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Heide L Ford - One of the best experts on this subject based on the ideXlab platform.
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Targeting the SIX1/Eya transcriptional complex for ovarian cancer therapy
2015Co-Authors: Rui Zhao, Heide L Ford, Kian BehbakhtAbstract:Abstract : Background: The SIX1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is downregulated after organ development is complete. Its expression is low or undetectable in most normal adult tissues, but it is over-expressed in almost 70% of ovarian cancers, where its expression correlates with shortened survival. SIX1 is critical for proliferation and survival of ovarian cancer cells, which has been linked to both metastasis and resistance to chemotherapy. Since SIX1 does not have an intrinsic activation or repression domain, it requires the Eya coactivator proteins to activate transcription. Similar to SIX1, Eya proteins are also not expressed in most normal adult tissues, but are re-expressed in cancers. Intriguingly, the Eya2 protein is amplified in 15% of epithelial ovarian cancers (EOCs), is overexpressed in as many as 94% of epithelial ovarian cancers, and like SIX1, is associated with shortened survival in ovarian cancers. Hypothesis: We hypothesized that the SIX1/Eya interaction is critical for ovarian tumor growth and metastasis and that disrupting this interaction would significantly inhibit ovarian tumor growth and metastasis. Progress made is as follows: Aim 1. Determine the role of the SIX1/Eya interaction in tumor growth and peritoneal metastasis in EOC using a genetic approach. We have created stable ovarian cancer cell lines in which SIX1 or Eya are knocked down, and others in which Eya is overexpressed (WT or mutations that do not allow it to interact with SIX1 or to have Tyr Phosphatase activity). We have demonstrated that the interaction of SIX1 and Eya is critical for proliferation of ovarian cancer cells as well as for migration and alterations in adhesion. Aim 2. Determine whether small molecules targeting the SIX1/Eya interaction inhibit the proliferation and survival of EOC cells. We have tested our most promising SIX1/Eya interaction inhibitors in ovarian cancer proliferation assays
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MicroRNA-30a regulates zebrafish myogenesis through targeting the transcription factor SIX1.
Journal of cell science, 2014Co-Authors: Jenean H O'brien, Laura Hernandez-lagunas, Kristin Bruk Artinger, Heide L FordAbstract:Precise spatiotemporal regulation of the SIX1 homeoprotein is required to coordinate vital tissue development, including myogenesis. Whereas SIX1 is downregulated in most tissues following embryogenesis, it is re-expressed in numerous cancers, including tumors derived from muscle progenitors. Despite crucial roles in development and disease, the upstream regulation of SIX1 expression has remained elusive. Here, we identify the first direct mechanism for SIX1 regulation in embryogenesis, through microRNA30a (miR30a)-mediated repression. In zebrafish somites, we show that miR30a and SIX1a and SIX1b (hereafter SIX1a/b) are expressed in an inverse temporal pattern. Overexpression of miR30a leads to a reduction in SIX1a/b levels, and results in increased apoptosis and altered somite morphology, which phenocopies SIX1a/b knockdown. Conversely, miR30a inhibition leads to increased SIX1 expression and abnormal somite morphology, revealing a role for endogenous miR30a as a muscle-specific miRNA (myomiR). Importantly, restoration of SIX1a in miR30a-overexpressing embryos restores proper myogenesis. These data demonstrate a new role for miR30a at a key node in the myogenic regulatory gene network through controlling SIX1 expression.
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Structure-function analyses of the human SIX1-EYA2 complex reveal insights into metastasis and BOR syndrome.
Nature Structural & Molecular Biology, 2013Co-Authors: Aaron N. Patrick, Heide L Ford, Joshua H. Cabrera, Anna L. Smith, Xiaojiang S. Chen, Rui ZhaoAbstract:SIX1–EYA form a bipartite transcription factor, essential for development, whose overexpression is linked to metastasis. The crystal structure of SIX1 bound to EYA2 reveals that the interaction is mediated by an α-helix of SIX1, and a single mutation can disrupt this association and SIX1's ability to promote EMT and metastasis.
