Gene Summary

Gene:HOXA4; homeobox A4
Aliases: HOX1, HOX1D
Summary:In vertebrates, the genes encoding the class of transcription factors called homeobox genes are found in clusters named A, B, C, and D on four separate chromosomes. Expression of these proteins is spatially and temporally regulated during embryonic development. This gene is part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:homeobox protein Hox-A4
Source:NCBIAccessed: 15 March, 2017


What does this gene/protein do?
Show (8)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 15 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Biomarkers, Tumor
  • Cell Movement
  • Neoplasm Proteins
  • Cell Adhesion
  • Chromosome Aberrations
  • Leukemic Gene Expression Regulation
  • DNA-Binding Proteins
  • Gene Expression Regulation
  • Up-Regulation
  • fms-Like Tyrosine Kinase 3
  • Lung Cancer
  • Cancer Gene Expression Regulation
  • Integrin beta3
  • DNA Methylation
  • Immunohistochemistry
  • Neoplasm Invasiveness
  • Multigene Family
  • Leukaemia
  • Homeobox Genes
  • Acute Myeloid Leukaemia
  • Chromosome 7
  • RT-PCR
  • Urokinase-Type Plasminogen Activator
  • Reproducibility of Results
  • Messenger RNA
  • Gene Expression Profiling
  • Gene Expression
  • Western Blotting
  • Transcriptional Regulator ERG
  • Oligonucleotide Array Sequence Analysis
  • Prostate Cancer
  • Signal Transduction
  • Mutation
  • Homeodomain Proteins
  • Transcription Factors
  • Epigenetics
  • Transfection
  • Transforming Growth Factor beta2
  • Nuclear Proteins
  • Neoplasm Metastasis
Tag cloud generated 15 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: HOXA4 (cancer-related)

Wang Y
Identifying key stage-specific genes and transcription factors for gastric cancer based on RNA-sequencing data.
Medicine (Baltimore). 2017; 96(4):e5691 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: To identify gastric cancer (GC)-associated genes and transcription factors (TFs) using RNA-sequencing (RNA-seq) data of Asians.
MATERIALS AND METHODS: The RNA-seq data (GSE36968) were downloaded from Gene Expression Omnibus database, including 6 noncancerous gastric tissue samples, 5 stage I GC samples, 5 stage II GC samples, 8 stage III GC samples, and 6 stage IV GC samples. The gene expression values in each sample were calculated using Cuffdiff. Following, stage-specific genes were identified by 1-way analysis of variance and hierarchical clustering analysis. Upstream TFs were identified using Seqpos. Besides, functional enrichment analysis of stage-specific genes was performed by DAVID. In addition, the underlying protein-protein interactions (PPIs) information among stage IV-specific genes were extracted from STRING database and PPI network was constructed using Cytoscape software.
RESULTS: A total of 3576 stage-specific genes were identified, including 813 specifically up-regulated genes in the normal gastric tissues, 2224 stage I and II-specific genes, and 539 stage IV-specific genes. Also, a total of 9 and 11 up-regulated TFs were identified for the stage I and II-specific genes and stage IV-specific genes, respectively. Functional enrichment showed SPARC, MMP17, and COL6A3 were related to extracellular matrix. Notably, 2 regulatory pathways HOXA4-GLI3-RUNX2-FGF2 and HMGA2-PRKCA were obtained from the PPI network for stage IV-specific genes. In the PPI network, TFs HOXA4 and HMGA2 might function via mediating other genes.
CONCLUSION: These stage-specific genes and TFs might act in the pathogenesis of GC in Asians.

Chen F, Sun G, Peng J
RNAi-mediated HOXD3 knockdown inhibits growth in human RKO cells.
Oncol Rep. 2016; 36(4):1793-8 [PubMed] Free Access to Full Article Related Publications
Numerous studies have shown that the multifunctional Homeobox-containing (HOX) D3 gene is involved in various physiological and pathological processes. To elucidate the role and mechanism of HOXD3 in colorectal cancer (CRC), we measured its expression in five CRC cell lines. After determining that HOXD3 was highly expressed in the human RKO cancer cell line, we used lentiviral-mediated small interfering RNAs (siRNAs) to knock down HOXD3 expression and assessed proliferation, cell cycle distribution, apoptosis and colony formation using cell proliferation, flow cytometric, and colony formation assays. The expression of HOXD3 was strongly suppressed in the RKO cells infected with the lentiviruse expressing an HOXD3 short hairpin RNA (shRNA). The downregulation of HOXD3 expression in RKO cells significantly decreased proliferation and colony formation, and increased apoptosis in vitro, compared to the cells infected with the mock control (p<0.01). Moreover, specific downregulation of HOXD3 led to the accumulation of cells at the G2 phase of the cell cycle. Our findings revealed that the HOXD3 gene promotes CRC cell growth and plays a pivotal role in the development and survival of malignant human colorectal cancer cells.

Mustafa M, Lee JY, Kim MH
CTCF negatively regulates HOXA10 expression in breast cancer cells.
Biochem Biophys Res Commun. 2015; 467(4):828-34 [PubMed] Related Publications
HOX genes not only play important roles in defining body patterning during embryonic development, but also control numerous cellular events in adult cells. Deregulated HOX gene expression in different cancers including breast cancer is now increasingly being reported. Given that human HOXA cluster is marked with several CTCF binding sites, we investigated whether the presence of CTCF is associated directly with expression of HOXA genes in breast cancer cells. Several HOX genes, such as HOXA4, HOXA5 and HOXA10, were deregulated by CTCF overexpression and knockdown in MCF-7 cells. Among these genes, HOXA10 is an emerging tumor suppressor for its role in activation of p53 and in countering tumorigenesis in breast cancer. Here we provided evidences that CTCF functions as a negative regulator of HOXA10 in breast cancer cells. The putative promoter region of HOXA10 lies between 5.3 and 6.1 kb upstream of its start codon and its promoter activity was negatively regulated by CTCF. Together with in-silico analysis and in vitro mutation assay we identified a 20 bp CTCF binding motif flanking with core promoter element of HOXA10. HOXA10 promoter region was kept inactivated by maintaining H3K27me3 inactivation marks in the presence of CTCF. Epigenetic silencing of HOXA10 by CTCF in breast cancer cells may contribute towards tumorigenesis by decreasing apoptosis and promoting metastasis.

