Microarray analysis identifies HoxA9 as a novel breast cancer progression gene

This study was designed to identify and characterize novel progression genes with differential expression between normal breast tissue and invasive ductal carcinoma tissue from the same individual. Fresh frozen tissue was collected and macrodissected with a pathologist to identify tissue that was 85% or greater homogeneous normal breast tissue or invasive carcinoma. Five matched tumors and normal pairs were examined using oligonucleotide expression microarrays. Extensive pathway mining identified HoxA9 as a novel progression target whose expression was significantly lower in the tumors compared to the matched normal adjacent tissue. RT-PCR confirmed a reduction in HoxA9 expression in 90% of breast cancers tested in a larger panel of normal and cancerous tissue and breast cancer cell lines.{09} Heteroduplex mutation testing did not identify any variants in the splice sites or coding regions of HoxA9 in the samples tested for HoxA9 gene expression. However, bisulfite sequencing and treatment of MCF-7 cells with a demethylation reagent indicates expression of HoxA9 may be regulated at least in part, by methylation and that the gene is partially methylated in normal breast tissues, suggesting imprinting. Overexpression of HoxA9 in MDA-MB-231 cells (which do not express HoxA9) led to the altered expression of a variety of genes, including some involved in cell cycle, cell adhesion and oncogenesis. Most interestingly, there was a dramatic increase in the expression of BRCA1 in cells overexpressing HoxA9. Further genomic analysis pinpointed a Hox consensus sequence in the promoter of this gene. Finally, MDA-MBA 231 cell lines overexpressing HoxA9 had a 2.9 fold decrease in invasiveness compared to cell lines containing a vector only construct. These findings combined with previous studies suggest that HoxA9 may play a role in tumor suppression, perhaps through a pathway involving BRCA1.