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Mucosal healing is associated with better clinical outcomes in patients with inflammatory bowel diseases (IBDs). Unresolved injury and inflammation, on the other hand, increases pathological fibrosis and the predisposition to cancer. Loss of Smad4, a tumor suppressor, is known to increase colitis-associated cancer in mouse models of chronic IBD. Since common biological processes are involved in both injury repair and tumor growth, we sought to investigate the effect of Smad4 loss on the response to epithelial injury. To this end, Smad4 was knocked out specifically in the intestinal epithelium and transcriptomic and morphological changes compared between wild type mice and Smad4 knock out mice after DSS-induced injury. We find that Smad4 loss alleviates pathological fibrosis and enhances mucosal repair. The transcriptomic changes specific to epithelium indicate molecular changes that affect epithelial extracellular matrix (ECM) and promote enhanced mucosal repair. These findings suggest that the biological processes that promote wound healing alleviate the pathological fibrotic response to DSS. Therefore, these mucosal repair processes could be exploited to develop therapies that promote normal wound healing and prevent fibrosis. NEW AND NOTEWORTHY: We show that transcriptomic changes due to Smad4 loss in the colonic epithelium alleviates the pathological fibrotic response to DSS in an IBD mouse model of acute inflammation. Most notably, we find that collagen deposition in the epithelial ECM, as opposed to that in the lamina propria, correlates with epithelial changes that enhance wound healing. This is the first report on a mouse model providing alleviated fibrotic response in a DSS-IBD mouse model in vivo .
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Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfß pathway.
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The gut epithelium has a remarkable ability to recover from damage. We employed a combination of high-throughput sequencing approaches, mouse genetics, and murine and human organoids and identified a role for TGFB signaling during intestinal regeneration following injury. At 2 days following irradiation (IR)-induced damage of intestinal crypts, a surge in TGFB1 expression is mediated by monocyte/macrophage cells at the location of damage. The depletion of macrophages or genetic disruption of TGFB signaling significantly impaired the regenerative response. Intestinal regeneration is characterized by the induction of a fetal-like transcriptional signature during repair. In organoid culture, TGFB1 treatment was necessary and sufficient to induce the fetal-like/regenerative state. Mesenchymal cells were also responsive to TGFB1 and enhanced the regenerative response. Mechanistically, pro-regenerative factors, YAP/TEAD and SOX9, are activated in the epithelium exposed to TGFB1. Finally, pre-treatment with TGFB1 enhanced the ability of primary epithelial cultures to engraft into damaged murine colon, suggesting promise for cellular therapy.
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Mucosa Intestinal , Intestinos , Animais , Humanos , Camundongos , Colo , Mucosa Intestinal/metabolismo , Organoides/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Hypoxia-inducible factor 2α (HIF2α) directly regulates a battery of genes essential for intestinal iron absorption. Interestingly, iron deficiency and overload disorders do not result in increased intestinal expression of glycolytic or angiogenic HIF2α target genes. Similarly, inflammatory and tumor foci can induce a distinct subset of HIF2α target genes in vivo These observations indicate that different stimuli activate distinct subsets of HIF2α target genes via mechanisms that remain unclear. Here, we conducted a high-throughput siRNA-based screen to identify genes that regulate HIF2α's transcriptional activity on the promoter of the iron transporter gene divalent metal transporter-1 (DMT1). SMAD family member 3 (SMAD3) and SMAD4 were identified as potential transcriptional repressors. Further analysis revealed that SMAD4 signaling selectively represses iron-absorptive gene promoters but not the inflammatory or glycolytic HIF2α or HIF1α target genes. Moreover, the highly homologous SMAD2 did not alter HIF2α transcriptional activity. During iron deficiency, SMAD3 and SMAD4 expression was significantly decreased via proteasomal degradation, allowing for derepression of iron target genes. Several iron-regulatory genes contain a SMAD-binding element (SBE) in their proximal promoters; however, mutation of the putative SBE on the DMT1 promoter did not alter the repressive function of SMAD3 or SMAD4. Importantly, the transcription factor forkhead box protein A1 (FOXA1) was critical in SMAD4-induced DMT1 repression, and DNA binding of SMAD4 was essential for the repression of HIF2α activity, suggesting an indirect repressive mechanism through DNA binding. These results provide mechanistic clues to how HIF signaling can be regulated by different cellular cues.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas Reguladoras de Ferro/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Animais , Células Cultivadas , Humanos , Proteínas Reguladoras de Ferro/genética , Camundongos , Camundongos Knockout , Proteína Smad3/deficiência , Proteína Smad4/deficiênciaRESUMO
The cell of origin of colon cancer is typically thought to be the resident somatic stem cells, which are immortal and escape the continual cellular turnover characteristic of the intestinal epithelium. However, recent studies have identified certain conditions in which differentiated cells can acquire stem-like properties and give rise to tumors. Defining the origins of tumors will inform cancer prevention efforts as well as cancer therapies, as cancers with distinct origins often respond differently to treatments. We report here a new condition in which tumors arise from the differentiated intestinal epithelium. Inactivation of the differentiation-promoting transcription factor SMAD4 in the intestinal epithelium was surprisingly well tolerated in the short term. However, after several months, adenomas developed with characteristics of activated WNT signaling. Simultaneous loss of SMAD4 and activation of the WNT pathway led to dedifferentiation and rapid adenoma formation in differentiated tissue. Transcriptional profiling revealed acquisition of stem cell characteristics, and colabeling indicated that cells expressing differentiated enterocyte markers entered the cell cycle and reexpressed stem cell genes upon simultaneous loss of SMAD4 and activation of the WNT pathway. These results indicate that SMAD4 functions to maintain differentiated enterocytes in the presence of oncogenic WNT signaling, thus preventing dedifferentiation and tumor formation in the differentiated intestinal epithelium.Significance: This work identifies a mechanism through which differentiated cells prevent tumor formation by suppressing oncogenic plasticity. Cancer Res; 78(17); 4878-90. ©2018 AACR.
