Hox Gene Expression Research Articles

Molecular mechanisms in the control of limb regeneration: the role of homeobox genes.

Axolotls are unique among vertebrates in their ability to regenerate lost appendages as adults. They provide the opportunity to study the mechanism of regeneration in vertebrates and are an inspiration to pursue the goal of appendage regeneration in humans. In this article, we review data on the role of homeobox-containing genes in the regulation of limb regeneration. As a group, these genes are important in pattern formation in the primary body axis, developing limbs and regenerating limbs. To date, a total of 22 homeobox genes have been identified as being expressed in regenerating limbs. Nearly all of these are also expressed during limb regeneration, further supporting the view that limb development and regeneration involve similar regulatory mechanisms. Our recent results on the expression of HoxA genes demonstrate that once a blastema has formed, subsequent outgrowth and pattern formation are similar to those of limb development. In contrast to developing limbs, reexpression of the HoxA genes in regeneration occurs by a non-colinear mechanism that likely is related to the necessity of mature limb cells to undergo dedifferentiation in order to give rise to the blastema. These studies also indicate that the pattern is respecified by a distal-first mechanism during regeneration in contrast to the apparent proximal-to-distal sequence observed in developing limbs. Expression of the HoxA genes is altered coordinately in response to retinoic acid in a manner consistent with the transformation of a distal blastema to a proximal blastema. Given the recent increase in studies of the molecules involved in regeneration, it is likely that many of the functionally important regeneration genes will be identified and characterized in the near future.

Functional analysis of HOXA10 and HOXB4 in human medulloblastoma cell lines.

Medulloblastoma (MB) is a malignant childhood brain tumor which at molecular level is classified into at least four major subtypes: WNT, SHH, groupC and groupD differing in response to treatment. Previous studies have associated changes in expression levels and activation of certain HOX genes with MB development. In the present study, we investigate the role of HOX genes in two attributes acquired by tumor cells: migration and proliferation potential, as well as, invivo tumorigenic potential. We analyzed UW402, UW473, DAOY and ONS-76 human pediatric MB cell lines and cerebellum primary cultures. Two-color microarray-based gene expression analysis was used to identify differentially expressed HOX genes. Among the various HOX genes significantly overexpressed in DAOY and ONS-76 cell lines compared to UW402 and UW473 cell lines, HOXA10 and HOXB4 were selected for further analysis. The expression levels of these HOX genes were validated by real-time PCR. A mouse model was used to study the effect of the HOXA10 and HOXB4 genes on the invivo tumorigenic potential and the invitro proliferative and migration potential of MB cell lines. Our results show that the inhibition of HOXA10 in DAOY cell line led to increased invitro cell migration while invitro cell proliferation or invivo tumorigenic potential were unaffected. We also observed that induced expression of HOXB4 in the UW473 cell line significantly reduced invitro cell proliferation and migration capability of UW473 cells with no effect on the invivo tumorigenicity. This suggests that HOXA10 plays a role in migration events and the HOXB4 gene is involved in proliferation and migration processes of medulloblastoma cells, however, it appears that these genes are not essential for the tumorigenic process of these cells.

Deep time perspective on turtle neck evolution: chasing the Hox code by vertebral morphology

The unparalleled ability of turtle neck retraction is possible in three different modes, which characterize stem turtles, living side-necked (Pleurodira), and hidden-necked (Cryptodira) turtles, respectively. Despite the conservatism in vertebral count among turtles, there is significant functional and morphological regionalization in the cervical vertebral column. Since Hox genes play a fundamental role in determining the differentiation in vertebra morphology and based on our reconstruction of evolutionary genetics in deep time, we hypothesize genetic differences among the turtle groups and between turtles and other land vertebrates. We correlated anterior Hox gene expression and the quantifiable shape of the vertebrae to investigate the morphological modularity in the neck across living and extinct turtles. This permitted the reconstruction of the hypothetical ancestral Hox code pattern of the whole turtle clade. The scenario of the evolution of axial patterning in turtles indicates shifts in the spatial expression of HoxA-5 in relation to the reduction of cervical ribs in modern turtles and of HoxB-5 linked with a lower morphological differentiation between the anterior cervical vertebrae observed in cryptodirans. By comparison with the mammalian pattern, we illustrate how the fixed count of eight cervical vertebrae in turtles resulted from the emergence of the unique turtle shell.

