The p53-induced Wig-1 protein : identification of mRNA targets and role as a survival factor in development and cancer

Abstract

The tumor suppressor p53 is activated in response to a variety of stress conditions. Upon activation, p53 can trigger apoptosis or cell cycle arrest, or regulate metabolism and other cellular processes by transactivation of its targets genes. Wig-1 (also named ZMAT3) is a p53 target gene, and both Wig-1 mRNA and protein levels increase upon p53 activation. Wig-1 is a zinc finger protein that binds to double strand RNA. It is an AU-rich element (ARE) binding proteins (ARE-BPs) and acts as a regulator of mRNA stability via direct binding to AREs. The Wig-1 gene is localized to the long arm of chromosome 3(3q26), a region that is frequently amplified in cancer.

In this thesis, I aimed to identify new Wig-1 mRNA targets and investigate the biological implications of the regulation of selected targets. I also explored Wig-1 protein expression in tumors as well as its clinical relevance, and determined the role of Wig-1 in mouse development.

In paper I, we performed a microarray analysis of HCT116 colon cancer cells with or without Wig-1 knockdown and identified Wig-1 regulated mRNAs. We also discovered that Wig-1 promotes cell cycle arrest rather than cell death upon cellular stress through regulation of p53 targets FAS and 14-3-3σ. Wig-1 regulates FAS mRNA negatively through binding to 3’UTR AREs in FAS mRNA.

In paper II, we studied Wig-1 expression in cervical carcinoma samples and found that the Wig-1 protein expression pattern in tumors is associated with patient survival. Patients with moderate nuclear Wig-1 staining and positive cytoplasmic Wig-1 staining in their tumors show better survival than patients with high nuclear Wig-1 staining and negative cytoplasmic Wig-1 staining.

In paper III, we showed that Wig-1 null mice embryos die before the blastocyst stage. We also found that Wig-1 knockdown in mouse embryonic stem cells (mESCs) leads to a reduction in proliferation rate. Wig-1 binds to and regulates both c-Myc and N-Myc mRNA in mESCs. Since Myc has essential roles during embryonic development, we suggest that deficient regulation of Myc in absence of Wig-1 may explain the observed embryonic lethality.

In paper IV, we performed RNA-immunoprecipitation (RIP) followed by high throughput RNA sequencing in HCT116 and Saos2 cells. We identified Wig-1 bound mRNAs and found a significant enrichment of mRNAs with AREs in their 3’UTRs as compared to unbound mRNAs.

In summary, this thesis greatly expands theWig-1 targets repertoire and further explores the role of Wig-1 as a survival factor in cell growth and early embryonic development. Our findings also suggest that Wig-1 may serve as a molecular biomarker together with other conventional clinical markers for cervical cancer prognosis.