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Adenoma/genética , Diferenciação Celular/genética , Neoplasias do Colo/genética , Proteína Smad4/genética , Adenoma/patologia , Animais , Carcinogênese/genética , Desdiferenciação Celular/genética , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Enterócitos/metabolismo , Enterócitos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Camundongos , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Via de Sinalização Wnt/genéticaRESUMO
Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.
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Diferenciação Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Predisposição Genética para Doença , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Fator de Transcrição CDX2/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Epitélio/metabolismo , Epitélio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Intestinos/patologia , Masculino , Camundongos Mutantes , Proteína Smad4/metabolismo , Via de Sinalização WntRESUMO
UNLABELLED: Disruption of the gene encoding Protein Tyrosine Kinase 6 (Ptk6) delayed differentiation and increased growth in the mouse intestine. However, Ptk6-null mice were also resistant to azoxymethane-induced colon tumorigenesis. To further explore functions of PTK6 in colon cancer, expression of epithelial and mesenchymal markers, as well as proliferation, migration, and xenograft tumor growth, was examined in human colon tumor cell lines with knockdown or overexpression of PTK6. PTK6 protein, transcript, and activation were also examined in a human colon tumor tissue array, using immunohistochemistry and qRT-PCR. Knockdown of PTK6 led to the epithelial-mesenchymal transition (EMT) in SW480 and HCT116 cells, whereas overexpression of PTK6 in SW620 cells restored an epithelial phenotype in a kinase-independent manner. PTK6 knockdown also increased xenograft tumor growth of SW480 cells, suggesting tumor suppressor functions. In clinical specimens, PTK6 expression was highest in normal differentiated epithelial cells and reduced in tumors. In contrast, overexpression of constitutively active PTK6 promoted STAT3 and ERK5 activation in colon cancer cells, and endogenous PTK6 promoted cell survival and oncogenic signaling in response to DNA-damaging treatments. These data indicate that PTK6 has complex, context-specific functions in colon cancer; PTK6 promotes the epithelial phenotype to antagonize the EMT in a kinase-independent manner, whereas activation of PTK6 promotes oncogenic signaling. IMPLICATIONS: Understanding context-specific functions of PTK6 is important, because although it promotes cell survival and oncogenic signaling after DNA damage, expression of PTK6 in established tumors may maintain the epithelial phenotype, preventing tumor progression. Mol Cancer Res; 14(6); 563-73. ©2016 AACR.
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Neoplasias do Colo/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas Tirosina Quinases/genética , Transdução de SinaisRESUMO
BACKGROUND: Aberrant DNA methylation at the 5-carbon on cytosine residues (5mC) in CpG dinucleotides is probably the most extensively characterized epigenetic modification in colon cancer. It has been suggested that the loss of adenomatous polyposis coli (APC) function initiates tumorigenesis and that additional genetic and epigenetic events are involved in colon cancer progression. We aimed to study the genome-wide DNA methylation profiles of intestinal tumorigenesis in Apc(min/+) mice. RESULTS: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was used to determine the global profile of DNA methylation changes in Apc(min/+) mice. DNA was extracted from adenomatous polyps from Apc(min/+) mice and from normal intestinal tissue from age-matched Apc(+/+) littermates, and the MeDIP-seq assay was performed. Ingenuity Pathway Analysis (IPA) software was used to analyze the data for gene interactions. A total of 17,265 differentially methylated regions (DMRs) displayed a ≥ 2-fold change (log2) in methylation in Apc(min/+) mice; among these DMRs, 9,078 (52.6 %) and 8,187 (47.4 %) exhibited increased and decreased methylation, respectively. Genes with altered methylation patterns were mainly mapped to networks and biological functions associated with cancer and gastrointestinal diseases. Among these networks, several canonical pathways, such as the epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin pathways, were significantly associated with genome-wide methylation changes in polyps from Apc(min/+) mice. The identification of certain differentially methylated molecules in the EMT and Wnt/ß-catenin pathways, such as APC2 (adenomatosis polyposis coli 2), SFRP2 (secreted frizzled-related protein 2), and DKK3 (dickkopf-related protein 3), was consistent with previous publications. CONCLUSIONS: Our findings indicated that Apc(min/+) mice exhibited extensive aberrant DNA methylation that affected certain signaling pathways, such as the EMT and Wnt/ß-catenin pathways. The genome-wide DNA methylation profile of Apc(min/+) mice is informative for future studies investigating epigenetic gene regulation in colon tumorigenesis and the prevention of colon cancer.
