HIF-2α regulates PD-L1 expression in RCC

Tumour cells can create immunosuppressive microenvironments by hijacking natural mechanisms such as PD-L1 expression to impair T-cell function. Several new immunotherapy treatments target the PD-1/PD-L1 pathway and have produced some long-lasting responses in patients (Motzer et al., 2015) but not all patients respond. High expression of PD-L1 in clear cell RCC (ccRCC) has been shown to correlate with metastasis and poor outcome (Thompson et al., 2007). New research from Messai et al. (2015) has assessed PD-L1 expression in ccRCC samples and cell lines carrying VHL mutations to identify meaningful correlations.

Messai et al. initially assessed PD-L1 expression in 32 ccRCC samples – 11 from VHL patients and 21 sporadic tumours – which had been sequenced for VHL gene mutations, loss of function (LOF) and loss of heterogeneity (LOH). Significantly higher PD-L1 expression was found in the tumours which had two altered VHL alleles (21/32), complete LOF (14/32) or had LOH at the VHL locus (23/32). This suggests a link between VHL status and PD-L1 expression in ccRCCs.

To further investigate the role of VHL in regulating PD-L1 expression Messai et al. reintroduced various mutated VHL constructs into VHL-deficient 786-O cells – these cells overexpress HIF-2α but not HIF-1α.  The different pVHL mutants resulted in a gradient expression of HIF-2α which positively correlated with PD-L1 mRNA and protein expression levels. In cells retransfected with wildtype VHL the expression of PD-L1 was significantly lower than those carrying VHL-mutants or no VHL constructs. This further confirms the correlation between VHL mutations and PD-L1 expression seen in the tumour samples.

Increased HIF-2α signalling resulting from VHL loss plays a role in tumourigenesis altering cellular metabolism and increasing angiogenesis. To determine the role of HIF-2α in the correlation between VHL and PD-L1 expression Messai et al. used siRNA to knockdown HIF-2α in 786-0 and A498 cells (also VHL-deficient); resulting in reduced PD-L1 mRNA, protein and surface expression. In addition overexpression of HIF-2α in cells with functional VHL results in increased PD-L1 expression. This suggests that PD-L1 expression is being regulated by HIF-2α signalling, which is mediated by VHL status.

Analysis of the PD-L1 promoter identified several putative Hypoxia Response Elements (HRE). Based on ChIP and luciferase assays, HIF-2α specifically binds to HRE4 and is transcriptionally active. Previously HIF-1α was shown to regulate PD-L1 expression in tumour infiltrating MDSCs, also through binding to HRE4 (Norman et al., 2014). To determine if HIF-1α has a similar role in ccRCC cells Messai et al. assessed PD-L1 expression in RRC4 cells which overexpress both HIF-1α and HIF-2α.  Knockdown of either HIF-1α or HIF-2α, or both resulted in a decrease in PD-L1 expression supporting a direct role for HIF signalling in PD-L1 regulation.

Further studies will be needed to determine the validity of VHL mutation status as a biomarker for PD-L1 expression and if there is any correlation to immunotherapy responses. Increased HIF signalling is generally associated with tumourigenesis and is also seen in BHD tumours and cell lines (Preston et al., 2011, Nishii et al., 2013). Greater understanding of the role of HIF signalling and PD-L1 expression in response to various treatments including immunotherapies could help identify the optimal combination or sequential treatment plans for a range of RCC patients.

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