Group Title: Breast Cancer Research
Title: Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion
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Title: Protein kinase D1 regulates matrix metalloproteinase expression and inhibits breast cancer cell invasion
Series Title: Breast Cancer Research
Physical Description: Book
Language: English
Creator: Eiseler, Tim
Doppler, Heike
Yan, Irene
Goodison, Steve
Storz, Peter
Publication Date: 2009
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Abstract: INTRODUCTION:The biological and molecular events that regulate the invasiveness of breast tumour cells need to be further revealed to develop effective therapies that stop breast cancer from expanding and metastasising.METHODS:Human tissue samples of invasive breast cancer and normal breast, as well as breast cancer cell lines, were evaluated for protein kinase D (PKD) expression, to test if altered expression could serve as a marker for invasive breast cancer. We further utilised specific PKD1-shRNA and a system to inducibly-express PKD1 to analyse the role of PKD1 in the invasive behaviour of breast cancer cell lines in two-dimensional (2D) and three-dimensional (3D) culture. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs), so we also determined if PKD1 regulates the expression and activity of these enzymes.RESULTS:We found that the serine/threonine kinase, PKD1, is highly expressed in ductal epithelial cells of normal human breast tissue, but is reduced in its expression in more than 95% of all analysed samples of human invasive breast tumours. Additionally, PKD1 is not expressed in highly invasive breast cancer cell lines, whereas non-invasive or very low-invasive breast cancer cell lines express PKD1. Our results further implicate that in MDA-MB-231 cells PKD1 expression is blocked by epigenetic silencing via DNA methylation. The re-expression of constitutively-active PKD1 in MDA-MB-231 cells drastically reduced their ability to invade in 2D and 3D cell culture. Moreover, MCF-7 cells acquired the ability to invade in 2D and 3D cell culture when PKD1 expression was knocked-down by shRNA. PKD1 also regulated the expression of breast cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, providing a potential mechanism for PKD1 mediation of the invasive phenotype.CONCLUSIONS:Our results identify decreased expression of the PKD1 as a marker for invasive breast cancer. They further suggest that the loss of PKD1 expression increases the malignant potential of breast cancer cells. This may be due to the function of PKD1 as a negative regulator of MMP expression. Our data suggest re-expression of PKD1 as a potential therapeutic strategy.
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Research article

Protein kinase D1 regulates matrix metalloproteinase expression

and inhibits breast cancer cell invasion
Tim Eiseler1, Heike DOppler1, Irene K Yan1, Steve Goodison2 and Peter Storz~,3


1Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Griffin Building, 4500 San Pablo Road, Jacksonville, FL 32224, USA
2Department of Surgery, University of Florida, 653 West 8th Street, Jacksonville, FL 32209, USA
3Mayo Clinic, Griffin Room 306, 4500 San Pablo Road, Jacksonville, FL 32224, USA

Corresponding author: Peter Storz, storz.peter@mayo.edu

Received: 8 Sep 2008 Revisions requested: 7 Oct 2008 Revisions received: 10 Feb 2009 Accepted: 25 Feb 2009 Published: 25 Feb 2009

Breast Cancer Research 2009, 11:R13 (doi:10.1186/bcr2232)
This article is online at: http://breast-cancer-research.com/content/11/1/R13
2009 Eiseler et al.; licensee BioMed Central Ltd.
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.


Abstract


Introduction The biological and molecular events that regulate
the invasiveness of breast tumour cells need to be further
revealed to develop effective therapies that stop breast cancer
from expanding and metastasising.

Methods Human tissue samples of invasive breast cancer and
normal breast, as well as breast cancer cell lines, were evaluated
for protein kinase D (PKD) expression, to test if altered
expression could serve as a marker for invasive breast cancer.
We further utilised specific PKD1-shRNA and a system to
inducibly-express PKD1 to analyse the role of PKD1 in the
invasive behaviour of breast cancer cell lines in two-dimensional
(2D) and three-dimensional (3D) culture. Invasive behaviour in
breast cancer cell lines has been linked to matrix
metalloproteinases (MMPs), so we also determined if PKD1
regulates the expression and activity of these enzymes.

Results We found that the serine/threonine kinase, PKD1, is
highly expressed in ductal epithelial cells of normal human
breast tissue, but is reduced in its expression in more than 950/0
of all analysed samples of human invasive breast tumours.


Additionally, PKD1 is not expressed in highly invasive breast
cancer cell lines, whereas non-invasive or very low-invasive
breast cancer cell lines express PKD1. Our results further
implicate that in MDA-MB-231 cells PKD1 expression is
blocked by epigenetic silencing via DNA methylation. The re-
expression of constitutively-active PKD1 in MDA-MB-231 cells
drastically reduced their ability to invade in 2D and 3D cell
culture. Moreover, MCF-7 cells acquired the ability to invade in
2D and 3D cell culture when PKD1 expression was knocked-
down by shRNA. PKD1 also regulated the expression of breast
cancer cell MMPs, MMP-2, MMP-7, MMP-9, MMP-10, MMP-11,
MMP-13, MMP-14 and MMP-15, providing a potential
mechanism for PKD1 mediation of the invasive phenotype.

Conclusions Our results identify decreased expression of the
PKD1 as a marker for invasive breast cancer. They further
suggest that the loss of PKD1 expression increases the
malignant potential of breast cancer cells. This may be due to
the function of PKD1 as a negative regulator of MMP
expression. Our data suggest re-expression of PKD1 as a
potential therapeutic strategy.


