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High-throughput microarray technologies were used to study DNA methylation accompanied by transcriptional changes in follicular lymphoma (FL). Using Methylated CpG Island Amplification with Microarrays (MCAM) to study CpG Island (CGI) DNA methylation in follicular lymphoma (FL) we discovered widespread hypermethylation of homeobox genes and previously identified targets of polycomb repressive complex 2 (PRC2) in cell lines and primary tumors, but not in benign follicular hyperplasia (BFH). DNA methylation for HOXA11, HOXD10, HOXB7, HOXC12, PAX6, LHX9, SFMBT2, EN2 and PAX7 was independently validated in the RL cell line and HOXA11, HOXD10, PAX6 and EN2 in primary tumors. Combined Bisulfite Restriction Analysis (COBRA) also established DNA methylation for the previously identified PRC2 targets DCC, DES, GAD2, AQP5, GPR61, GRIA4, GJD2 and AMPH in FL but not in BFH. Gene expression analyses revealed 411 genes that were hypermethylated and transcriptionally repressed in RL, 74% of which were re-activated by the demethylating agent 5-aza-2′-deoxycytidine (5-azaD) plus or minus the histone deacetylase inhibitor trichostatin A (TSA). Forty genes were also down-regulated in primary FL. Our results suggest that extensive hypermethylation in promoters of polycomb target genes is a characteristic of FL and that loss of expression of certain SUZ12 target genes could be functionally relevant for lymphomagenesis.

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.

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]

A central question in neural development is how the broad diversity of neurons is generated in the vertebrate CNS. We have investigated the function of Hoxc10 and Hoxd10 in mouse lumbar motoneuron development. We show that Hoxc10 and Hoxd10 are initially expressed in most newly generated lumbar motoneurons, but subsequently become restricted to the lateral division of the lateral motor column (lLMC). Disruption of Hoxc10 and Hoxd10 caused severe hindlimb locomotor defects. Motoneurons in rostral lumbar segments were found to adopt the phenotype of thoracic motoneurons. More caudally the lLMC and dorsal-projecting axons were missing, yet most hindlimb muscles were innervated. The loss of the lLMC was not due to decreased production of motoneuron precursors or increased apoptosis. Instead, presumptive lLMC neurons failed to migrate to their normal position, and did not differentiate into other motoneurons or interneurons. Together, these results show that Hoxc10 and Hoxd10 play key roles in establishing lumbar motoneuron columnar, divisional and motor pool identity.

As patient survival drops precipitously from early-stage cancers to late-stage and metastatic cancers, microRNAs that promote relapse and metastasis can serve as prognostic and predictive markers as well as therapeutic targets for chemoprevention. Here we show that miR-1269a promotes colorectal cancer (CRC) metastasis and forms a positive feedback loop with TGF-β signalling. miR-1269a is upregulated in late-stage CRCs, and long-term monitoring of 100 stage II CRC patients revealed that miR-1269a expression in their surgically removed primary tumours is strongly associated with risk of CRC relapse and metastasis. Consistent with clinical observations, miR-1269a significantly increases the ability of CRC cells to invade and metastasize in vivo. TGF-β activates miR-1269 via Sox4, while miR-1269a enhances TGF-β signalling by targeting Smad7 and HOXD10, hence forming a positive feedback loop. Our findings suggest that miR-1269a is a potential marker to inform adjuvant chemotherapy decisions for CRC patients and a potential therapeutic target to deter metastasis.

Hoxd10 is expressed in the posterior spinal cord and hindlimbs of the mouse. Hoxd10, along with other Hox transcription factors, is thought to regulate the activity of genes involved in nervous system patterning and motor neuron development, but little is known about the downstream targets regulated by this gene. cDNA microarrays were used to investigate the transcriptional network regulated by Hoxd10 in homozygous knockout animals. Sixty-nine genes were identified with altered expression levels in mutant spinal cords. Among these were genes involved in such diverse cellular events as cellular communication, cell cycle control, development and differentiation, and neuronal survival. The expression of some of these genes was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. Nine genes showed changes in expression of the same sign and similar magnitude using RT-PCR in Hoxd10 single mutant animals, with additional changes in expression seen in Hoxa10/Hoxd10 double mutant animals. In situ hybridization studies also demonstrated changes in expression consistent with microarray results. Analysis of putative promoter regions for Hox protein binding sites suggested that some genes may be direct Hoxd10 targets, whereas others likely are regulated through intermediate steps. Using cDNA microarrays to study a single gene knockout during critical developmental stages has identified a large number of genes regulated by Hoxd10, many of which would not have been approached as candidates for Hox gene regulation based on function or expression. © 2004 Wiley-Liss, Inc.

Glioblastoma multiforme (GBM), the most common primary brain tumor, is highly lethal despite the use of radiation and temozolamide-based (TMZ) chemotherapy. Even with therapy, median survival for patients remains approximately 15 months following diagnosis. While current treatments are effective in treating primary tumors, recurrent tumors are nearly universally fatal due to their increased aggressiveness and therapeutic resistance. Our understanding of the processes governing recurrence remains incomplete. GBM cells alter gene expression by epigenetic changes in response to therapy. These changes are thought to participate in generating recurrence. Here, we use genome-wide chromatin immunoprecipitation (ChIP) in parallel with DNA sequencing analyses (ChIP-seq) to show that EZH2, a powerful epigenetic regulator of histone methylation, preferentially binds to the homeobox D10 (HOXD10) promoter region following treatment with TMZ. This binding reduces the transcription and the protein level of HOXD10, an established tumor suppressor known to influence cell proliferation and invasion. Decreased HOXD10 expression correlates with elevated levels of MMP14 and MMP9, two well-established players in cancer cell invasion and motility. Thus, we propose that treatment with TMZ induces specific changes in EZH2 binding, which in turn elevates expression of MMP14 (2.8 fold increased p=0.007) and MMP9 (3 fold increased p=0.02) to enhance the invasive potential of GBM. This epigenetic process may underlie the genesis of recurrent GBM tumors. Our work demonstrates a novel interaction between EZH2 and HOXD10 and further highlights the importance of epigenetics in GBM recurrence.