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expression of SIX1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal regulated kinase and transforming growth factor beta signaling pathways
Breast Cancer Research, 2012Co-Authors: Ritsuko Iwanaga, Chu-an Wang, Paul Jedlicka, Douglas S. Micalizzi, S M Farabaugh, Chuck J Harrell, Carol A Sartorius, Peter Kabos, Andrew P Bradford, Heide L FordAbstract:Introduction: Mammary-specific overexpression of SIX1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. SIX1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that SIX1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. Methods: We examined gene expression datasets to determine the breast cancer subtypes with SIX1 overexpression, and then examined its expression in the CD24 low /CD44 + putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. SIX1 overexpression, or knockdown, was performed in different systems to examine how SIX1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which SIX1 influences TICs using genetic/ inhibitor approaches in vitro and in vivo. Finally, we examined the expression of SIX1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. Results: High levels of SIX1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal
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Abstract C127: Crystal structure of the SIX1/EYA transcriptional complex reveals ways to inhibit SIX1-mediated breast tumorigenesis/metastasis.
Protein-Protein Interaction, 2011Co-Authors: Aaron N. Patrick, Rui Zhao, Joshua H. Cabrera, Heide L FordAbstract:The SIX1 homeoprotein and members of the Eya family of co-activators are thought to form a bipartite transcription factor that is important in development and disease. SIX1 and Eya are highly expressed during development, but their expression is lost in most adult tissues. Interestingly, SIX1 and Eya are re-expressed in a variety of cancers, where they are known to promote both transformation and metastasis. However, a means to target the complex has remained elusive due to the recognized difficulties in targeting transcription factors. Thus, the molecular details of the protein-protein interface and an understanding of whether complex formation is necessary for tumorigenesis/metastasis are critical to the development of novel anti-cancer therapies targeting the SIX1/Eya complex. Here we describe the 1.9 A resolution crystal structure of the SIX1/Eya complex revealing unexpected protein binding interfaces. The interaction resembles that of the p53/HDM2 complex, with a single helix of SIX1 binding in a hydrophobic groove in Eya, both distant from sites originally thought to be involved in the SIX1/Eya interaction. Remarkably, we show that a single amino acid substitution in SIX1, involving a residue in the Eya binding interface, is sufficient to inhibit complex formation and to inhibit properties associated with SIX1-induced tumorigenesis/metastasis. Since SIX1 and Eya are regulators of embryonic development and are scarcely expressed in adult tissues, inhibition of the SIX1/Eya pathway likely will lead to limited side effects in the adult. In addition, since 50–90% of breast cancer patients over-express SIX1, therapies targeting the complex are likely to benefit a significant proportion of breast cancer patients, and an even greater proportion of patients with metastatic disease. Together, our findings suggest that inhibiting the SIX1-Eya helical interface is a potential mechanism to develop novel, anti-breast cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C127.
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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SIX1 is required for mouse dental follicle cell and human periodontal ligament-derived cell proliferation
Development Growth & Differentiation, 2016Co-Authors: Tatsuki Kawasaki, Hiroshi Yajima, Masanori Takahashi, Yoshiyuki Mori, Kiyoshi KawakamiAbstract:The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor SIX1 expression in mouse DFCs. However, the role of SIX1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-2'-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in SIX1-deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals.
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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.
Rui Zhao - One of the best experts on this subject based on the ideXlab platform.
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Targeting the SIX1/Eya transcriptional complex for ovarian cancer therapy
2015Co-Authors: Rui Zhao, Heide L Ford, Kian BehbakhtAbstract:Abstract : Background: The SIX1 homeobox gene encodes a transcription factor that is crucial for the development of many organs but is downregulated after organ development is complete. Its expression is low or undetectable in most normal adult tissues, but it is over-expressed in almost 70% of ovarian cancers, where its expression correlates with shortened survival. SIX1 is critical for proliferation and survival of ovarian cancer cells, which has been linked to both metastasis and resistance to chemotherapy. Since SIX1 does not have an intrinsic activation or repression domain, it requires the Eya coactivator proteins to activate transcription. Similar to SIX1, Eya proteins are also not expressed in most normal adult tissues, but are re-expressed in cancers. Intriguingly, the Eya2 protein is amplified in 15% of epithelial ovarian cancers (EOCs), is overexpressed in as many as 94% of epithelial ovarian cancers, and like SIX1, is associated with shortened survival in ovarian cancers. Hypothesis: We hypothesized that the SIX1/Eya interaction is critical for ovarian tumor growth and metastasis and that disrupting this interaction would significantly inhibit ovarian tumor growth and metastasis. Progress made is as follows: Aim 1. Determine the role of the SIX1/Eya interaction in tumor growth and peritoneal metastasis in EOC using a genetic approach. We have created stable ovarian cancer cell lines in which SIX1 or Eya are knocked down, and others in which Eya is overexpressed (WT or mutations that do not allow it to interact with SIX1 or to have Tyr Phosphatase activity). We have demonstrated that the interaction of SIX1 and Eya is critical for proliferation of ovarian cancer cells as well as for migration and alterations in adhesion. Aim 2. Determine whether small molecules targeting the SIX1/Eya interaction inhibit the proliferation and survival of EOC cells. We have tested our most promising SIX1/Eya interaction inhibitors in ovarian cancer proliferation assays
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Structure-function analyses of the human SIX1-EYA2 complex reveal insights into metastasis and BOR syndrome.
