Intercellular interactions and progression of hormonal resistance of breast cancer cells

The main goal of the study is the analysis of the role of cell-cell interactions in the formation of the tumor cell resistance to hormonal drugs. About 70 % of breast tumors contain estrogen receptor (ER), a key molecular target for hormone (endocrine) therapy. However, the efficiency of endocrine therapy of breast cancer is limited by the development of hormone resistance which leads to progression of tumor cells to hormone-independent phenotype, increase in tumor malignancy and worse prognosis. Hormonal independence may be accompanied with the loss of the receptors, as well as with the another mechanisms including ligand-independent receptor activation, disbalance between receptor activators and repressors, stimulation of hormone-independent pathways. It is less known about the role of the intercellular interactions in the progression of hormonal resistance. Several studies demonstrate the involvement of cell junctions in the mediating of cell response to (anti) estrogens, however the significance of cell-cell contacts in the formation of hormonal resistance still not clear. Here we have hypothesized that the formation of the hormone resistance of tumors may be based, at least in part, on the transferring of the resistant phenotype from the resistant to hormone-sensitive cells – as a result of the secretion of the specific factors acting in the paracrine manner or via the direct cell-cell contacts. Using the estrogen-dependent breast cancer cells MCF-7 and the resistant subline MCF-7 / T developed by long-term cultivation of MCF-7 cells in the presence of antiestrogen tamoxifen, we investigated the possible changes in the hormonal sensitivity of these cells caused by the co-cultivation in vitro. To discern the cell cultures, the MCF-7 / T cells were previously transfected with the plasmid containing the gene of the green fluorescent protein (GFP), and GFP-positive hormone-resistant subline MCF-7 / T / GFP+ was developed. We showed that the co-cultivation of the parent and resistant cells lead to increase in the resistance of the parent cell to tamoxifen. To further explore the mechanism of such resistance, the analysis of the biological activity of exosomes prepared from the estrogen-sensitive and resistant cells was performed. The exosome preparations isolated from the resistant MCF-7 / T cells were found to induce the similar resistance in the recipient MCF-7 cells – in contrast to the control exosomes isolated from the MCF-7 cells. The subsequent cloning of these newly formed resistant cells showed that the cells retain the resistant phenotype for at least 80 days of cultivation. Totally, the results presented demonstrate the important role of cell-cell interactions in the progression of hormonal resistance, opening a new perspectives in the development of probable targets for breast cancer therapy.

breast cancer, hormonal resistance, tamoxifen, exosomes, microRNA, estrogen receptor, intercellular interactions

1. Jensen E.V., DeSombre E.R. Estrogenreceptor interaction. Science 1973;182(4108):126–34.

2. Красильников М.А. Современные подходы к изучению механизма эстрогеннезависимого роста опухолей молочной железы. Вопросы онкологии 2004; 50(4):399–405. [Krasilnikov M.A. Stateof-art approaches to studying of the mechanism of estrogen-independent growth of breast tumors. Voprosy onkologii = Oncology Issues 2004;50(4):399–405. (In Russ.)].

3. Clarke R., Liu M.C., Bouker K.B. et al. Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling. Oncogene 2003;22(47):7316–39.

4. Lee M., Lee C.S., Tan P.H. Hormone receptor expression in breast cancer: postanalytical issues. J Clin Pathol 2013; 66(6):478–84.

5. Normanno N., Di Maio M., De Maio E. et al. Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 2005;12(4):721–47.

6. Jordan V.C. Targeting antihormone resistance in breast cancer: a simple solution. Ann Oncol 2003;14(7):969–70.

7. Jalava P., Kuopio T., Huovinen R. et al. Immunohistochemical staining of estrogen and progesterone receptors: aspects for evaluating positivity and defining the cutpoints. Anticancer Res 2005;25(3c): 2535–42.

