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1.  Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms 
BMC Physiology  2011;11:9.
The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.
We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).
Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.
PMCID: PMC3125234  PMID: 21627813
2.  Elevated HOXA1 expression correlates with accelerated tumor cell proliferation and poor prognosis in gastric cancer partly via cyclin D1 
HOXA1 is a member of the Homeobox gene family, which encodes a group of highly conserved transcription factors that are important in embryonic development. However, it has been reported that HOXA1 exhibits oncogenic properties in many malignancies. This study focused on the expression and clinical significance of HOXA1 in gastric cancer (GC).
To assess the mRNA and protein expression of HOXA1 and cyclin D1 in GC tissues, we utilized qRT-PCR and western blotting, respectively. The effects of HOXA1 on GC cell proliferation, migration, and invasion, as well as xenograft tumor formation and the cell cycle were investigated in our established stable HOXA1 knockdown GC cell lines. The protein expression of HOXA1 and cyclin D1 was examined by immunohistochemistry using GC tissue microarrays (TMA) to analyze their relationship on a histological level. The Kaplan-Meier method and cox proportional hazards model were used to analyze the relationship of HOXA1 and cyclin D1 expression with GC clinical outcomes.
HOXA1 mRNA and protein expression were upregulated in GC tissues. Knockdown of HOXA1 in GC cells not only inhibited cell proliferation, migration, and invasion in vitro but also suppressed xenograft tumor formation in vivo. Moreover, HOXA1 knockdown induced changes in the cell cycle, and HOXA1 knockdown cells were arrested at the G1 phase, the number of cells in S phase was reduced, and the expression of cyclin D1 was decreased. In GC tissues, high cyclin D1 mRNA and protein expression were detected, and a significant correlation was found between the expression of HOXA1 and cyclin D1. Survival analysis indicated that HOXA1 and cyclin D1 expression were significantly associated with disease-free survival (DFS) and overall survival (OS). Interestingly, patients with tumors that were positive for HOXA1 and cyclin D1 expression showed worse prognosis. Multivariate analysis confirmed that the combination of HOXA1 and cyclin D1 was an independent prognostic indicator for OS and DFS.
Our data show that HOXA1 plays a crucial role in GC development and clinical prognosis. HOXA1, alone or combination with cyclin D1, may serve as a novel prognostic biomarker for GC.
PMCID: PMC4721151  PMID: 26791264
HOXA1; cyclin D1; Gastric cancer; Biomarker; Prognosis
3.  The Truncated Hoxa1 Protein Interacts with Hoxa1 and Pbx1 in Stem Cells 
Journal of cellular biochemistry  2009;106(3):427-443.
Hox genes contain a homeobox encoding a 60-amino acid DNA binding sequence. The Hoxa-1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1-993), and another protein lacking this domain (Hoxa1-399). The functions of Hoxa1-399 are unknown. We detected Hoxa1-993 and Hoxa1-399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1-399 functions in cellular differentiation we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1-993 and its cofactor, Pbx1, bind to the Hoxb1 SOct-R3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1-399 (the F9-399 line) show that Hoxa1-399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1-399 reduces Hoxa1-993/Pbx1 binding to the Hoxb1 SOct-R3 region. GST-pull down experiments suggest that Hoxa1-399, Hoxa1-993, and Pbx1 form a trimer. However, the F9-399 line exhibits no differences in RA-induced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wild type.
PMCID: PMC3923656  PMID: 19115252
Homeobox; Transcription Factor; Splice Variant; cell differentiation; F9 cells; retinoids; Hoxb1; Pbx; teratocarcinoma; stem cell; vitamin A
4.  HOXA5-Induced Apoptosis in Breast Cancer Cells Is Mediated by Caspases 2 and 8 
Molecular and Cellular Biology  2004;24(2):924-935.
HOXA5 is a transcriptional factor whose expression is lost in more than 60% of breast carcinomas. Our previous work demonstrated that the overexpression of HOXA5 in MCF7 cells resulted in cell death through a p53-dependent apoptotic pathway. To determine whether p53-independent apoptotic pathways are involved in HOXA5-induced cell death, we engineered a p53-mutant breast cancer cell line, Hs578T, to inducibly express HOXA5. Induction of HOXA5 expression led to cell death with features typical of apoptosis within 24 h, and the expression levels of mutant p53 and its target genes either decreased or remained unchanged. To decipher apoptotic pathways, the HOXA5-expressing cells were treated with a variety of apoptotic inhibitors. Besides a general caspase inhibitor, caspase 2- and 8-specific inhibitors largely abolished HOXA5-induced apoptosis, whereas caspase 1-, 3-, 6-, and 9-specific inhibitors had no significant effects. Western blot analysis further confirmed that caspases 2 and 8 were activated after the induction of HOXA5 expression. Further, several small interfering RNAs which specifically silenced caspase 2 and caspase 8 expression significantly blocked HOXA5-induced apoptosis. HOXA5 expression could also sensitize cells to tumor necrosis factor alpha-induced apoptosis by at least 100-fold. These results indicate that expression of HOXA5 can induce apoptosis through an apoptotic mechanism mediated by caspases 2 and 8.