Musialik E, Bujko M, Kober P, et al.
Promoter DNA methylation and expression levels of HOXA4, HOXA5 and MEIS1 in acute myeloid leukemia.
Mol Med Rep. 2015; 11(5):3948-54 [PubMed] Related Publications
HOXA genes encode transcription factors, which are crucial for embryogenesis and tissue differentiation and are involved in the early stages of hematopoiesis. Aberrations in HOXA genes and their cofactor MEIS1 are found in human neoplasms, including acute myeloid leukemia (AML). The present study investigated the role of HOXA4, HOXA5 and MEIS1 promoter DNA methylation and mRNA expression in AML. Samples from 78 AML patients and 12 normal bone marrow (BM) samples were included. The levels of promoter DNA methylation were determined using quantitative methylation‑specific polymerase chain reaction (PCR; qMSP) and the relative expression levels were measured using reverse transcription quantitative PCR in Ficoll‑separated BM mononuclear cells and in fluorescent activated cell sorting‑sorted populations of normal hematopoietic progenitors. In total, 38.1 and 28.9% of the patients exhibited high methylation levels of HOXA4 and HOXA5, respectively, compared with the control samples, and MEIS1 methylation was almost absent. An inverse correlation between HOXA4 methylation and expression was identified in a group of patients with a normal karyotype (NK AML). An association between the genes was observed and correlation between the DNA methylation and expression levels of the HOXA gene promoter with the expression of MEIS1 was observed. Patients with favorable chromosomal aberrations revealed a low level of HOXA4 methylation and decreased expression levels of HOXA5 and MEIS1 compared with the NK AML and the adverse cytogenetic risk patients. The NK AML patients with NPM1 mutations exhibited elevated HOXA4 methylation and expression levels of HOXA5 and MEIS1 compared with the NPM1 wild‑type patients. Comparison of the undifferentiated BM‑derived hematopoietic CD34+CD38low, CD34+CD38+ and CD15+ cells revealed a gradual decrease in the expression levels of these three genes and an increase in HOXA4 promoter methylation. This differentiation‑associated variability was not observed in AML, which was classified according to the French‑American‑British system.

Litovkin K, Joniau S, Lerut E, et al.
Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer.
J Cancer Res Clin Oncol. 2014; 140(11):1849-61 [PubMed] Related Publications
PURPOSE: To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa).
METHODS: A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR.
RESULTS: A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89).
CONCLUSIONS: Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

Chen LN, Rubin RS, Othepa E, et al.
Correlation of HOXD3 promoter hypermethylation with clinical and pathologic features in screening prostate biopsies.
Prostate. 2014; 74(7):714-21 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Molecular markers that can discriminate indolent cancers from aggressive ones may improve the management of prostate cancer and minimize unnecessary treatment.Aberrant DNA methylation is a common epigenetic event in cancers and HOXD3 promoter hypermethylation (H3PH) has been found in prostate cancer. Our objective was to evaluate the relationship between H3PH and clinicopathologic features in screening prostate biopsies.
METHODS: Ninety-two patients who underwent a prostate biopsy at our institution between October 2011 and May 2012 were included in this study. The core with the greatest percentage of the highest grade disease was analyzed for H3PH by methylation-specific PCR. Correlational analysis was used to analyze the relationship between H3PH and various clinical parameters. Chi-square analysis was used to compare H3PH status between benign and malignant disease.
RESULTS: Of the 80 biopsies with HOXD3 methylation status assessable, 66 sets were confirmed to have cancer. In the 14 biopsies with benign disease there was minimal H3PH with the mean percentage of methylation reference (PMR) of 0.7%. In contrast, the HOXD3 promoter was hypermethylated in 16.7% of all cancers and in 50% of high risk tumors with an average PMR of 4.3% (P=0.008). H3PH was significantly correlated with age (P=0.013), Gleason score (P=0.031) and the maximum involvement of the biopsy core (P=0.035).
CONCLUSIONS: H3PH is associated with clinicopathologic features. The data indicate that H3PH is more common in older higher risk patients. More research is needed to determine the role of this marker in optimizing management strategies in men with newly diagnosed prostate cancer.