Nature Structural & Molecular Biology, 2013Co-Authors: Aaron N. Patrick, Heide L Ford, Joshua H. Cabrera, Anna L. Smith, Xiaojiang S. Chen, Rui ZhaoAbstract:SIX1–EYA form a bipartite transcription factor, essential for development, whose overexpression is linked to metastasis. The crystal structure of SIX1 bound to EYA2 reveals that the interaction is mediated by an α-helix of SIX1, and a single mutation can disrupt this association and SIX1's ability to promote EMT and metastasis.
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Abstract C127: Crystal structure of the SIX1/EYA transcriptional complex reveals ways to inhibit SIX1-mediated breast tumorigenesis/metastasis.
Protein-Protein Interaction, 2011Co-Authors: Aaron N. Patrick, Rui Zhao, Joshua H. Cabrera, Heide L FordAbstract:The SIX1 homeoprotein and members of the Eya family of co-activators are thought to form a bipartite transcription factor that is important in development and disease. SIX1 and Eya are highly expressed during development, but their expression is lost in most adult tissues. Interestingly, SIX1 and Eya are re-expressed in a variety of cancers, where they are known to promote both transformation and metastasis. However, a means to target the complex has remained elusive due to the recognized difficulties in targeting transcription factors. Thus, the molecular details of the protein-protein interface and an understanding of whether complex formation is necessary for tumorigenesis/metastasis are critical to the development of novel anti-cancer therapies targeting the SIX1/Eya complex. Here we describe the 1.9 A resolution crystal structure of the SIX1/Eya complex revealing unexpected protein binding interfaces. The interaction resembles that of the p53/HDM2 complex, with a single helix of SIX1 binding in a hydrophobic groove in Eya, both distant from sites originally thought to be involved in the SIX1/Eya interaction. Remarkably, we show that a single amino acid substitution in SIX1, involving a residue in the Eya binding interface, is sufficient to inhibit complex formation and to inhibit properties associated with SIX1-induced tumorigenesis/metastasis. Since SIX1 and Eya are regulators of embryonic development and are scarcely expressed in adult tissues, inhibition of the SIX1/Eya pathway likely will lead to limited side effects in the adult. In addition, since 50–90% of breast cancer patients over-express SIX1, therapies targeting the complex are likely to benefit a significant proportion of breast cancer patients, and an even greater proportion of patients with metastatic disease. Together, our findings suggest that inhibiting the SIX1-Eya helical interface is a potential mechanism to develop novel, anti-breast cancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C127.
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Biochemical and Functional Characterization of Six SIX1 Branchio-oto-renal Syndrome Mutations
Journal of Biological Chemistry, 2009Co-Authors: Aaron N. Patrick, Barbara J. Schiemann, Kui Yang, Rui Zhao, Heide L FordAbstract:Abstract Branchio-oto-renal syndrome (BOR) is an autosomal dominant developmental disorder characterized by hearing loss, branchial arch defects, and renal anomalies. Recently, eight mutations in the SIX1 homeobox gene were discovered in BOR patients. To characterize the effect of SIX1 BOR mutations on the EYA-SIX1-DNA complex, we expressed and purified six of the eight mutants in Escherichia coli. We demonstrate that only the most N-terminal mutation in SIX1 (V17E) completely abolishes SIX1-EYA complex formation, whereas all of the other mutants are able to form a stable complex with EYA. We further show that only the V17E mutant fails to localize EYA to the nucleus and cannot be stabilized by EYA in the cell. The remaining five SIX1 mutants are instead all deficient in DNA binding. In contrast, V17E alone has a DNA binding affinity similar to that of wild type SIX1 in complex with the EYA co-factor. Finally, we show that all SIX1 BOR mutants are defective in transcriptional activation using luciferase reporter assays. Taken together, our experiments demonstrate that the SIX1 BOR mutations contribute to the pathology of the disease through at least two different mechanisms that involve: 1) abolishing the formation of the SIX1-EYA complex or 2) diminishing the ability of SIX1 to bind DNA. Furthermore, our data demonstrate for the first time that EYA: 1) requires the N-terminal region of the SIX1 Six domain for its interaction, 2) increases the level of the SIX1 protein within the cell, and 3) increases the DNA binding affinity of SIX1.