PMCID: PMC343791  PMID: 14701762
5.  Two isoforms of HOXA9 function differently but work synergistically in human MLL-rearranged leukemia 
Blood cells, molecules & diseases  2012;49(2):102-106.
HOXA9 plays a critical role in both normal hematopoiesis and leukemogenesis, particularly in the development and maintenance of mixed lineage leukemia (MLL)-rearranged leukemia. Through reverse transcription polymerase chain reaction (RT-PCR) analysis of HOXA9 transcripts in human leukemia and normal bone marrow samples, we identified a truncated isoform of HOXA9, namely HOXA9T, and found that both HOXA9T and canonical HOXA9 were highly expressed in leukemia cell lines bearing MLL rearrangements, relative to human normal bone marrow cells or other subtypes of leukemia cells. A frameshift in HOXA9T in exon I causes a premature stop codon upstream of the PBX binding domain and the homeodomain, which leads to the generation of a non-homeodomain-containing protein. Unlike the canonical HOXA9, HOXA9T alone cannot transform normal bone marrow progenitor cells. Moreover, HOXA9T cannot cooperate with MEIS1 to transform cells, despite the presence of a MEIS1-binding domain. Remarkably, although the truncated isoforms of many proteins function as dominant-negative competitors or inhibitors of their full-length counterparts, this is not the case for HOXA9T; instead, HOXA9T synergized with HOXA9 in transforming mouse normal bone marrow progenitor cells through promoting self-renewal and proliferation of the cells. Collectively, our data indicate that both truncated and full-length forms of HOXA9 are highly expressed in human MLL-rearranged leukemia, and the truncated isoform of HOXA9 might also play an oncogenic role by cooperating with canonical HOXA9 in cell transformation and leukemogenesis.
PMCID: PMC3399022  PMID: 22633751
HOXA9; HOXA9T; isoforms; leukemia
6.  Comprehensive analysis of the long noncoding RNA HOXA11-AS gene interaction regulatory network in NSCLC cells 
Long noncoding RNAs (lncRNAs) are related to different biological processes in non-small cell lung cancer (NSCLC). However, the possible molecular mechanisms underlying the effects of the long noncoding RNA HOXA11-AS (HOXA11 antisense RNA) in NSCLC are unknown.
HOXA11-AS was knocked down in the NSCLC A549 cell line and a high throughput microarray assay was applied to detect changes in the gene profiles of the A549 cells. Bioinformatics analyses (gene ontology (GO), pathway, Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses) were performed to investigate the potential pathways and networks of the differentially expressed genes. The molecular signatures database (MSigDB) was used to display the expression profiles of these differentially expressed genes. Furthermore, the relationships between the HOXA11-AS, de-regulated genes and clinical NSCLC parameters were verified by using NSCLC patient information from The Cancer Genome Atlas (TCGA) database. In addition, the relationship between HOXA11-AS expression and clinical diagnostic value was analyzed by receiver operating characteristic (ROC) curve.
Among the differentially expressed genes, 277 and 80 genes were upregulated and downregulated in NSCLC, respectively (fold change ≥2.0, P < 0.05 and false discovery rate (FDR) < 0.05). According to the degree of the fold change, six upregulated and three downregulated genes were selected for further investigation. Only four genes (RSPO3, ADAMTS8, DMBT1, and DOCK8) were reported to be related with the development or progression of NSCLC based on a PubMed search. Among all possible pathways, three pathways (the PI3K-Akt, TGF-beta and Hippo signaling pathways) were the most likely to be involved in NSCLC development and progression. Furthermore, we found that HOXA11-AS was highly expressed in both lung adenocarcinoma and squamous cell carcinoma based on TCGA database. The ROC curve showed that the area under curve (AUC) of HOXA11-AS was 0.727 (95% CI 0.663–0.790) for lung adenocarcinoma and 0.933 (95% CI 0.906–0.960) for squamous cell carcinoma patients. Additionally, the original data from TCGA verified that ADAMTS8, DMBT1 and DOCK8 were downregulated in both lung adenocarcinoma and squamous cell carcinoma, whereas RSPO3 expression was upregulated in lung adenocarcinoma and downregulated in lung squamous cell carcinoma. For the other five genes (STMN2, SPINK6, TUSC3, LOC100128054, and C8orf22), we found that STMN2, TUSC3 and C8orf22 were upregulated in squamous cell carcinoma and that STMN2 and USC3 were upregulated in lung adenocarcinoma. Furthermore, we compared the correlation between HOXA11-AS and de-regulated genes in NSCLC based on TCGA. The results showed that the HOXA11-AS expression was negatively correlated with DOCK8 in squamous cell carcinoma (r = −0.124, P = 0.048) and lung adenocarcinoma (r = −0.176, P = 0.005). In addition, RSPO3, ADAMTS8 and DOCK8 were related to overall survival and disease-free survival (all P < 0.05) of lung adenocarcinoma patients in TCGA.