Stott-Miller M, Zhao S, Wright JL, et al.
Validation study of genes with hypermethylated promoter regions associated with prostate cancer recurrence.
Cancer Epidemiol Biomarkers Prev. 2014; 23(7):1331-9 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: One challenge in prostate cancer is distinguishing indolent from aggressive disease at diagnosis. DNA promoter hypermethylation is a frequent epigenetic event in prostate cancer, but few studies of DNA methylation in relation to features of more aggressive tumors or prostate cancer recurrence have been completed.
METHODS: We used the Infinium HumanMethylation450 BeadChip to assess DNA methylation in tumor tissue from 407 patients with clinically localized prostate cancer who underwent radical prostatectomy. Recurrence status was determined by follow-up patient surveys, medical record review, and linkage with the Surveillance, Epidemiology, and End Results (SEER) registry. The methylation status of 14 genes for which promoter hypermethylation was previously correlated with advanced disease or biochemical recurrence was evaluated. Average methylation level for promoter region CpGs in patients who recurred compared with those with no evidence of recurrence was analyzed. For two genes with differential methylation, time to recurrence was examined.
RESULTS: During an average follow-up of 11.7 years, 104 (26%) patients recurred. Significant promoter hypermethylation in at least 50% of CpG sites in two genes, ABHD9 and HOXD3, was found in tumors from patients who recurred compared with those without recurrence. Evidence was strongest for HOXD3 (lowest P = 9.46 × 10(-6)), with higher average methylation across promoter region CpGs associated with reduced recurrence-free survival (P = 2 × 10(-4)). DNA methylation profiles did not differ by recurrence status for the other genes.
CONCLUSIONS: These results validate the association between promoter hypermethylation of ADHB9 and HOXD3 and prostate cancer recurrence.
IMPACT: Tumor DNA methylation profiling may help to distinguish patients with prostate cancer at higher risk for disease recurrence.

Olkhov-Mitsel E, Zdravic D, Kron K, et al.
Novel multiplex MethyLight protocol for detection of DNA methylation in patient tissues and bodily fluids.
Sci Rep. 2014; 4:4432 [PubMed] Free Access to Full Article Related Publications
Aberrant DNA methylation is a hallmark of cancer and is an important potential biomarker. Particularly, combined analysis of a panel of hypermethylated genes shows the most promising clinical performance. Herein, we developed, optimized and standardized a multiplex MethyLight assay to simultaneously detect hypermethylation of APC, HOXD3 and TGFB2 in DNA extracted from prostate cancer (PCa) cell lines, archival tissue specimens, and urine samples. We established that the assay is capable of discriminating between fully methylated and unmethylated alleles with 100% specificity and demonstrated the assay as highly accurate and reproducible as the singleplex approach. For proof of principle, we analyzed the methylation status of these genes in tissue and urine samples of PCa patients as well as PCa-free controls. These data show that the multiplex MethyLight assay offers a significant advantage when working with limited quantities of DNA and has potential applications in research and clinical settings.

Kang JU
Characterization of amplification patterns and target genes on the short arm of chromosome 7 in early-stage lung adenocarcinoma.
Mol Med Rep. 2013; 8(5):1373-8 [PubMed] Related Publications
Chromosomal alterations are a predominant genomic force contributing to the development of lung adenocarcinoma (ADC). High density genomic arrays were conducted to identify critical genetic landmarks that may be important mediators in the formation or progression of early‑stage ADC. In this study, the most noteworthy and consistent observation was a copy number gain on the short arm of chromosome 7, which was detected in 85.7% (12/14) of cases. Notably, three distinct regions of amplification were identified between the 7p22.3 and q11.2 regions in 28.6% (4/14) of cases; at a size of 4.1 Mbp (7p22.3‑p21.1), 2.6 Mbp (7p15.2-p14.1) and 1.5 Mbp (7p12.3‑p11.2). Variations of the 7p11.2 locus that encodes EGFR are known to be oncogenic. Furthermore, potential target genes were identified that were previously not assumed to be involved in the pathogenesis of ADC, including CALM1P2 (7p11.2), HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, HOXA10, HOXA11 and HOXA13 (7p15.2) and LOC442586, LOC442589, LOC442282, FAM20C and LOC442651 (7p22.3). The present study determined critical regions on the 7p arm of chromosome 7, which were implicated in ADC. The pattern of rearrangements on the 7p arm may be a consequence of the high density of potential targets and the identified genes at the 7p regions may aid in the development of therapeutic targets for ADC.

Bhatlekar S, Addya S, Salunek M, et al.
Identification of a developmental gene expression signature, including HOX genes, for the normal human colonic crypt stem cell niche: overexpression of the signature parallels stem cell overpopulation during colon tumorigenesis.
Stem Cells Dev. 2014; 23(2):167-79 [PubMed] Free Access to Full Article Related Publications
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.

Elias MH, Baba AA, Husin A, et al.
HOXA4 gene promoter hypermethylation as an epigenetic mechanism mediating resistance to imatinib mesylate in chronic myeloid leukemia patients.
Biomed Res Int. 2013; 2013:129715 [PubMed] Free Access to Full Article Related Publications
Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients has emerged as a significant clinical problem. The observation that increased epigenetic silencing of potential tumor suppressor genes correlates with disease progression in some CML patients treated with IM suggests a relationship between epigenetic silencing and resistance development. We hypothesize that promoter hypermethylation of HOXA4 could be an epigenetic mechanism mediating IM resistance in CML patients. Thus a study was undertaken to investigate the promoter hypermethylation status of HOXA4 in CML patients on IM treatment and to determine its role in mediating resistance to IM. Genomic DNA was extracted from peripheral blood samples of 95 CML patients (38 good responders and 57 resistant) and 12 normal controls. All samples were bisulfite treated and analysed by methylation-specific high-resolution melt analysis. Compared to the good responders, the HOXA4 hypermethylation level was significantly higher (P = 0.002) in IM-resistant CML patients. On comparing the risk, HOXA4 hypermethylation was associated with a higher risk for IM resistance (OR 4.658; 95% CI, 1.673-12.971; P = 0.003). Thus, it is reasonable to suggest that promoter hypermethylation of HOXA4 gene could be an epigenetic mechanism mediating IM resistance in CML patients.

