatedwith MEKorPI3Kinhibitorfor5daysandthefrequencyofSPcellswereexaminedbySPanalysis.Datarepresentsthefoldchangeinmean(SD)of SPfrequency.( D )ABCG2expressionuponinhibitortreatmentasdetectedbywesternblotting.( E )SPcellfrequencyinNSCLCcelllines transfectedwithspecificsiRNAsagainstSrcorAKT.( F )ABCG2expressioninsiRNAtransfectedcellsasdetectedbywesternblotting.( G and H )SP cellsweresortedandplatedforself-renewalassayinthepresenceorabsenceofindicateddrugs.Averagenumberofspheresgeneratedfrom 1000cellsisplotted(meanSD).Phasecontrastmicroscopyimagesofthespheresinpresenceorabsenceofdrugsarepresented.*represent thepvalueof<0.05;**representthepvalueof<0.01;***representthepvalueof<0.001. Singh etal.MolecularCancer 2012, 11 :73 Page8of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 9

hasbeenreportedinearlierstudieswherePI3-KinasesignalingwasdemonstratedtoregulatetheERKphosphorylationinT-cell-receptor(TCR)signaling[31]and PDGFRmediatedsignaling[32].However,asshownin Figure5B,inhibitionofMEKactivitydidnotaffectthe levelsofSox2whilethePI3-kinaseinhibition,markedly reduceditslevelswithcorrespondingreductioninSPfrequency(Figure5C)andABCG2expression(Figure5D). TheseresultswereconfirmedusingsiRNAstoSrcand Akt.AsshowninFigure5E,SPfrequencywassignificantlydownregulatedinbothAktandSrcsiRNAtransfectedA549,H1650andH1975cellsascomparedtothe controlsiRNAtransfectedcells,withacorrespondingreductioninABCG2expression(Figure5F).Similarinhibitoryeffectswereobserveduponsilencingoftwoother Srcfamilymembers,FynandYes(datanotshown). TodeterminewhetherSrcorAktsignalingfacilitates self-renewalofSPcells,sphereformationassaywasconductedonSPcellsinpresenceorabsenceofSrcinhibitorsDasatiniborPP2,MEKinhibitorPD98059aswell asAktinhibitorLY294002.AsshowninFigures5Gand 5H,Src-kinaseinhibitorsdasatiniborPP2,aswellas PI3K/AktinhibitorLY294002showedasignificant(5 – 7 fold;p<0.001)decreaseinsphereformation;MEKinhibitionbyPD98059didnothaveanysignificanteffect onself-renewal.Theaveragesizeofthespheresformed wasfoundtobe7 – 10foldssmallerthantheuntreated cells.Collectively,thesedataindicatedthatinhibitionof EGFR/Src/AktsignalingresultsindepletionofSox2expressionanddecreasedself-renewalofSPcells. SuppressionofSox2expressionissufficienttoinhibitthe self-renewalofSPcells SinceinhibitionofEGFR/Src/Aktsignalingspecifically downregulatedtheexpressionofSox2,weexaminedthe contributionofSox2totheself-renewalofH165SP-Adh cells.TransienttransfectionofEGFRandSrcsiRNAin H1650-SPadhcellsreducedEGFRexpressionby60% andSrcexpressionby50%.ReductioninEGFRorSrc expressiondecreasedthelevelsofSox2by50%and40% respectively;theexpressionofOct4andNanogwasnot altered(Figure6A).Inaddition,depletionofEGFRor Figure6 Sox2isnecessaryformaintainingtheself-renewalofSPcells. ( A )H1650-SPAdhcellsweretransfectedwithnon-targetingcontrol siRNA,orsiRNAagainstEGFR,Src,orSox2andwesternblotanalysiswasperformedforSox2,Oct4,EGFRandSrcexpression.( B )siRNAtransfected cellswereplatedforself-renewalassayandanalyzedafter5daysofplating.Averagenumberofspheresgeneratedperwellfrom1000cellsis plotted(meanSD).( C )NSCLCcelllinesweretransfectedwithcontrolorSox2siRNAsandSPfrequencywasevaluated.Averagefoldchangein SP-frequency(SD)isplotted.( D )DecreaseinABCG2expressioninSox2siRNAtransfectedcellswasdetectedbywesternblotting.TheSox2 expressionwasevaluatedbyqRT-PCRandresultsarepresentedasbardiagram. Singh etal.MolecularCancer 2012, 11 :73 Page9of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 10

SrcbysiRNAsuppressedthesphereformationby2 – 3 folds(Figure6B) Tofurtherexplorethefunctionof Sox2inself-renewalofSPcells,wedepletedSox2expressioninH1650-SPadhcells.Transienttransfectionof Sox2siRNAreducedtheexpressionofSox2by60% (Figure6A).DepletionofSox2expressiondidnotsignificantlyaltertheexpressionofOct4orNanogexpressioninH1650-SPadhcells(Figure6AB),andreduced thesphereformationbyapproximately2.5folds(Figure6)withacorrespondingreductionintheaveragesize (p=0.003).DepletionofSox2expressionresultedina pronounceddecreaseinthefrequencyofSPcells (Figure6C)aswellasABCG2expression(Figure6D)in A549,H1650andH1975cellscomparedtocontrol siRNAtransfectedcells.Similarresultswereobtained whenadifferentsiRNAtoSox2wasused(Additional file1:FigureS3).Collectively,theseresultssuggestthat Sox2genehasadirectroleinmaintainingcancerstem cellcharacteristicsandself-renewalofSPcellsfrom NSCLC.Sox2isexpressedinNSCLCandisassociatedwith metastaticprogressionOurdatashowingthatdepletionofSox2affectstheselfrenewalpropertiesofstem-likecells,wenextexamined Sox2expressioninapanelofNSCLCtumorsamples obtainedfromstageI/IIorstageIVpatientsontissue microarrays(TMAs)byimmunohistochemistry.Samples from193patientswithNSCLC-stageI/IIdiseaseincluding73withadenocarcinomawereononeTMA;samples from103stageIV-NSCLCpatientsincluding45with adenocarcinomafromprimarysiteand17adenocarcinomasamplesfromthemetastaticsiteswereonthesecondTMA.Inaccordancewithearlierreports,Sox2was stronglyexpressedinsquamouscellcarcinoma(SCC) samplesforbothstageI/IIandIVpatients(Figure7A(i andii)).IncontrasttoSCCs,adenocarcinomasamples hadsignificantlylowerexpressionofSox2.Sox2positive cellswereheterogeneouslydistributedinadenocarcinomasamplesforbothstageI/IIandIVpatients (Figure7A(iiiandiv).Whiletherewasnosignificant differenceinSox2expressionbetweendifferentgrades oftumors,elevatedexpressionofSox2waspositively associatedwithmetastaticprogression.Representative imagesforadenocarcinoma-metastasesareshownin Figure7A(v-viii).Approximately67%ofstageI/II (n=73)and73%ofstageIV(primarysite,n=45) tumorsweredetectedaspositiveforSox2expression (score 1)usingasemi-quantitativescoringsystem. ComparedtotheprimarysitetumorforstageIV patients,highernumbersofmetastasizedtumorswere positiveforSox2(Figure7B).ThemedianscoreforSox2 expressionisrepresentedashistogram(Figure7C).The averagescoreforSox2expressionwasfoundtobe significantlyhigher( p =0.01)inmetastasizedtumorsas comparedtotheprimarysiteorlowerstagetumors. Overall,Sox2wasexpressedinallstagesofadenocarcinomaanditslevelsweresignificantlyhigherinmetastatic lesions.DiscussionInthecurrentstudy,weusedtheSPphenotypetoidentifyandenrichasubpopulationofNSCLCswiththe propertiesascribedtoCSCs.Thestudiespresentedhere demonstratesaspecificandsignificantroleforEGFR signalingcascadeinfacilitatingtheself-renewalgrowth andexpansionofthesidepopulationcellsfrom NSCLCs. Ourstudy,inaccordancewithearlierstudies[11], [33],confirmedthepresenceofSPcellsinestablished humanNSCLCcelllinesandinhumantumorxenograftswiththepropertiesofCSCs.ComparingtheselfrenewalabilityofSPandMPcellsisolatedfromhuman tumorxenografts,wefoundthatapproximately0.2%SP cellswereabletoself-renewandformspheres,whereas MPcellswereunabletoself-renew.ComparingthepercentageofsphereformingcellsinSPcells,weestimate thatapproximately1-2%ofSPcellsfromestablishedcell linesmayhavestem-likeproperties;therefore,SPphenotypemaynotbetheexclusivemarkerforCSCs,butcan beusedtoenrichstem-likecellsfromNSCLCs. SPcellswerefoundtobemoretumorigenic invivo confirmingtheenrichmentoftumorinitiatingcellsin SPcompartment.Thesecellswereabletoproduce highlyinvasivediseaseuponimplantationintothelungs. Also,thedirectassociationofstem-likecellswithgenerationofmetastaticdiseasemaybesupportedbyourobservationwhereasignificantcorrelationwasobserved betweenhighSox2expressionsinthemetastatictumors oflungadenocarcinomapatients.Recentreportsindicate thatthenormalepithelialcellsacquiretheCSCspropertiesuponinductionofEMTgovernedbyvariouscytokinesandgrowthfactorsfromstromalcells[10,25].Our resultsdemonstratethatSP-cellsintrinsicallyexhibitloss ofepithelialmarkersand/orthegainofmesenchymal markersascomparedtoMPcellsandcouldbedueto thehigherexpressionoftranscriptionfactorsTwist,Slug andSnail,whichareknowntobeinvolvedinmaintainingthemesenchymalphenotype.Togetherwiththeexpressionofembryonicstemcelltranscriptionfactorslike Oct4,Sox2,andNanogalongwiththeexhibitionof EMTlikefeaturesandorthotopictumor-formingability, collectivelysuggestthatSPcellsisolatedfromNSCLC celllinesandtumorshavestem-likeproperties.TheobservationthatEGFRsignalingaffectsstem-likefunctions ofSPcellsisintriguing,giventhatseveralEGFR tyrosine-kinaseinhibitorshaveefficacyagainstNSCLCs [34,35].Interestingly,EGFRappearstoregulateSox2Singh etal.MolecularCancer 2012, 11 :73 Page10of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 11

levels,throughtheSrc-Aktpathway;Sox2hasbeen showntoberegulatedbyAktinEScells,throughtheinhibitionofproteasomaldegradation[36].Consistent withtheseresults,ourobservationsuggestthatinhibitionofEGFR-Src-AktsignalingdownregulatesSox2 levelsalongwithareductioninABCG2levels.ThisdecreaseinABCG2expressionuponEGFRinhibitionis probablyacausaleffectofSox2depletion-mediateddifferentiationofSPintoMPcells. ThefactthatEGFR-pathwayinhibitionresultedinspecificdepletionofSox2withoutanysignificanteffecton Oct4orNanogexpressionsuggeststhattheirexpression mayberegulatedthroughindependentmechanismsin NSCLCSPcells.Ourresultsaswellasanearlierreport [37]suggestthatSox2isexpressedinbothlowaswell ashighstageadenocarcinomasirrespectiveoftheir grades.However,Oct4orNanogexpressionwasfound tobeassociatedonlywiththehighgradelungadenocarcinomaandnotexpressedinlowgradetumors[17,37]. Therefore,wepredictthattheEGFRpathwayinhibition mayexertitsfavorableeffectsonlyforthosetumors whereSox2isthemajordeterminantincontrollingthe self-renewalofCSCs.Interestingly,arecentstudy showedthattheectopicoverexpressionofOct4and NanogincreasesthetumorinitiatingpropertyofA549 cells[17].Inagreementwiththesereports,wefindthat specificandindependentdepletionofOct4orNanog alsoresultedindecreaseinSPphenotypebutinacell typedependentfashion(Datanotshown).Tworecent reportsdemonstratethatectopicexpressionofSox2 increasedthefrequencyofsidepopulationcellsand tumorformationinmouseandhumanNSCLCcelllines [33,38].ThesereportsstronglysuggestthatSox2expressingcellsharborthestemcell-likeproperties.Ourobservationfurtherstrengthensthispostulationwherewe demonstratethatSox2depletionwassufficienttoinhibit theself-renewingpropertySPcellsinallthethree NSCLCcelllines. InadditiontothemutationinEGFRsignaling,perturbationofp53activityisanotherimportantevent occursininitiationandprogressionofNSCLCs[39].Recently,p53isshowntohavecertainrolesinpromoting thedifferentiationofhumanembryonicstemcell throughrepressionoffactorslikeOct4,Klf4,Lin28A, andSox2[40].However,thereisnotmuchinformation availableonthedirectroleofp53transcriptional Figure7 Sox2expressioncorrelateswithmetastaticprogressionofadenocarcinoma. ( A )Sox2expressioninLungsquamouscells carcinoma,adenocarcinomaandmetastaticadenocarcinomawasanalyzedfromstageI/IIorstageIVpatientssamplesbyimmunohistochemistry. ( B )Semi-quantitativescoringwasperformedforadenocarcinomasamplesandthetumorswithscoreofoneormorewasconsideredpositivefor Sox2expressionandlisted.( C )MedianscoreforSox2expressionwascalculatedandplottedfordifferentstagesofNSCLCprogression.Themean (SD)ofscoreismentionedinparenthesis.Metastatictumorsshowedsignificantly( p=0.01 )higherexpressionofSox2. Singh etal.MolecularCancer 2012, 11 :73 Page11of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 12

