Hypoxia Inducible Factor-independent functions for the von Hippel-Lindau tumor suppressor gene

Abstract

Inactivating mutations of the von Hippel-Lindau gene (VHL) on chromosome 3p have been associated with the autosomal dominant VHL disease, characterized by extensively vascularized tumors and cysts in different organs, as well as the majority of conventional kidney cancers. The VHL gene product regulates the ubiquitination and subsequent degradation of hypoxia inducible factor (HIF), an important transcriptional activator of the genetic program triggered by hypoxia. The absence of functional VHL, thus, artificially stabilizes HIF and induces a cellular response to perceived low-oxygen tension. This illegitimate HIF signalling under normoxic conditions is likely to contribute to the extensive vascularization observed in VHL-related lesions. In the first chapter we examine the arguments for alternative function for the VHL related tumors.

The data presented in the second chapter contributes the concept of a HIF-independent function for the VHL gene. Here we present the outcome of a microarray analysis of VHL-positive vs. VHL-negative cells. In these experiments we find a known VHL-interacting partener: fibronectin that is regulated by VHL at the mRNA level, independently of hypoxia. The third, fourth and fifth chapters address what the molecular mechanism of the VHL-mediated HIF-independent function could be. As discussed in the first chapter our "prime suspect" to determine the HIF-independent function for VHL lies within the N-terminal acidic domain, which has not previously been assigned a function. We show that the acidic domain of VHL can be phosphorylated and that this modification is essential for tumor suppression in vivo. The acidic domain is also involved in binding the kinesin-2 complex and this interaction between the kinesin-2 complex and VHL plays a role in promoting microtubule stability in the cell periphery. Furthermore, we show that the kinesin-2 complex and VHL cooperate in the formation of primary cilia. These data represent the first pieces of experimental evidence that the acidic domain has a function. The model that arises from the data presented in these chapters is that the acidic domain plays an important role in determining the HIF-independent role for VHL; however, the exact molecular mechanism needs to be elucidated. The sixth chapter deals with a somewhat different aspect of VHL biology being the regulation of VHL expression and its role in the physiology of the intestine. We found that the expression of VHL was regulated by active wnt-signaling and displays the expression pattern of a typical wnt-signaling target in the intestinal mucosa. Interestingly there was reciprocal expression of HIF under normoxic conditions. These data show for the first time that regulation of expression of VHL has functional consequences and can lead to normoxic activation of HIF.

The HIF-independent role of VHL has been made plausible with the data represented in this thesis, however the molecular nature has not yet been clarified fully. For the optimal treatment of patients with VHL disease and those carrying sporadic renal cell carcinomas with VHL mutations, the elucidation of this mechanism is essential. These insights will allow us to design therapies that interfere with this mechanism and thereby target the tumor in a more specific way.