BACKGROUND: Long noncoding RNAs (lncRNAs) have been indicated to play critical roles in cancer development and progression. LncRNA HOXD cluster antisense RNA1 (HOXD-AS1) has recently been found to be dysregulated in several cancers. However, the expression levels, cellular localization, precise function and mechanism of HOXD-AS1 in colorectal carcinoma (CRC) are largely unknown.
METHODS: Real-time PCR and in situ hybridization were used to detect the expression of HOXD-AS1 in CRC tissue samples and cell lines. Gain- and loss-of-function experiments were performed to investigate the biological roles of HOXD-AS1 in CRC cell line. RNA pull down, RNA immunoprecipitation and chromatin immunoprecipitation assays were conducted to investigate the mechanisms underlying the functions of HOXD-AS1 in CRC.
RESULTS: We observed that HOXD-AS1 was located in the nucleus of CRC cells and that nuclear HOXD-AS1 was downregulated in most CRC specimens and cell lines. Lower levels of nuclear HOXD-AS1 expression were associated with poor outcomes of CRC patients. HOXD-AS1 downregulation enhanced proliferation and migration of CRC cells in vitro and facilitated CRC tumourigenesis and metastasis in vivo. Mechanistic investigations revealed that HOXD-AS1 could suppress HOXD3 transcription by recruiting PRC2 to induce the accumulation of the repressive marker H3K27me3 at the HOXD3 promoter. Subsequently, HOXD3, as a transcriptional activator, promoted Integrin β3 transcription, thereby activating the MAPK/AKT signalling pathways.
CONCLUSION: Our results reveal a previously unrecognized HOXD-AS1-HOXD3-Integrin β3 regulatory axis involving in epigenetic and transcriptional regulation constitutes to CRC carcinogenesis and progression.

BACKGROUND: Lung cancer is one of the most leading causes of cancer-related deaths in adults worldwide. Non-Small Cell Lung Cancer (NSCLC), which comprises 80 to 85% of all lung cancers, is the most lethal subtype of lung cancer with a 5-year survival of less than 13%. In this study, we identified a poorly-studied kinase PDK4 as the most up-regulated kinase encoding gene in Cisplatin resistant lung adenocarcinoma.
METHODS: In vitro cell viability assay and in vivo tumor xenograft assay were used in the detection of cell proliferation. RNA isolation, quantitative Real-Time PCR, Western blot analysis, immunohistochemistry were used to investigate the expression of RNA and protein. Lentivirus infection was used to regulate gene expression. Luciferase assays were used to monitor EPAS1 promoter activity.
RESULTS: In vivo PDK4 expression was elevated in a Cisplatin-resistant population of lung adenocarcinoma cells, PDK4-dependent Cisplatin-resistance promotes tumor growth of lung adenocarcinoma in vivo and in vitro, clinically PDK4 expression was associated with poor prognosis in lung adenocarcinoma patients, mechanically PDK4 promoted cell growth and Cisplatin-resistance of lung adenocarcinoma via transcriptional regulation of endothelial PAS domain-containing protein 1 (EPAS1).
CONCLUSION: PDK4 is the most up-regulated kinase encoding gene in Cisplatin resistant lung adenocarcinoma and PDK4-dependent Cisplatin-resistance promotes tumor growth of lung adenocarcinoma mainly through transcriptional regulation of EPAS1. Enriched PDK4 expression was correlated with the poor prognosis of lung cancer patients, indicating that PDK4 could be a potential therapeutic target for Cisplatin-resistant lung adenocarcinoma.

