| dc.description.abstract | Abstract Ovarian cancer is the deadliest of all female gynecological cancers and the fifth leading cause of cancer-related death in women. Lack of early detection and treatment failure are the major contributors to ovarian cancer death in women. Understanding ovarian cancer at the molecular level will help provide better treatment options, such as targeted therapies. Nearly sixty percent of women are diagnosed at late stage ovarian cancer. TP53 is the most commonly mutated gene in ovarian cancer as well as in all other cancers. However, there are subsets of ovarian cancer patients with wild-type TP53, and we would like to understand the molecular and cellular mechanisms that disable the transcriptional functions of p53 in those patients. Wild-type p53 is the transcription factor that activates sets of genes to respond to various types of cellular and molecular stress and to maintain genomic stability in normal cells. The major function of wild-type p53 is the transcriptional activation of genes that are involved in cell cycle arrest to repair DNA damage and cell death if the damage is beyond repair in cells. However, the p53 transcriptional functions of cell cycle arrest and cell death are not observed in those ovarian cancer patients with wild-type TP53 and the genes inhibiting transcriptional activities of p53 are not well studied. Additionally, our understanding of negative regulators of p53 in cell activity is not complete, and identifying those regulators may provide new insights into how the p53 pathway can be deregulated in tumors without mutations in TP53. For that purpose, we performed forward genetic screening using a patient-derived pool cDNA library constructed with pRetro-LIB vector in wild-type TP53 ovarian cancer cells to identify the potential negative regulators of p53. First, we tested our method of screening in wild-type TP53 ovarian cancer cells to prove the concept of our experimental approach. We found that using modified 293T cells (Phoenix AMPHO) with spin-fection method overcame the low transfection efficiency of retroviral particles in wild-type TP53 ovarian cancer cells (A2780). In normal cells, p53 is present at relatively low levels because of the negative feedback loop between Mdm2 and p53. To induce the p53 level in ovarian cancer cells with wild-type TP53, we used the small molecule inhibitor, Nutlin-3a. Nutlin-3a blocks the interaction between Mdm2 and p53, thereby allowing the stabilization of p53 in ovarian cancer cells. Stabilized p53 transactivates genes that initiate cell cycle arrest or cell death. Accordingly, Nutlin-3a suppresses the growth of cancer cells with wild-type TP53. We used this system to screen for exogenous genes from the patient-derived cDNA library that block Nutlin-3a-mediated growth suppression in A2780 cancer cells. The screen identified three candidate genes (NIFK, GXYLT 1, and SACS) that prevent the Nutlin-3a-induced growth suppression in A2780 cancer cells, and NIFK (also known as Nucleolar Protein Interacting with the FHA Domain of MKI67 or MKI67IP) was identified in four independent screening experiments. NIFK encodes a protein that interacts with the forkhead-associated domain of the Ki-67 antigen and has been implicated in ribosome biogenesis, e.g., pre-ribosomal RNA processing and ribosome assembly. NIFK also associates with pre-mRNAs. However, the potential association between NIFK and p53 in ovarian cancer is unknown. We evaluated the level of NIFK, p53, and Mdm2 in NIFK-overexpressing ovarian cancer cells. We found that the dose-dependent effect of NIFK expression in preventing the Nutlin-3a-induced growth suppression in A2780 cells. Our results suggest that NIFK overcomes the growth arrest by functional p53 induced by Nutlin-3a, and NIFK negatively regulates p53 pathway in ovarian cancer cells. In summary, our studies showed that NIFK overexpression cells play a role in cell survival, proliferation and the negative regulation of p53 in ovarian cancer cells, and this should be further explored to understand the molecular function of NIFK. | |
