Abstract
The von Hippel-Lindau (VHL) tumor suppressor gene is inactivated in the vast majority of human clear cell renal carcinomas. The pathogenesis of VHL loss is currently best understood to occur through stabilization of the hypoxia-inducible factors, activation of hypoxia-induced signaling pathways, and transcriptional reprogramming towards a pro-angiogenic and pro-growth state. However, hypoxia also drives other pro-tumorigenic processes, including the development of genomic instability via down-regulation of DNA repair gene expression. Here, we find that DNA repair genes involved in double-strand break repair by homologous recombination (HR) and in mismatch repair, which are down-regulated by hypoxic stress, are decreased in VHL-deficient renal cancer cells relative to wild type VHL-complemented cells. Functionally, this gene repression is associated with impaired DNA double-strand break repair in VHL-deficient cells, as determined by the persistence of ionizing radiation-induced DNA double-strand breaks and reduced repair activity in a homology-dependent plasmid reactivation assay. Furthermore, VHL deficiency conferred increased sensitivity to PARP inhibitors, analogous to the synthetic lethality observed between hypoxia and these agents. Finally, we discovered a correlation between VHL inactivation and reduced HR gene expression in a large panel of human renal carcinoma samples. Together, our data elucidate a novel connection between VHL-deficient renal carcinoma and hypoxia-induced down-regulation of DNA repair, and identify potential opportunities for targeting DNA repair defects in human renal cell carcinoma.
Highlights
The von Hippel-Lindau (VHL) gene is mutated, deleted, or silenced in 60–80% of human clear cell renal cell carcinomas, the most common type of kidney cancer [1]
We have found that VHL-deficient human renal carcinoma cells have reduced protein and mRNA expression of key homologous recombination (HR) and mismatch repair (MMR) genes down-regulated by hypoxia, including BRCA1, RAD51, FANCD2, and MLH1
We repeated this experiment in the RCC4 matched-pair cell lines and found a 30–40% reduction in FANCD2 levels in RCC4VHL–/– cells compared to RCC4+VHLWT cells under normoxic conditions, with FANCD2 levels equalizing between the two cell lines after 48 h of hypoxia (Figure 1B)
Summary
The von Hippel-Lindau (VHL) gene is mutated, deleted, or silenced in 60–80% of human clear cell renal cell carcinomas (ccRCC), the most common type of kidney cancer [1]. The best-characterized tumor suppressor function of the VHL protein (pVHL) is as the substrate-recognition subunit of an E3 ubiquitin ligase that targets hypoxiainducible factor (HIF) α-subunits for degradation under normoxic conditions [3, 4]. The PHDs are inhibited, preventing recognition of the HIF α-subunits by pVHL. The stabilized HIF α-subunits dimerize with www.impactjournals.com/oncotarget a constitutively-expressed HIF β-subunit, translocate to the nucleus, and induce changes in gene transcription by recruiting the p300/CBP transcriptional activators to consensus genetic sequences termed hypoxia response elements. HIF stabilization and activation of target genes is critical in the pathogenesis of ccRCC as HIF-2 has been shown to be both necessary and sufficient for tumor growth in VHLdeficient RCC cell lines [8,9,10,11]
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