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Epigenetics; Tumour-suppressor proteins Epigenética; Proteínas supresoras de tumores Epigenètica; Proteïnes supresores de tumors Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC. This project was supported by NHMRC project grant (1107831), a Cancer Council Victoria Grant (1164674) and the Operational Infrastructure Support Programme, Victorian Government, Australia. JMM was supported by a NHMRC Senior Research Fellowship (1046092). IYL was supported by F J Fletcher Research Scholarship and Randal and Louisa Alcock Scholarship from the University of Melbourne. LJJ was supported by La Trobe University Australian Postgraduate Awards. IN was supported by La Trobe University Postgraduate Research Scholarship. JWTT was supported by the University of Melbourne Australian Postgraduate Awards. OMS is a National Health and Medical Research Council (NHMRC) Senior Research Fellow (APP1136119). Open Access funding enabled and organized by CAUL and its Member Institutions.

SummaryFascin1 expression in colorectal carcinomas (CRCs) is linked to a clinically aggressive disease with poor prognosis. Despite that fascin1’s role in the etiology of CRCs has not been directly investigated. We show fascin1 expression in one-third of all CRCs underscoring the critical need to identify fascin1’s function in colorectal carcinogenesis. Here, we identify for the first time, fascin1’s role as a mechanosensor that modulates CRC cell adherens junction (AJ) plasticity to induce tumor growth and metastasis. We show that fascin1 expression drives protein sorting to transform AJ mechanotransduction and we reveal how these force-sensitive pathways activate oncogenic signaling in CRC cells. We made the novel finding that AJ remodeling by fascin1 also controls “collective plasticity” and bidirectional cell migration. Few studies have examined AJ plasticity in cancer cells, which remains poorly understood and has not been therapeutically targeted. Our findings could have widespread implications for understanding and treating metastatic carcinomas.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf−/−) or specifically in the gut epithelium. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf−/− mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf−/−mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

BackgroundEts homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) transcription factors. EHF is specifically expressed in epithelial tissues, however its role in development and epithelial homeostasis is largely uncharacterized.MethodsWe generated a novel mouse strain in which the Ets DNA binding domain (exon 8) of Ehf was flanked by loxP sites (EhfLox/Lox). To inactivate Ehf in the whole body, EhfLox/Lox mice were crossed to CMVCre mice, which were then bred out to generate germline Ehf null (Ehf−/−) mice. To inactivate Ehf specifically in the intestinal epithelium, EhfLox/Lox mice were bred to tamoxifen-inducible VillinCre-ERT2 mice. EhfLox/Lox mice were also crossed to tamoxifen-inducible Cdx2CreERT2; ApcLox/+ mice to determine the impact of Ehf deletion on Apc-initiated colon cancer development.ResultsTranscripts encoding the Ets binding domain of EHF were effectively deleted in all tissues in Ehf−/− mice. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain and developed a series of pathologies during their lifespan that led the majority of Ehf−/− mice to reach an ethical endpoint within one year of age. Most prominent of these were the development of papillomas in the chin, and abscesses in the preputial glands (males) or vulvae (females) which showed evidence of Staphylococcus and Proteus infection. Consistent with the development of papillomas, the epidermis of Ehf−/− mice showed evidence of mild hyperplasia. A subset of Ehf−/− mice also developed cataracts and corneal ulcers. EHF is highly expressed in the colonic epithelium and Ehf−/− mice displayed increased susceptibility to dextran sodium sulphate-induced colitis. This phenotype was confirmed in intestinal-specific Ehf knockout mice, and histopathological analyses revealed reduced numbers of goblet cells and extensive transcriptional reprogramming in the colonic epithelium. Finally, colon-specific deletion of Ehf enhanced Apc-initiated adenoma development, unveiling a novel, tumour suppressive role for EHF in colorectal cancer.ConclusionThe Ets DNA-binding domain of EHF is essential for post-natal homeostasis of the epidermis and colonic epithelium, and functions as a tumour suppressor in the colon.

Histone deacetylase 3 (Hdac3) regulates the expression of lipid metabolism genes in multiple tissues, however its role in regulating lipid metabolism in the intestinal epithelium is unknown. Here we demonstrate that intestine-specific deletion of Hdac3 (Hdac3IKO) protects mice from diet induced obesity. Intestinal epithelial cells (IECs) from Hdac3IKO mice display co-ordinate induction of genes and proteins involved in mitochondrial and peroxisomal β-oxidation, have an increased rate of fatty acid oxidation, and undergo marked remodelling of their lipidome, particularly a reduction in long chain triglycerides. Many HDAC3-regulated fatty oxidation genes are transcriptional targets of the PPAR family of nuclear receptors, Hdac3 deletion enhances their induction by PPAR-agonists, and pharmacological HDAC3 inhibition induces their expression in enterocytes. These findings establish a central role for HDAC3 in co-ordinating PPAR-regulated lipid oxidation in the intestinal epithelium, and identify intestinal HDAC3 as a potential therapeutic target for preventing obesity and related diseases., Histone deacetylase 3 (HDAC3) is a regulator of lipid homeostasis in several tissues, however, its role in intestinal lipid metabolism was not yet known. Here the authors study intestine specific HDAC3 knock out mice and report that these animals have increased fatty acid oxidation and undergo remodeling of the intestinal epithelial cell lipidome.

