High-grade serous ovarian cancer (HGSOC) is characterized by chromosomal instability, DNA damage, oxidative stress, and high metabolic demand that exacerbate misfolded, unfolded, and damaged protein burden resulting in increased proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. This study reports that the neuronal deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), is overexpressed in HGSOC and maintains protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions). High UCHL1 levels correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced HGSOC cell proliferation and invasion, as well as significantly decreased the in vivo metastatic growth of ovarian cancer xenografts. Transcriptional profiling of UCHL1-silenced HGSOC cells revealed downregulation of genes implicated with proteasome activity along with upregulation of endoplasmic reticulum stress-induced genes. Reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH), upon silencing of UCHL1, resulted in a significant decrease in proteasome activity, impaired protein degradation, and abrogated HGSOC growth. Furthermore, the accumulation of polyubiquitinated proteins in the UCHL1silenced cells led to attenuation of mTORC1 activity and protein synthesis, and induction of terminal unfolded protein response. Collectively, these results indicate that UCHL1 promotes HGSOC growth by mediating protein homeostasis through the PSMA7-APEH-proteasome axis.Implications: This study identifies the novel links in the proteostasis network to target protein homeostasis in HGSOC and recognizes the potential of inhibiting UCHL1 and APEH to sensitize cancer cells to proteotoxic stress in solid tumors.

High-grade serous ovarian cancer (HGSOC) is characterized by genomic instability, DNA damage, oxidative stress, and high metabolic demand that contribute to misfolded proteins and proteotoxicity. However, the underlying mechanisms that maintain protein homeostasis to promote HGSOC growth remain poorly understood. In this study, we report that the neuronal deubiquitinating enzyme, UCHL1 (ubiquitin C-terminal hydrolase L1) is overexpressed in HGSOC and regulates protein homeostasis. UCHL1 expression was markedly increased in HGSOC patient tumors and serous tubal intraepithelial carcinoma (HGSOC precursor lesions) and correlated with higher tumor grade and poor patient survival. UCHL1 inhibition reduced proliferation and invasion of HGSOC cells as well as significantly reduced the in vivo metastatic growth of the ovarian cancer xenografts. Transcriptional profiling of UCHL1 silenced HGSOC cells revealed down-regulation of genes implicated with proteasome activity along with the upregulation of endoplasmic reticulum (ER) stress-mediated genes. Silencing UCHL1 resulted in reduced expression of proteasome subunit alpha 7 (PSMA7) and acylaminoacyl peptide hydrolase (APEH) leading to a decrease in proteasome activity, accumulation of polyubiquitinated proteins, and reduced mTORC1 activity and protein synthesis. This induced ER-stress mediated pro-apoptotic factors, including ATF3 (activating transcription factor 3). In addition, the growth of HGSOC cells was significantly reduced upon silencing PSMA7 and APEH or inhibiting proteasome activity. Together, these results indicate that the UCHL1-PSMA7-APEH axis mediates the degradation of misfolded proteins, salvage amino acids for protein synthesis, and maintain protein homeostasis. This study highlights protein homeostasis as a therapeutic vulnerability in HGSOC and identifies UCHL1 and APEH inhibitors as novel therapeutic strategies.

High-grade serous ovarian cancer (HGSOC) is the most lethal subtype of ovarian cancer with high case-to-fatality ratio. However, not much advancement has been seen in the field in terms of novel adjuvant therapies. Somatic or germline mutations in HGSOC along with copy number variations results in intratumoral genetic heterogeneity, which leads to tumor growth and development of chemoresistant clones and treatment resistance. Moreover, molecular heterogeneity also leads to the accumulation of aberrant peptides and protein complexes with altered stoichiometry, causing extensive proteotoxic stress in cancer cells and adding to the known misfolded proteins stress in cancer cells due to enhanced protein synthesis, thus making these cancer cells more dependent on the protein clearance mechanisms. Not much is known about the molecular targets from the proteostasis network and their therapeutic potential in HGSOC. Here we explore the regulation and role of different components of ubiquitin proteasome system (UPS) in HGSOC and investigate the therapeutic effect of small-molecule inhibitors of UPS. We identified overexpression of deubiquitinating enzyme, ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in HGSOC, and high UCHL1 levels were correlated with the disease stage, tumor grade, and poor clinical outcome. UCHL1 overexpression in HGSOC was driven by mutant p53-mediated H3K4 trimethylation at the UCHL1 regulatory region. Mutant p53-UCHL1 axis transcriptionally promoted the expression of proteasomal subunit alpha 7 (PSMA7) and acylaminoacyl-peptide hydrolase (APEH), resulting in increased proteasome activity in HGSOC. Silencing UCHL1 or inhibiting proteasome activity in vitro and in vivo remarkably reduced HGSOC growth. Together, these results highlight the novel mechanisms of regulation of proteostasis in HGSOC and potential therapies targeting regulators of proteasome pathway. Citation Format: Kinzie Lighty, Yonghyun Shin, Fahmi Mesmer, Harikrishna Nakshatri, Marcin Iwanicki, Sumegha Mitra. Targeting new links in the proteostasis network as novel therapies in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A54.