Introduction for PKD enzymes in the regulation of cell adhesion, vesicle
Protein kinase D (PKD) belongs to the calcium/calmodulin- transport and cell survival [3-8]. There is also increasing evi-
regulated kinase family of serine/threonine kinases [1]. The dence that PKD enzymes are involved in pathways that inhibit
PKD family consists of three members, PKD1/PKCui, PKD2 apoptosis in tumours of the pancreas and cervix [5,8].
and PKD3/PKCv, which share a unique molecular architecture
[2]. Depending on the cancer cell type and the activation A potential mechanism for PKD1 regulation of cell survival is
mechanism, recent reports have revealed important functions via activation of the anti-apoptotic transcription factor nuclear


2D: two-dimensional; 3D: three-dimensional; ATCC: American Type Culture Collection; BSA: bovine serum albumin; ECM: extracellular matrix; ER:
oestrogen receptor; HDAC: histone deacetylase; HRP: horseradish peroxidase; MMP: matrix-metalloproteinase; MT: membrane type; NaCI: sodium
chloride; NF-KB: nuclear factor-KB; PBS: phosphate buffered saline; PKC: protein kinase C; PKD: protein kinase D; PR: progesterone receptor;
pTNM: pathological tumour-node-metastasis; RT-PCR: reverse transcription polymerase chain reaction; TIMP: tissue inhibitors of metalloproteinases;
TMA: tissue microarray.


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Breast Cancer Research Vol 11 No 1 Eiseler et al.


factor (NF) KB [8,9]. PKD1 was also recently implicated in the
inhibition of cell migration of pancreatic cancer cells [10]. In
line with its negative regulatory effects on cell motility, PKD1
can be activated by the RhoGTPase RhoA [11], which in its
active state has also been implicated in the inhibition of cell
migration [12,13]. PKD1 expression is downregulated in
androgen-independent prostate cancer [14] and the PKD1
promoter is epigenetically-silenced by methylation events in
gastric cancer [15]. To date, there was little known on the
expression and function of PKD1 in breast cancer. Breast can-
cer cells invade surrounding tissues by breaking through the
basal membrane using invadopodia, which participate in pro-
teolytic matrix degradation. In some breast cancer cells, PKD
forms a complex with cortactin and paxillin, which are both
associated with invadopodia membranes [4]. However, the
function and the activation status of PKD1 in this complex are
not known.

An important step in the complex regulation of tumour expan-
sion and metastasis is the degradation of extracellular matrix
(ECM) which allows cells to migrate and invade surrounding
areas and into peripheral tissues or enter the bloodstream
[16,1 7]. Matrix metalloproteinases (MMPs) are collagenases
(e.g. MMP-1, MMP-13), stromelysins (e.g. MMP-10, MMP-12),
gelatinases (e.g. MMP-2, MMP-9) or membrane-type enzymes
(e.g. MMP-14, MMP-16) and have been recognized as impor-
tant mediators of ECM degradation. In almost all human can-
cers the increased expression of certain MMPs correlates with
tumour expansion, increased invasiveness and poor prognosis
[16,18]. MMPs and MMP inhibitors have been extensively
investigated in human breast cancer clinical studies [19-22].

The tissue levels of at least MMP-1, MMP-2, MMP-9, MMP-11,
membrane type (MT) 1 -MMP, tissue inhibitors of metalloprotei-
nases (TIMP) 1 and TIMP-2 have been correlated with poor
outcome of breast cancer patients [20,23,24]. Furthermore,
MMP-1 and MMP-2 have been described as genes that selec-
tively mediate lung metastasis in the MDA-MB-231 xenograft
model of breast cancer [25] and are members of a lung metas-
tasis gene signature for human breast cancers [26]. Recent
data also show that tumour-derived, rather than stromal fibrob-
last-derived, MMP-13 correlates with aggressive breast
tumour types and is inversely correlated with the overall sur-
vival of breast cancer patients [22]. The regulation of MMP
expression is complex, involving a multitude of transcription
factors and histone deacetylases [27-31]; however, no infor-
mation is available regarding the negative regulation of MMP
genes in mechanisms that reduce ECM degradation.

Here we show that PKD1 expression is decreased in invasive
breast cancer tissue and that PKD1 expression is silenced in
invasive breast cancer cell lines. The re-expression of active
PKD1 in highly invasive breast cancer cells blocks cell inva-
sion and the reduction of PKD1 expression in very low-invasive
breast cancer cells increases the invasive ability of these cells.


We also identify PKD1 as an inhibitor of the expression of
matrix-metalloproteinases, such as MMP-2, MMP-7, MMP-9,
MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15, all of
which have been implicated in the progression of breast can-
cer. Our findings show that PKD1 inhibits breast tumour cell
invasion and thus may influence tumour cell dissemination and
metastasis, the most lethal aspect of breast cancer.

Materials and methods
Cell lines, DNA constructs, reagents and antibodies
All cell lines were purchased from the American Type Culture
Collection (ATCC, Manassas, VA, USA) and were maintained
according to information provided by ATCC. pcDNA3-based
expression constructs for HA-tagged wildtype PKD1, kinase-
dead PKD1 (PKDinactive, PKD1.K612W) and constitutively-
active PKD1 (PKDactive, PKD1.Y463E) have been described
previously [8]. The doxycycline-regulated expression system
for mammalian cells was from Invitrogen (Carlsbad, CA, USA).
MDA-MB-231 cells were first stably transfected with
pcDNA6/TR and selected with blasticidin to generate a MDA-
MB-231-TR cell line. Constitutively-active PKD1 (PKD1 active,
PKD1.Y463E) was cloned into pcDNA4/TO-B via BamHI and
Xhol sites and verified by DNA sequencing.