Summary Biliary tract cancers (BTCs) currently have no approved targeted therapies. Although genomic profiling of primary BTCs has identified multiple potential drug targets, accurate models are needed for their evaluation. Genomic profiling of 22 BTC cell lines revealed they harbor similar mutational signatures, recurrently mutated genes, and genomic alterations to primary tumors. Transcriptomic profiling identified two major subtypes, enriched for epithelial and mesenchymal genes, which were also evident in patient-derived organoids and primary tumors. Interrogating these models revealed multiple mechanisms of MAPK signaling activation in BTC, including co-occurrence of low-activity BRAF and MEK mutations with receptor tyrosine kinase overexpression. Finally, BTC cell lines with altered ERBB2 or FGFRs were exquisitely sensitive to specific targeted agents, whereas surprisingly, IDH1-mutant lines did not respond to IDH1 inhibitors in vitro. These findings establish BTC cell lines as robust models of primary disease, reveal specific molecular disease subsets, and highlight specific molecular vulnerabilities in these cancers., Graphical Abstract, Highlights • BTC cell lines harbor similar genomic alterations to primary tumors • Transcriptomic profiling of BTC cell lines identified two molecular subtypes • MAPK signaling is activated in BTC via multiple mechanisms • BTC lines with deregulated ERBB2 or FGFRs respond to specific targeted therapies, Biological Sciences; Genetics; Genomics; Cancer

The epithelium-specific ETS (ESE) transcription factors (ELF3, ELF5, EHF and SPDEF) are defined by their highly conserved ETS DNA binding domain and predominant epithelial-specific expression profile. ESE transcription factors maintain normal cell homeostasis and differentiation of a number of epithelial tissues, and their genetic alteration and deregulated expression has been linked to the progression of several epithelial cancers. Herein we review the normal function of the ESE transcription factors, the mechanisms by which they are dysregulated in cancers, and the current evidence for their role in cancer progression. Finally, we discuss potential therapeutic strategies for targeting or reactivating these factors as a novel means of cancer treatment.

Objective: Poor tumor differentiation status is associated with worse patient outcome in colorectal cancer. However, the molecular basis for loss of differentiation in colon cancer is not well understood. We have found that the transcription factor Ets homologous factor (EHF) is highly expressed in the normal human and mouse colonic epithelium and is down-regulated in poorly-differentiated colorectal cancer cell lines. The aim of this study was to investigate the role of EHF in regulating normal colonic epithelial cell differentiation in vivo. Methods: A novel mouse model was generated in which the Ets DNA binding domain (exon 8) of EHF was flanked by loxP sites (EHFlox/lox). To inactive EHF in the intestinal epithelium, EHFlox/lox mice were crossed to Villin-CreERT2 mice and recombination induced by tamoxifen treatment. Tamoxifen and vehicle treated mice were monitored weekly for up to one year. At each endpoint, tissue was collected and the effect of EHF deletion on intestinal cell proliferation and differentiation assessed by qRT-PCR and immunohistochemistry. Results: Targeted inactivation of EHF in the small intestinal and colonic epithelium following tamoxifen treatment was confirmed at the DNA and RNA level. EHF inactivation in the intestinal epithelium had minimal effect on survival, weight gain and overall health of the animals. Furthermore, loss of EHF did not markedly affect cell proliferation or the overall architecture of the intestinal epithelium. Finally, EHF inactivation also had minimal effect on markers of absorptive, enteroendocrine or Paneth cell differentiation but did reduce the number of goblet cells in the colonic epithelium. Conclusion: EHF does not play a major role in regulating cell proliferation in the mouse intestine but may play a role in regulating goblet cell differentiation. The role of EHF in intestinal tumourigenesis and response to stress are currently being investigated. Citation Format: Camilla M. Reehorst, Ian Y. Luk, Rebecca Nightingale, Mercedes Davalos-Salas, Amardeep S. Dhillon, John M. Mariadason. Investigating the in vivo role of the EHF transcription factor in intestinal epithelial differentiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3529. doi:10.1158/1538-7445.AM2017-3529