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Lester Andrews - One of the best experts on this subject based on the ideXlab platform.
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infrared spectra and density functional calculations for singlet ch2 SIX2 and triplet hc six3 and xc six3 intermediates in reactions of laser ablated silicon atoms with di tri and tetrahalomethanes
Inorganic Chemistry, 2016Co-Authors: Lester AndrewsAbstract:Reactions of laser-ablated silicon atoms with di-, tri-, and tetrahalomethanes in excess argon were investigated, and the products were identified from the matrix infrared spectra, isotopic shifts, and density functional theory energy, bond length, and frequency calculations. Dihalomethanes produce planar singlet silenes (CH2═SIX2), and tri- and tetrahalomethanes form triplet halosilyl carbenes (HC–SiX3 and XC–SiX3). The Si-bearing molecules identified are the most stable, lowest-energy product in the reaction systems. While the C–Si bond in the silene is a true double bond, the C–Si bond in the carbene is a shortened single bond enhanced by hyperconjugation of the two unpaired electrons on C to σ*(Si–X) orbitals, which contributes stabilization through a small amount of π-bonding and reduction of the HCSi or XCSi angles. The C–Si bond lengths in these carbenes (1.782 A for HC–SiF3) are between the single-bond length in the unobserved first insertion intermediate (1.975 A for CHF2–SiF) and the double-bond...
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Infrared Spectra and Density Functional Calculations for Singlet CH2═SIX2 and Triplet HC–SiX3 and XC–SiX3 Intermediates in Reactions of Laser-Ablated Silicon Atoms with Di-, Tri-, and Tetrahalomethanes
Inorganic Chemistry, 2016Co-Authors: Lester AndrewsAbstract:Reactions of laser-ablated silicon atoms with di-, tri-, and tetrahalomethanes in excess argon were investigated, and the products were identified from the matrix infrared spectra, isotopic shifts, and density functional theory energy, bond length, and frequency calculations. Dihalomethanes produce planar singlet silenes (CH2═SIX2), and tri- and tetrahalomethanes form triplet halosilyl carbenes (HC–SiX3 and XC–SiX3). The Si-bearing molecules identified are the most stable, lowest-energy product in the reaction systems. While the C–Si bond in the silene is a true double bond, the C–Si bond in the carbene is a shortened single bond enhanced by hyperconjugation of the two unpaired electrons on C to σ*(Si–X) orbitals, which contributes stabilization through a small amount of π-bonding and reduction of the HCSi or XCSi angles. The C–Si bond lengths in these carbenes (1.782 A for HC–SiF3) are between the single-bond length in the unobserved first insertion intermediate (1.975 A for CHF2–SiF) and the double-bond...
Kiyoshi Kawakami - One of the best experts on this subject based on the ideXlab platform.
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Mice doubly deficient in Six4 and Six5 show ventral body wall defects reproducing human omphalocele
Disease models & mechanisms, 2018Co-Authors: Masanori Takahashi, Shigeru Sato, Masaru Tamura, Kiyoshi KawakamiAbstract:Omphalocele is a human congenital anomaly in ventral body wall closure and may be caused by impaired formation of the primary abdominal wall (PAW) and/or defects in abdominal muscle development. Here, we report that mice doubly deficient in homeobox genes Six4 and Six5 showed the same ventral body wall closure defects as those seen in human omphalocele. SIX4 and SIX5 were localized in surface ectodermal cells and somatic mesoderm-derived mesenchymal and coelomic epithelial cells (CECs) in the PAW. Six4-/-;Six5-/- fetuses exhibited a large omphalocele with protrusion of both the liver and intestine, or a small omphalocele with protrusion of the intestine, with complete penetrance. The umbilical ring of Six4-/-;Six5-/- embryos was shifted anteriorly and its lateral size was larger than that of normal embryos at the E11.5 stage, before the onset of myoblast migration into the PAW. The proliferation rates of surface ectodermal cells in the left and right PAW and somatic mesoderm-derived cells in the right PAW were lower in Six4-/-;Six5-/- embryos than those of wild-type embryos at E10.5. The transition from CECs of the PAW to rounded mesothelial progenitor cells was impaired and the inner coelomic surface of the PAW was relatively smooth in Six4-/-;Six5-/- embryos at E11.25. Furthermore, Six4 overexpression in CECs of the PAW promoted ingression of CECs. Taken together, our results suggest that Six4 and Six5 are required for growth and morphological change of the PAW, and the impairment of these processes is linked to the abnormal positioning and expansion of the umbilical ring, which results in omphalocele.
