Elsevier

Urology

Volume 79, Issue 4, April 2012, Pages 966.e1-966.e7
Urology

Basic and Translational Science Abstract
TGFBI-promoted Adhesion, Migration and Invasion of Human Renal Cell Carcinoma Depends on Inactivation of von Hippel-Lindau Tumor Suppressor

https://doi.org/10.1016/j.urology.2011.12.011Get rights and content

Objective

To investigate the role of transforming growth factor-β-induced (TGFBI) in metastasis of renal cell carcinoma (RCC) and the associations between TGFBI expression and von Hippel-Lindau (VHL) status.

Methods

In null type VHL cells stably transfected with the VHL vector, the expression of VHL in cells with wild type VHL was decreased by siRNA. We investigated the effects of hypoxia-inducible transcription factor (HIF) on TGFBI in RCC cells by decreasing the expression levels of HIF-1α and HIF-2α through siRNA. The secretion of transforming growth factor-β1 (TGF-β1) in RCC cells with different VHL status was analyzed by enzyme-linked immunosorbent assay. The role of TGFBI in metastasis and the effect of VHL activation on TGFBI-induced adhesion, migration, and invasion in RCC cells were examined using matrigel, chemotaxis, and the transwell system, respectively.

Results

Our results suggested that TGF-β1 and TGFBI might be targets of VHL, and the suppression of TGFBI by VHL is not by way of the HIF-1α or HIF-2α pathway. The expression of TGFBI was significantly enhanced by TGF-β1 in VHL-inactive RCC cells compared with VHL-active cells. In addition, these results indicate that TGFBI participated in the adhesion, migration, and invasion of RCC cells, which are dependent on the inactivation of VHL.

Conclusion

The results of the present study suggest that TGFBI-promoted metastasis of RCC cells depends on inactivation of the VHL tumor suppressor and that TGFBI could be a therapeutic target against RCC in the future.

Section snippets

Cell Culture and Agents

Two RCC cell lines, ACHN (wild-type VHL) and A498 (null-type VHL), were obtained from American Type Culture Collection (Manassas, VA) and cultured in complete medium consisting of Roswell Park Memorial Institute-1640 medium (Gibco, Bio-cult, Glasgow, Scotland) supplemented with 25 mM HEPES, 2 mM l-glutamine, 1% nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum. The cell lines were maintained as monolayers in 10-cm plastic dishes

VHL Inactivation Upregulated TGFBI Expression

To detect the association of VHL status and TGFBI expression, A498 cells were stably transfected with either the VHL vector or the blank vector lacking the VHL insert. The ACHN cells were transfected with either VHL siRNA or negative siRNA controls. Protein expression was evaluated by Western blots (Fig. 1A). The VHL-active RCC cell lines had high levels of VHL expression and low levels of HIF-1α, HIF-2α, and TGFBI. In contrast, the VHL-inactive RCC cell lines had low levels of VHL expression

Comment

The protein TGFBI was first detected in a human lung adenocarcinoma cell line after stimulation by TGF-β15 and has been found immunohistochemically in such human tissues as the cornea, skin, lung, bone, bladder, and kidney.16 Additionally, TGFBI is involved in such human diseases as corneal dystrophies,17 melorheostosis, osteogenesis,18 diabetic angiopathy, atherothrombosis, and restenosis.19 The protein TGFBI elicits numerous changes in cellular behavior that include differentiating epithelial

Conclusions

Both TGF-β1 and TGFBI expression could be suppressed by VHL. Because VHL mutations occur early in RCC development, inactivating VHL induces an important secondary genetic event in the TGFBI signaling pathway that could lead to the metastasis of RCC cells. These results indicate that blocking TGFBI might provide a novel treatment strategy and that the TGF-β1-TGFBI pathway might be an appropriate target to treat RCC with inactive VHL.

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    Funding Support: Our research was supported by the National Natural Science Foundation of China (grant 30801139) and Basic-Clinical Research Foundation of Capital Medical University.

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    Donghao Shang and Yuting Liu contributed equally to this work.

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