Hypomethylated RRBP1 Potentiates Tumor Malignancy and Chemoresistance in Upper Tract Urothelial Carcinoma

Abstract

Ribosome-binding protein 1 (RRBP1) is a potential oncogene in several cancer types. However, the correlation between RRBP1 expression and the prognosis of patients with upper tract urothelial carcinoma (UTUC) remains unclear. In this study, we identified that RRBP1 is associated with carcinogenesis and metastasis in UTUC using a methylation profiling microarray. High correlations between RRBP1 and cancer stages, nodal metastasis status, molecular subtypes, and prognosis in bladder urothelial cancer (BLCA) were found. Aberrant DNA methylation in the gene body region of RRBP1 was determined in UTUC tissues by methylation-specific PCR. RRBP1 expression was significantly increased in UTUC tissues and cell lines, as determined by real-time PCR and immunohistochemistry. RRBP1 depletion significantly reduced BFTC909 cell growth induced by specific shRNA. On the other hand, molecular subtype analysis showed that the expression of RRBP1 was associated with genes related to cell proliferation, epithelial-mesenchymal transition, and basal markers. A patient-derived organoid model was established to analyze patients' responses to different drugs. The expression of RRBP1 was related to chemoresistance. Taken together, these results provide the first evidence that RRBP1 gene body hypomethylation predicts RRBP1 high expression in UTUC. The data highlight the importance of RRBP1 in UTUC malignancy and chemotherapeutic tolerance.

Keywords: RRBP1; chemoresistance; hypomethylation; oncogene; patient-derived organoid; upper tract urothelial carcinoma.

Figure 1

Methylation profiling analysis identified RRBP1 as an oncogene in UTUC. (A,B) The diagram shows the process of analyzing the DNA methylation profiles of normal and UTUC tumors. The DNA methylation was determined in UTUC tumor tissues from high-stage/high-grade patients and morphologically normal adjacent tissues (NAT) from low-stage/low-grade patients. The analysis of DNA methylation was conducted using a methyl-CpG binding-domain-based (MBD) protein microarray chip. (C–F) Gene oncology (GO) analysis of gene expression in molecular function, biological process, and cellular component. (G) Statistical analysis of gene expression in cellular component. (H) Venn diagram of RRBP1 satisfies the intersection conditions of hypomethylation, upregulated genes, poor survival, and cellular membrane component of GO analysis. Hypomethylation genes (133), upregulation genes (61), high expression with poor survival (5), and GO analysis: membrane (1473).

Figure 2

RRBP1 is hypomethylated in BLCA and is correlated with tumor malignancy. (A) High expression of RRBP1 transcripts in BLCA tumors. (B) High expression of RRBP1 is associated with lower survival rate of BLCA patients (log-rank test, p = 0.013). (C) High expression of RRBP1 in individual cancer stages of BLCA. (D) High expression of RRBP1 in metastatic nodal. (E) High expression of RRBP in basal squamous and luminal-infiltrated subtypes. (F) Low level of methylation in RRBP1 promoter. (* p < 0.05; ** p < 0.01; *** p < 0.001.) Student t-test with two-tailed p-value.

Figure 3

Low methylation of RRBP1 in tumors of UTUC. (A) RRBP1 is encoded on chromosome 20. Predicted CpG islands of RRBP1 in the region of exon 1 to intron 3 (chromosome GRCh38:17,613,678-17,682,283) are represented. Illustration represents the design of the methylation-specific PCR assay and the position of the primer sets in CpG islands. (B) Low methylation in CpG island of RRBP1 in N/T pairs of UTUC tumors. (** p < 0.01.) Student t-test with two-tailed p-value. (C) Methylation intensity of RRBP1 in N/T pairs of UTUC tumors. (** p < 0.01.) Paired t-test with two-tailed p-value. (D) Sequence of predicted primer sets for site 2 CpG island. (E) Differential level of methylation in site 2 CpG islands of RRBP1 from N/T pairs of UTUC. (F) Quantification of methylation in site 2 CpG islands of RRBP1 from N/T pairs of UTUC. (** p < 0.01; *** p < 0.001.) Statistical analysis was performed by using two-way ANOVA with Tukey’s multiple comparison test.