Shaoqiang C, Yue Z, Yang L, et al.
Expression of HOXD3 correlates with shorter survival in patients with invasive breast cancer.
Clin Exp Metastasis. 2013; 30(2):155-63 [PubMed] Related Publications
Hox genes encode a family of homeodomain-containing transcription factors that determine cellular identity during development and which are also expressed in some types of cancer. The HOXD3 gene, a member of the Hox gene family, has been demonstrated to be expressed in several tumor cell lines, which exhibit enhanced invasion and metastasis through coordinate expression of metastasis-associated factors. However, the clinical impact of HOXD3 in breast cancer remains unclear. In the current study, we examined the expression of HOXD3 and integrin β3 by immunohistochemical staining in patients with invasive breast cancer. We found that HOXD3 expression was significantly frequent in high histopathological grade and hormone-receptor negative breast cancer patients. The expression of HOXD3 was closely associated with integrin β3 expression. Furthermore, patients with high HOXD3 expression levels in their breast tumors had significantly shorter survival times than patients in which HOXD3 was weakly expressed in breast tumors. Univariate and multivariate analyses confirmed that increased HOXD3-expression was an independent and significant factor in predicting poor prognosis for patients with breast cancer. In conclusion, HOXD3 expression is a significant unfavorable prognostic factor for patients with invasive breast cancer and as such is a potentially useful prognostic marker for breast cancer.

Tholouli E, MacDermott S, Hoyland J, et al.
Quantitative multiplex quantum dot in-situ hybridisation based gene expression profiling in tissue microarrays identifies prognostic genes in acute myeloid leukaemia.
Biochem Biophys Res Commun. 2012; 425(2):333-9 [PubMed] Related Publications
Measurement and validation of microarray gene signatures in routine clinical samples is problematic and a rate limiting step in translational research. In order to facilitate measurement of microarray identified gene signatures in routine clinical tissue a novel method combining quantum dot based oligonucleotide in situ hybridisation (QD-ISH) and post-hybridisation spectral image analysis was used for multiplex in-situ transcript detection in archival bone marrow trephine samples from patients with acute myeloid leukaemia (AML). Tissue-microarrays were prepared into which white cell pellets were spiked as a standard. Tissue microarrays were made using routinely processed bone marrow trephines from 242 patients with AML. QD-ISH was performed for six candidate prognostic genes using triplex QD-ISH for DNMT1, DNMT3A, DNMT3B, and for HOXA4, HOXA9, Meis1. Scrambled oligonucleotides were used to correct for background staining followed by normalisation of expression against the expression values for the white cell pellet standard. Survival analysis demonstrated that low expression of HOXA4 was associated with poorer overall survival (p=0.009), whilst high expression of HOXA9 (p<0.0001), Meis1 (p=0.005) and DNMT3A (p=0.04) were associated with early treatment failure. These results demonstrate application of a standardised, quantitative multiplex QD-ISH method for identification of prognostic markers in formalin-fixed paraffin-embedded clinical samples, facilitating measurement of gene expression signatures in routine clinical samples.

Kron K, Liu L, Trudel D, et al.
Correlation of ERG expression and DNA methylation biomarkers with adverse clinicopathologic features of prostate cancer.
Clin Cancer Res. 2012; 18(10):2896-904 [PubMed] Related Publications
PURPOSE: Fusion of the TMPRSS2 gene with the ERG oncogene and aberrant DNA methylation patterns are commonly found in prostate cancer. The aim of this study was to analyze the relationship between ERG expression, DNA methylation of three biomarkers, and clinicopathologic features of prostate cancer.
EXPERIMENTAL DESIGN: Immunohistochemistry for ERG protein was conducted as a surrogate for TMPRSS2-ERG fusions. We analyzed methylation of CYP26A1, TBX15, and HOXD3 in 219 prostatectomy specimens by the quantitative MethyLight assay. DNA methylation was compared between ERG-positive and -negative cases and correlations of ERG and DNA methylation with clinicopathologic features were analyzed using χ(2), Spearman correlation, logistic regression, and Cox regression.
RESULTS: ERG expression varied according to Gleason pattern (almost absent in pattern II, highest in pattern III, and lower in pattern IV/V) and showed a strong positive correlation with methylation levels of CYP26A1, TBX15, and HOXD3 (Spearman P < 0.005). TBX15 and HOXD3 methylation were significantly associated with pathologic stage, Gleason score, and Gleason pattern (P ≤ 0.015). In multivariate regression analysis, PSA, TBX15 high methylation, and HOXD3 high methylation were significantly associated with stage (P < 0.05), whereas ERG expression was negatively correlated with Gleason score (P = 0.003). In univariate time-to-recurrence analysis, a combination of HOXD3/TBX15 high methylation predicted recurrence in ERG-positive and -negative cases (P < 0.05).
CONCLUSIONS: CYP26A1, TBX15, and HOXD3 are methylation markers of prostate cancer associated with ERG expression and clinicopathologic variables, suggesting that incorporation of these markers may be useful in a pre- and posttreatment clinical setting.

Kim HJ, Roh MS, Son CH, et al.
Loss of Med1/TRAP220 promotes the invasion and metastasis of human non-small-cell lung cancer cells by modulating the expression of metastasis-related genes.
Cancer Lett. 2012; 321(2):195-202 [PubMed] Related Publications
Med1/TRAP220 is an essential component of the TRAP/Mediator complex. In this study, we present a novel function of Med1 in human non-small-cell lung cancer (NSCLC) progression. We found that the loss of Med1 expression was strongly associated with increased rates of invasion and metastasis in NSCLC patients. Consistent with lung cancer patient data, the knockdown of Med1 in NSCLC cell lines led to an increase in cell migration and invasion. Med1-depleted cells displayed an increase in metastasis in a xenograft tumor model and in an in vivo metastasis assay. Moreover, a microarray analysis revealed that the mRNA levels of the metastasis-related genes uPAR, ID2, ID4, PTP4A1, PKP3, TGM2, PLD1, TIMP2, RGS2, and HOXA4 were altered upon Med1 knockdown. Collectively, these results suggest that the loss of Med1 increases the invasive potential of human NSCLC cells by modulating the expression of metastasis-related genes.