To generate stable cell lines which allow doxycycline-depend-
ent inducible expression of constitutively-active PKD1, the
pcDNA4/TO-B-PKD1.Y463E construct was stably trans-
fected into the MDA-MB-231-TR cells and selected with
Zeocin to generate the MDA-MB-231-TR-PKD1.Y463E cell
lines. All transfections were performed with Lipofectamine
2000 from Invitrogen (Carlsbad, CA, USA). MCF-7 cells were
infected with PKD1-shRNA lentivirus to generate the MCF-7-
PKD1 .RNAi cell lines. Clonal selection for stably infected cells
was performed with puromycin. The rabbit polyclonal antibody
specific for PKD1 was raised against a H2N-MAECQNDS-
GEMQDP-amide peptide corresponding to amino acids 372-
385 in human PKD1 (21 Century Biochemicals, Marlboro,
MA). The rabbit polyclonal antibody for PKD2 was from
Upstate (Charlottesville, VA, USA), and the rabbit polyclonal
antibody for PKD3 was from Bethyl Laboratories (Mont-
gomery, TX, USA). All these antibodies were specific for the
respective PKD isoenzyme and were not cross-reactive with
other PKD family members (data not shown). Anti-FLAG, anti-
HA and anti-Actin were from Sigma (St. Louis, MO, USA).
Anti-MMP-9 was from BD Biosciences (San Jose, CA, USA)
and anti-MMP-2 from Epitomics (Burlingame, CA, USA). The
secondary horseradish peroxidase (HRP) linked anti-mouse or
anti-rabbit antibodies were from Roche (Indianapolis, IN,
USA). The DNA methyltransferase inhibitor RG108 (2-(1,3-
Dioxo-1.3-dihydro-2H-isoindol-2-yl)-3-(1 H-indol-3-yl)propi-
onic acid) was from Sigma (St. Louis, MO, USA).


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Tissue samples, immunohistochemistry and statistical
analysis
Tissue microarray (TMA) slides containing histologically-con-
firmed human breast cancer and normal human breast tissue
samples were purchased from Imgenex (San Diego, CA,
USA). The TMAs were deparaffinised (one hour at 600C), de-
waxed in xylene (five times for four minutes) and gradually re-
hydrated with ethanol (100%, 95%, 75%, twice with each
concentration for three minutes). The rehydrated TMAs were
rinsed in water and subjected to antigen retrieval in citrate
buffer (pH 6.0) as described by the manufacturer (DAKO,
Carpinteria, CA, USA).

Slides were treated with 3% hydrogen peroxide (five minutes)
to reduce endogenous peroxidase activity and washed with
PBS containing 0.5% Tween 20. PKD1, PKD2 and PKD3
were detected using specific antibodies at a dilution of
1:2000, 1:1000 and 1:200, respectively, in PBS/Tween and
visualised using the Envision Plus Dual Labeled Polymer Kit
following the manufacturer's instructions (DAKO, Carpinteria,
CA, USA). Images were captured using ImagePro software
(Media Cybernetics, Bethesda, MD, USA). The TMAs were
scored independently by three different experienced scien-
tists. Uniform pre-established criteria were used.

Immunoreactivity was graded semiquantitatively by consider-
ing the intensity of the staining of the ductal cells. A histologi-
cal score was obtained from each sample, which ranged from
0 (no immunoreaction) to 5 (maximum immunoreactivity as
seen in normal ductal tissue). All normals were scored
between 4 and 5, with an average of all samples at 4.57.
Immunostaining was assessed by considering the percentage
of positive cells because the positivity was homogeneous in
each sample. Reproducibility of the scoring method between
three observers was greater than 90%. In the remaining cases,
in which discrepancies had been noted, differences were set-
tled by consensus review of corresponding slides. Statistical
analysis (student's t-test) was performed with GraphPad Soft-
ware (GraphPad Software, La Jolla, CA, USA).

Reverse transcription PCR
Cellular mRNA isolation was performed using RNA-Bee (TEL-
TEST, Friendswood, TX, USA) according to the manufac-
turer's instructions and was transcribed into cDNA using
Superscript II (Invitrogen, Carlsbad, CA, USA). For the tran-
scription reaction, 1 uLg Oligo dT(18) primer (New England
Biolabs, Beverly, MA, USA) and 1 uLg RNA were incubated in
a total volume of 10 pl water at 700C for 10 minutes. Then, 5x
buffer, 40 U RNAsin (Roche, Mannheim, Germany), 200 1LM
dNTP (New England Biolabs, Beverly, MA, USA), 10 mM DTT,
300 U Superscript II reverse transcriptase were then added to
a total volume of 20 uil. The reaction was carried out at 45C
for 60 minutes and then heat inactivated at 950C for five min-
utes. The resulting cDNA pool was subjected to PCR analysis
using specific primer sets. Primers for human PKD1 were


Available online http://breast-cancer-research.com/content/11/1/R13



TTCTCCCACCTCAGGTCATC and TGCCAGAGCACAT-
AACGAAG, PKD2 were CAACCCACACTGCTTTGAGA and
CACACAGCTTCACCTGAGGA, and PKD3 were TCATT-
GACAAACTGCGCTTC and GTACATGAT-
CACGCCCACTG. Primers for human MMPs and TIMPs are
described elsewhere [32]. The primers for actin were
CCTCGCCTTTGCCGATCC and GGATCTTCATGAGG-
TAGTCAGTC. Reaction conditions for the PCR reaction
were: one minute annealing at 550C, one minute amplification
at 720C, with 20, 35 and 40 cycles.

Lentiviral shRNA expression
The Lentiviral shRNA expression system to knock-down PKD1
expression is commercially available from Sigma (SHDNA
MISSION shRNA Plasmid DNA; St. Louis, MO, USA). The
chosen sequences for siRNA were specific, as judged by
BLASTn searches of the all GenBank+RefSeq Nucle-
otides+EMBL+DDBJ+PDB sequences and the human sub-
set of GenBank+EMBL+DDBJ sequences. Sequences are
available from Sigma (NM_002742.x-2498slc1 and
NM_002742.x-2978slc1; St. Louis, MO, USA). The ViraP-
ower Lentiviral Expression System (Invitrogen, Carlsbad, CA,
USA) was used for an optimised mix of packaging plasmids
which supplies the structural and replication proteins that
were required to produce Lentivirus in 293FT cells.

Cell lysates, immunoprecipitation and immunostaining
Cells were lysed in lysis buffer (50 mM Tris/HCI pH 7.4, 1%
TritonX-100, 150 mM sodium chloride (NaCI), 5 mM EDTA)
plus Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA)
and either lysates were used for immunoblot analysis or pro-
teins of interest were immunoprecipitated by a one-hour incu-
bation with the respective antibody (2 fLg) followed by a 30
minute incubation with protein A/G-agarose (Santa Cruz Bio-
technology, Santa Cruz, CA, USA). Immune complexes were
washed five times with TBS (50 mM Tris/HCI pH 7.4, 150 mM
NaCI), resolved by SDS-PAGE, transferred to nitrocellulose
and analysed by immunostaining.