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Activation of Six1 Expression in Vertebrate Sensory Neurons
PLOS ONE, 2015Co-Authors: Shigeru Sato, Yasuhide Furuta, Keiko Ikeda, Hiroshi Yajima, Kiyoshi KawakamiAbstract:SIX1 homeodomain protein is one of the essential key regulators of sensory organ development. Six1-deficient mice lack the olfactory epithelium, vomeronasal organs, cochlea, vestibule and vestibuloacoustic ganglion, and also show poor neural differentiation in the distal part of the cranial ganglia. Simultaneous loss of both Six1 and Six4 leads to additional abnormalities such as small trigeminal ganglion and abnormal dorsal root ganglia (DRG). The aim of this study was to understand the molecular mechanism that controls Six1 expression in sensory organs, particularly in the trigeminal ganglion and DRG. To this end, we focused on the sensory ganglia-specific Six1 enhancer (Six1-8) conserved between chick and mouse. In vivo reporter assays using both animals identified an important core region comprising binding consensus sequences for several transcription factors including nuclear hormone receptors, TCF/LEF, SMAD, POU homeodomain and basic-helix-loop-helix proteins. The results provided information on upstream factors and signals potentially relevant to Six1 regulation in sensory neurons. We also report the establishment of a new transgenic mouse line (mSix1-8-NLSCre) that expresses Cre recombinase under the control of mouse Six1-8. Cre-mediated recombination was detected specifically in ISL1/2-positive sensory neurons of Six1-positive cranial sensory ganglia and DRG. The unique features of the mSix1-8-NLSCre line are the absence of Cre-mediated recombination in SOX10-positive glial cells and central nervous system and ability to induce recombination in a subset of neurons derived from the olfactory placode/epithelium. This mouse model can be potentially used to advance research on sensory development.
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Six1 is a key regulator of the developmental and evolutionary architecture of sensory neurons in craniates
BMC Biology, 2014Co-Authors: Hiroshi Yajima, Shigeru Sato, Keiko Ikeda, Makoto Suzuki, Haruki Ochi, Ken Ichi Yamamura, Hajime Ogino, Naoto Ueno, Kiyoshi KawakamiAbstract:Various senses and sensory nerve architectures of animals have evolved during adaptation to exploit diverse environments. In craniates, the trunk sensory system has evolved from simple mechanosensory neurons inside the spinal cord (intramedullary), called Rohon-Beard (RB) cells, to multimodal sensory neurons of dorsal root ganglia (DRG) outside the spinal cord (extramedullary). The fish and amphibian trunk sensory systems switch from RB cells to DRG during development, while amniotes rely exclusively on the DRG system. The mechanisms underlying the ontogenic switching and its link to phylogenetic transition remain unknown. In Xenopus, Six1 overexpression promoted precocious apoptosis of RB cells and emergence of extramedullary sensory neurons, whereas Six1 knockdown delayed the reduction in RB cell number. Genetic ablation of Six1 and Six4 in mice led to the appearance of intramedullary sensory neuron-like cells as a result of medial migration of neural crest cells into the spinal cord and production of immature DRG neurons and fused DRG. Restoration of SIX1 expression in the neural crest-linage partially rescued the phenotype, indicating the cell autonomous requirements of SIX1 for normal extramedullary sensory neurogenesis. Mouse Six1 enhancer that mediates the expression in DRG neurons activated transcription in Xenopus RB cells earlier than endogenous six1 expression, suggesting earlier onset of mouse SIX1 expression than Xenopus during sensory development. The results indicated the critical role of Six1 in transition of RB cells to DRG neurons during Xenopus development and establishment of exclusive DRG system of mice. The study provided evidence that early appearance of SIX1 expression, which correlated with mouse Six1 enhancer, is essential for the formation of DRG-dominant system in mice, suggesting that heterochronic changes in Six1 enhancer sequence play an important role in alteration of trunk sensory architecture and contribute to the evolution of the trunk sensory system.