Liao SJ, Zhou YH, Yuan Y, et al.
Triggering of Toll-like receptor 4 on metastatic breast cancer cells promotes αvβ3-mediated adhesion and invasive migration.
Breast Cancer Res Treat. 2012; 133(3):853-63 [PubMed] Related Publications
Triggering of Toll-like receptor 4 (TLR4) on tumor cells has been found to promote tumor progression by promoting tumor cell proliferation and survival. So far, however, the effect of TLR4 signaling on tumor metastasis has not been well elucidated. Here, we report that triggering of TLR4 on metastatic breast cancer cells could reciprocally regulate the expression of αvβ3 and the expressions of TPM1 and maspin, and promote αvβ3-mediated adhesion and invasive migration of the cells. In metastatic breast cancer cells, TLR4 signaling increased the expression of integrin αvβ3 by activating NF-κB, resulting in the increased adhesion capacity of tumor cells to the ligand for αvβ3, and the increased polymerization of actin and production of MMP-9 in tumor cells in response to ECM. HoxD3 was required for the up-regulation of αv and β3 expressions by NF-κB. Moreover, TLR4 signaling increased the expression of miR-21 in breast cancer cells by activating NF-κB. Accordingly, the expressions of TPM1 and maspin were decreased at protein level, whereas the transcription activity of these genes was not influenced. Consistent with the promoting effect on αvβ3-mediated adhesion and invasive migration, TLR4 signaling promoted the arrest of metastatic breast cancer cells in circulation and following invasion. The effect of TLR4 signaling could be abrogated by inhibiting NF-κB. These findings suggest that metastatic breast cancer cells could acquire higher metastatic potential due to triggering of TLR4 and activation of NF-κB in the cells, and that both TLR4 and NF-κB could be therapeutic targets for preventing metastasis of breast cancer cells.

Gray S, Pandha HS, Michael A, et al.
HOX genes in pancreatic development and cancer.
JOP. 2011; 12(3):216-9 [PubMed] Related Publications
The HOX genes are a family of homeodomain-containing transcription factors that determine cellular identity during development and which are subsequently re-expressed in many types of cancer. Some recent studies have shown that HOX genes may have key roles both in pancreatic development and in adult diseases of the pancreas, including cancer. In this review we consider recent advances in elucidating the role of HOX genes in these processes, how they may connect early developmental events to subsequent adult disease, and their potential both as diagnostic markers and therapeutic targets.

Liu L, Kron KJ, Pethe VV, et al.
Association of tissue promoter methylation levels of APC, TGFβ2, HOXD3 and RASSF1A with prostate cancer progression.
Int J Cancer. 2011; 129(10):2454-62 [PubMed] Related Publications
Aberrant promoter methylation is known to silence tumor-suppressor genes in prostate cancer (PCa). We correlated quantitative promoter methylation levels of APC, TGFβ2 and RASSF1A in 219 radical prostatectomies diagnosed between 1998 and 2001 with clinicopathological follow-up data available including Gleason Pattern (GP), Gleason Score (GS) and pathological stage and explored their potential in predicting biochemical recurrence using univariate and multivariate analyses. We observed that the average methylation levels of APC increased significantly from GS ≤ 6 to GS7, and pT2 to pT3a, and that of TGFβ2 increased from GS ≤ 6 to GS7, but not for RASSF1A. PCa samples were also stratified into high methylation (HM) and low methylation (LM) groups based on the PMR scores of all cases analyzed for each marker. The HM frequency of APC was greater in pT3a than pT2, and in GS ≥ 8 than GS ≤ 6. The HM frequency also increased significantly from GP3 to GP4 for APC, TGFβ2 and RASSF1A. APC methylation level was a significant predictor of biochemical recurrence in univariate analysis (p-value = 0.028). Finally, we combined methylation data of these three genes with the previously reported novel methylation biomarker HOXD3. Quantitative methylation assessment of a multiplex panel of markers, consisting of APC, HOXD3 and TGFβ2, outperforms any single marker for the prediction of biochemical recurrence (p-value = 0.017). Our study demonstrated that quantitative increase in promoter methylation levels of APC, HOXD3 and TGFβ2 are associated with PCa progression.

Irving L, Mainou-Fowler T, Parker A, et al.
Methylation markers identify high risk patients in IGHV mutated chronic lymphocytic leukemia.
Epigenetics. 2011; 6(3):300-6 [PubMed] Free Access to Full Article Related Publications
Chronic lymphocytic leukemia (CLL) exhibits a very variable clinical course. Altered DNA methylation of genes has shown promise as a source of novel prognostic makers in a number of cancers. Here we have studied the potential utility of a panel of methylation markers (CD38, HOXA4 and BTG4) in 118 CLL patients. Each of the three loci assessed exhibited frequent methylation, as determined by COBRA analysis, and individually correlated with either good (CD38, BTG4 methylation) or poor (HOXA4 methylation) prognosis. Using a combined approach to produce an overall methylation score, we found that methylation score was significantly associated with time to first treatment in CLL patients. Multivariate Cox regression analysis revealed that methylation score was the strongest predictor of time to first treatment, and was independent of IGHV gene mutational status and CD38 expression. This study provides proof of principle that a panel of methylation markers can be used for additional risk stratification of CLL patients.