Transwell assay
Migration and invasion assays were performed as previously
described [33] using Transwell chambers. Transwell cham-
bers were coated with standard Matrigel (2 fig/well,) from
Fisher (Pittsburgh, PA, USA). For assays with transient trans-
fected cells, cells were co-transfected with the constructs of
interest and a p-Gal reporter plasmid (pCS2-(n)p-gal) at a ratio
of 5:1 for 24 hours. Inserts of transwell plates were coated,
dried over night and re-hydrated for one hour with 40 pl of tis-
sue culture media. Cells were harvested, washed once with
media containing 1% BSA, re-suspended in media containing
0.1% BSA (106 cells/ml) and seeded on the Transwells (105
cells). NIH-3T3 conditioned medium served as a chemoat-
tractant in the lower chamber. Remaining cells were used to
analyse the transfection efficiency and/or the expression of
proteins of interest. After 16 hours, cells on top of the transwell


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Breast Cancer Research Vol 11 No 1 Eiseler et al.


insert were removed and cells that had migrated to the lower
surface of the filters were fixed in 4% paraformaldehyde and
stained with X-Gal staining solution. Cells which stained posi-
tive for 1-Galactosidase expression were counted. The mean
of triplicate assays for each experimental condition was used
as percentage relative invasion.

Multicellular spheroids/3D cell culture assay
Three-dimensional (3D) analysis of morphology was per-
formed as described previously [12]. In brief, cell culture
dishes (24-well plates) were precoated with undiluted phenol
red-free Matrigel (10 mg/ml). In 200 1l PBS, 104 cells (per well
of a 24-well plate) were suspended and then mixed with 100
p l of cold Matrigel (10 mg/ml). The cell suspension was added
dropwise over the bottom layer to cover it. After the cell layer
was set complete, culture media was added over the top.
Media was changed every two days, without disturbing the
cell/matrix layer. Photos were taken after indicated days using
a 10x magnification for an overview and 40x to document
structure.

Cell proliferation assays
Cells were seeded at a density of 2500 cells/well in clear bot-
tom black 96-well tissue culture plates. After adhesion over-
night, the respective t = 0 plate was washed once with 1 x
PBS, tapped dry and then frozen at -80C. The same proce-
dure was used to process the respective t = 24 hour and t =
48 hour time-point plates. After all plates had been acquired,
cell proliferation was measured using a CytoQuant cell prolif-
eration assay kit (Invitrogen, Carlsbad, CA, USA). Cells were
lysed with 200 p lx cell-lysis buffer with CyQuant GR dye
(1:400 dilution) per well. CytoQuant GR fluorescence was
measured using a SpectraMAX M5 plate reader (Molecular
Devices/MDS, Toronto, Canada) by exiting the dye at 485 nm
and reading emission at 538 nm.

Zymography
Zymography was performed as previously described [34].
Briefly, 48 hours after transfection culture media was har-
vested. Samples were mixed with 2 x loading buffer (50 mM
Tris-HCI pH 6.8, 10% (v/v) glycerol, 1% (w/v) SDS, 0.01%
(w/v) bromophenol blue) and resolved on an SDS-PAGE con-
taining 0.12 mg/ml gelatin (porcine skin type A, bloom 300).
Gels were soaked for one hour in 2.5% Triton X-100, then
washed twice with collagenase buffer (50 mM Tris-HCI pH
7.6, 0.2 M NaCI, 5 mM calcium chloride, 0.2% Brij-35) and
incubated at 370C for 18 hours. Gels were then washed with
distilled water and incubated in Coomassie brilliant blue stain-
ing solution (40% methanol, 10% acetic acid/0.025%0
Coomassie Brilliant Blue R-250) at room temperature for two
hours. Gels were then washed for 24 hours in distilled water
and scanned using an Agfa Duoscan T1200 Scanner (Agfa-
Gevaert, Mortsel, Belgium).


Results
PKD1 expression is reduced in invasive ductal carcinoma
We analysed TMAs including 10 normal breast tissue sam-
ples, 40 invasive ductal carcinoma of the breast and 10 meta-
static invasive ductal carcinoma samples from lymph nodes for
the expression of the PKD family kinases, PKD1, PKD2 and
PKD3. We found that PKD1 is highly expressed in epithelial
ductal tissue of human normal breast samples, but is reduced
in its expression in more than 95% of invasive human breast
tumour samples (representative pictures of normal and tumour
tissue are in Figures lal to 1a4). When compared with normal
breast tissue the tumour samples revealed an approximate
60% reduction in PKD1 expression in both, invasive ductal
carcinoma and metastatic invasive ductal carcinoma (Figure
1 b). PKD1 may have functions in both the cytosol and the
nucleus [35]. PKD1 staining was observed in normal breast
tissue in the nuclei, as well as in the cytosol. Breast cancer
samples of invasive ductal carcinoma and metastatic invasive
ductal carcinoma showed both a decrease of cytosolic stain-
ing and nuclear staining. Interestingly, the two other PKD fam-
ily members, PKD2 and PKD3, showed no significant
difference in their expression or localisation in infiltrating ductal
carcinoma and normal breast tissue (Figures 1 cl to 1 d2), indi-
cating a potential function for PKD1 in invasive breast cancer.
For all samples, sex, age, diagnosis, pathological tumour-
node-metastasis (pTNM), stage, lymph node (positive lymph
nodes/examined lymph nodes) as well as progesterone recep-
tor (PR) and oestrogen receptor (ER) expression status were
available. In all 50 samples of invasive ductal cancer we
observed a significant reduction of PKD1 expression as com-
pared with the normal ductal tissue regardless of stage, ER,
PR or other above markers. A similar downregulation of PKD1
was also recently described for other cancers such as pros-
tate [14] and gastric cancer [15].

