VHL Mediates Aurora Kinase A Degradation To Regulate Ciliogenesis in Renal Cell Carcinoma

Saturday, February 13, 2016
Elshad Hasanov, Hacettepe University Cancer Institute, Ankara, Turkey
Background: Loss of heterozygosity at the von Hippel Lindau (VHL) tumor suppressor gene locus on chromosome 3p is commonly associated with renal cell carcinoma (RCC).  VHL is an E3 ubiquitin ligase, most famed for its role in proteasome-mediated degradation of hypoxia inducible factor alpha (HIFa), although more recently VHL has been implicated in stabilization of microtubules.  Consistent with VHL’s role in microtubule stabilization, VHL loss results in loss of the primary cilium, and the development of renal cysts and tumors, categorizing VHL disease as a ciliopathy. Aurora Kinase A (AURKA), in a novel non-mitotic role caused disassembly of the primary cilium. The objective of this study was to investigate the role of VHL in modulating AURKA and regulating ciliogenesis. Methods: In vitro and in vivo ubiquitination assays were performed to establish AURKA as a direct target of VHL. Immunoblot analyses were used to determine AURKA levels in human RCC patient material and VHL deficient cell lines. Immunofluorescence was utilized to implicate VHL-mediated AURKA ubiquitination in regulating ciliogenesis. Results: In this study, we show that AURKA is a novel target for VHL’s E3-ligase activity.  VHL directly regulates AURKA expression by promoting AURKA degradation via the 26S proteasome.  In vitro and in vivo ubiquitination assays showed enhanced AURKA ubiquitination in the presence of VHL.  We found that VHL mediates AURKA degradation via a multi-monoubiquitin chain linkage, in contrast to the more traditional and abundant K48-linkage of proteins targeted for proteasome-mediated degradation.  Biochemical studies revealed that unlike HIF1 recognition and degradation by VHL, which requires proline hydroxylation, VHL interacted with, and degraded AURKA independent of its hydroxylation status, suggesting an alternate recognition motif on AURKA. Importantly, we observed increased AURKA ubiquitination in cells induced to ciliate, suggesting that VHL degradation of AURKA potentially modulates primary cilia formation by blocking the cilia disassembly pathway.  Pharmacologic inhibition of AURKA and HDAC6 restored the ability of cells with an acute loss of VHL to form primary cilia.   Conclusion: Thus, our data identify a novel target of VHL’s E3 ligase activity, and establish a role for VHL in regulating ciliogenesis. Importantly, these data present therapeutic strategies targeting the epithelial cell defect associated with VHL-null renal cell carcinoma.