Our results showed that the gene profiles were significantly changed after HOXA11-AS knock-down in NSCLC cells. We speculated that HOXA11-AS may play an important role in NSCLC development and progression by regulating the expression of various pathways and genes, especially DOCK8 and TGF-beta pathway. However, the exact mechanism should be verified by functional experiments.
PMCID: PMC5133743  PMID: 27980454
HOXA11-AS; NSCLC; Microarray assay; GO; KEGG; Pathway
7.  Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes 
eLife  null;5:e09540.
The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation.
eLife digest
The nucleus of a eukaryotic cell (which includes plant and animal cells) contains most of the cell’s genetic material in the form of carefully packaged strands of DNA. Genes are stretches of DNA that contain the instructions needed to produce the proteins and RNA molecules that the cell needs to survive. These molecules move across the membrane that surrounds the nucleus through pores made of proteins. One of these pore-forming proteins is called Nup98. The gene that produces Nup98 is frequently mutated in leukemia, where part of it becomes fused to regions of other unrelated genes. The proteins made from these combined genes are known as “fusion proteins”.
The Nup98-HoxA9 fusion protein has been well studied, and appears to cause leukemia by interfering with the process called (“cell differentiation”) by which stem cells specialize to form different types of blood cells. During cell differentiation, cells change which sets of genes they activate to become specific types of cells. A family of genes called Hox genes (to which the gene for HoxA9 belongs) is critical in cell differentiation and thus must be fine-tuned. It is also known that the Hox genes form clusters, and its activation is partly controlled by how tightly the DNA is packaged.
Previous studies have shown that the Nup98-HoxA9 fusion protein takes on the form of small dots in the nucleus. Oka et al. have now tracked how these proteins are distributed inside of the nucleus, and examined which part of the DNA they bind to, in more detail. This revealed that the dots of Nup98-HoxA9 tend to associate with tightly packed DNA, especially on Hox cluster genes, and activate these genes.
Oka et al. further found that a protein called Crm1, which is well known as a nuclear export factor that carries molecules out of the nucleus through the pore, is already bound to the Hox cluster genes in the nucleus and recruits the Nup98-HoxA9 protein. This interaction may change how the Hox gene is packaged in the nucleus. A future challenge will be to reveal how the Nup98-HoxA9 fusion protein and Crm1 on Hox cluster genes control gene expression.
PMCID: PMC4718815  PMID: 26740045
Nucleoporin; Nup98; Crm1; Leukemia; Hox; Chromatin; Human; Mouse
8.  Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells 
Homeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited.
To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay.
Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect.
Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.
PMCID: PMC2904782  PMID: 20540809
9.  HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes 
PLoS ONE  2009;4(9):e6845.
During the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.
PMCID: PMC2731163  PMID: 19727442
10.  Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis 
Oncogenesis  2014;3(12):e135-.
Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.
PMCID: PMC4275563  PMID: 25531430
11.  Cardiovascular defects in a mouse model of HOXA1 syndrome 
Human Molecular Genetics  2011;21(1):26-31.
Congenital heart disease is one of the most common human birth defects, yet many genes and pathways regulating heart development remain unknown. A recent study in humans revealed that mutations in a single Hox gene, HOXA1 (Athabascan Brainstem Dysgenesis Syndrome, Bosley-Salih-Alorainy Syndrome), can cause severe cardiovascular malformations, some of which are lethal without surgical intervention. Since the discovery of the human syndromes, there have been no reports of any Hox mouse mutants with cardiac defects, hampering studies to explore the developmental causes of the human disease. In this study, we identify severe cardiovascular malformations in a Hox mouse model, which mimic the congenital heart defects in HOXA1 syndrome patients. Hoxa1 null mice show defects such as interrupted aortic arch, aberrant subclavian artery and Tetralogy of Fallot, demonstrating that Hoxa1 is required for patterning of the great arteries and outflow tract of the heart. We show that during early embryogenesis, Hoxa1 is expressed in precursors of cardiac neural crest cells (NCCs), which populate the heart. We further demonstrate that Hoxa1 acts upstream of several genes, important for neural crest specification. Thus, our data allow us to suggest a model in which Hoxa1 regulates heart development through its influence on cardiac NCCs, providing insight into the mechanisms underlying the human disease.