Kron KJ, Liu L, Pethe VV, et al.
DNA methylation of HOXD3 as a marker of prostate cancer progression.
Lab Invest. 2010; 90(7):1060-7 [PubMed] Related Publications
DNA methylation in gene promoters causes gene silencing and is a common event in cancer development and progression. The ability of aberrant methylation events to serve as diagnostic and prognostic markers is being appreciated for many cancers, including prostate cancer. Using quantitative MethyLight technology, we evaluated the relationship between HOXD3 methylation and clinicopathological parameters including biochemical recurrence, pathological stage, Gleason score (GS), and Gleason pattern in a series of 232 radical prostatectomies performed between 1998 and 2001. HOXD3 methylation was significantly greater in GS 7 cancers vs GS < or = 6 cancers (P-value <0.001) as well as pT3/pT4 vs pT2 cancers (P-value <0.001). The proportion of cases with high methylation in GS 7 vs < or = GS 6 and pT3/pT4 vs pT2 were also significantly different (P-values=0.002 and 0.005, respectively). There were also significant increases in methylation from Gleason pattern 2-3 and from pattern 3 to 4/5 (paired t-test P-values=0.01 and <0.001, respectively), whereas methylation from lymph node metastases was decreased when compared with matched tumor tissue (P-value=0.029). HOXD3 methylation was associated with biochemical recurrence in univariate analysis (P-value=0.043) and showed evidence for interaction with pathological stage as a predictor variable in Cox regression analysis (P-value=0.028). The results indicate that HOXD3 methylation distinguishes low-grade prostate cancers from intermediate and high-grade ones and may also have prognostic value when considered together with pathological stage.

Bach C, Buhl S, Mueller D, et al.
Leukemogenic transformation by HOXA cluster genes.
Blood. 2010; 115(14):2910-8 [PubMed] Related Publications
HOX homeobox genes are important regulators of normal and malignant hematopoiesis. Abdominal-type HOXA genes like HOXA9 are highly leukemogenic. However, little is known about transformation by anterior HOXA genes. Here we performed a comprehensive assessment of the oncogenic potential of every HOXA gene in primary hematopoietic cells. With exception of HOXA2 and HOXA5, all HOXA genes caused a block or delay of hematopoietic differentiation and cooperated with Meis1. No evidence for the alleged tumor-suppressor function of HOXA5 could be found. Whereas all active HOXA genes immortalized mixed granulocytic/monocytic populations, HOXA13 preferentially specified monocytoid development. The anterior HOXA genes HOXA1, HOXA4, and HOXA6 transformed cells, generating permanent cell lines, although they did so less potently than HOXA9. Upon transplantation these lines induced myeloproliferation and acute myeloid leukemia in recipient animals. Kinetic studies with inducible HOX derivatives demonstrated that anterior HOXA genes autonomously contributed to cellular transformation. This function was not mediated by endogenous Hoxa9, which was persistently expressed in cells transformed by anterior HOX genes. In summary our results demonstrate a hitherto unexpected role of anterior HOXA genes in hematopoietic malignancy.

Klausen C, Leung PC, Auersperg N
Cell motility and spreading are suppressed by HOXA4 in ovarian cancer cells: possible involvement of beta1 integrin.
Mol Cancer Res. 2009; 7(9):1425-37 [PubMed] Related Publications
HOX genes are transcription factors that control morphogenesis, organogenesis and differentiation. Increasing evidence suggests that HOX genes play a role in ovarian cancer progression; however few studies have defined functional roles and mechanisms of action. We showed previously that HOXA4 expression is increased in invasive, compared to noninvasive, epithelial ovarian tumors. However, HOXA4 suppressed cell migration suggesting that elevated HOXA4 expression in invasive tumors constitutes a homeostatic response. In the present study, we used siRNA and forced-expression in multiple cell lines to define the role of HOXA4 in the regulation of transwell migration/invasion and cellular/colony morphology. Knockdown of endogenous HOXA4 increased migration, but not Matrigel invasion, of OVCAR-8 and OVCAR-3 cells. HOXA4 knockdown also increased cell spreading on plastic or fibronectin, reduced cell-cell adhesion, and increased filopodia in two- and three-dimensional cultures. These changes were not associated with significant changes in alphaV or beta3 integrin and E- or N-cadherin. However, down-regulation of HOXA4 significantly reduced beta1 integrin protein levels within cell colonies and cell aggregates, but not of single, nonadherent cells. It had no effect on beta1 integrin, alpha5 integrin, or fibronectin mRNA levels. Conversely, overexpression of HOXA4 in CaOV-3 cells suppressed transwell migration and increased beta1 integrin protein levels. Our results confirm that HOXA4 inhibits cell motility, show that it suppresses cell spreading and filopodia formation while enhancing cell-cell adhesion, and suggest a role for beta1 integrin in mediating these changes. These observations support the hypothesis that overexpression of HOXA4 in invasive ovarian tumors is a homeostatic, invasion-suppressive response.