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homeoproteins six1 and six4 regulate male sex determination and mouse gonadal development
Developmental Cell, 2013Co-Authors: Yuka Fujimoto, Hiroki Kobayashi, Satomi S Tanaka, Yasuka L Yamaguchi, Shunsuke Kuroki, Makoto Tachibana, Mai Shinomura, Yoshiakira Kanai, Kenichirou Morohashi, Kiyoshi KawakamiAbstract:The Y-linked gene Sry regulates mammalian sex determination in bipotential embryonic gonads. Here, we report that the transcription factors Six1 and Six4 are required for male gonadal differentiation. Loss of Six1 and Six4 together, but neither alone, resulted in a male-to-female sex-reversal phenotype in XY mutant gonads accompanied by a failure in Sry activation. Decreased gonadal precursor cell formation at the onset of Sry expression and a gonadal size reduction in both sexes were also found in mutant embryos. Forced Sry transgene expression in XY mutant gonads rescued testicular development but not the initial disruption to precursor growth. Furthermore, we identified two downstream targets of Six1/Six4 in gonadal development, Fog2 (Zfpm2) and Nr5a1 (Ad4BP/Sf1). These two distinct Six1/Six4-regulated pathways are considered to be crucial for gonadal development. The regulation of Fog2 induces Sry expression in male sex determination, and the regulation of Nr5a1 in gonadal precursor formation determines gonadal size.
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Development of gustatory papillae in the absence of Six1 and Six4
Journal of Anatomy, 2011Co-Authors: Yuko Suzuki, Keiko Ikeda, Kiyoshi KawakamiAbstract:Six family genes encode homeobox transcription factors, and a deficiency in them leads to abnormal structures of the sensory organs. In a previous paper, Six1 was reported to be expressed in the taste bud-bearing lingual papillae of mice, and loss of Six1 affected the development of these gustatory papillae. We show here that embryos lacking both Six1 and Six4 revealed more severe abnormalities than those lacking Six1 alone during morphogenesis of their gustatory papillae. By in situ hybridization, Six4 was shown to be broadly distributed in the epithelium of the lateral lingual swellings at embryonic day (E) 11.5, and in the tongue epithelium, mesenchyme, and muscles at E12.5. From E14, Six4 was similar in expression pattern to Six1, as previously reported. In the fungiform papillae, Six4 was expressed in the epithelium at E14–E16.5. In the circumvallate and foliate papillae, Six4 expression was observed in the trench wall of these papillae at E15.5-P0. Although Six4-deficient mice had no abnormalities, Six1/Six4-deficient mice showed distinct morphological changes: fusion of the lateral lingual swellings was delayed, and the tongue was poorly developed. The primordia of fungiform papillae appeared earlier than those in the wild-type or Six1-deficient mice, and the papillae rapidly increased in size; thus fusion of each papilla was evident. The circumvallate papillae showed severe defects; for example, invagination of the trenches started asymmetrically, which resulted in longer and shorter trenches. The foliate papillae elevated initially, and showed stunted trenches. Therefore, Six1 and Six4 function synergistically to form gustatory papillae during development of the tongue.
Guillermo Oliver - One of the best experts on this subject based on the ideXlab platform.
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regulation of a remote shh forebrain enhancer by the six3 homeoprotein
Nature Genetics, 2008Co-Authors: Yongsu Jeong, Kenia B Eljaick, Anastasia Yocum, Erich Roessler, Maximilian Muenke, Federico Coluccio Leskow, Christèle Dubourg, Xin Geng, Xue Li, Guillermo OliverAbstract:In humans, SHH haploinsufficiency results in holoprosencephaly (HPE), a defect in anterior midline formation. Despite the importance of maintaining SHH transcript levels above a critical threshold, we know little about the upstream regulators of SHH expression in the forebrain. Here we describe a rare nucleotide variant located 460 kb upstream of SHH in an individual with HPE that resulted in the loss of Shh brain enhancer-2 (SBE2) activity in the hypothalamus of transgenic mouse embryos. Using a DNA affinity-capture assay, we screened the SBE2 sequence for DNA-binding proteins and identified members of the Six3 and Six6 homeodomain family as candidate regulators of Shh transcription. Six3 showed reduced binding affinity for the mutant compared to the wild-type SBE2 sequence. Moreover, Six3 with HPE-causing alterations failed to bind and activate SBE2. These data suggest a direct link between Six3 and Shh regulation during normal forebrain development and in the pathogenesis of HPE.
