Abstract 645: Class V β-tubulin expression as biomarker in solid tumors

Abstract

Abstract Microtubules are a component essential of the cytoskeleton and mediate a complex network of functions, including the assembly of mitotic spindle but also the intracellular trafficking of signaling proteins. In order to achieve their functions, composition of microtubules is tightly regulated, particularly at the (+) end, exposing the β-tubulin subunit. At least seven independent genes encode for β-tubulin, whose expression is diverse in several tissues. Functionally β-tubulin isotypes can be divided in constitutive isotypes and regulatory isotypes. Regulatory isotypes include class III β-tubulin (TUBB3), whose role as factor of resistance to chemotherapy has been demonstrated in a large number of solid tumors. Some structural motifs of TUBB3 are shared with class V β-tubulin (TUBB6), but since no commercial antibodies are available for this isotype its role as an additional factor of chemoresistance is not known. In this work we have developed a polyclonal anti-TUBB6 antibody. Since the highest degree of dishomology between the β-tubulin isotypes is concentrated at the C-terminus, all the tails of the β-tubulin isotypes were cloned in E. coli in a carrier vector encoding MBP (maltose-binding protein). Recombinant proteins were expressed in E. coli and then purified through columns filled with Amylose resin column, able to entrap MBP. The negative control was represented by MBP alone. The developed anti-TUBB6 antibody was validated since it demonstrated specific reactivity only against TUBB6. Therefore, the profile of expression of TUBB6 was assessed in a panel of normal and cancer tissues. In normal tissues, immunostaining of TUBB6 was confined to given cellular elements and in a focal pattern such as in ciliary cells of bronchus, salpinx, thereby suggesting that class V is focally associated to the cilium complex. In contrast to the focal expression noticed in normal tissues, in cancer specimens TUBB6 exhibited a diffuse staining pattern, with the highest expression detectable in colon and prostate carcinoma. In order to have some insights on the role of TUBB6 as a biomarker, we studied TUBB6 expression in a cohort of 56 ovarian cancer patients with a median follow up of 54 months. There was no statistically significant difference in terms of Time to Progression (TTP) in cases with high versus low TUBB6 expression (p value= 0.4). On the other hand, cases with high TUBB6 expression showed a worse Overall Survival (OS) with respect to cases with low TUBB6 expression (median OS= 50 months versus median OS= 96 months, respectively, p value=0.047). In summarizing these results, patients expressing high levels of TUBB6 are featured by biological aggressiveness and poor response in terms of OS to standard chemotherapy. These results suggest that TUBB6 could be useful as a biomarker for ovarian cancer patients and possibly other tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 645.