Zangenberg M, Grubach L, Aggerholm A, et al.
The combined expression of HOXA4 and MEIS1 is an independent prognostic factor in patients with AML.
Eur J Haematol. 2009; 83(5):439-48 [PubMed] Related Publications
HOXA4 gene expression is a predictor for outcome in normal karyotypic acute myeloid leukaemia (AML) patients. Given that Meis1 is a co-factor for Hox genes, we hypothesized that the combined expression of HOXA4 and MEIS1 might add prognostic information in these patients. When diagnostic samples from 246 AML patients were divided into three main groups based on gene expression levels of HOXA4 combined with MEIS1 we found that within the group of patients exhibiting low levels of HOXA4, those with a high expression of MEIS1 had a significantly worse outcome than those exhibiting low MEIS1 expression (P = 0.025). Moreover, this prediction was independent of cytogenetics, mutational status of the NPM1 and FLT3 genes as well as upon WBC and age. To evaluate the possible contribution of regulatory events underlying these observations, 157 patient samples were subjected to promoter hypermethylation analysis. We observed that 77% were HOXA4- and 15%MEIS1 hypermethylated and that this epigenetic alteration was highly correlated to the gene expression level (MEIS1: P = 0.001; HOXA4: P = 0.007). Finally, we found a higher expression level and a higher frequency of hypermethylation of HOXA4 among patients with NPM1 mutations. In conclusion, our data show that the combination of low HOXA4 and low MEIS1 gene expression is a favourable predictor for outcome in all AML patients and that the expression levels are governed by the methylation state of these genes.

Yang YC, Wang SW, Wu IC, et al.
A tumorigenic homeobox (HOX) gene expressing human gastric cell line derived from putative gastric stem cell.
Eur J Gastroenterol Hepatol. 2009; 21(9):1016-23 [PubMed] Related Publications
GOAL: Study the mechanism of gastric tumor development.
BACKGROUND: We have generated and characterized a novel human gastric cell line, KMU-CS12 (CS12), from an immortal cell line, KMU-CSN (CSN; formerly named as GI2CS) which was derived from putative human gastric stem cell/progenitor cell clone, KMU-GI2.
STUDY: The characterization of the CS12 cell line includes gene expression by immunocytochemical staining, cell proliferation and differentiation potential, cyotogenetic analysis by Giemsa banding and spectral karyotype analysis (SKY), and tumorigenicity in immune-deficient congenic inbred, nude mice (BALB/cAnN-Foxn1nu/CrlNarl). The Agilent Human 1A oligo-array and RT-PCR were also employed to analyze the expression of homeobox (HOX) genes.
RESULTS: The CS12 gastric cell line showed cancer cell phenotypes, i.e. the ability of anchorage-independent growth high frequency (44%) and to the expression of Oct-4, a transcription factor expressed in embryonic stem cells and many types of cancer cells, and tumor development in immune deficient mice. SKY analysis indicated a characteristic duplication of the short arm of chromosome 7 to chromosome 12. Agilent Human 1A oligo-array analysis showed that the expression of 1145 genes was upregulated while that of 890 genes was downregulated in CS12 cells. RT-PCR revealed that homeobox genes (HOXA4, HOXA5, HOXA7, HOXA9, and HOXA13) were highly expressed in CS12 cells in culture, as well as tumor tissues developed by CS12 cells in immunodeficient mice for six to eight weeks.
CONCLUSION: Except for the duplication of the short arm of Chromosome 7 on Chromosome12, the karyotype of the tumorigenic CS12 cells is similar to the parental GI2 cells which are non-tumorigenic and normal in karyotype. This chromosomal change could be the cause for the high expression of HOXA genes and tumorigenicity of these cells found in this study. Thus HOXA genes might play an important role in gastric carcinogenesis.

Kron K, Pethe V, Briollais L, et al.
Discovery of novel hypermethylated genes in prostate cancer using genomic CpG island microarrays.
PLoS One. 2009; 4(3):e4830 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Promoter and 5' end methylation regulation of tumour suppressor genes is a common feature of many cancers. Such occurrences often lead to the silencing of these key genes and thus they may contribute to the development of cancer, including prostate cancer.
METHODOLOGY/PRINCIPAL FINDINGS: In order to identify methylation changes in prostate cancer, we performed a genome-wide analysis of DNA methylation using Agilent human CpG island arrays. Using computational and gene-specific validation approaches we have identified a large number of potential epigenetic biomarkers of prostate cancer. Further validation of candidate genes on a separate cohort of low and high grade prostate cancers by quantitative MethyLight analysis has allowed us to confirm DNA hypermethylation of HOXD3 and BMP7, two genes that may play a role in the development of high grade tumours. We also show that promoter hypermethylation is responsible for downregulated expression of these genes in the DU-145 PCa cell line.
CONCLUSIONS/SIGNIFICANCE: This study identifies novel epigenetic biomarkers of prostate cancer and prostate cancer progression, and provides a global assessment of DNA methylation in prostate cancer.

Ota T, Klausen C, Salamanca MC, et al.
Expression and function of HOXA genes in normal and neoplastic ovarian epithelial cells.
Differentiation. 2009; 77(2):162-71 [PubMed] Related Publications
We studied the roles of three HOXA genes in cultured normal ovarian surface epithelial (OSE) cells and ovarian cancer cells. They included HOXA4 and HOXA7 because, by cDNA microarray analysis, these were more highly expressed in invasive ovarian carcinomas than in benign or borderline (noninvasive) ovarian tumors, and HOXA9 because it characterizes normal oviductal epithelium, which resembles ovarian serous adenocarcinomas. The three HOXA genes were more highly expressed when OSE cells were dividing and motile than when they were confluent and stationary, and also when they dispersed in response to EGF treatment or to reduced calcium concentrations in culture media. The expression of the HOXA genes varied among ovarian cancer cell lines, but was highest in lines with compact epithelial morphologies. We focused on HOXA4 as the most highly expressed in the ovarian carcinoma array. HOXA4 expression did not parallel proliferative activities of either OSE or ovarian cancer lines. Moreover, modifying HOXA4 expression in ovarian cancer cell lines did not alter either E-cadherin expression or CA125 secretion. However, HOXA4 downregulation enhanced EGFR phosphorylation and migration in serum-starved OSE and ovarian cancer cells in response to EGF, and enhanced migration of all ovarian cancer lines in 5% serum even without EGF treatment. Thus, HOXA4 expression does not correlate with proliferation or with epithelial differentiation, but it increases in response to OSE cell dispersion and negatively regulates EGFR activation and the motility of OSE and of ovarian cancer cells. HOXA4 expression was highest in cancer lines with compact epithelial growth patterns, suggesting, again, an anti-dispersion function. In summary, increased HOXA4 expression in ovarian cancer appears to constitute a tumor-suppressive, homeostatic response to aberrant cell behavior, and, in particular, to cell dispersion and migration.

