Abstract HOX genes are thought to play a role in regulating stem cells (SCs) during embryological development. Aberrant expression of HOX genes is seen in many cancers, including colorectal cancer (CRC). However, the mechanisms leading to aberrant HOX gene expression in CRC are not known. Because SCs are key to colon tumorigenesis, we hypothesized that (i) expression of specific HOX genes is different in malignant colonic SCs than in normal SCs and (ii) aberrant expression of these HOX genes is necessary for the tumor initiating ability of colon cancer SCs. Using real time PCR-based array analysis, we found that expression of many HOX genes are upregulated in colon carcinomas relative to normal colonic epithelium. We previously reported a ‘unique HOX gene signature’ for the SC enriched population; two-color microarray analysis showed that two HOX genes, HOXA4 and HOXD10 are expressed in the bottom part of the colonic crypt where SCs reside. Here, we confirmed this finding, and then, using immunohistochemistry, we validated it. HOXA4 and HOX D10 were expressed at the bottom part of the normal colonic crypt. Increased expression of these genes was also seen in colon carcinoma tissues. The results for colon carcinoma tissues were validated using real time PCR. As an independent method to validate the differential expression of HOX genes, we used flow cytometry and ALDEFLUOR to isolate, from a colon cancer cell line, SCs and non-SCs. HOXA4 and HOXD10 expression was then studied using real time PCR. HOXA4 was upregulated in SCs vs non-SCs. To determine whether these genes are differentially expressed in carcinomas relative to normal tissue, we co-stained tissues with antibodies against HOXA4 or HOXD10, and with SC markers, ALDH1 or CD166. We found that SCs express these HOX genes in the bottom part of the colonic crypt compared to non-SCs. In colon carcinoma tissues, not only was there co-staining, but also, the co-staining was increased relative to normal colonic epithelium. Our results implicate expression of HOXA4 and HOXD10 genes not only in the functioning of normal SCs, but also in the contribution of colon SCs to colon tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-102. doi:1538-7445.AM2012-LB-102