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BACKGROUND & AIMS: We previously reported that colon epithelial cell silencing of Smad4 increased epithelial expression of inflammatory genes, including the chemokine c-c motif chemokine ligand 20 (CCL20), and increased susceptibility to colitis-associated cancer. Here, we examine the role of the chemokine/receptor pair CCL20/c-c motif chemokine receptor 6 (CCR6) in mediating colitis-associated colon carcinogenesis induced by SMAD4 loss. METHODS: In silico analysis of SMAD4, CCL20, and CCR6 messenger RNA expression was performed on published transcriptomic data from human ulcerative colitis (UC), and colon and rectal cancer samples. Immunohistochemistry for CCL20 and CCR6 was performed on human tissue microarrays comprising human UC-associated cancer specimens, Mice with conditional, epithelial-specific Smad4 loss with and without germline deletion of the Ccr6 gene were subjected to colitis and followed for up to 3 months. Tumors were quantified histologically, and immune cell populations were analyzed by flow cytometry and immunostaining. RESULTS: In human UC-associated cancers, loss of epithelial SMAD4 was associated with increased CCL20 expression and CCR6+ cells. SMAD4 loss in mouse colon epithelium led to enlarged gut-associated lymphoid tissues and recruitment of immune cells to the mouse colon epithelium and stroma, particularly T regulatory, Th17, and dendritic cells. Loss of CCR6 abrogated these immune responses and significantly reduced the incidence of colitis-associated tumors observed with loss of SMAD4 alone. CONCLUSIONS: Regulation of mucosal inflammation is central to SMAD4 tumor suppressor function in the colon. A key downstream node in this regulation is suppression of epithelial CCL20 signaling to CCR6 in immune cells. Loss of SMAD4 in the colon epithelium increases CCL20 expression and chemoattraction of CCR6+ immune cells, contributing to greater susceptibility to colon cancer.
Assuntos
Carcinoma , Neoplasias Associadas a Colite , Colite , Humanos , Camundongos , Animais , Receptores CCR6/genética , Quimiocina CCL20/metabolismo , Ligantes , Inflamação , Colite/complicações , RNA Mensageiro , Proteína Smad4/genética , Proteína Smad4/metabolismoRESUMO
Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGFß signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFß and intestinal barrier function. Here, we examine the role of TGFß signaling via SMAD4 in modulation of colon barrier function. The Smad4 gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), Western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Mice lacking intestinal expression of Smad4 demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in Cldn2/Claudin 2 and Cldn8/Claudin 8, and decreases in Cldn3, Cldn4, and Cldn7/Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFß1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. Cldn2, Cldn4, Cldn7, and Cldn8 are regulated by canonical TGFß signaling, and TGFß-dependent regulation of these genes is dependent on nascent RNA transcription (Cldn2, Cldn4, Cldn8) but not nascent protein translation (Cldn4, Cldn8). Human IBD/colon cancer specimens demonstrate decreased SMAD4, CLDN4, CLDN7, and CLDN8 and increased CLDN2 compared with healthy controls. Canonical TGFß signaling modulates the expression of tight junction proteins and barrier function in mouse colon.NEW & NOTEWORTHY We demonstrate that canonical TGFß family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.
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Colo/metabolismo , Células Epiteliais/metabolismo , Transdução de Sinais/fisiologia , Proteínas de Junções Íntimas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteínas de Junções Íntimas/genética , Junções Íntimas/metabolismoRESUMO
A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked inhibitor of apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its antiapoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it monoubiquitylates Groucho (Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing ß-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.