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The intestinal stem cell fuels the highest rate of tissue turnover in the body and has been implicated in intestinal disease and cancer; understanding the regulatory mechanisms controlling intestinal stem cell physiology is of great importance. Here, we provide evidence that the transcription factor YY1 is essential for intestinal stem cell renewal. We observe that YY1 loss skews normal homeostatic cell turnover, with an increase in proliferating crypt cells and a decrease in their differentiated villous progeny. Increased crypt cell numbers come at the expense of Lgr5(+) stem cells. On YY1 deletion, Lgr5(+) cells accelerate their commitment to the differentiated population, exhibit increased levels of apoptosis, and fail to maintain stem cell renewal. Loss of Yy1 in the intestine is ultimately fatal. Mechanistically, YY1 seems to play a role in stem cell energy metabolism, with mitochondrial complex I genes bound directly by YY1 and their transcript levels decreasing on YY1 loss. These unappreciated YY1 functions broaden our understanding of metabolic regulation in intestinal stem cell homeostasis.
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Divisão Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Intestinos/citologia , Mitocôndrias/metabolismo , Células-Tronco/fisiologia , Fator de Transcrição YY1/metabolismo , Animais , Imunoprecipitação da Cromatina , Perfilação da Expressão Gênica , Camundongos , Camundongos Knockout , Análise em Microsséries , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Fator de Transcrição YY1/genéticaRESUMO
BACKGROUND & AIMS: Protein tyrosine kinase 6 (PTK6) is expressed in epithelial linings of the gastrointestinal tract. PTK6 sensitizes the nontransformed Rat1a fibroblast cell line to apoptotic stimuli. The aim of this study was to determine if PTK6 regulates apoptosis in vivo after DNA damage in the small intestine. METHODS: Wild-type and Ptk6(-/-) mice were subjected to gamma-irradiation; intestinal tissues were collected, protein was isolated, and samples were fixed for immunohistochemical analyses at 0, 6, and 72 hours after the mice were irradiated. Expression of PTK6 was examined in the small intestine before and after irradiation. Apoptosis and proliferation were compared between wild-type and Ptk6(-/-) mice. Expression and activation of prosurvival signaling proteins were assessed. RESULTS: Irradiation induced PTK6 in crypt epithelial cells of the small intestine in wild-type mice. Induction of PTK6 corresponded with DNA damage-induced apoptosis in the wild-type small intestine. Following irradiation, the apoptotic response was impaired in the intestinal crypts of Ptk6(-/-) mice. Increased activation of AKT and extracellular signal-regulated kinase (ERK)1/2 and increased inhibitory phosphorylation of the proapoptotic protein BAD were detected in Ptk6(-/-) mice after irradiation. In response to the induction of apoptosis, compensatory proliferation increased in the small intestines of wild-type mice but not in Ptk6(-/-) mice at 6 hours after irradiation. CONCLUSIONS: PTK6 is a stress-induced kinase that promotes apoptosis by inhibiting prosurvival signaling. After DNA damage, induction of PTK6 is required for efficient apoptosis and inhibition of AKT and ERK1/2.
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Apoptose/fisiologia , Dano ao DNA/fisiologia , Mucosa Intestinal/patologia , Quinases da Família src/fisiologia , Animais , Apoptose/efeitos da radiação , Caspase 3/metabolismo , Proliferação de Células , Sobrevivência Celular , Dano ao DNA/efeitos da radiação , Ativação Enzimática , Raios gama , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efeitos da radiação , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
The epithelial linings of the small and large intestine are rapidly turned over and provide an ideal system for exploring links between differentiation and regulation of cell cycle exit. We utilized wild type, p21-/-, p27-/- and p21/p27-/- mice to address contributions of the Cdk inhibitors p21 and p27 to proliferation and differentiation in the mouse gastrointestinal tract. We did not detect any significant differences in proliferation, and all differentiated epithelial cell lineages were represented in all four genotypes. These data indicate that p21 and p27 do not play essential roles in the regulation of normal epithelial renewal in the intestine. These Cdk inhibitors are not needed in vivo for either assembly of Cdk/Cyclin complexes that drive active proliferation, or inhibition of Cdk/Cyclin complexes during cell cycle exit. However, expression of Cyclin D2 and to a lesser degree Cyclin D3 was reduced in p27-/- and p21/p27-/- mice, indicating a unique role for p27 in the regulation of these specific D-type Cyclins in vivo. In the absence of p27, reduced levels of Cyclin D2 and D3 may help to counteract increased proproliferative signals in the intestine.