E.A. Avilova Aspirant, N.N. Blokhin Cancer Research Center, Moscow E-mail: k_avilova@mail.ru E.B. Kuznetsova Ph.D. (Biol.), Leading Research Scientist, I.M. Sechenov First Moscow State Medical University, Moscow M.V. Nemtsova Dr.Sc. (Biol.), Professor, Leading Research Scientist, Research Centre of Medical Genetics, Moscow E-mail: nemtsova_m_v@mail.ru A.M. Scherbakov Ph.D. (Biol.), Senior Research Scientist, N.N. Blokhin Cancer Research Center, Moscow E-mail: alex.scherbakov@gmail.com V.A. Shatskaya Ph.D. (Biol.), Senior Research Scientist, N.N. Blokhin Cancer Research Center, Moscow М.А. Krasil-nikov Dr.Sc. (Biol.), Professor, N.N. Blokhin Cancer Research Center, Moscow E-mail: krasilnikovm@main.crc.umos.ru

The mechanism of the regulation of PAK1, one of the key signaling proteins, in the estrogen-dependent and estrogen-independent breast cancer cells was studied. We showed that estrogen-dependent MCF-7 cells were characterized with the low PAK1 expression in comparison with estrogen-dependent cell lines (HBL-100, MDA-MB-231, SKBR-3). We found for the first time that such difference in PAK1 content was correlated with the methylation of PAK1 promoter: it was methylated in MCF-7 cells, and demethylated in estrogen-independent cells. PAK1 proliferative and protective activities were revealed; the involvement of PAK1 in the breast cancer cells adaptation to hypoxia was demonstrated. The study of PAK1 methylation showed that constitutive PAK1 hyperexpression in estrogen-independent cells was accompanied by PAK1 demethylation, whereas physiological PAK1 activation in MCF-7 cells, in particular - under hypoxia, was not correlated with PAK1 demethylation, being based, probably, on the activation of upstream regulators of PAK1. In general, the results obtained show the possible usage of PAK1 determination as the marker of cancer growth and progression of hormonal resistance as well as the potential object of target therapy of breast tumors.

 

