RESUMO
The histone deacetylase inhibitor (HDACi) sodium butyrate promotes differentiation of colon cancer cells as evidenced by induced expression and enzyme activity of the differentiation marker intestinal alkaline phosphatase (ALPi). Screening of a panel of 33 colon cancer cell lines identified cell lines sensitive (42%) and resistant (58%) to butyrate induction of ALP activity. This differential sensitivity was similarly evident following treatment with the structurally distinct HDACi, MS-275. Resistant cell lines were significantly enriched for those harboring the CpG island methylator phenotype (p = 0.036, Chi square test), and resistant cell lines harbored methylation of the ALPi promoter, particularly of a CpG site within a critical KLF/Sp regulatory element required for butyrate induction of ALPi promoter activity. However, butyrate induction of an exogenous ALPi promoter-reporter paralleled up-regulation of endogenous ALPi expression across the cell lines, suggesting the presence or absence of a key transcriptional regulator is the major determinant of ALPi induction. Through microarray profiling of sensitive and resistant cell lines, we identified KLF5 to be both basally more highly expressed as well as preferentially induced by butyrate in sensitive cell lines. KLF5 overexpression induced ALPi promoter-reporter activity in resistant cell lines, KLF5 knockdown attenuated butyrate induction of ALPi expression in sensitive lines, and butyrate selectively enhanced KLF5 binding to the ALPi promoter in sensitive cells. These findings demonstrate that butyrate induction of the cell differentiation marker ALPi is mediated through KLF5 and identifies subsets of colon cancer cell lines responsive and refractory to this effect.
Assuntos
Fosfatase Alcalina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Fosfatase Alcalina/genética , Benzamidas/farmacologia , Sítios de Ligação/genética , Western Blotting , Ácido Butírico/farmacologia , Diferenciação Celular/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ilhas de CpG/genética , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Fatores de Transcrição Kruppel-Like/genética , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Colorectal cancers (CRCs) are classified as having microsatellite instability (MSI) or chromosomal instability (CIN); herein termed microsatellite stable (MSS). MSI colon cancers frequently display a poorly differentiated histology for which the molecular basis is not well understood. Gene expression and immunohistochemical profiling of MSS and MSI CRC cell lines and tumors revealed significant down-regulation of the intestinal-specific cytoskeletal protein villin in MSI colon cancer, with complete absence in 62% and 17% of MSI cell lines and tumors, respectively. Investigation of 577 CRCs linked loss of villin expression to poorly differentiated histology in MSI and MSS tumors. Furthermore, mislocalization of villin from the membrane was prognostic for poorer outcome in MSS patients. Loss of villin expression was not due to coding sequence mutations, epigenetic inactivation, or promoter mutation. Conversely, in transient transfection assays villin promoter activity reflected endogenous villin expression, suggesting transcriptional control. A screen of gut-specific transcription factors revealed a significant correlation between expression of villin and the homeobox transcription factor Cdx-1. Cdx-1 overexpression induced villin promoter activity, Cdx-1 knockdown down-regulated endogenous villin expression, and deletion of a key Cdx-binding site within the villin promoter attenuated promoter activity. Loss of Cdx-1 expression in CRC lines was associated with Cdx-1 promoter methylation. These findings demonstrate that loss of villin expression due to Cdx-1 loss is a feature of poorly differentiated CRCs.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Animais , Biomarcadores Tumorais/genética , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA/genética , DNA de Neoplasias/genética , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Estimativa de Kaplan-Meier , Camundongos , Camundongos SCID , Proteínas dos Microfilamentos/genética , Instabilidade de Microssatélites , Repetições de Microssatélites , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Prognóstico , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Células Tumorais CultivadasRESUMO
