Abstract 2426: SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death

Abstract

Abstract The purpose of this study was to determine if squamous cell carcinoma antigen (SCCA), also known as SERPINB3, protects cervical cancer cells from ionizing radiation (IR)-induced death, and to determine the mechanism(s) of IR-induced cell death with or without SERPINB3. Cervical cancer remains a leading cause of cancer death in women worldwide despite advances in screening, vaccination and treatment. Recurrence after definitive chemoradiation occurs in up to 30-50% of patients with locally advanced disease. We and others have demonstrated that elevated serum SCCA portends poor prognosis in cervical cancer. In addition, CRISPR-Cas9-mediated knock out of SERPINB3 significantly sensitizes cervical tumor cells to IR in vitro. We hypothesize that SERPINB3 promotes radioresistance by protecting cells against lysosome damage and lysosomal mediated necrosis. Two cervical cancer cell lines with high SERPINB3 expression were edited using CRISPR-Cas9 technology at the SERPINB3 locus resulting in knock out (KO). B3-KO cells were significantly more radiation sensitive compared to control cells at all doses and time points evaluated. Cell death morphology in the B3-KO cells was necrotic, with large cytoplasmic single membraned vesicles, many of which were ruptured, consistent with that seen in lysosome-mediated necrosis. Live-cell time-lapse imaging showed loss of lysosome integrity in the hours leading up to cell death (propidium iodide nuclear staining). Western blot analysis showed low levels of caspase-3 and caspase-7 cleavage only at high doses and late time points after IR, with no evidence of phospho-MLKL or phospo-RIPK3 (markers of necroptosis), or gasdermin-D cleavage (marker of pyroptosis). Necrostatin, ferrostatin, liproxstatin and YVAD-Cho had little to no effect on cell death following IR, while pan-caspase inhibitors decreased IR-induced cell death in both control and B3-KO cells, and E64D decreased IR-induced cell death in B3-KO cells to a greater degree than control cells. Additionally, cervical tumor cell lines that had no detectable expression of SERPINB3 were engineered to expressed high levels of SERPINB3 (B3-wt) or SERPINB3 containing the P14 mutation A351R (B3-P14m), which lacks protease inhibitory function. B3-wt expressing cells displayed increased radiation resistance in clonogenic survival assays and mouse tumor models. Growth rate and radiation response of B3-p14m expressing tumors was similar to vector control. These data suggest that SERPINB3 is an important mediator of radiation response in cervical cancer and protects cells by inhibiting cysteine protease-dependent cell death, likely via lysosome-mediated necrosis. These findings support SERPINB3 as a potential target for novel radiosensitizing therapies. Citation Format: Songyan Wang, Clifford Luke, Victoria Shi, Perry Grigsby, Gary Silverman, Stephanie Markovina. SERPINB3 promotes radiation resistance in cervical cancer by inhibiting lysosome-mediated cell death [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2426.