activitiesinregulatingSox2expressioninstem-likecells incancer,andwouldbeinterestingtoexploreinfuture. Conclusions Figure8summarizestheroleofSox2inSPcellbiology andtumorgrowth.Whilecertainfrequencyofisolated SPcellsfromNSCLCexhibitstemcell-likeproperties andcanformmetastatictumors,moredifferentiatedMP cellsaregreatlyimpairedintheirabilitytogenerate tumors.Further,inhibitionofEGFRpathwayincluding SrcandPI3-kinasecouldstronglyinhibittheexpression ofSox2,suppressingtheself-renewalpropertiesofSP cells.Therefore,relativeSox2expressionandfunctions withinthetumor-CSCsmaybeamajordeterminantin EGFR-targetedtherapyagainstNSCLCs.Thisinformationmightalsobepotentiallyusefultoovercomethe acquiredresistancetoEGFRtherapies,bytargeting downstreamtargetsofEGFRsignaling,includingSox2. Additionalinvestigationsinthisdirectionmightleadto thedevelopmentofmoreeffectivetherapeuticagentsto combatNSCLC,especiallythoseharboringEGFR mutations. Materialsandmethods Celllinesandtumorsamples H1650,andH1975celllineswereobtainedfrom ATCCandmaintainedinRPMIorDMEMcontaining10%fetalbovineserum(FBS;Mediatech)in5% CO 2 at37C.Humantumorxenograftswereobtained fromSAlaboratory. Inhibitors,siRNAsandantibodies Gefitinib,Erlotinib,BIBW2992andDasatinibwerepurchasedfromLClaboratories.PP2andFumitremorginC (FTC)werepurchasedfromSigmaInc.Inthepresent study,Gefitiniborerlotinibisusedat500nM,dasatinib orBIBW2992isusedat200nMandPP2isusedat 1 Mdose.siRNAagainstEGFR,Srcfamilykinases,Akt andSox2,Oct4andNanogwaspurchasedfromSanta CruzBiotechnologyorOriGeneTechnologyInc.PrimaryantibodiesagainstSox2(#3579),Oct4(#2750), Nanog(#4903),Phos-Src-pY 416 (#2101),pERK1/2 (#4376)andphospho-AKT-pS 473 (#4058)werepurchasedfromCellSignalingTechnology;Phos-EGFRpY 1068 (#44788G)fromInvitrogen;EGFRneutralizing antibody(#05-101)fromMiliporeandisotypematched mouseIgGwerepurchasedfromBiolegend. RNApreparationandqRTPCRanalysis RNApreparationandRT-PCRanalysiswasperformed asdescribedearlier[41].Foldinductionswerecalculated usingtheformula2 – (ddCt) usingGAPDHasinternalcontrolgene.Thegene-specificprimerpairswereasfollows. ABCG2(F)5 ’ -CACAAGGAAACACCAATGGCT-3 ’ ABCG2(R)5 ’ -ACAGCTCCTTCAGTAAATGCC TTC-3 ’ ;Oct4(F)5 ’ -ACATCAAAGCTCTGCAGA AAGAAC-3 ’ ,Oct4(R)5 ’ -CTGAATACCTTCCCA AATAGAACCC-3 ’ ,Sox2(F)5 ’ -GGGAAATGGGAG GGGTGCAAAAGA-3 ’ ,Sox2(R)5 ’ -TTGCGTGAG TGTGGATGGGATTGG-3 ’ ,Nanog(F)5 ’ -AGAAGG CCTCAGCACCTA-3 ’ ,Nanog(R)5 ’ -GGCCTGATT GTTCCAGGATT-3 ’ ;Twist(F)5 ’ -CTCGGACAA Figure8 AschematicdepictionofthesignalingeventsthatregulatethebiologyofSPcells. IsolatedSPcellsformmetastatictumorsbut MPcellsdonot.InhibitingEGFR,SrcorPI3-kinasesignificantlyimpairstheself-renewalpropertiesofSPcells. Singh etal.MolecularCancer 2012, 11 :73 Page12of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 13

GCTGAGCAAGATTCAGA-3 ’ ,Twist(R)5 ’ -CGT GAGCCACATAGCTGCAGC-3 ’ ,Slug(F)5 ’ -ACA CATTACCTTGTGTTTGCAAGATCT-3 ’ ,Slug(R) 5 ’ -TGTCTGCAAATGCTCTGTTGCAGTG-3 ’ Snail(F)5 ’ -CCTCAAGATGCACATCCGAAG CCAC-3 ’ ,Snail(R)5 ’ -CCGGACATGGCCTTGTAG CAGC-3 ’ ,GAPDH(F)5 ’ -GGTGGTCTCCTCTGA CTTCAACA-3 ’ ,GAPDH(R)5 ’ -GTTGCTGTAGCC AAATTCGTTGT-3 ’ .Hoechst33342dyeeffluxassayforSPanalysisandcell sortingAdherentcellswereharvestedusingaccutasereagent (SigmaInc).HumanTumortissuegrowninathymic nudemicewasminced,enzymaticallydigestedwith 0.2%collagenaseIV(WorthingtonBiochemicalCorporation)preparedin10%FBScontainingmediumfor 60minat37C.Thedigestwasfurtherdisaggregated bypassingthrough10mlpipetteseveraltimesandfilteredthrough100/70mcellstrainertoobtainasinglecellsuspension.Cellswerewashedandresuspended inHBSSat1X106cells/mldensityandincubatedwith 4 g/mlofHoechst33342dye(Invitrogen)for90min at370Cinpresenceorabsenceof1 MFTC,as describedbyGoodelletal.[21].Cellswereincubated with2 g/mlPropidiumiodide(PI;SigmaInc)before analysistovisualizeandexcludethenon-viablecells. TheHoechst33342dyewasexcitedat350nmusing UVlaseranditsfluorescencewasanalyzedusing400 – 500nmBPfilterforblueemissionand640 – 680nm BPfilterincombinationwith655nmLP-filterforred emission.FlowcytometersfromBDBioscienceswere usedfordataacquisition.Datawereacquiredusing LSRIIorFACSVantage(DiVa),andsortedusingFACS Vantage(DiVa)cellsorter.Dataanalysesweredone usingFlowJosoftware(TreeStar).Cellcycleanalyses forfixedcellswereperformedforPIstainedcellsusing Vindelovmethodwithsimilarprotocolasdescribed earlier[41].SphereformationorSelfrenewalassaySortedSPorMPcellswereplatedin96wellplatesat thedensityof10,000cells/ml(1000cells/wellin100 l medium)inserumfreestemcellselectivemedia (DMEM/F12K(1:1)(Invitrogen),supplementedwith1XN2supplement(Invitrogen),10ng/mlEGFand10ng/ mlbFGF(Sigma))andallowedtogrowasspheresfor 10days.Imagesofthespheresweretakenusingphase contrastmicroscope(Nikon)andtotalnumberswere counted.Tostudytheeffectofdrugsontheself-renewal ofSPcells,drugswereaddedtotherespectivewellson day1and5andsizeandnumberofthesphereswere analyzedonday10.ImmunofluorescenceForimmunostaining,spheresweretransferredtopoly D-lysine/Laminincoatedglasssurfacefor18h.For monolayercultures,cellsweredirectlyplatedoverthe polyD-lysin/Laminincoatedglasssurfaceandcultured ortreatedinstemcellselectivemediaasindicated.Immunofluorescencestainingwasperformedasdescribed previously[42].CellswereobservedusingaLeicaTCS SP5confocalmicroscope(LeicaMicrosystems)at630 magnification.ImmunohistochemistryHumanlungcancertissuemicroarray(TMA)slideswith stageI/IIorstageIVNSCLCpatientswereobtained throughLungCancerSpecializedProgramofResearch Excellence(SPORE).TMAslidewithstageI/IItumor samplescontainedusablecoresfrom193patients,and TMAslidewithstageIVtumorsamplescontainedusable coresfrom103patientsincluding17adenocarcinoma samplesfromthemetastaticsites.TheImmunohistochemicalstainingwasperformedasdescribed[42].The sampleswerescoredbyapathologist(D.Coppola).The semiquantitativescorewasreachedbytakingintoconsiderationbothcellularityandintensityofexpression(semiquantitativescore=cellularityintensity).Cellularitywas scoredasfollows:ascoreof3equalstogreaterthan66% cellularity,ascoreof2equalsto34% – 65%cellularity,and ascoreof1equalstolessthan33%cellularity.Intensity wasscoredasfollows:ascoreof3equalstostrongintensity,ascoreof2equalstomoderateintensity,andascore of1equalstoweakintensity[42].Thescoreof1orabove wasconsideredaspositiveexpressionofSox2.Theimages werecapturedat200magnification.Invivo tumorformationassayandbioluminescence imaging5-weeks-oldfemaleSCID-beigemicewereusedforthese experimentsunderanIACUCapprovedprotocol.For orthotopicimplantationoftumorcells,sortedSPorMP cellsfromA549celllinestablyexpressingluciferase gene(A549-Luc)werewashedwithserum-freeDMEMF12Kmediumandresuspendedatindicatednumbersin HBSScontaining500 g/mlgrowthfactorreduced Matrigel.Surgicalprocedurefororthotopiclungimplantationwasfollowedassuggestedearlierforintrapulmonaryimplantationoftumorcellswithsomemodifications [43].Specifically,cellswereinoculatedwith1mlsyringes with30-gaugehypodermicneedlesinanopentechnique underdirectvisualizationintotherightlungtissueof SCIDmiceanesthetizedbygasanesthesia(3%isoflurane).Tumorgrowth/metastaseswereimagedweekly usingbioluminescencebyIVIS-200imagingsystemfrom CaliperCorporation.Micewereanesthetizedand30mg/ KgofD-luciferininPBSwasadministeredbySingh etal.MolecularCancer 2012, 11 :73 Page13of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 14

