GOLPH3 is a potential therapeutic target and a prognostic indicator of poor survival in bladder cancer treated by cystectomy

Abstract

Golgi phosphoprotein 3 (GOLPH3) has been reported to be involved in the development of several human cancers. However, its clinical significance and biological role in bladder cancer remains unclear. In this study, we sought to analyze the GOLPH3 expression in bladder cancer samples and cells, and explore its clinical significance and biological role. We found that GOLPH3 was significantly increased in bladder cancer tissues and cells. Overexpression of GOLPH3 had significant correlation with poorer survival for bladder cancer patients treated by cystectomy. Knockdown of GOLPH3 inhibited the proliferation, migration and invasion of cancer cells, and tumor growth in a xenograft mouse model. GOLPH3 silencing inhibited AKT/m-TOR signaling, increased the cyclin-dependent kinase (CDK) inhibitor p27 and decreased the CDK regulator cyclin D1 and matrix metallopeptidase 9 (MMP9). Thus, GOLPH3 is likely to play important roles in bladder cancer progression via modulating AKT/mTOR signaling, and it is a novel prognostic biomarker and promising therapeutic target for bladder cancer.

Keywords: AKT/mTOR signalling; GOLPH3; bladder cancer; prognosis; survival.

Figure 1. GOLPH3 expression is increased in human bladder cancer tissues and is related to the expression of Ki67 and the prognosis of patients

Western blot (18 paired) A. and real-time PCR (12 paired) B. analysis of GOLPH3 expression in paired primary bladder cancer tissues (T), adjacent noncancerous tissues (ANT) and Normal bladder tissues (NB). IHC staining analysis of the association of GOLPH3 and Ki67 protein expression in paraffin-embedded, archived bladder cancer tissues C. Kaplan-Meier survival analysis of overall survival, cancer-specific survival and progression-free survival D. for all 137 bladder cancer patients with low GOLPH3-expressing (n = 72) versus high GOLPH3-expressing tumors (n = 65). The log-rank test was used to calculate P-values.

Figure 2. GOLPH3 silencing inhibited the proliferation and tumorigenicity of bladder cancer cells in vitro

Western blot and real-time PCR analysis A. of GOLPH3 expression in the immortalized normal human uroepithelium cell line SV-HUC-1, and 7 other bladder cancer cells. GOLPH3 expression of negative control siRNA-transfected and GOLPH3-silenced T24 and J82 cells was also analyzed by western blot and real-time PCR B. analysis. CCK8 assays C. indicated that the growth rates decreased in GOLPH3-silenced cells. Inhibition of T24 and J82 cells anchorage independent colony formation capacity by GOLPH3 siRNA D. Flow cytometric analysis E. indicated that GOLPH3 stimulates the G1-S phase transition of bladder cancer cells, and representative micrographs (left) and quantification (right) are shown. Western blot analysis F. of cell-cycle regulators (Cyclin D1 and P27) in GOLPH3 siRNA-infected cells and 5 paired primary bladder cancer tissues (T) and adjacent noncancerous tissues (ANT).

Figure 3. GOLPH3 inhibition attenuated the invasion and migration capability of T24 and J82 cells in vitro

A. Matrigel invasion assays showed that GOLPH3 siRNA-transfected cells resulted in low penetration through Matrigel-coated membranes compared with control cells. B. A scratch migration assay demonstrated that GOLPH3 decreased tumor cell migration. Western blot analysis C. of the metastasis associated protein MMP9 in GOLPH3 siRNA-infected cells and 5 paired primary bladder cancer tissues (T) and adjacent noncancerous tissues (ANT).

Figure 4. The effects of GOLPH3 inhibition on AKT/mTOR signaling

Western blot analysis of AKT/mTOR signaling (p-AKT, total AKT, p-mTOR, total mTOR, p-p70S6K, total p70S6K), p-ERK1/ERK2, and total ERK in GOLPH3 siRNA-infected cells A. and 5 paired primary bladder cancer tissues (T) and adjacent noncancerous tissues (ANT) B.

Figure 5. GOLPH3 silencing inhibits the tumorigenicity of bladder cancer cells in vitro and in vivo

A. The establishment of stable T24 bladder cancer cell lines expressing GOLPH3 and negative control shRNAs. The GFP expression ratio of T24 cells was greater than 90% 24 h post-cotransfection. B. GOLPH3 expression in stable T24 cells expressing GOLPH3 and negative control shRNAs was analyzed by western blot analysis. CCK8 assays C. indicated that the growth rates decreased in GOLPH3-silenced T24 cells. Flow cytometric analysis D. indicated that GOLPH3 stimulates the G1-S-phase transition of bladder cancer cells, and representative micrographs (left) and quantification (right) are shown. Stable knockdown of GOLPH3 by Lv-shRNA-GOLPH3 F. inhibits the growth of T24-derived xenografts in nude mice E. and G. Macrographic images show that the tumor size of the Lv-shRNA-GOLPH3 group was markedly smaller on the 40th day than those of the Lv-shRNA-NC group. The growth curves of the tumor xenografts and the final tumor weights of the Lv-shRNA-GOLPH3 group was decreased compared with those of the Lv-shRNA-NC group H. The tumor inhibition effect induced by GOLPH3 silencing in vivo correlate with the inhibition of AKT/mTOR signaling and Cyclin D1, MMP9 and increased p27 expression (F).

Figure 6. GOLPH3 may as a promising therapeutic target for bladder cancer in vivo

A. Knockdown of GOLPH3 by the intratumoral injection of siRNA inhibits the growth of T24-derived xenografts in nude mice. Top, macrographic image showing that the tumor size in the GOLPH3 siRNA group was markedly smaller on the 45th day after tumor cells inoculation than that of the groups injected with PBS and control siRNA; middle, growth curves of the tumor xenografts; bottom, the final tumor weights of the GOLPH3 siRNA group were decreased compared with that of the PBS and negative control siRNA groups. Intratumoral injection of GOLPH3 siRNA inhibited the protein expression of GOLPH3, p-mTOR and Ki67 as determined by western blotting B. and IHC C. analysis in T24 cell derived xenografts.