Abstract P4-01-11: UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation
Authors: Shoji Yoshiaki , Yokoe Takamichi , Bostick Peter J , Shiloh Yosef , Hoon Dave SB , Bustos Matias A 
Published: March 2022

Abstract
Background: Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer (BC) subtype. Cisplatin is one of the broad FDA-approved drugs to treat TNBC patients with recurrent and unresectable disease. However the treatment response is limited, and patients frequently develop resistance and recur locally and distantly. The aim of this study is to find molecular mechanisms driving cisplatin-resistance in TNBC. The ubiquitin proteasomal system (UPS) is a universal and conserved mechanism that helps to maintain cellular homeostasis. The aberrant expression of certain components of the UPS have been associated with cancer onset, progression, and metastasis as we and other groups have shown. UBQLN4 is part of the UPS and play a major role in controlling ubiquitinated proteins in response to genotoxic stress. BCL2-associated athanogene-6 (BAG6), alternately known as BAT3, is a chaperone that complex with other chaperones and ubiquitin ligases to regulate protein stability and insertion of tail-anchored membrane proteins into the endoplasmic reticulum. This is a critical step that reduce mislocalized proteins and proteotoxic stress. We hypothesized that UBQLN4 upregulation determine cisplatin-resistance in BC patients by targeting BAT3 for proteasomal degradation to reduce proteotoxic stress. Methods: TCGA BRCA database was utilized to determine clinical associations and outcomes in BC patients. A BC tissue microarray (TMA) was stained by IHC and protein levels quantified to confirm associations between UBQLN4 and TNBC patients. TNBC cell lines were CRISPR-engineered to generate UBQLN4 knockout (KO), and lentivirus transduction was used to recover the KO phenotype. Functional assays and confocal imaging were performed in BRCA mutant and wildtype human TNBC cell lines. Results: Increased UBQLN4 mRNA expression was observed in the TCGA BRCA database in primary BC tissues compared to normal adjacent tissues (p&lt;0.0001). Using PAM50 classification, the BC patients were stratified and compared for UBQLN4 expression. BC patients with basal-like tumors showed significantly higher UBQLN4 expression than normal-like subtype (p&lt; 0.0001). In the assessment of 5-years relapse-free survival (RFS), patients with high levels of UBQLN4 (n=221) showed significantly lower RFS rates compared to those with low UBQLN4 (n=221) (HR=1.67(1.21-2.3), log-rank test p&lt;0.0016). These results were further evaluated by IHC using a BC TMA. Patients with TNBC showed a significantly increased UBQLN4 H-scores values compared to normal adjacent breast tissues. KO of UBQLN4 significantly increased cisplatin sensitivity in TNBC cell lines (p&lt;0.0001). UBQLN4 overexpression restored the cisplatin-resistance in UBQLN4-KO cell line. In TGCA BRCA database, BAT3 was significantly upregulated in BC patients with basal-like tumors compared to normal-like subtype. Knockdown of BAT3 significantly increased cisplatin sensitivity in TNBC cell lines (p&lt;0.0001). In TCGA BRCA database, BAT3 and UBQLN4 mRNA levels positively correlated (r= 0.26, p&lt;0.0001). By using co-immunoprecipitation, endogenous BAT3 and UBQLN4 proteins interact in parental TNBC cell line, but only during cisplatin-treatment. These results were further validated using reciprocal co-immunoprecipitation and confocal imaging in TNBC cell lines with UBQLN4 overexpression. Conclusions: UBQLN4 expression controls cisplatin-resistance in TNBC cell lines. UBLQN4 interacts with BAT3 under cisplatin treatment. During cisplatin treatment, UBQLN4 targets BAT3 for proteasomal degradation to reduce proteotoxic stress induced by genotoxic cisplatin treatment. The UBQLN4 protein levels may represent a prognostic biomarker to predict RFS in TNBC patients.
Citation Format: Yoshiaki Shoji, Takamichi Yokoe, Peter J Bostick, Yosef Shiloh, Dave SB Hoon, Matias A Bustos. UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-11.