Tamoxifen induces stem-like phenotypes and multidrug resistance by altering epigenetic regulators in ERα+ breast cancer cells

Aparna Kalyanaraman; Dhanavathy Gnanasampanthapandian; Prasad Shanmughan; Puneet Kishore; Satish Ramalingam; Rathnaswami Arunachalam; Selvaraj Jayaraman; Ilango Kaliappan; Ganesh Munuswamy-Ramanujam; Ilangovan Ramachandran; Yuvaraj Sambandam; Muralidharan Anbalagan; Parthasarathy Chandrakesan; Kanagaraj Palaniyandi

doi:10.21037/sci-2020-020

Tamoxifen induces stem-like phenotypes and multidrug resistance by altering epigenetic regulators in ERα+ breast cancer cells

Stem Cell Investig. 2020 Nov 3:7:20. doi: 10.21037/sci-2020-020. eCollection 2020.

Authors

Affiliations

¹ Cancer Science Laboratory, Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India.
² Department of Genetic Engineering, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, Kancheepuram, India.
³ Department of Surgical Gastroenterology, SRM Medical College Hospital and Research Center, Kattankulathur, Kancheepuram, India.
⁴ Department of Biochemistry, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Velappanchavadi, Velappanchavadi, Chennai, India.
⁵ Departmemt of Pharmaceutical Chemistry, SRM College of Pharmacy, SRM Institute of Science and Technology, Kattankulathur, India.
⁶ Interdisciplinary Institute of Indian System of Medicine, SRM Institute of Science and Technology, Kattankulathur, India.
⁷ Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani campus, Chennai, India.
⁸ Department of Structural and Cellular Biology, Tulane University, New Orleans, LA, USA.
⁹ Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.

Abstract

Background: To understand the mechanism underlying tamoxifen-induced multidrug resistance (MDR) and stem-like phenotypes in breast cancer cells, we treated the MCF-7 cells with 4-hydroxy-tamoxifen (TAM) for 6 months continuously and established MCF-7 tamoxifen resistance (TR) phenotypes.

Methods: In the present study, the following methods were used: cell viability assay, colony formation, cell cycle analysis, ALDEFLUOR assay, mammosphere formation assay, chromatin immunoprecipitation (ChIP) assay, PCR array, western blot analysis and quantitative reverse transcription polymerase chain reaction (QRT-PCR).

Results: The expression of ERα was significantly higher in MCF7-TR cells when compared with parental MCF-7 cells. MCF7-TR cells exposed to TAM showed a significant increase in the proliferation and rate of colony formation. The number of cancer stem cells was higher in MCF7-TR cells as observed by the increase in the number of ALDH+ cells. Furthermore, the number of mammospheres formed from the FACS-sorted ALDH+ cells was higher in MCF7-TR cells. Using PCR array analysis, we were able to identify that the long-term exposure of TAM leads to alterations in the epigenetic and MDR stem cell marker genes. Furthermore, western blot analysis demonstrated elevated levels of Notch-1 expression in MCF-TR cells compared with MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay revealed that Notch-1 enhanced the cyclin D1 expression significantly in these cells. In addition, we observed that MCF7-TR cells were resistant to doxorubicin but not the MCF-7 cells.

Conclusions: In the present study, we conclude that the treatment with tamoxifen induces multiple epigenetic alterations that lead to the development of MDR and stem-like phenotypes in breast cancers. Therefore, our study provides better insights to develop novel treatment regime to control the progression of breast cancer.

Keywords: Notch-1; Tamoxifen resistance (TR); breast cancer stem cells; epigenetics; multidrug resistance (MDR).