Increased HIF-1α activity in RCC modulated by p62

Clear cell Renal Cell Carcinoma (ccRCC) is frequently associated with the loss of VHL, but gain of chromosome 5q35.3 – which contains the oncogene SQSTM1 – was also found in approximately 70% of tumours (Li et al., 2013). The SQSTM1 protein, p62, is a scaffold protein involved in cellular processes and signalling pathways related to malignant growth. New research from Chen et al., (2015) determined a role for p62 in regulating glucose metabolism, through HIF-1α signalling.

Expression of p62 is upregulated in various malignancies and can promote tumourigenic cell proliferation and growth by enhancing NF-kB, mTORC1 and NRF2 signalling. Additionally, p62 binds both ubiquitinated proteins and LC3 to promote autophagosome formation. Dunlop et al., (2014) identified increased levels of p62 in BHD tumours and cell lines associated with the dysregulation of autophagy. An additional trademark of cancer cells is altered metabolism; the Warburg effect. Chen et al. assessed glycolytic enzyme expression, glucose uptake and lactate production following sh-RNA knockdown of p62 in ACHN cells: all were reduced and the cells showed reduced cell growth and viability.

In various forms of inherited and sporadic RCC changes in metabolism are regulated by HIF-1α signalling. Chen et al., report that knockdown of p62 results in decreased HIF-1α, but not HIF-2α, expression which was rescued when p62 was restored. In addition to regulating HIF-1α expression, overexpression of p62 also increases HIF-1α transcriptional activity. HIF signalling can be altered in different ways: in VHL and HLRCC there is an increase in HIF-1α stability (Bratslavsky et al., 2007, Isaacs et al., 2005); in TSC HIF signalling is upregulated via mTOR; and in BHD increased AMPK activity, via PGC1A-mediated ROS production, results in increased HIF-1α activity (Yan et al., 2014). Overexpression of p62, commonly seen in these tumours, could also be contributing.

Both mTORC1 signalling and NF-kB signalling can upregulate HIF-1α signalling. Inhibition of either mTOR signalling using rapamycin, or NF-kB by knockdown of the p65 subunit, reduced HIF-1α levels in cells expressing p62. Additionally, p62 has previously been shown to coimmunoprecipitate with VHL E3 ligase complex component CUL2. Chen et al. determined that p62 also coimmunoprecipitates with the other complex components including pVHL, but not with HIF-1α. Using deletion mutations this interaction was mapped to the TRAF6 binding domain in p62 and the intact β-domain in pVHL – the same domain that binds HIF proteins. Chen et al. propose that p62 competes with HIF-1α at this site, blocking HIF-1α ubquitination and reducing protein degradation. VHL E3 ligase activity is further reduced due to p62 blocking the neddylation of CUL2.

To confirm whether p62-promoted aerobic glycolysis due to increased HIF-1α signalling Chen et al. used shRNAs to knockdown either p62 or HIF-1α. Knockdown of HIF-1α impaired p62-mediated increases in glucose uptake and lactate production, but HIF-1α overexpression could reverse the reduced uptake caused by p62 silencing. p62 therefore promotes enhanced HIF-1α signalling both by increasing expression and reducing degradation, which leads to altered cellular metabolism.

Altered mTORC1, NF-kB and NRF2 signalling has been reported in several tumour type and the loss of VHL E3 ligase activity is characteristic of ccRCC. If increased p62 can disrupt VHL E3 ligase in cells with functional pVHL then the same pathways could be perturbed in other tumour types, such as those associated with BHD and FLCN mutations, contributing to the enhanced HIF-1α activity. Understanding how all these different signalling pathways interact is important to further elucidate RCC tumourigenesis and develop more effective treatments that can target the key pathways.

 

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