Recurrent, chemotherapy-resistant epithelial ovarian cancer (EOC) accounts for the most lethality among gynecologic cancers. Unfortunately, during the past 30 years, patients with EOC have experienced little improvement in overall survival, and standard treatment has not advanced beyond PT-based combination chemotherapy. Recent literature suggests that molecular and phenotypic associations exist between chemoresistance and the establishment of an epithelial-to-mesenchymal transition (EMT)-like program, by regulating the cancer cell plasticity and the tumor spreading. How this phenotype is linked to an alteration of the DNA damage response (DDR) activated by chemotherapy is still unclear and the possible molecular mediators connecting EMT and DDR are still undisclosed.
Using an unbiased screening with shRNAs targeting genes belonging to apoptosis and DDR pathways, we identified ubiquitin specific protease-1 (USP1) as a key mediator of PT sensitivity in EOC. USP1 is a de-ubiquitinase previously implicated in the control of both DDR and EMT via the de-ubiquitination of FANCD2-FANCI and ID proteins, respectively. Here we report that USP1 impairment, using both genetic and pharmacologic approaches, significantly reduced PT-IC50 of different EOC cells encompassing the three most common histotypes (i.e., serous COV-362 and OVCAR8, endometrioid MDAH-2774,and clear cell TOV-21G). Moreover, USP1 regulates expression of several differentiation status-associated genes and controls spheroids formation in both untreated and PT-treated cells, the evasion from 3D-matrix, and the invasion of the mesothelial cell monolayer.
Mechanistically, we identify the EMT transcription factor SNAIL as a new USP1 substrate.
We demonstrated that upon PT treatment, USP1 is phosphorylated by the ATM and ATR kinases and this modification favors the binding of USP1 to SNAIL. USP1, by directly de-ubiquitinating SNAIL, stabilizes its expression and confers higher cell plasticity and resistance to PT-induced cell death. Accordingly, CRISPR-mediated USP1 knockout (KO) or its pharmacologic inhibition strongly decreased the ability of EOC cells to metastasize within the abdominal cavity and increased in vivo PT-induced cell death. Reintroduction of USP1 wild-type protein in USP1 KO cells restored their ability to spread in the abdomen and to resist PT therapy. Conversely, a USP1 mutant not phosphorylable by ATM or ATR failed to properly grow in the abdomen and was highly sensitive to PT treatment.
Finally, we demonstrated that the ability of USP1 to regulate SNAIL is associated with a transcriptional program able to predict worse prognosis of EOC patients.
Overall our work unveils several new relevant insights in the regulation of PT resistance in EOC. First, we report for the first time that a DUB is directly involved in the stabilization of an EMT transcription factor (i.e., SNAIL). Then we demonstrated that USP1 for its ability to link at molecular level EMT and DDR represents a particularly powerful oncoprotein that affects drug response and metastatic dissemination. Finally, we provide evidence that genetic and/or pharmacologic inhibition of USP1 could represent a promising strategy that might positively impact the management of EOC patients.
Citation Format: Maura Sonego, Ilenia Pellarin, Alice Costa, Gian Luca Rampioni Vinciguerra, Michela Coan, Sara D’Andrea, Yohann Coutè, Riccardo Spizzo, Andrea Vecchione, Barbara Belletti, Monica Schiappacassi, Gustavo Baldassarre. Stabilization of SNAIL by USP-1 mediates chemoresistance and cell plasticity in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A68.