1981

Septin 9 (Sept9) is a microtubule associated protein whose function in normal and cancer cells remains largely unknown. Our previous comparative cytogenetic analysis of Sept9 demonstrated that its locus was amplified in the form of double minute chromosomes and that it was overexpressed in mouse mammary tumors. The Sept9 locus is also amplified in human breast and ovarian tumors. These findings indicate that Sept9 is a putative oncogene. Sept9 is expressed as multiple isoforms that may result from alternative splicing of its mRNA. Based on the ability of Sept9 to bind microtubules and microfilaments, and the genomic amplification observed in cancer cells, we hypothesized that Sept9 might play a role in drug resistance. In particular, Sept9 could be involved in resistance to microtubule interacting drugs, such as Taxol that is used in the treatment of ovarian and breast cancers. Genomic and proteomic analyses on two pairs of Taxol-sensitive and -resistant human cell lines were performed. Using bi-dimensional gel electrophoresis and Western blotting, we found that the complex patterns of Sept9 isoform expression were significantly altered in the resistant cell lines when compared to their sensitive counterparts. The two resistant cell lines displayed almost superimposable patterns of Sept9 expression, despite their selection with two different microtubule-stabilizing agents and their different tissue origin (A540.EpoB40 selected with epothilone B, non-small lung carcinoma and MDA-MB-231.K20T, selected with Taxol, breast carcinoma). FISH analyses indicated no copy number gain compared to the ploidy of the cells. Because we did not detect any significant difference in total Sept9 expression levels in the resistant vs sensitive cells, differential expression of isoforms may have a role in the resistance phenotype. We are currently investigating, at the transcriptional and post-transcriptional levels, how these alterations occurred and if they alter sensitivity to Taxol. Based on a detailed bioinformatic examination of the Sept9 genomic locus, we identified stretches of high density CG di-nucleotides, that mapped at isoform transcription start sites, which could undergo cytosine methylation. Pyrosequencing analysis is being performed to precisely quantify differential levels of methylation in sensitive vs resistant cell lines. Identification by mass spectrometry of Sept9 isoforms expressed in these cells is ongoing. Our findings identify a novel candidate, Sept9, that may be involved in mechanisms of drug resistance and suggest that epigenetic mechanisms could regulate isoform expression in cancer resistant cells. These experiments will reveal critical insights into the regulation of Sept9 isoform expression and its role in cancer development and drug resistance.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA