Birinapant Enhances Gemcitabine's Antitumor Efficacy in Triple-Negative Breast Cancer by Inducing Intrinsic Pathway-Dependent Apoptosis

Abstract

Triple-negative breast cancer (TNBC) is the most aggressive subgroup of breast cancer, and patients with TNBC have few therapeutic options. Apoptosis resistance is a hallmark of human cancer, and apoptosis regulators have been targeted for drug development for cancer treatment. One class of apoptosis regulators is the inhibitors of apoptosis proteins (IAPs). Dysregulated IAP expression has been reported in many cancers, including breast cancer, and has been shown to be responsible for resistance to chemotherapy. Therefore, IAPs have become attractive molecular targets for cancer treatment. Here, we first investigated the antitumor efficacy of birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspases (SMACs), in TNBC. We found that birinapant as a single agent has differential antiproliferation effects in TNBC cells. We next assessed whether birinapant has a synergistic effect with commonly used anticancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), erlotinib (EGFR inhibitor), and gemcitabine, in TNBC. Among these tested drugs, gemcitabine showed a strong synergistic effect with birinapant. Birinapant significantly enhanced the antitumor activity of gemcitabine in TNBC both in vitro and in xenograft mouse models through activation of the intrinsic apoptosis pathway via degradation of cIAP2 and XIAP, leading to apoptotic cell death. Our findings demonstrate the therapeutic potential of birinapant to enhance the antitumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.

Figure 1.. Birinapant increases sensitivity of TNBC cells to gemcitabine in vitro.

A, Cell viability was determined using CellTiter-Blue assay at 72 h following treatment with gemcitabine alone (0.0001-1 μM for SUM149 cells and 0.005-20 μM for other tested cells) or gemcitabine (at the same concentrations) plus birinapant at IC₂₀ or 5 μM when IC₂₀ was greater than 10 μM. B, Anchorage-independent growth was determined using soft agar colony formation assay at 3 weeks following treatment with birinapant alone, gemcitabine alone, or birinapant plus gemcitabine. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed Student t-test. G, gemcitabine; B, birinapant.

Figure 2.. Birinapant increases sensitivity of TNBC cells to gemcitabine by inducing apoptosis.

A-C, Cells were treated with birinapant alone, gemcitabine alone, or birinapant plus gemcitabine for 72 h and then subjected to cell cycle analysis (A) for sub-G1 fraction and (B) for S-phase fraction and (C) stained with annexin V and 7-AAD to assess apoptosis by flow cytometry analysis. G, gemcitabine; B, birinapant. The experiments were repeated twice, with no replicates for each treatment.

Figure 3.. Birinapant increases sensitivity of TNBC cells to gemcitabine by activating the intrinsic apoptotic pathway.

A, Cells were treated with birinapant alone, gemcitabine alone, or birinapant plus gemcitabine for 72 h and then subjected to Western blot analysis for cleavage of PARP and caspases. B, Cells were pre-treated with Z-VAD-FMK and then with birinapant plus gemcitabine. On day 5 after treatment, cell viability was determined using CellTiter-Blue assay. *P < 0.001 by unpaired Student t-test. C, Cells were treated with birinapant alone, gemcitabine alone, or birinapant plus gemcitabine for 72 h and then subjected to Western blot analysis for IAP degradation.

Figure 4.. Birinapant increases sensitivity of SUM149 and MDA-MB-231 xenografts to gemcitabine in vivo by inducing apoptosis.

A and B, Birinapant synergizes with gemcitabine in (A) SUM149 and (B) MDA-MB-231 xenograft mouse models. SUM149 or MDA-MB-231 cells (4 × 10⁶) were injected into 1 of the mammary fat pads of female nude mice. When the tumors were about 75-150 mm³, the mice were treated with vehicle, birinapant alone, gemcitabine alone, or birinapant plus gemcitabine for 21 days in the SUM149 model or 38 days in the MDA-MB-231 model. **P < 0.01, ***P < 0.001, ****P < 0.0001 for gemcitabine alone vs. combination; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 for birinapant alone vs. combination by a 2-tailed Student t-test. C and D, Immunohistochemical staining showing expression levels of Ki-67, XIAP, cIAP2, cleaved caspase 3, and cleaved PARP in tumors from mice implanted with (C) SUM149 cells or (D) MDA-MB-231 cells and treated with vehicle, birinapant alone, gemcitabine alone, or birinapant plus gemcitabine. Images were taken at 20× magnification. Scale bars, 200 μm. E and F, Measurement of the intensity of immunohistochemical staining for Ki-67, XIAP, cIAP2, cleaved caspase 3, and cleaved PARP in tumors from mice implanted with (E) SUM149 cells or (F) MDA-MB-231 cells and treated with vehicle, birinapant alone, gemcitabine alone, or birinapant plus gemcitabine. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-tailed Student t-test.