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Proteínas Correpressoras/metabolismo , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Ubiquitinação , Via de Sinalização Wnt , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , Animais , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose/fisiologia , Modelos Genéticos , Interferência de RNA , Proteínas Wnt/metabolismo , Proteína Wnt1/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Xenopus , Proteínas de Xenopus/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: The World Health Organization (WHO) 2010 has classified GI neuroendocrine neoplasms into neuroendocrine tumor (NET) and high-grade neuroendocrine carcinoma (NEC). The genetic underpinnings of NEC are poorly understood. The aim of the study was to perform genomic profiling of NEC to better characterize this aggressive disease. METHODS: We identified nine patients with colonic NEC between January 1, 2005 and June 30, 2013. Whole exome sequencing (WES) was performed on tumor DNA from two patients with ≥80% tumor cellularity and matched normal tissue available. Focused BRAF mutational analysis was performed on an additional seven patients via sanger sequencing of BRAF exons 11 and 15. RESULTS: We identified BRAF exon 15 mutations (c.A1781G: p.D594G and c.T1799A: p.V600E) by WES in two patients. Upon additional screening of seven colonic NECs for BRAF exon 11 and 15 mutations, we identified BRAF V600E mutations in two of seven specimens (29%). Overall, BRAF exon 15 mutations were present in four of nine colonic NECs. CONCLUSION: Colonic NEC is a rare but aggressive tumor with high frequency (44%) of BRAF mutations. Further investigation is warranted to ascertain the incidence of BRAF mutations in a larger population as BRAF inhibition may be a potential avenue of targeted treatment for these patients.
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Biomarcadores Tumorais/genética , Carcinogênese/patologia , Neoplasias do Colo/patologia , DNA de Neoplasias/genética , Mutação , Tumores Neuroendócrinos/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , Carcinogênese/genética , Estudos de Casos e Controles , Neoplasias do Colo/genética , Análise Mutacional de DNA , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/genética , PrognósticoRESUMO
Ewing sarcomas are rare mesenchymal-derived bone and soft tissue tumors in children. Afflicted children with distant metastases have poor survival despite aggressive therapeutics. Epithelial-to-mesenchymal transition in epithelial carcinomas is associated with loss of E-cadherin and resistance to apoptosis. ML327 is a novel small molecule that we have previously shown to reverse epithelial-to-mesenchymal transition features in both epithelial and neural crest-derived cancers. Herein, we sought to evaluate the effects of ML327 on mesenchymal-derived Ewing sarcoma cells, hypothesizing that ML327 initiates growth arrest and sensitizes to TNF-related apoptosis-inducing ligand. ML327 induced protein expression changes, increased E-cadherin and decreased vimentin, consistent with partial induction of mesenchymal-to-epithelial transition in multiple Ewing Sarcoma cell lines (SK-N-MC, TC71, and ES-5838). Induction of epithelial features was associated with apoptosis, as demonstrated by PARP and Caspase 3 cleavage by immunoblotting. Cell cycle analysis validated these findings by marked induction of the subG0 cell population. In vitro combination treatment with TRAIL demonstrated additive induction of apoptotic markers. Taken together, these findings establish a rationale for further in vivo trials of ML327 in cells of mesenchymal origin both alone and in combination with TRAIL.
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Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Isoxazóis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Niacinamida/análogos & derivados , Bibliotecas de Moléculas Pequenas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Antígenos CD , Antineoplásicos/química , Caderinas/genética , Caderinas/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Isoxazóis/química , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Niacinamida/química , Niacinamida/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/química , Vimentina/genética , Vimentina/metabolismoRESUMO
AIMS: Colorectal carcinoma (CRC) with micropapillary (MP) features has only been described recently and is still being characterized. METHODS AND RESULTS: We reviewed the clinicopathological and molecular features of 42 CRC with MP features. Twenty-nine cases were also evaluated for immunohistochemical evidence of epithelial-mesenchymal transition (EMT). The extent of MP features within our cohort ranged from 5% (13 cases) to 100% (one case). Twenty-seven cases featured prominent cribriforming with dirty necrosis in the non-MP component; nine displayed mucinous features. Twenty-four of 29 cases (83%) demonstrated evidence of EMT. Thirty-six cases (86%) showed advanced T-category (pT3 or pT4), 31 (74%) had lymph node metastases and 23 (55%) had distant metastases. Median overall follow-up was 36 months. Seventeen patients (40%) died of disease, with median survival of 23 months. Mutations were seen in 17 of 31 tested cases (55%), including 11 KRAS mutations and four BRAF V600E mutations. Microsatellite instability testing was performed on 21 cases; all were microsatellite-stable. Compared to a cohort of 972 conventional CRC, MP CRC was more likely to present as stage IV disease (P < 0.001), but patients with MP CRC showed no significant differences in overall survival after adjusting for stage. CONCLUSIONS: Micropapillary features in CRC portend a high likelihood of advanced local disease and distant metastases. MP CRC is often associated with a cribriform pattern elsewhere in the tumour and cystic nodal metastases with prominent necrosis. They also show frequent mutations in KRAS and BRAF. Immunohistochemical evidence of EMT is common in MP CRC.