Inhibition of HOX/PBX dimer formation leads to necroptosis in acute myeloid leukemia cells.

The HOX genes encode a family of transcription factors that have key roles in both development and malignancy. Disrupting the interaction between HOX proteins and their binding partner, PBX, has been shown to cause apoptotic cell death in a range of solid tumors. However, despite HOX proteins playing a particularly significant role in acute myeloid leukemia (AML), the relationship between HOX gene expression and patient survival has not been evaluated (with the exception of HOXA9), and the mechanism by which HOX/PBX inhibition induces cell death in this malignancy is not well understood. In this study, we show that the expression of HOXA5, HOXB2, HOXB4, HOXB9, and HOXC9, but not HOXA9, in primary AML samples is significantly related to survival. Furthermore, the previously described inhibitor of HOX/PBX dimerization, HXR9, is cytotoxic to both AML-derived cell lines and primary AML cells from patients. The mechanism of cell death is not dependent on apoptosis but instead involves a regulated form of necrosis referred to as necroptosis. HXR9-induced necroptosis is enhanced by inhibitors of protein kinase C (PKC) signaling, and HXR9 combined with the PKC inhibitor Ro31 causes a significantly greater reduction in tumor growth compared to either reagent alone.

Segmental arithmetic: summing up the Hox gene regulatory network for hindbrain development in chordates.

Organization and development of the early vertebrate hindbrain are controlled by a cascade of regulatory interactions that govern the process of segmentation and patterning along the anterior-posterior axis via Hox genes. These interactions can be assembled into a gene regulatory network that provides a framework to interpret experimental data, generate hypotheses, and identify gaps in our understanding of the progressive process of hindbrain segmentation. The network can be broadly separated into a series of interconnected programs that govern early signaling, segmental subdivision, secondary signaling, segmentation, and ultimately specification of segmental identity. Hox genes play crucial roles in multiple programs within this network. Furthermore, the network reveals properties and principles that are likely to be general to other complex developmental systems. Data from vertebrate and invertebrate chordate models are shedding light on the origin and diversification of the network. Comprehensive cis-regulatory analyses of vertebrate Hox gene regulation have enabled powerful cross-species gene regulatory comparisons. Such an approach in the sea lamprey has revealed that the network mediating segmental Hox expression was present in ancestral vertebrates and has been maintained across diverse vertebrate lineages. Invertebrate chordates lack hindbrain segmentation but exhibit conservation of some aspects of the network, such as a role for retinoic acid in establishing nested Hox expression domains. These comparisons lead to a model in which early vertebrates underwent an elaboration of the network between anterior-posterior patterning and Hox gene expression, leading to the gene-regulatory programs for segmental subdivision and rhombomeric segmentation. WIREs Dev Biol 2017, 6:e286. doi: 10.1002/wdev.286 For further resources related to this article, please visit the WIREs website.

Commentary on Some Recent Theses Relevant to Combating Aging: August 2017.

Theses reviewed in this issue include "Engineering Cellular Input-Output for the Robust Control of Mammalian Cell-Based Therapies"; "Enzyme-Instructed Self-Assembly (EISA) Selectively Targets Cancer Cells"; "Exploration of helminth-derived immunoregulatory molecules as options for therapeutic intervention in allograft rejection and autoimmune disease"; "Expression of the Medial HOXA genes is indispensible for self-renewal in human hematopoietic stem cells"; "Gamma frequency entrainment attenuates amyloid load and modifies microglia"; and "Heterogeneous Distribution of Microvascular Blood Flow Contributes to Impaired Skeletal Muscle Oxygenation in Diabetes".

Comprehensive analyses of hox gene expression in Xenopus laevis embryos and adult tissues.

From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount insitu hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.

Polycomb and Trithorax Group Genes in Drosophila.

Polycomb group (PcG) and Trithorax group (TrxG) genes encode important regulators of development and differentiation in metazoans. These two groups of genes were discovered in Drosophila by their opposing effects on homeotic gene (Hox) expression. PcG genes collectively behave as genetic repressors of Hox genes, while the TrxG genes are necessary for HOX gene expression or function. Biochemical studies showed that many PcG proteins are present in two protein complexes, Polycomb repressive complexes 1 and 2, which repress transcription via chromatin modifications. TrxG proteins activate transcription via a variety of mechanisms. Here we summarize the large body of genetic and biochemical experiments in Drosophila on these two important groups of genes.

