Abstract 442: Serine protease inhibitor Kazal type 1 (SPINK1) drives anoikis resistance in ovarian clear cell carcinoma

Abstract

Abstract Patients diagnosed with ovarian cancer face an average 5 year survival rate of 46%; low survival is driven by early and ongoing intraperitoneal dissemination and metastasis of the tumor. Among the different histotypes of ovarian cancer, ovarian clear cell carcinoma (OCCC) has a particularly poor prognosis when diagnosed in late stage, as these tumors tend to be chemoresistant, leaving no effective therapeutic options. Intraperitoneal metastasis requires cells to become anoikis resistant, where apoptosis normally induced by loss of attachment is suppressed. We seek to better understand the mechanisms involved in anoikis resistance in OCCC, which in the future could lead to new therapeutic strategies for these patients. We have found that Serine Protease Inhibitor Kazal type 1 (SPINK1), an endogenous inhibitor of trypsin like serine proteases, is also an important regulator of anoikis resistance in some ovarian cancers. In this study, we aimed to dissect the role of SPINK1 specifically in OCCC, defining its contribution to anoikis resistance using cultured OCCC cell lines and elucidating its mechanism of action. We compared anoikis resistance of cells with endogenous SPINK1 expression versus cells in which SPINK1 was silenced using lentiviral shRNA constructs, when culturing cells on ultra-low attachment plates to mimic cell detachment from the extracellular matrix. We found that knockdown of SPINK1 reduced survival and stimulated apoptotic pathways in OCCC cells grown under detached conditions, implicating SPINK1 in anoikis resistance of OCCC cells. As a secreted protease inhibitor, SPINK1 may be expected to confer anoikis resistance by inhibiting an extracellular serine protease involved in triggering anoikis. Because many proteases represent potential targets of SPINK1, we have designed a strategy using Activity Based Protein Profiling (ABPP) to discover the targets of SPINK1 from among this large pool of candidates. In pilot studies, we have successfully used a novel activity-based probe to covalently label the active sites of serine proteases secreted from OCCC cells. In ongoing efforts, we are optimizing methods to identify labeled proteases with high sensitivity using tandem mass spectrometry. We will then identify those proteases regulated by SPINK1 through comparison of labeled proteomes from OCCC cells with and without SPINK1 treatment,, and candidate proteases will be further analyzed for their role in triggering anoikis. The identification of SPINK1 regulated proteases that are responsible for mediating anoikis in our models will give insight into important mechanisms whereby tumor cells acquire resistance to anoikis in OCCC. In-depth understanding of anoikis resistance, a critical component of ovarian cancer progression and metastasis, may lead to novel biomarkers of disease progression as well as important therapeutic targets. Citation Format: Christine Mehner, Mathew A. Coban, Alexandra Hockla, Derek C. Radisky, Evette S. Radisky. Serine protease inhibitor Kazal type 1 (SPINK1) drives anoikis resistance in ovarian clear cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 442.