Abstract LB-352: p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells
Authors: Yang-Hartwich Yang , Tedja Roslyn , Bingham Jamie , Gurrea Soteras Marta , Alvero Ayesha B. , Mor Gil 
Published: July 2016

Background: Tumor metastasis is the primary cause of mortality in patients with advanced ovarian cancer. The molecular mechanism behind ovarian cancer metastasis is still not clear. Twist1 is associated with the increased ability of migration and tumor-initiation of ovarian cancer cells; therefore, understanding the regulation of Twist is critical for the prevention of metastatic disease. Previous studies have shown an interaction between Twist1 and the tumor suppressor p53. We propose that p53 functions as a normal inhibitor of Twist1. We hypothesize that through their interaction, p53 promotes the proteasome-dependent degradation of Twist1 and consequently inhibits epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer cells. p53 mutations, which are common in advanced ovarian cancer patients, may impair p53's ability to inhibit Twist1, leading to enhanced EMT and metastasis.

Method: p53, Twist1, Pirh2, and Ubiquitin were overexpressed in HEK293T or epithelial ovarian cancer (EOC) cells by transfection. Co-immunoprecipitation 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.

Result: Wild type p53 overexpression reduces Twist1 protein level and enhances the ubiquitination of Twist1 without affecting Twist1 mRNA level. Mutant p53 (R175H, R148W, and R273H) failed to regulate Twist1 degradation. E3 ligase, Pirh2, forms complex with p53 and Twist1 to induce Twist1 degradation. In EOC cells, Twist1 is suppressed by proteasome-dependent degradation. During in vitro EMT, Twist1 and p53 protein levels negatively correlated. 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. The 9 samples with high levels of wild type p53 all showed low or no Twist1 expression.

Conclusion: Our data revealed a mechanism by which a crucial EMT inducer, Twist1, is regulated by wild type p53 through enhancing its proteasome-dependent degradation. p53 promotes the formation of a Twist1-p53-Pirh2 complex and facilitates 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. p53-promoted Twist1 degradation inhibits EMT in ovarian cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-352.