Two sides to every story: the HIF-dependent and HIF-independent functions of pVHL

Abstract

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by inherited mutations that inactivate the VHL tumour suppressor gene. The VHL locus encodes pVHL, whose best studied function is to bind to and down-regulate the hypoxia-inducible factor (HIF) family of oxygen-dependent transcription factors. Early efforts have established the fundamental role of HIF in VHL-defective tumorigenesis and in particular renal cell carcinoma. However, recent findings have revealed an alternate side to the story, the HIF-independent tumour suppressor functions of pVHL. These include pVHL's ability to regulate apoptosis and senescence as well as its role in the maintenance of primary cilium and orchestrating the deposition of the extracellular matrix. To what extent these HIF-dependent and HIF-independent functions cooperate in VHL-defective tumorigenesis remains to be determined.

Fig 1

Clinical manifestations of VHL disease. Summary of the spectrum of benign and malignant tumours seen in association with VHL disease. Special notes: Retinal haemangiomas are found in up to 70% of VHL patients who survive to age 60 years. Haemangioblastomas are the most common lesions associated with VHL disease and are found most frequently in the cerebellum and spinal cord. Lung cysts appear to be rare, occurring in <1% of VHL patients. Pancreatic neuroendocrine tumours are relatively rare (9%) and are typically nonfunctional. The incidence of broad ligament papillary cystadenomas in women is not known secondary to their asymptomatic nature.

Fig 2

pVHL controls HIF via oxygen-sensitive hydroxylation. HIFα subunits have both an NTAD and a CTAD transactivation domain. When O₂ levels are high, HIFα is hydroxylated on one or both conserved prolyl (Pro) residues located within the NTAD by the oxygen-dependent PHD2. This prolyl hydroxylation event generates a high affinity binding site for the pVHL E3 ubiquitin ligase complex composed of Cullin 2 (Cul2), Elongin B (EloB), Elongin C (EloC) and Rbx1. The pVHL complex polyubiquitinates HIFα, leading to its destruction by the proteasome. When O₂ levels are intermediate, HIFα is hydroxylated by factor inhibiting HIF (FIH1) at a conserved asparaginyl (Asn) residue located in the CTAD, inhibiting HIF's interaction with the transcriptional co-activators p300/CBP. When O₂ levels are low, HIFα subunits are stabilized, able to heterodimerize with the constitutively stable HIFβ, interact with p300/CBP and promote the transcription of downstream target genes. In cells lacking VHL, PHD2 and FIH1 remain active but HIFα subunits are not polyubquitinated and therefore allowed to accumulate.

Fig 3

pVHL inactivation mediates both HIFdependent and HIF-independent pathways. HIF-responsive gene products play important roles in tumorigenesis. EGFR and TGFα promote cell proliferation and survival. VEGF, PDGF and connective tissue growth factor (CTGF) stimulate angiogenesis. Some proteins encoded by HIF-targeted gene products are responsible for regulating glucose uptake and metabolism, such as GLUT1, 6-PFK and pyruvate dehydrogenase kinase (PDK). CXCR4 and its ligand SDF1 stimulate chemotaxis and may also contribute to tumour cell invasion and metastases. MMP1 and lysyl oxidase (encoded by LOX) are implicated in ECM breakdown and tumour cell invasion/migration. Finally, dysregulation of TWIST (TWIST1 and TWIST2) and activation of HGFR (encoded by c-MET) are involved in EMT. pVHL has a number of HIF-independent functions as well. pVHL interacts with MDM2 and suppresses its ability to ubiqutinate p53, resulting in p53 accumulation and apoptosis. It can also act as an adaptor to bind CK2, which inactivates the NF-kB agonist CARD9, leads to inhibition of NF-kB signalling and overall inhibits cell survival. pVHL also down-regulates atypical protein kinase C (aPKC), which secondarily results in decreased levels of JUNB (an antagonist of JUN) thus permitting JUN-dependent neuronal apoptosis. Acute pVHL loss causes a senescent-like phenotype. It appears that pVHL increases p400 activity, which results in inactivation (hypophosphorylation) of the retinoblastoma protein (pRb) and prevents senescence. pVHL also interacts with collagen IV (Col IV), Kinesin 2 and fibronectin to ensure proper ECM deposition. Finally, pVHL plays an important role in primary cilium function by promoting microtubule stabilization and binding with aPKC and the polarity proteins Par3 and Par6.