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MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that microRNA-7 (miR-7) inhibits p21-activated kinase 1 (Pak1) expression, a widely up-regulated signaling kinase in multiple human cancers, by targeting the 3′-untranslated region (UTR) of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor, HoxD10, the loss of which leads to an increased invasiveness. HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively up-regulated whereas those of miR-7 and its upstream activator HoxD10 are progressively down-regulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased miR-7 expression but reduced Pak1 3′-UTR-luciferase activity and reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth, and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 plays a regulatory role in modifying the expression of miR-7 and, consequently, the functions of the miR-7-Pak1 pathway in human cancer cells. [Cancer Res 2008;68(20):8195–200]

Homeobox (Hox) genes are master regulatory genes that direct organogenesis and maintain differentiated tissue function. We previously reported that HoxD10 helps to maintain a quiescent, differentiated phenotype in endothelial cells by suppressing expression of genes involved in remodeling the extracellular matrix and cell migration. Here we investigated whether HoxD10 could also promote or maintain a differentiated phenotype in epithelial cells. We observed that HoxD10 expression is progressively reduced in epithelial cells as malignancy increases in both breast and endometrial tumors. Retroviral gene transfer to restore expression of HoxD10 in the malignant breast tumor cells MDA-MB-231 significantly impaired migration, and when these cells were cultured in a three-dimensional laminin-rich basement membrane (3DlrBM) model, they formed polarized, acinar structures. This phenotypic reversion was accompanied by decreased α3 integrin expression and reduced proliferation. Importantly, expression of HoxD10 in the MDA-MB-231 cells inhibited their ability to form tumors in mouse xenografts. Taken together, our results suggest that HoxD10 has tumor-suppressive functions for mammary epithelial cells.

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

We sought to elucidate the role of a potentially oncogenic microRNA, microRNA-10b (mir-10b), in mediating the pathology of glioblastoma multiforme (GBM). MicroRNAs are small noncoding RNAs that play a critical role in developmental and physiological processes and are implicated in the pathogenesis of several human diseases including cancer. MicroRNAs function by regulating target gene expression post-transcriptionally. We have previously assessed the expression pattern of 241 mature human microRNAs in the NCI60 panel of cell lines and in a set of corresponding normal tissues (Gaur et al., 2007, Cancer Research. 67(6):2456-68). We found that microRNA expression patterns mark specific biological characteristics of tumors and/or mediate biological activities important for their pathobiology. In CNS tumor-derived cell lines, we identified mir-10b as a candidate oncogene, as it is expressed at higher levels in human GBM lines an primary GBM tissue than it is in normal brain tissue. Using several target prediction algorithms we identified a member of the homeobox family, Homeobox D10 (HOXD10), as a potential target of mir-10b. Our in vitro studies demonstrate that down regulation of mir-10b in GBM-derived cell lines results in increased expression levels of HOXD10. Additionally, our data indicate that down-regulation of mir-10b in human GBM lines leads to decreased proliferation and decreased anchorage independent colony formation in soft agar. Importantly, using a xenograft model in immune-deficient nude mice, we have observed human that GBM lines in which mir-10b expression is inhibited exhibit decreased tumor formation and growth in vivo. Furthermore, the decreased colony formation in soft agar observed as a result of mir-10b inhibition in GBM-derived cell lines is significantly antagonized by expression of siRNAs complementary to HOXD10 mRNA. Also, over expression of HOXD10 in GBM cell lines results in differentiation and decreased proliferation. Further experiments are ongoing to identify the specific role of HOXD10 in mediating the pathologic characteristics of GBM. An understanding of the pathways mediated by HOXD10 signaling may provide novel opportunities for therapeutic intervention. The National Brain Tumor Society (ABG) and the Theodora B. Betz Foundation (MAI) supported this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2100.