RESUMO
Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, we identified transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell-cell adhesion, including the cell polarity genes Crumbs3, HUGL2 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promotor activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour-host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. Our data show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.
Assuntos
Neoplasias da Mama/patologia , Diferenciação Celular , Polaridade Celular , Neoplasias do Colo/patologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Adulto , Idoso , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Imunoprecipitação da Cromatina , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Progressão da Doença , Regulação para Baixo , Epitélio/metabolismo , Epitélio/patologia , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Immunoblotting , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Pessoa de Meia-Idade , Invasividade Neoplásica/patologia , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
The insulin-like growth factor (IGF)2/IGF1 receptor (IGF1R) signaling axis has an important role in intestinal carcinogenesis and overexpression of IGF2 is an accepted risk factor for colorectal cancer (CRC) development. Genetic amplifications and loss of imprinting contribute to the upregulation of IGF2, but insufficiently explain the extent of IGF2 expression in a subset of patients. Here, we show that IGF2 was specifically induced in the tumor stroma of CRC and identified cancer-associated fibroblasts (CAFs) as the major source. Further, we provide functional evidence that stromal IGF2, via the paracrine IGF1R/insulin receptor axis, activated pro-survival AKT signaling in CRC cell lines. In addition to its effects on malignant cells, autocrine IGF2/IGF1R signaling in CAFs induced myofibroblast differentiation in terms of alpha-smooth muscle actin expression and contractility in floating collagen gels. This was further augmented in concert with transforming growth factor-ß (TGFß) signaling suggesting a cooperative mechanism. However, we demonstrated that IGF2 neither induced TGFß/smooth muscle actin/mothers against decapentaplegic (SMAD) signaling nor synergized with TGFß to hyperactivate this pathway in two dimensional and three dimensional cultures. IGF2-mediated physical matrix remodeling by CAFs, but not changes in extracellular matrix-modifying proteases or other secreted factors acting in a paracrine manner on/in cancer cells, facilitated subsequent tumor cell invasion in organotypic co-cultures. Consistently, colon cancer cells co-inoculated with CAFs expressing endogenous IGF2 in mouse xenograft models exhibited elevated invasiveness and dissemination capacity, as well as increased local tumor regrowth after primary tumor resection compared with conditions with IGF2-deficient CAFs. In line, expression of IGF2 correlated with elevated relapse rates and poor survival in CRC patients. In agreement with our results, high-level coexpression of IGF2 and TGFß was predicting adverse outcome with higher accuracy than increased expression of the individual genes alone. Taken together, we demonstrate that stroma-induced IGF2 promotes colon cancer progression in a paracrine and autocrine manner and propose IGF2 as potential target for tumor stroma cotargeting strategies.
Assuntos
Neoplasias Colorretais/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Animais , Comunicação Autócrina , Células CACO-2 , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Progressão da Doença , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Células HCT116 , Xenoenxertos , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Camundongos Endogâmicos NOD , Comunicação Parácrina , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , TransfecçãoRESUMO
Scp160p (Saccharomyces cerevisiae protein involved in the control of ploidy), a polypeptide with a molecular mass of around 160 kDa, is associated with the nuclear envelope and the endoplasmic reticulum. The most noteworthy phenotype of SCP160 deletion mutants is a decrease in viability and an increased number of chromosomes in the surviving cells [Wintersberger, U., Kühne, C. & Karwan, A. (1995) Yeast 11, 929-944]. Scp160p contains 14 KH domains, conserved motifs that have lately been identified in a variety of RNA-binding proteins. In this report, we demonstrate that the Scp160p sequence shows nearly perfect colinearity with the putative gene product of C08H9.2 from the nematode Caenorhabditis elegans as well as with the vigilins, vertebrate RNA-binding proteins with a cellular location similar to that of Scp160p. Moreover, we found that Scp160p contains a potential nuclear-export signal (NES) near its N-terminus and a potential nuclear-localization signal (NLS) between KH domains 3 and 4. To determine whether the protein is able to bind to RNA, we purified Scp160p from yeast cell extract by DNA-cellulose and anti-Scp160p affinity chromatography. In northwestern blotting experiments, the electrophoretically homogeneous protein bound to ribohomopolymers and ribosomal RNA as well as to single-stranded and double-stranded DNA. Subcellular fractionation studies revealed that the major part of Scp160p is membrane associated via ionic interactions and can be released from the membrane fraction under conditions that lead to a dissociation of ribosomes. Together, our findings suggest that Scp160p is the yeast homologue of the vigilins, and point to a role for Scp160p in nuclear RNA export or in RNA transport within the cytoplasm.