Distinct roles for VHL and hypoxia in RCC gene expression and metabolism

As discussed in last week’s blog renal cell carcinoma (RCC) cells show altered metabolism favouring lactate fermentation as the major energy source. Such metabolic changes can be a response to hypoxia or mutations in genes, such as VHL, that disrupt HIFα-proteasomal degradation. HIF signalling, directly and indirectly, regulates over 2% of human genes including those involved in angiogenesis, survival, proliferation and metabolism (Manalo  et al., 2005). Hypoxia and VHL-loss are generally thought to result in the same changes in gene and protein expression, however, Leisz et al., (2015) have found differential effects in RCC cellular models.

Differential effects of hypoxia and VHL-loss were assessed in three VHL-deficient cell lines – 786-O, RRC4 and RCC10 – alongside VHL transfectant counterparts, cultured in normoxic and hypoxic conditions. VHL expression in the transfectant lines was confirmed in both conditions, and HIFα-degradation in normoxia but not hypoxia indicated that the pVHL was functional. Leisz et al. used cDNA microarrays and 2-DE-based proteomics to identify changes in gene expression and protein levels in these cells.

Microarray results identified the greatest changes in gene expression between normoxic VHL+ cells and normoxic VHL- cells representative of the significant impact of VHL-loss. However, there were still differences in expression between hypoxic VHL- cells and normoxic VHL- cells suggesting that there are some VHL-independent hypoxia-dependent changes. In total 662 putative VHL-regulated genes and 194 putative hypoxia-regulated genes were identified. Proteomic analysis identified 76 differentially expressed proteins which could also be segregated into VHL-dependent and hypoxia-dependent targets.

In response to hypoxia cell metabolism switches to aerobic glycolysis with a high glucose influx and increased lactate production – the same changes are found in cancer cells (Warburg, 1956). The largest proportion of differentially expressed genes and proteins identified by Leisz et al. were metabolic – 24% of genes and 30% of proteins regulated by VHL-expression and 28% genes and 38% of proteins regulated by hypoxia.

VHL+ cells in normoxia showed increased basal oxygen consumptions and mitochondrial dehydrogenase activity compared to hypoxic VHL+ cells or VHL- cells indicative of greater levels of mitochondrial respiration. Hypoxic VHL+ cells and VHL- cells instead show increased expression of glucose transporter GLUT1, increased glycolysis enzyme activity, lactate dehydrogenase activity and lactate production – in line with proteasomal analysis of primary RCC tumours (Lichtenfels et al., 2009) – indicative of a shift towards lactate fermentation. This indicates that the downregulation of glycolysis enzymes is VHL-dependent whilst upregulation is hypoxic-dependent.

Lee et al., (2015) recently described a HIF-independent lactate-induced hypoxia response mediated by NDRG3 that alters expression of numerous genes. This secondary hypoxia response would also be induced in VHL- cells due to reduced post-transcriptional VHL-mediated ubiquitination of NDRG3, thereby contribute to the changes in expression in both situations.

Inoperable RCC tends to be resistant to chemotherapy and radiotherapy. Whilst advances in targeted treatments have increased patient survival, tumours often develop resistance. Leisz et al. found that although a large number of genes are concordantly regulated by VHL-status and hypoxia, there are some VHL-dependent, VHL-independent, hypoxia-dependent and hypoxia independent genes and proteins that have differential effects on the metabolic switch in RCC. Identifying the different impact of hypoxia-responses based on true oxygen deprivation and those due to mutations in genes such as VHL and FLCN, which artificially increase HIF-2a signalling, could be important for the development of new treatments for RCC.

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