Abstract 3094: Microtubule-dependent regulation of HIF-1α

Abstract

Abstract Hypoxia inducible factor-1α (HIF-1α) is a pro-angiogenic transcription factor over-expressed in over 70% of human cancers. We have shown that microtubule-targeting drugs (MTDs) such as the taxanes, inhibit HIF-1α protein translation and transcriptional activity. The underlying mechanism involves trafficking of HIF-1α mRNA on dynamic microtubules as a requirement for its active translation. MTD-induced microtubule disruption resulted in polysome release of HIF-1α mRNA, followed by its enrichment to Argonaute-2 containing P-bodies, where HIF-specific miRNAs were also accumulated and repressed its translation. This process was reversible following microtubule repolymerization, and both Argonaute-2 knock-down and removal of HIF's UTR regions abrogated Taxol's inhibitory activity. Together these results suggest that microtubule dynamics tightly regulate HIF-1α's translation by altering its mRNA localization. To identify additional, similarly regulated mRNAs, we are currently isolating Argonaute-2 bound mRNAs from untreated versus MTD-treated cells and analyzing them by RNA Seq. We are also trying to identify the “zip-code” sequence within the UTR regions of HIF-1α that confers microtubule sensitivity. Interestingly, preliminary data from our lab have also shown that microtubule disruption inhibits the hypoxia-induced nuclear accumulation of HIF-1α protein and the subsequent activation of HIF response element (HRE)-containing genes, such as VEGF. Immunofluorescence followed by confocal microscopy revealed that HIF-1α protein colocalized with the microtubule cytoskeleton. This result is further supported by transmission electron microscopy (TEM), co-immunoprecipitation, and microtubule co-sedimentation assays. Inhibition of the minus-end directed motor protein, dynein, prevented HIF-1α nuclear accumulation, further implicating microtubule dynamics in HIF-1α protein trafficking and activity. Thus far, we have shown that HIF-1α is regulated by microtubules at both the level of protein synthesis and protein trafficking. To date, the only example where HIF-1α activity is not microtubule-dependent is in Renal Cell Carcinoma (RCC), where HIF-1α is constitutively active due to VHL mutations. MTD treatment failed to inhibit HIF-1α in VHL-null RCCs at concentrations that disrupted microtubules, while reintroduction of wild-type VHL did not restore the ability of MTDs to inhibit HIF-1α. Furthermore, MTD treatment of RCC cells did not release HIF-1α mRNA from polysomes, and HIF-1α protein did not bind microtubules. These results suggest that the regulation of HIF-1α has become independent of the microtubule cytoskeleton, which may contribute to the chemoresistant nature of RCCs. Further understanding of the microtubule dependent regulation of HIF-1α, and lack thereof in RCCs, will help us better elucidate the biology of HIF-1α translation and will have important therapeutic implications given the wide use of MTDs in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3094. doi:10.1158/1538-7445.AM2011-3094