Douglas S. Micalizzi - One of the best experts on this subject based on the ideXlab platform.
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expression of SIX1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal regulated kinase and transforming growth factor beta signaling pathways
Breast Cancer Research, 2012Co-Authors: Ritsuko Iwanaga, Chu-an Wang, Paul Jedlicka, Douglas S. Micalizzi, S M Farabaugh, Chuck J Harrell, Carol A Sartorius, Peter Kabos, Andrew P Bradford, Heide L FordAbstract:Introduction: Mammary-specific overexpression of SIX1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. SIX1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that SIX1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. Methods: We examined gene expression datasets to determine the breast cancer subtypes with SIX1 overexpression, and then examined its expression in the CD24 low /CD44 + putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. SIX1 overexpression, or knockdown, was performed in different systems to examine how SIX1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which SIX1 influences TICs using genetic/ inhibitor approaches in vitro and in vivo. Finally, we examined the expression of SIX1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. Results: High levels of SIX1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal
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Eya2 is required to mediate the pro-metastatic functions of SIX1 via the induction of TGF-β signaling, epithelial–mesenchymal transition, and cancer stem cell properties
Oncogene, 2012Co-Authors: S M Farabaugh, Paul Jedlicka, Douglas S. Micalizzi, R Zhao, H L FordAbstract:SIX1 is a critical regulator of embryonic development that requires interaction with the Eya family of proteins (Eya1-4) to activate the transcription of genes involved in neurogenesis, myogenesis and nephrogenesis. Although expression of SIX1 and Eya family members is predominantly observed in development, their overexpression is observed in numerous cancers. Importantly, both SIX1 and Eya have independently been shown to mediate breast cancer metastasis, but whether they functionally interact during tumor progression has not been explored. Herein, we demonstrate that knockdown of Eya2 in MCF7 mammary carcinoma cells reverses the ability of SIX1 to induce transforming growth factor-β signaling, as well as to induce characteristics associated with epithelial–mesenchymal transition and cancer stem cells, suggesting that SIX1 is dependent on Eya2 to mediate numerous pro-metastatic characteristics. The importance of the SIX1–Eya interaction in human breast cancer is underscored by the finding that high levels of SIX1 correlate with shortened time to relapse and metastasis as well as decreased survival only when co-expressed with high levels of Eya2. Overall, these data implicate Eya2 as a necessary co-factor for many of the metastasis promoting functions of SIX1, suggesting that targeting the SIX1–Eya interaction may inhibit breast cancer progression. As SIX1 and Eya2 are not highly expressed in most adult tissues, the SIX1–Eya interaction may be a valuable future therapeutic target whose inhibition would be expected to impair breast cancer progression while conferring limited side effects.