1. Zhao Z.S., Manser E. PAK family kinases: Physiological roles and regulation // Cellular Logistics. 2012. V. 2. № 2. P. 59-68.
2. Balasenthil S.et al. p21 activated kinase 1 signaling mediates cyclin D1 expression in mammary epithelial and cancer cells // J. Biol. Chem. 2004. V. 279. P. 1422-1428.
3. Arias-Romero L.E., Chernoff J. p21 activated kinases in Erbb2 positive breast cancer: A new therapeutic target - // Small GTPases. 2010. V. 1. P. 124-128.
4. Arias-Romero L.E.,Chernoff J. A tale of two Paks // Biol. Cell. 2008. V. 100. P. 97-108.
5. Wang Z. et al. p21 Activated Kinase 1 (PAK1) Can Promote ERK Activation in a Kinase-independent Manner // J. Biol. Chem. 2013. V. 288. P. 20093-20099.
6. Shrestha Y. et al. PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling // Oncogene. 2012. V. 31. P. 3397-3408.
7. Ghosh A., Awasthi S., Peterson J.R., Hamburger A.W. Regulation of tamoxifen sensitivity by a PAK1-EBP1 signaling pathway in breast cancer // British Journal of Cancer. 2013. V. 108. P. 557-563.
8. Kok M., Zwart W., Holm C. et al. PKA-induced phosphorylation of ERα at serine 305 and high PAK1 levels is associated with sensitivity to tamoxifen in ER-positive breast cancer // Breast Cancer Research and Treatment. 2011. V. 125. № 1. P. 1-12.
9. Bostner J., Skoog L., Fornander T., Nordenskjold B., Stal O. Estrogen receptor-α phosphorylation at serine 305, nuclear p21-activated kinase 1 expression, and response to tamoxifen in postmenopausal breast cancer // Clinical Cancer Research. 2010. V. 16. № 5. P. 1624-1633.
10. Lundgren K., Holm K., Nordenskjold B., Borg A., Landberg G. Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer // Breast Cancer Res. 2008. V. 10. P. R81.
11. Radu M., Semenova G., Kosoff R., Chernoff J. PAK signalling during the development and progression of cancer // Nat Rev Cancer. 2014. V. 14. № 1. P. 13-25.
12. Holm C., Rayala S., Jirstrom K., Stal O., Kumar R., Landberg G. Association between Pak1 expression and subcellular localization and tamoxifen resistance in breast cancer patients // J. Natl. Cancer. Inst. 2006. V. 98. № 10. P. 671-680.
13. Bostner J., Ahnstrom Waltersson M., Fornander T., Skoog L., Nordenskjold B., Stal O. Amplification of CCND1 and PAK1 as predictors of recurrence and tamoxifen resistance in postmenopausal breast cancer // Oncogene. 2007. V. 26. № 49. P. 6997-7005.
14. Zhang X., Lv C., Liu X., Hao D., Qin J., Tian H., Li Y., Wang X. Genome-wide analysis of DNA methylation in rat lungs with lipopolysaccharide‑induced acute lung injury // Molecular Medicine Reports 7.5. 2013. P. 1417-1424.
15. Yi C. et al. Validation of the p21-Activated Kinases as Targets for Inhibition in Neurofibromatosis Type 2 // Cancer research. 2008. V. 68. № 19. P. 7932-7937.
16. Kiseleva N.P., Katargin A.N., Gra D.V. EHpigenomika i kancerogenez: unikalnost patterna metilirovanija DNK opukholevojj kletki // Voprosy onkologii. 2007. № 1. S. 14.
17. Merlin J.L.,Azzi S., Lignon D. et al. MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy // Eur. J. Cancer. 1992. P. 1452-1458.
18. Lobanova Y.S.,Scherbakov A.M., Shatskaya V.A., Evteev V.A., Krasil\'nikov M.A. NF-kappa B suppression provokes the sensitization of hormone-resistant breast cancer cells to estrogen apoptosis // Molecular and cellular biochemistry. 2009. V. 324. № 1-2.
19. Li L.C.,Dahiya R. MethPrimer: designing primers for methylation PCRs // Bioinformatics. 2002. V. 18. № 11. P. 1427-1431.
20. Kuznecova E.B. Kekeeva T.V., Larin S.S., Zemljakova V.V., Babenko O.V., Nemcova M.V., Zaletaev D.V., Strelnikov V.V. Novye markery metilirovanija i ehkspressii genov pri rake molochnojj zhelezy // Molekuljarnaja biologija. 2007. T. 41. № 4. S. 624-633.
21. Katargin A., Pavlova L., Kisseljov F., Kisseljova N. Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer // BMC Medical Genomics. 2009. V. 2. P. 30.
22. Wang R.A.,Mazumdar A., Vadlamudi R.K., Kumar R. P21-activated kinase-1 phosphorylates and transactivates estrogen receptor-alpha and promotes hyperplasia in mammary epithelium // EMBO J., 2002. № 21. P. 5437-5447.
23. Michalides R.,Griekspoor A., Balkenende A. et al. Tamoxifen resistance by a conformational arrest of the estrogen receptor α after PKA activation in breast cancer // Cancer Cell. 2004. V. 5. № 6. P. 597-605.
24. Rayala S.K., Talukder A.H., Balasenthil S. et al. P21-activated kinase 1 regulation of estrogen receptor-α activation involves serine 305 activation linked with serine 118 phosphorylation // Cancer Research. 2006. V. 66. № 3. P. 1694-1701.
25. Avilova E.A., Andreeva O.E., SHatskaja V.A., Krasilnikov M.A. Znachenie proteinkinazy PAK1 v reguljacii ehstrogennezavisimogo rosta kletok raka molochnojj zhelezy // Biomedicinskaja khimija. 2014. T. 60. № 3. C. 322-331.
26. SHHerbakov A.M., JAkushina I.A., Semina S.E., Gudkova M.V., Krasilnikov M.A. Mekhanizm adaptacii kletok raka molochnojj zhelezy k gipoksii: rol AMRK/mTOR-signalnogo puti // Kletochnye tekhnologii v biologii i medicine. 2015 (v pechati).