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Adenocarcinoma Papilar/patologia , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal , Adenocarcinoma Papilar/genética , Adenocarcinoma Papilar/mortalidade , Adulto , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-IdadeRESUMO
E-cadherin is a ubiquitous trans-membrane protein that has important functions in cellular contacts and has been shown to play a role in the epithelial mesenchymal transition. We have previously reported the use of an HTS screen to identify compounds that are capable of restoring e-cadherin in cancer cells. Here, we report the additional medicinal chemistry optimization of these molecules, resulting in new molecules that restore e-cadherin expression at low micromolar concentrations. Further, we report preliminary pharmacokinetic data on a compound, ML327, that can be used as a probe of e-cadherin restoration.
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Caderinas/biossíntese , Isoxazóis/farmacologia , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Isoxazóis/síntese química , Isoxazóis/química , Camundongos , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.
Assuntos
Carcinoma Ductal Pancreático/imunologia , Macrófagos/fisiologia , Pâncreas/patologia , Neoplasias Pancreáticas/imunologia , Células Estreladas do Pâncreas/fisiologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Metaplasia , Camundongos , Neoplasias Pancreáticas/patologia , Receptor Cross-TalkRESUMO
Identifying patients with stage II and III colon cancer who will benefit from 5-fluorouracil (5-FU)-based adjuvant chemotherapy is crucial for the advancement of personalized cancer therapy. We employ a semi-supervised machine learning approach to analyze a large dataset with 933 stage II and III colon cancer samples. Our analysis leverages gene regulatory networks to discover an 18-gene prognostic signature and to explore a 10-gene signature that potentially predicts chemotherapy benefits. The 10-gene signature demonstrates strong prognostic power and shows promising potential to predict chemotherapy benefits. We establish a robust clinical assay on the NanoString nCounter platform, validated in a retrospective formalin-fixed paraffin-embedded (FFPE) cohort, which represents an important step toward clinical application. Our study lays the groundwork for improving adjuvant chemotherapy and potentially expanding into immunotherapy decision-making in colon cancer. Future prospective studies are needed to validate and establish the clinical utility of the 10-gene signature in clinical settings.
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BACKGROUND & AIMS: Mutational inactivation of adenomatous polyposis coli (APC) is an early event in colorectal cancer (CRC) progression that affects the stability and increases the activity of ß-catenin, a mediator of Wnt signaling. Progression of CRC also involves inactivation of signaling via transforming growth factor ß and bone morphogenetic protein (BMP), which are tumor suppressors. However, the interactions between these pathways are not clear. We investigated the effects of loss of the transcription factor Smad4 on levels of ß-catenin messenger RNA (mRNA) and Wnt signaling. METHODS: We used microarray analysis to associate levels of Smad4 and ß-catenin mRNA in colorectal tumor samples from 250 patients. We performed oligonucleotide-mediated knockdown of Smad4 in human embryonic kidney (HEK293T) and in HCT116 colon cancer cells and transgenically expressed Smad4 in SW480 colon cancer cells. We analyzed adenomas from (APC(Δ1638/+)) and (APC(Δ1638/+)) × (K19Cre(ERT2)Smad4(lox/lox)) mice by using laser capture microdissection. RESULTS: In human CRC samples, reduced levels of Smad4 correlated with increased levels of ß-catenin mRNA. In Smad4-depleted cell lines, levels of ß-catenin mRNA and Wnt signaling increased. Inhibition of BMP or depletion of Smad4 in HEK293T cells increased binding of RNA polymerase II to the ß-catenin gene. Expression of Smad4 in SW480 cells reduced Wnt signaling and levels of ß-catenin mRNA. In mice with heterozygous disruption of Apc(APC(Δ1638/+)), Smad4-deficient intestinal adenomas had increased levels of ß-catenin mRNA and expression of Wnt target genes compared with adenomas from APC(Δ1638/+) mice that expressed Smad4. CONCLUSIONS: Transcription of ß-catenin is inhibited by BMP signaling to Smad4. These findings provide important information about the interaction among transforming growth factor ß, BMP, and Wnt signaling pathways in progression of CRC.
