Use Of Independent Genome-Wide Assays To Discover Hoxa Signature In Colon And Rectal Cancers And Validate A Role In Tumorigenesis

e15014 Background: Rectal cancer differs from colon cancer in terms of prognosis. Here we present genome-wide expression analysis of 79 colon and rectal tumors and gene dosage analysis of 34 archival colorectal tumors in paraffin (FFPE). Methods: Expression data were acquired ( http://expo.intgen.org/geo/home.do ) from Affymetrix experiments analyzed with GeneSpring software version 7.3 (Silicon Genetics, CA). Expression data were normalized ’per chip’ normalized to the 50th percentile of all values; and ’per gene’ normalized to the median expression level across all samples. Expression Project for Oncology (expO) samples included colon (n=50) and rectum (n=29). Differentially expressed genes were identified by parametric test for which variances were not assumed equal (Welch ANOVA). Array comparative genomic hybridization (aCGH) were performed separately on an independent set of FFPE colorectal tumors (n=34) by 244 K microarrays for CGH, Agilent Technologies, CA and with platform embedded analyses tools of CGH Analytics software and ADM-1 bioinformatics. Functional assay by knockout of HOXA9 promoter by lentiviral sh-RNAi construct was performed on colon cancer cell lines shown to have increased HOXA9 dosage and expression. Results: The analysis of expO colon and rectal cancers generated 42 genes with significant differential expression, 26 showed an increased expression of > 2 fold in colon versus rectal samples. Transcription factor family HOXA9 was the most highly expressed (3.8 fold) in colon versus rectal cancer. The aCGH data showed a low level gain of the HOXA gene to be the most frequent dosage alteration (38%). Validation by IHC and qRT-PCR showed 80% and 76% concordance, respectively. Functional assay by sh-RNAi in cell lines (compared to vehicle only) showed marked decrease in cellular viability (40 to 60%), marked morphologic change and a significantly increased apoptotic rate. Conclusion: Multiple genome-wide assays have identified HOXA9 as differentially dosed and expressed in colon and rectal tumors. Compelling functional data from sh-RNAi experiments suggests a tumorigenic role for HOXA9 in altered apoptosis. No significant financial relationships to disclose.