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Published on: February 8, 2020
JunB promotes cell invasion and angiogenesis in VHL-defective renal cell carcinoma
1Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Abstract:
Inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene causes both hereditary and sporadic clear-cell renal-cell carcinoma (ccRCC). Although the best-characterized function of the VHL protein (pVHL) is regulation of hypoxia-inducible factor-α (HIFα), pVHL also controls the development of pheochromocytoma through HIF-independent pathways by regulating JunB. However, it is largely unknown how these pathways contribute to the development and progression of ccRCC. In the present study, we confirmed that JunB was upregulated in VHL-defective ccRCC specimens by immunostaining. Short-hairpin RNA (shRNA)-mediated knockdown of JunB in 786-O and A498 VHL null ccRCC cells suppressed their invasiveness. In addition, JunB knockdown significantly repressed tumor growth and microvessel density in xenograft tumor assays. Conversely, forced expression of wild-type, but not dimerization-defective, JunB in a VHL-restored 786-O subclone promoted invasion in vitro and tumor growth and vessel formation in vivo. Quantitative PCR array analysis revealed that JunB regulated multiple genes relating to tumor invasion and angiogenesis such as matrix metalloproteinase-2 (MMP-2), MMP-9 and chemokine (C-C motif) ligand-2 (CCL2) in 786-O cells. JunB knockdown in these cells reduced the proteolytic activity of both MMPs in gelatin zymography and the amount of CCL2 in the culture supernatant. Moreover, shRNA-mediated knockdown of MMP-2 or inhibition of CCL2 activity with a neutralizing antibody repressed xenograft tumor growth and angiogenesis. Collectively, these results suggest that JunB promotes tumor invasiveness and enhances angiogenesis in VHL-defective ccRCCs.
Insights
JunB promotes clear-cell renal-cell carcinoma (ccRCC) progression by increasing tumor invasiveness and blood vessel formation. Targeting JunB, MMP-2, or CCL2 may offer new therapeutic strategies for VHL-defective ccRCC.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Genetics
Background:
- Von Hippel-Lindau (VHL) gene inactivation is a key driver of clear-cell renal-cell carcinoma (ccRCC).
- While VHL protein (pVHL) typically regulates hypoxia-inducible factor-α (HIFα), it also influences pheochromocytoma via HIF-independent pathways involving JunB.
- The precise role of these HIF-independent pathways, particularly JunB, in ccRCC development remains largely unexplored.
Purpose of the Study:
- To investigate the role of JunB in the development and progression of VHL-defective ccRCC.
- To elucidate the molecular mechanisms by which JunB influences ccRCC invasiveness and angiogenesis.
Main Methods:
- Immunostaining of ccRCC specimens to assess JunB levels.
- Short-hairpin RNA (shRNA)-mediated knockdown of JunB in ccRCC cell lines (786-O, A498).
- In vitro invasion assays and in vivo xenograft tumor models.
- Quantitative PCR array analysis to identify JunB-regulated genes.
- Gelatin zymography and ELISA to measure MMP and CCL2 activity/levels.
- Pharmacological inhibition of MMP-2 and CCL2 pathways.
Main Results:
- JunB was upregulated in VHL-defective ccRCC specimens.
- JunB knockdown suppressed ccRCC cell invasiveness, tumor growth, and microvessel density in vivo.
- Forced expression of JunB promoted invasion, tumor growth, and angiogenesis.
- JunB regulated genes involved in invasion and angiogenesis, including MMP-2, MMP-9, and CCL2.
- Knockdown of JunB reduced MMP activity and CCL2 levels; inhibiting MMP-2 or CCL2 repressed tumor growth and angiogenesis.
Conclusions:
- JunB plays a significant role in promoting tumor invasiveness in VHL-defective ccRCC.
- JunB enhances angiogenesis in ccRCC by regulating key factors like MMP-2 and CCL2.
- Targeting JunB or its downstream effectors (MMP-2, CCL2) represents a potential therapeutic strategy for ccRCC.
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