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Adenocarcinoma/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Proteína Smad4/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/prevenção & controle , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Polipose Adenomatosa do Colo/prevenção & controle , Idoso , Animais , Sítios de Ligação , Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Regulação para Baixo , Feminino , Genes APC , Células HCT116 , Células HEK293 , Humanos , Microdissecção e Captura a Laser , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteína Smad4/deficiência , Proteína Smad4/genética , Via de Sinalização Wnt/genética , beta Catenina/genéticaRESUMO
AIMS: Despite continual efforts to develop prognostic and predictive models of colorectal cancer by using clinicopathological and genetic parameters, a clinical test that can discriminate between patients with good or poor outcome after treatment has not been established. Thus, the authors aim to uncover subtypes of colorectal cancer that have distinct biological characteristics associated with prognosis and identify potential biomarkers that best reflect the biological and clinical characteristics of subtypes. METHODS: Unsupervised hierarchical clustering analysis was applied to gene expression data from 177 patients with colorectal cancer to determine a prognostic gene expression signature. Validation of the signature was sought in two independent patient groups. The association between the signature and prognosis of patients was assessed by Kaplan-Meier plots, log-rank tests and the Cox model. RESULTS: The authors identified a gene signature that was associated with overall survival and disease-free survival in 177 patients and validated in two independent cohorts of 213 patients. In multivariate analysis, the signature was an independent risk factor (HR 3.08; 95% CI 1.33 to 7.14; p=0.008 for overall survival). Subset analysis of patients with AJCC (American Joint Committee on Cancer) stage III cancer revealed that the signature can also identify the patients who have better outcome with adjuvant chemotherapy (CTX). Adjuvant chemotherapy significantly affected disease-free survival in patients in subtype B (3-year rate, 71.2% (CTX) vs 41.9% (no CTX); p=0.004). However, such benefit of adjuvant chemotherapy was not significant for patients in subtype A. CONCLUSION: The gene signature is an independent predictor of response to chemotherapy and clinical outcome in patients with colorectal cancer.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Quimioterapia Adjuvante , Análise por Conglomerados , Estudos de Coortes , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Feminino , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND & AIMS: Expression of the tight junction protein claudin-1 is dysregulated in colon tumors and associates with their progression. Up-regulation of claudin-1 reduces expression of E-cadherin. We investigated the mechanisms by which claudin-1 regulates E-cadherin expression and its effects in colon cancer cells. MATERIALS AND METHODS: We used gene expression analysis, immunoblotting, and reverse transcription polymerase chain reaction to associate expression of the repressor of transcription Zinc Finger E-box binding homeobox-box1 (ZEB-1) with claudin-1. We analyzed SW480 colon cancer cells that overexpressed claudin-1, or SW620 cells in which claudin-1 expression was repressed, to determine the effects on ZEB-1 and E-cadherin expression, invasive activity, and resistance to anoikis. We studied cells that expressed constitutively active or dominant negative forms of factors in the Wnt or phosphotidylinositol-3-kinase signaling pathways and used pharmacologic inhibitors of these pathways to study their role in claudin-1-dependent regulation of ZEB-1. We used microarray analysis to examine gene expression patterns in 260 colorectal tumor and normal colon samples. RESULTS: Claudin-1 down-regulates E-cadherin expression by up-regulating expression of ZEB-1. Claudin-1 activates Wnt and phosphotidylinositol-3-kinase/Akt signaling. ZEB-1 mediates claudin-1-regulated changes in cell invasion and anoikis. Expression of claudin-1 correlated with that of ZEB-1 in human colon tumor samples. In the progression from normal colonic epithelium to colon adenocarcinoma, levels of E-cadherin decreased, whereas levels of claudin-1 and ZEB-1 increased. Down-regulation of E-cadherin and up-regulation of ZEB-1 in colon tumors were associated with shorter survival times. CONCLUSIONS: Claudin-1 up-regulates the repressor ZEB-1 to reduce expression of E-cadherin in colon cancer cells, increasing their invasive activity and reducing anoikis. This pathway is associated with colorectal cancer progression and patient survival.