Komeno Y, Kitaura J, Kitamura T
Molecular bases of myelodysplastic syndromes: lessons from animal models.
J Cell Physiol. 2009; 219(3):529-34 [PubMed] Related Publications
Myelodysplastic syndrome (MDS) is a clonal disorder of hematopietic stem cells characterized by ineffective hematopoiesis, peripheral blood cytopenia, morphologic dysplasia, and susceptibility to acute myeloid leukemia. Several mechanisms have been suggested as causes of MDS: unbalanced chromosomal abnormalities reflecting a gain or loss of chromosomal material, point mutations of transcription factors, and inactivation of p53. However, appropriate animal models that mimic MDS have long been lacking. We recently reported a novel murine model of MDS that recapitulates trilineage dysplasia and transformation to AML. In this review, we summarize the animal models of MDS and discuss the molecular bases of MDS as well as those of leukemia and myeloproliferative disorders (MPD). J. Cell. Physiol. 219: 529-534, 2009. (c) 2009 Wiley-Liss, Inc.

Grubach L, Juhl-Christensen C, Rethmeier A, et al.
Gene expression profiling of Polycomb, Hox and Meis genes in patients with acute myeloid leukaemia.
Eur J Haematol. 2008; 81(2):112-22 [PubMed] Related Publications
The Polycomb group (PcG) of genes is important for differentiation and cell-cycle regulation and is aberrantly expressed in several cancers. To analyse the role of deregulated PcG genes in acute myeloid leukaemia (AML), we determined by RQ-PCR the expression of the PcG genes BMI-1, MEL18, SCML2, YY1 and EZH2, and the downstream PcG targets HOXA4, HOXA9 and MEIS1 in diagnostic bone marrow samples from 126 AML patients. There was a general overexpression of the genes in AML patients compared to 20 healthy donors, except of HOXA4 and MEL18, which both displayed a wide range of expression levels within the AML subgroups. Among the AML patients with normal karyotype, a low HOXA4 level was associated with a shorter overall survival (P = 0.005). In addition, expression levels of MEL18 and EZH2 were significantly (P < 0.025) higher in patients with complex karyotype and lower in CBF-mutated patients. The t(8;21) vs. inv(16) positive patients showed significantly different expression of SCML2, BMI-1, YY1, HOXA9 and MEIS1 (P < or = 0.01). Comparisons between the PcG and PcG-regulated genes and a number of clinical and molecular data revealed correlations to genes involved in DNA methylation (DNMT1, DNMT3B), apoptosis (BAX, CASPASE 3) and multidrug-resistance (MDR1, MRP ) (P < 0.01). In conclusion, our data suggest that the role of PcG and PcG-regulated genes in leukaemogenesis varies between, as well as within karyotypic subgroups.

Strathdee G, Holyoake TL, Sim A, et al.
Inactivation of HOXA genes by hypermethylation in myeloid and lymphoid malignancy is frequent and associated with poor prognosis.
Clin Cancer Res. 2007; 13(17):5048-55 [PubMed] Related Publications
PURPOSE: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are key regulators of embryonic development, hematopoietic differentiation, and leukemogenesis. We aimed to study the role of DNA methylation as an inducer of HOX gene silencing in leukemia.
EXPERIMENTAL DESIGN: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis and pyrosequencing of bisulfite-modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model.
RESULTS: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations with important clinical variables, most notably in CML, in which hypermethylation of both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis cells resulted in the induction of markers of granulocytic differentiation.
CONCLUSION: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.

Ohta H, Hamada J, Tada M, et al.
HOXD3-overexpression increases integrin alpha v beta 3 expression and deprives E-cadherin while it enhances cell motility in A549 cells.
Clin Exp Metastasis. 2006; 23(7-8):381-90 [PubMed] Related Publications
We have previously shown that transduction of HOXD3, one of homeobox genes, into human lung cancer A549 cells enhances cell motility, invasion and metastasis. In the present study, we examined the roles of integrin beta3 which was up-regulated by HOXD3-overexpression in the HOXD3-induced motility of A549 cells. We first established integrin beta3-transfectants and compared their motile activity to those of the HOXD3-transfected, control-transfected and parental cells by three different assays. The integrin beta3-transfectants as well as the HOXD3-transfectants formed heterodimer with integrin alphav subunit, and showed highly motile activities assessed by haptotaxis or phagokinetic track assay compared to the control transfectants or parental cells. In vitro wound-healing assay revealed that migratory activities were graded as the HOXD3-transfectants > the integrin beta3-transfectants > the control transfectants or parental cells. E-cadherin was expressed in the integrin beta3-transfectants but not expressed in the HOXD3-transfectants. An addition of function-blocking antibody to E-cadherin into the wound-healing assay promoted the migratory activity of the integrin beta3-transfectants, suggesting that E-cadherin prevented the cells from dissociating from the wound edges. These results indicate that increased expression of integrin alphav beta3 and loss of E-cadherin by HOXD3-overexpression are responsible for the enhanced motility and dissociation.

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