Anti-tumor effect and destabilization of mutant p53 by CB002, a p53-pathway restoring small molecule that stimulates autophagy

Open Access
Hernandez Borrero, Liz Janice
Graduate Program:
Biomedical Sciences
Master of Science
Document Type:
Master Thesis
Date of Defense:
July 28, 2016
Committee Members:
  • Wafik El-Deiry, Thesis Advisor
  • Scot R Kimball, Committee Member
  • Lisa M Shantz, Committee Member
  • Rosalyn Bryson Irby, Committee Member
  • mutant p53
  • apoptosis
  • autophagy
  • p53 pathway restoration
  • small molecules
  • NOXA
Tumor suppressor p53 mediates genotoxic and cellular stress signals by controlling cell fate through transcriptional activation of genes involved in DNA repair, cell cycle arrest, cell senescence, and apoptosis. p53 is mutated in over half of human cancers and this is associated with tumor development and chemotherapy resistance. Mutations in p53 are mostly found in the DNA-binding domain and they prevent p53 to exert its normal tumor suppressive functions. Furthermore, p53 mutations can result in gain-of-function activity, acquiring oncogenic characteristics. Therefore, altering the stability of mutant p53 protein is an attractive therapeutic strategy in cancer cells. The current project aims to identify compounds that restore the p53 pathway and modulate mutant p53 protein. Accordingly, we used a luciferase based p53 reporter to screen for small molecules that restore the p53 pathway in mutant p53-bearing cancer cells. We identified a small molecule, CB002, as a candidate for restoration of the p53 pathway in mutant p53-harboring cancer cells. Colorectal cancer cell lines SW480 and DLD-1 were treated with different concentrations of CB002 at various time points. Cell lines exposed to CB002 showed an increase in p53 target gene expression (i.e. NOXA/DR5/P21) and apoptotic cell death markers (i.e. cleaved caspases and PARP), as early as 16 hrs. Moreover, we showed that CB002 selectively results in apoptotic cell death in cancer cells and not in WI38 normal human lung fibroblast cells as indicated by the Sub-G1 content. To explore the importance of the p53 target genes in apoptotic cell death, we knocked-down DR5 and NOXA proteins. Stable knockdown of NOXA abolished apoptotic cell death whereas DR5 did not, indicating that NOXA is required for CB002-mediated cell death. Following CB002 treatment, we observed an increase in the formation of vacuoles within treated cells. Thus, NOXA induction together with the formation of vacuoles prompted us to investigate if autophagy was playing a role in degradation of mutant p53. Autophagy induction was confirmed by LC3B conversion in cell lysates. CB002 decreased the stability of mutant p53 R175H in HCT116 and RXF393 cells. Although blocking autophagy did not rescue mutant p53 protein expression, induction of autophagy was required for cell death. We further investigated if CB002 was mediating mutant R175H p53 protein degradation through the ubiquitin proteasome system. R175H p53 mutant protein expression was largely rescued by the co-treatment of CB002 with MG132, a proteasomal inhibitor, implicating a role for the ubiquitin proteasome system. Altogether, our data suggests that CB002 stimulates apoptosis through the induction of NOXA and degrades mutant p53 R175H through the ubiquitin proteasome system. In addition, induction of autophagy by CB002 appears to be required for cell death. Hence, our results provide insight into effective p53 pathway activation through the use of small molecules.