intraperitoneal(i.p.)injection.Tenminutesafterinjection,bioluminescencewasimagedwithacharge-coupled devicecamera(Caliper)withanimagingtimeof2min. Attheendoftheexperiment,orwhenmicebecome moribund,allofthemicewereeuthanizedandindividual organsharvestedforevaluationoftumorsize;distant metastaseswasdeterminedbybioluminescenceofluciferaseexpressingcells.StatisticalmethodsDatawerepresentedasthemeanstandarddeviation (SD).Toassessthestatisticalsignificanceofdifferences, student ’ s t testwasperformed.Thedatawereconsideredstatisticallysignificantwhenthe P valuewasless than0.05.AdditionalfileAdditionalfile1: FigureS1. BIBW2992inhibitsEGFRphophorylation. H1975cellsweretreatedwith500nMgefitinibor200nMBIBW2992for 5days.EGFRphosphorylationandtotalEGFRexpressionwasdetectedin presenceorabsenceofdrugtreatment. FigureS2 .Downregulationof Sox2expressionafterEGFRandSrcinhibition.H1650-SPAdhcellswere treatedplatedoverPDL-Laminincoatedglasssurfaceandtreatedwith indicateddrugsfor4days.(A)ExpressionofSox2wasmonitoredby immunofluorescenceconfocalimaging.Isotypeantibodywasusedto showthespecificstainingofSox2.(B)NumberofSox2positivecellsfor eachtreatmentconditionwereconvertedintopercentageandplotted.P valueswerecalculatedfromthreedifferentexperimentsandsuggesteda significantdecreaseinSox2positivecellsafterEGFRandSrcinhibition. (C)Undersimilartreatm,entconditionscellswerestainedwithNanog specificantibodies.DrugtreatmentdidnotaltertheexpressionofNanog inH1650-SPAdhcells. FigureS3 .DepletionofSox2expression suppressesSPfrequency.(A)A549,H1650andH1975cellswere transientlytransfectedwithsecondsetsiRNA(purchasedfromOrigene). 48haftertransfection,cellswereanalyzedforSPfrequency.Similarto firstsetofsiRNA(purchasedfromSantaCruz),depletionofSox2resulted insignificantdecreaseinSPfrequencyinNSCLCs.(B)NSCLCcellswere transfectedwithSox2SIRNAandABCG2expressionwasdetectedby westernblotting. -Actinwasusedasinternalcontrolforequalloading.* p<0.05. Competinginterest Wedonothaveanyconflictofinterest. Authors ’ contributions SSconductedtheexperimentsandwrotetheinitialversionofthe manuscript;JTandNBSconductedcertainexperiments;DCdidpathological analysisofthesamples;EHprovidedintellectualinput;SAprovidedhuman tumorxenograftsandinput;SCdirectedtheprojectandfinalizedthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgments WethankJenniferGemmerfortechnicalsupport.Thesestudieswere supportedbythegrantCA127725fromtheNCItoSPC.SupportoftheLung CancerSPORE,NationalFunctionalGenomicsCenterandtheCoreFacilities attheMoffittCancerCenterisgreatlyappreciated. Authordetails1DepartmentofTumorBiology,H.LeeMoffittCancerCenterandResearch Institute,12902MagnoliaDrive,Tampa,FL33612,USA.2Departmentof AnatomicPathology,H.LeeMoffittCancerCenterandResearchInstitute, 12902MagnoliaDrive,Tampa,FL33612,USA.3DepartmentofThoracic Oncology,H.LeeMoffittCancerCenterandResearchInstitute,12902 MagnoliaDrive,Tampa,FL33612,USA.4Currentaddress:NationalInstituteof BiomedicalGenomics,2ndfloor,NetajiSubashSanatorium,Kalyani741251, India.5Currentaddress:DepartmentofSurgery,UniversityofFlorida,1600 SWArcherRd,Rm6175,Gainesville,FL32610-0109,USA. Received:14June2012Accepted:18September2012 Published:25September2012 References1.ParkinDM,BrayF,FerlayJ,PisaniP: Globalcancerstatistics,2002. CA CancerJClin 2005, 55: 74 – 108. 2.JemalA,SiegelR,WardE,HaoY,XuJ,MurrayT,ThunMJ: Cancerstatistics, 2008. CACancerJClin 2008, 58: 71 – 96. 3.JemalA,ThunMJ,RiesLA,HoweHL,WeirHK,CenterMM,WardE,WuXC, EhemanC,AndersonR, etal : Annualreporttothenationonthestatusof cancer,1975 – 2005,featuringtrendsinlungcancer,tobaccouse,and tobaccocontrol. JNatlCancerInst 2008, 100: 1672 – 1694. 4.SiegelR,NaishadhamD,JemalA: Cancerstatistics,2012. CACancerJClin 2012, 62: 10 – 29. 5.BonnetD,DickJE: Humanacutemyeloidleukemiaisorganizedasa hierarchythatoriginatesfromaprimitivehematopoieticcell. NatMed 1997, 3: 730 – 737. 6.ClarkeMF,DickJE,DirksPB,EavesCJ,JamiesonCH,JonesDL,VisvaderJ, WeissmanIL,WahlGM: Cancerstemcells-perspectivesoncurrentstatus andfuturedirections:AACRWorkshoponcancerstemcells. CancerRes 2006, 66: 9339 – 9344. 7.CleversH: Stemcells,asymmetricdivisionandcancer. NatGenet 2005, 37: 1027 – 1028. 8.MorrisonSJ,KimbleJ: Asymmetricandsymmetricstem-celldivisionsin developmentandcancer. Nature 2006, 441: 1068 – 1074. 9.SalnikovAV,GladkichJ,MoldenhauerG,VolmM,MatternJ,HerrI: CD133is indicativeforaresistancephenotypebutdoesnotrepresenta prognosticmarkerforsurvivalofnon-smallcelllungcancerpatients. IntJCancer 2010, 126: 950 – 958. 10.SinghA,SettlemanJ: EMT,cancerstemcellsanddrugresistance: anemergingaxisofevilinthewaroncancer. Oncogene 2010, 29: 4741 – 4751. 11.HoMM,NgAV,LamS,HungJY: Sidepopulationinhumanlungcancer celllinesandtumorsisenrichedwithstem-likecancercells. CancerRes 2007, 67:4827 – 4833. 12.GolebiewskaA,BronsNH,BjerkvigR,NiclouSP: Criticalappraisalofthe sidepopulationassayinstemcellandcancerstemcellresearch. Cell StemCell 2011, 8: 136 – 147. 13.WuC,AlmanBA: Sidepopulationcellsinhumancancers. CancerLett 2008, 268: 1 – 9. 14.ZhouS,SchuetzJD,BuntingKD,ColapietroAM,SampathJ,MorrisJJ, LagutinaI,GrosveldGC,OsawaM,NakauchiH,SorrentinoBP: TheABC transporterBcrp1/ABCG2isexpressedinawidevarietyofstemcellsand isamoleculardeterminantoftheside-populationphenotype. NatMed 2001, 7: 1028 – 1034. 15.KimJ,ChuJ,ShenX,WangJ,OrkinSH: Anextendedtranscriptional networkforpluripotencyofembryonicstemcells. Cell 2008, 132: 1049 – 1061. 16.SchoenhalsM,KassambaraA,DeVosJ,HoseD,MoreauxJ,KleinB: Embryonicstemcellmarkersexpressionincancers. BiochemBiophysRes Commun 2009, 383: 157 – 162. 17.ChiouSH,WangML,ChouYT,ChenCJ,HongCF,HsiehWJ,ChangHT, ChenYS,LinTW,HsuHS,WuCW: CoexpressionofOct4andNanog enhancesmalignancyinlungadenocarcinomabyinducingcancerstem cell-likepropertiesandepithelial-mesenchymaltransdifferentiation. CancerRes 2010, 70: 10433 – 10444. 18.JinX,YinJ,KimSH,SohnYW,BeckS,LimYC,NamDH,ChoiYJ,KimH: EGFR-AKT-Smadsignalingpromotesformationofgliomastem-likecells andtumorangiogenesisbyID3-drivencytokineinduction. CancerRes 2011, 71: 7125 – 7134. 19.SoedaA,InagakiA,OkaN,IkegameY,AokiH,YoshimuraS,NakashimaS, KunisadaT,IwamaT: Epidermalgrowthfactorplaysacrucialrolein mitogenicregulationofhumanbraintumorstemcells. JBiolChem 2008, 283: 10958 – 10966. 20.HuQ,ZhangL,WenJ,WangS,LiM,FengR,YangX,LiL: TheEGF receptor-sox2-EGFreceptorfeedbacklooppositivelyregulatesthe self-renewalofneuralprecursorcells. StemCells 2010, 28: 279 – 286.Singh etal.MolecularCancer 2012, 11 :73 Page14of15 http://www.molecular-cancer.com/content/11/1/73

PAGE 15

21.GoodellMA,BroseK,ParadisG,ConnerAS,MulliganRC: Isolationand functionalpropertiesofmurinehematopoieticstemcellsthatare replicating invivo JExpMed 1996, 183: 1797 – 1806. 22.RobeyRW,SteadmanK,PolgarO,MorisakiK,BlayneyM,MistryP,BatesSE: PheophorbideaisaspecificprobeforABCG2functionandinhibition. CancerRes 2004, 64: 1242 – 1246. 23.PastranaE,Silva-VargasV,DoetschF: Eyeswideopen:acriticalreviewof sphere-formationasanassayforstemcells. CellStemCell 2011, 8: 486 – 498. 24.SinghSK,HawkinsC,ClarkeID,SquireJA,BayaniJ,HideT,HenkelmanRM, CusimanoMD,DirksPB: Identificationofhumanbraintumourinitiating cells. Nature 2004, 432: 396 – 401. 25.ManiSA,GuoW,LiaoMJ,EatonEN,AyyananA,ZhouAY,BrooksM, ReinhardF,ZhangCC,ShipitsinM, etal : Theepithelial-mesenchymal transitiongeneratescellswithpropertiesofstemcells. Cell 2008, 133: 704 – 715. 26.Moreno-BuenoG,PortilloF,CanoA: Transcriptionalregulationofcell polarityinEMTandcancer. Oncogene 2008, 27: 6958 – 6969. 27.PolyakK,WeinbergRA: Transitionsbetweenepithelialandmesenchymal states:acquisitionofmalignantandstemcelltraits. NatRevCancer 2009, 9: 265 – 273. 28.PollardSM,YoshikawaK,ClarkeID,DanoviD,StrickerS,RussellR,BayaniJ, HeadR,LeeM,BernsteinM, etal : Gliomastemcelllinesexpandedin adherentculturehavetumor-specificphenotypesandaresuitablefor chemicalandgeneticscreens. CellStemCell 2009, 4: 568 – 580. 29.ZhangJ,KalyankrishnaS,WislezM,ThilaganathanN,SaigalB,WeiW,MaL, WistubaII,JohnsonFM,KurieJM: SRC-familykinasesareactivatedinnonsmallcelllungcancerandpromotethesurvivalofepidermalgrowth factorreceptor-dependentcelllines. AmJPathol 2007, 170: 366 – 376. 30.KimLC,SongL,HauraEB: Srckinasesastherapeutictargetsforcancer. NatRevClinOncol 2009, 6: 587 – 595. 31.RobertsonLK,MireauLR,OstergaardHL: Aroleforphosphatidylinositol 3-kinaseinTCR-stimulatedERKactivationleadingtopaxillinphosphorylationandCTLdegranulation. JImmunol 2005, 175 :8138 – 8145. 32.WangCC,CiritM,HaughJM: PI3K-dependentcross-talkinteractions convergewithRasasquantifiableinputsintegratedbyErk. MolSystBiol 2009, 5: 246. 33.NakatsugawaM,TakahashiA,HirohashiY,TorigoeT,InodaS,MuraseM, AsanumaH,TamuraY,MoritaR,MichifuriY, etal : SOX2isoverexpressedin stem-likecellsofhumanlungadenocarcinomaandaugmentsthe tumorigenicity. LabInvest 2011, 91: 1796 – 1804. 34.GazdarAF,MinnaJD: DeregulatedEGFRsignalingduringlungcancer progression:mutations,amplicons,andautocrineloops. CancerPrevRes 2008, 1: 156 – 160. 35.AmannJ,KalyankrishnaS,MassionPP,OhmJE,GirardL,ShigematsuH, PeytonM,JuroskeD,HuangY,StuartSalmonJ, etal : Aberrantepidermal growthfactorreceptorsignalingandenhancedsensitivitytoEGFR inhibitorsinlungcancer. CancerRes 2005, 65: 226 – 235. 36.JeongCH,ChoYY,KimMO,KimSH,ChoEJ,LeeSY,JeonYJ,LeeKY,YaoK, KeumYS, etal : PhosphorylationofSox2cooperatesinreprogrammingto pluripotentstemcells. StemCells 2010, 28: 2141 – 2150. 37.ShollLM,BarlettaJA,YeapBY,ChirieacLR,HornickJL: Sox2protein expressionisanindependentpoorprognosticindicatorinstageIlung adenocarcinoma. AmJSurgPathol 2010, 34: 1193 – 1198. 38.XiangR,LiaoD,ChengT,ZhouH,ShiQ,ChuangTS,MarkowitzD,Reisfeld RA,LuoY: DownregulationoftranscriptionfactorSOX2incancerstem cellssuppressesgrowthandmetastasisoflungcancer. BrJCancer 2011, 104: 1410 – 1417. 39.MogiA,KuwanoH: TP53mutationsinnonsmallcelllungcancer. JBiomedBiotechnol 2011, 2011: 583929. 40.JainAK,AlltonK,IacovinoM,MahenE,MilczarekRJ,ZwakaTP,KybaM, BartonMC: p53regulatescellcycleandmicroRNAstopromote differentiationofhumanembryonicstemcells. PLoSBiol 2012, 10: e1001268. 41.SinghS,DavisR,AlamandaV,PiredduR,PernazzaD,SebtiS,LawrenceN, ChellappanS: Rb-Raf-1interactiondisruptorRRD-251inducesapoptosis inmetastaticmelanomacellsandsynergizeswithdacarbazine. Mol CancerTher 2010, 9:3330 – 3341. 42.DasguptaP,RizwaniW,PillaiS,DavisR,BanerjeeS,HugK,LloydM,Coppola D,HauraE,ChellappanSP: ARRB1-mediatedregulationofE2Ftarget genesinnicotine-inducedgrowthoflungtumors. JNatlCancerInst 2011, 103: 317 – 333. 43.DokiY,MurakamiK,YamauraT,SugiyamaS,MisakiT,SaikiI: Mediastinal lymphnodemetastasismodelbyorthotopicintrapulmonary implantationofLewislungcarcinomacellsinmice. BrJCancer 1999, 79: 1121 – 1126.doi:10.1186/1476-4598-11-73 Citethisarticleas: Singh etal. : EGFR/Src/AktsignalingmodulatesSox2 expressionandself-renewalofstem-likeside-populationcellsin non-smallcelllungcancer. MolecularCancer 2012 11 :73. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Singh etal.MolecularCancer 2012, 11 :73 Page15of15 http://www.molecular-cancer.com/content/11/1/73