MicroRNAs are emergent players of epigenetics that function as oncogenes or tumor suppressors and that have been implicated in regulating diverse cellular pathways. MiR-10b is an oncogenic microRNA involved in tumor invasion and metastasis in various cancers. Our data have shown that miR-10b is overexpressed in colorectal cancer samples in comparison with non-tumorous adjacent mucosa (p = 0.0025) and that it is associated with severe features such as tumor size >5 cm (p = 0.023), distant metastasis (p = 0.0022), non-differentiated tumors (p = 0.016), and vascular invasion (p = 0.01). Regarding the regulation of its expression, positive correlation between the loss of miR-10b and aberrant DNA methylation (p = 0.02) as well as a loss of TWIST-1 messenger RNA (p = 0.018) have been observed. Furthermore, expression analysis of the downstream miR-10b targets has shown that there are associations between low HOXD10 messenger RNA and E-cadherin protein levels (p

Single microRNA (miRNA) can regulate expression of several or multiple principal targets in a specific microenvironment. In different cellular contexts, the same miRNA may exhibit diverse functions, depending on the repertoire and stoichiometry of its direct mRNA targets. For instance, in breast cancer, microRNA-10b (miR-10b) promotes invasion and metastasis of tumor cells through post-transcriptional regulation of HOXD10. In contrast, in glioblastoma (GBM), the most common and malignant primary brain tumor, miR-10b promotes proliferation and prevents death of cancer cells by targeting cell cycle inhibitors and pro-apoptotic genes. Here, we discuss a unique role of miR-10b in cancer cell survival, in diverse tumor microenvironments.

// Daniel J. Sharpe 1 , Katy S. Orr 1 , Michael Moran 1 , Sharon J. White 2 , Stephen McQuaid 3 , Terence R. Lappin 1 , Alexander Thompson 1 and Jacqueline A. James 1,3 1 Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast 2 Pathology Department, Ninewells Hospital & Medical School, Dundee 3 The Northern Ireland Molecular Pathology Laboratory, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast and The Belfast Trust, Belfast City Hospital, Lisburn Road, Belfast Correspondence: Jacqueline A James, email: // Keywords : Head and Neck cancer; HOXD10; HOXD11; POU2F1; Biomarker Received : July 16, 2014 Accepted : September 15, 2014 Published : September 16, 2014 Abstract HOX genes are master regulators of organ morphogenesis and cell differentiation during embryonic development, and continue to be expressed throughout post-natal life. To test the hypothesis that HOX genes are dysregulated in head and neck squamous cell carcinoma (HNSCC) we defined their expression profile, and investigated the function, transcriptional regulation and clinical relevance of a subset of highly expressed HOXD genes. Two HOXD genes, D10 and D11 , showed strikingly high levels in HNSCC cell lines, patient tumor samples and publicly available datasets. Knockdown of HOXD10 in HNSCC cells caused decreased proliferation and invasion, whereas knockdown of HOXD11 reduced only invasion. POU2F1 consensus sequences were identified in the 5’ DNA of HOXD10 and D11 . Knockdown of POU2F1 significantly reduced expression of HOXD10 and D11 and inhibited HNSCC proliferation. Luciferase reporter constructs of the HOXD10 and D11 promoters confirmed that POU2F1 consensus binding sites are required for optimal promoter activity. Utilizing patient tumor samples a significant association was found between immunohistochemical staining of HOXD10 and both the overall and the disease-specific survival, adding further support that HOXD10 is dysregulated in head and neck cancer. Additional studies are now warranted to fully evaluate HOXD10 as a prognostic tool in head and neck cancers.

Background:\ud HOX gene expression is altered in many cancers; previous microarray revealed changes in HOX gene expression in head and neck squamous cell carcinoma (HNSCC), particularly HOXD10.\ud \ud Methods:\ud HOXD10 expression was assessed by qPCR and immunoblotting in vitro and by immunohistochemistry (IHC) in tissues. Low-expressing cells were stably transfected with HOXD10 and the phenotype assessed with MTS, migration and adhesion assays and compared with the effects of siRNA knockdown in high-HOXD10-expressing cells. Novel HOXD10 targets were identified using expression microarrays, confirmed by reporter assay, and validated in tissues using IHC.\ud \ud Results:\ud HOXD10 expression was low in NOKs, high in most primary tumour cells, and low in lymph node metastasis cells, a pattern confirmed using IHC in tissues. Overexpression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with knockdown causing reciprocal effects. There was no consistent effect on apoptosis. Microarray analysis identified several putative HOXD10-responsive genes, including angiomotin (AMOT-p80) and miR-146a. These were confirmed as HOXD10 targets by reporter assay. Manipulation of AMOT-p80 expression resulted in phenotypic changes similar to those on manipulation of HOXD10 expression.\ud \ud Conclusions:\ud HOXD10 expression varies by stage of disease and produces differential effects: high expression giving cancer cells a proliferative and migratory advantage, and low expression may support invasion/metastasis, in part, by modulating AMOT-p80 levels.

microRNAs are a family of endogenous, short, non-coding RNAs that play critical roles in regulating gene expression for key cellular processes in normal and abnormal physiology. microRNA-7 is a 23 nucleotide miRNA whose expression is tightly regulated and restricted predominantly to the brain, spleen and pancreas. Reduced levels of miR-7 have been linked to the development of cancer and metastasis. As a tumor suppressor, miR-7 functions to co-ordinately downregulate a number of direct (e.g. the epidermal growth factor receptor) and indirect (e.g. phospho-Akt) growth promoting targets to decrease tumor growth in vitro and in vivo. In addition, miR-7 can increase the sensitivity of treatment-resistant cancer cells to therapeutics and inhibit metastasis. These data suggest that replacement of miR-7 ('miRNA replacement therapy') for specific human cancers could represent a new treatment approach. This article is part of a Directed Issue entitled: The Non-coding RNA Revolution.

