The long-term goal of the proposed research is to decipher the microtubule-regulated pathways in interphase and their potential aberrations in cancer in order to better understand the molecular basis of clinical sensitivity and resistance to taxanes and other microtubule inhibitors used in cancer chemotherapy. We have recently identified a novel mechanism by which the microtubule cytoskeleton, and its perturbation by taxanes regulates HIF-1? signaling axis and activity. Briefly, we showed that microtubule disruption stalls the translation of HIF- 1? by targeting HIF mRNA to cytoplasmic P-bodies which are sites of translational suppression. The objective here, is to identify what other cancer-specific mRNAs are regulated by microtubules as well as the signaling cascade that originates with microtubule damage and culminates with translational suppression. Our central hypothesis is that the clinical activity of taxanes stems primarily from their effects on interphase microtubules and the downstream pathways that become affected following drug-induced microtubule perturbation. In this proposal we focus on the interphase microtubule-dependent pathway of translational regulation. Elucidation of these pathways will not only help us understand the molecular basis of clinical taxane sensitivity and resistance, but it will also lead to the identification of novel targets for cancer therapy, whose therapeutic exploitation can greatly synergize with existing microtubule-based chemotherapy.
Specific Aim I : Identify which other mRNAs, in addition to HIF-1 ?, require dynamic microtubules for their translation and as such represent novel downstream targets of taxane activity. To do so we will: Ia. Perform polysome and ribosome profiling coupled with RNA-Seq in untreated and taxane-treated cells to identify which mRNAs change translational status, and how, following drug treatment. Ib. Perform RNA-Seq of Ago2-associated mRNAs in order to identify which mRNAs are enriched in P-body sites following taxane treatment. Ic. Perform bioinformatic analyses of the microtubule-regulated mRNAs in order to identify the underlying """"""""microtubule-susceptibility-recognition"""""""" sequence.
Specific Aim II : Identify the signaling pathways that link microtubule damage to P-body-HIF mRNA targeting. IIa. Perform HIF mRNA pull-down coupled with TOF/TOF-Mass Spectrometry in untreated and taxane-treated cells in order to identify the proteins that target HIF-1? mRNA to P-bodies following microtubule disruption. IIb. Assess the role of the identified proteins in taxane sensitivity and resistance by pathway perturbation, knock-down and over-expression experiments that will allow modulation of the microtubule-P-body-mRNA axis. IIc. Evaluate the cytoplasmic polyadenylation element binding protein (CPEB) as one candidate protein involved in HIF-1? mRNA P-body targeting.
Taxanes and other microtubule inhibitors are used for the treatment of a variety of solid tumors, however, the molecular basis of clinical drug resistance is not understood. With this proposal we seek to identify all proteins that are regulated by microtubules at the level of translation and as such represent downstream targets of taxanes. We will also investigate how these proteins/pathways are deregulated in patient samples in an effort to understand and correct clinical drug resistance.
|Antonarakis, Emmanuel S; Tagawa, Scott T; Galletti, Giuseppe et al. (2017) Randomized, Noncomparative, Phase II Trial of Early Switch From Docetaxel to Cabazitaxel or Vice Versa, With Integrated Biomarker Analysis, in Men With Chemotherapy-Naïve, Metastatic, Castration-Resistant Prostate Cancer. J Clin Oncol 35:3181-3188|