xml version 1.0 encoding utf-8 standalone no
mets ID sort-mets_mets OBJID sword-mets LABEL DSpace SWORD Item PROFILE METS SIP Profile xmlns http:www.loc.govMETS
xmlns:xlink http:www.w3.org1999xlink xmlns:xsi http:www.w3.org2001XMLSchema-instance
xsi:schemaLocation http:www.loc.govstandardsmetsmets.xsd
metsHdr CREATEDATE 2012-11-14T12:05:32
agent ROLE CUSTODIAN TYPE ORGANIZATION
name BioMed Central
dmdSec sword-mets-dmd-1 GROUPID sword-mets-dmd-1_group-1
mdWrap SWAP Metadata MDTYPE OTHER OTHERMDTYPE EPDCX MIMETYPE textxml
xmlData
epdcx:descriptionSet xmlns:epdcx http:purl.orgeprintepdcx2006-11-16 xmlns:MIOJAVI
http:purl.orgeprintepdcxxsd2006-11-16epdcx.xsd
epdcx:description epdcx:resourceId sword-mets-epdcx-1
epdcx:statement epdcx:propertyURI http:purl.orgdcelements1.1type epdcx:valueURI http:purl.orgeprintentityTypeScholarlyWork
http:purl.orgdcelements1.1title
epdcx:valueString EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer
http:purl.orgdctermsabstract
Abstract
Background
Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling.
Results
SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples.
Conclusions
Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.
http:purl.orgdcelements1.1creator
Singh, Sandeep
Trevino, Jose
Bora-Singhal, Namrata
Coppola, Domenico
Haura, Eric
Altiok, Soner
Chellappan, Srikumar P
http:purl.orgeprinttermsisExpressedAs epdcx:valueRef sword-mets-expr-1
http:purl.orgeprintentityTypeExpression
http:purl.orgdcelements1.1language epdcx:vesURI http:purl.orgdctermsRFC3066
en
http:purl.orgeprinttermsType
http:purl.orgeprinttypeJournalArticle
http:purl.orgdctermsavailable
epdcx:sesURI http:purl.orgdctermsW3CDTF 2012-09-25
http:purl.orgdcelements1.1publisher
BioMed Central Ltd
http:purl.orgeprinttermsstatus http:purl.orgeprinttermsStatus
http:purl.orgeprintstatusPeerReviewed
http:purl.orgeprinttermscopyrightHolder
Sandeep Singh et al.; licensee BioMed Central Ltd.
http:purl.orgdctermslicense
http://creativecommons.org/licenses/by/2.0
http:purl.orgdctermsaccessRights http:purl.orgeprinttermsAccessRights
http:purl.orgeprintaccessRightsOpenAccess
http:purl.orgeprinttermsbibliographicCitation
Molecular Cancer. 2012 Sep 25;11(1):73
http:purl.orgdcelements1.1identifier
http:purl.orgdctermsURI http://dx.doi.org/10.1186/1476-4598-11-73
fileSec
fileGrp sword-mets-fgrp-1 USE CONTENT
file sword-mets-fgid-0 sword-mets-file-1
FLocat LOCTYPE URL xlink:href 1476-4598-11-73.xml
sword-mets-fgid-1 sword-mets-file-2 applicationpdf
1476-4598-11-73.pdf
sword-mets-fgid-3 sword-mets-file-3 applicationvnd.openxmlformats-officedocument.wordprocessingml.document
1476-4598-11-73-S1.DOCX
structMap sword-mets-struct-1 structure LOGICAL
div sword-mets-div-1 DMDID Object
sword-mets-div-2 File
fptr FILEID
sword-mets-div-3
sword-mets-div-4


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1476-4598-11-73
ji 1476-4598
fm
dochead Research
bibl
title
p EGFR/Src/Akt signaling modulates Sox2 expression and self-renewal of stem-like side-population cells in non-small cell lung cancer
aug
au id A1 snm Singhfnm Sandeepinsr iid I1 I4 email SS5@nibmg.ac.in
A2 TrevinoJoseI5 Jose.Trevino@surgery.ufl.edu
A3 Bora-SinghalNamrataNamrata.Borasinghal@moffitt.org
A4 CoppolaDomenicoI2 Domenico.Coppola@moffitt.org
A5 HauraEricI3 Eric.Haura@moffitt.org
A6 AltiokSonerSoner.Altiok@moffitt.org
A7 ca yes Chellappanmi PSrikumarSrikumar.Chellappan@moffitt.org
insg
ins Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL, 33612, USA
Department of Anatomic Pathology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL, 33612, USA
Department of Thoracic Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL, 33612, USA
Current address: National Institute of Biomedical Genomics, 2nd floor, Netaji Subash Sanatorium, Kalyani, 741251, India
Current address: Department of Surgery, University of Florida, 1600 SW Archer Rd, Rm 6175, Gainesville, FL, 32610-0109, USA
source Molecular Cancer
issn 1476-4598
pubdate 2012
volume 11
issue 1
fpage 73
url http://www.molecular-cancer.com/content/11/1/73
xrefbib pubidlist pubid idtype doi 10.1186/1476-4598-11-73pmpid 23009336
history rec date day 14month 6year 2012acc 1892012pub 2592012
cpyrt 2012collab Singh et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd Cancer stem-like cells
Side-population cells
Self-renewal
EGFR
Sox2
abs
sec
st
Abstract
Background
Cancer stem cells are thought to be responsible for the initiation and progression of cancers. In non-small cell lung cancers (NSCLCs), Hoechst 33342 dye effluxing side population (SP) cells are shown to have stem cell like properties. The oncogenic capacity of cancer stem-like cells is in part due to their ability to self-renew; however the mechanistic correlation between oncogenic pathways and self-renewal of cancer stem-like cells has remained elusive. Here we characterized the SP cells at the molecular level and evaluated its ability to generate tumors at the orthotopic site in the lung microenvironment. Further, we investigated if the self-renewal of SP cells is dependent on EGFR mediated signaling.
Results
SP cells were detected and isolated from multiple NSCLC cell lines (H1650, H1975, A549), as well as primary human tumor explants grown in nude mice. SP cells demonstrated stem-like properties including ability to self-renew and grow as spheres; they were able to generate primary and metastatic tumors upon orthotopic implantation into the lung of SCID mice. In vitro study revealed elevated expression of stem cell associated markers like Oct4, Sox2 and Nanog as well as demonstrated intrinsic epithelial to mesenchymal transition features in SP cells. Further, we show that abrogation of EGFR, Src and Akt signaling through pharmacological or genetic inhibitors suppresses the self-renewal growth and expansion of SP-cells and resulted in specific downregulation of Sox2 protein expression. siRNA mediated depletion of Sox2 significantly blocked the SP phenotype as well as its self-renewal capacity; whereas other transcription factors like Oct4 and Nanog played a relatively lesser role in regulating self-renewal. Interestingly, Sox2 was elevated in metastatic foci of human NSCLC samples.
Conclusions
Our findings suggest that Sox2 is a novel target of EGFR-Src-Akt signaling in NSCLCs that modulates self-renewal and expansion of stem-like cells from NSCLC. Therefore, the outcome of the EGFR-Src-Akt targeted therapy may rely upon the expression and function of Sox2 within the NSCLC-CSCs.
bdy
Introduction
Despite significant therapeutic advances, lung cancer causes the maximum number of cancer related deaths worldwide abbrgrp
abbr bid B1 1
. In the United States, ~85% of the patients diagnosed with NSCLCs, die within five years
B2 2
B3 3
B4 4
, thus, highlight a need for better understanding of the cellular and molecular events underlying the genesis of this disease. Cancer stem cell model has emerged as a viable explanation for the initiation and progression of the aggressive cancers like NSCLCs.Cancer stem cell model suggests that cancer stem-like cells (CSCs) are a subpopulation of cells within the tumor that have the deregulated properties of normal stem cells with sustained self-renewal, and can generate secondary tumors that recapitulate the heterogeneity and diversity of original tumor
B5 5
B6 6
B7 7
B8 8
. CSCs are considered to be responsible for tumor initiation, propagation, recurrence and resistance to therapy
B9 9
B10 10
. Hoechst 33342 dye excluding cells, termed side-population (SP) cells, have been described as CSCs in a variety of tumor types, including NSCLCs
B11 11
, where they have been shown to display increased tumorigenicity when transplanted into immunocompromised mice
B12 12
B13 13
as compared to major population (MP) cells. SP phenotype is dependent on the differential ability of cells to efflux the Hoechst 33342 dye via the ATP-binding cassette (ABC) family of transporter protein, mainly ABCG2 (breast cancer resistance protein, BRCP1) which is specifically expressed on the cell membrane of stem cell populations
B14 14
. Earlier studies have demonstrated the existence of SP cells in various established human NSCLC cell lines
11
but their ability to generate tumors in lung microenvironment as well as the signaling pathways governing their stem-like properties remain to be elucidated.The transcription factors Oct4, Sox2 and Nanog have been identified as core regulators that maintain the self-renewal of embryonic stem cells
B15 15
. These factors are overexpressed in various cancers and are associated with malignant progression and poor prognosis including NSCLCs
B16 16
B17 17
, suggesting that the core regulators that govern normal stem cell self-renewal may also maintain the stem-like properties of CSCs in cancers. However, the influence of NSCLC specific oncogenic pathways on the expression of these factors remains relatively unknown. Alterations in EGFR-gene like copy number gains and/or mutant allele-specific amplifications are associated with NSCLC pathogenesis. In addition, activation of EGFR signaling increases the self-renewal capacity of neural precursor cells and brain tumor stem cells
B18 18
B19 19
B20 20
b In this study, we provide biochemical and biological evidence that SP cells isolated from established human NSCLC cell lines and tumors are highly enriched in NSCLC-CSCs and EGFR-Src-Akt signaling axis contributes significantly to the self-renewal of SP cells. Interestingly, Sox2 transcription factor is the predominant downstream target of EGFR signaling in these cells and plays a major role in self-renewal growth and expansion of SP cells, independent of Oct4 and Nanog.
Results
SP cells are enriched with tumorigenic cells and produce highly invasive tumors
In an attempt to identify NSCLC stem-like cells, SP analysis was conducted on four primary human NSCLC explants grown in athymic nude mice. SP cells appeared as a well separated population as described previously
B21 21
. As shown in Figure figr fid F1 1A, a specific inhibitor of ABCG2
B22 22
, Fumitremorgin C (FTC) could block the appearance of SP phenotype. All the four tumor samples displayed the presence of SP cells with varying frequency ranging from 0.6-3%, and could be significantly blocked by FTC (Figure 1B).
fig Figure 1caption SP cells exhibit stem-like propertiestext
SP cells exhibit stem-like properties. (A) FACS analysis on single cell suspension of human NSCLC xenograft stained with Hoechst 33342 dye showing SP cells. Fumitremorgin C (FTC) inhibited the efflux of the dye and caused the disappearance of SP cells. (B) SP cell frequency in presence or absence of FTC in four different human tumor xenografts. (C) Sphere formation assay on SP or MP cells grown in stem cell selective media for 10 days. The pictures of representative spheres are presented. The bar diagram show the average (±SD) number of spheres formed from 2000 cells. (D) SP or MP cells from H1650 and H1975 cell lines were plated in serum free medium supplemented with EGF and bFGF for 10 days under self-renewal assay conditions. The pictures of representative spheres are depicted (E) Indicated number of SP and MP cells from A549-Luc cell line were implanted into the right lung tissue of SCID mice. The tumor incidence and metastasis was monitored for 12 weeks by bioluminescence imaging. (F) Ex-vivo images of the lung, liver, kidney/adrenals and brain captured at the end of experiment. Metastasis was prevalent in SP cells implanted mice. (G) H&E, pan-keratin (brown staining) and mucicarmine (pink staining, indicated by arrow head) staining of whole lungs from mice implanted with SP or MP cells.
graphic file 1476-4598-11-73-1 Self-renewing normal or cancer-stem-like cells can be expanded as non-adherent spheres when cultured at low density in serum free, stem cell selective medium; differentiated cells do not grow or form spheres under these conditions
6
B23 23
B24 24
. The self-renewal property of SP cells was examined by performing sphere formation assay on sorted SP and MP cells isolated from human tumor xenografts. While sorted SP cells were able to grow as spheres, MP cells had markedly less capacity to grow under identical conditions (Figure 1C).Attempts were then made to assess the presence of SP cells in human NSCLC cell lines. As shown in later sections, A549 (K-Ras mutant; wild type EGFR amplification), H1650 (EGFR mutant; Exon 20 deletion: delE746-A750) and H1975 (EGFR mutant; L858R and T790M mutations), contained SP-cells with varying frequency. Appearance of SP cells was completely blocked by FTC. Sorted SP cells were able to grow as spheres whereas MP cells showed markedly reduced capability (Figure 1D) suggesting that NSCLC-SP cells are enriched with CSCs.The stem cell like property of NSCLC-SP cells was verified by evaluating its ability to form tumors in the lung microenvironment. Sorted SP and MP cells from A549 cells stably expressing the luciferase gene were orthotopically implanted into the left lung of SCID mice and tumor growth was monitored for 12 weeks. As shown in Figure 1E, SP cells generated primary tumors in the lung more efficiently than MP cells. At the end of the experiment, lungs, liver, kidney and brain were excised from each mouse and ex-vivo images were examined for the presence of metastasized luciferase positive cells. Mice injected with SP cells demonstrated substantial tumor burden throughout the lungs and showed luminescent metastatic loci in liver, kidney (adrenals) and brain (Figure 1F). In contrast, MP cells formed only one luminescent focus in the lung of one mouse (out of five) injected with 50 000 MP cells and there was no metastasis (Figure 1F). These results were confirmed by H&E staining; further, tumors formed within the lung from SP cells, recapitulated the histopathology of adenocarcinoma as confirmed by positive staining with pan-keratin antibody as well as mucicarmine dye (Figure 1G). These data suggested that SP cells are enriched with tumorigenic cells and can develop metastatic tumors it in vivo.
SP cells exhibit molecular markers of stem-like cells
Recent reports suggest that epithelial cells acquire cancer stem cell properties upon induction of epithelial to mesenchymal transition (EMT)
B25 25
. To evaluate whether SP cells show features of EMT, SP and MP cells from A549, H1650 and H1975 were examined for the levels of EMT markers like E-cadherin, Vimentin and Fibronectin. As shown in Figure F2 2A, ABCG2 expression was significantly higher in the SP fraction in all the three cell lines. The levels of E-cadherin was lower in H1650-SP cells as compared to MP cells, however, it was undetectable in A549 and unchanged in H1975 cells. Fibronectin was detected at higher levels in A549 and H1975-SP cells, but undetectable in H1650 cells. Vimentin level was higher in A549-SP cells, but low in H1975 and H1650 SP cells. Although the levels vary in a cell type dependent manner, these results suggest that, SP cells express proteins indicative of EMT without any external stimuli to the cells (Figure 2A). The molecular basis for the differential expression of the EMT markers was then examined. Transcription factors like Twist, Slug and Snail have been demonstrated to be capable of coordinating the EMT program during embryonic development and in cancers
B26 26
B27 27
. Therefore, we next assessed the expression of these transcription factors in SP and MP cells. Real-time PCR analysis revealed that Twist, Slug and Snail transcription factors are expressed at higher levels in SP cells in all the three NSCLC cell lines (Figure 2B, C, D).
Figure 2SP cells express stem cell-like markers
SP cells express stem cell-like markers. (A) Western blot analysis on SP and MP cells from A549, H1650 and H1975 cell lines for the indicated proteins. β-Actin was used as the loading control. (B, C, D) SP and MP cells were analyzed by real time qRT-PCR method for the expression of Twist, Slug and Snail genes. Average (±SD) fold change between MP and SP cells were plotted. (E, F, G) SP and MP cells were analyzed by real time qPCR method for the expression of ABCG2, Oct4, Sox2 and Nanog genes. Average (±SD) fold change between MP and SP cells were plotted. (H) The spheres formed in self-renewal assays of H1650 SP cells were stained for the expression of ABCG2, Oct4, Sox2 and Nanog. Confocal image of one of the z-stacks for a sphere is presented. DAPI was used to stain the nucleus.
1476-4598-11-73-2 The expression of Oct4, Sox2 and Nanog transcription factors was next examined in SP cells. Real-time PCR analysis showed elevated levels of ABCG2, Oct4, Sox2, and Nanog in the SP fraction in all the three cell lines. (Figure 2E, F, G). Further, SP cells from H1650 cells growing as spheres showed expression of ABCG2, Oct4, Sox2 and Nanog proteins by fluorescence microscopy (Figure 2H), indicating the undifferentiated growth of self-renewing SP cells within the spheres.
EGFR tyrosine kinase inhibitors downregulate self-renewal and SP phenotype
Experiments were conducted to explore the molecular mechanisms involved in the self-renewal of SP cells. Since aberrant EGFR signaling is implicated with the initiation and progression of lung cancer, we first assessed SP frequency and expression of ABCG2 in the presence of an antagonistic antibody against EGFR. Cells were mixed with 10 μg/ml anti-EGFR antibody or an isotype control and plated in 2% FBS containing media for 5 days. Blocking EGF-receptors resulted in a significant decrease in SP frequency in both A549 and H1650 cells (Figure F3 3A, B), along with decreased EGFR phosphorylation as well as ABCG2 expression in both the cell lines (Figure 3C). Confirming these results, depletion of EGFR expression by a siRNA resulted in decreased SP frequency and ABCG2 expression in A549, H1650 and H1975 cells (Figure 3D, E).
Figure 3Inhibition of EGFR suppresses SP frequency and self-renewal
Inhibition of EGFR suppresses SP frequency and self-renewal. (A) H1650 and A549 cells revealed a significant decrease in SP frequency after treatment with 10 μg/ml EGFR-neutralizing antibodies for 5 days. (B) Frequency of SP cells in control antibody (1) and EGFR-neutralizing antibodies (2) treated cells. (C) Western blot analysis for ABCG2 and phospho-EGFR after EGFR-neutralizing antibody treatment. (D) SP frequency in NSCLC cells transfected with siRNA against EGFR or a control siRNA. (E) Expression of ABCG2 and EGFR in antibody treated cells. (F and G) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated per well from 1000 cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. represent the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.
1476-4598-11-73-3 To further evaluate whether EGFR signaling contributed to the self-renewal property of H1650 SP cells, sphere formation assay was conducted in the presence or absence of EGFR inhibitors Gefitinib or Erlotinib. As shown in Figure 3F, inhibition of EGFR-kinase activity by 500 nM of Gefitinib or Erlotinib, demonstrated a 5–7 fold (p < 0.001) decrease in the number of spheres; further the size of the spheres was also significantly reduced.A secondary point mutation in exon 20 of EGFR (T790M) is associated with acquired resistance to gefitinib or Erlotinib, but this can be overcome by the irreversible EGFR-tyrosine kinase inhibitor BIBW2992 (BIBW). We tested the effect of 500 nM of gefitinib and 200 nM of BIBW on EGFR phosphorylation and self-renewal growth of SP cells from H1975 cell line, which harbors gefitinib-resistant-T790M mutation along with Gefitinib responsive-L858R mutation in exon 21. Western blot analysis showed that tyrosine phosphorylation of EGFR was insensitive to 500 nM concentration of gefitinib, whereas significant downregulation occurred after treatment with 200 nM of BIBW in H1975 cells (Additional file supplr sid S1 1: Figure S1). Consistent with this, BIBW could significantly inhibit the self-renewal of SP cells from H1975 cells (Figure 3G).
suppl
Additional file 1
Figure S1. BIBW2992 inhibits EGFR phophorylation. H1975 cells were treated with 500 nM gefitinib or 200 nM BIBW2992 for 5 days. EGFR phosphorylation and total EGFR expression was detected in presence or absence of drug treatment. Figure S2. Downregulation of Sox2 expression after EGFR and Src inhibition. H1650-SPAdh cells were treated plated over PDL-Laminin coated glass surface and treated with indicated drugs for 4 days. (A) Expression of Sox2 was monitored by immunofluorescence confocal imaging. Isotype antibody was used to show the specific staining of Sox2. (B) Number of Sox2 positive cells for each treatment condition were converted into percentage and plotted. P values were calculated from three different experiments and suggested a significant decrease in Sox2 positive cells after EGFR and Src inhibition. (C) Under similar treatm,ent conditions cells were stained with Nanog specific antibodies. Drug treatment did not alter the expression of Nanog in H1650-SPAdh cells. Figure S3. Depletion of Sox2 expression suppresses SP frequency. (A) A549, H1650 and H1975 cells were transiently transfected with second set siRNA (purchased from Origene). 48 h after transfection, cells were analyzed for SP frequency. Similar to first set of siRNA (purchased from SantaCruz), depletion of Sox2 resulted in significant decrease in SP frequency in NSCLCs. (B) NSCLC cells were transfected with Sox2 SIRNA and ABCG2 expression was detected by western blotting. β-Actin was used as internal control for equal loading. p<0.05.
name 1476-4598-11-73-S1.docx
Click here for file
Adherent cultures of SP cells maintain stem-like properties
To conduct further molecular studies on SP cells, we attempted to establish adherent cell cultures of isolated SP cells from A549, H1975 and H1650 cell lines, as suggested for glioma stem cells
B28 28
. Isolated SP cells were plated on uncoated or Poly-D Lysine + Laminin coated culture plates in serum free, stem cell media. While A549-SP and H1975-SP cells detached from the surface, H1650-SP cells grew as an adherent culture. As shown in Figure 3A, H1650-SP cells cultured on uncoated surface failed to maintain SP phenotype with high frequency (Figure F4 4A, (i)), but 80% of the cells maintained as SP cells even after 5 passages when plated on PDL + laminin coated surface (Figure 4A, (ii); H1650-SPAdh cells). H1650-SPAdh cells cultured back in 5% FBS containing medium for 10 days could recapitulate the proportion of SP and MP cells found in parental H1650 cells (Figure 4A, (iii)), with a concomitant reduction in expression of ABCG2 (Figure 4B), as well as Oct4, Sox2 and Nanog mRNA as seen by R-PCR (Figure 4C). Cell cycle analysis showed that H1650-SPAdh cells were slow cycling compared to parental cells (Figure 4D), having approximately 20% higher number of cells in Gsub 0/G1 phase; but upon serum-induced differentiation, H1650-SPAdh cells acquired cell-cycle phase distribution comparable to H1650-parental cells (Figure 4D).
Figure 4Establishment and characterization of H1650-SPAdh cells
Establishment and characterization of H1650-SPAdh cells. (A) SP cells from H1650 cell line was plated on normal tissue culture plate (i) or poly-D-lysine-laminin (PDL-Laminin) coated surface (ii) in serum free medium containing N2-supplement, EGF and bFGF. H1650-SPAdh cells growing in self-renewing condition was cultured in serum for 5 days to induce differentiation, and reanalyzed for SP frequency (iii). (B) Serum induces differentiation of SPAdh cells as seen by ABCG2 expression and (C) real time qPCR analysis for stem cell markers, ABCG2, Oct4, Sox2, Nanog. (D) Cell cycle analysis for parental-H1650 or H1650-SPAdh and serum differentiated H1650-SPAdh cells grown on PDL-Laminin coated surface. Histograms were plotted using ModFit program. (E) The average number of spheres generated from 1000 H1650-SPAdh cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of indicated drugs.
1476-4598-11-73-4 Treatment of H1650-SPAdh cells with 200 nM BIBW significantly suppressed the number as well as the size of spheres (Figure 4E); at the same time, treatment with 30 μM cisplatin did not affect the number or the size of the spheres formed by H1650-SP cells, suggesting enhanced chemoresistance of these cells. Further, the sphere-formation ability of SP was not altered by the ABCG2 inhibitor, FTC, suggesting that self-renewal of SP cells was independent of ABCG2 activity (Figure 4E).
Inhibition of EGFR-Src-Akt signaling downregulates Sox2 expression
Experiments were conducted to examine the downstream signaling events from EGFR that modulates self-renewal of SP cells and whether these pathways impinge transcription factors associated with stemness. Role of c-Src in the process was first examined since Src is altered in NSCLC
B29 29
B30 30
. H1650-SPAdh cells were treated with EGFR or Src TKIs and the levels of Oct4 and Sox2 was assessed by western blotting (Figure F5 5A). EGFR inhibition by 500 nM gefitinib or 200 nM BIBW as well as inhibition of Src activity by 200 nM dasatinib or 1 μM PP2 markedly reduced Sox2 expression; Oct4 level was not affected (Figure 5A). These results were verified by immunoflorescence experiments. Similar to Oct4, there was no significant difference in Nanog expression; however, the number Sox2 positive cells were significantly decreased in response to the treatment of EGFR- and Src-TKIs (Additional file 1: Figure S2).
Figure 5Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors for 4 days and western blot analysis was performed for the indicated proteins
Inhibition of EGFR signaling downregulates Sox2 expression (A) H1650-SPAdh cells were treated with EGFR or Src inhibitors for 4 days and western blot analysis was performed for the indicated proteins. (B) H1650-SPAdh cells were treated with MEK or PI3K inhibitor for 4 days and levels of Sox2 and activation of Akt or ERK was evaluated by western blot. (C) A549 and H1975 cells were treated with MEK or PI3K inhibitor for 5 days and the frequency of SP cells were examined by SP analysis. Data represents the fold change in mean (± SD) of SP frequency. (D) ABCG2 expression upon inhibitor treatment as detected by western blotting. (E) SP cell frequency in NSCLC cell lines transfected with specific siRNAs against Src or AKT. (F) ABCG2 expression in siRNA transfected cells as detected by western blotting. (G and H) SP cells were sorted and plated for self-renewal assay in the presence or absence of indicated drugs. Average number of spheres generated from 1000 cells is plotted (mean ± SD). Phase contrast microscopy images of the spheres in presence or absence of drugs are presented. represent the p value of <0.05; ** represent the p value of <0.01; *** represent the p value of <0.001.
1476-4598-11-73-5 Inhibition of EGFR as well as Src signaling resulted in decreased phosphorylation of EGFR, Src, ERK and Akt (Figure 5A). Contribution of ERK and Akt pathways to EGFR mediated induction of Sox2 was next examined in H1650SPAdh cells. Phosphorylation of ERK was suppressed by MEK inhibitor PD98059 and AKT-phosphorylation was suppressed by the PI3-kinase inhibitor, LY294002. However, PI3-Kinase inhibited H1650SPAdh cells also resulted in slight inhibition in ERK phosphorylation (Figure 5B). A similar observation has been reported in earlier studies where PI3-Kinase signaling was demonstrated to regulate the ERK phosphorylation in T-cell-receptor (TCR) signaling
B31 31
and PDGFR mediated signaling
B32 32
. However, as shown in Figure 5B, inhibition of MEK activity did not affect the levels of Sox2 while the PI3-kinase inhibition, markedly reduced its levels with corresponding reduction in SP frequency (Figure 5C) and ABCG2 expression (Figure 5D). These results were confirmed using siRNAs to Src and Akt. As shown in Figure 5E, SP frequency was significantly downregulated in both Akt and Src siRNA transfected A549, H1650 and H1975 cells as compared to the control siRNA transfected cells, with a corresponding reduction in ABCG2 expression (Figure 5F). Similar inhibitory effects were observed upon silencing of two other Src family members, Fyn and Yes (data not shown).To determine whether Src or Akt signaling facilitates self-renewal of SP cells, sphere formation assay was conducted on SP cells in presence or absence of Src inhibitors Dasatinib or PP2, MEK inhibitor PD98059 as well as Akt inhibitor LY294002. As shown in Figures 5G and 5H, Src-kinase inhibitors dasatinib or PP2, as well as PI3K/Akt inhibitor LY294002 showed a significant (5–7 fold; p < 0.001) decrease in sphere formation; MEK inhibition by PD98059 did not have any significant effect on self-renewal. The average size of the spheres formed was found to be 7–10 folds smaller than the untreated cells. Collectively, these data indicated that inhibition of EGFR/Src/Akt signaling results in depletion of Sox2 expression and decreased self-renewal of SP cells.
Suppression of Sox2 expression is sufficient to inhibit the self-renewal of SP cells
Since inhibition of EGFR/Src/Akt signaling specifically downregulated the expression of Sox2, we examined the contribution of Sox2 to the self-renewal of H165SP-Adh cells. Transient transfection of EGFR and Src siRNA in H1650-SPadh cells reduced EGFR expression by 60% and Src expression by 50%. Reduction in EGFR or Src expression decreased the levels of Sox2 by 50% and 40% respectively; the expression of Oct4 and Nanog was not altered (Figure F6 6A). In addition, depletion of EGFR or Src by siRNA suppressed the sphere formation by 2–3 folds (Figure 6B). To further explore the function of Sox2 in self-renewal of SP cells, we depleted Sox2 expression in H1650-SPadh cells. Transient transfection of Sox2 siRNA reduced the expression of Sox2 by 60% (Figure 6A). Depletion of Sox2 expression did not significantly alter the expression of Oct4 or Nanog expression in H1650-SPadh cells (Figure 6AB), and reduced the sphere formation by approximately 2.5 folds (Figure 6) with a corresponding reduction in the average size (p = 0.003). Depletion of Sox2 expression resulted in a pronounced decrease in the frequency of SP cells (Figure 6C) as well as ABCG2 expression (Figure 6D) in A549, H1650 and H1975 cells compared to control siRNA transfected cells. Similar results were obtained when a different siRNA to Sox2 was used (Additional file 1: Figure S3). Collectively, these results suggest that Sox2 gene has a direct role in maintaining cancer stem cell characteristics and self-renewal of SP cells from NSCLC.
Figure 6Sox2 is necessary for maintaining the self-renewal of SP cells
Sox2 is necessary for maintaining the self-renewal of SP cells. (A) H1650-SPAdh cells were transfected with non-targeting control siRNA, or siRNA against EGFR, Src, or Sox2 and western blot analysis was performed for Sox2, Oct4, EGFR and Src expression. (B) siRNA transfected cells were plated for self-renewal assay and analyzed after 5 days of plating. Average number of spheres generated per well from 1000 cells is plotted (mean ± SD). (C) NSCLC cell lines were transfected with control or Sox2 siRNAs and SP frequency was evaluated. Average fold change in SP-frequency (± SD) is plotted. (D) Decrease in ABCG2 expression in Sox2 siRNA transfected cells was detected by western blotting. The Sox2 expression was evaluated by qRT-PCR and results are presented as bar diagram.
1476-4598-11-73-6
Sox2 is expressed in NSCLC and is associated with metastatic progression
Our data showing that depletion of Sox2 affects the self-renewal properties of stem-like cells, we next examined Sox2 expression in a panel of NSCLC tumor samples obtained from stage I/II or stage IV patients on tissue microarrays (TMAs) by immunohistochemistry. Samples from 193 patients with NSCLC-stage I/II disease including 73 with adenocarcinoma were on one TMA; samples from 103 stage IV-NSCLC patients including 45 with adenocarcinoma from primary site and 17 adenocarcinoma samples from the metastatic sites were on the second TMA. In accordance with earlier reports, Sox2 was strongly expressed in squamous cell carcinoma (SCC) samples for both stage I/II and IV patients (Figure F7 7A (i and ii)). In contrast to SCCs, adenocarcinoma samples had significantly lower expression of Sox2. Sox2 positive cells were heterogeneously distributed in adenocarcinoma samples for both stage I/II and IV patients (Figure 7A (iii and iv). While there was no significant difference in Sox2 expression between different grades of tumors, elevated expression of Sox2 was positively associated with metastatic progression. Representative images for adenocarcinoma-metastases are shown in Figure 7A (v-viii). Approximately 67% of stage I/II (n = 73) and 73% of stage IV (primary site, n = 45) tumors were detected as positive for Sox2 expression (score ≥ 1) using a semi-quantitative scoring system. Compared to the primary site tumor for stage IV patients, higher numbers of metastasized tumors were positive for Sox2 (Figure 7B). The median score for Sox2 expression is represented as histogram (Figure 7C). The average score for Sox2 expression was found to be significantly higher (p = 0.01) in metastasized tumors as compared to the primary site or lower stage tumors. Overall, Sox2 was expressed in all stages of adenocarcinoma and its levels were significantly higher in metastatic lesions.
Figure 7Sox2 expression correlates with metastatic progression of adenocarcinoma
Sox2 expression correlates with metastatic progression of adenocarcinoma. (A) Sox2 expression in Lung squamous cells carcinoma, adenocarcinoma and metastatic adenocarcinoma was analyzed from stage I/II or stage IV patients samples by immunohistochemistry. (B) Semi-quantitative scoring was performed for adenocarcinoma samples and the tumors with score of one or more was considered positive for Sox2 expression and listed. (C) Median score for Sox2 expression was calculated and plotted for different stages of NSCLC progression. The mean (± SD) of score is mentioned in parenthesis. Metastatic tumors showed significantly (p = 0.01) higher expression of Sox2.
1476-4598-11-73-7
Discussion
In the current study, we used the SP phenotype to identify and enrich a subpopulation of NSCLCs with the properties ascribed to CSCs. The studies presented here demonstrates a specific and significant role for EGFR signaling cascade in facilitating the self-renewal growth and expansion of the side population cells from NSCLCs.Our study, in accordance with earlier studies
11
,
B33 33
, confirmed the presence of SP cells in established human NSCLC cell lines and in human tumor xenografts with the properties of CSCs. Comparing the self-renewal ability of SP and MP cells isolated from human tumor xenografts, we found that approximately 0.2% SP cells were able to self-renew and form spheres, whereas MP cells were unable to self-renew. Comparing the percentage of sphere forming cells in SP cells, we estimate that approximately 1-2% of SP cells from established cell lines may have stem-like properties; therefore, SP phenotype may not be the exclusive marker for CSCs, but can be used to enrich stem-like cells from NSCLCs.SP cells were found to be more tumorigenic in vivo, confirming the enrichment of tumor initiating cells in SP compartment. These cells were able to produce highly invasive disease upon implantation into the lungs. Also, the direct association of stem-like cells with generation of metastatic disease may be supported by our observation where a significant correlation was observed between high Sox2 expressions in the metastatic tumors of lung adenocarcinoma patients. Recent reports indicate that the normal epithelial cells acquire the CSCs properties upon induction of EMT governed by various cytokines and growth factors from stromal cells
10
25
. Our results demonstrate that SP-cells intrinsically exhibit loss of epithelial markers and/or the gain of mesenchymal markers as compared to MP cells and could be due to the higher expression of transcription factors Twist, Slug and Snail, which are known to be involved in maintaining the mesenchymal phenotype. Together with the expression of embryonic stem cell transcription factors like Oct4, Sox2, and Nanog along with the exhibition of EMT like features and orthotopic tumor-forming ability, collectively suggest that SP cells isolated from NSCLC cell lines and tumors have stem-like properties. The observation that EGFR signaling affects stem-like functions of SP cells is intriguing, given that several EGFR tyrosine-kinase inhibitors have efficacy against NSCLCs
B34 34
B35 35
. Interestingly, EGFR appears to regulate Sox2 levels, through the Src-Akt pathway; Sox2 has been shown to be regulated by Akt in ES cells, through the inhibition of proteasomal degradation
B36 36
. Consistent with these results, our observation suggest that inhibition of EGFR-Src-Akt signaling downregulates Sox2 levels along with a reduction in ABCG2 levels. This decrease in ABCG2 expression upon EGFR inhibition is probably a causal effect of Sox2 depletion-mediated differentiation of SP into MP cells.The fact that EGFR-pathway inhibition resulted in specific depletion of Sox2 without any significant effect on Oct4 or Nanog expression suggests that their expression may be regulated through independent mechanisms in NSCLC SP cells. Our results as well as an earlier report
B37 37
suggest that Sox2 is expressed in both low as well as high stage adenocarcinomas irrespective of their grades. However, Oct4 or Nanog expression was found to be associated only with the high grade lung adenocarcinoma and not expressed in low grade tumors
17
37
. Therefore, we predict that the EGFR pathway inhibition may exert its favorable effects only for those tumors where Sox2 is the major determinant in controlling the self-renewal of CSCs. Interestingly, a recent study showed that the ectopic overexpression of Oct4 and Nanog increases the tumor initiating property of A549 cells
17
. In agreement with these reports, we find that specific and independent depletion of Oct4 or Nanog also resulted in decrease in SP phenotype but in a cell type dependent fashion (Data not shown). Two recent reports demonstrate that ectopic expression of Sox2 increased the frequency of side population cells and tumor formation in mouse and human NSCLC cell lines
33
B38 38