STUDY QUESTION Are other HOX genes, in addition to HOXA10, involved in endometrial receptivity? SUMMARY ANSWER The highly expressed HOXA9, HOXA11 and HOXD10 genes also appear to be involved in endometrial receptivity. WHAT IS KNOWN ALREADY Within the HOX family of homeobox transcription factor genes are the leading candidates for the regulation of embryonic implantation. A crucial role of HOXA10 in endometrial receptivity has been well established. STUDY DESIGN, SIZE, DURATION To identify HOX candidate genes, we performed data mining on all 39 human HOX genes in the 'Human body index' gene expression database of normal human tissue. The temporal and spatial expression pattern of four highly expressed HOX genes in the human endometrium was determined. To further investigate the function of these Hox genes, we used a robust in vivo mouse model in which we blocked maternal Hox gene expression. PARTICIPANTS/MATERIALS, SETTING AND METHODS Analysis of a gene expression profile set in the public domain consisting of 504 samples representing 95 different normal human tissues, showed that in addition to HOXA10, also HOXA9, HOXA11, HOXB6 and HOXD10 mRNA showed increased expression in the human endometrium (16 samples). The temporal and spatial expression pattern of these four HOX genes throughout the menstrual cycle was determined in the endometrium from 27 female patients eligible for IVF-embryo transfer with a normal cycle by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The role of maternal Hoxa9, Hoxa11 and Hoxd10 was assessed in a mouse implantation model by expression knockdown using RNA interference. Forty mice were transfected with Hoxa9-, Hoxa11- or Hoxd10-specific small hairpin RNA (shRNA) constructs or a vector control by injection into the uterine horn at Day 2 after vaginal plug detection (Day 1) (160 mice in total). The effects were examined by qRT-PCR and western blot at Day 4 and litter sizes counted at Day 9 of pregnancy. MAIN RESULTS AND THE ROLE OF CHANCE HOXA10, HOXA9, HOXA11 and HOXD10 all showed increased expression during the mid-secretory phase of the menstrual cycle (P < 0.01). Knockdown of Hoxa9, Hoxa11 and Hoxd10 in the murine uterus resulted in significantly reduced average implantation rates (P < 0.01) and, with regard to four Hox target genes, also correlated with a significantly increased empty spiracles homolog 2 (Emx2) and insulin-like growth factor binding protein-1 (Igfbp1), and decreased integrin β3 (Itgb3) and leukemia inhibitory factor (Lif), expression (P < 0.01). LIMITATIONS, REASONS FOR CAUTION Menstrual cycle stage was not confirmed by serum hormone analysis. We verified the absence of significant differences in stage-specific expression of the reference genes used in our study (ACTB/Actb and GAPDH/Gapdh) and therefore possible limitations of this approach were minimized. In addition, the translatability of our data from a mouse model to patients needs to be investigated further. WIDER IMPLICATIONS OF THE FINDINGS We provide evidence that three other HOX genes in addition to HOXA10 are involved in endometrial receptivity, and that part of their function is asserted through several known HOX target genes, suggesting the presence of a central HOX signal transduction pathway.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by the National Natural Science Foundation of China. The authors declare no conflict of interest.

Background The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. Results We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Conclusion Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.

Although they were discovered only in the early 1990s, many regulatory functions of microRNAs — naturally occurring short RNA sequences — have already been reported. The latest news is that they mediate cancer spread. A microRNA that may facilitate the movement of cells from one part of the embryo to another in its normal role has been found to be highly expressed in aggressive human breast cancers and to mediate breast cancer cell migration, invasion and metastasis. MicroRNAs, naturally occurring single-stranded RNA molecules involved in gene regulation, have been implicated previously in causing cancers, but this is the first report of a link to metastasis. The functional target of the microRNA appears to be the HOXD10 gene, one of the Hox gene family involved in directing embryo development.

OBJECTIVE Hox genes encode DNA transcription regulatory proteins that contain a conserved 61 amino acid protein called the homeodomain. Although best known for their role in cellular differentiation during embryonic development, aberrant expression of these genes has been associated with hematologic and solid neoplasms. The purpose of this study was to determine the relative expression of HOXD10 in human endometrial adenocarcinomas. METHODS mRNA was isolated from 7 normal endometrial specimens and 28 endometrial adenocarcinoma specimens. cDNA was synthesized using random hexamer primers. The expression of HOXD10 relative to beta-tubulin (internal control) was assessed by densitometric comparison of co-amplified Phosphorus-32 (32P) labeled gene products separated by agarose gel electrophoresis. Direct sequencing of purified HOXD10 polymerase chain reaction product was also performed. RESULTS The sequence of the purified HOXD10 product corresponds to the known DNA sequence reported in the National Institute of Health Gene Bank. mRNA expression of HOXD10 relative to beta-tubulin is significantly lower in endometrial carcinomas than in normal endometrium. Furthermore, the ratio of HOXD10 to beta-tubulin expression varies inversely with the histologic grade of the tumor (P = .0009). CONCLUSION Cancer is a multistep process involving the aberrant expression of genes that regulate cell growth and differentiation. Human HOXD10 gene expression is altered in endometrial carcinoma and varies with the histologic grade of differentiation. This observation supports the theory that homeobox genes play a role in oncogenesis.

