Abstract 2595: Deubiquitinase USP9X inhibition induces apoptosis in metastatic breast cancer cells

Abstract

Abstract Highly metastatic Triple Negative (ER/PR and HER2 negative) breast cancer (TNBC) comprises 20% of all breast cancers but accounts for a disproportionate share of mortality due to its aggressive nature and limited therapeutic options. Thus novel metastatic breast cancer targets and inhibitors need to be defined. Usp9x is a deubiquitinase that has been shown to regulate the stability and function of many signaling, cell cycle regulatory and survival proteins and has emerged as a negative prognosticator and potential therapeutic target in several cancers. Usp9x functions in polarization and survival of tumor cells derived from various sites of origin. Additional studies have shown that Usp9x expression is required to maintain growth of glioblastoma and medulloblastomas and it suppresses the apoptotic effects of targeted-, chemo- and radio-therapy in a broad spectrum of tumors. Previous studies also demonstrated that Usp9x and Mcl-1 protein levels are highly elevated in ductal breast carcinoma when compared to normal tissue. Thus, we hypothesized that Usp9x may play a role in tumor and metastasis promoting functions in breast cancer. We first assessed Usp9x activity and total protein expression in a panel of benign (HME, MCF10A), tumorigenic (MCF7 and T47D) and highly metastatic (SUM149 and MDAMB231) human breast cell lines and found up-regulation of Usp9x enzymatic activity and total protein in tumorigenic and metastatic breast cancers as compared to benign breast cells. Increased Usp9x expression also correlated with increased levels of Mcl-1. Knockdown (KD) of Usp9x in four TNBC cell lines [MDAMB231, MDAMB436, BT549 and SUM159] triggered phenotypic apoptotic events like membrane blebbing and nuclear fragmentation, which were further confirmed by the presence of activated caspases, cleaved PARP and reduced Mcl-1 protein expression. Importantly Usp9x KD specifically induced apoptosis in metastatic breast cancer cells with a mesenchymal stem cell-like phenotype (EpCAM+/CD24-/CD44+ population of cells) and fully suppressed TNBC colony growth and acinar structures when grown on Matrigel. Usp9x KD in tumorigenic MCF7 cells resulted in growth inhibition with G2 arrest, without evidence of apoptosis, whereas Usp9x KD had no effect on the growth or survival of benign breast cells. To examine the therapeutic potential of Usp9x inhibitors in breast cancer we employed EOAI3402143 (G9), a small molecule Usp9x inhibitor developed in our laboratory with efficacy in myeloma tumor regression in mice. G9 inhibited Usp9x activity, reduced Mcl-1 levels and induced apoptosis in TNBC cells within 4h of inhibitor treatment. G9 also phenocopied the effect of Usp9x KD in 3D culture with nM activity. Collectively, our data shows that Usp9x plays a role in breast tumor growth and survival and its inhibition in tumorigenic and highly metastatic breast cancers may provide a novel therapy for the treatment of metastatic breast cancer. Citation Format: Anupama Pal, Ming Luo, Max Wicha, Moshe Talpaz, Nicholas Donato. Deubiquitinase USP9X inhibition induces apoptosis in metastatic breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2595. doi:10.1158/1538-7445.AM2014-2595