Figure 4

High expression of RRBP1 in tumors from patients with UTUC. (A) Levels of RRBP1 mRNA are higher than nontumor in UTUC. The mRNA expression of RRBP1 was analyzed based on the unlinked database from National Health Institute. (** p < 0.01.) Student t-test with two-tailed p-value. (B) High expression of RRBP1 in tissues from UTUC tumors. Tissues of nontumor and tumor were fixed and embedded in paraffin. Immunohistochemistry staining of tissue sections was performed with RRBP1 antibody. (C) Analysis of survival within five years according to the immunohistochemistry staining in tumor tissue microarray (n = 197). (* p < 0.05.) Log-rank test. (D) Relative expression of RRBP1 in cell lines. mRNA of SV-HUV1, J82, T24, and BFTC909 cells were collected for quantitative PCR assay targeting RRBP1 (** p < 0.01; *** p < 0.001). (E) Protein expression of RRBP1 in cell lines. Cell lysates were harvested and protein expression was detected by Western blotting assay with RRBP1 antibody. (F) Silencing of RRBP1 in BFTC909 cell. BFTC909 cells were transfected with shRNA for RRBP1#1 and RRBP1#2. (G) The mRNA was collected for quantitative PCR analysis targeting RRBP1 (RPL37A: internal control) (** p < 0.01). (H,I) Silencing of RRBP1 reduced cell proliferation. BFTC909-shLuc, shRRBP1#1, and shRRBP1#2 cells were seeded in a 96-well plate and cultured for 24, 48, and 72 h. The cell number was counted or determined by the WST-1 assay (** p < 0.01). (J–L) Silencing of RRBP1 reduced cell migration and invasion. BFTC909-shLuc, shRRBP1#1, and shRRBP1#2 cells were seeded in the Transwell inserts, and cell migration or invasion was performed with incubation at 37 °C for 18 h. The crystal-violet-stained cells were lyzed and analyzed (** p < 0.01; *** p < 0.001.). Statistical analysis was performed by using one-way ANOVA with Dunnett’s test. Scale bar = 100 µm.

Figure 5

Analysis of mRNA expression of molecular subtypes according to the level of RRBP1 in UTUC. Heatmap analysis of mRNA expression represented a high correlation between RRBP1 and chemotherapy. RNA expression of molecular subtypes was analyzed according to the level of RRBP1. Ten samples were analyzed using the unlinked database from the National Health Institute. (A) Clustering of genes for the proliferation and EMT biomarkers. (B) Clustering of genes for the basal and luminal biomarkers. (C) Clustering of genes for bladder-cancer-associated and YAP1-mediated chemoresistant genes.

Figure 6

RRBP1 shows potential for drug combination therapy. (A) UTUC patient-derived organoid culture system mimics the cell morphology similar to the parental tumor. The cells were extracted from the patient’s tumor and then embedded in Matrigel to establish organoid cultures. HE staining was used to detect sections from the parental tumor and organoid. Scale bar = 100 µm. (B) Representative images for the four primary UTUC organoids. The organoids were constructed from the patient’s tumor and cultured for two weeks. Scale bar = 100 µm. (C) The organoids were treated with 100 µM drugs for 7 days. Scale bar = 100 µm. (D) The organoids were seeded overnight and then treated with drugs for 7 days. The viability of organoids was determined by the 3D organoid viability assay. (E) The levels of RRBP1 mRNA in three organoids were measured. Data represent the mean ± SD. (F–H) The level of RRBP1 mRNA was correlated to the cytotoxicity effect of drugs in organoids. The organoids were treated with 100 µM cisplatin, gemcitabine, and 0.8 µM epirubicin for 7 days, and then the viability was determined. Data represent the mean ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001.) Statistical analysis was performed by using one-way ANOVA with Dunnett’s test.