Deciphering the dynamics of alternative pre‐mRNA processing of glutaminase in ovarian cancer

Ovarian carcinoma is the fifth leading cause of cancer death among women in the United States and has the highest mortality rate of all gynecologic malignancies. Patients with ovarian cancer are often diagnosed with advanced stage, metastatic disease due to lack of specific symptoms and adequate reliable tumor specific biomarkers for early disease detection. An emerging hallmark of cancer is the metabolic reprogramming of transformed cells to support the energy and biosynthetic needs of the rapidly dividing and metastatic tumor phenotype. These metabolic changes facilitate increased glucose and glutamine uptake and metabolism. Recent studies suggest that highly invasive ovarian cancer cells show a remarkable dependence on glutamine, making glutamine metabolism a viable therapeutic target in ovarian cancer. There are several clinical trials focused on inhibiting glutaminase, the enzyme that catalyzes the first step of glutaminolysis and converts glutamine to glutamate. There are two genes that code for glutaminase in the human genome, glutaminase 1 (GLS1) located on chromosome 2 and glutaminase 2 (GLS2) encoded by chromosome 12. GLS1 is the enzyme most associated with tumor metabolism. There are two annotated alternative isoforms of GLS1, GAC and KGA, which result from alternative splicing. Different tumors express either or both isoforms. Under normal conditions the production of the KGA isoform is repressed by miR‐23 which binds to the long 3′UTR of KGA and silences its expression. We recently found that depletion of a 3′end processing factor, CFIm25, results in alternative polyadenylation of the KGA isoform resulting in a novel KGA transcript that has a truncated 3′UTR and is able to evade miR‐23 regulation. In addition, knockdown of CFIm25 also switched splicing to preferentially facilitate splicing to KGA instead of the GAC isoform. Our goal is to determine the different glutaminase isoforms in metastatic ovarian cancer cells and determine how they are regulated for therapeutic targeting using isoform specific siRNAs. We have developed isoform specific amplicons to measure levels of GAC, long KGA (with long 3′UTR) and short KGA (with short 3′UTR). Using qRT‐PCR we show that the highly metastatic ovarian cancer cell lines express high levels of the GAC isoform. The cisplatin resistant cell line SKOV3 also expresses KGA in addition to GAC and we detected this at both the mRNA and protein level. Inhibition of GLS1 using inhibitors resulted in decreased cell viability of highly metastatic ovarian cancer cell lines. Based on our findings, we hypothesize that expressing both the KGA and GAC isoforms facilitates the chemotherapeutic drug resistance of highly metastatic ovarian tumors. Support or Funding Information Funding: Butler University Holcomb research award, Senior Research Grant from the Indiana Academy of Sciences