Gasdermin C sensitizes tumor cells to PARP inhibitor therapy in cancer models

Abstract

Several poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are approved by FDA to treat cancer with BRCA mutations. BRCA mutations are considered to fuel a PARPi killing effect by inducing apoptosis. However, resistance to PARPi is frequently observed in the clinic due to an incomplete understanding on the molecular basis of PARPi function and a lack of good markers, beyond BRCA mutations, to predict response. Here, we show that gasdermin C (GSDMC) sensitized tumor cells to PARPi in vitro and in immunocompetent mice and caused durable tumor regression in an immune-dependent manner. A high expression level of GSDMC predicted better response to PARPi treatment in patients with triple-negative breast cancer (TNBC). PARPi treatment triggered GSDMC/caspase-8-mediated cancer cell pyroptosis (CCP) that enhanced PARPi killing of tumor cells. GSDMC-mediated CCP increased memory CD8+ T cell population in lymph node (LN), spleen, and tumor and, thus, promoted cytotoxic CD8+ T cell infiltration in the tumor microenvironment. T cell-derived granzyme B (GZMB) activated caspase-6, which subsequently cleaved GSDMC to induce pyroptosis. Interestingly, IFN-γ induced GSDMC expression, which, in turn, enhanced the cytotoxicity of PARPi and T cells. Importantly, GSDMC promoted tumor clearance independent of BRCA deficiency in multiple cancer types with PARPi treatment. This study identifies a general marker and target for PARPi therapy and offers insights into the mechanism of PARPi function.

Keywords: Cancer; Drug therapy; Oncology; T cells; Therapeutics.

Figure 1. GSDMC-mediated CCP enhances the cytotoxicity of PARPi.

(A) Representative IHC staining results for GSDMC in human TNBC tissues. Scale bar: 50 μm. (B) MDA-MB-436 and HCC1937 cells harboring an empty vector (vector) or expressing WT GSDMC (GSDMC-WT) or the D365A mutant (GSDMC-mut). Immunoblotting demonstrating caspase-8 cleavage of GSDMC in indicated cells treated with olaparib (20μM) for 48 hours. (C) Cells in B were treated with olaparib (20 μM) for 72 hours. Cell death measured by LDH release (LDH-released cell death) is shown (n = 3). (D) Same treatment as in C; representative images of dying cell morphology. Red arrows indicate cell swelling with large bubbles. Scale bar: 20 μm. (E) Cells in B were treated with the indicated concentrations of olaparib for 72 hours and subjected to a cell viability assay (n = 3). (F) MDA-MB-157 and Hs578t cells with deletion of GSDMC or caspase-8 were treated with olaparib (100 μM) for 72 hours. LDH-released cell death is shown (n = 3). (G) Cells in F were treated with the indicated concentrations of olaparib for 72 hours and subjected to a cell viability assay (n = 3). Data represent mean ± SD. 1-way ANOVA was used. **P < 0.01, ***P < 0.001.

Figure 2. GSDMC-increased sensitivity of PARPi is immune-mediated in vivo.

(A) MDA-MB-436 stable cells as indicated were inoculated into the mammary fat pad of nude mice (n = 10). Mice were administered olaparib. Tumor growth was shown. (B and C) 4TO7 cells harboring an empty vector (vector) or expressing WT mouse Gsdmc (Gsdmc-WT) or the caspase-8 cleavage site D263A mutant (Gsdmc-mut) were inoculated into the mammary fat pad of nude mice (B) or immunocompetent BALB/c mice (C) (n = 10). Mice were administered olaparib. Tumor growth was shown. (D) LDH level in tumor slurry of tumors indicated in C was measured. (E and F) Same as B and C, except that stable transfectants were established in 4TO7-Brca KO instead of 4TO7 parental cells and injected (n = 10). (G) LDH level in slurry of tumors indicated in F was measured. (H) Parental 4TO7 cells mixed with 0%, 15%, or 30% 4TO7-Brca–KO Gsdmc-WT cells were inoculated into BALB/c mice (n = 10). Mice were administered olaparib. Tumor growth was shown. (I) BALB/c mice with 4TO7-Brca–KO Gsdmc-WT or vector tumors were administered olaparib and durable tumor regression was monitored (n = 10). (J and K) 4TO7-Gsdmc-WT (J) or 4TO7-Brca–KO Gsdmc-WT (K) cells were inoculated into the mammary fat pad of nude mice and immunocompetent BALB/c mice (n = 10). Mice were administered olaparib. Survival curves were shown. Data represent mean ± SD. 1-way ANOVA was used for A–H. The log-rank test was used for J and K. ***P < 0.001.

Figure 3. GSDMC-mediated CCP increases the population and tumor infiltration of memory T cell.