PKD1 is not expressed in invasive breast cancer cell lines
We next determined the PKD1 expression status in a subset
of breast cancer cell lines. We found that PKD1 expression at
the mRNA level is absent in the highly invasive breast cancer
cell lines SKBR3, T47D and MDA-MB-231 (Figure 2a). Non-
invasive or very low-invasive breast cancer cell lines such as
BT-474 and MCF-7 and a normal breast cell line MCF-10A
showed moderate PKD1 expression. No distinct pattern was
detectable for PKD2 and PKD3 expression when cells with
high invasive potential were compared with cell lines with low
invasive capacity (Figure 2a). We also analysed the 1-HMT-
3522 cell progression model, in which the subclone S1
retains a more benign phenotype, and the subclone T4/2 has
a more invasive character [36]. T4/2 cells showed less PKD1
mRNA expression as compared with S1 cells, whereas PKD2,
PKD3 and actin mRNA levels were similar (Figure 2b). These
data suggest that PKD1 expression is decreased when cells
achieve a more aggressive state.


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Available online http://breast-cancer-research.com/content/11/1/R13


Figure 1

(al)


.4,

a i


(a2; ##PKD1
(a2) !~J


'.0IS-


norma


infi rating ductal carcinoma


(a4) PK1no






tini ng cuctal carcinomj


a. 100

8
0 60


U5 40

_m 20
0


P < 0.0001
P < 0.0001


Tumor


Sinfiltrang ductal carcinoma normal infiltrating ductal carcinoma

PKD1 expression is reduced in invasive ductal carcinoma. (a to d) Tissue microarrays (TMAs) including 10 normal breast tissue samples, 40 inva-
sive ductal carcinoma of the breast and 10 metastatic invasive ductal carcinoma samples from lymph nodes were analysed for the expression of the
protein kinase D (PKD) family kinases PKD1, PKD2 and PKD3, using isoform specific antibodies. Representative pictures of normal and tumour tis-
sue are depicted. (b) Statistical analysis of the normal and tumour samples. Error bars shown represent standard deviation. P values were acquired
with the student's t-test using Graph Pad software. P values indicate extreme statistical significance. IDC = invasive ductal carcinoma; mlDC = met-
astatic invasive ductal carcinoma.


Further, as expected, loss of PKD1 mRNA correlated with a
lack of PKD1 protein expression in highly invasive cells lines
(Figure 2c). In gastric cancer it was recently shown that PKD1
is downregulated in its expression by DNA methyltransferases
[15]. Epigenetic silencing of genes by DNA methylation is also
a common mechanism involved in gene silencing in breast
cancer. We found that in MDA-MB-231 cells the PKD1 gene
is also epigenetically silenced by DNA methylation, because
treatment of these cells with agents that inhibit DNA methyl-
transferases such as RG108 (Figure 2d) or Decitabine (data
not shown) led to the re-expression of PKD1.

Knockdown of PKD1 increases cell invasion
We next analysed if the decreased expression of PKD1 is one
of the means by which breast tumour cells may increase their
invasive potential. To test this we utilised the very low-invasive
breast cancer cell line MCF-7 which we have shown moder-
ately expresses PKD1 (Figures 2a,c). We transfected MCF-7
cells stably with control shRNA or two different PKD1 -specific
shRNA sequences to knockdown PKD1 expression (Figure
3a). Both shRNA sequences led to an approximate 80%


reduction of PKD1 expression. The knockdown of PKD1
expression had no effect on cell proliferation because all three
cell lines showed similar proliferation rates (Figure 3b). We
next analysed if the knockdown of PKD1 had an impact on the
invasiveness of the cells. Interestingly, cellular invasion in
Matrigel Transwell assays was increased three to four-fold
when PKD1 was knocked down (Figure 3c). Finally, we ana-
lysed the invasive potential of control and PKD1 -shRNA MCF-
7 cells in 3D cell culture. Cells were embedded in Matrigel and
invasive growth was monitored over a period of 60 days. We
observed that the multicellular MCF-7 spheroids showed a
more invasive phenotype when PKD1 expression was reduced
(Figure 3d). These results clearly indicate that the loss of
PKD1 in very low-invasive tumour cells increases their invasive
potential in two-dimensional (2D) and 3D culture systems.

Active PKD1 inhibits breast tumour cell invasion
We next determined if the re-expression of constitutively-
active PKD1 impairs the invasive phenotype of the highly-inva-
sive MDA-MB-231 cells. First, we transiently-transfected
MDA-MB-231 cells with wildtype, constitutively-active


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Breast Cancer Research Vol 11 No 1 Eiseler et al.


Figure 2


/



RT-PCR: PKD1


RT-PCR: PKD2


RT-PCR: PKD3


RT-PCR: Actin


4 PKD1
munoblot: a-PKD1

4 Actin


mm
RT-PCR: PKD1


RT-PCR: PKD2


RT-PCR: PKD3


RT-PCR: Actin



(d)

RG108 (250 nM)

0 24 hours

1 11 -4 PKD1
IP: a-PKD1, Blot: a-PKD1

1 Lysa Actin
Lysates: a-Actin


PKD1 is not expressed in invasive breast cancer cell lines. (a,b) MCF-10A cells and BT-474, MCF-7, SKBR3, T47D, MDA-MB-231, 1-HMT-3522-
S1 or 1 -HMT-3522-T4/2 breast cancer cell lines were cultivated under normal growth conditions. mRNA was isolated and the expression of protein
kinase D (PKD) 1, PKD2, PKD3 and actin was detected by RT-PCR. (c) MCF-1 OA cells and BT-474, MCF-7, SKBR3, T47D, MDA-MB-231, 1 -HMT-
3522-S1 or 1 -HMT-3522-T4/2 breast cancer cell lines were cultivated under normal growth conditions. Cells were lysed and lysates were analysed
for PKD1 expression by western blotting. Actin served as loading control. (d) MDA-MB-231 cells were either left untreated or treated with RG108
(250 nM, for 24 hours). PKD1 was immunoprecipitated (u-PKD1 antibody) and immunoprecipitates were analysed for PKD1 expression. Western
blotting of lysates for actin (a-actin antibody) served as a control.


