1 Effect of Kinase Inhibitors on DAXX and FASN Localization in Triple Negative Breast Cancer Cells Jennifer Clees University of Florida Department of Chemistry April 19 2018
2 Abstract This thesis outlines efforts to determine the effect of vario us kinase inhibitors on DAXX and FASN localization in triple negative breast cancer. Triple negative breast cancer (TNBC) is characterized by its lack of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (H ER2) amplification. Patients with this aggressive, invasive form of breast cancer (BC) have a nearly one in three chance of relapse within five years of chemotherapy. The poor prognosis associated with TNBC acts as an impetus for researchers to discover better, more targeted treatment option s. This study has used the TNBC cell line MDA M B 231, a p53 mutant line, to investigate the effects of various novel small molecule kinase inhibitors. Kinases are involved in phosphorylating a large variety o f molecules active in signaling transduction pathways, the cell cycle, and apoptosis, or programmed cell death. In particular, inhibitors to certain kinases act on the cellular localization of two proteins of interest: death domain associated protein 6 (D AXX) and fatty acid synthase (FASN), and as a result, the pro liferative abilities of TNBCs. DAXX functions as a transcr iption represso r in the nucleus and is involved in apoptosis whi le the enzyme FASN catalyzes a terminal step in de novo lipogenesis.
3 Acknowledgements I would like to express my utmost gratitude to Dr. Daiqing Liao for his guidance and wisdom throughout my experience in the research laboratory and his mentorship during my time as an undergraduate student. I would also like to thank Mr. Iqbal Mahmud and the members of the Liao Lab, past and present, for their patience, support and willingness to teach me as a novice researcher.
4 Table of Cont ents Abstract Acknowledgements Chapter 1: Introduction Chapter 2: Methods Chapter 3: Results and Discussion Chapter 4: Conclusion References 2 3 5 8 9 12 13
5 Chapter 1: Introduction Background Triple negative breast cancer (TNBC) is nam ed so because, based on immunohistochemistry (IHC), it lacks estrogen receptor (ER) and progesterone receptor (PR), along with having no overexpression or amplification of human epidermal growth factor receptor 2 (HER2). 1,2,3 ,4 In addition to this triple negative character resulting in a current lack of specific targeted therapies, TNBC is associated with a poor prognosis because it is highly aggressive and invasive, with its invasiveness mediated by the proteolytic degradation of the extracellular matrix (ECM). 1,2,3 ,4 While chemotherapy remains an option, nearly one in three TNBC patients relapse within five years, which is shorter than the period of time to relapse and/or death in patients with other subtypes of breast cancer (BC). 3 Proteins of Interes t in TNBC In the medical research laboratory setting, TNBC is commonly modeled using MDA MB 231, a p53 mutant cell line. 4 Mutations to p53, the most commonly mutated gene in cancer cells, not only suspend its normal tumor suppressor activities, but may al so contribute to the malignant progression of cancer cells by gaining new oncogenic activities. 5 Mutant p53 can prevent the transcription of the cyclin dependent kinase inhibitor p21 which normally acts as a stop signal during the G1 and S phase of the ce ll cycle, and a modulator in DNA repair and apoptosis. 5 Moreover, mutant p53 combined with the high phospholipase D (PLD) activity of MDA MB 231 cells causes the cells to express survival signals to suppress apoptosis when serum growth factors are absent. 5 of tumor to survive during the stressful process of metastasizing, when conditions, as modeled by serum free medium in the laboratory, would normally promote apoptosis.
