An RNA Interference Lethality Screen of the Human Druggable Genome to Identity Molecular Vulnerabilities in Epithelial Ovarian Cancer
Authors:   , Sethi Geetika , Godwin Andrew 
Published: July 2024

Epithelial ovarian cancer (EOC) is the second most common gynecological cancer and the fifth leading cause of death among women. Estimates predict that 22,240 women will be diagnosed with ovarian cancer in 2013 while 14,030 will succumb to the disease (http://seer.cancer.qov/statfacts/html/ovary ). The overall survival rate for women with ovarian cancer has not changed significantly over the last 30 years. Thus a new armament of therapies is required. However, in order to improve treatment we must continue to expand our understanding of the biology of the disease and the essential factors that drive tumorigenesis. We used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (UDC) cell lines. The top 300 "hits" affecting the viability of A1847 cells were rescreened across six additional EOC cell lines and three non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three (53) gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly overexpressed in serous adenocarcinomas. Among the three top priority candidates identified, Pleiotrophin (PTN) was further explored for its role in the pathogenesis of ovarian cancer. Although, it is well-known that PTN elicits its protumorigenic effects through its receptor, Protein Tyrosine Phosphatase Receptor Z1 (PTPRZ1), not much is known about this pathway in ovarian cancer. Using immunoblotting, we found that PTN was overexpressed in serous ovarian cancers, the most common and deadly form of the disease, relative to normal ovarian specimens by immunoblotting. We further demonstrated that EOC cell lines produce and secrete PTN, which can also be detected in the serum of ovarian cancer patients by ELISA. We show that PTPRZ1 is differentially expressed at the mRNA (by RT-PCR) and protein (by immunoblotting) levels across a panel of EOC cell lines. Using immunohistochemical approaches, PTPRZ1 was found to be significantly overexpressed in ovarian cancer tissues (n = 54) as compared to the cell of origin, i.e., ovarian surface epithelial (OSE) cells. Moreover, we found that similar to PTN, PTPRZ1 is a molecular vulnerability in EOC cells. siRNA mediated knockdown of PTPRZ1 decreased EOC cell viability and significantly induced apoptosis with little detectable effect on the cell cycle phase distribution. In order to dissect the mechanism(s) behind the role of PTN in pro-survival, we evaluated the expression patterns of survival related genes in EOC cells following PTN silencing using RNAi approaches. Through integrated computational and experimental approach we found that the ERK1/2 (members of the mitogen-activated protein kinase family) signaling pathway was a mediator of the PTN in EOC cells. Our results provide the first experimental evidence that PTN and its signaling components may be of significance in the pathogenesis of epithelial ovarian cancer and provide a rationale for clinical evaluation of MAPK inhibitors in PTN and/or PTPRZ1 expressing ovarian tumors.