Abstract C32: Stathmin depletion from cells lacking p53 delays mitotic entry by increasing microtubule stability during interphase
Authors: Bruce Carney, Victoria Caruso, Lynne Cassimeris
Journal: Cancer Research (2011)

Abstract Microtubules (MTs) have been a successful target for control of cell proliferation; drugs such as vinblastine or taxol block cells in mitosis and induce apoptosis, but these drugs are indiscriminate, affecting both normal and cancerous cells. We are examining a pathway induced by depletion of the MT destabilizing protein, stathmin, that results in slowed proliferation and apoptosis only in cancer cells and thus has potential for targeted killing of cancer cells. Depletion of stathmin caused slower cell proliferation, a cell cycle delay during G2 of the cell cycle, and cell death by apoptosis only in cells lacking p53 (Carney and Cassimeris. 2010. Cancer Biology and Therapy 9: 699–709). We demonstrated this synergy in both Hela cells, where p53 could be restored by depletion of the HPV E6 protein, or in matched HCT116 colon cancer lines differing only in p53 genotype. We are working to identify the signal immediately downstream of stathmin depletion that is responsible for the G2 delay by addressing whether the cell cycle delay is signaled by increased MT stability, or by one of two other stathmin-binding proteins, STAT3 or p27Kip1. In support of increased MT stability serving as a signal relay, we find that a brief incubation in nocodazole, a MT-depolymerizing drug, is sufficient to abrogate the G2 cell cycle delay in both Hela and HCT116p53-/- cell lines. Expression of full-length stathmin or stathmin truncations, combined with depletion of endogenous stathmin, was used to further test the role of increased MT stability in causing a G2 delay. Expression of full length stathmin-GFP, or stathmin N-terminus (deleted of amino acids 101–149)-GFP was sufficient to reduce MT density and abrogate the G2 delay. In contrast, a truncation missing 20 amino acids at the N terminus (deleted of amino acids 5–25) did not depolymerize MTs and did not abrogate the G2 delay. Others previously demonstrated that STAT3 and p27Kip1 bind to stathmin's C-terminus, and this region was not necessary to abrogate the delay, making it unlikely that these proteins act downstream of stathmin depletion. Instead, our results indicate that increased MT stability, in cells lacking p53, stalls cells in G2 and delays entry into mitosis. The stathmin depletion-induced G2 delay is independent of a mitotic defect, since interphase duration, but not mitotic duration is lengthened significantly in stathmin-depleted cells. We find that interphase is approximately 5 hours longer in stathmin-depleted cells, measured either by the time between mitoses for cells growing asynchronously or by the time for cells released from an S phase block to reach mitosis. While our results point to increased MT stability as a signal delaying cell cycle progression, the nature of the signal is not yet known. Working upstream from the delay at mitotic entry, we find that stathmin depletion does not activate Chk1 and does not increase Wee1 level, making the pathways where these proteins function as unlikely contributors to delayed G2 progression. Instead, we find that Aurora A, Plk1 and Pak1 have reduced activity, as measured by reduced levels of activating phosphorylations. These data point to a model where increased MT stability blocks full activation of RhoA and its downstream targets to delay mitotic entry. Increased stable MTs may sequester components necessary for RhoA activation, or these stable MTs may be more difficult to reorganize into a mitotic spindle. Unraveling the remaining steps between stathmin depletion and cell cycle delay and/or apoptosis should identify targets for selective control of cancer cell proliferation and explain why MT stability caused by stathmin depletion and taxol addition induce delays at different cell cycle steps. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C32.