Anatomical integration of the sacral-hindlimb unit coordinated by GDF11 underlies variation in hindlimb positioning in tetrapods.

Elucidating how body parts from different primordia are integrated during development is essential for understanding the nature of morphological evolution. In tetrapod evolution, while the position of the hindlimb has diversified along with the vertebral formula, the mechanism responsible for this coordination has not been well understood. However, this synchronization suggests the presence of an evolutionarily conserved developmental mechanism that coordinates the positioning of the hindlimb skeleton derived from the lateral plate mesoderm with that of the sacral vertebrae derived from the somites. Here we show that GDF11 secreted from the posterior axial mesoderm is a key factor in the integration of sacral vertebrae and hindlimb positioning by inducing Hox gene expression in two different primordia. Manipulating the onset of GDF11 activity altered the position of the hindlimb in chicken embryos, indicating that the onset of Gdf11 expression is responsible for the coordinated positioning of the sacral vertebrae and hindlimbs. Through comparative analysis with other vertebrate embryos, we also show that each tetrapod species has a unique onset timing of Gdf11 expression, which is tightly correlated with the anteroposterior levels of the hindlimb bud. We conclude that the evolutionary diversity of hindlimb positioning resulted from heterochronic shifts in Gdf11 expression, which led to coordinated shifts in the sacral-hindlimb unit along the anteroposterior axis.

Overexpression of HOXA4 and HOXA9 genes promotes self-renewal and contributes to colon cancer stem cell overpopulation.

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.

Increased methylation and decreased expression of homeobox genes TLX1, HOXA10 and DLX5 in human placenta are associated with trophoblast differentiation

Homeobox genes regulate embryonic and placental development, and are widely expressed in the human placenta, but their regulatory control by DNA methylation is unclear. DNA methylation analysis was performed on human placentae from first, second and third trimesters to determine methylation patterns of homeobox gene promoters across gestation. Most homeobox genes were hypo-methylated throughout gestation, suggesting that DNA methylation is not the primary mechanism involved in regulating HOX genes expression in the placenta. Nevertheless, several genes showed variable methylation patterns across gestation, with a general trend towards an increase in methylation over gestation. Three genes (TLX1, HOXA10 and DLX5) showed inverse gains of methylation with decreasing mRNA expression throughout pregnancy, supporting a role for DNA methylation in their regulation. Proteins encoded by these genes were primarily localised to the syncytiotrophoblast layer, and showed decreased expression later in gestation. siRNA mediated downregulation of DLX5, TLX1 and HOXA10 in primary term villous cytotrophoblast resulted in decreased proliferation and increased expression of differentiation markers, including ERVW-1. Our data suggest that loss of DLX5, TLX1 and HOXA10 expression in late gestation is required for proper placental differentiation and function.

Abstract 1373: The role of the ASH1L SET domain in leukemogenesis

Abstract ASH1L (absent, small, or homeotic-like 1) is a histone lysine methyltransferase (KMTase) specific for histone 3, lysine 36 that has an emerging role in multiple types of cancer. During development, ASH1L activates HOX genes and regulates self-renewal potential of long-term hematopoietic stem cells. Interestingly, HOX genes are oncogenic drivers in a variety of cancer types. For example, overexpression of HOXA9 is highly associated with a poor prognosis in acute myeloid leukemia (AML). In addition, ASH1L is overexpressed in a variety of cancers, including breast and thyroid cancer, but its role in cancer remains poorly characterized. Recent data suggest that ASH1L promotes leukemogenesis in mixed-lineage leukemia by recruiting the MLL epigenetic regulator to target genes. However, ASH1L is a large protein with multiple chromatin-interacting domains, and which regions of the ASH1L protein are important in leukemia has not been reported. Specifically, the role of ASH1L’s KMTase activity in leukemogenesis remains unknown. To further investigate the function of ASH1L in leukemia, we knocked down expression of ASH1L in leukemia cell lines. We found that ASH1L knockdown slowed cell growth and caused a decrease in expression of HOXA genes and their collaborator MEIS1. To specifically investigate the role of ASH1L KMTase activity in leukemia, we used a mouse model with a targeted in-frame deletion of the ASH1L SET domain (ΔSET). ΔSET cells express a shorter version of ASH1L that does not contain the SET domain. We transduced bone marrow cells (BMCs) isolated from ΔSET and WT mice with several oncogenes relevant to AML to determine the role of the ASH1L SET domain in leukemic transformation. We found that ΔSET BMCs were significantly impaired in leukemic transformation compared to WT BMCs, as manifested by decreased colony-forming ability and reduced expression of HOXA genes. These findings suggest that the ASH1L SET domain plays an important role in leukemogenesis mediated by multiple different oncogenes. Future studies will further characterize ASH1L’s role in leukemia by identifying the complete set of genetic loci that it methylates and regulates, and will determine whether the ASH1L SET domain represents a viable therapeutic target. Citation Format: David Rogawski, Hongzhi Miao, Katarzyna Kempinska, Lili Chen, Jen Chase, Ann Friedman, Xiaomei Yan, Kenichi Nishioka, Gang Huang, Patricia Ernst, Andy Muntean, Ivan Maillard, Tomasz Cierpicki, Jolanta Grembecka. The role of the ASH1L SET domain in leukemogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1373. doi:10.1158/1538-7445.AM2017-1373