The homeobox gene extradenticle (exd) acts as a cofactor of Hox function both in Drosophila and vertebrates. It has been shown that the distribution of the Exd protein is developmentally regulated at the post-translational level; in the regions where exd is not functional Exd is present only in the cell cytoplasm, whereas it accumulates in the nuclei of cells requiringexd function. We show that the subcellular localization of Exd is regulated by the BX-C genes and that each BX-C gene can prevent or reduce nuclear translocation of Exd to different extents. In spite of this negative regulation, two BX-C genes, Ultrabithorax andabdominal-A, require exd activity for their maintenance and function. We propose that mutual interactions between Exd and BX-C proteins ensure the correct amounts of interacting molecules. As theHoxd10 gene has the same properties as Drosophila BX-C genes, we suggest that the control mechanism of subcellular distribution of Exd found in Drosophila probably operates in other organisms as well.

Glioma is the most frequent primary brain tumor. Recently, the upregulation of microRNA (miR)-23a was found to be associated with glioma, but the molecular mechanism by which miR-23a promotes glioma growth remains to be unveiled. In the present study, we found that miR-23a was significantly upregulated in glioma tissues compared to their matched adjacent tissues. miR-23a was also highly expressed in glioma cell lines SHG44, U251 and U87 cells. Moreover, we identified homeobox D10 (HOXD10) as a novel target for miR-23a. The expression of HOXD10 was significantly reduced in glioma tissues and cell lines and miR-23a negatively regulates the protein expression of HOXD10 in U251 and U87 cells. We further showed that miRNA-23a promoted U251 and U87 cell invasion, at least partially, by directly targeting HOXD10 and further modulating MMP-14. These findings suggest that miR-23a may serve as a promising therapeutic target for glioma.

Our goal was to identify a unique gene expression signature for human colonic stem cells (SCs). Accordingly, we determined the gene expression pattern for a known SC-enriched region--the crypt bottom. Colonic crypts and isolated crypt subsections (top, middle, and bottom) were purified from fresh, normal, human, surgical specimens. We then used an innovative strategy that used two-color microarrays (∼18,500 genes) to compare gene expression in the crypt bottom with expression in the other crypt subsections (middle or top). Array results were validated by PCR and immunostaining. About 25% of genes analyzed were expressed in crypts: 88 preferentially in the bottom, 68 in the middle, and 131 in the top. Among genes upregulated in the bottom, ∼30% were classified as growth and/or developmental genes including several in the PI3 kinase pathway, a six-transmembrane protein STAMP1, and two homeobox (HOXA4, HOXD10) genes. qPCR and immunostaining validated that HOXA4 and HOXD10 are selectively expressed in the normal crypt bottom and are overexpressed in colon carcinomas (CRCs). Immunostaining showed that HOXA4 and HOXD10 are co-expressed with the SC markers CD166 and ALDH1 in cells at the normal crypt bottom, and the number of these co-expressing cells is increased in CRCs. Thus, our findings show that these two HOX genes are selectively expressed in colonic SCs and that HOX overexpression in CRCs parallels the SC overpopulation that occurs during CRC development. Our study suggests that developmental genes play key roles in the maintenance of normal SCs and crypt renewal, and contribute to the SC overpopulation that drives colon tumorigenesis.

Homeobox genes, including HOX and non-HOX genes, have been identified to be expressed aberrantly in solid tumors. In gastrointestinal (GI) cancers, most studies have focused on the function of non-HOX genes including caudal-related homeobox transcription factor 1 (CDX1) and CDX2. CDX2 is a crucial factor in the development of pre-cancerous lesions such as Barrett’s esophagus or intestinal metaplasia in the stomach, and its tumor suppressive role has been investigated in colorectal cancers. Recently, several HOX genes were reported to have specific roles in GI cancers; for example, HOXA13 in esophageal squamous cell cancer and HOXB7 in stomach and colorectal cancers. HOXD10 is upregulated in colorectal cancer while it is silenced epigenetically in gastric cancer. Thus, it is essential to examine the differential expression pattern of various homeobox genes in specific tumor types or cell lineages, and understand their underlying mechanisms. In this review, we summarize the available research on homeobox genes and present their potential value for the prediction of prognosis in GI cancers.

Background: Four posterior Hoxd genes, from Hoxd13 to Hoxd10, are collectively regulated during the development of tetrapod digits. Besides the well-documented role of Hoxd13, the function of the neighboring genes has been difficult to evaluate due to the close genetic linkage and potential regulatory interferences. We used a combination of five small nested deletions in cis, involving from two to four consecutive genes of the Hoxd13 to Hoxd9 loci, in mice, to evaluate their combined functional importance. Results: We show that deletions leading to a gain of function of Hoxd13, via regulatory re-allocation, generate abnormal phenotypes, in agreement with the dominant negative role of this gene. We also show that Hoxd10, Hoxd11, and Hoxd12 all seem to play a genuine role in digit development, though less compelling than that of Hoxd13. In contrast, the nearby Hoxd9 contributed no measurable function in digits. Conclusions: We conclude that a slight and transient deregulation of Hoxd13 expression can readily affect the relative lengths of limb segments and that all posterior Hoxd genes likely contribute to the final limb morphology. We discuss the difficulty to clearly assess the functional share of individual genes within such a gene family, where closely located neighbors, coding for homologous proteins, are regulated by a unique circuitry and all contribute to shape the distal parts of our appendages. Developmental Dynamics 241:792-802, 2012. © 2012 Wiley Periodicals, Inc.

