cp-31398 and Colonic-Neoplasms

The p53 tumor suppressor gene encodes a homotetrameric transcription factor which is activated in response to a variety of cellular stressors, including DNA damage and oncogene activation. p53 mutations occur in >50% of human cancers. Although p53 has been shown to regulate Wnt signaling, the underlying mechanisms are not well understood. Here we show that silencing p53 in colon cancer cells led to increased expression of Aha1, a co-chaperone of Hsp90. Heat shock factor-1 was important for mediating the changes in Aha1 levels. Increased Aha1 levels were associated with enhanced interactions with Hsp90, resulting in increased Hsp90 ATPase activity. Moreover, increased Hsp90 ATPase activity resulted in increased phosphorylation of Akt and glycogen synthase kinase-3β (GSK3β), leading to enhanced expression of Wnt target genes. Significantly, levels of Aha1, Hsp90 ATPase activity, Akt, and GSK3β phosphorylation and expression of Wnt target genes were increased in the colons of p53-null as compared with p53 wild type mice. Using p53 heterozygous mutant epithelial cells from Li-Fraumeni syndrome patients, we show that a monoallelic mutation of p53 was sufficient to activate the Aha1/Hsp90 ATPase axis leading to stimulation of Wnt signaling and increased expression of Wnt target genes. Pharmacologic intervention with CP-31398, a p53 rescue agent, inhibited recruitment of Aha1 to Hsp90 and suppressed Wnt-mediated gene expression in colon cancer cells. Taken together, this study provides new insights into the mechanism by which p53 regulates Wnt signaling and raises the intriguing possibility that p53 status may affect the efficacy of anticancer therapies targeting Hsp90 ATPase.

Topics: Adenosine Triphosphatases; Animals; Colonic Neoplasms; Disease Models, Animal; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Li-Fraumeni Syndrome; Mice; Mice, Transgenic; Molecular Chaperones; Pyrimidines; Tumor Suppressor Protein p53; Wnt Signaling Pathway

The cancer stem cell hypothesis suggests that rare populations of tumor-initiating cells may be resistant to therapy, lead to tumor relapse and contribute to poor prognosis for cancer patients. We previously demonstrated the feasibility of p53 pathway restoration in p53-deficient tumor cell populations using small molecules including ellipticine or its derivatives. We now establish a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in human DLD1 colon tumor cells expressing mutant p53 protein. We use these p53-EGFP reporter DLD1 cells to investigate the status of p53 transcriptional activity in putative colon cancer stem cell populations following exposure to p53 pathway-restoring drugs and/or classical chemotherapy. We demonstrate induction of p53-specific EGFP reporter fluorescence following overexpression of p53 family member p73 by an Adenovirus vector. We further show that p53-reporter activity is induced in DLD1 putative cancer stem cell side-populations analyzed by their Hoechst dye efflux properties following treatment with the p53 pathway restoring drug ellipticine. Combination of ellipticine with the cytotoxic agent 5-fluorouracil resulted in increased cytotoxicity as compared to either agent alone and this was associated with depletion of putative cancer stem cell populations as compared with 5-FU alone treatment. Our results support the feasibility of therapeutic targeting of mutant p53 in putative cancer stem cells as well as the potential to enhance cytotoxic chemotherapy.

Topics: Adenocarcinoma; Antineoplastic Agents; Colonic Neoplasms; DNA-Binding Proteins; Drug Synergism; Ellipticines; Fluorouracil; Genes, p53; Genes, Reporter; Genes, Synthetic; Genetic Vectors; Humans; Mutation; Neoplasm Proteins; Neoplastic Stem Cells; Nuclear Proteins; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Recombinant Fusion Proteins; Tumor Protein p73; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

CP-31398, a styrylquinazoline, emerged from a high throughput screen for therapeutic agents that restore a wild-type-associated epitope (monoclonal antibody 1620) on the DNA-binding domain of the p53 protein. We found that CP-31398 can not only restore p53 function in mutant p53-expressing cells but also significantly increase the protein level and promote the activity of wild-type p53 in multiple human cell lines, including ATM-null cells. Cells treated with CP-31398 undergo either cell cycle arrest or apoptosis. Further investigation showed that CP-31398 blocks the ubiquitination and degradation of p53 but not in human papillomavirus E6-expressing cells. Of note, CP-31398 does not block the physical association between p53 and MDM2 in vivo. Moreover, unlike the DNA-damaging agent adriamycin, which induces strong phosphorylation of p53 on serines 15 and 20, CP-31398 exposure leads to no measurable phosphorylation on these sites. We found that CP-31398 could also stabilize exogenous p53 in p53 mutant, wild-type, and p53-null human cells, even in MDM2-null p53(-/-) mouse embryonic fibroblasts. Our results suggest a model wherein CP-31398-mediated stabilization of p53 may result from reduced ubiquitination, leading to high levels of transcriptionally active p53. Further understanding of this mechanism may lead to novel strategies for p53 stabilization and tumor suppression in cancers, even those with absent ARF or high MDM2 expression.

Topics: Adenocarcinoma; Animals; Ataxia Telangiectasia Mutated Proteins; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Damage; DNA-Binding Proteins; Doxorubicin; Female; Fibroblasts; Gene Expression Regulation; Genes, p53; Humans; Lung Neoplasms; Lymphocytes; Mice; Mice, Knockout; Neoplasm Proteins; Nuclear Proteins; Oncogene Proteins, Viral; Ovarian Neoplasms; Phosphorylation; Phosphoserine; Protein Binding; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Repressor Proteins; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Ubiquitin

p53 is considered the guardian of the genome and has a number of biological functions, including cell cycle arrest, DNA repair, and apoptosis. In a recent study by Foster and colleagues, the pharmacological compound CP-31398 was found to stabilize wild-type p53 to enhance its transcriptional activity and inhibit tumor growth in mice. We hypothesize that CP-31398 induces apoptosis by stabilizing the p53 protein and activating the mitochondrial-mediated pathway. Using the wild-type p53 HCT116+/+ and the p53-deficient HCT116-/- colon carcinoma cell lines, we demonstrate here that CP-31398 induces apoptosis in a dose-, time-, and p53-dependent manner. CP-31398 dramatically elevated p53 and p21(Waf1) protein levels in HCT116+/+, while a smaller p53-independent p21(Waf1) induction by CP-31398 in HCT116-/- cells was also observed. Moreover, we also found that CP-31398 increased Bax expression, altered mitochondrial membrane potential causing the release of cytochrome c, and induced the cleavage of caspases-9 and -3. Taken together, our results indicate that CP-31398 induces p53-dependent apoptosis by activating the Bax/mitochondrial/caspase-9 pathway. Elucidating the mechanism by which CP-31398 induces cell death may establish it as an anticancer agent.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma; Caspase 3; Caspase 9; Caspases; Cell Division; Cell Transformation, Neoplastic; Colonic Neoplasms; Cytochrome c Group; Eukaryotic Cells; Humans; Membrane Potentials; Mitochondria; Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Tumor Cells, Cultured; Tumor Suppressor Protein p53