(PKDlactive, PKD1.Y463E mutant) or kinase-inactive
(PKDlinactive, PKD1.K610W mutant) PKD1 alleles and
measured their invasiveness in Matrigel Transwell assays. We
found that the expression of constitutively-active PKD1 signif-
icantly inhibited cell invasion through Matrigel (Figure 4a).
Wildtype PKD1 moderately decreased and kinase-inactive
PKD1 slightly increased cell invasiveness (Figure 4a). To test
long-term effects of expression of active PKD1 on cell invasion
in the same cell line, we then generated a MDA-MB-231 cell
line that allowed inducible expression of a constitutively-active
PKD1 via doxycyclin. Doxycyclin induced the expression of
constitutively-active PKD1 (PKDlactive) within 24 hours (Fig-
ure 4b) and we did not see any leakage of this system. In
Matrigel Transwell assays, the induction of constitutively-
active PKD1 inhibited tumour cell invasion in a similar way
shown for cells transiently-transfected with active PKD1 (data
not shown).


We then utilised this inducible system to determine if active
PKD1 affects the invasive behaviour of MDA-MB-231 cells
growing in 3D cell culture. MDA-MB-231 cells growing in 3D
culture in Matrigel within 12 days form multicellular spheroids
with a size of approximately 80 iLm (Figure 4c1). We found
that from approximately day 18 this phenotype changes to a
more stellate morphology, with projections of invasive cells
emanating from a central multicellular spheroid (Figure 4c2).
However, when spheroids were treated with doxycyclin at day
12 to induce the expression of active PKD1, the outgrowth of
these invasive projections was blocked until day 18 (Figure
4c3). This indicates that the expression of PKD1 indeed
blocks the invasive phenotype. The effect of PKD1 expression
on cell invasiveness became even more apparent when cells
were cultivated without doxycyclin for 24 days, where massive
invasion from the spheroid into the surrounding ECM was
observed (Figure 4c4). On the other hand, when cells were


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Figure 3


(a)



1~949




jwj --jJ4 PKD2

;;I 4Actin
mmn-tIot o-Act,f



MC


0-
wJ

6~ Z
M.
-Q


S "-W MCF-7 (control)
-A MCF-7 PKDI-RNAI Seq.1
0- MCF-7 PKD1-RNAi Seq.2
0 24 48
Time (hrs)


Control RNAi


PKD1-RNAi


P 0 003


1-
C)


U^


Transwell/Matdgel Assay)


The knockdown of PKD1 increases cell invasion. (a) MCF-7 cells were stably transduced with lentivirus coding for two different human shRNA
sequences for protein kinase D (PKD) 1 (PKD1 -RNAi Seq.1 and Seq.2) or for a non-target sequence which served as control. Cells were lysed and
analysed for PKD1 expression by western blotting. Immunostaining for PKD2 and actin expression served as controls. (b) MCF-7 control and PKD1-
RNAi clones were subjected to a cell proliferation assay using the CyQuant cell proliferation assay kit. (c) MCF-7 control and PKD1 -RNAi clones
were seeded on Matrigel-coated Transwell filters and Transwell invasion assays were performed over a time period of 16 hours. (d) MCF-7 cells sta-
bly-transfected with control RNAi or PKD1 -RNAi were grown in 3D/Matrigel cell culture over a period of 60 days. Cells were photographed at day
60. Bars indicate the size of the spheroids. For experiments shown in b and c, error bars represent standard deviation. P values were acquired with
the student's t-test using Graph Pad software. All P values indicate statistical significance.


cultivated without doxycyclin for 12 days and then treated with
doxycyclin to induce the expression of active PKD1 (12 days
without and 12 days with doxycyclin), we observed signifi-
cantly less cell invasion into the surrounding matrix (Figure
4c5). These results indicate that active PKD1 inhibits the inva-
sion of breast cancer cells.

Active PKD1 regulates the expression and activity of
invasion-relevant MMPs
It is known that breast tumour cells actively produce MMPs to
facilitate tumour cell invasion. We therefore aimed to find out
if PKD1 regulates the expression of a panel of MMPs. MDA-
MB-231 cells are an established model for malignant human
breast cancer cell invasion in vitro, and it has been shown that
multiple MMPs, including MMP-1, MMP-2, MMP-7, MMP-9,
MMP-11, MMP-12, MMP-13, MMP-14 and MMP-1 7, enhance
cell invasiveness [37-39]. To test whether PKD1 impacts the
expression of MMPs in MDA-MB-231 cells, we transfected
them with constitutively-active PKD1 (PKDlactive) and ana-
lysed the expression of multiple MMPs and TIMP. Using


reverse transcription (RT) PCR, we analysed the expression of
MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10,
MMP-11, MMP12, MMP-13, MMP-14, MMP-15, MMP-16,
TIMP1 and TIMP2, as well as actin which served as a loading
control (Figure 5a). We found that the expression of constitu-
tively-active PKD1 downregulated mRNA transcripts of MMP-
2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14
and MMP-15. Of these MMPs, all but MMP-15 are known to
enhance the invasiveness of MDA-MB-231 cells [37-39]. We
did not observe differences in the expression of MMP-1, MMP-
8, MMP-16, TIMP1, TIMP2 or actin. Further, the expression of
MMP-3 was increased by active PKD1. This is interesting,
because MMP-3 was previously shown to inhibit cell invasion
of MDA-MB-231 [40].

We then performed gelatin zymographic analysis to test if the
decreased expression of MMPs can relay to decreased MMP
activity. Therefore, MDA-MB-231 cells were either transfected
with vector control or with constitutively-active PKD1. We
observed a significant decrease in MMP activity when consti-


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200 Pm 200 pm







Breast Cancer Research Vol 11 No 1 Eiseler et al.