Background: Hox transcription factors play a critical role in the specification of motoneuron subtypes within the spinal cord. Our previous work showed that two orthologous members of this family, Hoxd10 and Hoxd11, exert opposing effects on motoneuron development in the lumbosacral (LS) spinal cord of the embryonic chick: Hoxd10 promotes the development of lateral motoneuron subtypes that project to dorsal limb muscles, while Hoxd11 represses the development of lateral subtypes in favor of medial subtypes that innervate ventral limb muscles and axial muscles. The striking degree of homology between the DNA-binding homeodomains of Hoxd10 and Hoxd11 suggested that non-homeodomain regions mediate their divergent effects. In the present study, we investigate the relative contributions of homeodomain and non-homeodomain regions of Hoxd10 and Hoxd11 to motoneuron specification. Results: Using in ovo electroporation to express chimeric and mutant constructs in LS motoneurons, we find that both the homeodomain and non-homeodomain regions of Hoxd10 are necessary to specify lateral motoneurons. In contrast, non-homeodomain regions of Hoxd11 are sufficient to repress lateral motoneuron fates in favor of medial fates. Conclusions: Together, our data demonstrate that even closely related Hox orthologues rely on distinct combinations of homeodomain-dependent and -independent mechanisms to specify motoneuron identity. Developmental Dynamics 241:718–731, 2012. © 2012 Wiley Periodicals, Inc.

Hox genes, a highly conserved subgroup of the homeobox superfamily, have crucial roles in development, regulating numerous processes including apoptosis, receptor signalling, differentiation, motility and angiogenesis. Aberrations in Hox gene expression have been reported in abnormal development and malignancy, indicating that altered expression of Hox genes could be important for both oncogenesis and tumour suppression, depending on context. Therefore, Hox gene expression could be important in diagnosis and therapy.

MicroRNAs (miRNAs) are increasingly implicated in regulating metastasis. Despite progress in silencing miRNAs in normal tissues of rodents and non-human primates, the development of effective approaches for sequence-specific inhibition of miRNAs in fast-growing tumors remains a significant scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirs – a class of chemically modified anti-miRNA oligonucleotides – suppresses breast cancer metastasis. Silencing of miR-10b both in vitro and in vivo with antagomirs significantly decreases miR-10b levels and increases levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but instead markedly suppresses formation of lung metastases. This metastasis-suppressing effect is sequence-specific. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate and a starting point for the development of new anti-metastasis agents.

Small non-coding microRNAs (miRNAs) contribute to cancer development and progression, and are differentially expressed in normal tissues and cancers. However, the specific role of miRNAs in the metastatic process is still unknown. To seek a specific miRNA expression signature characterizing the metastatic phenotype of solid tumours, we performed a miRNA microarray analysis on 43 paired primary tumours (ten colon, ten bladder, 13 breast, and ten lung cancers) and one of their related metastatic lymph nodes. We identified a metastatic cancer miRNA signature comprising 15 overexpressed and 17 underexpressed miRNAs. Our results were confirmed by qRT-PCR analysis. Among the miRNAs identified, some have a well-characterized association with cancer progression, eg miR-10b, miR-21, miR-30a, miR-30e, miR-125b, miR-141, miR-200b, miR-200c, and miR-205. To further support our data, we performed an immunohistochemical analysis for three well-defined miRNA gene targets (PDCD4, DHFR, and HOXD10 genes) on a small series of paired colon, breast, and bladder cancers, and one of their metastatic lymph nodes. We found that the immunohistochemical expression of these targets significantly follows the corresponding miRNA deregulation. Our results suggest that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets.

MicroRNAs (miRNAs) are small noncoding RNAs with regulatory functions, which play an important role in breast cancer. Several studies have shown that miRNAs can act either as tumor suppressors or as oncogenes, and that measurement of miRNA expression in malignancies may have diagnostic and prognostic implications. This article highlights a series of three recent studies that prove the involvement of miRNAs in breast cancer metastases. The first proves that miR-10b indirectly activates the pro-metastatic gene RHOC by suppressing HOXD10, thus leading to tumor invasion and metastasis. The second proves that miR-373 and miR-520c can also promote tumor invasion and metastasis, at least in part by regulating the gene CD44. The third identifies miR-335, miR-206, and miR-126 as suppressors of breast cancer metastasis. Loss of miR-335 leads to the activation of SOX4 and TNC (encoding tenascin C), which are responsible for the acquisition of metastatic properties. Altogether, these remarkable findings are important for our understanding of malignant transformation in the breast and may have implications for the management of patients with advanced breast cancer. The use of miRNAs as anticancer therapeutic agents is promising, and such fine molecular studies certainly help in bringing miRNAs closer to clinical practice.