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SIX2 defines and regulates a multipotent self renewing nephron progenitor population throughout mammalian kidney development
Cell Stem Cell, 2008Co-Authors: Akio Kobayashi, Guillermo Oliver, Todd M Valerius, Joshua W Mugford, Thomas J Carroll, Michelle Self, Andrew P McmahonAbstract:Nephrons, the basic functional units of the kidney, are generated repetitively during kidney organogenesis from a mesenchymal progenitor population. Which cells within this pool give rise to nephrons and how multiple nephron lineages form during this protracted developmental process are unclear. We demonstrate that the SIX2-expressing cap mesenchyme represents a multipotent nephron progenitor population. SIX2-expressing cells give rise to all cell types of the main body of the nephron during all stages of nephrogenesis. Pulse labeling of SIX2-expressing nephron progenitors at the onset of kidney development suggests that the SIX2-expressing population is maintained by self-renewal. Clonal analysis indicates that at least some SIX2-expressing cells are multipotent, contributing to multiple domains of the nephron. Furthermore, SIX2 functions cell autonomously to maintain a progenitor cell status, as cap mesenchyme cells lacking SIX2 activity contribute to ectopic nephron tubules, a mechanism dependent on a Wnt9b inductive signal. Taken together, our observations suggest that SIX2 activity cell-autonomously regulates a multipotent nephron progenitor population.
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six3 mediated auto repression and eye development requires its interaction with members of the groucho related family of co repressors
Development, 2002Co-Authors: Michael A Dyer, Masanori Uchikawa, Oleg Lagutin, Hisato Kondoh, Guillermo OliverAbstract:Recent findings suggest that Six3, a member of the evolutionarily conserved So/Six homeodomain family, plays an important role in vertebrate visual system development. However, little is known about the molecular mechanisms by which this function is accomplished. Although several members of the So/Six gene family interact with members of the eyes absent (Eya) gene family and function as transcriptional activators, Six3 does not interact with any known member of the Eya family. Here, we report that Grg4 and Grg5, mouse counterparts of the Drosophila transcriptional co-repressor Groucho, interact with mouse Six3 and its closely related member Six6, which may also be involved in vertebrate eye development. The specificity of the interaction was validated by co-immunoprecipitation of Six3 and Grg4 complexes from cell lines. We also show that the interaction between Six3 and Grg5 requires the Q domain of Grg5 and a conserved phenylalanine residue present in an eh1-like motif located in the Six domain of Six3. The pattern of Grg5 expression in the mouse ventral forebrain and developing optic vesicles overlapped that previously reported for Six3 and Six6 . Using PCR, we identified a specific DNA motif that is bound by Six3 and we demonstrated that Six3 acts as a potent transcriptional repressor upon its interaction with Groucho-related members. We also demonstrated that this interaction is required for Six3 auto repression. The biological significance of this interaction in the retina and lens was assessed by overexpression experiments using either wild type full-length Six3 cDNA or a mutated form of this gene in which the interaction with Groucho proteins was disrupted. Overexpression of wild type Six3 by in vivo retroviral infection of newborn rat retinae led to an altered photoreceptor phenotype, while the in ovo electroporation of chicken embryos resulted in failure of lens placode invagination and production of δ-crystallin-negative cells within the placode. These specific alterations were not seen when the mutated form of Six3 cDNA was used in similar experimental approaches, indicating that Six3 interaction with Groucho proteins plays an essential role in vertebrate eye development.
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six3 promotes the formation of ectopic optic vesicle like structures in mouse embryos
Developmental Dynamics, 2001Co-Authors: Oleg Lagutin, Yasuhide Furuta, David H Rowitch, Andrew P Mcmahon, Guillermo OliverAbstract:A few years ago, three novel murine homeobox genes closely related to the Drosophila sine oculis (so) gene (Six1-3) were isolated and were all included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional ortholog of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue. Here, we show that similar to Drosophila, where the eyeless/Pax6 gene regulates the eye-specific expression of so, Six3 expression in the murine lens placodal ectoderm is also controlled by Pax6. We also show that ectopic Six3 expression promotes the formation of ectopic optic vesicle-like structures in the hindbrain-midbrain region of developing mouse embryos. © 2001 Wiley-Liss, Inc.