PMCID: PMC3235008  PMID: 21940751
12.  Endothelin Type A Receptor (ETA) Expression Is Regulated by HOXA10 in Human Endometrial Stromal Cells 
Endothelin type A receptor (ETA) is a member of the superfamily of G protein-coupled receptors. Our laboratory conducted a microarray screen that identified ETA as target of HOXA10 transcriptional control in endometrium. Here, we confirm HOXA10-regulated ETA expression in endometrium. Endometrial biopsies were obtained from fertile reproductive-age individuals, and first trimester decidual samples were obtained at the time of elective termination. Immunohistochemistry (IHC) was used to identify ETA protein in endometrium as well as first trimester decidua. ETA was expressed in endometrial stromal cells throughout the menstrual cycle. ETA was also highly expressed in first trimester decidual cells. The regulatory relationship between HOXA10 and ETA was established by transient transfection analysis. The human endometrial stromal cell line (HESC) and the human endometrial epithelial cell line (Ishikawa) were transfected with pcDNA/HOXA10, HOXA10 small interfering RNA (siRNA), or respective controls. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to determine expression levels of HOXA10 and ETA in each group. ETA gene expression increased 9-fold (P < .05) after pcDNA/HOXA10 transfection of HESC. ETA was not regulated by HOXA10 in Ishikawa cells. We conclude that ETA is expressed in normal endometrium and decidua. Expression of this receptor is regulated by an essential mediator of endometrial receptivity, HOXA10. ETA may enhance the proliferative potential of endometrial cells in a manner similar to that seen in vascular smooth muscle cells. ETA likely acts as a molecular mechanism by which HOXA10 promotes stromal cell growth and prostaglandin production in both the implantation window and decidua.
PMCID: PMC3107845  PMID: 20371740
ETA; HOXA10; endometrium; decidua
13.  A Hoxa13: Cre Mouse Strain for Conditional Gene Manipulation in Developing Limb, Hindgut, and Urogenital System 
Genesis (New York, N.Y. : 2000)  2015;53(6):366-376.
The developing limb is a useful model for studying organogenesis and developmental processes. Although Cre alleles exist for conditional loss- or gain-of-function in limbs, Cre alleles targeting specific limb subdomains are desirable. Here we report on the generation of the Hoxa13:Cre line, in which the Cre gene is inserted in the endogenous Hoxa13 gene. We provide evidence that the Cre is active in embryonic tissues/ regions where the endogenous Hoxa13 gene is expressed. Our results show that cells expressing Hoxa13 in developing limb buds contribute to the entire autopod (hand/feet) skeleton and validate Hoxa13 as a distal limb marker as far as the skeleton is concerned. In contrast, in the limb musculature, Cre-based fate mapping shows that almost all muscle masses of the zeugopod (forearm) and part of the triceps contain Hoxa13-expressing cells and/or their descendants. Besides the limb, the activity of the Cre is detectable in the urogenital system and the hindgut, primarily in the epithelium and smooth muscles. Together our data show that the Hoxa13:Cre allele is a useful tool for conditional gene manipulation in the urogenital system, posterior digestive tract, autopod and part of the limb musculature.
PMCID: PMC4486385  PMID: 25980463 CAMSID: cams4764
genetics; process; mammal; organism; organogenesis; process; fate specification; process; limb/wing/appendage; tissue; muscle; tissue; skeletal; tissue; reproductive; tissue; gut; tissue
14.  The long non-coding RNA HOTTIP promotes progression and gemcitabine resistance by regulating HOXA13 in pancreatic cancer 
The human genome encodes many long non-coding RNAs (lncRNAs). However, their biological functions, molecular mechanisms, and the prognostic value associated with pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. Here, we identify a fundamental role for the lncRNA HOXA transcript at the distal tip (HOTTIP) in the progression and chemoresistance of PDAC.
High-throughput microarrays were performed to detect the expression profiles of lncRNAs and messenger RNAs in eight human PDAC tissues and four pancreatic tissues. Quantitative real-time PCR was used to determine the levels of HOTTIP and HOXA13 transcripts in PDAC cell lines and 90 PDAC samples from patients. HPDE6 cells (immortalized human pancreatic ductal epithelial cells) and corresponding adjacent non-neoplastic tissues were used as controls, respectively. The functions of HOTTIP and HOXA13 in cell proliferation, invasion, and epithelial-mesenchymal transition were evaluated by targeted knockdown in vitro. CCK-8 assays, colony formation assays, and xenografts in nude mice were used to investigate whether targeted silencing of HOTTIP could sensitize pancreatic cancer cells to gemcitabine. Immunohistochemistry was performed to investigate the relationship between HOXA13 expression and patient outcome.
Microarray analyses revealed that HOTTIP was one of the most significantly upregulated lncRNAs in PDAC tissues compared with pancreatic tissues. Quantitative PCR further verified that HOTTIP levels were increased in PDAC cell lines and patient samples compared with controls. Functionally, HOTTIP silencing resulted in proliferation arrest by altering cell-cycle progression, and impaired cell invasion by inhibiting epithelial-mesenchymal transition in pancreatic cancer. Additionally, inhibition of HOTTIP potentiated the antitumor effects of gemcitabine in vitro and in vivo. Furthermore, knockdown of HOXA13 by RNA interference (siHOXA13) revealed that HOTTIP promoted PDAC cell proliferation, invasion, and chemoresistance, at least partly through regulating HOXA13. Immunohistochemistry results revealed that higher HOXA13 expression was correlated with lymph node metastasis, poor histological differentiation, and decreased overall survival in PDAC patients.