. These reports strongly suggest that Sox2 expressing cells harbor the stem cell-like properties. Our observation further strengthens this postulation where we demonstrate that Sox2 depletion was sufficient to inhibit the self-renewing property SP cells in all the three NSCLC cell lines.In addition to the mutation in EGFR signaling, perturbation of p53 activity is another important event occurs in initiation and progression of NSCLCs
B39 39
. Recently, p53 is shown to have certain roles in promoting the differentiation of human embryonic stem cell through repression of factors like Oct4, Klf4, Lin28A, and Sox2
B40 40
. However, there is not much information available on the direct role of p53 transcriptional activities in regulating Sox2 expression in stem-like cells in cancer, and would be interesting to explore in future.
Conclusions
Figure F8 8 summarizes the role of Sox2 in SP cell biology and tumor growth. While certain frequency of isolated SP cells from NSCLC exhibit stem cell-like properties and can form metastatic tumors, more differentiated MP cells are greatly impaired in their ability to generate tumors. Further, inhibition of EGFR pathway including Src and PI3-kinase could strongly inhibit the expression of Sox2, suppressing the self-renewal properties of SP cells. Therefore, relative Sox2 expression and functions within the tumor-CSCs may be a major determinant in EGFR-targeted therapy against NSCLCs. This information might also be potentially useful to overcome the acquired resistance to EGFR therapies, by targeting downstream targets of EGFR signaling, including Sox2. Additional investigations in this direction might lead to the development of more effective therapeutic agents to combat NSCLC, especially those harboring EGFR mutations.
Figure 8A schematic depiction of the signaling events that regulate the biology of SP cells
A schematic depiction of the signaling events that regulate the biology of SP cells. Isolated SP cells form metastatic tumors but MP cells do not. Inhibiting EGFR, Src or PI3-kinase significantly impairs the self-renewal properties of SP cells.
1476-4598-11-73-8
Materials and methods
Cell lines and tumor samples
H1650, and H1975 cell lines were obtained from ATCC and maintained in RPMI or DMEM containing10% fetal bovine serum (FBS; Mediatech) in 5% CO2 at 37°C. Human tumor xenografts were obtained from SA laboratory.
Inhibitors, siRNAs and antibodies
Gefitinib, Erlotinib, BIBW2992 and Dasatinib were purchased from LC laboratories. PP2 and Fumitremorgin C (FTC) were purchased from Sigma Inc. In the present study, Gefitinib or erlotinib is used at 500 nM, dasatinib or BIBW2992 is used at 200 nM and PP2 is used at 1 μM dose. siRNA against EGFR, Src family kinases, Akt and Sox2, Oct4 and Nanog was purchased from Santa Cruz Biotechnology or OriGene Technology Inc. Primary antibodies against Sox2 (#3579), Oct4 (#2750), Nanog (#4903), Phos-Src-pYsup 416 (#2101), pERK1/2 (#4376) and phospho-AKT-pS473 (#4058) were purchased from Cell Signaling Technology; Phos-EGFR-pY1068 (#44788G) from Invitrogen; EGFR neutralizing antibody (#05-101) from Milipore and isotype matched mouse IgG were purchased from Biolegend.
RNA preparation and qRT PCR analysis
RNA preparation and RT-PCR analysis was performed as described earlier
B41 41
. Fold inductions were calculated using the formula 2–(ddCt) using GAPDH as internal control gene. The gene-specific primer pairs were as follows. ABCG2 (F) 5’-CAC AAG GAA ACA CCA ATG GCT-3’, ABCG2 (R) 5’-ACA GCT CCT TCA GTA AAT GCC TTC-3’; Oct4 (F) 5’-ACA TCA AAG CTC TGC AGA AAG AAC-3’, Oct4 (R) 5’-CTG AAT ACC TTC CCA AAT AGA ACC C-3’, Sox2 (F) 5’-GGG AAA TGG GAG GGG TGC AAA AGA-3’, Sox2 (R) 5’-TTG CGT GAG TGT GGA TGG GAT TGG-3’, Nanog (F) 5’-AGA AGG CCT CAG CAC CTA-3’, Nanog (R) 5’-GGC CTG ATT GTT CCA GGA TT-3’; Twist (F) 5’-CTC GGA CAA GCT GAG CAA GAT TCA GA-3’, Twist (R) 5’-CGT GAG CCA CAT AGC TGC AGC-3’, Slug (F) 5’- ACA CAT TAC CTT GTG TTT GCA AGA TCT-3’, Slug (R) 5’- TGT CTG CAA ATG CTC TGT TGC AGT G-3’, Snail (F) 5’- CCT CAA GAT GCA CAT CCG AAG CCA C-3’, Snail (R) 5’- CCG GAC ATG GCC TTG TAG CAG C-3’, GAPDH (F) 5’-GGT GGT CTC CTC TGA CTT CAA CA-3’, GAPDH (R) 5’-GTT GCT GTA GCC AAA TTC GTT GT-3’.
Hoechst 33342 dye efflux assay for SP analysis and cell sorting
Adherent cells were harvested using accutase reagent (Sigma Inc). Human Tumor tissue grown in athymic nude mice was minced, enzymatically digested with 0.2% collagenase IV (Worthington Biochemical Corporation) prepared in 10% FBS containing medium for 60 min at 37°C. The digest was further disaggregated by passing through 10 ml pipette several times and filtered through 100/70-μm cell strainer to obtain a single cell suspension. Cells were washed and resuspended in HBSS at 1X106 cells/ml density and incubated with 4 μg/ml of Hoechst 33342 dye (Invitrogen) for 90 min at 370C in presence or absence of 1 μM FTC, as described by Goodell et al.
21
. Cells were incubated with 2 μg/ml Propidium iodide (PI; Sigma Inc) before analysis to visualize and exclude the non-viable cells. The Hoechst 33342 dye was excited at 350 nm using UV laser and its fluorescence was analyzed using 400–500 nm BP filter for blue emission and 640–680 nm BP filter in combination with 655 nm LP-filter for red emission. Flow cytometers from BD Biosciences were used for data acquisition. Data were acquired using LSRII or FACS Vantage (DiVa), and sorted using FACS Vantage (DiVa) cell sorter. Data analyses were done using FlowJo software (Tree Star). Cell cycle analyses for fixed cells were performed for PI stained cells using Vindelov method with similar protocol as described earlier
41
.
Sphere formation or Self renewal assay
Sorted SP or MP cells were plated in 96 well plates at the density of 10,000 cells/ml (1000 cells/well in 100 μl medium) in serum free stem cell selective media (DMEM/F12K (1:1) (Invitrogen), supplemented with 1X-N2 supplement (Invitrogen), 10 ng/ml EGF and 10 ng/ml bFGF (Sigma)) and allowed to grow as spheres for 10 days. Images of the spheres were taken using phase contrast microscope (Nikon) and total numbers were counted. To study the effect of drugs on the self-renewal of SP cells, drugs were added to the respective wells on day 1 and 5 and size and number of the spheres were analyzed on day 10.
Immunofluorescence
For immunostaining, spheres were transferred to poly D-lysine/Laminin coated glass surface for 18 h. For monolayer cultures, cells were directly plated over the poly D-lysin/Laminin coated glass surface and cultured or treated in stem cell selective media as indicated. Immunofluorescence staining was performed as described previously
B42 42
. Cells were observed using a Leica TCS SP5 confocal microscope (Leica Microsystems) at × 630 magnification.
Immunohistochemistry
Human lung cancer tissue microarray (TMA) slides with stage I/II or stage IV NSCLC patients were obtained through Lung Cancer Specialized Program of Research Excellence (SPORE). TMA slide with stage I/II tumor samples contained usable cores from 193 patients, and TMA slide with stage IV tumor samples contained usable cores from 103 patients including 17 adenocarcinoma samples from the metastatic sites. The Immunohistochemical staining was performed as described
42
. The samples were scored by a pathologist (D. Coppola). The semiquantitative score was reached by taking into consideration both cellularity and intensity of expression (semiquantitative score = cellularity × intensity). Cellularity was scored as follows: a score of 3 equals to greater than 66% cellularity, a score of 2 equals to 34%–65% cellularity, and a score of 1 equals to less than 33% cellularity. Intensity was scored as follows: a score of 3 equals to strong intensity, a score of 2 equals to moderate intensity, and a score of 1 equals to weak intensity
42
. The score of 1 or above was considered as positive expression of Sox2. The images were captured at × 200 magnification.
In vivo tumor formation assay and bioluminescence imaging
5-weeks-old female SCID-beige mice were used for these experiments under an IACUC approved protocol. For orthotopic implantation of tumor cells, sorted SP or MP cells from A549 cell line stably expressing luciferase gene (A549-Luc) were washed with serum-free DMEM-F12K medium and resuspended at indicated numbers in HBSS containing 500 μg/ml growth factor reduced Matrigel. Surgical procedure for orthotopic lung implantation was followed as suggested earlier for intrapulmonary implantation of tumor cells with some modifications
B43 43
. Specifically, cells were inoculated with 1 ml syringes with 30-gauge hypodermic needles in an open technique under direct visualization into the right lung tissue of SCID mice anesthetized by gas anesthesia (3% isoflurane). Tumor growth/metastases were imaged weekly using bioluminescence by IVIS-200 imaging system from Caliper Corporation. Mice were anesthetized and 30 mg/Kg of D-luciferin in PBS was administered by intraperitoneal (i.p.) injection. Ten minutes after injection, bioluminescence was imaged with a charge-coupled device camera (Caliper) with an imaging time of 2 min. At the end of the experiment, or when mice become moribund, all of the mice were euthanized and individual organs harvested for evaluation of tumor size; distant metastases was determined by bioluminescence of luciferase expressing cells.
Statistical methods
Data were presented as the mean ± standard deviation (SD). To assess the statistical significance of differences, student’s t test was performed. The data were considered statistically significant when the P value was less than 0.05.
Competing interest
We do not have any conflict of interest.
Authors’ contributions
SS conducted the experiments and wrote the initial version of the manuscript; JT and NBS conducted certain experiments; DC did pathological analysis of the samples; EH provided intellectual input; SA provided human tumor xenografts and input; SC directed the project and finalized the manuscript. All authors read and approved the final manuscript.
bm
ack
Acknowledgments
We thank Jennifer Gemmer for technical support. These studies were supported by the grant CA127725 from the NCI to SPC. Support of the Lung Cancer SPORE, National Functional Genomics Center and the Core Facilities at the Moffitt Cancer Center is greatly appreciated.
refgrp Global cancer statistics, 2002ParkinDMBrayFFerlayJPisaniPCA Cancer J Clin20055574lpage 10810.3322/canjclin.55.2.7415761078Cancer statistics, 2008JemalASiegelRWardEHaoYXuJMurrayTThunMJCA Cancer J Clin200858719610.3322/CA.2007.0010link fulltext 18287387Annual report to the nation on the status of cancer, 1975–2005, featuring trends in lung cancer, tobacco use, and tobacco controlJemalAThunMJRiesLAHoweHLWeirHKCenterMMWardEWuXCEhemanCAndersonRetal J Natl Cancer Inst20081001672169410.1093/jnci/djn389pmcid 263929119033571Cancer statistics, 2012SiegelRNaishadhamDJemalACA Cancer J Clin201262102910.3322/caac.2013822237781Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cellBonnetDDickJENat Med1997373073710.1038/nm0797-7309212098Cancer stem cells-perspectives on current status and future directions: AACR Workshop on cancer stem cellsClarkeMFDickJEDirksPBEavesCJJamiesonCHJonesDLVisvaderJWeissmanILWahlGMCancer Res2006669339934410.1158/0008-5472.CAN-06-312616990346Stem cells, asymmetric division and cancerCleversHNat Genet2005371027102810.1038/ng1005-102716195718Asymmetric and symmetric stem-cell divisions in development and cancerMorrisonSJKimbleJNature20064411068107410.1038/nature0495616810241CD133 is indicative for a resistance phenotype but does not represent a prognostic marker for survival of non-small cell lung cancer patientsSalnikovAVGladkichJMoldenhauerGVolmMMatternJHerrIInt J Cancer201012695095819676044EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancerSinghASettlemanJOncogene2010294741475110.1038/onc.2010.215317671820531305Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cellsHoMMNgAVLamSHungJYCancer Res2007674827483310.1158/0008-5472.CAN-06-355717510412Critical appraisal of the side population assay in stem cell and cancer stem cell researchGolebiewskaABronsNHBjerkvigRNiclouSPCell Stem Cell2011813614710.1016/j.stem.2011.01.00721295271Side population cells in human cancersWuCAlmanBACancer Lett20082681910.1016/j.canlet.2008.03.04818487012The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotypeZhouSSchuetzJDBuntingKDColapietroAMSampathJMorrisJJLagutinaIGrosveldGCOsawaMNakauchiHSorrentinoBPNat Med200171028103410.1038/nm0901-102811533706An extended transcriptional network for pluripotency of embryonic stem cellsKimJChuJShenXWangJOrkinSHCell20081321049106110.1016/j.cell.2008.02.03918358816Embryonic stem cell markers expression in cancersSchoenhalsMKassambaraADe VosJHoseDMoreauxJKleinBBiochem Biophys Res Commun200938315716210.1016/j.bbrc.2009.02.15619268426Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiationChiouSHWangMLChouYTChenCJHongCFHsiehWJChangHTChenYSLinTWHsuHSWuCWCancer Res201070104331044410.1158/0008-5472.CAN-10-263821159654EGFR-AKT-Smad signaling promotes formation of glioma stem-like cells and tumor angiogenesis by ID3-driven cytokine inductionJinXYinJKimSHSohnYWBeckSLimYCNamDHChoiYJKimHCancer Res2011717125713410.1158/0008-5472.CAN-11-133021975932Epidermal growth factor plays a crucial role in mitogenic regulation of human brain tumor stem cellsSoedaAInagakiAOkaNIkegameYAokiHYoshimuraSNakashimaSKunisadaTIwamaTJ Biol Chem2008283109581096610.1074/jbc.M70420520018292095The EGF receptor-sox2-EGF receptor feedback loop positively regulates the self-renewal of neural precursor cellsHuQZhangLWenJWangSLiMFengRYangXLiLStem Cells20102827928619882665Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivoGoodellMABroseKParadisGConnerASMulliganRCJ Exp Med19961831797180610.1084/jem.183.4.179721925118666936Pheophorbide a is a specific probe for ABCG2 function and inhibitionRobeyRWSteadmanKPolgarOMorisakiKBlayneyMMistryPBatesSECancer Res2004641242124610.1158/0008-5472.CAN-03-329814973080Eyes wide open: a critical review of sphere-formation as an assay for stem cellsPastranaESilva-VargasVDoetschFCell Stem Cell2011848649810.1016/j.stem.2011.04.00721549325Identification of human brain tumour initiating cellsSinghSKHawkinsCClarkeIDSquireJABayaniJHideTHenkelmanRMCusimanoMDDirksPBNature200443239640110.1038/nature0312815549107The epithelial-mesenchymal transition generates cells with properties of stem cellsManiSAGuoWLiaoMJEatonENAyyananAZhouAYBrooksMReinhardFZhangCCShipitsinMCell200813370471510.1016/j.cell.2008.03.027272803218485877Transcriptional regulation of cell polarity in EMT and cancerMoreno-BuenoGPortilloFCanoAOncogene2008276958696910.1038/onc.2008.34619029937Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traitsPolyakKWeinbergRANat Rev Cancer2009926527310.1038/nrc262019262571Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screensPollardSMYoshikawaKClarkeIDDanoviDStrickerSRussellRBayaniJHeadRLeeMBernsteinMCell Stem Cell2009456858010.1016/j.stem.2009.03.01419497285SRC-family kinases are activated in non-small cell lung cancer and promote the survival of epidermal growth factor receptor-dependent cell linesZhangJKalyankrishnaSWislezMThilaganathanNSaigalBWeiWMaLWistubaIIJohnsonFMKurieJMAm J Pathol200717036637610.2353/ajpath.2007.060706176270717200208Src kinases as therapeutic targets for cancerKimLCSongLHauraEBNat Rev Clin Oncol2009658759510.1038/nrclinonc.2009.12919787002A role for phosphatidylinositol 3-kinase in TCR-stimulated ERK activation leading to paxillin phosphorylation and CTL degranulationRobertsonLKMireauLROstergaardHLJ Immunol20051758138814516339552PI3K-dependent cross-talk interactions converge with Ras as quantifiable inputs integrated by ErkWangCCCiritMHaughJMMol Syst Biol20095246265753519225459SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicityNakatsugawaMTakahashiAHirohashiYTorigoeTInodaSMuraseMAsanumaHTamuraYMoritaRMichifuriYLab Invest2011911796180410.1038/labinvest.2011.14021931300Deregulated EGFR signaling during lung cancer progression: mutations, amplicons, and autocrine loopsGazdarAFMinnaJDCancer Prev Res2008115616010.1158/1940-6207.CAPR-08-0080Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancerAmannJKalyankrishnaSMassionPPOhmJEGirardLShigematsuHPeytonMJuroskeDHuangYStuart SalmonJCancer Res20056522623515665299Phosphorylation of Sox2 cooperates in reprogramming to pluripotent stem cellsJeongCHChoYYKimMOKimSHChoEJLeeSYJeonYJLeeKYYaoKKeumYSStem Cells2010282141215010.1002/stem.54020945330Sox2 protein expression is an independent poor prognostic indicator in stage I lung adenocarcinomaShollLMBarlettaJAYeapBYChirieacLRHornickJLAm J Surg Pathol2010341193119810.1097/PAS.0b013e3181e5e024292381920631605Downregulation of transcription factor SOX2 in cancer stem cells suppresses growth and metastasis of lung cancerXiangRLiaoDChengTZhouHShiQChuangTSMarkowitzDReisfeldRALuoYBr J Cancer20111041410141710.1038/bjc.2011.94310194421468047TP53 mutations in nonsmall cell lung cancerMogiAKuwanoHJ Biomed Biotechnol20112011583929303536021331359p53 regulates cell cycle and microRNAs to promote differentiation of human embryonic stem cellsJainAKAlltonKIacovinoMMahenEMilczarekRJZwakaTPKybaMBartonMCPLoS Biol201210e100126810.1371/journal.pbio.1001268328960022389628Rb-Raf-1 interaction disruptor RRD-251 induces apoptosis in metastatic melanoma cells and synergizes with dacarbazineSinghSDavisRAlamandaVPiredduRPernazzaDSebtiSLawrenceNChellappanSMol Cancer Ther201093330334110.1158/1535-7163.MCT-10-0442305823821139044ARRB1-mediated regulation of E2F target genes in nicotine-induced growth of lung tumorsDasguptaPRizwaniWPillaiSDavisRBanerjeeSHugKLloydMCoppolaDHauraEChellappanSPJ Natl Cancer Inst201110331733310.1093/jnci/djq541303972821212384Mediastinal lymph node metastasis model by orthotopic intrapulmonary implantation of Lewis lung carcinoma cells in miceDokiYMurakamiKYamauraTSugiyamaSMisakiTSaikiIBr J Cancer1999791121112610.1038/sj.bjc.6690178236225410098745