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Abstract 1463: SIX1 induces lymphatic and distant metastasis by upregulating VEGF-C and stimulating lymphangiogenesis in breast cancer
Tumor Biology, 2011Co-Authors: Chu-an Wang, Paul Jedlicka, Kimberly L. Christensen, Douglas S. Micalizzi, J. Chuck Harrell, Heide L FordAbstract:The homeoprotein SIX1 is expressed during embryogenesis and re-expressed in 50% of primary and in 90% of metastatic breast cancer lesions.Using an orthotopic xenograft model of breast cancer, SIX1 overexpression results in a 40% increased in lymphatic metastases as compared to controls.SIX1 correlates with positive lymph node status in human breast cancer, further suggesting that it influences lymphatic metastasis, perhaps through stimulating lymphangiogenesis.Microarray analysis revealed that the critical lymphangiogenic regulator, VEGF-C, may be a target of SIX1.Real-time PCR and VEGF-C promoter-luciferase assays demonstrated that VEGF-C upregulation is mediated transcriptionally by SIX1.Chromatin immunoprecipitation analysis demonstrates in vivo binding of SIX1 to the VEGF-C promoter.Increased levels of VEGF-C mRNA in response to SIX1 overexpression result in increased intracellular and secreted VEGF-C protein.We quantified the number of intra- and peri-tumoral vessels in MCF7-SIX1 versus MCF7-Control tumors and the MCF7-SIX1 tumors display increased lymphangiogenesis and a 5-fold increase in the frequency of lymphatic invasion compared to the MCF7-Ctrl tumors.To determine whether SIX1 depends on VEGF-C to increase lymphangiogenesis and lymphatic metastasis, we knocked down VEGF-C in MCF7-SIX1 cells and injected the cells into the mammary gland of nude mice.We observed decreased lymph node metastases in the mice bearing MCF7-SIX1/VEGF-C KD tumors compared to the MCF7-SIX1 tumors, suggesting SIX1-induced lymphatic metastasis is in part through upregulation of VEGF-C.We also used a complementary model, the 66c14 mammary carcinoma cell line which metastasizes primarily through the lymphatic system and expresses high levels of SIX1, to study the role of SIX1 in lymphatic metastasis.SIX1 knockdown (KD) in 66c14 cells resulted in decreased levels of Vegf-c and strikingly, the SIX1 KD tumors also contained less lymphatic vessel formation, suggesting a reduction of lymphangiogensis. Finally, SIX1 KD dramatically decreased lung metastases in the 66c14 model as seen using bioluminescent imaging. When we rescued Vegf-c expression in 66c14 in the context of SIX1 KD, we observed a restoration of lymphatic metastasis, but the rescue failed to restore lung metastasis, suggesting SIX1 regulates additional factor/pathways required to accomplish the later stages of metastasis.Regulation of VEGF-C by SIX1 is supported by clinical observations that correlation of SIX1 and VEGF-C can be extended broadly to breast cancer cell lines (oncomine) and to human breast cancer tissues (tissue array).In conclusion, we demonstrate that SIX1 mediates lymphangiogenesis and lymphatic metastasis in multiple mouse models via its ability to regulate VEGF-C, and extend this observation to human tissues where SIX1 and VEGF-C overexpression are significantly correlated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1463. doi:10.1158/1538-7445.AM2011-1463
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homeoprotein SIX1 increases tgf β type i receptor and converts tgf β signaling from suppressive to supportive for tumor growth
Cancer Research, 2010Co-Authors: Douglas S. Micalizzi, Chu-an Wang, S M Farabaugh, William P Schiemann, Heide L FordAbstract:The SIX1 homeodomain protein is a developmental transcription factor that has been implicated in tumor onset and progression. Our recent work shows that SIX1 overexpression in human breast cancer cell lines is sufficient to induce epithelial-to-mesenchymal transition (EMT) and metastasis. Importantly, SIX1-induced EMT and metastasis are dependent on TGF-β signaling. The TGF-β pathway plays a dual role in cancer, acting as a tumor suppressor in early lesions but enhancing metastatic spread in more advanced tumors. Our previous work indicated that SIX1 may be a critical mediator of the switch in TGF-β signaling from tumor suppressive to tumor promotional. However, the mechanism by which SIX1 impinges on the TGF-β pathway was, until now, unclear. In this work, we identify the TGF-β type I receptor (TβRI) as a target of SIX1 and a critical effector of SIX1-induced TGF-β signaling and EMT. We show that SIX1-induced upregulation of TβRI is both necessary and sufficient to activate TGF-β signaling and induce properties of EMT. Interestingly, increased TβRI expression is not sufficient to induce experimental metastasis, providing in vivo evidence that SIX1 overexpression is required to switch TGF-β signaling to the prometastatic phenotype and showing that induction of EMT is not sufficient to induce experimental metastasis. Together, these results show a novel mechanism for the activation of TGF-β signaling, identify TβRI as a new target of SIX1, and implicate SIX1 as a determinant of TGF-β function in breast cancer. Cancer Res; 70(24); 10371–80. ©2010 AACR.
Kim E. Creek - One of the best experts on this subject based on the ideXlab platform.