Abstract 3042: MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression

Abstract MIR142 mutations have been identified in acute myeloid leukemia (AML) and non-Hodgkins lymphoma. In AML, all MIR142 mutations localize to the miR-142-3p seed sequence. We show that mutated MIR142 is unable to suppress several well-known targets of miR-142-3p. Interestingly, the mutations of miR-142-3p result in their preferential loading into the RNA-induced silencing complex, leading to the degradation of miR-142-5p. Accordingly, miR-142-5p expression is decreased in MIR142 mutated AML. Hence, MIR142 mutations in AML disrupt both miR-142-3p/5p functions. Thus, we modeled the effect of MIR142 mutations on hematopoiesis using Mir142-/- mice. We show that loss of miR142 results in significant increases in myeloid hematopoietic stem/progenitor cells (HSPCs), including granulocyte-macrophage progenitors, myeloid-biased multipotent progenitors (CD150- CD48+ Flk2- Kit+ Sca+ lineage-) and CD229- myeloid-biased HSCs (CD150+ CD48- Kit+ Sca+ lineage-). In contrast, there are significant decreases of megakaryocyte-erythroid progenitors and erythroid precursors. Although the number of HSCs is normal in Mir142-/- mice, HSC transplantation suggest that they are myeloid-biased. In AML, MIR142 mutations are commonly found in conjunction with mutations of IDH1/2. To assess the importance of this association, we transduced wildtype or Mir142-/- HSPCs with retrovirus expressing IDH2 R172K and then transplanted into lethally irradiated recipients. Expression of IDH2 R172K alone was sufficient to induce a lethal myeloproliferative neoplasm (MPN). In contrast, Mir142-/- alone did not result in MPN. However, loss of Mir142 cooperates with IDH2 R172K to produce a more severe MPN, with increased CD34+ blasts and more severe anemia. Moreover, secondary transplantation shows that Mir142-/- x IDH2 R172K cells but not IDH2 R172K cells efficiently engraft and induce MPN, suggesting that loss of miR142 increases leukemia-initiating activity. We identify the histone methyltransferase ASH1L as a target gene of miR142 that contributes to altered hematopoiesis in Mir142-/- mice. The 3’-untranslated region of ASH1L has four miR-142-3p binding sites, and luciferase reporter assay shows that miR142 suppresses its translation by 80%. Consistent with this observation, Ashl1 protein expression is 3-fold higher in Mir142-/- bone marrow. ASH1L is a key positive regulator of HOX gene expression. Accordingly, we observed markedly (5-10 fold) increased HoxA9/A10 expression in myeloid progenitors in Mir142-/- mice. Likewise, HoxA9/A10 expression is increased in CD34+ blasts from Mir142-/- x IDH2 R172K transplanted mice. Of note, increased HoxA9 expression has been shown to cooperate with mutant IDH1 to induce AML in mice. Together, these findings support a model in which loss-of-function mutations of MIR142 contribute to hematopoietic malignancies by derepressing ASH1L and inducing HOXA9/10 gene expression. Citation Format: Juo-Chin Yao, Terrence N. Wong, Maria Trissal, Rahul Ramaswamy, Yaping Sun, Pavan R. Reddy, Daniel C. Link. MIR142 loss-of-function mutations promote leukemogenesis through derepression of ASH1L resulting in increased HOX gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2017-3042

Abstract LB-224: A novel model of pediatric spinal ependymoma using conditionally reprogrammed cells from a primary tumor demonstrates aberrant expression of HMGA, HOX, MYC and other Wnt target genes