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Six3 promotes the formation of ectopic optic vesicle‐like structures in mouse embryos
Developmental Dynamics, 2001Co-Authors: Oleg Lagutin, Yasuhide Furuta, David H Rowitch, Andrew P Mcmahon, Guillermo OliverAbstract:A few years ago, three novel murine homeobox genes closely related to the Drosophila sine oculis (so) gene (Six1-3) were isolated and were all included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional ortholog of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue. Here, we show that similar to Drosophila, where the eyeless/Pax6 gene regulates the eye-specific expression of so, Six3 expression in the murine lens placodal ectoderm is also controlled by Pax6. We also show that ectopic Six3 expression promotes the formation of ectopic optic vesicle-like structures in the hindbrain-midbrain region of developing mouse embryos. © 2001 Wiley-Liss, Inc.
Heide L Ford - One of the best experts on this subject based on the ideXlab platform.
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Abstract 5001: Identification of a SIX2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization
Tumor Biology, 2018Co-Authors: Michael U. J. Oliphant, Ahwan Pandey, Katherine R. Johnson, Rani K. Powers, Matthew D. Galbraith, James C. Costello, Heide L FordAbstract:Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor SIX2 in promoting metastatic burden. SIX2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that SIX2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and SIX2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of SIX2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that SIX2 OE leads to an increase, whereas SIX2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that SIX2 regulates tumor initiation. In addition, we show that SIX2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that SIX2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which SIX2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that SIX2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by SIX2. In human breast cancer gene expression datasets, SIX2 significantly positively correlates with both Sox2 and Nanog, and combined OE of SIX2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of SIX2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that SIX2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel SIX2/Sox2/Nanog axis that is critical for metastatic colonization. Citation Format: Michael U. Oliphant, Ahwan Pandey, Katherine Johnson, Rani Powers, Matthew Galbraith, James Costello, Heide L. Ford. Identification of a SIX2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization [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 5001.
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abstract 5001 identification of a SIX2 sox2 nanog stem cell axis that promotes breast cancer metastatic colonization
Cancer Research, 2018Co-Authors: Michael U. J. Oliphant, Ahwan Pandey, Katherine R. Johnson, Rani K. Powers, Matthew D. Galbraith, James C. Costello, Heide L FordAbstract:Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor SIX2 in promoting metastatic burden. SIX2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that SIX2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and SIX2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of SIX2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that SIX2 OE leads to an increase, whereas SIX2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that SIX2 regulates tumor initiation. In addition, we show that SIX2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that SIX2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which SIX2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that SIX2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by SIX2. In human breast cancer gene expression datasets, SIX2 significantly positively correlates with both Sox2 and Nanog, and combined OE of SIX2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of SIX2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that SIX2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel SIX2/Sox2/Nanog axis that is critical for metastatic colonization. Citation Format: Michael U. Oliphant, Ahwan Pandey, Katherine Johnson, Rani Powers, Matthew Galbraith, James Costello, Heide L. Ford. Identification of a SIX2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization [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 5001.
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Abstract 2705: Novel functions of the homeoprotein SIX2 in mediating anchorage independence and metastasis in breast cancer.
Tumor Biology, 2013Co-Authors: Chu-an Wang, Paul Jedlicka, Aik Choon Tan, Vadym Zaberezhnyy, Heide L FordAbstract:Homeobox genes encode for transcription factors that are master regulators of embryogenesis, and their misexpression has been implicated in multiple cancers. The role of the homeoprotein SIX2 in developing kidney has been well demonstrated; however, its role in cancer progression is largely unknown. Here, we demonstrate, for the first time, that SIX2 is causally involved in mammary tumor progression. When SIX2 is knocked down (KD) in the 66cl4 mammary carcinoma cells, lung metastasis is significantly decreased compared to control KD; however, SIX2 KD conferred no significant effect on growth of the primary tumor or on tumor-associated angiogenesis/lymphangiogenesis, in contrast to its closely related family member, Six1, which has been implicated in all the aforementioned properties; suggesting that