Assuntos
Caderinas/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Membrana/fisiologia , Fatores de Transcrição/metabolismo , Anoikis , Caderinas/genética , Linhagem Celular Tumoral , Claudina-1 , Neoplasias do Colo/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Análise em Microsséries , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética , Regulação para Cima , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: Colorectal cancer is the second-leading cause of cancer-related mortality in the United States and a leading cause of cancer-related mortality worldwide. Loss of SMAD4, a critical tumor suppressor and the central node of the transforming growth factor-beta superfamily, is associated with worse outcomes for colorectal cancer patients; however, it is unknown whether an RNA-based profile associated with SMAD4 expression could be used to better identify high-risk colorectal cancer patients. AIM: Identify a gene expression-based SMAD4-modulated profile and test its association with patient outcome. METHODS AND RESULTS: Using a discovery dataset of 250 colorectal cancer patients, we analyzed expression of BMP/Wnt target genes for association with SMAD4 expression. Promoters of the BMP/Wnt genes were interrogated for SMAD-binding elements. Fifteen genes were implicated and three tested for modulation by SMAD4 in patient-derived colorectal cancer tumoroids. Expression of the 15 genes was used for unsupervised hierarchical clustering of a training dataset and two resulting clusters modeled in a centroid model. This model was applied to an independent validation dataset of stage II and III patients. Disease-free survival was analyzed by the Kaplan-Meier method. In vitro analysis of three genes identified in the SMAD4-modulated profile (JAG1, TCF7, and MYC) revealed modulation by SMAD4 consistent with the trend observed in the profile. In the training dataset (n = 553), the profile was not associated with outcome. However, among stage II and III patients (n = 461), distinct clusters were identified by unsupervised hierarchical clustering that were associated with disease-free survival (p = .02, log-rank test). The main model was applied to a validation dataset of stage II/III CRC patients (n = 257) which confirmed the association of clustering with disease-free survival (p = .013, log-rank test). CONCLUSIONS: A SMAD4-modulated gene expression profile identified high-risk stage II and III colorectal cancer patients, can predict disease-free survival, and has prognostic potential for stage II and III colorectal cancer patients.
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Neoplasias Colorretais/genética , Perfil Genético , Proteína Smad4/metabolismo , Idoso , Biomarcadores Tumorais/genética , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Medição de RiscoRESUMO
BACKGROUND & AIMS: Staging inadequately predicts metastatic risk in patients with colon cancer. We used a gene expression profile derived from invasive, murine colon cancer cells that were highly metastatic in an immunocompetent mouse model to identify patients with colon cancer at risk of recurrence. METHODS: This phase 1, exploratory biomarker study used 55 patients with colorectal cancer from Vanderbilt Medical Center (VMC) as the training dataset and 177 patients from the Moffitt Cancer Center as the independent dataset. The metastasis-associated gene expression profile developed from the mouse model was refined with comparative functional genomics in the VMC gene expression profiles to identify a 34-gene classifier associated with high risk of metastasis and death from colon cancer. A metastasis score derived from the biologically based classifier was tested in the Moffitt dataset. RESULTS: A high score was significantly associated with increased risk of metastasis and death from colon cancer across all pathologic stages and specifically in stage II and stage III patients. The metastasis score was shown to independently predict risk of cancer recurrence and death in univariate and multivariate models. For example, among stage III patients, a high score translated to increased relative risk of cancer recurrence (hazard ratio, 4.7; 95% confidence interval, 1.566-14.05). Furthermore, the metastasis score identified patients with stage III disease whose 5-year recurrence-free survival was >88% and for whom adjuvant chemotherapy did not increase survival time. CONCLUSION: A gene expression profile identified from an experimental model of colon cancer metastasis predicted cancer recurrence and death, independently of conventional measures, in patients with colon cancer.
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Adenocarcinoma/genética , Neoplasias Colorretais/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Testes Genéticos , Análise de Sequência com Séries de Oligonucleotídeos , Adenocarcinoma/mortalidade , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Idoso , Animais , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Intervalo Livre de Doença , Feminino , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Seleção de Pacientes , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Procedimentos DesnecessáriosRESUMO
Obtaining protein profiles from a homogeneous cell population in tissues can significantly improve our capability in protein biomarker discovery. In this study, unique protein profiles from the top and bottom sections of mouse crypts and Apc(Min+/-) adenomas were obtained using laser capture microdissection (LCM) combined with MALDI MS. Statistically significant protein peaks with differential expression were selected, and a set of novel protein biomarkers were identified. Immunohistochemistry was performed to confirm the differentially expressed protein biomarkers found by LCM combined with MALDI MS. To validate the relevance of the findings in human colorectal cancer (CRC), S100A8 was further confirmed in human CRC using immunohistochemistry. In addition, S100A8 was found to have an increased expression at different human CRC stages (Duke's A-D) compared with controls at both protein (n = 168 cases) and RNA (n = 215 cases) levels. Overall LCM combined with MALDI MS is a promising method to identify intestinal protein biomarkers from minute amounts of tissue. The novel protein biomarkers identified from the top and bottom crypts will increase our knowledge of the specific protein changes taking place during cell migration from the crypt bottom to top. In addition, the identified cancer protein biomarkers will aid in the exploration of colorectal tumorigenesis mechanisms as well as in the advancement of molecularly based diagnosis of colorectal cancer.