6 D eath domain associated prote in 6 (DAXX) is a multifunctional protein known to act as a histone chaperone and a transcription corepressor, be involved in apoptosis, and reside in the nucleus predominantly, and the cytoplasm. 6,7 modulated in the nucleus by localization to compartments such as, the nucleolus and nuclear bodies. 6,7 DAXX, a novel negative regulator of p53, possesses an acidic domain th at binds to the carboxy terminal domain (CTD) of p53. 7 This binding interaction is dependen t upon positive charges being present in the CTD with p53 mutations of lysine (Lys) to positively charged arginine (Arg) preserving the interaction with a preference for Lys, while Lys to neutral alanine ( Ala ) or serine ( Ser ) to negatively charged glutama te ( Glu ) inhibit the interaction. 7 Additionally, posttranslational modification of the CTD of p53 in the form of acetylation and phosphorylation can prevent the DAXX p53 interaction from occurring. 7 Furthermore, DAXX expression has been shown to be incr eased in TNBC and to promote de novo lipogenesis (DNL), the enzymatic pathway for converting the acetyl CoA from carbohydrates to fatty acids which in turn promotes cancer cell survival and proliferation 8, 9 Fatty acid synthase ( FASN ) is an enzyme tha t catalyze s the synthesis of palmitate from acetyl CoA and malonyl CoA during lipogenesis, and, like DAXX is overexpressed in breast cancer. 10 11 ,12 Thus, FASN aids the cancer cell in synthesizing cell membranes, acylating proteins, and proceeding th rough the cell cycle. 9 12 Kinase Inhibito rs This project focuses on the effect of kinase inhibitors, which prevent phosphorylation from occurring, on DAXX/FASN localization within MDA MB 231 cells in the absence of serum. PD 0332991 inhibits cyclin depe ndent kinase s (CDK) 4 and 6 which are serine/threonine kinases that interact with cyclin D 1 to phosphorylate the retinoblastoma protein
7 ( pRb), thus inactivating it and releasing transcription factors enabli ng progression from G 1 to S through a cell cycle checkpoint that normally prevents abnormal replication 1 3 AZD6244 inhibits mitogen activated protein kinase (MAPK)/ extracellular signal regulated kinase ( ERK) kinase s (MEK ) 1 and 2 th u s preventing the completion of the MAPK/ERK signal cascade involved in gene expression a nd cancer cell proliferation. 1 4 BMS 754807 inhibits insulin like growth factor 1 receptor (IGF 1R) and insulin re ceptor (InsR) (shown to be overexpressed in BC) tyrosine kinases which init iate cascades leading to activation of MAPK signaling and the antiapoptotic/survival p hosphatidylinositol 3 kinase protein kinase B (PI3 K Akt) pathway 1 5 AZD8055 inhibits rapamycin (mTOR) kinase a serine/ t hreonine kinase belonging to the PI3K like kinase (PIKK ) superfamily which is activated by PI3K and Akt signals and forms the mTORC1 a nd mTORC2 multiprotein complexes which phosphorylate downstream proteins involved in protein translation and cell growth 1 6 Cancer cells are known to demonstrate increased signaling through the mTOR pathway. 1 6 GD C0941 is a pan class I PI3K inhibitor that prevents PI3K from pro ducing the second messenger phosphatidyl inositol 3,4,5 triphosphate (PIP 3 ) and from activating Akt and mTORC1 1 7 PI 3K regulates many aspects involved in cancer progression including cell growth, survival, and metastases and its deregulation has been implicated in BC. 1 7
8 Chapter 2: Methods The protocol outlined is generally consistent for cell fixation and immunofluorescent staining in the Liao Lab. MDA MB 231 cells were seeded (1/15 of a 10 cm dish per well) on glass coverslips in a six well plate in 1000 L in media containing bovine serum albumin (BSA) and incub ated at 37 C After 24 h, cells were washed once with 1 mL phosphate buffered saline ( PBS ), then 1mL serum free Dulbecco's Modified Eagle Medium ( DMEM ) was added 4h before fixation, kinase inhibitor was added. The wells contained as follows: (1) 1 L di methyl sulfoxide (DMSO) as control, (2) 1 L of 100 M PD 0332991, (3) 1 L of 100 M AZD6244, (4) 1 L of 100 M BMS 754807, (5) 1 L of 100 M AZD8055, and (6) 1 L of 100 M GDC0941. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2 % Triton x 100 Cells incubated in the dark at room temperature for 30min with primary antibody: 320 L of blocking buffer plus 30 L anti DAXX 5G11 (mouse) and 0.5 L anti FASN (rabbit) After PBS wash, cells incubated in the dark at room temperature fo r 30min with secondary antibody: 350 L blocking buffer plus 0.5 L goat anti mouse Alexa Fluor 594 (A 11005) and 0.5 L goat anti rabbit 488 (A11008) Cells were mounted to slides using 25 L VectaShield, anti fade mounting medium with diamidino 2 p henylindole (DAPI). After 24h, the cells are visualized on the Zeiss microscope at 20x (optivar at 1.25x). All cell nuclei are stained blue by the DAPI in the mounting medium with the exposure time set to 0.1s, while DAXX is stained red (3.0s) and FASN i s stained green (1.0s). Microscope pictures are formed by merg ing these three colored channel photographs into one photo and ten (merged) photos were taken per slide
9 Chapter 3: Results and Discussion When the cells incubate in serum free medium, it was expected for FASN expression demonstrated a bright green FASN signal in the cytoplasm and red DAXX s ignal localized to the nucleus (Figure 1) Figure 1. Treatment with 1 L dimet hyl sulfoxide (DMSO) pictured right, as control 1 8 Note on scale : MDA MB 231 heterogeneous cell population, nuclei width 9 m In sl ide 2, treatment with the CDK 4/6 inhibitor, PD 0332991 did not produce noticeably different expression levels or cellular localization (Figure 2) 13 This drug was previously studied with ER positive BC y to tamoxifen 13 Figure 2. Treatment with 0.1 M PD 0332991 pictured right 1 8
10 Slides 3 and 4 demonstrated very weak FASN and DAXX signals, but both proteins maintained similar cellular localization to slides 1 and 2 (Figure s 3, 4) Such a larg e decrease in signal compared to slides 1, 2, 5, and 6 could perhaps be attributable to an error in the to capture the emitted light. Such a failure is correctible using software to brighten the signals while decreasing the background, but repetition of the experiment would give a better source for expression level comparison between slides. It is also possible that the inhibition of the target ed kinases affected the protein stability of DAXX and FASN resulting in reduced signal intensities Figure 3. Treatment with 0.1 M AZD6244 pictured right 1 8 Figure 4. Treatment with 0.1 M BMS 754807 pictured right 1 8
11 Distinct from the previous slides Slide 5 possessed nuclear bodies of DAXX/FASN colocalization, perhaps indicating transcriptional activity and an interact ion between the two (Figure 5) AZD8055 is currently in clinical trials as the first mTOR inhibitor known to inhibit both mTORC1 and mTORC2 complexes, and it has been shown to overcome tamoxifen resistance in ER postive BC. 16 Figure 5. Treatment wi th 0.1 M AZD8055 pictured right 1 8 In Slide 6, besides the red DAXX nuclear signal being incredibly bright the signal and localization of FASN and DAXX followed closely with that of Slide 5 above. The pan class I PI3K inhibitor GDC0941 is also in clinical trials. Figure 6. Treatment with 0.1 M GDC0941 pictured right 1 8
12 Chapter 4: Conclusion While testing of these five kinase inhibitors produced similar results in terms of FASN expression and local ization, DAXX expression notably increased and localized more specifically to nuclear bodies with FASN in slides 5 and 6. Possible future experiments could work to correlate the levels of DAXX and FASN expression with the potency of these kinase inhibitor s to induce transcriptional changes or effect apoptosis In addition, using an adjusted set of methods to better quantify changes in protein expressio n levels could prove beneficial. Further study is necessary to determine how exactly DAXX and FASN inter act in the ab s ence of serum growth factors, particularly how the FASN/DAXX localization to nuclear bodies that might be impacted by the signal transduction pathways regulated by the kinases investigated here affects TNBC proliferation.