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HPV16-transformed human keratinocytes depend on SIX1 expression for proliferation and HPV E6/E7 gene expression
Virology, 2019Co-Authors: Maria Hosseinipour, Kim E. Creek, Fang Wan, Diego Altomare, Lucia PirisiAbstract:Abstract The homeodomain transcription factor SIX1 plays a critical role in embryogenesis, is not expressed in normal adult tissue, but is expressed in many malignancies, including cervical cancer. SIX1 drives the progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward malignancy: HKc/HPV16 express high levels of SIX1 mRNA and protein; overexpression of SIX1 in HKc/HPV16 produces pre-malignant, differentiation-resistant lines (HKc/DR); SIX1 overexpression in HKc/DR induces tumorigenicity. In this paper, we explore the consequences of inhibition of SIX1 expression in premalignant HKc/DR. Only partial inhibition of SIX1 expression could be obtained in HKc/DR by RNA interference. Decreased SIX1 expression (up to 80%) in HKc/DR resulted in slower proliferation, decreased HPV16-E6/E7 mRNA levels, and increased p53 protein levels. Gene expression changes induced in HKc/DR by anti-SIX1 shRNA were indicative of mesenchymal-epithelial transition (MET) and changes in TGF-beta signaling. We conclude that HPV16-transformed cells depend on SIX1 for survival, HPV16 E6/E7 gene expression and epithelial-mesenchymal transition.
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SIX1 promotes colorectal cancer growth and metastasis by stimulating angiogenesis and recruiting tumor-associated macrophages.
Carcinogenesis, 2017Co-Authors: Yu Zhang, Lucia Pirisi, Maria Marjorette O. Peña, Kim E. CreekAbstract:The homeoprotein SIX1 is overexpressed in many human cancers and is associated with increased tumor progression and metastasis. Recent studies have shown that SIX1 is associated with poorer overall survival in advanced-stage colorectal cancer (CRC). In the current study, we explored the functional changes and molecular events associated with SIX1 overexpression in a mouse model of CRC. An orthotopic model and a splenic injection metastasis model were used to investigate the role of SIX1 in CRC tumor growth and metastasis using mouse colon adenocarcinoma MC38 cells overexpressing SIX1. We found that overexpression of SIX1 dramatically promotes CRC tumor growth and metastasis in vivo. SIX1 overexpression in MC38 increased protein levels of aldehyde dehydrogenase-1 and expanded CD44+/CD166+ populations, indicating SIX1 increased features of cancer stem cells. In addition, SIX1 overexpression stimulated angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF). SIX1-overexpressing tumor cells recruited tumor-associated macrophages (TAM) by increasing the expression of macrophage-specific colony stimulating factor, chemokine (C-C motif) ligand 2/5 and VEGF, further facilitating CRC tumor growth and metastasis. Furthermore, we determined that SIX1 activated mitogen-activated protein kinase (MAPK) signaling in CRC cells. In summary, our studies strongly suggest that SIX1 overexpression promotes CRC growth and metastasis and remodels tumor stroma by stimulating angiogenesis and recruiting TAM. MAPK activation may be a pivotal event in SIX1-associated tumor progression, which may provide opportunities for pharmacologic intervention.
