Glucocorticoid-Inducible Kinase 2 Promotes Bladder Cancer Cell Proliferation, Migration and Invasion by Enhancing β-catenin/c-Myc Signaling Pathway

Abstract

Background: Bladder cancer is one of the most common malignancies in urologic system. The glucocorticoid-inducible kinase 2 (SGK2) expression and function were largely unknown in cancers. Current study was aimed to investigate the role of SGK2 in bladder cancer and its potential mechanisms. Methods: SGK2 expression was quantified by western blot (WB) in multiple bladder cancer cell lines (T24, 5637, J82 and UMUC3) compared with normal urothelial cell line (SVHUC). SGK2 knocking down and overexpression model were established by lentivirus transfection. MTT, colony formation, wound healing and transwell assay were used to assess the tumor cell proliferation, migration and invasion abilities, respectively. In addition, molecular function analysis was performed using FunRich software V3. Immunoprecipitation (IP) assay was applied to investigate the interaction between SGK2 and β-catenin at protein level. TCGA database was retrieved to verify the association between these genes and clinical tumor stage as well as prognosis among bladder cancer patients. Results: SGK2 expression was significantly upregulated in multiple bladder cancer cell lines compared with SVHUC at protein level. Cell proliferation, migration and invasion abilities were significantly decreased after knocking down SGK2 in J82 and UMUC3 cell lines. Inversely, cell aggressive phenotypes were significantly increased after overexpressing SGK2 in T24 cell line. Furthermore, functional analyses of SGK2 based on TCGA database showed that SGK2 related genes were involved in receptor activity, ATP binding, DNA repair protein, trans-membrane receptor activity and lipid binding. In addition, protein interaction analysis identified c-Myc was significantly enriched in SGK2 positively associated genes. The prediction was validated by WB and IP assay that SGK2 could directly bind with β-catenin at protein level to regulate their downstream gene c-Myc expression in bladder cancer to influence tumor progression. And clinical data generated from TCGA database also identified these downstream genes were significantly associated with tumor stage and survival status of bladder cancer patients. Conclusion: Taken together, our findings suggest SGK2 promotes bladder cancer progression via mediating β-catenin/c-Myc signaling pathway, which may serve as a potential therapeutic target for bladder cancer patients.

Keywords: bladder cancer; c-Myc; glucocorticoid-inducible kinase 2; β-catenin.

Figure 1

SGK2 expression in bladder cancer patients and cell lines. (A). The SGK2 mRNA expression between bladder tumor vs. para-tumor tissues. Patients' data was retrieved from TCGA database. (B). Western blot (WB) showed SGK2 protein expression in several bladder cancer cell lines (T24, 5637, J82, UMUC3) compared with normal epithelial cell line SVHUC. (C). WB showed the SGK2 knocking down efficacy in UMUC3 and J82 cell lines. (D). WB showed the SGK2 overexpression efficacy in T24 cell line.

Figure 2

SGK2 promotes bladder cancer cell growth and colony formation (CF) abilities in vitro. (A). MTT assays indicated cell growth was inhibited after knocking down SGK2 in UMUC3 and J82 cell lines (P < 0.05). (B). Cell growth was significantly increased after overexpressing SGK2 in T24 cell line using MTT assay (P < 0.05). (C). The colony formation (CF) assay indicated knocking down SGK2 significantly decreased CF ability in UMUC3 (P < 0.01) and J82 cells (P < 0.05). (D). CF ability was significantly increased after overexpressing SGK2 in T24 cell line (P < 0.05). Data are representative of three independent experiments. * indicated P < 0.05. ** indicated P < 0.01.

Figure 3

SGK2 promotes bladder cancer cells migration and invasion abilities in vitro. (A). Wound healing assays indicated cell migration was inhibited after knocking down SGK2 in UMUC3 and J82 cell lines (P < 0.05). Inversely, cell migration was significantly increased after overexpressing SGK2 in T24 cell line (P < 0.01). (B). Transwell assays indicated cell migration was inhibited after knocking down SGK2 in UMUC3 and J82 cell lines (P < 0.01). Inversely, cell migration was significantly increased after overexpressing SGK2 in T24 cell line (P < 0.05). (C). Transwell assays indicated cell invasion was inhibited after knocking down SGK2 in UMUC3 and J82 cell lines (P < 0.01). Inversely, cell invasion was significantly increased after overexpressing SGK2 in T24 cell line (P < 0.05). Data are representative of three independent experiments. * indicated P < 0.05. ** indicated P < 0.01.

Figure 4

Molecular function analysis of SGK2. (A). Molecular function analysis suggested that positive related genes of SGK2 were mainly enriched in receptor activity, ATP binding, DNA repair protein, transmembrane receptor activity and lipid binding. (B). Biological process analysis indicated SGK2 positive related genes were enriched in immune response, regulation of immune response, protein folding, signal complex formation and regulation of gene expression epigenetic. (C). Biological pathway analysis indicated SGK2 positive related genes were enriched in Mitotic M-M/G1 phases, G1/S Transition, Cell cycle mitotic, HIV infection, Cross-presentation of soluble exogenous antigens.

Figure 5

SGK2 regulates c-Myc/β-catenin gene expression. (A). WB results showed the protein levels of β-catenin and c-Myc were significantly decreased after down-regulating SGK2 expression in UMUC3 and J82 cells. (B). WB results showed the protein levels of β-catenin and c-Myc were significantly increased after overexpressing SGK2 in T24 cell. (C). Immunoprecipitation (IP) assay indicated SGK2 could directly interact with β-catenin protein in T24 cell. (D). MG132 (10 µM) abrogated shSGK2 induced inhibition of β-catenin protein expression in UMUC3 cell. (E). A time-dependent decrease in β-catenin protein expression in T24-vector and T24-oeSGK2 cell lines exposed to CHX (10 µg/mL). However, the decrease speed was marked slower in T24-oeSGK2 than T24-vector group. (F). The expression of c-Myc and CTNNB1 gene (encoding β-catenin protein) were significantly associated with tumor stage of bladder cancer (P < 0.05). (G). c-Myc expression was significantly associated with overall survival (OS), recurrence-free survival (RFS) in bladder cancer patients (P < 0.05). Upper and middle indicated protein expression analysis and lower indicated gene expression analysis.