13 References 1. Yao, H.; He, G.; Yan, S.; Chen, C.; Song, L; Rosol., T. J.; Deng, X. Oncotarget 2017, 8 (1) 1913 1924 2. Aysola, K.; Desai, A.; Welch, C.; Xu, J.; Qin, Y.; Reddy, V.; Matthews, R.; Owens, C.; Okoli, J.; Beech, D. J.; Piyathilake, C.J.; Reddy, S. P.; Rao, V. N. Hereditary Genet 2013, 2013 ( Sup pl 2 ) 1 3. 3. Hudis, C. A.; Gianni, L. Oncologist 2011, 16 ( Suppl 1 ) 1 11. 4. Cell line profile MDA MB 231 (ECACC catalogue no. 92020424) https://www.phe culturecollections.org.uk/media/133182/mda mb 231 cell line profile.pdf (accessed April 13, 2018). 5. Hui, L.; Zheng, Y.; Yan, Y.; Bargonetti, J.; Foster D. A. Oncogene 2006 25 7305 7310. 6. NCBI: DAXX death domain associated protein [ Homo sapiens (human) ] https://www.ncbi.nlm.nih.gov/gene/1616 (accessed April 13, 2018). 7. Zhao, L. Y.; Liu, J.; Sidhu, G. S.; Niu, Y.; Wamg, R.; Liao, D. J. Biol. Chem. 2004, 279 50566 50579. 8. Z aid i, N. ; Lupien, L.; Kuemmerle, N. B.; Kinlaw, W. B.; Swinnen, J. V.; Smans, K. Prog Lipid Res 2013, 52 (4) 585 589. 9. Gir Perafita, A.; Sarrats, A.; Prez Bueno, F.; Oliveras, G.; Bux, M.; Brunet, J.; Vias, G.; Miquel, T. P. Oncotarget 2017, 8 (43) 74391 74405 10. Gir Perafita, A.; Palomeras, S.; Lum, D. H.; Blancafort, A.; Vias, G.; Oliveras, G.; Prez Bueno, F.; Sarrats, A.; Welm, A.L.; Puig, T. Clin Cancer Res. 2016, 22 (18) 4687 46 97
14 11. N CBI: FASN fatty acid synthase [ Homo sapiens (human) ] https: //www.ncbi.nlm.nih.gov/gene/2194 (accessed April 13, 2018). 12. Fla vin, R.; Peluso, S.; Nguyen, P.L.; Loda, M. Future Oncol. 2010, 6(4) 551 62. 13. Finn, R. S.; Dering, J. ; Conklin, D.; Kalous, O.; Cohen, D. J.; Desai, A. J.; Ginther, C.; Atefi, M.; Chen, I.; Fowst, C; Los, G.; Slamon, D. J. Breast Cancer Res. 2009, 11(5) R77. 14. NCBI: PubChem Compound Summary for CID 10127622 https://pubchem.ncbi.nlm.nih.gov/compound/Selumetini b#section=Top (accessed April 18, 2018). 15. Carboni, J. M.; W ittman, M.; Yang, Z.; Lee, F.; Greer, A.; Hurlburt, W.; Hillerman, S.; Cao, C.; Cantor, G. H.; Dell John, J.; Chen, C.; Discenza, L.; Menard, K.; Li, A. ; Trainor, G.; Vya s, D.; Kramer, R.; Attar, R. M.; Gottardis, M. M. Mol Cancer Ther. 2009, 8(12), 3341 334 9. 16. C hresta, C. M.; Davies, B. R.; Hickson, I.; Harding, T.; Cosulich, S.; Critchlow, S. E.; Vincent, J. P.; Ellston, R.; Jones, D.; Sini, P; James, D.; Howard, Z.; Dudley, P; Hughes, G.; Smith, L.; Maguire, S.; Hummersone, M.; Malagu, K.; Menear, K.; Jenkins, R.; Jacobsen, M.; Smith, G. C.; Guichard, S.; P ass, M. Cancer Res. 2010, 70(1), 288 2 98. 17. Sarker, D.; Ang, J. E.; Baird, R.; Kristeleit, R.; Shah, K.; Moreno, V.; Clarke, P. A.; Raynaud, F. I.; Levy, G.; Ware, J. A.; Mazina, K.; Lin, R.; Wu, J.; Fredrickson, J.; Spoerke, J. M.; Lackner, M. R.; Yan, Y.; Friedman, L. S.; Kaye, S. B. ; Derynck M. K.; Workman, P.; de Bono, J. S Clin Cancer Res. 2015, 21(1), 77 86. 18. NCBI: Pub Chem Compound, https://www.ncbi.nlm.nih.gov/pccompound (accessed April 13, 2018).