CTSV (cathepsin V) promotes bladder cancer progression by increasing NF-κB activity

Abstract

Chronic inflammation is positively associated with the development of urinary bladder cancer. However, its detailed regulatory mechanism remains elusive. The quantitative real-time polymerase chain reaction was used to measure mRNA levels of relative genes. The protein levels were monitored by western blotting. Cell proliferation and viability were evaluated by the cell counting Kit 8 (CCK8) and colony formation assays, respectively. The dual-luciferase reporter assay was performed to assay the transcriptional activity. In vivo experiments were implemented in nude mice as well. The TCGA database analysis suggested that the aberrant expression of cathepsin V (CTSV) was related to a poor outcome in bladder cancer patients. CTSV boosted the inflammation reaction, which facilitated the development of bladder cancer. The overexpression of CTSV increased the proliferation and viability of bladder cancer cells. On the contrary, the deletion of CTSV significantly inhibited the proliferation and viability of bladder cancer cells. The tumor repression resulting from CTSV deficiency in vitro was also verified in vivo. Moreover, multiple cancer-associated luciferase screening showed that the overexpression of CTSV triggered the inflammatory signaling pathway, which could be restored by introducing the NF-κB inhibitor. CTSV is upregulated and promotes proliferation through the NF-κB pathway in bladder cancer and may be a potential target in inflammation-associated bladder cancer.

Keywords: CTSV; NF-κB; bladder cancer; inflammation.

Graphical abstract

Figure 1.

Aberrant expression of CTSV in bladder cancer. A. The relative mRNA expression of CTSV in the bladder cancer tissues (n = 404) and normal tissues (n = 26). B. A Kaplan-Meier survival curve showing a significant association between the high levels of CTSV and poor overall survival of bladder cancer patients (p = 0.013). Low CTSV (n = 101) and high CTSV (n = 101). C. A Kaplan-Meier survival curve showing a significant association between the high levels of CTSV and poor survival in disease-free bladder cancer patients (p = 0.0086). Low CTSV (n = 101) and high CTSV (n = 101). Statistical significance was determined by Student’s t-test. *, p < 0.01.

Figure 2.

Overexpression of CTSV facilitates bladder cancer cell viability. A and B. The qPCR and western blotting of the relative CTSV mRNA expression and protein levels, respectively in different bladder cancer cell lines, including EJ, UMUC3, 5637, and T24. UMUC3 was used to construct the CTSV overexpression cell line. C. The CTSV expression in UMUC3 cell lines tested by immunoblotting using an anti-Flag antibody. D. Colony formation assay performed both in wild-type cells and CTSV overexpression stable cell line with quantification of the colony numbers. E. Cell growth curve assay performed in wild-type cells and CTSV overexpression stable cell line. The relative cell number was measured at 450 nm using the CCK8 method on the indicated days. Error bars and mean ± SD obtained from three replicates. Statistical significance was determined by Student’s t-test or one-way ANOVA, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Figure 3.

CTSV deficiency curbed in vivo xenograft tumor growth of T24 cells. CTSV-deficient T24 cells or the negative control were implanted into the right flank of each nude mouse to generate tumors. A. The photography of xenograft tumors after 31 days subcutaneous implantation of the T24 cells. B. CTSV expression in xenograft tumor tissues was detected by western blot. C.Weight of xenografted tumors at 31 days post-transplantation. D. Subcutaneous tumor growth curves. ****, p < 0.0001.

Figure 4.

CTSV knockout represses tumorigenesis in vitro. A. CTSV gene inactivation in T24 bladder cancer cell line via CRISPR/Cas9 mediated somatic cell knockout method. The expression of CTSV protein in wild-type and knockout cells was determined by immunoblotting. B. Colony formation assay performed in wild-type cells and targeted knockout CTSV cells and the quantification of their colony numbers. C. Cell growth curve assay performed in wild-type cells and targeted knockout CTSV cells. The relative cell number was measured at 450 nm by the CCK8 method on the indicated days. Error bars and mean ± SD obtained from three replicates. Statistical significance was determined by Student’s t-test or one-way ANOVA, **, p < 0.05, **, p < 0.01.

Figure 5.

CTSV resulting in the NF-κB signaling pathway. A. 293 T cells were transfected using 200 ng of NF-κB luciferase reporter plasmid along with the CTSV expression plasmid in different doses or equivalent empty vector. Whole-cell extracts were used for the luciferase assay. B. NF-κB luciferase reporter plasmid was introduced into CTSV-deficient cells. Whole-cell extracts were used for the luciferase assay. C. qPCR analysis of the relative IκBα in wild-type and targeted knocked-out CTSV-T24 cells treated with TNFα (20 ng/ml, abclone, cat#: RP00001) at different times. D. RT-qPCR analysis of TNFα mRNA expression in wild-type and targeted knocked-out CTSV-T24 cells treated with TNFα (20 ng/ml, abclone, cat#: RP00001) at different times.E. Immunoblotting of the indicated protein in CTSV-overexpressed UMUC3 cells with and without PDTC. F. Colony formation assays in both wild-type cells and CTSV-overexpressed UMUC3 cells with and without PDTC with the quantification of colony numbers. Error bars and mean ± SD obtained from three replicates. Statistical significance was determined by Student’s t-test or one-way ANOVA, *, p < 0.05, **, p < 0.01, ***, p < 0.001.