As a crucial tumor promoter, HOTTIP promotes cell proliferation, invasion, and chemoresistance by modulating HOXA13. Therefore, the HOTTIP/HOXA13 axis is a potential therapeutic target and molecular biomarker for PDAC.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0442-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4372045  PMID: 25889214
Pancreatic cancer; HOTTIP; Oncogenic; Epithelial-mesenchymal transition; Chemoresistance
15.  Changing homeodomain residues 2 and 3 of Hoxa1 alters its activity in a cell-type and enhancer dependent manner 
Nucleic Acids Research  2002;30(12):2663-2668.
The second and third amino acid residues of the N-terminal arm of most Hox protein homeodomains are basic (lysine or arginine), whereas they are asparagine and alanine, respectively, in the Hoxa1 homeodomain. Previous reports pinpointed these residues as specificity determinants in the function of Hoxa1 when it is acting as a monomer. However, in vitro data supported that these residues do not influence the target specificity of Hoxa1 in Pbx1a–Hoxa1 heterodimers. Here, we have analysed the transcriptional activity of a Hoxa1(NA-KR) mutant for which the asparagine and alanine residues of the homeodomain have been replaced by lysine and arginine, respectively. Comparison between the wild-type and mutant Hoxa1 reveals that they show distinct activity on the TSEII enhancer of the somatostatin gene, but that they are equally active in the presence of Pbx and Prep cofactors. This therefore corroborates the biochemical evidence having shown that the second and third residues of the homeodomain do not contribute to the DNA binding of Hoxa1–Pbx dimers. However, on the hoxb1 autoregulatory enhancer, Hoxa1 and Hoxa1(NA-KR) may display distinct activity despite the presence of Pbx, in a cell-type dependent manner. Therefore, our data suggest that, depending on the enhancer, these residues may contribute to the functional specificity of Hoxa1 and that this contribution may not be abrogated by the interaction with Pbx.
PMCID: PMC117285  PMID: 12060683
16.  Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of HoxA Genes in Developing Limbs 
PLoS Genetics  2013;9(12):e1004018.
HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.
Author Summary
Hox genes encode transcription factors with crucial roles during development. These genes are grouped in four different clusters names HoxA, B, C, and D. Mutations in genes of the HoxA and D clusters have been found in several human syndromes, affecting in some cases limb development. Despite their essential role and contrary to the genes of the HoxD cluster, little is known about how the HoxA genes are regulated. Here, we identified a large set of regulatory elements controlling HoxA genes during limb development. By studying spatial chromatin organization at the HoxA region, we found that the regulatory elements are spatially clustered regardless of their activity. Clustering of enhancers define tissue-specific chromatin domains that interact specifically with each other and with active genes in the limb. Our findings give support to the emerging concept that chromatin architecture defines the functional properties of genomes. Additionally, our study suggests a common constraint of the chromatin topology in the evolution of HoxA and HoxD regulation in the emergence of the hand/foot, which is one of the major morphological innovations in vertebrates.
PMCID: PMC3873244  PMID: 24385922
17.  Targeted germ line disruptions reveal general and species-specific roles for paralog group 1 hox genes in zebrafish 
The developing vertebrate hindbrain is transiently segmented into rhombomeres by a process requiring Hox activity. Hox genes control specification of rhombomere fates, as well as the stereotypic differentiation of rhombomere-specific neuronal populations. Accordingly, germ line disruption of the paralog group 1 (PG1) Hox genes Hoxa1 and Hoxb1 causes defects in hindbrain segmentation and neuron formation in mice. However, antisense-mediated interference with zebrafish hoxb1a and hoxb1b (analogous to murine Hoxb1 and Hoxa1, respectively) produces phenotypes that are qualitatively and quantitatively distinct from those observed in the mouse. This suggests that PG1 Hox genes may have species-specific functions, or that anti-sense mediated interference may not completely inactivate Hox function in zebrafish.
Using zinc finger and TALEN technologies, we disrupted hoxb1a and hoxb1b in the zebrafish germ line to establish mutant lines for each gene. We find that zebrafish hoxb1a germ line mutants have a more severe phenotype than reported for Hoxb1a antisense treatment. This phenotype is similar to that observed in Hoxb1 knock out mice, suggesting that Hoxb1/hoxb1a have the same function in both species. Zebrafish hoxb1b germ line mutants also have a more severe phenotype than reported for hoxb1b antisense treatment (e.g. in the effect on Mauthner neuron differentiation), but this phenotype differs from that observed in Hoxa1 knock out mice (e.g. in the specification of rhombomere 5 (r5) and r6), suggesting that Hoxa1/hoxb1b have species-specific activities. We also demonstrate that Hoxb1b regulates nucleosome organization at the hoxb1a promoter and that retinoic acid acts independently of hoxb1b to activate hoxb1a expression.
We generated several novel germ line mutants for zebrafish hoxb1a and hoxb1b. Our analyses indicate that Hoxb1 and hoxb1a have comparable functions in zebrafish and mouse, suggesting a conserved function for these genes. In contrast, while Hoxa1 and hoxb1b share functions in the formation of r3 and r4, they differ with regards to r5 and r6, where Hoxa1 appears to control formation of r5, but not r6, in the mouse, whereas hoxb1b regulates formation of r6, but not r5, in zebrafish. Lastly, our data reveal independent regulation of hoxb1a expression by retinoic acid and Hoxb1b in zebrafish.