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Abstract 2025: SIX1 overexpression extends the lifespan of normal human keratinocytes and promotes epithelial-mesenchymal transition
Molecular and Cellular Biology Genetics, 2016Co-Authors: Maria Hosseinipour, Kim E. Creek, Diego Altomare, Aspasia Amiridis, Lucia PirisiAbstract:SIX1, a homeodomain-containing transcription factor, has a critical role in the expansion of progenitor cells during embryogenesis. The overexpression of SIX1 contributes to tumorigenesis, promoting malignant transformation and metastasis. SIX1 promotes tumor progression by induction of epithelial-to-mesenchymal transition (EMT). In HPV16-transformed human keratinocytes (HKc), SIX1 overexpression produces EMT and a differentiation-resistant phenotype at early stages of in-vitro progression, and transforms cells to malignancy at late stages. We transfected normal HKc with a plasmid encoding human-SIX1 in order to determine to what extent SIX1 overexpression may induce growth, differentiation and EMT changes in normal cells. We also studied the effects of expressing the HRas-V12 oncogene while downregulating the p53 tumor suppressor gene in normal HKc expressing SIX1, in order to push these keratinocytes to a precancerous state. Normal HKc overexpressing SIX1 (HKc/SIX1) or a combination of SIX1, HRas-V12 and p53i (HKc/ALL3) grow continuously while their vector-transfected controls senesced. Continuous growth past the end of the normal lifespan was also observed in normal human fibroblasts overexpressing SIX1. HKc/SIX1 and HKc/ALL3 exhibit a spindle-shape and fibroblastic appearance rather than the cuboidal morphology of epithelial cells. They are also morphologically distinct from SIX1-overexpressing human fibroblasts. HKc /SIX1 and HKc/ALL3 grow best in KSFM supplemented with 10% FBS and 1mM CaCl2, while normal HKc grow best in KSFM with 0.1 mM CaCl2. RT-PCR confirmed that mRNA levels of SIX1 were significantly increased and p53 levels were significantly decreased, and that HRasV12 was expressed in HKc/ALL3. The growth rates of HKc/SIX1 and HKc/ALL3 are comparable to those of early-stage HPV16-immoratlized cells (HKc/HPV16). Invasion assays demonstrated that HKc/SIX1 are more invasive than normal HKc, and their invasiveness is comparable to that of HKc/HPV16. The invasiveness of HKc/ALL3 is greater than that of late-stage, differentiation-resistant HPV-immortalized cells (HKc/DR), which are about 2.5-fold more invasive than HKc/HPV16. In conclusion, SIX1 promotes continued proliferation and invasiveness, signs indicative of cellular transformation, even in the background of normal HKc. Currently, we are exploring the gene expression changes induced by SIX1 in normal cells. Additionally, we plan to investigate whether these cells express stem cell markers. The results of these studies suggest that SIX1 facilitates the establishment of primary human keratinocytes into immortal cell lines, or perhaps into stem-like cells with extended proliferation potential. Citation Format: Maria Hosseinipour, Aspasia Amiridis, Diego Altomare, Kim E. Creek, Lucia Pirisi. SIX1 overexpression extends the lifespan of normal human keratinocytes and promotes epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2025.
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SIX1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT.
Virology, 2015Co-Authors: Lucia Pirisi, Kim E. CreekAbstract:Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of SIX1 at early stages of HPV16-mediated transformation by overexpressing SIX1 in HKc/HPV16. We found that SIX1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of SIX1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing SIX1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that SIX1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • SIX1 expression increases during HPV16-mediated transformation. • SIX1 overexpression causes differentiation resistance in HPV16-immortalized cells. • SIX1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • SIX1 overexpression reduces Smad-dependent TGF-β signaling.
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SIX1 promotes epithelial mesenchymal transition and malignant conversion in human papillomavirus type 16 immortalized human keratinocytes
Carcinogenesis, 2014Co-Authors: Yu Zhang, Lucia Pirisi, Maria Marjorette O. Peña, Fang Wan, Diego Altomare, Kim E. CreekAbstract:SIX1, a member of the Six family of homeodomain transcription factors, is overexpressed in various human cancers, and SIX1 overexpression is associated with tumor progression and metastasis. SIX1 messenger RNA levels increase during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we show that HKc/DR-overexpressing SIX1 exhibited a more mesenchymal phenotype, as characterized by a fibroblastic appearance and increased invasion. We utilized Whole Human Genome Microarrays to explore the gene expression changes associated with SIX1 overexpression in HKc/DR. We found that overexpression of SIX1 downregulated epithelial-related genes and upregulated mesenchymal-related genes, which suggests that SIX1 overexpression induces epithelial–mesenchymal transition (EMT). Pathway analysis of the microarray data showed alterations in the transforming growth factor-beta (TGF-β) pathway, including enhanced expression of the TGF-β receptor type II (TβRII), and activation of the mitogen-activated protein kinase (MAPK) pathway in HKc/DR-overexpressing SIX1, suggesting that Smad-independent pathways of TGF-β signaling may be involved in SIX1-mediated EMT. p38 MAPK activation was required for sustained SIX1-induced EMT and TβRII overexpression. Finally, we determined that SIX1 overexpression in HKc/DR resulted in malignant conversion and increased the cancer stem cell (CSC)-like population. Thus, SIX1 overexpression promotes EMT, CSCs properties and malignant conversion in HKc/DR through MAPK activation, which supports the possible use of p38-TβRII inhibitors for the treatment of cancers overexpressing SIX1.
