Differential response in p53 wild-type cells to p53 activation by DNA damage and Nutlin-3

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild-type tumor cell lines, such as HCT116 and U2OS, are sensitive to inhibition of Chk1 due to attenuated p21waf1 induction upon DNA damage. This study determined the cause of this attenuated p21waf1 protein induction. Neither the induction of p21waf1 mRNA nor protein half-life is sufficient to explain the low p21waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have suppressed p21waf1 mRNA translation. The level of p21waf1 was inversely related to expression of miR-93, miR-106b and miR-130b. This represents a novel mechanism for disruption of the p53-p21waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21waf1 expression may render some p53 wild-type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.;This attenuated p21waf1 induction was overcome in HCT116 and U2OS cells with the non-DNA damaging agent Nutlin-3, which induces p53 by disrupting binding to its negative regulator MDM2. Nutlin-3 circumvented the attenuated translation of p21waf1 mRNA by increasing the protein half-life which led to G1 and G2 arrest. Interestingly, the p21 waf1 protein half-life remained short in p53 wild-type MCF10A cells incubated with Nutlin-3; these cells achieve high p21waf1 levels through transcriptional upregulation. Consequently, all three p53 wild-type cells but not p53 mutant MDA-MB-231 cancer cells were protected from subsequent incubation with a combination of DNA damage plus a checkpoint inhibitor.;This thesis provides a molecular basis for how the p53 pathway may be rewired in p53 wild-type tumor cells to facilitate tumorigenesis. By understanding how the p53 pathway can be manipulated by tumor cells to their own advantage, these studies may identify novel targets or therapies for the treatment of cancer.