Role Of HOXA5 Research Articles

Abstract 2669: A role for HOXA5 in the transcriptional response of ovarian and fallopian tube surface epithelial cells to norepinephrine

Abstract Chronic stress has been suggested to play a role in tumor initiation, progression and patient survival. Studies have shown that activation of the sympathetic nervous system and increased levels of stress-associated hormones such as norepinephrine can enhance migration, invasion and growth of ovarian cancer cells. However, the extent to which stress contributes to tumor initiation remains unknown. The purpose of this study was to explore the effects of norepinephrine on the transcriptional program of immortalized normal cell lines presumed to be the origin of epithelial ovarian cancers. We treated normal immortalized ovarian (iOSE) and fallopian tube (iFTE) surface epithelial cells with 10uM norepinephrine for 15 min, 1h and 4h and generated global transcriptomic profile of treated vs untreated cells by RNA-Seq. The most significant gene expression changes (P(adj) < 0.05) were observed at the 1h time point; with an overlap of 45 differentially expressed genes in both iOSE and iFTE cells. Genes in immune-related and developmental pathways were found to be highly enriched in the norepinephrine treated samples set. By the 4h time point, the number of differentially expressed genes had increased to 313 genes from 234 at 1h in iFTE cells, while it reduced to 34 genes from 53 in iOSE cells. Additionally, the number of differentially expressed genes common to iOSE and iFTE cells had reduced to 14. Gene ontology analysis of the differentially expressed genes revealed ‘transcription factor’ to be the most significantly overrepresented class (based on FDR value) at both 1h and 4h time-points. Promoter enrichment analysis identified HOXA5, GFI1 and FOXI1 transcription factor binding sites to be highly enriched (based on Z-score and Fisher-Score) at both time points. HOXA5 ranked consistently at the top in both cell lines and at both 1h and 4h time points. These observations suggest that HOXA5 may play a central role in regulating the early response to norepinephrine in normal ovarian and fallopian tube cells. We will further assess the function of HOXA5 through ChIP-Seq and shRNA- mediated silencing. Citation Format: Sweta Dash, Anxhela Gjyshi, Ling Cen, Chia-Ho Cheng, Chaomei Zhang, Sean J. Yoder, Jamie K. Teer, Guillermo N. Armaiz-Pena, Alvaro N. Monteiro. A role for HOXA5 in the transcriptional response of ovarian and fallopian tube surface epithelial cells to norepinephrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2669.

Abstract 4467: The role of HOXA5 in the malignant progression of engineered isogenic MCF10A sublines harboring common breast cancer mutations

Abstract Background and Aims: HOXA5 behaves as a tumor suppressor gene in breast cancer and regulates E-cadherin, CD24, progesterone receptor and p53. Retinoic acid activates transcription of HOXA5 by binding to RAR elements in the HOXA5 locus. The goal of this study was to establish whether the loss of HOXA5 cooperates with loss or mutations in common tumor suppressor genes and oncogenes in isogenic untransformed breast epithelial MCF10A sublines to induce tumorigenesis. Materials and Methods: Isogenic MCF10A sublines with a single knock in mutation in p53 (R248W), or double knock in mutations in both HER2 (V777L) and PIK3CA (E545) generated by Cre recombinase mediated excision/insertion by the Park lab were used. Genomic DNA extraction followed by PCR, gel extraction and Sanger sequencing was used to confirm genotype. Western blotting and quantitative real time PCR was used to analyze gene expression. All-trans Retinal- ATAL (Sigma #R2500) (1 μM for 7 days) was used treat p53 R248W cell line. Colony formation assay was performed and assessed using 0.5% crystal violet staining. XTT assay was used for cell proliferation assays. Immunocytochemical images were taken. For xenograft studies, 2 X 106 cells in 80% growth factor reduced Matrigel and 20% PBS were implanted subcutaneously. Results: Stable knock down (KD) of HOXA5 using shRNAs in the V777L DKI cell line (HER2 V777L and PIK3CA E545K) resulted in a more spindle like, elongated morphology. Elevated levels of mesenchymal markers N-cadherin, Slug and Vimentin, and reduced levels of epithelial marker E-cadherin, at both the protein and RNA levels were seen. Ectopic overexpression of HOXA5 in the p53 R248W cell line caused reduced proliferation, colony formation and wound healing relative to vector transfected cells. ATAL treatment of p53 R248W restored expression of HOXA5, E-Cadherin, progesterone receptor and other HOXA5 targets. Subcutaneous injection of V777L DKI SCR cells into NSG mice resulted in small growths of ductal structures with large lumens surrounded by a single layer of cells, while V777L DKI-HOXA5 knock down cells formed structures with smaller lumens surrounded by multiple layers of cells, resembling hyperplastic outgrowths. Conclusions: Knock down of HOXA5 expression in the partially transformed V777L DKI cell line results in a more mesenchymal phenotype accompanied by an increase in EMT marker expression, and a transformation from normal ductal structures to hyperplastic nodules when implanted in NSG mice. Whether the hyperplastic nodules will progress to invasive cancer as the tumor grows is under study. Conversely, ectopic expression of HOXA5 in the p53 R248W cell line causes a reduction in functional properties associated with a transformed phenotype: proliferation, colony formation and wound healing. Citation Format: Priya Pai, Guannan Wang, Saraswati Sukumar. The role of HOXA5 in the malignant progression of engineered isogenic MCF10A sublines harboring common breast cancer mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4467.

