title: Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma.
abstract: Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor invitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.
authors.affiliation: Department of Dermatology, University of Colorado Denver, Aurora, CO 80045, USA. adil.anwar@ucdenver.edu
authors.forename: Adil
authors.initials: A
authors.lastname: Anwar
authors.forename: David A
authors.initials: DA
authors.lastname: Norris
authors.forename: Mayumi
authors.initials: M
authors.lastname: Fujita
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005047:Etoposide / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects; D017353:Gene Deletion; D019942:Genes, p16; D006207:Half-Life; D006801:Humans; D007783:Lactones / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008544:Melanocytes / Q000378:metabolism; D008545:Melanoma / Q000201:enzymology / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D055550:Protein Stability / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D007783:Lactones; D007976:Leupeptins; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C098869:clasto-lactacystin beta-lactone; D005047:Etoposide; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde