Src and caveolin-1 reciprocally regulate metastasis via a common downstream signaling pathway in bladder cancer

Abstract

In bladder cancer, increased caveolin-1 (Cav-1) expression and decreased Src expression and kinase activity correlate with tumor aggressiveness. Here, we investigate the clinical and functional significance, if any, of this reciprocal expression in bladder cancer metastasis. We evaluated the ability of tumor Cav-1 and Src RNA and protein expression to predict outcome following cystectomy in 257 patients enrolled in two independent clinical studies. In both, high Cav-1 and low Src levels were associated with metastasis development. We overexpressed or depleted Cav-1 and Src protein levels in UMUC-3 and RT4 human bladder cancer cells and evaluated the effect of this on actin stress fibers, migration using Transwells, and lung metastasis following tail vein inoculation. Cav-1 depletion or expression of active Src in metastatic UMUC-3 cells decreases actin stress fibers, cell migration, and metastasis, while Cav-1 overexpression or Src depletion increased the migration of nonmetastatic RT4 cells. Biochemical studies indicated that Cav-1 mediates these effects via its phosphorylated form (pY14), whereas Src effects are mediated through phosphorylation of p190RhoGAP and these pathways converge to reduce activity of RhoA, RhoC, and Rho effector ROCK1. Treatment with a ROCK inhibitor reduced UMUC-3 lung metastasis in vivo, phenocopying the effect of Cav-1 depletion or expression of active Src. Src suppresses whereas Cav-1 promotes metastasis of bladder cancer through a pharmacologically tractable common downstream signaling pathway. Clinical evaluation of personalized therapy to suppress metastasis development based on Cav-1 and Src profiles seems warranted.

Figure 1. Expression of Cav-1 and Src in bladder cancer tissues and cell lines

A) The relationship of Src and Cav-1 expression to tumor stage in human bladder cancer (25, 26). (from www.oncomine.org). B) Scatter plot and linear regression analysis of 65 bladder cancer specimens on HG-U133A Gene Chip array (Affymetrix)(20). Y-axis shows normalized Src probe expression (Src HG-U133A expression divided by β-actin HG-U133A expression level) against normalized Cav-1 probe expression on X-axis. C) Kaplan Meier curves showing overall survival of 109 bladder cancer patients following cystectomy as a function of Cav-1 and Src RNA expression determined by Sanchez-Carbayo et. al (26). D) (i) Representative immunohistochemical (IHC) evaluation and scoring of Cav-1 and Src as described in methods. Numbers indicate compound score (see methods) for each example. (ii) Kaplan Meier curves showing overall survival of 151 bladder cancer patients as a function of Cav-1 and Src expression determined by immunohistochemistry. Src High Score >5; Cav-1 High Score >8. * Log-rank for comparison to other two groups.

Figure 2. Effect of Cav-1 depletion and Src expression on transwell migration and the actin cytoskeleton

A) UMUC-3 cells were transfected with GL-2 and Cav-1 siRNA duplex. (i) Lysates were collected 24, 48 and 72 hours after transfection and analyzed for Cav-1 protein expression. Data shown are representative blots of three separate experiments. (ii) Transwell migration was performed as previously described (43) on cells collected 24, 48 and 72 hours post siRNA transfection. Columns, mean; bar, ± SD. *p <0.05 significantly different from control GL-2 transfected cells at each time point. B). Transwell migration. *p<0.05 significantly different from control UMUC-3 pcDNA transfected cells. **p<0.05 significantly different from UMUC-3 GL-2 transfected cells 72 hours after transfection. Inset: Western analysis for Cav-1, pY14Cav-1, pY418 Src, total Src and tubulin expression was carried out on lysates collected from cells used for transwell migration at 72 hours after transfection. C) Actin cytoskeletal staining with phalloidin-AlexaFluor 594 (red) 72 hours after transfection with Cav-1 siRNA or GL-2 duplex. Magnification indicated.

Figure 3. Effect of Src up-regulation and Cav-1 down regulation on Rho-ROCK pathway in UMUC-3 cells

A) Rho A, RhoC and ROCK1 activation assays and western blots for lysates collected from cells 72 hours post transfection. B) RhoA-GTP and RhoC -GTP from Figure 3A were quantified relative to total RhoA and RhoC respectively by densitometry. *, p< 0.01 compared to their respective controls. C&D) Rho A and C activation assays and western blots for lysates collected from cells 72 hours post transfection.

Figure 4. Effect of Src inhibition and Cav-1 introduction on migration of RT4

A) Transwell migration. *p <0.05 significantly different from RT4 and RT4 pcDNA transfected cells at each time point. B) Transwell migration *p <0.05 significantly different from RT4 GL-2 and RT4 pcDNA transfected cells. C & D) Western blotting and Rho activation assays as described. Data shown are representative blots of three separate experiments.

Figure 5. Effect of Src and Cav-1 on migration is mediated via Rho-ROCK pathway

A) Transwell migration. *p <0.05 significantly different from RT4 pcDNA transfected cells. **p<0.05 significantly different from RT4 pcDNA transfected cells ***p<0.05 significantly different from RT4 pcDNA+PP2 transfected cells #p<0.05 significantly different from RT4-Cav-1 transfected cells. Inset: Corresponding ROCK1 activity. B) Transwell migration. *p <0.05 significantly different from UMUC-3 vehicle treated cells. Inset: Corresponding ROCK1 activity. C) Proposed Cav-1 and Src mediated regulation of Rho/ROCK in bladder cancer. Cav-1 at Tyr 14 results in GTPase activation of RhoA and RhoC leading to ROCK1 activation subsequently resulting in increased stress fiber formation, migration and metastasis. Activated Src leads to 190RhoGAP phosphorylation resulting in the inhibition of Rho/ROCK pathway leading to loss of stress fibers, reduced migration and metastasis. * PP2 inhibits the activity of other members of the Src family kinases (SFK) and hence these may also be involved.

Figure 6. Effect of Src upregulation and Cav-1 downregulation on growth and lung colonization of UMUC-3 cells

A) In vivo subcutaneous growth of cells in Figure 5a. 10⁶ cells were injected subcutaneously in 10 mice per group. Mean tumor volume ± SEM (44). *p <0.036 compared to controls at 21 days. B) In vivo lung metastasis by 12p quantitative PCR in mice injected UMUC-3 cells. Inset: Appearance of lungs with metastatic nodules (arrow) found at necropsy. C) In vivo lung metastasis of mice injected 8 weeks previously with Lul-2 cells with or without Y27632 treatment. i) Representative Xenogen images (D-luciferase) of vehicle treated and Y27632 treated animals ii) Representative gross lung pictures at 8 weeks. Arrows indicate lung metastases. iii) 12p quantitative PCR from vehicle treated and Y27632 treated mice at 8 weeks.