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla.

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self-renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real-time RT-PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16INK4A and p18INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16INK4A , p18INK4C , and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental-tissue-derived MSCs.

Abstract 1526: HOXA5 inhibits cell plasticity and stemness in breast cancer cells by reinforcing epithelial traits

Abstract Members of the Retinoic acid receptor (RAR) and HOX gene families are known to be associated with cellular differentiation. Nonetheless, how HOX genes drive differentiation in mammalian cells is poorly understood. HOX genes are transcription factors that orchestrate body segmentation during embryogenesis. But the continued expression of several members in adulthood indicates the necessity of their biological functions in specifying cell fate in adulthood. The mammary gland is an organ that continues to go through multiple cycles of development during life. Here, loss of specific HOX genes could be detrimental to normal development. We have previously shown that expression of HOXA5, a downstream target of the retinoic acid signaling pathway, is often lost in breast cancer. Loss of HOXA5 expression correlates with increasing pathological grades of breast cancer. Expression array analysis of HOXA5-depleted MCF10A (immortalized line of human breast epithelium) revealed its potential role in regulating several traits of the epithelial lineage, including expression of E-cadherin and CD24. Depleting HOXA5 in MCF10A enhanced cell motility, invasiveness and increased stem-cell population (CD24lo/CD44hi). Using an inducible HOXA5-depletion system, differentiation of the stem cells in MCF10A was shown to be mediated by HOXA5. Conversely, ectopic expression of HOXA5 in SUM149 breast cancer cells significantly reduced plasticity and stem cell population, confirmed by in vivo limiting dilution assays in mice. MCF10A-KRAS-HOXA5 depleted cells formed invasive structures in 3D matrigel cultures, and formed larger tumors, implanted orthotopically in mice. MCF10A-KRAS-tumors were histologically dedifferentiated and lost expression of luminal cell markers such as E-cadherin and cytokeratin 18. Collectively, our findings demonstrate the role of HOXA5 in retinoid-induced cell differentiation by regulating cell fate specific surface adhesion molecules. Citation Format: Wei Wen Teo, Vanessa Merino, Sean Cho, Preethi Korangath, Neil M. Neumann, Andrew J. Ewald, Saraswati Sukumar. HOXA5 inhibits cell plasticity and stemness in breast cancer cells by reinforcing epithelial traits. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1526. doi:10.1158/1538-7445.AM2015-1526

α5-GABAA receptors negatively regulate MYC-amplified medulloblastoma growth

Neural tumors often express neurotransmitter receptors as markers of their developmental lineage. Although these receptors have been well characterized in electrophysiological, developmental and pharmacological settings, their importance in the maintenance and progression of brain tumors and, importantly, the effect of their targeting in brain cancers remains obscure. Here, we demonstrate high levels of GABRA5, which encodes the α5-subunit of the GABAA receptor complex, in aggressive MYC-driven, "Group 3" medulloblastomas. We hypothesized that modulation of α5-GABAA receptors alters medulloblastoma cell survival and monitored biological and electrophysiological responses of GABRA5-expressing medulloblastoma cells upon pharmacological targeting of the GABAA receptor. While antagonists, inverse agonists and non-specific positive allosteric modulators had limited effects on medulloblastoma cells, a highly specific and potent α5-GABAA receptor agonist, QHii066, resulted in marked membrane depolarization and a significant decrease in cell survival. This effect was GABRA5 dependent and mediated through the induction of apoptosis as well as accumulation of cells in S and G2 phases of the cell cycle. Chemical genomic profiling of QHii066-treated medulloblastoma cells confirmed inhibition of MYC-related transcriptional activity and revealed an enrichment of HOXA5 target gene expression. siRNA-mediated knockdown of HOXA5 markedly blunted the response of medulloblastoma cells to QHii066. Furthermore, QHii066 sensitized GABRA5 positive medulloblastoma cells to radiation and chemotherapy consistent with the role of HOXA5 in directly regulating p53 expression and inducing apoptosis. Thus, our results provide novel insights into the synthetic lethal nature of α5-GABAA receptor activation in MYC-driven/Group 3 medulloblastomas and propose its targeting as a novel strategy for the management of this highly aggressive tumor.

