Alternative and aberrant messenger RNA splicing of the mdm2 oncogene in invasive breast cancer

mdm2 is part of a complex mechanism that regulates the expression of p53 as well as the function of Rb, p19ARF, and other genes. In humans, mdm2 dysre gulation is associated with gene amplification. This study was undertaken t o characterize altered mdm22 expression in a cohort of 38 invasive breast c ancers and 9 normal breast specimens. Reverse-transcription PCR with primer s spanning the entire open reading frame of the mdm2 gene in breast tissue RNA samples generated PCR products of full-length mdm2 (1526 bp) as web as smaller products (653, 281, 254, and 219 bp). Sequence analysis demonstrate d that the 653-bp product was an alternatively spliced product (defined as splicing at the exon/intron boundary consensus sites), whereas the 281, 254 , and 219 bp mdm2 products were aberrantly spliced products (splicing at si tes not considered to be exon/intron boundary sites). Reverse-transcription -PCR with normal breast tissue RNA samples yielded only the 1526-bp product in five samples and the 1526-bp product and the 653-bp product in four sam ples. The 653-bp alternatively spliced product was expressed in 21% of brea st cancers, and the smaller, aberrantly spliced mRNA products (281 bp, 254 bp, and/or 219 bp) were expressed in 26% of breast cancers. The protein pro ducts predicted by the alternatively spliced mRNAs and the aberrantly splic ed mRNAs lacked either the entire binding domain for p53 of the majority of the binding domain for p53, Immunohistochemical analysis of HER2/neu (c-er bB2), estrogen receptor, progesterone receptor, epidermal growth factor rec eptor, and p53 protein was performed. p53 sequence alterations were identif ied by mismatch detection and confirmed by p53 oligonucleotide microarray t echnology, An association was demonstrated between the expression of aberra ntly and/or alternatively spliced mdm2 mRNAs and a lack of progesterone: re ceptor. An association was also demonstrated between mdm2 aberrantly and/or alternatively expression products and the presence of p53 tumor suppressor gene mutations. mdma is transcribed from two different promoters: one, p53 -dependent, and the other, p53-independent. The 5' untranslated region of t he transcripts was evaluated to determine the promoter usage in each breast cancer specimen, No correlation was observed between mdm22 splice products and promoter usage. The presence of aberrant expression products of mdm2 i n breast cancer specimens was correlated with a shortened overall patient s urvival, These observations suggest that mdm2 expression is altered in inva sive breast cancer and is associated with more aggressive disease.