Stathmin Depletion from Cells Lacking p53 Leads to Delayed Cell Cycle Progression and Apoptosis Due to Increased Microtubule Stability

Control of cell proliferation is greatly diminished in cancer cells. In order to understand how to prevent the uncontrolled proliferation of cancer cells, it is first important to understand how cells normally control proliferation. Stathmin, a microtubule regulatory protein, is over-expressed in many cancers and required for survival of several cancer lines. In a study of breast cancer cell lines, Alli et al. (Oncogene. 26:1003--12) proposed that stathmin is required for survival of cells lacking p53, but this hypothesis was not tested directly. Here we tested their hypothesis by examining cell survival in cells depleted of stathmin, p53 or both proteins. Comparing HCT116 colon cancer cell lines differing in TP53 genotype, stathmin depletion resulted in significant cell cycle delay and death only in cells lacking p53. As a second experimental system, we compared the effects of stathmin depletion from HeLa cells, which normally lack detectable levels of p53 due to expression of the HPV E6 protein. Stathmin depletion caused a large percentage of HeLa cells to both delay and die via apoptosis. Restoring p53, by depletion of HPV E6, rescued HeLa cells from stathmin-depletion induced delay and death. The stathmin-dependent survival of cells lacking p53 was not confined to cancerous cells because both proteins were required for survival of normal human fibroblasts.;Although, both p53 and stathmin must be lacking to lead to a G2 cell cycle delay, it is still unclear as to why we see this synergy. We hypothesize that stathmin depletion relays a signal via stabilization of the microtubule cytoskeleton, specifically in cells lacking p53. Alternatively, stathmin could have a function separate from its microtubule regulatory activity, and loss of this additional function could lead to cell cycle delay. As a test of our hypothesis, we examined whether microtubule stability is greatest in cells lacking both stathmin and p53. Results showed that HeLa cells depleted of both stathmin and p53 showed a large increase in acetylated microtubules (a marker of microtubule stability) and the rate of microtubule nucleation. Restoring p53 in these cells reduces both the amount of acetylated microtubules and the microtubule nucleation rate in stathmin depleted cells. To further test the link between stathmin depletion in cells lacking p53 and microtubules, the microtubule network was manipulated by either complete depolymerization, with nocodazole, or by restoring the microtubule network to normal levels by over-expressing stathmin truncations that affect the microtubule array (stathmin Delta101-149). Both of these treatments allowed cells to escape the G2 delay, while over-expression of a stathmin truncation that does not affect the microtubule array (stathmin Delta5-25) did not affect the G2 delay.;Our results demonstrate that stathmin is required for cell survival in cells lacking p53, suggesting that stathmin depletion could be used therapeutically to induce a G2 cell cycle delay and apoptosis in tumors without functional p53. Our results also indicate that the interphase microtubule cytoskeleton is a novel target for control of cell proliferation, particularly for the greater than 50% of human cancers that lack p53 because of either inactivating mutations or viral infection. Together the results described in this dissertation could lead to new methods to treat and kill cancer cells that specifically lack p53, while leaving healthy cells relatively unaffected.