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Biomarcadores Tumorais/análise , Lasers , Microdissecção/métodos , Proteínas de Neoplasias/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Adenoma/metabolismo , Adenoma/patologia , Animais , Calgranulina A/metabolismo , Estudos de Casos e Controles , Humanos , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Proteoma/análise , Proteômica , Reprodutibilidade dos TestesRESUMO
The cyclooxygenase-2 (COX-2) enzyme catalyzes the rate-limiting step of prostaglandin formation in inflammatory states, and COX-2 overexpression plays a key role in carcinogenesis. To understand the mechanisms regulating COX-2 expression, we examined its posttranscriptional regulation mediated through the AU-rich element (ARE) within the COX-2 mRNA 3'-untranslated region (3'UTR). RNA binding studies, performed to identify ARE-binding regulatory factors, demonstrated binding of the translational repressor protein TIA-1 to COX-2 mRNA. The significance of TIA-1-mediated regulation of COX-2 expression was observed in TIA-1 null fibroblasts that produced significantly more COX-2 protein than wild-type fibroblasts. However, TIA-1 deficiency did not alter COX-2 transcription or mRNA turnover. Colon cancer cells demonstrated to overexpress COX-2 through increased polysome association with COX-2 mRNA also showed defective TIA-1 binding both in vitro and in vivo. These findings implicate that TIA-1 functions as a translational silencer of COX-2 expression and support the hypothesis that dysregulated RNA-binding of TIA-1 promotes COX-2 expression in neoplasia.
Assuntos
Regulação Enzimológica da Expressão Gênica , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Biossíntese de Proteínas , Proteínas , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Ciclo-Oxigenase 2 , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Isoenzimas/genética , Proteínas de Membrana/genética , Camundongos , Proteínas de Ligação a Poli(A) , Prostaglandina-Endoperóxido Sintases/genética , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Elementos Silenciadores Transcricionais , Antígeno-1 Intracelular de Células T , Células Tumorais CultivadasRESUMO
Colon cancer progression is characterized by activating mutations in Ras and by the emergence of the tumor-promoting effects of transforming growth factor-beta (TGF-beta) signaling. Ras-inducible rat intestinal epithelial cells (RIE:iRas) undergo a well-described epithelial to mesenchymal transition and invasive phenotype in response to H-RasV12 expression and TGF-beta treatment, modeling tumor progression. We characterized global gene expression profiles accompanying Ras-induced and TGF-beta-induced epithelial to mesenchymal transition in RIE:iRas cells by microarray analysis and found that the regulation of gene expression by the combined activation of Ras and TGF-beta signaling was associated with enrichment of a class of mRNAs containing 3' AU-rich element (ARE) motifs known to regulate mRNA stability. Regulation of ARE-containing mRNA transcripts was validated at the mRNA level, including genes important for tumor progression. Ras and TGF-beta synergistically increased the expression and mRNA stability of vascular endothelial growth factor (VEGF), a key regulator of tumor angiogenesis, in both RIE:iRas cells and an independent cell culture model (young adult mouse colonocyte). Expression profiling of human colorectal cancers (CRC) further revealed that many of these genes, including VEGF and PAI-1, were differentially expressed in stage IV human colon adenocarcinomas compared with adenomas. Furthermore, genes differentially expressed in CRC are also significantly enriched with ARE-containing transcripts. These studies show that oncogenic Ras and TGF-beta synergistically regulate genes containing AREs in cultured rodent intestinal epithelial cells and suggest that posttranscriptional regulation of gene expression is an important mechanism involved in cellular transformation and CRC tumor progression.