The phase III MAX clinical trial randomised patients with metastatic colorectal cancer (mCRC) to receive first-line capecitabine chemotherapy alone or in combination with the anti-VEGF-A antibody bevacizumab (± mitomycin C). We utilised this cohort to examine whether single nucleotide polymorphisms (SNPs) in VEGF-A, VEGFR1, and VEGFR2 are predictive of efficacy outcomes with bevacizumab or the development of hypertension. Genomic DNA extracted from archival FFPE tissue for 325 patients (69% of the MAX trial population) was used to genotype 16 candidate SNPs in VEGF-A, VEGFR1, and VEGFR2, which were analysed for associations with efficacy outcomes and hypertension. The VEGF-A rs25648 'CC' genotype was prognostic for improved PFS (HR 0.65, 95% CI 0.49 to 0.85; P = 0.002) and OS (HR 0.70, 95% CI 0.52 to 0.94; P = 0.019). The VEGF-A rs699947 'AA' genotype was prognostic for shorter PFS (HR 1.32, 95% CI 1.002 to 1.74; P = 0.048). None of the analysed SNPs were predictive of bevacizumab efficacy outcomes. VEGFR2 rs11133360 'TT' was associated with a lower risk of grade ≥ 3 hypertension (P = 0.028). SNPs in VEGF-A, VEGFR1 and VEGFR2 did not predict bevacizumab benefit. However, VEGF-A rs25648 and rs699947 were identified as novel prognostic biomarkers and VEGFR2 rs11133360 was associated with less grade ≥ 3 hypertension.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Austrália/epidemiologia , Carcinoma/genética , Carcinoma/mortalidade , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Feminino , Humanos , Hipertensão/genética , Masculino , Pessoa de Meia-Idade , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/genética , Nucléolo Celular/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismoRESUMO
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a negative regulator of phosphatidylinositol 3-kinase (PI3K) signaling that is frequently inactivated in colorectal cancer through mutation, loss of heterozygosity, or epigenetic mechanisms. The aim of this study was to determine the effect of intestinal-specific PTEN inactivation on intestinal epithelial homeostasis and tumorigenesis. PTEN was deleted specifically in the intestinal epithelium, by crossing PTEN(Lox/Lox) mice with villin(Cre) mice. PTEN was robustly expressed in the intestinal epithelium and maximally in the differentiated cell compartment. Targeted inactivation of PTEN in the intestinal epithelium of PTEN(Lox/Lox)/villin(Cre) mice was confirmed by genotyping, immunohistochemistry, and qPCR. While intestinal-specific PTEN deletion did not have a major effect on cell fate determination or proliferation in the small intestine, it did increase phosphorylated (p) protein kinase B (AKT) expression in the intestinal epithelium, and 19% of animals developed small intestinal adenomas and adenocarcinomas at 12 mo of age. These tumors demonstrated pAKT and nuclear ß-catenin staining, indicating simultaneous activation of the PI3K/AKT and Wnt signaling pathways. These findings demonstrate that, while PTEN inactivation alone has a minimal effect on intestinal homeostasis, it can facilitate tumor promotion upon deregulation of ß-catenin/TCF signaling, further establishing PTEN as a bona fide tumor suppressor gene in intestinal cancer.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Células Epiteliais/metabolismo , Neoplasias Intestinais/metabolismo , Intestino Delgado/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/genética , Adenoma/patologia , Animais , Células Epiteliais/patologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
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 ß-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.
