Potentiating doxorubicin activity through BCL-2 inhibition in p53 wild-type and mutated triple-negative breast cancer

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive sub-type of breast cancer that is associated with higher rates of recurrent disease. Chemotherapy with an anthracycline is an integral part of curative therapy but resistance remains a clinical problem. Cellular senescence is a terminal cell fate that has been observed in models of doxorubicin resistance. Identifying novel combinations with doxorubicin to eliminate senescent cells and promote apoptosis may lead to improved clinical outcomes. The purpose of this study was to investigate the combination of doxorubicin with the pro-apoptotic BCL-2 inhibitor venetoclax in TNBC cell lines and to assess the role of p53 in cellular senescence and apoptosis.

Methods: TNBC cell lines with wild-type (WT), mutated or knocked-down (KD) p53 were treated with doxorubicin, venetoclax or the combination in vitro and evaluated for impacts on viability, proliferation, apoptosis, and senescence. Down-stream markers of apoptosis were also assessed to evaluate cellular mechanistic changes. An in vivo TNBC MDA-MB-231 murine model was used to assess tumor growth, cellular proliferation, and senescence changes following treatment with doxorubicin, venetoclax or combination.

Results: Venetoclax with doxorubicin had synergistic antiproliferative activity against TNBC cell lines and increased apoptosis. The addition of venetoclax to doxorubicin reduced senescent cells in a p53-independent manner. In vivo, the addition of venetoclax to doxorubicin improved tumor growth inhibition and reduced senescent cells.

Conclusion: The combination of doxorubicin with venetoclax is promising for the treatment of p53-WT and mutated TNBC and this work supports further investigation.

Keywords: TNBC; doxorubicin; resistance; senescence; venetoclax.

Figure 1

Doxorubicin + venetoclax has synergistic effects in TNBC cell lines. (A) Percentage of viable cells as measured by CellTiter-Glo after exposure to varying doses of doxorubicin (DOX) and venetoclax (VEN) for 72 hours. (B) Loewe synergy plots calculated from CellTiter-Glo analysis. (C) Anti-proliferative effects following 48 hours of dosing with no drug, 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN. Comparisons were performed using an ordinary one-way ANOVA with Tukey correction (**P < 0.01, ****P < 0.0001). Error bars represent standard error of the mean (SEM). All experiments were done in triplicate.

Figure 2

Apoptotic effects on TNBC cell lines. Cells were dosed with no drug (ND), 0.5 µM of doxorubicin (DOX), 25 µM of venetoclax (VEN), or 0.5 µM of DOX in combination with 25 µM of VEN. (A) Flow cytometry analysis of Annexin V/PI following 24 hours of drug exposure. (B) Percentage of total Annexin V+ following 24 hours of drug exposure. (C) Flow cytometry analysis of Annexin V/PI following 48 hours of drug exposure. (D) Percentage of total Annexin V+ following 48 hours of drug exposure. Comparisons were performed using an ordinary one-way ANOVA with Tukey correction. (*P < 0.05, **P < 0.01). Error bars represent standard error of the mean (SEM). All experiments were done in triplicate.

Figure 3

Senescence and cell cycle analysis of TNBC cell lines. (A) β-Gal staining indicating senescence following 6 days of treatment with no drug (ND), 0.1 µM of doxorubicin (DOX), 25 µM of venetoclax (VEN), or 0.1 µM of DOX in combination with 25 µM of VEN. (B) Percentage area of β-Gal positive cells. (C) Cell cycle analysis following 24 hours of exposure to ND, 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN. Comparisons were performed using an ordinary one-way ANOVA with Tukey correction. (**P < 0.01, ***P <0.001, ****P < 0.0001). Error bars represent standard error of the mean (SEM). All experiments were done in triplicate.

Figure 4

Western blot analysis. Downstream effects following 24 hours of treatment with no drug (ND), 0.5 µM of doxorubicin (DOX), 25 µM of venetoclax (VEN), or 0.5 µM of DOX in combination with 25 µM of VEN (COMBO) in CAL-51, MDA-MB-231, Hs 578T, and CAL-120 TNBC cell lines. All experiments were done in triplicate. kDa: kilodalton.

Figure 5

Synergistic and apoptotic effects in CAL-51 p53 knock-down and scramble cell lines. (A) Percentage of viable cells using CellTiter-Glo analysis after 72 hours of drug exposure to varying doses of doxorubicin (DOX) and venetoclax (VEN). (B) Loewe synergy plots calculated from CellTiter-Glo analysis. (C) Anti-proliferative effects following 48 hours of dosing with no drug (ND), 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN. (D) Flow cytometry analysis of Annexin V/PI following 24 hours of drug exposure. Cells were dosed with ND, 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN. (E) Percentage of total Annexin V+ following 24 hours of drug exposure. Cells were dosed with ND, 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN. (F) Western blot analysis of downstream effects following 24 hours of treatment with ND, 0.5 µM of DOX, 25 µM of VEN, or 0.5 µM of DOX in combination with 25 µM of VEN (COMBO). (G) β-Gal staining indicating senescence following 6 days of treatment with ND, 0.1 µM of DOX, 25 µM of VEN, or 0.1 µM of DOX in combination with 25 µM of VEN. (H) Percentage area of β-Gal positive cells. Comparisons were performed using an ordinary one-way ANOVA with Tukey correction (*P < 0.05, **P < 0.01, ***P <0.001, ****P < 0.0001). Error bars represent standard error of the mean (SEM). All experiments were done in triplicate.

Figure 6

Assessment of the combination of doxorubicin + venetoclax in vivo. (A) Tumor volumes of MDA-MB-231 murine models treated with vehicle, doxorubicin (DOX), venetoclax (VEN) or combination over 37 days. (B) SGR at day 37 depicting overall tumor growth inhibition. (C) Staining for H&E, Ki-67, and β-Gal on frozen tumor tissue collected at end of treatment, day 37. Representative images were taken at 20x magnification. Scale for H&E and Ki-67 is 100 µm. Scale for β-Gal is 50 µm. (D) Percentage area of Ki-67 positive cells. (E) Percentage area of β-Gal positive cells. Comparisons were performed using an ordinary one-way ANOVA with Tukey correction (*= P < 0.05, **P < 0.01). There were 8-9 sub-cutaneous tumors per group with tumor volumes ~50-100mm³.