Abstract 5858: Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness

Abstract

Abstract High-grade serous ovarian cancer (HGSOC) is the most predominant and lethal subtype of ovarian cancer. While most patients initially respond to standard-of-care treatment, the majority will eventually relapse. Despite improved treatment strategies, the prognosis has not improved significantly and the overall 5-year survival rate remains ~31%. To understand the underlying mechanisms that govern the distinctions between tumors with good and poor prognoses, we implemented a proteogenomic approach to analyze gene expression, proteomic and phosphoproteomic profiles of HGSOC tumors available in the TCGA and the CPTAC studies. Our analyses identified upregulated hematopoietic cell kinase (HCK) signaling along with innate immunity related and ERBB activity in poor prognostic tumors. These results were confirmed using independent datasets to demonstrate HCK up-regulation in tumor samples compared to borderline tumors and/or normal samples. Further analyses of micro-dissected HGSOC tumor tissue determined that HCK was specifically upregulated in tumor epithelial cells. Importantly, differential analysis of CPTAC phospho-proteomic data identified significant enrichment of phosphorylated HCK in the primary tumors of patients with tumor recurrence within 1 year compared to tumor-free patients. Interestingly, in-silico analyses suggest that HCK may play a dichotomous tumorigenic role as HCK was strongly associated with pro-survival signaling pathways as well as enrichment of pro-tumorigenic immune signaling. Consistent with these results, in vitro studies in NIH-OVCAR3 and CAOV3 cell lines with high endogenous HCK expression demonstrated that HCK silencing decreased cell proliferation, invasion, and colony formation. Conversely, HCK overexpression significantly induced proliferation and clonogenic capacity in OVSAHO cells (low HCK). These results suggest that HCK is essential for HGSOC proliferation and survival. Reverse phase protein array (RPPA) analysis was next used to identify potential mechanisms by which HCK modulates HGSOC tumorigenesis. Our analyses identified a number of candidate proteins that are significantly downregulated following shRNA-mediated silencing of HCK including PD-L1, CD44, and NOTCH3. Further in silico analyses demonstrated significant enrichment of PD-L1 protein expression in HGSOC tumors with high HCK activity suggesting a potential role for HCK in regulating the tumor microenvironment. These latter analyses also identified a significant correlation between HCK and CD44 mRNA and protein expression in human tumors. Importantly, experimental studies in CAOV3 cells confirmed that shRNA silencing of HCK inhibited PD-L1, CD44 and NOTCH3 protein expression; these results were confirmed by lentiviral overexpression of HCK which resulted in increased CD44 and NOTCH3 expression in OVSAHO cells. Given that CD44 and NOTCH3 signaling have been shown to mediate tumor cell proliferation and survival, our data, which indicate that CD44 and NOTCH3 expression are regulated by HCK activity, suggest that these genes may contribute to HCK mediated HGSOC tumorigenesis. Our data collectively implicate HCK as a novel driver of HGSOC development and progression and may represent a novel therapeutic opportunity in this subset of patients. Citation Format: Christen A. Khella, Michael L. Gatza. Multiplatform analyses identify upregulated hematopoietic cell kinase activity in poorly prognostic high-grade serous ovarian cancer and its role in regulating tumor aggressiveness [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 5858.