The Loss of Hoxa5 Function Causes Estrous Acyclicity and Ovarian Epithelial Inclusion Cysts

Hox genes encode transcription factors that play essential roles during embryo morphogenesis and organogenesis. Expression of several Hox members persists at the adult age, indicating a wide spectrum of action from embryonic to postnatal life. In the present study, we reported that in adult mice, the Hoxa5 gene shows a dynamic expression profile in the ovary that depends on the estrous cycle, the gestational status, and the age of the female, suggesting that Hoxa5 may have distinct physiological functions in the ovary. Consistent with a role for Hoxa5 in ovarian function, Hoxa5(-/-) nulliparous females exhibit precocious puberty and an early onset of estrous acyclicity. They show a prolonged estrous cycle with increased metestrus-diestrus length, a phenotype that worsens with age. Older mutant females also develop ovarian epithelial inclusion cysts reminiscent of human endosalpingiosis. Immunolabeling studies suggest that these cysts originate from the ovarian surface epithelium, a source of epithelial ovarian carcinomas. Staining of the Hoxa5(-/-) ovarian cysts by the ovarian cancer markers paired box gene 8 (PAX8) and Wilms' tumor 1 (WT1) further strengthens the notion that these cysts may constitute preneoplastic lesions. Moreover, the deregulation of the estrous cycle and the presence of ovarian epithelial cysts in Hoxa5(-/-) older females correlate with a reduced expression of specific epidermal growth factor receptor signaling components, namely Egfr, Areg, and Btc. Altogether, our data unveil that Hoxa5, a stroma-specific gene, plays a significant role in ovarian biology and may be involved in ovarian cancer predisposition.

Elucidating the role of Hoxa-5 in the development of the chick axial skeleton

Elucidating the role of Hoxa-5 in the development of the chick axial skeleton

Elucidating the role of Hoxa-5 in development of the chick axial skeleton

Elucidating the role of Hoxa-5 in development of the chick axial skeleton

A Role for Hox A5 in Regulating Angiogenesis and Vascular Patterning

Homeobox (Hox) genes are transcriptional regulators which modulate embryonic morphogenesis and pathological tissue remodeling in adults via regulation of genes associated with cell-cell or cell extracellular matrix (ECM) interactions. We previously showed that while Hox 3 genes promote angiogenesis, Hox D10 inhibits this process. Here we show that another Hox family gene, Hox A5, also blocks angiogenesis but accomplishes this by targeting different downstream genes than Hox D10. Sustained expression of Hox A5 leads to down regulation of many pro-angiogenic genes including VEGFR2, ephrin A1, Hif1alpha and COX-2. In addition, Hox A5 also upregulates expression of anti-angiogenic genes including Thrombospondin-2. Furthermore, we show that while Hox A5 mRNA is expressed in quiescent endothelial cells (EC), its expression is diminished or absent in active angiogenic EC found in association with breast tumors or in proliferating infantile hemangiomas. Together our results suggest that restoring Hox A5 expression may provide a novel means to limit breast tumor growth or expansion of hemangiomas.

Characterization of HOX Gene Expression During Myelopoiesis: Role of HOX A5 in Lineage Commitment and Maturation

During the process of normal hematopoiesis, proliferation is tightly linked to maturation. The molecular mechanisms that lead to production of mature effector cells with a variety of phenotypes and functions from a single multipotent progenitor are only beginning to be elucidated. It is important to determine how these maturation events are regulated at the molecular level, because this will provide significant insights into the process of normal hematopoiesis as well as leukemogenesis. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. In this study, we focused on identification and characterization of homeobox genes of the HOX family that are important in regulating normal human myeloid differentiation induced by the hematopoietic growth factor, granulocyte-macrophage colony-stimulating factor (GM-CSF). We have identified three homeobox genes, HOX A5, HOX B6, and HOX B7, which are expressed during early myelopoiesis. Treating bone marrow cells with antisense oligodeoxynucleotides to HOX A5 resulted in inhibition of granulocytic/monocytic hematopoiesis and increased the generation of erythroid progenitors. Also, overexpression of HOX A5 inhibited erythroid differentiation of the K562 cell line. Based on these observations, we propose that HOX A5 functions as an important regulator of hematopoietic lineage determination and maturation.