Abstract Pediatric spinal ependymomas are rare tumors of the central nervous system with limited treatment options. Successful outcomes depend primarily upon the extent of surgical resection, although significant impairment is common, particularly in children with developing nervous systems. Because these tumors are uncommon, there are no established tumor models nor are there many studies to define underlying molecular lesions. Here, we describe an innovative spinal ependymoma (SE) tumor model based on the recent discovery that primary carcinoma cells can be cultured indefinitely ex vivo on fibroblast feeder cells in the presence of a rho kinase inhibitor; these cells are denoted conditionally reprogrammed cells (CRCs). In prior studies, CRCs form spheres in vitro, replicate tumor pathology as xenografts in vivo, and predict drug responses to the primary tumor. We therefore adopted this strategy to generate a cell line from a 12 year-old girl with a spinal myxopapillary ependymoma. The cell line and primary tumor are of human origin and identical with respect to short tandem repeat (STR) analysis. Using targeted gene expression analysis, we discovered that genes encoding the of High Mobility Group Protein AT-hook 1 (HMGA1), High Mobility Group Protein AT-hook 2 (HMGA2), cMYC, HOXB13, and HOXA10 proteins are all up-regulated compared to their expression in spinal cord tissue from a normal individual. In primary pediatric tumors, HMGA1 gene expression was also higher in spinal ependymomas as compared to supratentorial or posterior fossa ependymomas. There was also a significant positive correlation between HMGA1 and HOX genes, including HOXA10, HOXD13, HOXD1, and HOXB7 in primary tumors. In addition, HMGA1 and cMYC, together with other Wnt target genes, were positively correlated in primary spinal ependymoma. Pathway analysis showed that HMGA1 expression correlated with the following pathways: 1) metabolic pathways, 2) osteoclast differentiation, 3) MAPK signaling, and, 4) Neurotropin signaling. Studies are underway to generate additional cell lines from primary tumor samples and to elucidate the mechanisms that account for up-regulation in HMGA1, HOX and Wnt genes. We will harness this information to identify novel therapeutic targets and agents for children and young adults with spinal ependymoma. Citation Format: Li Z. Luo, Ewa Krawczyk, Anbarasu Lourdusamy, Lisa C. Storer, Lingling Xian, Kenneth J. Cohen, Richard Schlegel, Richard Grundy, Linda Resar. A novel model of pediatric spinal ependymoma using conditionally reprogrammed cells from a primary tumor demonstrates aberrant expression of HMGA, HOX, MYC and other Wnt target genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-224. doi:10.1158/1538-7445.AM2017-LB-224

Unexpected UBX expression in the maxilliped of the mystacocarid crustacean Derocheilocharis remanei-evidence for a different way of making a maxilliped?

In terms of morphology, crustacean maxillipeds are hybrid appendages. They arise in anterior thoracic segments and display characteristics of both locomotory (thoracic) and feeding (gnathal) appendages. Maxillipeds are functionally integrated with the anterior gnathal appendages. Hox gene expression patterns and immunolabeling with the FP6.87 antibody, which detects conserved epitopes of UBX and ABD-A proteins, reveal that maxillipeds are consistently associated with a shift in the expression of the homeotic gene Ubx. Ubx transcription products or proteins only appear in thoracic segments with a typical locomotory thoracopod and are consistently absent in the maxilliped. This pattern is found in various crustaceans: the copepod Mesocyclops, the mysid Mysidium, the decapods Homarus and Periclimenes, the isopod Porcellio, and the amphipod Parhyale. In Parhyale, which possesses maxillipeds on the first thoracic segment, gene manipulation experiments have shown that a leg-like thoracic appendage can be recovered by mis-expressing Ubx in that segment and walking legs can be transformed into maxillipeds by Ubx-knockdown. This survey focuses on the expression of UBX/ABD-A proteins, studied using the FP6.87 antibody, in the larval stages of the mystacocarid crustacean Derocheilocaris remanei. Mystacocarids inhabit the intertidal meiofauna zone of sandy beaches and possess one pair of maxillipeds on the first thoracic segment. Strong UBX/ABD-A expression in the developing maxilliped makes Derocheilocaris unique among crustaceans. Our data might also show that the transformation from locomotory thoracopod to maxilliped cannot be accounted for by the mere presence or absence of UBX, because in mystacocarids, UBX is present in both kinds of limbs. The role of the other Hox gene known to be involved in this transformation, Sex combs reduced (Scr), is unclear. The results presented here may document a new example of a shift in Hox gene function in arthropods. The difference in UBX/ABD-A expression between D. remanei and the copepod maxillipeds is of particular interest because correspondences between the feeding apparatus-including the maxilliped-in mystacocarids and copepods have been suggested as being of phylogenetic significance.

Abstract 5708: HOXA4 and HOXB3 gene expression signature as a biomarker of tumor recurrence in patients with high-grade serous ovarian cancer (HGSOC) following primary cytoreductive surgery and first-line adjuvant chemotherapy

Abstract Background: Aberrant HOX gene expression has been observed in various malignancies; however, their role as a biomarker for recurrence in patients with HGSOC is unknown. We tested the hypothesis that a HOX gene signature is a biomarker for disease-free survival (DFS) in HGSOC patients following primary cytoreductive surgery and adjuvant platinum-based chemotherapy. Methods: A panel of HOX genes associated with DFS in a discovery cohort of 19 HGSOC patients with available RNA-Seq data, and/or previously reported as associated with survival endpoints in the literature, were selected for testing by qPCR using the Fluidigm platform in an independent training cohort of primary tumors from 73 HGSOC patients. Cox proportional hazards model was used to identify HOX genes significantly associated with DFS. A prognostic gene signature was developed based on the combined linear predictor from the final multivariable Cox model. Patients were stratified into risk groups using the optimal cutoff of the linear predictor risk score, whereby the optimal cutoff was defined as the value that maximized the log-rank test statistic. Risk group stratification was further tested for association with DFS in a larger HGSOC dataset (N=414) with similar clinical characteristics from The Cancer Genome Atlas (TCGA). The role of the identified HOX genes in influencing drug sensitivity was examined by overexpressing these genes in a HGSOC cell line, PEO1. Results: Of 23 HOX genes selected for testing in the training cohort, overexpression of HOXA4 (HR=1.21, 95% CI=1.08-1.35, P=0.001) and HOXB3 (HR=1.09, 95% CI=1.02-1.17, P=0.016) were significantly associated with decreased DFS in multivariate analysis. The median DFS in patients with a HOXA4/HOXB3 risk score ≤ the optimal cutoff was > 80 months (not yet reached), whereas the median DFS in patients with a HOXA4/HOXB3 risk score > the optimal cutoff was 16.9 months (HR=7.85, 95% CI=4.19-14.69, P<0.001). The HOXA4/HOXB3 risk score remained significantly associated with DFS in the HGSOC dataset from the TCGA (P=0.02). In vitro studies demonstrated that PEO1 cells overexpressing either HOXA4 or HOXB3 were significantly (P<0.05) more resistant to cisplatin and carboplatin, but not to paclitaxel, as compared to empty vector transfected cells. Conclusion: The HOXA4/HOXB3 risk score was significantly associated with DFS in a training cohort of HGSOC patients, and remained significantly associated in a larger independent HGSOC dataset from the TCGA. Further, in vitro studies suggest that HOXA4 and HOXB3 overexpression is associated with platinum resistance. Thus, overexpression of HOXA4 and HOXB3 in primary HGSOC may serve as a potential biomarker for recurrence and the risk score model developed warrants prospective validation in HGSOC patients. Citation Format: Jai N. Patel, Katherine Miller, James Symanowski, Jalid Sehouli, Chad Michener, Ioana Braicu, Darla Destephanis, Ashley P. Sutker, Eric J. Norris, David Tait, Wendell Jones, Qing Zhang, Chad Livasy, Charles Biscotti, Ram N. Ganapathi, Mahrukh K. Ganapathi. HOXA4 and HOXB3 gene expression signature as a biomarker of tumor recurrence in patients with high-grade serous ovarian cancer (HGSOC) following primary cytoreductive surgery and first-line adjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5708. doi:10.1158/1538-7445.AM2017-5708

Correlation between Hox code and vertebral morphology in the mouse: towards a universal model for Synapsida