Figure 4


150 O


0
100
0


2 Io


+ DOX

L41 PKD1 active

Blot: a-FLAG

Blot: -ActActin

Blot: a-Actin


Transwell/Matrigel Assay


12 days w/o DOX,
24 days wlo DOX then 12 days with DOX

(c4) (c5)








200 mrn 200 gm


12 days wlo DOX
then 6 days with DOX


Active PKD1 inhibits breast tumour cell invasion. (a) MDA-MB-231 cells were transiently transfected with wildtype protein kinase D (PKD) 1, kinase-
dead PKD1 (PKD1 inactive) or constitutively-active PKD1 (PKD1active). After 24 hours cells were seeded on Matrigel-coated Transwell filters and
Transwell invasion assays were performed over a time period of 16 hours. (b) Inducible expression of active PKD1 in MDA-MB-231 -TR-PKD1 active
(PKD1 .Y463E mutant) cells. Cells were treated with doxycyclin (DOX) for 16 hours. Cells were lysed and lysates were analysed by western blotting
for expression of constitutively-active PKD1 (anti-FLAG) or actin (loading control). (c) MDA-MB-231 -TR-PKD1 .Y463E cells were seeded in 3D cul-
ture and were either left untreated for 1 2 (cl), 18 (c2) and 24 days (c4), or were treated with doxycyclin after 12 days of normal growth to induce
the expression of active PKD1 (c3 and c5). Cells were photographed at day 12 (cl), day 18 (c2 and c3) or day 24 (c4 and c5). Arrows in c2 indi-
cate cells invading from the spheroid into the extracellular matrix. Bars indicate the size of the spheroids.


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Available online http://breast-cancer-research.com/content/11/1/R13


Figure 5


MMP-1



MMP-8



MMP-12



MMP-16


Ao





MMP-2



MMP-9



MMP-13



TIMP-1


"o



MMP-3



MMP-10




MMP-14



TIMP-2


MMP-7





MMP-11





Actin


~a0


175 kDa

83 kDa

62 kDa

47 kDa


32 kDa


Zymography


-115 kDa




- 64 kDa

* 49 kDa

* 37 kDa


Western Blot:
ca-MMP-9


Active PKD1 regulates the expression of invasion-relevant MMPs. (a) MDA-MB-231 cells were transfected with vector control or constitutively-active
protein kinase D (PKD) 1 (PKD1 active, PKD1 .Y463E mutant). After 16 hours mRNA was isolated and the expression of the matrix mettaloprotein-
ases (MMP) MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15 and MMP-16, tissue
inhibitors of metalloproteinases (TIMP) TIMP1 and TIMP2 and actin (control) was analysed by RT-PCR (shown as a PCR reaction with 35 cycles;
PCR reaction with 20 and 40 cycles showed similar results). (b,c) MDA-MB-231 cells were transfected with vector control or constitutively-active
PKD1 (PKD1 active, PKD1.Y463E mutant). After 48 hours supernatants were collected and used for (b) zymographic analysis or (c) western blot
analysis for MMP-9 expression.


tutively-active PKD1 is expressed. This is most likely to be
because of decreased MMP-2 (p72) and MMP-9 (p68) activity
as the MMP activity was detected at a molecular weight of
approximately 70 kDa (Figure 5b). Western blotting analysis
for MMP-9 and MMP-2 showed that MMP-2 is not detectable
in supernatants of MDA-MB-231 cells (data not shown), but
that MMP-9 is decreased in cells expressing active PKD1 (Fig-


ure 5c). This suggests that MMP-9 is the mainly expressed
MMP in MDA-MB-231 and that its expression is negatively-
regulated by PKD1, which directly translates to decreased
activity. Therefore, PKD1 mediates breast cancer cell invasion
through regulation of the expression of invasion-relevant
MMPs.


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Breast Cancer Research Vol 11 No 1 Eiseler et al.


Discussion
To develop effective therapies that stop breast cancer from
metastasising, the underlying biological and molecular events
need to be understood in further detail. We show here that the
PKD family members PKD1, PKD2 and PKD3 are all
expressed in ductal epithelial cells of the normal breast (Figure
1). We further show that decreased expression of PKD1 can
serve as a marker for invasive breast cancer, whereas PKD2
and PKD3 expression remain unchanged in normal breast and
invasive breast tumour tissue (Figure 1). However, all three
PKD enzymes are markers for breast epithelial cells (normal
and tumour) and may be utilised as markers to identify breast
epithelia-derived metastases. PKD1 expression was downreg-
ulated by approximately 60% in more than 95% of the ana-
lysed samples of invasive ductal carcinoma and distant lymph
node metastases (Figure 1 b). All 50 analysed tumours were
assessed by pathologists and stages were at a range from 0,
IIA, IIB, IlA, IIIB, IIIC. Further, additional information such as
sex, age, diagnosis, pTNM, lymph node stage (positive lymph
nodes/examined lymph nodes), as well as expression of the
PR, or the more-aggressive ER-negative, basal sub-type of
breast cancer were available. Downregulation of PKD1
expression occurred in more than 95% of the analysed cases
of invasive ductal cancer and no correlation was observed with
stage, ER, PR or other markers. Our results on PKD1 in inva-
sive breast cancer are in consensus with data obtained for
gastric cancer and prostate cancer, where decreased expres-
sion of PKD1 was described in most of the cases analysed
[14,15].

Our data showing reduced PKD1 protein expression in inva-
sive breast cancer is also in consensus with published tran-
scriptional microarray data profiling over 350 surgically
excised, advanced breast tumour tissues. In these arrays
PRKD1 gene expression was drastically reduced in most
cases analysed [41-44]. Our data show that reduced gene
expression invariably translates to decreased protein levels.
Investigation of other publicly available microarray datasets on
the NCBI Gene Expression Omnibus (GEO) showed that
PRKD1 is detected at appreciable levels in normal lobular and
ductal breast cells [GEO:GDS2635] [45], in atypical hyper-
plasia [GEO:GDS1250] [46] and in the cancerous lesions
invasive ductal and lobular carcinomas [GEO:GDS2635]
[45], suggesting that PKD1 expression is indeed decreased
with increased invasiveness of the tumours.