Gene expression profiling of rheumatoid arthritis (RA) and osteoarthritis (OA) joint tissue samples provides a unique insight into the gene signatures involved in disease development and progression. Fibroblast-like synovial (FLS) cells were obtained from RA, OA and control trauma joint tissues (non-RA, non-OA) and RNA was analyzed by Affymetrix microarray. Thirty-four genes specific to RA and OA FLS cells were identified (P0.05). HOXD10, HOXD11, HOXD13, CCL8 and LIM homeobox 2 were highly and exclusively expressed in RA and CLU, sarcoglycan-gamma, GPR64, POU3F3, peroxisome proliferative activated receptor-gamma and tripartite motif-containing 2 were expressed only in OA. The data also revealed expression heterogeneity for patients with the same disease. To address disease heterogeneity in RA FLS cells, we examined the effects of clinical disease parameters (Health Assessment Questionnaire (HAQ) score, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF)) and drug therapies (methotrexate/prednisone) on RA FLS cell gene expression. Eight specific and unique correlations were identified: human leukocyte antigen (HLA)-DQA2 with HAQ score; Clec12A with RF; MAB21L2, SIAT7E, HAPLN1 and BAIAP2L1 with CRP level; RGMB and OSAP with ESR. Signature RA FLS cell gene expression profiles may provide insights into disease pathogenesis and have utility in diagnosis, prognosis and drug responsiveness.

The genetic etiology of idiopathic clubfoot is unknown. There have been cases reported in which both clubfoot and vertical talus appears in the same family; therefore, the genes responsible for vertical talus are reasonable candidates for idiopathic clubfoot. A mutation in HOXD10 was previously identified in a family with isolated congenital vertical talus. To determine whether HOXD10 is involved in the etiology of idiopathic clubfoot, HOXD10 coding and 5' and 3' untranslated regions were resequenced in 190 patients (177 with clubfoot, 10 with sporadic vertical talus, and 3 with both clubfoot and vertical talus), and 160 ethnically matched control subjects. Rare nonsynonymous HOXD10 amino acid substitutions (Leu154Val, Asn202Lys, and Thr175Ala), likely benign variants, were all detected once in patients and control subjects. Nucleotide substitutions were also identified in HOXD10 intronic and 3' untranslated regions, but were not more frequent in cases compared to controls. To investigate the possibility that unsequenced regulatory regions play a role in this disorder, we performed linkage analysis with markers on chromosome 2q near HOXD10 in one large family. We found no evidence of linkage near the HOXD gene cluster on chromosome 2q, suggesting genes other than HOXD10 are responsible for idiopathic clubfoot.

Background Biologics are an important subset of drugs used to treat rheumatoid arthritis (RA); however, up to 40% of patients have an inadequate response. Precision medicine aims to identify biomarkers that predict sub-populations of patients likely to respond to a given drug in order to optimise patient benefit. MicroRNAs (miRNAs) are small non-coding post-transcriptional regulators of gene expression. Dysregulation of several miRNAs have been implicated in RA and as such have been suggested as potential markers of disease activity. However, little research has been conducted into the potential clinical use of such biomarkers in predicting treatment response. We hypothesise that miRNA expression in pre-treatment serum samples of RA patients will provide such a biomarker. Objectives To identify miRNA biomarkers in pre-treatment serum samples with sufficient power to differentiate between subsequent responder (R) and non-responder (NR) RA patients to the biologic drug, etanercept. Methods Fifteen patients about to undergo treatment with etanercept were selected based on their EULAR response at 3-months from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort. This equated to 6 extreme NR (disease activity score in 28-joints (DAS28) >5.1 and improvement 1.2 at follow-up); all were female and receiving concurrent methotrexate. MicroRNA was extracted from pre-treatment serum and expression of 754 miRNAs was measured on the TaqMan OpenArray Human miRNA Panel on the QuantStudio™ Real-Time qPCR System (Applied Biosystems). Raw data were processed using the Bioconductor package HTqPCR. Briefly, raw cycle threshold (C T ) values were normalised to an endogenous spike-in control miR-ath159a using the deltaCt method and differential expression (DE) was conducted between R and NR. Results Of the 754 miRNAs assayed, 405 were not expressed in any of the samples and were removed from further analysis; miRNAs were also excluded based on an amplification quality score (good amplification indicated by a score >1.1). This left 241 miRNAs for DE analysis. One circulating miRNA, miR-10b, was differentially expressed in pre-treatment samples (p-value =0.004), demonstrating higher relative expression in R vs. NR. A number of validated targets for miR-10b are pertinent to RA, including NOTCH1, a gene with a reported role in TNF induced proliferation of RA synoviocytes; SMRT, a negative regulator of interstitial MMP-1 synthesis and HOXD10, a transcription factor exclusively expressed in RA fibroblast-like synoviocytes (miRTarBase). Conclusions Pilot data from this preliminary study suggests higher expression of miR-10b in pre-treatment RA serum is predictive of subsequent response to etanercept by 3 months. Although the findings require replication, the presence of a miRNA which is known to interact with RA related target genes adds support that circulating miRNAs could be useful predictive markers of response to etanercept in RA patients. Acknowledgements SLS is a PhD student funded by an investigator-initiated award to AB from Pfizer (award WS1940162). Disclosure of Interest None declared

A spontaneous semidominant mutation (Ironside, Irn) was isolated in mice, leading to severe hindlimb paralysis following multiple deletions in cis at the HoxD locus. To understand its cellular and molecular etiology, we embarked on a comparative analysis using systematic HoxD cluster deletions, produced via targeted meiotic recombination (TAMERE). Different lines of mice were classified according to the severity of their paralyses, and subsequent analyses revealed that multiple causative factors were involved, alone or in combination, in the occurrence of this pathology. Among them are the loss of Hoxd10 function, the sum of remaining Hoxd gene activity, and the ectopic gain of function of the neighboring gene Evx2, all contributing to the mispositioning, the absence, or misidentification of specific lumbo-sacral pools of motoneurons, nerve root homeosis, and hindlimb innervation defects. These results highlight the importance of a systematic approach when studying such clustered gene families, and give insights into the function and regulation of Hox and Evx2 genes during early spinal cord development.