BackgroundThe importance of the cervical vertebrae as part of the skull–neck system in facilitating the success and diversity of tetrapods is clear. The reconstruction of its evolution, however, is problematic because of the variation in the number of vertebrae, making it difficult to identify homologous elements. Quantification of the morphological differentiation in the neck of diverse archosaurs established homologous units of vertebrae (i.e. modules) resulting from Hox gene expression patterns within the cervical vertebral column. The present study aims to investigate the modularity of the cervical vertebral column in the mouse and to reveal the genetic patterns and changes underlying the evolution of the neck of modern mammals and their extinct relatives. In contrast to modern mammals, non-mammalian synapsids are characterized by a variable cervical count, the presence of free cervical ribs and the presence of a separate CV1 centrum. How might these evolutionary modifications be associated with changes in the Hox code?ResultsIn combination with up-to-date information on cervical Hox gene expression including description of the vertebral phenotype of Hox knock-out mutants, the 3D landmark-based geometric morphometric approach demonstrates a correlation between Hox code and vertebral morphology in the mouse. There is evidence that the modularity of the neck of the mouse had already been established in the last common ancestor of mammals, but differed from that of non-mammalian synapsids. The differences that likely occurred during the evolution of synapsids include an anterior shift in HoxA-5 expression in relation to the reduction of cervical ribs and an anterior shift in HoxD-4 expression linked to the development of the highly differentiated atlas-axis complex, whereas the remaining Hox genes may have displayed a pattern similar to that in mammals on the basis of the high level of conservatism in the axial skeleton of this lineage.ConclusionThus, the mouse Hox code provides a model for understanding the evolutionary mechanisms responsible for the great morphological adaptability of the cervical vertebral column in Synapsida. However, more studies in non-model organisms are required to further elucidate the evolutionary role of Hox genes in axial patterning of the unique mammalian body plan.

Regulation of Hox gene expression during anteroposterior patterning involves collinear Hox-Hox interactions

Regulation of Hox gene expression during anteroposterior patterning involves collinear Hox-Hox interactions

Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells

Homeobox a1 (Hoxa1) is one of the most rapidly induced genes in ES cell differentiation and it is the earliest expressed Hox gene in the mouse embryo. In this study, we used genomic approaches to identify Hoxa1-bound regions during early stages of ES cell differentiation into the neuro-ectoderm. Within 2 h of retinoic acid treatment, Hoxa1 is rapidly recruited to target sites that are associated with genes involved in regulation of pluripotency, and these genes display early changes in expression. The pattern of occupancy of Hoxa1 is dynamic and changes over time. At 12 h of differentiation, many sites bound at 2 h are lost and a new cohort of bound regions appears. At both time points the genome-wide mapping reveals that there is significant co-occupancy of Nanog (Nanog homeobox) and Hoxa1 on many common target sites, and these are linked to genes in the pluripotential regulatory network. In addition to shared target genes, Hoxa1 binds to regulatory regions of Nanog, and conversely Nanog binds to a 3′ enhancer of Hoxa1. This finding provides evidence for direct cross-regulatory feedback between Hoxa1 and Nanog through a mechanism of mutual repression. Hoxa1 also binds to regulatory regions of Sox2 (sex-determining region Y box 2), Esrrb (estrogen-related receptor beta), and Myc, which underscores its key input into core components of the pluripotential regulatory network. We propose a model whereby direct inputs of Nanog and Hoxa1 on shared targets and mutual repression between Hoxa1 and the core pluripotency network provides a molecular mechanism that modulates the fine balance between the alternate states of pluripotency and differentiation.

HOXA7, HOXA9, and HOXA10 are differentially expressed in clival and sacral chordomas

Chordomas are rare tumours of the bone arising along the spine from clivus to sacrum. We compared three chordoma cell lines of the clivus region including the newly established clivus chordoma cell line, U-CH14, with nine chordoma cell lines originating from sacral primaries by morphology, on genomic and expression levels and with patient samples from our chordoma tissue bank. Clinically, chordomas of the clivus were generally smaller in size at presentation and patients with sacral chordomas had more metastases and more often recurrent disease. All chordoma cell lines had a typical physaliphorous morphology and expressed brachyury, S100-protein and cytokeratin. By expression analyses we detected differentially expressed genes in the clivus derived cell lines as compared to the sacral cell lines. Among these were HOXA7, HOXA9, and HOXA10 known to be important for the development of the anterior-posterior body axis. These results were confirmed by qPCR. Immunohistologically, clivus chordomas had no or very low levels of HOXA10 protein while sacral chordomas showed a strong nuclear positivity in all samples analysed. This differential expression of HOX genes in chordomas of the clivus and sacrum suggests an oncofetal mechanism in gene regulation linked to the anatomic site.