Metformin reverses PARP inhibitors-induced epithelial-mesenchymal transition and PD-L1 upregulation in triple-negative breast cancer

Abstract

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising targeted therapies for BRCA-mutated cancers by blocking repair of DNA double-strand breaks. However, resistance to PARP inhibitors (PARPi) has been described in some patients lowering the overall response rates. To investigate the underlying mechanisms of PARPi resistance, we developed the adaptive resistant clones in triple-negative breast cancer cell lines. We identified epithelial-mesenchymal transition (EMT) and upregulation of programmed death-ligand 1 (PD-L1) in resistant cells and further demonstrated the important role of Akt S473 phosphorylation in PARPi resistance. In addition, PARPi mediated EMT is independent of PD-L1 upregulation. Blocking the p-Akt S473 axis by metformin reversed EMT and PD-L1 expression which sensitized PARPi-resistant cells to cytotoxic T cells. Thus, a combination of metformin and PARP inhibitors may be a promising therapeutic strategy to increase the efficacy of PARP inhibitors and tumor sensitivity to immunotherapy.

Keywords: PARP; PD-L1; epithelial-mesenchymal transition; metformin; triple-negative breast cancer.

Figure 1

Olaparib induces scattering and morphologic changes in several triple-negative breast cancer (TNBC) cell lines. A. Representative phase-contrast microscopy images of four cell lines treated with olaparib (5 µM) or vehicle for 7 days. Scale bar: 200 µM. B. Cell number counted by ImageJ in PARPi treated group and control group. Two group data were analyzed by GraphPrism 9.0. *P<0.05; ***P<0.001 (Student’s t-test). Error bar represents standard and variation in 3 repeated experiments. C. Quantification of the aspect ratio between the area and circumference of TNBC cell lines, determined 7 days after treatment with or without olaparib. D. Scatter score of TNBC cell lines treated with or without olaparib. *P<0.05; **P<0.01 (Student’s t-test).

Figure 2

PARP inhibitors (PARPis) induce epithelial-mesenchymal transition (EMT) in several triple-negative breast cancer (TNBC) cell lines. A. Western blot analysis of EMT markers, E-cadherin, N-cadherin, and vimentin, in resting cells after treatment with olaparib (5 µM) for 72 hours. B. Western blot analysis of EMT markers, E-cadherin, N-cadherin, and vimentin, in resting HCC1806 (PARPi-resistant cell lines) and SUM149 (PARPi-sensitive cell lines) cells after treatment with the indicated PARP inhibitor. C. Western blot analysis of EMT markers in HCC1806 cells after treatment with olaparib at the indicated doses and duration. D. Representative images of tumors stained with E-cadherin, N-cadherin, vimentin, and 4’,6-diamidino-2-phenylindole (DAPI). Scale bar: 50 µM. E. mRNA levels of Vimentin in TNBC cells treated with or without PARP inhibitors (quantitative reverse-transcription PCR, qrt-PCR). F. mRNA levels of RAD51 in TNBC cells treated with or without PARP inhibitors (qrt-PCR). G. mRNA levels of N-cadherin in TNBC cells treated with or without PARP inhibitors (qrt-PCR). Error bars represent standard and variation in 3 repeated experiments.

Figure 3

PARP inhibitor-induced tumor cells exhibit stemness features. A. Flow cytometric analysis of stem-like markers (CD44 and CD24) in MDA-MB-468 and 4T1 cells treated with or without olaparib (CD24^-/low/CD44⁺ represents stem-like features). B. Open histograms, isotype IgG negative control. The mean fluorescence intensity of each cell population was quantified by FlowJo for comparison. Experiments were repeated three times. *P<0.05, ***P<0.001, Student’s t-test. C. Representative phase-contrast microscopy images of spheres growing from control cells or cells with epithelial-mesenchymal transition driven by PARP inhibitors. Scale bar: 400 µM. D. Open histograms represent the relative number of spheres (diameter > 50 μM) on the third and sixth day of treatment. *P<0.05, **P<0.01, Student’s t-test.