Assuntos
Enterócitos/metabolismo , Histona Desacetilases/genética , Metabolismo dos Lipídeos/genética , Obesidade/genética , Animais , Calorimetria , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Peroxidação de Lipídeos/genética , Lipidômica , Camundongos , Mitocôndrias/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/agonistas , Receptores Ativados por Proliferador de Peroxissomo/genética , Triglicerídeos/metabolismoRESUMO
The ERK signalling pathway regulates key cell fate decisions in the intestinal epithelium and is frequently dysregulated in colorectal cancers (CRCs). Variations in the dynamics of ERK activation can induce different biological outcomes and are regulated by multiple mechanisms, including activation of negative feedback loops involving transcriptional induction of dual-specificity phosphatases (DUSPs). We have found that the nuclear ERK-selective phosphatase DUSP5 is downregulated in colorectal tumours and cell lines, as previously observed in gastric and prostate cancer. The DUSP5 promoter is methylated in a subset of CRC cell lines and primary tumours, particularly those with a CpG island methylator phenotype (CIMP). However, this epigenetic change alone could not account for reduced DUSP5 expression in CRC cells. Functionally, DUSP5 depletion failed to alter ERK signalling or proliferation in CRC cell lines, and its transgenic overexpression in the mouse intestine had minimal impact on normal intestinal homeostasis or tumour development. Our results suggest that DUSP5 plays a limited role in regulating ERK signalling associated with the growth of colorectal tumours, but that methylation the DUSP5 gene promoter can serve as an additional means of identifying CIMP-high colorectal cancers.
Assuntos
Carcinogênese/genética , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Metilação de DNA/genética , Fosfatases de Especificidade Dupla/genética , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Ilhas de CpG/genética , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Intestinos/patologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Transgênicos , Fenótipo , Regiões Promotoras Genéticas/genéticaRESUMO
Most patients with BRAF-mutant metastatic melanoma display remarkable but incomplete and short-lived responses to inhibitors of the BRAF kinase or the mitogen-activated protein kinase kinase (MEK), collectively BRAF/MEK inhibitors. We found that inherent resistance to these agents in BRAF(V600)-mutant melanoma cell lines was associated with high abundance of c-JUN and characteristics of a mesenchymal-like phenotype. Early drug adaptation in drug-sensitive cell lines grown in culture or as xenografts, and in patient samples during therapy, was consistently characterized by down-regulation of SPROUTY4 (a negative feedback regulator of receptor tyrosine kinases and the BRAF-MEK signaling pathway), increased expression of JUN and reduced expression of LEF1. This coincided with a switch in phenotype that resembled an epithelial-mesenchymal transition (EMT). In cultured cells, these BRAF inhibitor-induced changes were reversed upon removal of the drug. Knockdown of SPROUTY4 was sufficient to increase the abundance of c-JUN in the absence of drug treatment. Overexpressing c-JUN in drug-naïve melanoma cells induced similar EMT-like phenotypic changes to BRAF inhibitor treatment, whereas knocking down JUN abrogated the BRAF inhibitor-induced early adaptive changes associated with resistance and enhanced cell death. Combining the BRAF inhibitor with an inhibitor of c-JUN amino-terminal kinase (JNK) reduced c-JUN phosphorylation, decreased cell migration, and increased cell death in melanoma cells. Gene expression data from a panel of melanoma cell lines and a patient cohort showed that JUN expression correlated with a mesenchymal gene signature, implicating c-JUN as a key mediator of the mesenchymal-like phenotype associated with drug resistance.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/genética , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Western Blotting , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
The Forkhead box P3 (FOXP3) transcription factor is the key driver of regulatory T cell (Treg cells) differentiation and immunosuppressive function. In addition, FOXP3 has been reported to be expressed in many tumors, including melanoma. However, its role in tumorigenesis is conflicting, with both tumor suppressive and tumor promoting functions described. The aim of the current study was to characterize the expression and function of FOXP3 in melanoma. FOXP3 expression was detected by immunohistochemistry (IHC) in 12% (18/146) of stage III and IV melanomas. However expression was confined to fewer than 1% of cells in these tumors. Stable over-expression of FOXP3 in the SK-MEL-28 melanoma cell line reduced cell proliferation and clonogenicity in vitro, and reduced xenograft growth in vivo. FOXP3 over-expression also increased pigmentation and the rate of apoptosis of SK-MEL-28 cells. Based on its infrequent expression in human melanoma, and its growth inhibitory and pro-apoptotic effect in over-expressing melanoma cells, we conclude that FOXP3 is not likely to be a key tumor suppressor or promoter in melanoma.