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

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.

List of scientific papers

I. Cinzia Bersani, Li-Di Xu, Anna Vilborg, Weng-Onn Liu, Klas G. Wiman. Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ. Oncogene. 2014 Aug 28; 33(35): 4407-17.
https://doi.org/10.1038/onc.2013.594

II. Li-Di Xu, Susanne Muller, Srinivasan R.Thoppe, Fredrik Hellborg, Lena Kanter, Mikael Lerner, Biying Zheng, Svetlana Bajalica Lagercrantz, Dan Grander, Keng Ling Wallin, Klas G. Wiman, Catharina Larsson, Sonia Andersson. Expression of the p53 target Wig-1 is associated with HPV status and patient survival in cervical carcinoma. PLoS ONE. 2014 Nov 7; 9 (11): e111125.
https://doi.org/10.1371/journal.pone.0111125

III. Li-Di Xu, Anna Vilborg, Sophia Ceder, Cinzia Bersani, Björn Rozell, Klas G. Wiman, Margareta T. Wilhelm. Complete lack of Wig-1 leads to embryonic lethality before the blastocyst stage. [Manuscript]

IV. Cinzia Bersani, Mikael Huss, Stefania Giacomello, Li-Di Xu, Julie Bianchi, Sofi Eriksson, Fredrik Jerhammar, Anna Vilborg, Andrey Alexeyenko, Weng-Onn Liu, Klas G. Wiman. Genome-wide identification of Wig-1 mRNA targets by RIP-Seq analysis. Oncotarget. [Submitted]