8. Берштейн Л.М. Современная эндокринология гормонозависимых опухолей. Вопросы онкологии 2002; 48(4):496–504. [Berstein L.M. Modern endocrinology of hormone-dependent tumors. Voprosy onkologii = Oncology Issues 2002;48(4):496–504. (In Russ.)].

9. Красильников М.А. Сигнальные пути, регулируемые фосфатидилинозит 3-киназой, и их значение для роста, выживаемости и злокачественной трансформации клеток. Биохимия 2000;65(1):68–78. [Krasilnikov M.A. Signal paths regulated with phosphatidylinosit-3-kinase and their significance for growth, survival, and malignant transformation of cells. Biokhimiya = Biochemistry 2000;65(1): 68–78. (In Russ.)].

10. Kurebayashi J. Endocrine-resistant breast cancer: underlying mechanisms and strategies for overcoming resistance. Breast Cancer 2003;10(2):112–9.

11. Roop R.P., Ma C.X. Endocrine resistance in breast cancer: molecular pathways and rational development of targeted therapies. Future Oncol 2012;8(3):273–92.

12. Красильников М.А., Щербаков А.М. Сигнальные пути, регулируемые эстрогенами, и их роль в опухолевой прогрессии: новые факты и направления поиска. Успехи молекулярной онкологии 2014;1:18–26.[Krasilnikov M.A., Shcherbakov A.M. Signal paths regulated with estrogens and their role in tumor progression: new facts and directions of search. Uspekhi molekulyarnoy onkologii = Advanses of Molecular Oncology 2014;1:18–26. (In Russ.)].

13. Oshima A. Structure and closure of connexin gap junction channels. FEBS Lett 2014;588(8):1230–7.

14. El-Saghir J.A., El-Habre E.T., El-Sabban M.E., Talhouk R.S. Connexins: a junctional crossroad to breast cancer. Int J Dev Biol 2011;55(7–9):773–80.

15. Rameshwar P. Potential novel targets in breast cancer. Curr Pharm Biotechnol 2009;10(2):148–53.

16. Taipale J., Beachy P.A. The Hedgehog and Wnt signalling pathways in cancer. Nature 2001;411(6835):349–54.

17. Adjei A.A., Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy. J Clin Oncol 2005;23:5386–403.

18. Niepel M., Hafner M., Pace E.A. et al. Analysis of growth factor signaling in genetically diverse breast cancer lines. BMC Biol 2014;12:20.

19. Kohlhapp F.J., Mitra A.K., Lengyel E., Peter M.E. MicroRNAs as mediators and communicators between cancer cells and the tumor microenvironment. Oncogene 2015;13.

20. Falcone G., Felsani A., D’Agnano I. Signaling by exosomal microRNAs in cancer. J Exp Clin Cancer Res 2015; 34:32.

21. Squadrito M.L., Baer C., Burdet F. et al. Endogenous RNAs modulate microRNA sorting to exosomes and transfer to acceptor cells. Cell Rep 2014;8(5): 1432–46.

22. Taylor D.D., Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecol Oncol 2008;110(1):13–21.

23. Corcoran C., Rani S., O’Brien K. et al. Docetaxel-resistance in prostate cancer: evaluating associated phenotypic changes and potential for resistance transfer via exosomes. PLoS One 2012;7(12).

24. Lv M.M., Zhu X.Y., Chen W.X. et al. Exosomes mediate drug resistance transfer in MCF-7 breast cancer cells and a probable mechanism is delivery of P-glycoprotein. Tumour Biol 2014;35(11):10773–9.

25. Harris D.A., Patel S.H., Gucek M. et al. Exosomes released from breast cancer carcinomas stimulate cell movement. PLoS One 2015;10(3).

26. Bonifacino J.S., Dasso M., Harford J.B. et al. Current Protocols in Cell Biology. John Wiley & Sons, 2004. 3178 p.

27. Scherbakov A.M., Stefanova L.B., Sorokin D.V. et al Snail/beta-catenin signaling protects breast cancer cells from hypoxia attack. Exp Cell Res 2013;319(20):3150–9.