BACKGROUND: Prevention of unnecessary biopsies and overtreatment of indolent disease remains a challenge in the management of prostate cancer. Novel non-invasive tests that can identify clinically significant (intermediate-risk and high-risk) diseases are needed to improve risk stratification and monitoring of prostate cancer patients. Here, we investigated a panel of six DNA methylation biomarkers in urine samples collected post-digital rectal exam from patients undergoing prostate biopsy, for their utility to guide decision making for diagnostic biopsy and early detection of aggressive prostate cancer.
RESULTS: We recruited 408 patients in risk categories ranging from benign to low-, intermediate-, and high-risk prostate cancer from three international cohorts. Patients were separated into 2/3 training and 1/3 validation cohorts. Methylation biomarkers were analyzed in post-digital rectal exam urinary sediment DNA by quantitative MethyLight assay and investigated for their association with any or aggressive prostate cancers. We developed a Prostate Cancer Urinary Epigenetic (ProCUrE) assay based on an optimal two-gene (HOXD3 and GSTP1) LASSO model, derived from methylation values in the training cohort, and assessed ProCUrE's diagnostic and prognostic ability for prostate cancer in both the training and validation cohorts. ProCUrE demonstrated improved prostate cancer diagnosis and identification of patients with clinically significant disease in both the training and validation cohorts. Using three different risk stratification criteria (Gleason score, D'Amico criteria, and CAPRA score), we found that the positive predictive value for ProCUrE was higher (59.4-78%) than prostate specific antigen (PSA) (38.2-72.1%) for all risk category comparisons. ProCUrE also demonstrated additive value to PSA in identifying GS ≥ 7 PCa compared to PSA alone (DeLong's test p = 0.039), as well as additive value to the PCPT risk calculator for identifying any PCa and GS ≥ 7 PCa (DeLong's test p = 0.011 and 0.022, respectively).
CONCLUSIONS: ProCUrE is a promising non-invasive urinary methylation assay for the early detection and prognostication of prostate cancer. ProCUrE has the potential to supplement PSA testing to identify patients with clinically significant prostate cancer.

Many non-small cell lung cancer (NSCLC) patients initially benefiting from gefitinib are confronted with acquired resistance. MiR-138 was previously stated as a growth inhibitor of several cancer cell lines including NSCLC cells and its expression level was significantly lower in gefitinib-resistant cells. The role of miR-138 in NSCLC cell lines PC9 and A549 was verified using methyl thiazolyl tetrazolium (MTT) assay and colony formation assay. Quantitative real-time PCR (RT-PCR) was employed to assess the level of miR-138 in gefitinib-sensitive PC9 cells and gefitinib-resistant PC9GR cells. Bioinformatic algorithms (TargetScan) and rVISTA 2.0 were used to predict binding sites on miR-138 and its target genes. MiR-138 inhibited cell proliferation of PC9 and A549 cells. In PC9GR cells, miR-138 expression was inhibited. Gefitinib treatment negatively regulated miR-138 in PC9 cells. Transfection of PC9GR cells with miR-138 mimics significantly reduced cell viability. MiR-138 was directly regulated by Homeobox A4 (HOXA4) via an HOXA4-binding site on the promoter region. TargetScan predicted numerous miR-138 target genes and EGFR was found to be the functional downstream effector of miR-138. We demonstrated that miR-138 is regulated by HOXA4 and exerts its functions via inhibiting EGFR expression in NSCLC cells.

