Abstract 2869: FLASH-dependent post-translational regulation of the EMT activator ZEB1

Understanding the mechanisms of metastasis initiation and tumor persistence despite aggressive treatments is paramount to developing new viable therapeutic targets. Critical to tumor progression is initiation of epithelial-to-mesenchymal transition (EMT), a cellular process characterized by loss of E-cadherin, a critical cell-cell adhesion molecule, gain of migratory properties and chemoresistance. Transcriptional repression of E-cadherin and other genes responsible for maintaining the epithelial phenotype by ZEB1/ZEB2 family is one of the most significant events occurring at the interface between non-metastatic/metastatic cancer and chemo-sensitive/chemo-resistant cancer. Using a novel interdisciplinary high-throughput screen methodology we uncovered novel regulators of E-cadherin. Caspase 8 - associated protein 2 (CASP8AP2 or FLASH) a protein involved in apoptosis and transcriptional control was identified through this screen and we discovered that FLASH depletion resulted in increased E-cadherin levels at the plasma membrane through loss of ZEB1 expression. Subsequent examination of ZEB1 protein expression revealed that ZEB1 is rapidly degraded in FLASH-depleted cells. This process is blocked by proteasome inhibitors suggesting that post-translational modifications (PTMs) and in particular ubiquitination of ZEB1 is responsible for its fast turnover and decreased protein expression in the absence of FLASH. Indeed, ubiquitinated ZEB1 was detected in FLASH-depleted cells in the presence of proteasome inhibitors. Although ZEB1 is degraded through the ubiquitin pathway, its rapid turnover in the absence of FLASH suggests that protective mechanisms are in place to delay and/or prevent this process. Even more, we determined that the atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 is involved in ZEB1 degradation in the absence of FLASH and is upregulated in cells depleted for FLASH. Altogether our data suggests that FLASH can stabilizes ZEB1 indirectly by transcriptional repression of the ubiquitin ligase responsible for its degradation and directly by binding to ZEB1 and masking the recognition site of the ubiquitin ligase. Even more, absence of FLASH reprograms the cells into a more epithelial-like phenotype which is less invasive and is resistant to TGFβ-induced loss of E-cadherin. Not only is FLASH-dependent post-translational regulation of ZEB1 novel, but is also conserved in multiple cells lines from various tissues, making this mechanism an original therapeutic target for cancer metastasis. Citation Format: Camille Abshire, Ana Maria Dragoi. FLASH-dependent post-translational regulation of the EMT activator ZEB1. [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 2869.