RESUMO
BACKGROUND & AIMS: Human colorectal carcinomas display an infiltrative front of invasion where tumor cells undergo an epithelomesenchymal transition associated with low survival. Epithelomesenchymal transition is regulated by a nuclear beta-catenin accumulation, and subsequently, activation of beta-catenin/TCF4 target genes similar to CYCLIN D(1). Unexpectedly, these tumor cells are characterized by low proliferation, which correlates with the expression of the cell cycle inhibitor p16(INK4A). Therefore, we investigated the molecular mechanism of the transcriptional regulation of p16(INK4A) in colorectal cancer and its correlation with survival. METHODS: Molecular biological techniques were used for investigating the transcriptional mechanisms of the p16(INK4A) gene regulation. Moreover, p16(INK4A) expression was correlated with the 10-year survival of patients with colorectal carcinomas. RESULTS: In colorectal carcinomas, expression of the p16(INK4A) gene is regulated by beta-catenin/TCF4 and correlates with low survival rates of patients with tumors displaying an infiltrative front of invasion. CONCLUSIONS: beta-catenin/TCF4 regulates cell cycle promoting (c-MYC, CYCLIN D(1)) and inhibiting genes (p16(INK4A)) at the same time in the mesenchymally differentiated tumor cells at the front of invasion. The function of p16(INK4A) seems to supersede in this context thus leading to low proliferation. Moreover, these tumor cells seem to govern the outcome of colorectal cancer independently of their proliferation.
Assuntos
Neoplasias Colorretais/mortalidade , Genes p16 , beta Catenina/fisiologia , Adulto , Idoso , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Regiões Promotoras Genéticas , Fator de Transcrição 4 , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Genetically, colorectal cancers (CRCs) can be subdivided into tumors with chromosomal instability (CIN) or microsatellite instability (MSI). In both types of CRCs genes that are involved in the degradation of beta-CATENIN are frequently mutated. Whereas in CIN CRCs APC (Adenomatous Polyposis Coli) is affected in most cases, high grade MSI (MSI-H) CRCs frequently display mutations in various genes, like the APC-, AXIN2- or CTNNBI (beta-CATENIN) gene itself. Recently in Wilms tumors, WTX (Wilms tumor gene on the X-chromosome) was discovered as another gene involved in the destruction of beta-CATENIN. As the WTX-gene harbors a short T6-microsatellite in its N-terminal coding region, we hypothesized that frameshift-mutations might occur in MSI-H CRCs in the WTX gene, thus additionally contributing to the stabilization of beta-CATENIN in human CRCs. METHODS: DNA was extracted from 632 formalin-fixed, paraffin-embedded metastatic CRCs (UICCIV) and analyzed for MSI-H by investigating the stability of the highly sensitive microsatellite markers BAT25 and BAT26 applying fluorescence capillary electrophoresis (FCE). Then, in the MSI-H cases, well described mutational hot spot regions from the APC-, AXIN2- and CTNNBI genes were analyzed for genomic alterations by didesoxy-sequencing while the WTX T6-microsatellite was analyzed by fragment analysis. Additionally, the PCR products of T5-repeats were subcloned and mutations were validated using didesoxy-sequencing. Furthermore, the KRAS and the BRAF proto-oncogenes were analyzed for the most common activating mutations applying pyro-sequencing. mRNA expression of WTX from MSI-H and MSS cases and a panel of colorectal cancer cell lines was investigated using reverse transcription (RT-) PCR and FCE. RESULTS: In our cohort of 632 metastatic CRCs (UICCIV) we identified 41 MSI-H cases (6.5%). Two of the 41 MSI-H cases (4.8%) displayed a frameshift mutation in the T6-repeat resulting in a T5 sequence. Only one case, a male patient, expressed the mutated WTX gene while being wild type for all other investigated genes. CONCLUSION: Mutations in the WTX-gene might compromise the function of the beta-CATENIN destruction complex in only a small fraction of MSI-H CRCs thus contributing to the process of carcinogenesis.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/secundário , Mutação da Fase de Leitura/genética , Regulação Neoplásica da Expressão Gênica , Instabilidade de Microssatélites , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , DNA de Neoplasias/genética , Feminino , Genes APC , Genótipo , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , beta Catenina/genéticaRESUMO
In colon cancer, CD133 has recently been used to enrich for a subset of tumour cells with tumour-initiating capabilities and was therefore suggested to mark colon cancer stem cells. However, this molecule has surprisingly been shown to lack functional importance for tumour initiation itself. Herein, we investigated whether CD133 may be relevant for colon cancer metastasis in patients, and as metastasis requires several additional biological characteristics besides tumour initiation, we examined the effects of knocking down CD133 expression in colon cancer cell lines on proliferation, migration, invasion, and colony formation. We demonstrate that high CD133 expression correlates strongly with synchronous liver metastasis in a matched case-control collection, while siRNA-mediated knock down of this factor has no significant effect on the mentioned biological characteristics. Thus, we conclude that CD133 expression is a marker with high prognostic impact for colon cancer, while it seems to have no obvious functional role as a driving force of this malignancy.
Assuntos
Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias do Colo/metabolismo , Glicoproteínas/metabolismo , Neoplasias Hepáticas/secundário , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Antígeno AC133 , Antígenos CD/genética , Antígenos CD/fisiologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicoproteínas/genética , Glicoproteínas/fisiologia , Humanos , Neoplasias Hepáticas/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Células-Tronco Neoplásicas/patologia , Peptídeos/genética , Peptídeos/fisiologia , Prognóstico , RNA Interferente Pequeno/genética , Ensaio Tumoral de Célula-TroncoRESUMO
Tumor cells are stressed by unfavorable environmental conditions like hypoxia or starvation. Driven by the resulting cellular stress tumor cells undergo epithelial-mesenchymal transition. Additionally, cellular stress is accompanied by endoplasmic reticulum-stress which induces an unfolded protein response. It is unknown if epithelial-mesenchymal transition and endoplasmic reticulum-stress are occurring as independent parallel events or if an interrelationship exists between both of them. Here, we show that in colorectal cancer cells endoplasmic reticulum-stress depends on the induction of ZEB-1, which is a main factor of epithelial-mesenchymal transition. In the absence of ZEB-1 colorectal cancer cells cannot mount endoplasmic reticulum-stress as a reaction on cellular stress situations like hypoxia or starvation. Thus, our data suggest that there is a hierarchy in the development of cellular stress which starts with the presence of environmental stress that induces epithelial-mesenchymal transition which allows finally endoplasmic reticulum-stress. This finding highlights the central role of epithelial-mesenchymal transition during the process of tumorigenesis as epithelial-mesenchymal transition is also associated with chemoresistance and cancer stemness. Consequently, endoplasmic reticulum-stress might be a well suited target for chemotherapy of colorectal cancers.
Assuntos
Neoplasias Colorretais/metabolismo , Estresse do Retículo Endoplasmático , Transição Epitelial-Mesenquimal , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Like Dickkopf-1 (DKK1), DKK4 is a target of beta-catenin/Tcf-4 in colorectal cancer. However, as a negative regulator of Wnt signalling its function in colorectal cancer cells is not well understood. We report that DKK4 is frequently down-regulated in colorectal cancer cell lines with deregulated beta-catenin/Tcf-4 and in primary colorectal cancers. Exposure of cancer cells to DKK4 strongly inhibits basal beta-catenin/Tcf-4 signalling activity, cancer cell growth and cell cycle progression. Therefore, loss of this negative feed-back loop provides Wnt factor expressing cancer cells with a growth advantage. Our data demonstrate that DKK4 is an important negative regulator of colon cancer cell growth.