Login to Access Video or Poster Abstract: MP60-14
Sources of Funding: CIHR, Vanier Canada Graduate Scholarships

Introduction

VHL is characterized as a tumour suppressor, being suppressed in up to 90% of clear cell Renal Cell Carcinomas (ccRCC). Canonically, VHL binds to and mediates degradation of hypoxia inducible factors (HIF). However, VHL has been discovered to bind other proteins, like p53, thereby altering the cell in more ways than just HIF degradation. Furthermore, there are conflicting reports on the impact of VHL on cancer proliferation and apoptosis, which can be regulated by p53 target genes, like p21 and PUMA. We hypothesize that VHL inhibits p53 activation paradoxically leading to increased proliferation and reduced apoptosis in cancer cells.

Methods

VHL-deficient cells were transiently (adenoviral) or stably (lentiviral) transduced with VHL. We generated VHL knockout cells using CRISPR/Cas9 genome editing technology. HIF overexpression was achieved using hypoxia or a non-degradable HIF-expressing adenovirus. Immunodeficient nude mice were injected with tumor cells bilaterally (left, -VHL; right, +VHL) and treated with weekly injections of doxorubicin (2mg/kg, IP). Co-immunoprecipitations, immunoblots, confocal imaging and siRNA transfections were performed using standard techniques.

Results

Transient and stable VHL overexpression robustly decreased, while knockout of VHL increased p21/PUMA mRNA and protein levels. VHL-deficient cells lacking HIF (via siRNA) had unaltered p21. Conversely, VHL and non-degradable HIF mutant co-expressing cells exhibited low p21 levels compared to VHL-deficient cells. Taken together, these data suggest that VHL-mediated p53 regulation is independent of HIF. VHL over-expression significantly attenuated induction of apoptosis in doxorubicin (p53 activator) treated cells. Prior to treatment, VHL-deficient tumors grew larger than VHL expressing tumors. However, doxorubicin resulted in a more significant reduction in tumor size in VHL-deficient tumors.

Conclusions

These results suggest an inhibitory interaction between VHL and p53, in which VHL mediates reduces p53 target gene expression. Furthermore, the attenuation of doxorubicin treatment in VHL-expressing cancer cells suggests that this chemotherapy may be more effective against VHL-deficient tumors or in combination with a VHL inhibitor. This work suggests that through a previously undescribed mechanism, a known tumor suppressor may paradoxically be capable of potentiating cancer growth and apoptosis resistance.

Funding

CIHR, Vanier Canada Graduate Scholarships