The taxanes are effective microtubule-stabilizing chemotherapy drugs used in the treatment of various solid tumors. However, the emergence of drug resistance hampers their clinical efficacy. The molecular basis of clinical taxane resistance remains poorly understood. Breast cancer 1, early onset gene, BRCA1, is a tumor-suppressor gene, whose expression has been correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer. However, the molecular mechanism underlying the relationship between BRCA1 (B1) expression and taxane activity remains unclear. To this end, we created a stable B1 knockdown A549 cell line (B1-KD) to investigate B1’s role in microtubule biology and response to taxane treatment. We show that B1-KD rendered A549 cells resistant to paclitaxel (PTX), phenocopying clinical studies showing that low B1 expression correlated with taxane resistance. As previously reported, we show that loss of B1 enhanced centrosomal γ-tubulin localization and microtubule nucleation. Interestingly, we found that the B1-KD cells exhibited increased microtubule dynamics as compared with parental A549 cells, as assessed by live-cell confocal microscopy using enhanced green fluorescent protein-tagged α-tubulin or EB1 protein. In addition, we showed that loss of B1 impairs the ability of PTX to induce microtubule polymerization using immunofluorescence microscopy and a cell-based tubulin polymerization assay. Furthermore, B1-KD cells exhibited significantly lower intracellular binding of a fluorescently labeled PTX to microtubules. Recent studies have shown that PTX-stabilized microtubules serves as a scaffold for pro-caspase-8 binding and induction of apoptosis downstream of induced-proximity activation of caspase-8. Here we show that loss of B1 reduces the association of pro-caspase-8 with microtubules and subsequently leads to impaired PTX-induced activation of apoptosis. Taken together, our data show that B1 regulates indirectly endogenous microtubule dynamics and stability while its loss leads to microtubules that are more dynamic and less susceptible to PTX-induced stabilization conferring taxane resistance.
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
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