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Background: Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor-2 over-expression. Compared to other subtypes of breast cancer, TNBC is associated with a higher risk for metastatic recurrence. While immunotherapy, PARP inhibitors, and sacituzumab govitecan have shown benefit in a subset of TNBC patients, chemotherapy with doxorubicin remains a mainstay of treatment. Senescence is a cellular phenomenon where cells are committed to an arrested state, however, senescent cells are able to secrete pro-tumorigenic factors which can help to promote tumor progression and invasion. It has been suggested that senescence is a potential mechanism of resistance in doxorubicin (dox) treated cells. Transitioning senescence mediated resistance to apoptosis is crucial to the treatment of TNBC. The objective of this study was to evaluate the combination BCL-2 and HDAC inhibitors with dox to overcome resistance and induce apoptosis. Experimental Procedures: TNBC cell lines resistant and sensitive to doxorubicin were identified using a CellTiter-Glo Viability Assay and resistant cell lines were selected for further analysis. To assess proliferation, dox resistant TNBC cell lines were plated in 96-well plates. Cells were exposed to vehicle control, dox, BCL-2 inhibitor, HDAC inhibitor or the combination of dox and BCL-2 inhibitor or HDAC inhibitor for 72 hours. Cellular proliferation was assessed using the BioSpa live cell analysis system. Apoptosis at 24 hours was analyzed by flow cytometry using Annexin V on cells treated in combination and as single agents. Immunoblotting was performed to evaluate the downstream effects of apoptosis and senescence of drugs as single agents and in combination. Results: The addition of a BCL-2 inhibitor and HDAC inhibitor to doxorubicin resulted in decreased proliferation in resistant TNBC cell lines compared to single agents and vehicle control by live cell microscopy. Furthermore, the combination of dox and a BCL-2 inhibitor resulted in increased apoptosis when compared to single agents and control using Annexin V staining. The cyclin dependent kinase inhibitors p21 and p16 were over-expressed in cells exposed to combination treatment. Apoptotic proteins BAD, PUMA, cleaved PARP and the anti-apoptotic protein BCL-2 were upregulated in cells treated with a BCL-2 inhibitor with or without dox. The pro-mitotic protein cyclin-B1 was downregulated in resistant cells treated with single agent BCL-2 inhibitor and in combination with dox. Additional mechanistic studies are ongoing. Conclusion: The combination of doxorubicin with BCL-2 and HDAC inhibitors resulted in decreased cellular proliferation and increased apoptosis. Potential senolytic drugs used with dox represent an exciting potential to overcome dox resistance and warrant further investigation. Citation Format: Anna R. Schreiber, Stephen Smoots, Betelehem W. Yacob, Adrian TA Dominguez, Cecilia Levandowski, Jennifer R. Diamond, Todd M. Pitts. Combination strategies to overcome doxorubicin induced senescence in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1063. |