BACKGROUND/AIMS: Homeobox D3 (HOXD3) is a member of the homeobox family of genes that is known primarily for its transcriptional regulation of morphogenesis in all multicellular organisms. In this study, we sought to explore the role that HOXD3 plays in the stem-like capacity, or stemness, and drug resistance of breast cancer cells.
METHODS: Expression of HOXD3 in clinical breast samples were examined by RT-PCR and immunohistochemistry. HOXD3 expression in breast cancer cell lines were analyzed by RT-PCR and western blot. Ability of drug resistance in breast cancer cells were elevated by MTT cell viability and colony formation assays. We examined stemness using cell fluorescent staining, RT-PCR and western blot for stem cell marker expression. Finally, activity of wnt signaling was analyzed by FOPflash luciferase assays. RT-PCR and western blot were performed for downstream genes of wnt signaling.
RESULTS: We demonstrated that HOXD3 is overexpressed in breast cancer tissue as compared to normal breast tissue. HOXD3 overexpression enhances breast cancer cell drug resistance. Furthermore, HOXD3 upregulation in the same cell lines increased sphere formation as well as the expression levels of stem cell biomarkers, suggesting that HOXD3 does indeed increase breast cancer cell stemness. Because we had previously shown that HOXD3 expression is closely associated with integrin β3 expression in breast cancer patients, we hypothesized that HOXD3 may regulate breast cancer cell stemness and drug resistance through integrin β 3. Cell viability assays showed that integrin β 3 knockdown increased cell viability and that HOXD3 could not restore cancer cell stemness or drug resistance. Given integrin β 3's relationship with Wnt/β-catenin signaling, we determine whether HOXD3 regulates integrin β 3 activity through Wnt/β-catenin signaling. We found that, even though HOXD3 increased the expression of Wnt/β-catenin downstream genes, it did not restore Wnt/β-catenin signaling activity, which was inhibited in integrin β3 knockdown breast cancer cells.
CONCLUSION: We demonstrate that HOXD3 plays a critical role in breast cancer stemness and drug resistance via integrin β3-mediated Wnt/β-catenin signaling. Our findings open the possibility for improving the current standard of care for breast cancer patients by designing targeted molecular therapies that overcome the barriers of cancer cell stemness and drug resistance.

Hepatocellular carcinoma (HCC), one of the most common aggressive tumors worldwide has a relatively high mortality rate among malignant tumors. MicroRNAs (miRNAs), acting as tumor suppressors, are involved in the regulation of invasion, metastasis, and angiogenesis of tumor cells. However, a potential role for miR-203a in HCC has not been described yet. In this study, we show that miR-203a markedly suppresses HCC cell migration, invasion, and angiogenesis. In addition, the transcription factor HOXD3 appears to be a direct target of miR-203a. HOXD3 knockdown substantially decreased HCC cell migration, invasion, and angiogenesis, effects similar to those seen for miR-203a expression. Rescuing the function of HOXD3 attenuated the effect of miR-203a overexpression in HCC cells. Furthermore, HOXD3 can directly target the promoter region of VEGFR and increase VEGFR expression. Taken together, our findings indicate that miR-203a inhibits HCC cell invasion, metastasis, and angiogenesis by negatively targeting HOXD3 and suppressing cell signaling through the VEGFR pathway, suggesting that miR-203a might represent a potential therapeutic target for HCC intervention.

