Identification of leucine-rich repeat-containing protein 59 (LRRC59) located in the endoplasmic reticulum as a novel prognostic factor for urothelial carcinoma

Abstract

Background: Urothelial carcinoma (UC) is one of the most common cancers worldwide. The biological heterogeneity of UCs causes considerable difficulties in predicting treatment outcomes and usually leads to clinical mismanagement. The identification of more sensitive and efficient predictive biomarkers is important in the diagnosis and classification of UCs. Herein, we report leucine-rich repeat-containing protein 59 (LRRC59) located in the endoplasmic reticulum as a novel predictive factor and potential therapeutic target for UCs.

Methods: Using whole-slide image analysis in our cohort of 107 UC samples, we performed immunohistochemistry to evaluate the prognostic value of LRRC59 expression in UCs. In vitro experiments using RNAi were conducted to explore the role of LRRC59 in promoting UC cell proliferation and migration.

Results: A significant correlation between LRRC59 and unfavorable prognosis of UCs in our cohort was demonstrated. Subsequent clinical analysis also revealed that elevated expression levels of LRRC59 were significantly associated with higher pathological grades and advanced stages of UC. Subsequently, knockdown of LRRC59 in UM-UC-3 and T24 cells using small interfering RNA significantly inhibited cell proliferation and migration, resulting in cell cycle arrest at the G1 phase. Conversely, the overexpression of LRRC59 in UC cells enhanced cell proliferation and migration. An integrated bioinformatics analysis revealed a significant functional network of LRRC59 involving protein misfolding, ER stress, and ubiquitination. Finally, in vitro experiments demonstrated that LRRC59 modulates ER stress signaling.

Conclusions: LRRC59 expression was significantly correlated with UC prognosis. LRRC59 might not only serve as a novel prognostic biomarker for risk stratification of patients with UC but also exhibit as a potential therapeutic target in UC that warrants further investigation.

Keywords: Endoplasmic reticulum stress; Leucine-rich repeat-containing protein; Prognosis; Ubiquitination; Urothelial carcinoma.

Fig. 1

Screening of unfavorable prognostic genes in UCs. (A) Venn diagram demonstrated the upregulated unfavorable prognostic genes in bladder cancer. (B) Correlation matrix of the upregulated unfavorable prognostic genes. (C) ROC plots for the prediction of the prognosis of bladder cancer in the testing dataset in TCGA/BLCA. (D, E) Survival curves for the comparisons of the high and low expression levels for the LRRC59 and KPNA2 genes identified in UCs.

Fig. 2

Representative images of LRRC59 expression in UC samples. (A) The expression patterns of LRRC59 in the adjacent normal urothelium and tumor tissue within the same sample. Scale bar: 50 um; magnification at 400 × . (B) LRRC59 was upregulated in tumor cells but not in umbrella cells.

Fig. 3

Elevated expression levels of LRRC59 were revealed as clinically relevant. (A) Representative images to indicate IHC staining intensities. Scale bar (top): 100 μm; magnification at 200 × . Scale bar (bottom): 25 μm; magnification at 800 × . (B) The elevated expression levels of LRRC59 were significantly correlated with poorer survival of UCs in our cohort (n = 107). Scale bar: 50 μm; magnification at 400 × . (C) Higher H-score was associated with poorer median survival compared with low H-score from our cohort. (D) Based on H-score, significant differences were found between high-grade UCs and low-grade UCs. (E) Based on H-score, significant differences were found between NMIBC and MIBC. NMIBC, non-muscle-invasive bladder cancer; MIBC, muscle-invasive bladder cancer. ****p < 0.0001.

Fig. 4

Multivariate Cox proportional hazards survival analysis of LRRC59. *p < 0.05. **p < 0.01.

Fig. 5

LRRC59 promoted the proliferation and migration of UC cells. (A, B) LRRC59 was markedly downregulated or upregulated after siLRRC59s or pcDNA3.1-LRRC59 transfection. (C, D) SiRNA knockdown of LRRC59 in T24 and UM-UC-3 cells significantly inhibited cell proliferation in CCK8 assay. (E, F) Overexpression of LRRC59 in T24 and UM-UC-3 cells markedly increased cell proliferation in CCK8 assay. (G, H) Colony-formation assays demonstrated that LRRC59 knockdown dramatically inhibited the size and number of colonies of T24 and UM-UC-3 cells. (I, J) The overexpression of LRRC59 increased the size and number of colonies of T24 and UM-UC-3 cells. (K, L) The silencing of LRRC59 reduced T24 and UM-UC-3 cell migration in the Transwell assay. (M, N) The expression of LRRC59 increased T24 and UM-UC-3 cell migration in the Transwell assay. *p < 0.05; **p < 0.01, ***p < 0.001.

Fig. 6

Enrichment analysis of LRRC59 co-expressed genes in microarray datasets of bladder cancer and flow cytometry analysis of cell cycle. (A) The heatmap of the co-expression network of LRRC59 in bladder cancer microarray datasets. (B) Enrichment analysis of the co-expression genes of LRRC59. (C) Top-ranked enrichment plots in GSEA analysis for the LRRC59-positive correlation pathways. (D, E) The silencing of LRRC59 increased the percentage of G1 phase in T24 cells. (F, G) The silencing of LRRC59 increased the percentage of G1 phase in UM-UC-3 cells.

Fig. 7

Knockdown of LRRC59 induced ER stress and dys-regulation of cell cycle genes in vitro. (A) The expression of HYOU1 was upregulated after tunicamycin (TM) stimulation, and the expression of HYOU1 in both siLRRC59 and TM treatment was higher than TM treatment alone. (B) The expression of XBP1 was upregulated after tunicamycin (TM) stimulation, and the expression of XBP1 in both siLRRC59 and TM treatment was higher than TM treatment alone. (C) The expression of LRRC59 was down-regulated in T24 and UM-UC-3 cells after siLRRC59s transfection. (D, E)The IC₅₀ of cisplatin in LRRC59-downregulated UM-UC-3 cells was decreased compared to negative control. *p < 0.05; **p < 0.01, ***p < 0.001.