Abstract 4421: The ability of the microtubule-targeting agent T0070907 to induce tubulin degradation in colorectal cancer cell lines is affected by the microtubule-destabilizing but not the tubulin sequestering functions of stathmin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The compound T0070907 is a microtubule-targeting agent (MTA) that, unlike standard MTAs, induces proteasomal degradation of tubulin. Strikingly, tubulin loss occurs preferentially in tumor cells. One potential candidate for a tumor-specific function that affects the ability of T0070907 to induce tubulin loss is the microtubule-associated protein stathmin. Stathmin normally destabilizes microtubules and also sequesters tubulin in complexes unavailable for degradation. We therefore examined whether T0070907-induced stathmin inactivation is required for tubulin loss. Methods: 50μM T0070907 was added to the colorectal cancer cell lines HT-29, HCT-15, and SW620, and microtubule polymerization, tubulin loss, and stathmin phosphorylation measured at multiple time points by Western blot. When applicable, microtubules were depolymerized with 0.1 μM vinblastine or nocodazole or polymerized with 0.1 μM paclitaxel before addition of T0070907. Microtubule polymerization was quantified by cell lysis in microtubule stabilizing buffer followed by centrifugation to separate the insoluble microtubules from the soluble tubulin heterodimers. Stathmin levels were reduced using chemically synthesized stathmin-specific siRNAs; responsiveness to T0070907 was assessed after 72h knockdown. Results: T0070907 induced tubulin loss starting 8h after compound addition. Depolymerizing the microtubules eliminated the lag time before tubulin loss, while polymerizing the microtubules with paclitaxel prevented loss. T0070907 alone or in the presence of 25μM of the proteasome inhibitor MG132 increased the amount of tubulin found in the soluble, degradable fraction. T0070907 also increased phosphorylation of the stathmin regulatory ser16 site that normally inactivates both microtubule destabilizing and sequestering functions. Stathmin knockdown slowed the kinetics of T0070907-induced tubulin loss, as was expected given that stathmin normally destabilizes microtubules. However, stathmin knockdown did not increase the rate of T0070907-induced tubulin loss when microtubules were fully depolymerized with vinblastine, as would be expected if inactivation of the sequestering function were critical. Conclusion: T0070907-induced degradation requires microtubule depolymerization of the microtubules. T0070907 increases depolymerization, and also inactivates stathmin. However, this inactivation does not contribute to the ability of T0070907 to degrade tubulin, as inactivation of the stathmin sequestering function does not seem to be critical to T0070907's ability to degrade tubulin, and inactivation of the microtubule destabilizing function antagonizes tubulin degradation. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 4421.