PAGE 1

1 Supplementary Figure s: Supplementary Figure S 1: BIBW 2992 inhibits EGFR phophorylation H1975 cells were treated with 500 nM gefitinib or 200 nM BIBW2992 for 5 days. EGFR phosphorylation and total EGFR expression was detected in presence or absence of drug treatment.

PAGE 2

2 Supplementary Figure S2 : Downregulation of Sox2 expression after EGFR and Src inhibition. H1 650 SPAdh cells were treated plated over PDL Laminin coated glass surface and treated with indicated drugs for 4 days. (A) Expression of Sox2 was monitored by immuno fluorescence confocal imaging. Isotype antibody was used to show the specific staining of Sox2. (B) Number of Sox2 posi tive cells for each treatment condition were converted into percentage and plotted. P values were calculated from three different experiments and

PAGE 3

3 suggested a significant decrease in Sox2 positive cells after EGFR and Src inhibition. (C) Under similar treat m,ent conditions cells were stained with Nanog specific antibodies. Drug treatment did not alter the expression of Nanog in H1650 SPAdh cells.

PAGE 4

4 Supplementary Figure S3 : Depletion of Sox2 expression suppresses SP frequency (A) A549, H1650 and H1975 ce lls were transiently transfected with second set siRNA (purchased from Origene). 48 hr after transfection, cells were analyzed for SP frequency. Similar to first set of siRNA (purchased from SantaCruz), depletion of Sox2 resulted in significant decrease in SP frequency in NSCLCs. (B) NSCLC cells were transfected with Sox2 SIRNA and ABCG2 expression was detected by western blotting. Actin was used as internal control for equal loading. p<0.05.