MDM2, MDM2-C, and mutant p53 expression influence breast cancer survival in a multiethnic population.

PURPOSE: The purpose of the study was to examine the association between expression of mutant p53 (mtp53), full-length MDM2 (MDM2), and MDM2 isoform C (MDM2-C) and survival in multiethnic breast cancer patients. METHODS: A total of 787 invasive breast tumors included in a clinically annotated multiethnic population-based tissue microarray (TMA) were screened utilizing commercially available antibodies to p53 and MDM2, and a newly developed monoclonal antibody recognizing MDM2-C. RESULTS: Mutant p53 (mtp53) was more common in younger (< 50 years) breast cancer patients. Among the 787 cases included in the study, mtp53, MDM2, and MDM2-C expression were not significantly associated with risk of overall or breast cancer-specific mortality. However when associations within individual racial/ethnic groups (White, Japanese, and Native Hawaiian) were examined, expression of MDM2-C was found to be associated with lower risk of breast cancer-specific mortality exclusively for White patients HR 0.32, 95% CI 0.15-0.69 and mtp53 expression was associated with higher overall mortality in Japanese patients (HR 1.63, 95% CI 1.02-2.59). Also, Japanese patients positive for the joint expression of MDM2-C and mtp53 had a greater than twofold risk of overall mortality (HR 2.15, 95% CI 1.04-4.48); and White patients with positive MDM2-C and wild-type p53 expression (HR 0.28, 95% CI 0.08-0.96) were at lower risk of mortality when compared to patients with negative MDM2-C and wild-type p53 expression in their respective racial/ethnic group. CONCLUSION: Racial/ethnic differences in expression profiles of mtp53, MDM2, and MDM2-C and associations with breast cancer-specific and overall mortality. MDM2-C may have a positive or negative role in breast tumorigenesis depending on mtp53 expression.

Endogenous human MDM2-C is highly expressed in human cancers and functions as a p53-independent growth activator.

Human cancers over-expressing mdm2, through a T to G variation at a single nucleotide polymorphism at position 309 (mdm2 SNP309), have functionally inactivated p53 that is not effectively degraded. They also have high expression of the alternatively spliced transcript, mdm2-C. Alternatively spliced mdm2 transcripts are expressed in many forms of human cancer and when they are exogenously expressed they transform human cells. However no study to date has detected endogenous MDM2 protein isoforms. Studies with exogenous expression of splice variants have been carried out with mdm2-A and mdm2-B, but the mdm2-C isoform has remained virtually unexplored. We addressed the cellular influence of exogenously expressed MDM2-C, and asked if endogenous MDM2-C protein was present in human cancers. To detect endogenous MDM2-C protein, we created a human MDM2-C antibody to the splice junction epitope of exons four and ten (MDM2 C410) and validated the antibody with in vitro translated full length MDM2 compared to MDM2-C. Interestingly, we discovered that MDM2-C co-migrates with MDM2-FL at approximately 98 kDa. Using the validated C410 antibody, we detected high expression of endogenous MDM2-C in human cancer cell lines and human cancer tissues. In the estrogen receptor positive (ER+) mdm2 G/G SNP309 breast cancer cell line, T47D, we observed an increase in endogenous MDM2-C protein with estrogen treatment. MDM2-C localized to the nucleus and the cytoplasm. We examined the biological activity of MDM2-C by exogenously expressing the protein and observed that MDM2-C did not efficiently target p53 for degradation or reduce p53 transcriptional activity. Exogenous expression of MDM2-C in p53-null human cancer cells increased colony formation, indicating p53-independent tumorigenic properties. Our data indicate a role for MDM2-C that does not require the inhibition of p53 for increasing cancer cell proliferation and survival.

Splicing up mdm2 for cancer proteome diversity.

Cancer cells often have high expression of Mdm2. However, in many cancers mdm2 is alternatively spliced, with more than 40 mRNA variants identified. Many of the alternative spliced mdm2 mRNAs have the potential to encode truncated Mdm2 isoforms. These putative Mdm2 isoforms can theoretically increase the diversity of the cancer proteome. The 3 best characterized are Mdm2-A, Mdm2-B, and Mdm2-C. As described in this review, the exogenous expression of these isoforms results in paradoxical phenotypes of transformation-associated growth as well as the inhibition of growth. Interestingly, these Mdm2 isoforms contribute tumor-promoting capacity in p53-null backgrounds. Herein we describe how alternative splicing of mdm2 may result in Mdm2 protein products that alter signal transduction to promote tumorigenesis. The tumor promoting capacity of Mdm2 isoforms is discussed in the context of functions that do not require the inhibition of p53. When N-terminal portions of Mdm2 are missing, the biochemical functions encoded by exon 12 are proposed to become more important. This may result in growth promoting functions when wild-type p53 is absent or compromised. The p53-independent tumor promoting activity of Mdm2 is proposed to result from C-terminal biochemical contributions of DNA binding, RNA binding, nucleolar localization, and nucleotide binding.

One-pot synthesis of azaindoles via palladium-catalyzed alpha-heteroarylation of ketone enolates.

A convenient, one-pot method for the construction of a variety of azaindoles using simple ketones and haloaminopyridines is described.

Disruption of the p53-Mdm2 complex by Nutlin-3 reveals different cancer cell phenotypes.

INTRODUCTION: Mdm2 inhibits p53 transactivation by forming a p53-Mdm2 complex on chromatin. Upon DNA damage-induced complex disruption, such latent p53 can be activated, but in cells overexpressing Mdm2 because of a homozygous single nucleotide polymorphism at position 309 (T --> G) of mdm2, the complex is highly stable and cannot be disrupted by DNA damage, rendering p53 inactive. METHODS: To determine whether the p53 response phenotype is influenced differentially in cells with variable mdm2 genotypes, we compared responses to DNA damage and targeted p53-Mdm2 complex disruption by Nutlin-3 in the following wild-type p53 human cancer cell lines: A875 and CCF-STTG-1 (G/G for mdm2 SNP309), SJSA-1 (mdm2 genomic amplification and T/T for mdm2 SNP309), MCF-7 (estrogen-induced Mdm2 overexpression and T/G for mdm2 SNP309), ML-1 and H460 (T/T for mdm2 SNP309), and K562 (p53-null and T/G for mdm2 SNP309). We also examined mdm2 gene-splicing patterns in these lines by cloning and sequencing analyses. RESULTS: While Mdm2-overexpressing G/G cells were resistant to p53 activation by DNA damage, they were sensitive to Nutlin-3. Strikingly, the p53 G1 checkpoint in G/G cells was activated by Nutlin-3 but not by etoposide, whereas in other Mdm2-overexpressing cells, both drugs activated p53 and subsequent G1 arrest or apoptosis. cDNA clones lacking exons 5-9 were generated at a high frequency in cells overexpressing Mdm2. CONCLUSION: Nutlin-3 and DNA damage distinguish a differential phenotype in human cancer cells with G/G mdm2 SNP309 from other Mdm2 overexpressers. Categorization of the Mdm2 isoforms produced and their influence on p53 activity will help in characterization and treatment development for different cancers.