Co-Inhibition of PARP and STAT3 as a Promising Approach for Triple-Negative Breast Cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive subtype known for its rapid metastatic potential. Despite its severity, treatment options for TNBC remain limited. Olaparib, an FDA-approved PARP inhibitor, has been used to treat germline BRCA-mutated TNBC in both metastatic and high-risk early-stage settings. However, acquired resistance to PARP inhibitors and their limited applicability in non-BRCA TNBCs are now two major growing clinical problems. Activation of the IL-6/STAT3 signaling cascade has been implicated in therapeutic resistance. In this study, we evaluated the combined effects of the PARP inhibitor olaparib and the STAT3 inhibitor LLL12B in human TNBC cell lines with both BRCA mutations and wild-type BRCA status. Our results demonstrate that the PARP inhibitor olaparib can induce increased interleukin-6 (IL-6) in TNBC cells, with ELISA showing a 2- to 39-fold increase across five cell lines. MTT assays revealed that knocking down or inhibiting STAT3, a key downstream effector of the IL-6/GP130 pathway, sensitizes TNBC cells to olaparib. Treatment with either olaparib or LLL12B alone reduced TNBC cell viability, migration, and invasion. Notably, their combined administration produced a markedly enhanced inhibitory effect compared to individual treatments, regardless of BRCA mutation status. These findings highlight the potential of dual PARP and STAT3 inhibition as a novel targeted therapeutic strategy for both BRCA-mutant and BRCA-proficient TNBC.

Keywords: BRCA; IL-6/STAT3 signaling; PARP inhibitor; STAT3 inhibition; triple-negative breast cancer (TNBC).

Figure 1

Olaparib induced IL-6 secretion in TNBC cells. SUM149, MDA-MB-231, MDA-MB-231 BoneM, MDA-MB-436, and HCC1937 cells were treated with increasing concentrations of olaparib (5 µM, 10 µM, 20 µM, and 30 µM; labeled as O5 → O30) for 6 days and IL-6 secretion was determined by ELISA assay. Data are presented as the mean ± standard error. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Knockdown of STAT3 inhibited TNBC cell viability. MDA-MB-436, HCC1937, SUM149, MDA-MB-231 and MDA-MB-231 BoneM cells were exposed to either control or STAT3-targeting siRNA for 72 h, Western blotting was used to examine the levels of phosphorylated STAT3 (Y705) and total STAT3 (A), with GAPDH included as a loading control. Cell viability was assessed using the MTT assay (B). Values are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. C, control siRNA; Si, STAT3 siRNA; O15, 15 µM olaparib. Original Western blot images can be found in the Supplementary File as Figure S1.

Figure 3

Effects of single and combined olaparib and LLL12B on TNBC cell viability. TNBC cell lines MDA-MB-436, HCC1937, SUM149, MDA-MB-231, and MDA-MB-231 BoneM were treated with varying concentrations of olaparib and/or LLL12B for 3 days. Two drug combination exhibited a synergistic effect, and inhibited cell viability more significantly than either single drug. Drug concentrations were optimized for each cell line to balance efficacy and minimize cytotoxicity. Values are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.1, 0.1 µM of LLL12B; L0.25, 0.25 µM of LLL12B; O10, 10 µM of olaparib; O15, 15 µM of Olaparib.

Figure 4

Inhibition of STAT3 activation by LLL12B in olaparib-treated TNBC cells. (A) Western blot analysis of phosphorylated STAT3 (P-STAT3) and total STAT3 in SUM149 and MDA-MB-231 cells following treatment with DMSO, olaparib, LLL12B, or the combination of olaparib and LLL12B for 24 h. GAPDH was used as a loading control. (B) Immunofluorescence staining of MDA-MB-231 cells treated with DMSO or olaparib (10 μM) for 24 h. Cells were stained with STAT3 primary antibody followed by Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Increased nuclear localization of STAT3 was observed in olaparib-treated cells. Scale bar: 20 μm. Original Western blot images can be found in the Supplementary File as Figure S2.

Figure 5

Effects of single and combined olaparib and LLL12B on TNBC cell migration. A wound healing assay was performed in (A) MDA-MB-436, (B) HCC1937, (C) SUM149, (D) MDA-MB-231, and (E) MDA-MB-231 BoneM cells. Cells were treated with olaparib, LLL12B, or their combination, and wound closure was monitored to assess migration. Combination treatment significantly inhibited migration compared to either single agent or DMSO. Doses were selected based on preliminary optimization for each assay system. Images were captured at a magnification of ×10. Data are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.25, 0.25 µM of LLL12B; L0.5, 0.5 µM of LLL12B; L1, 1 µM of LLL12B; O15, 15 µM of olaparib; O30, 30 µM of Olaparib.

Figure 5

Effects of single and combined olaparib and LLL12B on TNBC cell migration. A wound healing assay was performed in (A) MDA-MB-436, (B) HCC1937, (C) SUM149, (D) MDA-MB-231, and (E) MDA-MB-231 BoneM cells. Cells were treated with olaparib, LLL12B, or their combination, and wound closure was monitored to assess migration. Combination treatment significantly inhibited migration compared to either single agent or DMSO. Doses were selected based on preliminary optimization for each assay system. Images were captured at a magnification of ×10. Data are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.25, 0.25 µM of LLL12B; L0.5, 0.5 µM of LLL12B; L1, 1 µM of LLL12B; O15, 15 µM of olaparib; O30, 30 µM of Olaparib.

Figure 6

Inhibitory effects of Olaparib, LLL12B, and their combination on cell invasion. Invasion assays were performed using Matrigel-coated transwell chambers for (A) MDA-MB-436, (B) HCC1937, (C) SUM149, (D) MDA-MB-231, and (E) MDA-MB-231 BoneM cells treated with olaparib, LLL12B, or their combination. (F) Quantification of invasive inhibition percentages relative to DMSO controls in TNBC cells. Combination treatment resulted in a stronger suppression of cell invasion compared to monotherapy across most cell lines. Images were captured at a magnification of ×10. Drug concentrations were optimized to capture measurable inhibition while avoiding excessive cytotoxicity. Data are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.25, 0.25 µM of LLL12B; L0.5, 0.5 µM of LLL12B; L1, 1 µM of LLL12B; O30, 30 µM of olaparib; O35, 35 µM of olaparib.

Figure 6

Inhibitory effects of Olaparib, LLL12B, and their combination on cell invasion. Invasion assays were performed using Matrigel-coated transwell chambers for (A) MDA-MB-436, (B) HCC1937, (C) SUM149, (D) MDA-MB-231, and (E) MDA-MB-231 BoneM cells treated with olaparib, LLL12B, or their combination. (F) Quantification of invasive inhibition percentages relative to DMSO controls in TNBC cells. Combination treatment resulted in a stronger suppression of cell invasion compared to monotherapy across most cell lines. Images were captured at a magnification of ×10. Drug concentrations were optimized to capture measurable inhibition while avoiding excessive cytotoxicity. Data are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.25, 0.25 µM of LLL12B; L0.5, 0.5 µM of LLL12B; L1, 1 µM of LLL12B; O30, 30 µM of olaparib; O35, 35 µM of olaparib.

Figure 6

Inhibitory effects of Olaparib, LLL12B, and their combination on cell invasion. Invasion assays were performed using Matrigel-coated transwell chambers for (A) MDA-MB-436, (B) HCC1937, (C) SUM149, (D) MDA-MB-231, and (E) MDA-MB-231 BoneM cells treated with olaparib, LLL12B, or their combination. (F) Quantification of invasive inhibition percentages relative to DMSO controls in TNBC cells. Combination treatment resulted in a stronger suppression of cell invasion compared to monotherapy across most cell lines. Images were captured at a magnification of ×10. Drug concentrations were optimized to capture measurable inhibition while avoiding excessive cytotoxicity. Data are shown as mean ± SE. Statistical significance is indicated as follows: ns, no significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. L0.25, 0.25 µM of LLL12B; L0.5, 0.5 µM of LLL12B; L1, 1 µM of LLL12B; O30, 30 µM of olaparib; O35, 35 µM of olaparib.

Figure 7

Schematic model illustrating the mechanism by which combination therapy with LLL12B enhances therapeutic efficacy in TNBC cells. Olaparib treatment upregulates IL-6 secretion in TNBC cells, leading to activation of the STAT3 signaling pathway. Activated STAT3 translocates to the nucleus, where it promotes the transcription of downstream target genes involved in cancer cell survival, migration, and invasion. Inhibition of STAT3 activation by LLL12B sensitizes TNBC cells to olaparib, resulting in reduced viability, migration, and invasion. The combination therapy exhibits synergistic anti-tumor effects.