Abstract AP16: THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS

BACKGROUND: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism underlying metastasis is still not clear. Twist1 is a transcriptional factor that promotes epithelial-mesenchymal transition (EMT) and enhances the migration and tumor-initiation of ovarian cancer cells. It also has been associated with cancer stem cells and chemoresistance. The purpose of our study is to better understand the regulation of Twist1 and to identify molecular pathways that can be targeted in order to control metastatic diseases. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We hypothesize that through their interaction, p53 promotes the proteasomedependent degradation of Twist1 and consequently inhibits EMT in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p539s ability to inhibit Twist1, leading to enhanced EMT and metastasis. METHOD: In ubiquitin assay p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Coimmunoprecipitation and western blotting were used to detect protein interaction and ubiquitination. Protein lysate were prepared from 25 tumor samples (12 ovarian tumor, 10 ovarian tumor metastases, and 2 fallopian tube tumors) for western blotting. Plasmid constructs containing different Twist1 mutants were created to select the degradation resistant mutation. ΔC-Twist1 (C-terminal deletion) was used in in vivo study. RESULT: 1) In EOC cells, proteasome-dependent degradation suppressed Twist1 protein. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. In ovarian cancer cells, wild type p53 overexpression reduced Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to enhance Twist1 degradation. E3 ligase, Pirh2, formed complex with p53 and Twist1 to induce Twist1 degradation. 2) ΔC-Twist1 was resistant to p53-regulated degradation. In xenograft mouse model, EOC cells overexpressing ΔC-Twist1 could form more aggressive tumors with increased numbers of metastatic lesions than wild type Twist1 group. 3) In 25 ovarian tumor samples, 11 tumors with high levels of Twist1 had either a p53 mutation or very low levels of wild type p53. Nine samples with high levels of wild type p53 all showed low or no Twist1 expression. CONCLUSION: Our data revealed a mechanism by which Twist1 is regulated through p53-promoted proteasome-dependent degradation. p53 facilitates the formation of a Twist1-p53-Pirh2 complex and the Pirh2-mediated Twist1 degradation. Our data also demonstrated that three hotspot p53 mutants failed to promote Twist1 degradation, leading to the stabilization of Twist1 and the induction of EMT. This mechanism may be critical for controlling metastasis of ovarian tumors. The newly discovered role of Pirh2 as an E3 ligase in mediating Twist1 ubiquitination and degradation has expanded the known spectrum of Pirh2 function. In addition to providing new insights into metastatic process at the molecular and cellular levels, our data suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to inhibit Twist1 and control ovarian cancer metastasis. Citation Format: Yang Yang-Hartwich, Roslyn Tedja, Jamie Bingham, Marta Gurrea Soteras, Ayesha B. Alvero, Gil Mor. THE CRITICAL ROLE OF P53–TWIST1 AXIS IN OVARIAN CANCER METASTASIS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP16.