Figure 4

PARPi treated stable cells with mesenchymal function induced by PARP inhibitors. A. The migration in MCF10A and 4T1 cells cultured with or without olaparib was determined using the wound-healing assay (IncuCyte 96-well Scratch Wound Cell Migration assay). Representative phase-contrast microscopic images were obtained by IncuoCyte ZOOM microscope. Red line labels the migration line of cells. B. Comparison of PARPi on cell migration with wound distance percentage. C. Comparison of migration (relative wound density) between primary cells and treated cells (Student’s t-test). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. D. Transwell Matrigel invasion assay. Representative microscopic images of PARPi-treated stable cells (HCC1806 and SUM149) and untreated cells that invaded through the transwell in the Matrigel invasion assay. (Giemsa stain, magnification × 10). E. The box-and-whisker plot of cell number per visual field was shown and analyzed (magnification × 10) of 3 replicate wells. (Welch’s test, **P<0.01, ****P<0.0001).

Figure 5

Reversal of PARP inhibitor-mediated PD-L1 upregulation metformin and chemotherapy. A. Western blot analysis of EMT markers (E-cadherin, N-cadherin, and vimentin) and PD-L1 in MCF10A cells at the indicated times upon treatment with 5 µM olaparib. B. Flow cytometry analysis of PD-L1 expression levels in treated populations of MCF10A and HCC1806 cells. The three representative groups shown are IgG, untreated, rucaparib (10 µM), and metformin (10 µM). C. Representative phase-contrast image of PARPi-induced HCC1806 cells with a high dose of doxorubicin, epirubicin and daunorubicin for 24 h. CTRL, control; DOXO, doxorubicin (0.1µM); EPI, epirubicin (1 µM); DNR, daunorubicin (0.5 µM). Scale bars: 400 µM. D. The right panel shows a Western blot analysis of EMT markers (E-cadherin, N-cadherin, and vimentin) in resting cells after treatment with the indicated agents on a high-dose, short-term (24-hour) schedule. E. A relative number of cells after treatment with the indicated agents on a high-dose, short-term schedule. F. Cell viability was analyzed by trypan blue exclusion. G. Representative phase-contrast image of PARPi-induced HCC1806 cells with a low dose of doxorubicin, epirubicin and daunorubicin for 96 h. CTRL, control; DOXO, doxorubicin (0.01 µM); EPI, epirubicin (0.01 µM); DNR, daunorubicin (0.01 µM). Scale bars: 400 µM. H. The right panel shows Western blot analysis of EMT markers (E-cadherin, N-cadherin, and vimentin) in resting cells after treatment with the indicated agents on a low-dose, long-term schedule. I. A relative number of cells after treatment with the indicated agents on a low-dose, long-term (0.01 µM, 96-hour) schedule. J. Cell viability was also analyzed by trypan blue exclusion. Error bars represent standard and variation in 3 repeated experiments. *P<0.05, **P<0.01.

Figure 6

PARP inhibitors induce EMT and PD-L1 upregulation by enhancing p-Akt S473 activation. A. Western blot analysis of N-cadherin, E-cadherin, p-GSK3β, p-Akt S473, PD-L1 and β-actin in olaparib (5 μM) treated HCC1806 cells at indicated time points. B. Western blot analysis of EMT markers (E-cadherin, N-cadherin, and vimentin) and PD-L1 in resting cells after treatment with indicated agents. C. Western blot analysis of E-cadherin, N-cadherin, p-GSK3β, p-Akt, and PD-L1 in MDA-MB-231 shPD-L1 cells. D. A proposed working model. PARPi induces EMT through activation of p-Akt S473/mTOR axis pathway, and independently, transcriptional activation of PD-L1 responds to that pathway. Both can be blocked by metformin and sensitized to T cell. TNBC, triple-negative breast cancer; PARPi, PARP inhibitors.