Little is known about the role of PKD1 in regulating tumour cell
migration and invasion, important processes that regulate both
tumour expansion and metastasis. In order to investigate a
potential role for PKD1 in cell invasion, we first compared
PKD1 expression in very low-invasive and highly invasive
breast cancer cell lines (Figures 2a,c) and found that from the
three PKD family members only PKD1 showed a significant
expression pattern associated with the invasive phenotype.
PKD1 expression was absent in highly invasive breast cancer


cell lines including MDA-MB-231, T47D and SKBR3 (Figure
2). This is most likely because of epigenetic silencing medi-
ated by DNA methyltransferases (Figure 2d). Non-invasive or
very low-invasive breast cancer cell lines such as BT-474 or
MCF-7 and the normal breast cancer cell line MCF-10A mod-
erately expressed PKD1. Moreover, by analysing PKD1
expression in the 1-HMT-3522 breast cancer cell progression
model, we found that the T4/2 clone which shows increased
invasiveness as compared with the S1 clone also expressed
less PKD1 (Figure 2b).

We utilised two breast cancer model cell lines, MCF-7 and
MDA-MB-231, to investigate the role of PKD1 in cell invasion.
MCF-7 and MDA-MB-231 cells express comparable amounts
of PKD2 and PKD3, but differ in their expression of PKD1 (Fig-
ure 2). The depletion of PKD1 in MCF-7 cells resulted in
increased cell invasion in both 2D and 3D cell culture systems
(Figure 3).

On the other hand, the re-introduction of active PKD-1 in
MDA-MB-231 cells impaired their invasive behaviour in 2D
and 3D cell culture (Figure 4). Notably, the knockdown of
PKD1 in MCF-7 cells (Figure 3B) and the induction of consti-
tutively active PKD1 in MDA-MB-231 cells had no significant
effects on cell proliferation or cell death (data not shown). This
is interesting, because one of the PKD family members, PKD3,
was recently linked to increased tumour cell proliferation in
prostate cancer [47]. This implies that in different cancers the
three PKD family members may have different functions. A sim-
ilar phenomenon was recently demonstrated for the kinase
Akt, which, depending on the isoenzyme expressed, contrib-
utes to breast tumour cell survival and proliferation, or blocks
cell migration and invasion [48]. Cell proliferation, survival and
cell motility are not necessarily linked in cancer cells, and it is
generally accepted in the field that proliferation and invasive-
ness are independent of each other.

Our data further suggest that PKD1 inhibits breast cancer cell
invasion by regulating the expression of factors involved in the
degradation of ECM. The invasion of MDA-MB-231 cells in
Matrigel is dependent on MMPs. For example, MMP-2, MMP-
7, MMP-9, MMP-11, MMP-13 and MMP-14 are known to
enhance the invasiveness of MDA-MB-231 cells [37-39]. We
found that the expression of active PKD1 in MDA-MB-231
cells downregulated mRNA transcripts of MMP-2, MMP-7,
MMP-9, MMP-10, MMP-11, MMP-13, MMP-14 and MMP-15
(Figure 5a). Thus, PKD1 decreased the expression of all
MMPs so far implicated in the invasive phenotype of this cell
line. The mechanism of how PKD1 regulates such a multitude
of genes is not known yet. One explanation is that PKD1 may
regulate a common element in the promoter of these MMPs. In
this context histone deacetylases (HDACs) have been shown
to regulate the expression of MMPs [30,31]. PKD1 is known
to be a negative regulator of HDACs [49] and it is possible


Page 10 of 12
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that PKD1 exerts its effects on all the MMPs via regulation of
HDACs.

We did not observe any differences in the expression of MMP-
1, MMP-8, MMP-16, TIMP1 or TIMP2 by PKD1. Further, the
expression of MMP-3 was slightly increased by active PKD1.
This is interesting, because MMP-3 has been previously
shown to inhibit cell invasion of MDA-MB-231 [40]. MMP-3
expression was associated with benign and early stage breast
tumours but is frequently lost in advanced stage, aggressive
breast disease [40]. The events leading to the transition from
a benign to a metastatic tumour are not fully understood, but
are linked to ECM degradation and increased motility of cells.
It is possible that loss of PKD1 expression and the resulting
change in the expression of MMPs is part of the switch driving
the progression from a benign to an invasive, malignant
tumour.

Conclusions
Our results show that decreased PKD1 expression can serve
as a marker for invasive and metastatic breast cancer. They fur-
ther suggest that the loss of PKD1 expression increases the
malignant potential of breast cancer cells. This may be
because of the function of PKD1 as a negative regulator of
MMP expression. This knowledge can be applied to develop
new therapeutic avenues such as the re-expression of PKD1
as one potential strategy to ameliorate breast cancer
metastasis.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
TE, HD and IY performed the experiments. PS was responsi-
ble for the study concept and design and wrote the manu-
script. SG contributed to writing the manuscript and
performed microarray data analysis.

Acknowledgements
The authors would like to thank James N. Ingle and other members of the
Mayo Clinic Breast Cancer SPORE, and Dr. Edith Perez, as well as
members of the Storz laboratory for insightful discussions. We thank
Derek C. Radisky for the 1-HMT-3522-S1 and T4/2 cell lines and
Brandy H. Edenfield and Pamela Kreinest of the Mayo Clinic Jacksonville
Histopathology Facility for immunohistochemistry. This work was spon-
sored in part by funds from the Mayo Foundation and the Mayo Clinic
Comprehensive Cancer Center, the Mayo Clinic Breast Cancer SPORE
(CA116201-03DR4), a "Friends for an Earlier Breast Cancer Test"
Grant (all to PS) and NCI/NIH R01 CA108597 (SG).

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