(A) 4TO7-Brca–KO cells stably expressing an empty vector (vector) or WT mouse Gsdmc (Gsdmc-WT) or the D263A mutant (Gsdmc-mut) were inoculated into the mammary fat pad of immunocompetent BALB/c mice (n = 10). Mice were administered olaparib. Percentage of tumor-infiltrating CD8⁺ T cells was analyzed. (B) Overexpression of eGFP in the stable cells as indicated in A. Then cells and mice were treated same as in A. Mean numbers of eGFP tetramer⁺ (eGFP tet⁺) CD8⁺ T cells per gram of tumor (left). Percentage of IFN-γ⁺ (middle) or TNF-α⁺ (right) CD8⁺ T cells activated by eGFP peptide. (C) Frequency of memory T cell subsets in lymph node (LN), spleen, and tumors of A. Tex, exhausted T cell. (D) Initial tumor challenge and mice treatment were same as in A. Tumors were removed on day 18. Then tumor rechallenge of 4TO7 parental cells was performed 60 days after tumor removal. Tumor growth was shown (n = 10). (E) Stable cells as indicated in A were inoculated into the mammary fat pad of immunocompetent BALB/c mice (n = 10). 4TO7 parental cells were simultaneously injected into contralateral mammary fat pad. Mice were administered olaparib. Tumor growth of 4TO7 parental cells was monitored. (F and G) 4TO7-Brca–KO Gsdmc-WT cells were inoculated into BALB/c mice (n = 10). Mice were administered olaparib. Depletion of CD8⁺ T cell with anti-CD8. Curves of tumor growth (F) and survival (G). (H) Growth curve of 4TO7-Gsdmc-WT and 4TO7-vector tumors in BALB/c mice (n = 10) treated with olaparib or PD-1 antibody or the combination. Data represent mean ± SD. 1-way ANOVA was used for A, B, D, E, and H. Unpaired 2-tailed t test was used for F. Log-rank test was used for G. **P < 0.01, ***P < 0.001.

Figure 4. T cell–derived GZMB induces CCP by caspase-6 cleavage of GSDMC, and IFN-γ promotes GSDMC expression to enhance the killing effect of T cells and PARPi.

(A) GZMB-mediated GSDMC cleavage by caspase-6 in MDA-MB-157 and Hs578t cells. Caspase-6i, caspase-6 inhibitor; caspase-8i, caspase-8 inhibitor. (B) MDA-MB-436 cells expressing WT GSDMC (GSDMC-WT) or the D365A mutant (GSDMC-mut) were treated with GZMB or inhibitors of caspase-6 or caspase-8. Immunoblotting of GSDMC cleavage. (C) Same as B, except that cells were cocultured with T cells instead of GZMB treatment. (D) Cell death measured by LDH release (LDH-released cell death) induced by GZMB in MDA-MB-157 and Hs578t cells (n = 3). (E) LDH-released cell death induced by cytotoxic T cells in MDA-MB-157 and Hs578t cells treated with caspase-6 siRNA (6si) and/or caspase-8 siRNA (8si) (n = 3). (F) LDH-released cell death induced by cytotoxic T cell in MDA-MB-436 cells with expression of vector, GSDMC-WT, and GSDMC-mut (n = 3). (G) Quantification of cytokine levels by ELISA in tumors of Figure 2F. (H) GSDMC induction by cytokines indicated in BT549 and HCC38 cells. (I) IFN-γ enhanced LDH-released cell death induced by olaparib at indicated concentration in BT549 and HCC38 cells (n = 3). (J and K) IFN-γ enhanced LDH-released cell death in BT549 and HCC38 cells treated with cytosolic delivery of GZMB (J) or cocultured with cytotoxic T cells (K) (n = 3). Data represent mean ± SD. 1-way ANOVA was used for D–F. 2-way ANOVA was used for I. Unpaired 2-tailed t test was used for G, J, and K. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. GSDMC contributes to PARPi efficacy in both BRCA-proficient and -deficient tumors.

(A–F) MDA-MB-157 and Hs578t cells with deletion of BRCA. Cells were treated with the indicated concentrations of olaparib for 72 hours and subjected to a cell viability assay (n = 3) (A and B). Immunoblotting of GSDMC cleavage in cells treated with the indicated concentrations of olaparib for 72 hours (C and D). Cell death measured by LDH release (LDH-released cell death) induced by olaparib at the indicated concentrations (n = 3) (E and F). (G and H) 4TO7 parental or Brca-KO cells with ectopic expression of Gsdmc were inoculated into the mammary fat pad of immunocompetent BALB/c mice (n = 10). Mice were administered olaparib (50 mg/kg) 5 times per week for 18 days. Tumor growth (G) and survival (H) curves were shown. (I) Ectopic expression of Gsdmc in parental or Brca-KO cells of PanO2, MC38, Hepa-1-6, B16. Cells were inoculated into the mammary fat pad of immunocompetent C57BL/6 mice (n = 10). Mice were treated same as G. Tumor growth curves were shown. Data represent mean ± SD. Unpaired 2-tailed t test was used for A and B. 2-way ANOVA was used for E and F. 1-way ANOVA was used for G and I. Log-rank test was used for H. ***P < 0.001.