SIX2 may participate in later stages of the metastatic cascade. Expression of SIX2 in the 4TO7 mammary carcinoma cell line (a cell line that is syngeneic with 66cl4, but expresses very low levels of endogenous SIX2) led to changes in cell morphology, increased growth in soft agar, increased resistance to anoikis, and significantly enhanced lung metastasis in Balb/c mice. To determine the mechanism by which SIX2 mediates metastasis, microarray analysis was performed on 4TO7-control and SIX2 expressing cells. Interestingly, genes which have been implicated in lung metastasis (MMP, ANGPTL4, VCAM1) are significantly up-regulated in SIX2 overexpressing 4TO7 cells; while the epithelial marker, E-Cadherin, is dramatically decreased. Finally, analysis of SIX2 expression from public microarray datasets indicates that SIX2 is increased in breast cancers compared to normal breast tissue. In addition, high expression of SIX2 correlates with poor prognosis (distant metastasis free survival, overall survival and relapse free survival) in 1881 human breast tumors examined using the GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database, and upon further investigation we found that SIX2 expression is particularly associated with poor prognosis in luminal A, normal-like and ER-positive breast tumors. Together, our studies define a novel role of SIX2 in breast cancer metastasis. Citation Format: Chu-An Wang, Paul Jedlicka, Vadym Zaberezhnyy, Aik-Choon Tan, Heide Ford. Novel functions of the homeoprotein SIX2 in mediating anchorage independence and metastasis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2705. doi:10.1158/1538-7445.AM2013-2705
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Abstract PR04: The SIX2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin
Tumor Dormancy and Metastasis, 2013Co-Authors: Chu-an Wang, Paul Jedlicka, Aik Choon Tan, Catherine Pham, Vadym Zaberezhnvv, Heide L FordAbstract:Homeobox genes are master regulators of embryogenesis and it is widely known that misexpression of these transcription factors can lead to tumorigenesis/ metastasis. The Six1 homeoprotein has been implicated in tumor onset and metastasis of many different cancers, including breast cancer. However, little is known about whether related homeoproteins in the Six family can affect tumor onset or progression. Recent studies have shown that while Six1 knockdown dramatically decreases metastasis, knockdown cells that are capable of metastasizing have upregulated a related family member, SIX2. Interestingly, SIX2 regulates stem/ progenitor cell populations and maintains a mesenchymal progenitor phenotype in the developing kidney; however, the role of SIX2 in breast cancer progression has not been explored. Here, we demonstrate for the first time that up-regulation of SIX2 in breast cancer cells can promote metastasis in a mechanism that differs from its related family member, Six1. Knockdown (KD) of SIX2 in 66cl4 mouse mammary carcinoma cells significantly decreases lung metastasis in an immune-competent orthotopic mouse model, as compared to control KD. In contrast to what is seen with Six1 KD in the same orthotopic mouse model, no significant inhibition of primary tumor size was observed with SIX2 knockdown, suggesting that unlike Six1, the functions of SIX2 may be confined to later stages of tumor progression. Overexpression of SIX2 in the weakly metastatic 4TO7 mouse mammary carcinoma cell line led to changes in cell morphology, increased anchorage independent growth and significantly enhanced lung metastasis in an immune competent mouse model. Microarray analysis was performed to determine the mechanism by which SIX2 mediates metastasis and the epithelial marker E-Cadherin, whose loss has been implicated in the metastatic potential of many cancers, was dramatically downregulated in the 4TO7-SIX2 expressing cells. Importantly, restoration of E-cadherin expression reduced lung metastasis induced by SIX2 and increased survival of the animals. We further determine that repression of E-cadherin by SIX2 is mediated via multiple mechanisms. SIP1/Zeb2 (zinc finger E box binding homeobox2), an epithelial to mesenchymal transition (EMT) inducer, is increased in cells overexpressing SIX2, and Zeb2 KD in these cells reverts E-cadherin expression back to the level as that observed in the control cells and importantly leads to a reduction in metastasis in vivo. In addition, we find that SIX2 may also repress E-cadherin though an epigenetic mechanism, since repression of E-cadherin downstream of SIX2 can be reversed by inhibiting DNA methylation. Finally, analysis of SIX2 expression from public microarray datasets indicates that SIX2 is increased in breast cancers compared to normal breast tissue and that high expression of SIX2 correlates with poor prognosis (distant metastasis free survival, overall survival and relapse free survival) in 1881 human breast tumors examined using the GOBO (Gene Expression-Based Outcome for Breast Cancer Online) database. Moreover, a reverse correlation of E-cadherin and SIX2 in breast cancer datasets suggest that SIX2 promotes loss of E-cadherin in human breast cancer. Together, these data demonstrate that SIX2 is a specific regulator of the metastatic process, through regulating E-cadherin expression. This abstract is also presented as Poster B094. Citation Format: Chu-An Wang, Paul Jedlicka, Aik-Choon Tan, Catherine Pham, Vadym Zaberezhnvv, Heide Ford. The SIX2 homeoprotein mediates breast cancer metastasis via repressing E-cadherin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr PR04.