PMCID: PMC4061917  PMID: 24902847
Zinc finger nuclease; TALEN nuclease; Retinoic acid signaling; Hindbrain; Nucleosome positioning; Gene expression
18.  Effect of silencing HOXA5 gene expression using RNA interference on cell cycle and apoptosis in Jurkat cells 
Acute lymphocytic leukemia (ALL) is a common malignant tumor with a high morbidity rate among children, accounting for approximately 80% of leukemia cases. Although there have been improvements in the treatment of patients frequent relapse lead to a poor prognosis. The aim of the present study was to determine whether HOXA5 may be used as a target for gene therapy in leukemia in order to provide a new treatment. Mononuclear cells were extracted from the bone marrow according to the clinical research aims. After testing for ALL in the acute stage, the relative mRNA and protein expression of HOXA5 was detected in the ALL remission groups (n=25 cases per group) and the control group [n=20 cases, immune thrombocytopenia (ITP)]. Gene silencing by RNA interference (RNAi) was used to investigate the effect of silencing HOXA5 after small interfering RNA (siRNA) transfection to Jurkat cells. The HOXA5-specific siRNA was transfected to Jurkat cells using lipofectamine. The experiment was divided into the experimental group (liposomal transfection of HOXA5 targeting siRNA), the negative control group (liposomal transfection of cells with negative control siRNA) and the control group (plus an equal amount of cells and culture media only). Western blotting and quantitative fluorescent polymerase chain reaction (QF-PCR) were used to detect the relative HOXA5 mRNA expression and protein distribution in each cell group. Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined using flow cytometry. The expression of HOXA5 at the mRNA and protein levels in the acute phase of ALL was significantly higher than that in ALL in the remission and control groups. In cells transfected with HOXA5-specific siRNA, the expression of HOXA5 at the mRNA and protein levels decreased significantly (P<0.05). The distribution of cells in the cell cycle was also altered. Specifically, more cells were present in the G0/G1 phase compared to the S phase (P<0.05). In addition, the apoptotic rate was significantly higher in cells transfected with HOXA5-specific siRNA (P<0.05). In conclusion, high expression levels of HOXA5 mRNA and protein in children with ALL indicate that HOXA5 is closely associated with childhood ALL. In addition, HOXA5-specific siRNA effectively silences HOXA5 gene expression and induces apoptosis and cell-cycle arrest in Jurkat cells, thus inhibiting cell proliferation.
PMCID: PMC4771120  PMID: 26846409
HOXA5 gene; Jurkat cells; RNA interference; apoptosis; cell cycle
19.  Induction of p53-independent apoptosis by ectopic expression of HOXA5 in human liposarcomas 
Scientific Reports  2015;5:12580.
Dedifferentiated liposarcoma (DDLPS) is a highly malignant subtype of human liposarcoma (LPS), whose genomic profile is characterized by chromosomal amplification at 12q13-q22. miR-26a-2 is one of the most frequently amplified genes in the region, and inhibition of its downstream target genes likely contributes to LPS tumorigenesis. Our previous study of LPS predicted homeobox protein A5 (HOXA5) as a target of miR-26a-2, and here we explored further the function of HOXA5, and its relationship with miR-26a-2 in DDLPS cells. Compared to normal human adipocytes, all LPS cell lines showed significant downregulation of HOXA5 (p = 0.046), and inhibition of miR-26a-2 using anti-miR-26a-2 substantially upregulated HOXA5 expression in these LPS cells. Interestingly, overexpression of HOXA5 alone induced very strong apoptotic response of LPS cells. HOXA5-induced apoptosis was p53-independent and caspase-dependent. Surprisingly, overexpression of HOXA5 induced nuclear translocation of RELA (p65), which was not associated with the transcriptional activity of RELA. Rather, nucleolar sequestration of RELA was observed. Overall, our study demonstrated for the first time that the downregulation of HOXA5 in LPS cells, partly by overexpression of miR-26a-2 in DDLPS, confers LPS cells resistance to apoptotic death. Further studies are required to understand the relationship of HOXA5 and the NFκB pathway in LPS cells.
PMCID: PMC4518222  PMID: 26219418
20.  MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5 
BMC Cancer  2012;12:348.
MicroRNAs (miRNAs) are short, non-coding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Although miR-196a has been implicated in several other cancers, its role in non-small cell lung cancer (NSCLC) is unknown. The aim of the present study was to examine the expression pattern of miR-196a in NSCLC and its clinical significance, as well as its biological role in tumor progression.