Assuntos
Epitélio/patologia , Mesoderma/patologia , Proteína Oncogênica p21(ras)/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2/metabolismo , Progressão da Doença , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/genética , Ratos , Reprodutibilidade dos Testes , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have recently reported that the expression of a tight junction protein, claudin-1, is increased during colon carcinogenesis and particularly metastatic colorectal cancer. Manipulation of claudin-1 levels in colon cancer cells showed a positive correlation between claudin-1 expression and tumor growth and metastasis. However, the mechanisms underlying the increased claudin-1 expression in colorectal cancer remains unknown. The tumor suppressor Smad4 is a central intracellular signal transduction component of the transforming growth factor-beta (TGF-beta) family of cytokines. Loss of Smad4 protein expression is correlated with poor prognosis and is frequently observed in invasive and metastatic colorectal carcinoma. In the present study, we report an inverse relationship between Smad4 and claudin-1 expression in human colorectal carcinoma tumor samples and in human colon cancer cell lines. We found that the expression of Smad4 in Smad4-deficient but claudin-1-positive SW480 or HT29 colon cancer cell lines down-regulates claudin-1 expression through transcriptional repression by modulating beta-catenin/T-cell factor/lymphocyte enhancer factor activity. Furthermore, this Smad4-dependent inhibition of claudin-1 expression is independent of TGF-beta signaling because Smad4 expression alone is insufficient to restore TGF-beta signaling in the SW480 cells, and the selective TGF-beta receptor kinase inhibitor LY364947 did not prevent the Smad4 suppression of claudin-1 protein expression in either SW480 or HT29 cells. Taken together, these findings suggest a novel mechanism underlying Smad4 tumor-suppressive function through regulation of a potential metastatic modulator, claudin-1, in a TGF-beta-independent manner.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Membrana/biossíntese , Proteína Smad4/biossíntese , Animais , Linhagem Celular Tumoral , Claudina-1 , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HCT116 , Células HT29 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Invasividade Neoplásica , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Transdução de Sinais , Proteína Smad4/deficiência , Fatores de Transcrição TCF/metabolismo , Transcrição Gênica , Transfecção , Fator de Crescimento Transformador beta/metabolismo , beta Catenina/metabolismoRESUMO
TGF-ß superfamily signaling is responsible for many critical cellular functions including control of cell growth, cell proliferation, cell differentiation, and apoptosis. TGF-ß appears to be critical in gastrulation, embryonic development, and morphogenesis, and it retains pleiotropic roles in many adult tissues and cell types in a highly context-dependent manner. While TGF-ß signaling within leukocytes is known to have an immunosuppressive role, its immunomodulatory effects within epithelial cells and epithelial cancers is less well understood. Recent data has emerged that suggests TGF-ß pathway signaling within epithelial cells may directly modulate pro-inflammatory chemokine/cytokine production and resultant leukocyte recruitment. This immunomodulation by epithelial TGF-ß pathway signaling may directly impact tumorigenesis and tumor progression through modulation of the epithelial microenvironment, although causal pathways responsible for such an observation remain incompletely investigated. This review presents the published literature as it relates to the immunomodulatory effects of TGF-ß family signaling within intestinal epithelial cells and carcinomas.
RESUMO
BACKGROUND & AIMS: Activating mutation of the KRAS gene is common in some cancers, such as pancreatic cancer, but rare in other cancers. Chronic pancreatitis is a predisposing condition for pancreatic ductal adenocarcinoma (PDAC), but how it synergizes with KRAS mutation is not known. METHODS: We used a mouse model to express an activating mutation of Kras in conjunction with obstruction of the main pancreatic duct to recapitulate a common etiology of human chronic pancreatitis. Because the cell of origin of PDAC is not clear, Kras mutation was introduced into either duct cells or acinar cells. RESULTS: Although KrasG12D expression in both cell types was protective against damage-associated cell death, chronic pancreatitis induced p53, p21, and growth arrest only in acinar-derived cells. Mutant duct cells did not elevate p53 or p21 expression and exhibited increased proliferation driving the appearance of PDAC over time. CONCLUSIONS: One mechanism by which tissues may be susceptible or resistant to KRASG12D-initiated tumorigenesis is whether they undergo a p53-mediated damage response. In summary, we have uncovered a mechanism by which inflammation and intrinsic cellular programming synergize for the development of PDAC.