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Characterization of the Six1 homeobox gene in normal mammary gland morphogenesis
BMC Developmental Biology, 2010Co-Authors: Ricardo D Coletta, Kiyoshi Kawakami, Erica L Mccoy, Valerie Burns, James L Mcmanaman, John J Wysolmerski, Heide L FordAbstract:Background The Six1 homeobox gene is highly expressed in the embryonic mammary gland, continues to be expressed in early postnatal mammary development, but is lost when the mammary gland differentiates during pregnancy. However, Six1 is re-expressed in breast cancers, suggesting that its re-instatement in the adult mammary gland may contribute to breast tumorigenesis via initiating a developmental process out of context. Indeed, recent studies demonstrate that Six1 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas, and that its overexpression in xenograft models of mammary cancer leads to metastasis. These data demonstrate that Six1 is causally involved in both breast tumorigenesis and metastasis, thus raising the possibility that it may be a viable therapeutic target. However, because Six1 is highly expressed in the developing mammary gland, and because it has been implicated in the expansion of mammary stem cells, targeting Six1 as an anti-cancer therapy may have unwanted side effects in the breast. Results We sought to determine the role of Six1 in mammary development using two independent mouse models. To study the effect of Six1 loss in early mammary development when Six1 is normally expressed, Six1 ^-/- embryonic mammary glands were transplanted into Rag1 ^-/- mice. In addition, to determine whether Six1 downregulation is required during later stages of development to allow for proper differentiation, we overexpressed Six1 during adulthood using an inducible, mammary-specific transgenic mouse model. Morphogenesis of the mammary gland occurred normally in animals transplanted with Six1 ^-/- embryonic mammary glands, likely through the redundant functions of other Six family members such as SIX2 and Six4 , whose expression was increased in response to Six1 loss. Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation. Conclusions Six1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function. However, as both SIX2 and Six4 levels are increased in Six1 ^-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families. This data, in conjunction with recent findings that Six1 is capable of promoting breast cancer initiation and progression, suggest that Six1 may serve as a reasonable chemotherapeutic target in a clinical setting, particularly for those women diagnosed with breast cancer in their childbearing years.
Pascal Maire - One of the best experts on this subject based on the ideXlab platform.
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Crucial and Overlapping Roles of Six1 and SIX2 in Craniofacial Development.
Journal of dental research, 2019Co-Authors: Z. Liu, Pascal Maire, Yu Lan, Han Liu, X. Wang, Rulang JiangAbstract:SIX1 and SIX2 encode closely related transcription factors of which disruptions have been associated with distinct craniofacial syndromes, with mutations in SIX1 associated with branchiootic syndrome 3 (BOS3) and heterozygous deletions of SIX2 associated with frontonasal dysplasia defects. Whereas mice deficient in Six1 recapitulated most of the developmental defects associated with BOS3, mice lacking SIX2 function had no obvious frontonasal defects. We show that Six1 and SIX2 exhibit partly overlapping patterns of expression in the developing mouse embryonic frontonasal, maxillary, and mandibular processes. We found that Six1 -/- SIX2 -/- double-mutant mice were born with severe craniofacial deformity not seen in the Six1 -/- or SIX2 -/- single mutants, including skull bone agenesis, midline facial cleft, and syngnathia. Moreover, whereas Six1 -/- mice exhibited partial transformation of maxillary zygomatic bone into a mandibular condyle-like structure, Six1 -/-SIX2 +/- mice exhibit significantly increased penetrance of the maxillary malformation. In addition to ectopic Dlx5 expression at the maxillary-mandibular junction as recently reported in E10.5 Six1 -/- embryos, the E10.5 Six1 -/- SIX2 +/- embryos showed ectopic expression of Bmp4, Msx1, and Msx2 messenger RNAs in the maxillary-mandibular junction. Genetically inactivating 1 allele of either Ednra or Bmp4 significantly reduced the penetrance of maxillary malformation in both Six1 -/- and Six1 -/- SIX2 +/- embryos, indicating that Six1 and SIX2 regulate both endothelin and bone morphogenetic protein-4 signaling pathways to pattern the facial structures. Furthermore, we show that neural crest-specific inactivation of Six1 in SIX2 -/- embryos resulted in midline facial cleft and frontal bone agenesis. We show that Six1 -/- SIX2 -/- embryos exhibit significantly reduced expression of key frontonasal development genes Alx1 and Alx3 as well as increased apoptosis in the developing frontonasal mesenchyme. Together, these results indicate that Six1 and SIX2 function partly redundantly to control multiple craniofacial developmental processes and play a crucial neural crest cell-autonomous role in frontonasal morphogenesis.