Expression of miR-196a was analyzed in 34 NSCLC tissues and five NSCLC cell lines by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of DNA methylation on miR-196a expression was investigated by 5-aza-2-deoxy-cytidine treatment and bisulfite sequencing. The effect of miR-196a on proliferation was evaluated by MTT and colony formation assays, and cell migration and invasion were evaluated by transwell assays. Analysis of target protein expression was determined by western blotting. Luciferase reporter plasmids were constructed to confirm the action of miR-196a on downstream target genes, including HOXA5. Differences between the results were tested for significance using Student’s t-test (two-tailed).
miR-196a was highly expressed both in NSCLC samples and cell lines compared with their corresponding normal counterparts, and the expression of miR-196a may be affected by DNA demethylation. Higher expression of miR-196a in NSCLC tissues was associated with a higher clinical stage, and also correlated with NSCLC lymph-node metastasis. In vitro functional assays demonstrated that modulation of miR-196a expression affected NSCLC cell proliferation, migration and invasion. Our analysis showed that miR-196a suppressed the expression of HOXA5 both at the mRNA and protein levels, and luciferase assays confirmed that miR-196a directly bound to the 3’untranslated region of HOXA5. Knockdown of HOXA5 expression in A549 cells using RNAi was shown to promote NSCLC cell proliferation, migration and invasion. Finally, we observed an inverse correlation between HOXA5 and miR-196a expression in NSCLC tissues.
Our findings indicate that miR-196a is significantly up-regulated in NSCLC tissues, and regulates NSCLC cell proliferation, migration and invasion, partially via the down-regulation of HOXA5. Thus, miR-196a may represent a potential therapeutic target for NSCLC intervention.
PMCID: PMC3503718  PMID: 22876840
Non-small cell lung cancer; miR-196a; Proliferation; Invasion; HOXA5
21.  Oncogenic function of the homeobox A13-long noncoding RNA HOTTIP-insulin growth factor-binding protein 3 axis in human gastric cancer 
Oncotarget  2016;7(24):36049-36064.
To study the mechanisms of gastric tumorigenesis, we have established CSN cell line from human normal gastric mucosa, and CS12, a tumorigenic and invasive gastric cancer cell line from CSN passages. Many stem cell markers were expressed in both CSN and CS12 cells, but LGR5 and NANOG were expressed only in CS12 cells. Increased expression of homeobox A13 (HoxA13) and its downstream cascades was significant for the tumorigenic activity of CS12 cells, and was associated with recruitment of E2F-1 to HoxA13 promoter accompanied with increased trimethylation of histone H3 lysine 4 (H3K4me3) at the hypomethylated E2F motifs. Knockdown of HoxA13 caused the downregulation of long non-coding RNA HOTTIP and insulin growth factor-binding protein 3 (IGFBP-3) genes, indicating that both were targets of HoxA13. Concurrent regulation of HoxA13-HOTTIP was mediated by the mixed lineage leukemia-WD repeat domain 5 complex, which caused the trimethylation of H3K4 and then stimulated cell proliferation. HoxA13 transactivated the IGFBP-3 promoter through the HOX-binding site. Activation of IGFBP-3 stimulated the oncogenic potential and invasion activity. Increased expression of HoxA13 (63.2%) and IGFBP-3 (28.6%) was detected in human gastric cancer tissues and was found in the gastric cancer data of The Cancer Genome Atlas. Taken together, the HoxA13–HOTTIP–IGFBP-3 cascade is critical for the carcinogenic characteristics of CS12 cells.
PMCID: PMC5094982  PMID: 27144338
gastric cancer cells; HOTTIP; HoxA13; IGFBP-3; p53-E2F signaling
22.  HOXA5 Regulates hMLH1 Expression in Breast Cancer Cells1 
Neoplasia (New York, N.Y.)  2006;8(4):250-258.
Homeobox protein HOXA5 functions as a transcriptional factor for genes that are not only involved in segmentation identity but also in cell differentiation. Although HOXA5 has been shown to regulate the expression of the tumor-suppressor protein p53, its role in breast tumorigenesis is not well understood. Using yeast as a model system, we now demonstrate that overexpression of HOXA5 in yeast can be used to identify downstream target genes that are homologous in humans. One such identified gene was that of the mismatch repair pathway component MutL homolog 1. Analysis of the promoter region of the gene for human MutL homolog 1 (hMLH1) displayed several putative HOXA5-binding sites. In transient transfection experiments, the overexpression of HOXA5 transactivated the hMLH1 promoter-reporter construct. In addition, chromatin immunoprecipitation assay using a human breast cancer cell line MCF-7 demonstrated that HOXA5 binds to the hMLH1 promoter in vivo. Furthermore, we demonstrate that, in the presence of HOXA5, there is an increase in in vivo repair activity in MCF-7 cells. Taken together, our results indicate that HOXA5 is a transcriptional regulator of hMLH1 in breast cancer cells.
PMCID: PMC1600677  PMID: 16756717
Breast cancer; mismatch repair; homeotic gene; yeast; promoter analyses; HOX, homeotic; hMLH1, human MutL homolog 1; ChIP, chromatin immunoprecipitation; MMR, mismatch repair; Luc, luciferase
23.  HOXA genes cluster: clinical implications of the smallest deletion 
HOXA genes cluster plays a fundamental role in embryologic development. Deletion of the entire cluster is known to cause a clinically recognizable syndrome with mild developmental delay, characteristic facies, small feet with unusually short and big halluces, abnormal thumbs, and urogenital malformations. The clinical manifestations may vary with different ranges of deletions of HOXA cluster and flanking regions.
