Abstract 2896: 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity

Abstract

Abstract Taxane based chemotherapy is commonly used to treat a wide variety of solid tumors including breast, prostate, lung and ovarian. Taxanes bind to and stabilize microtubules inhibiting their function in interphase and mitosis, and leading to cancer cell apoptosis. Although taxanes are widely used clinically and their target, tubulin, is ubiquitously expressed, patient response is heterogeneous and the molecular mechanism of clinical drug resistance remains elusive. In addition to their antimitotic effects, taxanes affect several signaling pathways whose perturbation by microtubule stabilization may determine cell sensitivity or resistance. Our recent work has identified one such pathway by showing that Taxol treatment inhibits the translation of HIF-1α mRNA by inducing its sequestration to P-bodies where it is silenced. However, the signaling cascade that is initiated in response to microtubule damage leading to translation suppression of HIF-1α is not understood. Using polysome analysis we showed that Taxol treatment inhibited translation at the initiation step of protein synthesis, evidenced by the increase in the 80S peak. Post-translational modifications of various translation initiation factors are implicated in the regulation of translation initiation. Hence, we analyzed phosphorylation states of several initiation factors, including eIF4E, eIF2α, 4E-BP1, 4E-BP2 and rpS6. We didn't observe any change in the phosphorylation state of these initiation factors except for 4E-BP1. We observed that Taxol treatment led to 4E-BP1 hyper-phosphorylation, at a unique previously unrecognized, site. Using isogenic cell lines with acquired mutations at the taxane binding site we conclusively showed that microtubule disruption was required for the hyper-phosphorylation of 4E-BP1. Phosphorylation of 4E-BP1 is known to activate cap-dependent translation. Instead, our data using polysome profiling revealed that Taxol-induced 4E-BP1 hyper-phosphorylation was associated with translation suppression. We confirmed these results by performing the cap-binding assay, which demonstrated that Taxol treatment had no effect on cap-dependent translation. Taken together these results suggest a novel biological function for 4E-BP1 hyper-phosporylation. Further, treatment with the mTOR inhibitor, Torin-1, had no effect on Taxol-induced 4E-BP1 hyper-phosphorylation, suggesting mTOR pathway independence. Interestingly, treatment with the CDK inhibitor Roscovitine, prevented both Taxol-induced 4E-BP1 hyper-phosphorylation and Taxol-induced cell death. Although we do not yet know which of the three CDKs exerts the inhibitory effect on taxanes, our data suggest that there is a functional link between 4E-BP1 hyper-phosphorylation and taxane activity. We are currently investigating the role of CDK1, CDK2 and CDK5 in this pathway to decipher the novel biological function of 4E-BP1 hyper-phosphorylation. Citation Format: Prashant Khade, Paraskevi Giannakakou. 4E-BP1 hyper-phosphorylation senses microtubule damage and plays a critical role in taxane antitumor activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2896.