Clinical significance of elevated UBQLN4 expression with resistance to cisplatin in intrahepatic cholangiocarcinoma

Backgrounds & Aims: Intrahepatic cholangiocarcinoma (iCCA) is a devastating biliary tract cancer increasing in incidence worldwide with limited treatment options. We previously identified ubiquilin-4 ( UBQLN4 ) was associated with prognosis and chemotherapy response in breast and esophageal cancer. UBQLN4 is a ubiquitinated-binding protein which inhibits homologous recombination repair while promoting non-homologous end-joining repair in DNA damage repair. The objective of this study is to determine the clinical utility of UBQLN4 , as a potential prognostic biomarker associated with iCCA clinical outcomes. Methods: The public datasets from TCGA-CHOL (n=32), DepMap, and Fu-iCCA cohort (n=238), GSE107943 (n=30), and GSE32225 (n=148) were assessed for copy number variation (CNV) and/or mRNA expression analysis of UBQLN4 located on 1q22 chromosome. As validation, an independent cohort of iCCA patients (n=66) were analyzed for clinical significance of UBQLN4 . We finally investigated in-vitro analysis to evaluate the molecular features and cisplatin-resistance (CR) mechanism of UBQLN4. Results: GISTIC analysis of TCGA-CHOL dataset showed that 1q22 including UBQLN4 is one of the highest amplifications in all chromosomes. There is high correlation between CNV and mRNA expression of UBQLN4 gene in iCCA tissues from TCGA-CHOL, DepMap, and FuiCCA cohort. UBQLN4 was also found to be upregulated in iCCA tissues compared to normal liver tissues or biliary epithelium from TCGA-CHOL, GSE107943 and GSE32225 datasets. In an independent iCCA validation cohort, UBQLN4 mRNA upregulation was significantly associated with poor recurrence-free survival ( p = 0.025). UBQLN4 mRNA high expression was an independent recurrence risk factor in a multivariate cox regression analysis (Hazard risk = 2.26, 95% confidence interval 1.00 - 5.11, p = 0.048). In in-vitro using iCCA cell lines (RBE and HCCC9810), knockdown of UBQLN4 lead to high sensitivity for cisplatin compared to controls; however, CR-iCCA cell lines showed high UBQLN4 expression concomitant with increased phosphorylated ATM (pATM). Moreover, 3D spheroid assay revealed UBQLN4 and pATM are expressed significantly higher in CR-iCCA cell lines than respective parental cell lines. In clinically annotated iCCA specimens, non-responders to neoadjuvant chemotherapy including cisplatin had increased UBQLN4 and pATM protein levels in iCCA tissues as assessed by multiplex immunofluorescence, supporting that FuiCCA cohorts showed high UBQLN4 and pATM protein levels lead to worse prognosis (p=0.049). Conclusions: Upregulated UBQLN4 expression correlates with postoperative recurrence as well as resistance to cisplatin in iCCA patients, indicating that UBQLN4 is a strong prognostic biomarker related to cisplatin treatment of iCCA patients.