Case presentation
We report a girl with the smallest deletion reported to date involving the entire HOXA cluster at 7p15.2-p14.3. The patient was the third child born to a healthy and non-consanguineous Italian couple. She was born at the 34th week of gestation by caesarean section due to cholestasis of pregnancy. Her birth weight, length, and occipitofrontal circumference were 2,140 g (25-50th centile), 46 cm (50th centile), and 33 cm (75-90th centile), respectively. The Apgar scores were 8 at both the 1st and 5th minutes. The patient presented with typical mild facial anomalies, hand and feet abnormalities, urinary anomalies, and mild speech delay. Unexpectedly, the patient demonstrated complex unusual features of multiple episodes of oxyhemoglobin desaturation, laryngeal stridor and a branchial cyst. Chromosome analysis of the patient revealed an apparently normal karyotype at the 550 band level. Based on array comparative genomic hybridization, a 2.5 Mb interstitial deletion was detected at 7p15.2p14.3 (chr7: 26,333,553-28,859,312), involving the entire HOXA cluster and a small number of other genes as SNX10, SKAP2, EVX1, HIBADH, TAX1BP1, JAZF1, and CREB5.
This report improves our understanding of the genotype-phenotype correlations of HOXA genes cluster deletions via the identification and characterization of the smallest deletion (as well as critical region) reported to date. In particular we discuss the possible implications of preterm and haploinsufficiency in the pathogenesis of the unusual findings, furthermore opening new discussion and interpretation cues.
PMCID: PMC4395968  PMID: 25881986
HOXA; Speech delay; Hand-foot-genital syndrome; 7p15 deletion
24.  Hoxa1 lineage-tracing indicates a direct role for Hoxa1 in development of the inner ear, the heart and the third rhombomere 
Developmental biology  2010;341(2):499-509.
Loss of Hoxa1 function results in severe defects of the brainstem, inner ear and cranial ganglia in humans and mice as well as cardiovascular abnormalities in humans. Since Hoxa1 is expressed very transiently during an early embryonic stage, it has been difficult to determine whether Hoxa1 plays a direct role in the precursors of the affected organs or if all defects result from indirect effects due to mispatterning of the hindbrain. In this study we use a Hoxa1-IRES-Cre mouse to genetically label the early Hoxa1-expressing cells and determine their contribution to each of the affected organs, allowing us to conclude in which precursor tissue Hoxa1 is expressed. We found Hoxa1 lineage-labeled cells in all tissues expected to be derived from the Hoxa1 domain, such as the facial and abducens nuclei and nerves as well as r4 neural crest cells. Additionally, we detected the lineage in derivatives that were not thought to have expressed Hoxa1 during development. In the brainstem the anterior border of the lineage was found to be in r3, which is more anterior than previously reported. We also observed an interesting pattern of the lineage in the inner ear, namely a strong contribution to the otic epithelium with the exception of sensory patches. Moreover, lineage-labeled cells were detected in the atria and outflow tract of the developing heart. In conclusion, Hoxa1 lineage-tracing uncovered new domains of Hoxa1 expression in rhombomere 3, the otic epithelium and cardiac precursors, suggesting a more direct role for Hoxa1 in development of these tissues than previously believed.
PMCID: PMC2862106  PMID: 20171203
Hoxa1; hindbrain; inner ear; heart
25.  Isolated Hoxa9 overexpression predisposes to the development of lymphoid but not myeloid leukemia 
Experimental hematology  2013;41(6):518-529.e5.
Hoxa9 is expressed in hematopoietic stem and progenitor cells although this expression is usually diminished as these cells undergo differentiation. In addition, aberrant expression of Hoxa9 is strongly associated with both T-cell and myeloid leukemia in mice and humans. Despite this strong association, enforced expression of Hoxa9 in murine bone marrow or thymus has only shown a modest ability to transform cells. To investigate this question, we used Vav regulatory elements to generate a transgenic mouse that targets Hoxa9 overexpression to all hematopoietic tissues. High level expression of the Hoxa9 transgene in the hematopoietic compartment was associated with embryonic lethality, as no pups from founders that expressed high levels of the transgene were born live. However, offspring of an additional founder line, which expressed lower levels of Hoxa9, developed a precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL), accompanied by spontaneous Notch1 mutations. In contrast to most murine models of leukemia associated with Hoxa9 overexpression, the Vav-Hoxa9 mice did not overexpress other Hoxa cluster genes, mir196b (a microRNA which is embedded in the Hoxa locus), Meis1, or Pbx3. The Hoxa9 transgenic mouse reported here provides a suitable system for the study of Hoxa9 collaborators that drive myeloid and lymphoid malignant transformation.
PMCID: PMC3718276  PMID: 23435313
Hoxa9; pre-T LBL; embryonic lethal; Notch1

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