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.

Homeobox genes represent a class of transcription factors important in embryogenesis, organogenesis, cell growth and differentiation, and cell migration. However, there is little known about their role in regulating endothelial cell (EC) phenotype in response to proangiogenic factors secreted by breast cancer, although at least two homeobox genes (HOXD3 and HOXD10) have been implicated in inducing the angiogenic phenotype in ECs. We are therefore testing the hypothesis that the homeobox gene Gax regulates breast cancer-induced angiogenesis through its ability to regulate the expression of downstream target genes in ECs. Using in vitro tube formation assays, we have found that Gax expression inhibits in vitro angiogenesis. Moreover, by quantitative real time reverse transcriptase real time PCR, we have found that Gax expression is downregulated by proangiogenic factors, while cDNA micorarray analysis demonstrates that Gax downregulates pro-angiogenic adhesion molecules in ECs and upregulates the cyclin-dependent kinase inhibitor p19INK4D. More importantly, Gax expression downregulates NF(-x)B activity in ECs. These observations will allow us to study the mechanism of Gax-mediated activation or repression of their expression to be studied and will form the basis for future studies that will examine in more detail the mechanism by which Gax activates downstream target genes and the detailed signaling pathways involved in this activation. Given the profound effect Gax has on endothelial cell activation, it is likely that these studies will identify new molecular targets for the antiangiogenic therapy of breast cancer. Ultimately, these same techniques will be applied to other homeobox genes implicated in regulating EC phenotype during breast cancer-induced angiogenesis.

Expression from the mouse Ren-1(c) gene in As4.1 cells is dependent on a proximal promoter element (PPE) located at approximately -60 and a 241-base pair enhancer region located at -2625 relative to the transcription start site. The PPE (TAATAAATCAA) is identical to a consensus HOX.PBX binding sequence. Further, PBX1b has been shown to be a component of a PPE-specific binding complex present in nuclear extracts from As4.1 cells. The binding affinities of different paralog HOX members to the PPE were examined in the absence or presence of PBX1b. HOXB6, -B7, and -C8 failed to bind the PPE alone but showed weak affinity in the presence of PBX1b. In contrast, HOXD10 and to a lesser degree HOXB9 bound the PPE with high affinities regardless of whether PBX1b was present. Abd-B HOX members, including HOXD10, -A10, -A9, -B9, and -C9, are expressed in As4.1 cells. The ability of HOX and PBX1b to form a ternary complex with PREP1 on the PPE is also demonstrated both in vivo and in vitro. Point mutations in either the HOX or PBX half-site of the PPE disrupted the formation of the HOX.PBX complex and dramatically decreased transcriptional activity of the Ren-1(c) gene demonstrating that both the HOX and PBX half-sites are critical for mouse renin gene expression. These results strongly implicate Abd-B class Hox genes and their cofactors as major determinants of the sites of renin expression.

The most 5′ mouse Hoxa and Hoxd genes, which occupy positions 9–13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.

The five most 5′ HoxD genes, which are related to the Drosophila Abd-B gene, play an important role in patterning axial and appendicular skeletal elements and the nervous system of developing vertebrate embryos. Three of these genes, Hoxd11, Hoxd12, and Hoxd13, act synergistically to pattern the hindlimb autopod. In this study, we examine the combined effects of two additional 5′ HoxD genes, Hoxd9 and Hoxd10. Both of these genes are expressed posteriorly in overlapping domains in the developing neural tube and axial mesoderm as well as in developing limbs. Locomotor behavior in animals carrying a double mutation in these two genes was altered; these alterations included changes in gait, mobility, and adduction. Morphological analysis showed alterations in axial and appendicular skeletal structure, hindlimb peripheral nerve organization and projection, and distal hindlimb musculature. These morphological alterations are likely to provide the substrate for the observed alterations in locomotor behavior. The alterations observed in double-mutant mice are distinct from the phenotypes observed in mice carrying single mutations in either gene, but exhibit most of the features of both individual phenotypes. This suggests that the combined activity of two adjacent Hox genes provides more patterning information than activity of each gene alone. These observations support the idea that adjacent Hox genes with overlapping expression patterns may interact functionally to provide patterning information to the same regions of developing mouse embryos.

Homeobox D10 (HoxD10) plays important roles in the differentiation of embryonic cells and progression of breast cancer. Our previous report revealed that insulin-like growth factor binding protein-3 (IGFBP3) was regulated by HoxD10 in gastric cancer cells; however, the functional roles and underlying mechanisms of IGFBP3 in gastric cancer remain unclear. Here, we found that the expression of IGFBP3 were upregulated after ectopic expression of HoxD10 in gastric cancer cells. Chromatin immunoprecipitation assay showed that HoxD10 bound to three potential regions of IGFBP3 promoter. Exogenous HoxD10 significantly enhanced the activity of luciferase reporter containing these binding regions in gastric cancer cells. Further data showed that all of these binding sites had Hox binding element “TTAT”. Immunohistochemical staining results revealed that IGFBP3 expression was significantly downregulated in 86 gastric adenocarcinomas tissues relative to their adjacent non-cancerous tissues (p

