Abstract 4055: Class III β-tubulin and the cytoskeletal gateway of drug resistance in ovarian cancer

Abstract

Abstract Objective: Class III β-tublin (TUBB3) is a potent prognostic biomarker for ovarian cancer and other solid tumors. In this work we have discovered a novel role for TUBB3 as a gateway for cytoskeletal prosurvival signals. Methods: Through a genomic screening of a cell line made resistant to a drug whose activity is TUBB3 dependent, we have identified two GTPases (GBP1 and GNAI1) whose expression is strictly related to TUBB3 expression. Using the technique of Far Western Blotting, we have demonstrated that these GTPases physically interact with TUBB3. At variance of TUBB3, the two GTPAses can be overexpressed in vitro. Therefore, they served as prey in a high density protein microarray to identify protein kinases capable to interact with them. Results: Using transient overexpression the functional role of the two GTPases was ascertained, with GBP1 capable to activate the TUBB3 pathway and GNAI1 with an inhibitory function on its. The physical interaction between the two GTPases and cytoskeletal proteins were proven with Far Western Blot. At variance with TUBB3, the two GTPases can be expressed as recombinant proteins. Taken advantage of this fact, the two proteins served as prey on high density protein array containing over 9,000 human proteins. At least 18 protein kinases were capabe to interact with either GBP1 or GNAI1 or both. Among these kinases, we focused our attention on PIM1. PIM1:TUBB3 correlation was proven in ovarian cancer cell lines, both sensitive and resistant to chemotherapeutics, using coimmunoprecipiation experiments and again far western blotting. For the first time, we discovered that PIM1 is expressed in the microtubules and its expression is directly related to resistance to Paclitaxel and Cisplatin, but not to Epothilone-B. Importantly, PIM1 is overexpressed in cytoskeleton in microtubules when TUBB3 pathway is activated in hypoxic conditions. Finally, to analyze the translational value of our findings, we performed immunohistochemical analysis of TUBB3 and PIM1 expression in a clinical subset of 98 ovarian cancer patients. This approach revealed that double negative patients (TUBB3 and PIM1) have an overall survival (median 76 months) better than that noticeable in double positive (median 29 months). Conclusions: TUBB3 is commonly believed as a simple mechanism of Paclitaxel-resistance. Instead, this work demonstrates that TUBB3 is a gateway for prosurvival signals like PIM1 into the cytoskeleton. Ovarian cancer patients exhibiting expression of both TUBB3 and PIM1 have high probability of drug resistance and extreme short survival and should be carefully monitored for response to first line chemotherapy. 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 4055. doi:10.1158/1538-7445.AM2011-4055