OBJECTIVES: Aberrant homeobox (HOX) gene expression is reported in high-grade serous ovarian carcinoma (HGSOC), however, its prognostic significance remains unclear.
METHODS: HOX genes associated with progression-free survival (PFS) in a discovery cohort of primary HGSOC samples with RNA sequencing data, and those previously reported to be associated with clinical outcomes, were selected for qPCR testing in an independent training cohort of primary HGSOC samples (n=71). A prognostic model for PFS was developed using univariate and multivariate Cox regression. Patients were stratified into risk groups that optimized the test statistic. The model was tested in an independent HGSOC cohort from The Cancer Genome Atlas (TCGA) (n=320). The effect of selected HOX genes on drug sensitivity and reactive oxygen species (ROS) accumulation was examined in vitro.
RESULTS: Of 23 HOX genes tested in the training cohort, HOXA4 (HR=1.20, 95% CI=1.07-1.34, P=0.002) and HOXB3 (HR=1.09, 95% CI=1.01-1.17, P=0.027) overexpression were significantly associated with shorter PFS in multivariate analysis. Based on the optimal cutoff of the HOXA4/HOXB3 risk score, median PFS was 16.9months (95% CI=14.6-21.2months) and not reached (>80months) for patients with high and low risk scores, respectively (HR=8.89, 95% CI=2.09-37.74, P<0.001). In TCGA, the HOXA4/HOXB3 risk score was significantly associated with disease-free survival (HR=1.44, 95% CI=1.00-2.09, P=0.048). HOXA4 or HOXB3 overexpression in ovarian cancer cells decreased sensitivity to cisplatin and attenuated the generation of cisplatin-induced ROS (P<0.05).
CONCLUSIONS: HOXA4/HOXB3 gene expression-based risk score may be useful for prognostic risk stratification and warrants prospective validation in HGSOC patients.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive malignancy only cured by intensive therapy. However, many elderly and unfit patients cannot receive such treatment due to an unacceptable risk of treatment-related morbidity and mortality. Disease-stabilizing therapy is then the only possible strategy, one alternative being treatment based on all-trans retinoic acid (ATRA) combined with the histone deacetylase inhibitor valproic acid and possibly low-toxicity conventional chemotherapy.
METHODS: Primary AML cells were derived from 43 patients included in two clinical studies of treatment based on ATRA, valproic acid and theophyllamine; low toxicity chemotherapy (low-dose cytarabine, hydroxyurea, 6-mercaptopurin) was also allowed. Pretreatment leukemic cells were analyzed by mutation profiling of 54 genes frequently mutated in myeloid malignancies and by global gene expression profiling before and during in vivo treatment.
RESULTS: Patients were classified as responders and non-responders to the treatment, however response to treatment showed no significant associations with karyotype or mutational profiles. Significance analysis of microarray (SAM) showed that responders and non-responders significantly differed with regard to the expression of 179 different genes. The differentially expressed genes encoding proteins with a known function were further classified based on the PANTHER (protein annotation through evolutionary relationship) classification system. The identified genes encoded proteins that are involved in several important biological functions, but a main subset of the genes were important for transcriptional regulation. These pretherapy differences in gene expression were largely maintained during treatment. Our analyses of primary AML cells during in vivo treatment suggest that ATRA modulates HOX activity (i.e. decreased expression of HOXA3, HOXA4 and HOXA5 and their regulator PBX3), but altered function of DNA methyl transferase 3A (DNMT3A) and G-protein coupled receptor signaling may also contribute to the effect of the overall treatment.
CONCLUSIONS: Responders and non-responders to AML stabilizing treatment based on ATRA and valproic acid differ in the pretreatment transcriptional regulation of their leukemic cells, and these differences may be important for the clinical effect of this treatment.
TRIAL REGISTRATIONS: ClinicalTrials.gov no. NCT00175812 ; EudraCT no. 2004-001663-22, registered September 9, 2005 and ClinicalTrials.gov no. NCT00995332 ; EudraCT no. 2007-2007-001995-36, registered October 14, 2009.

Because HOX genes encode master regulatory transcription factors that regulate stem cells (SCs) during development and aberrant expression of HOX genes occurs in various cancers, our goal was to determine if dysregulation of HOX genes is involved in the SC origin of colorectal cancer (CRC). We previously reported that HOXA4 and HOXD10 are expressed in the colonic SC niche and are overexpressed in CRC. HOX gene expression was studied in SCs from human colon tissue and CRC cells (CSCs) using qPCR and immunostaining. siRNA-mediated knockdown of HOX expression was used to evaluate the role of HOX genes in modulating cancer SC (CSC) phenotype at the level of proliferation, SC marker expression, and sphere formation. All-trans-retinoic-acid (ATRA), a differentiation-inducing agent was evaluated for its effects on HOX expression and CSC growth. We found that HOXA4 and HOXA9 are up-regulated in CRC SCs. siRNA knockdown of HOXA4 and HOXA9 reduced: (i) proliferation and sphere-formation and (ii) gene expression of known SC markers (ALDH1, CD166, LGR5). These results indicate that proliferation and self-renewal ability of CRC SCs are reduced in HOXA4 and HOXA9 knockdown cells. ATRA decreased HOXA4, HOXA9, and HOXD10 expression in parallel with reduction in ALDH1 expression, self-renewal, and proliferation. Overall, our findings indicate that overexpression of HOXA4 and HOXA9 contributes to self-renewal and overpopulation of SCs in CRC. Strategies designed to modulate HOX expression may provide ways to target malignant SCs and to develop more effective therapies for CRC.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.