Glioblastoma multiforme (GBM) is a heterogeneous disease despite its seemingly uniform pathology. Deconvolution of The Cancer Genome Atlas's GBM gene expression data has unveiled the existence of distinct gene expression signature underlying discrete GBM subtypes. Recent conflicting findings proposed that microRNA (miRNA)-10b exclusively regulates glioma growth or invasion but not both. We showed that silencing of miRNA-10b by baculoviral decoy vectors in a glioma cell line resembling the mesenchymal subtype of GBM reduces its growth, invasion and angiogenesis while promoting apoptosis in vitro. In an orthotopic human glioma mouse model, inhibition of miRNA-10b diminishes the invasiveness, angiogenicity and growth of the mesenchymal subtype-like glioma cells in the brain and significantly prolonged survival of glioma-bearing mice. We demonstrated that the pleiotropic nature of miRNA-10b was due to its suppression of multiple tumor suppressors, including TP53, FOXO3, CYLD, PAX6, PTCH1, HOXD10 and NOTCH1. In particular, siRNA-mediated knockdown experiments identified TP53, PAX6, NOTCH1 and HOXD10 as invasion regulatory genes in our mesenchymal subtype-like glioma cells. By interrogating the REMBRANDT, we noted that dysregulation of many direct targets of miRNA-10b was associated with significantly poorer patient survival. Thus, our study uncovers a novel role for miRNA-10b in regulating angiogenesis and suggests that miRNA-10b may be a pleiotropic regulator of gliomagenesis.

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.

CONCLUSIONS: NF-kappaB inhibition at the time of reperfusion, by antagonism of both the proteasome and IKK-beta, attenuates myocardial infarction. These data support the emerging role of targeted NF-kappaB therapy not only during periods of controlled IR as seen with cardiac surgery, but its delayed use for patients presenting with unknown periods of ischemia as seen with acute coronary syndromes.

The acute myeloid leukemias (AMLs) are a heterogeneous group of hematological malignancies with diverse clinical outcomes. Pre-treatment karyotype analysis identifies biologically distinct subgroups and is currently used as a predictor of response to induction chemotherapy and risk of relapse. Cases may be stratified into one of three prognostic groups as follows: relatively favorable prognosis [t(8;21), t(15;17) and inv(16)]; adverse prognosis [−5/del(5q), −7, abnormalities of chromosome 3q and complex karyotype]; and intermediate prognosis [remainder including normal karyotype]. HOX genes encode master transcription factors which regulate key developmental processes including differentiation, proliferation and apoptosis. Humans have 39 HOX genes and multiple lines of evidence implicate their deregulated expression in the pathogenesis of AML. Drabkin et al. (Leukemia2002; 16: 186–95) have reported that AMLs with a relatively favorable prognostic karyotype are associated with low levels of HOX gene expression whereas AMLs with an adverse prognostic karyotype have higher levels of expression. To further characterize HOX gene expression in cytogenetic prognostic groups we determined the expression profiles of 26 HOX genes by real-time quantitative PCR (Q-PCR) in diagnostic samples, representative of the three prognostic groups, from 26 patients with de novo AML. Profiles were then analyzed using Artificial Neural Network based computational approaches to identify a subset of HOX genes which could discriminate between prognostic groups in a predictive fashion. Predictive models were developed for each prognostic group. Predictive classification performance for prognostic groups based on blind data of 88%, 92%, and 97% (with equal sensitivity and specificity) were achieved for the three prognostic groups. The models were interrogated to determine the nature of the relationship between the key HOX genes identified and prognostic group. The relatively favorable prognosis group was primarily defined by downregulation of HOXA5 and upregulation of HOXC4. The intermediate prognosis group was characterized by upregulation of HOXB3 and downregulation of HOXD10 and the adverse prognosis group by downregulation of both HOXC5 and HOXD3. Although the sample size is small, the results show that Artificial Neural Network based computational approaches are capable of further characterizing HOX gene expression within AML prognostic groups as determined by presenting karyotype and that measuring the expression levels of a small number of HOX genes at diagnosis can provide useful clinical information in cases where karyotype analysis has been unsuccessful.

Introduction: Homeobox genes are a large family of transcriptional mediators that are essential in the process of angiogenesis. The role of Hox gene expression after hindlimb ischemia is not known. The purpose of this study is to characterize the expression of pro-angiogenic (HoxD3) and anti-angiogenic (HoxD10) genes in a murine model of hindlimb ischemia. Methods: Femoral artery resection was performed in the left hindlimb of C57Bl/6 mice, with the right limb serving as control (n = 20). Hindlimb perfusion was determined using laser Doppler technique. Animals were sacrificed at 4, 7, 14, 28 days after surgery. Gastrocnemius samples were harvested for RNA isolation. RT-PCR was performed for HoxD3, HoxD10, B3-integrin and Col1A1. In situ hybridization was performed on histological sections of calf muscle to determine tissue expression of HoxD3. Results: Hindlimb ischemia was maximal at 4d (L/R ratio = 0.3), with recovery by 28d (L/R ratio = 0.8). No limb loss was observed. HoxD3 expression was significantly increased at 4d and 7d after the induction of ischemia, with decreased expression at 14d and 28d. Downstream genes, B3-integrin and Col1A1, show a similar timecourse of increased expression. HoxD10 shows a reciprocal decrease in expression after hindlimb ischemia. In situ hybridization confirmed the tissue expression of HoxD3, primarily localized to areas of new blood vessel formation. Table 1. PCR relative expression (left/right ratio) 4 days 7 days 14 days 28 days HoxD3 1.55∗ 1.76∗ 1.01 1.02 B3-integrin 2.24∗ 2.56∗ 1.01 1.07 Col1A1 2.00∗ 1.89∗ 0.98 1.01 HoxD10 0.35∗ 0.61∗ 0.84 0.86 ∗ p < 0.05 using student’s t-test (n = 4 per group). Conclusions: These findings support the hypothesis that Hox gene expression is important in the creation of a pro-angiogenic state following hindlimb ischemia.