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Six homeoproteins directly activate Myod expression in the gene regulatory networks that control early myogenesis
PLoS Genetics, 2013Co-Authors: F. Relaix, Christine Laclef, Josiane Demignon, Claire Niro, J. Pujol, M. Santolini, M. Lagha, D. Rocancourt, M. Buckingham, Pascal MaireAbstract:In mammals, several genetic pathways have been characterized that govern engagement of multipotent embryonic progenitors into the myogenic program through the control of the key myogenic regulatory gene Myod. Here we demonstrate the involvement of Six homeoproteins. We first targeted into a Pax3 allele a sequence encoding a negative form of Six4 that binds DNA but cannot interact with essential Eya co-factors. The resulting embryos present hypoplasic skeletal muscles and impaired Myod activation in the trunk in the absence of Myf5/Mrf4. At the axial level, we further show that Myod is still expressed in compound Six1/Six4:Pax3 but not in Six1/Six4:Myf5 triple mutant embryos, demonstrating that Six1/4 participates in the Pax3-Myod genetic pathway. Myod expression and head myogenesis is preserved in Six1/Six4:Myf5 triple mutant embryos, illustrating that upstream regulators of Myod in different embryonic territories are distinct. We show that Myod regulatory regions are directly controlled by Six proteins and that, in the absence of Six1 and Six4, SIX2 can compensate.
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Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome
Developmental Biology, 2010Co-Authors: Claire Niro, Josiane Demignon, Stéphane Vincent, Yubing Liu, Julien Giordani, Nicolas Sgarioto, Maryline Favier, Isabelle Guillet-deniau, Alexandre Blais, Pascal MaireAbstract:While the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis.
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six1 and six4 homeoproteins are required for pax3 and mrf expression during myogenesis in the mouse embryo
Development, 2005Co-Authors: Raphaelle Grifone, Mary J. Seller, Josiane Demignon, Christophe Houbron, Evelyne Souil, Claire Niro, Ghislaine Hamard, Pascal MaireAbstract:In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3 -deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.
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six1 is required for the early organogenesis of mammalian kidney
Development, 2003Co-Authors: Pinxian Xu, Weiming Zheng, Christine Laclef, Li Huang, Pascal Maire, Derek SilviusAbstract:The murine Six gene family, homologous to Drosophila sine oculis ( so ) which encodes a homeodomain transcription factor, is composed of six members ( Six1-6 ). Among the six members, only the SIX2 gene has been previously shown to be expressed early in kidney development, but its function is unknown. We have recently found that the Six1 gene is also expressed in the kidney. In the developing kidney, Six1 is expressed in the uninduced metanephric mesenchyme at E10.5 and in the induced mesenchyme around the ureteric bud at E11.5. At E17.5 to P0, Six1 expression became restricted to a subpopulation of collecting tubule epithelial cells. To study its in vivo function, we have recently generated Six1 mutant mice. Loss of Six1 leads to a failure of ureteric bud invasion into the mesenchyme and subsequent apoptosis of the mesenchyme. These results indicate that Six1 plays an essential role in early kidney development. In Six1 -/- kidney development, we have found that Pax2, SIX2 and Sall1 expression was markedly reduced in the metanephric mesenchyme at E10.5, indicating that Six1 is required for the expression of these genes in the metanephric mesenchyme. In contrast, Eya1 expression was unaffected in Six1 -/- metanephric mesenchyme at E10.5, indicating that Eya1 may function upstream of Six1 . Moreover, our results show that both Eya1 and Six1 expression in the metanephric mesenchyme is preserved in Pax2 -/- embryos at E10.5, further indicating that Pax2 functions downstream of Eya1 and Six1 in the metanephric mesenchyme. Thus, the epistatic relationship between Pax, Eya and Six genes in the metanephric mesenchyme during early kidney development is distinct from a genetic pathway elucidated in the Drosophila eye imaginal disc. Finally, our results show that Eya1 and Six1 genetically interact during mammalian kidney development, because most compound heterozygous embryos show hypoplastic kidneys. These analyses establish a role for Six1 in the initial inductive step for metanephric development.
