Aline Simoneti Fonseca1,2,3,4, Anelisa Ramão5,6, Matheus Carvalho Bürger6, Jorge Estefano Santana de Souza6, Dalila Lucíola Zanette5,6,7,8, Greice Andreotti de Molfetta5,6,7, Luiza Ferreira de Araújo5,6,7, Rafaela de Barros E Lima Bueno5,6, Graziela Moura Aguiar6, Jessica Rodrigues Plaça6, Cleidson de Pádua Alves6, Anemari Ramos Dinarte Dos Santos6, Daniel Onofre Vidal6, Gyl Eanes Barros Silva9, Rodrigo Alexandre Panepucci6, Fernanda Maris Peria10, Omar Feres11, José Joaquim Ribeiro da Rocha11, Marco Antonio Zago6, Wilson Araújo Silva12,13,14. 1. Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Av Bandeirantes, 3900, CEP: 14049-900, Monte Alegre, Ribeirão Preto, SP, Brazil. aline.fonseca@pelepequenoprincipe.org.br. 2. Center for Cell Based Therapy and National Institute of Science and Technology in Stem Cell and Cell Therapy, Ribeirão Preto, SP, Brazil. aline.fonseca@pelepequenoprincipe.org.br. 3. Center for Integrative Systems Biology - CISBi, NAP/USP, Ribeirão Preto, SP, Brazil. aline.fonseca@pelepequenoprincipe.org.br. 4. Research Institute Pelé Pequeno Príncipe, Av Silva Jardim, 1632, CEP: 80250-060, Água Verde, Curitiba, PR, Brazil. aline.fonseca@pelepequenoprincipe.org.br. 5. Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Av Bandeirantes, 3900, CEP: 14049-900, Monte Alegre, Ribeirão Preto, SP, Brazil. 6. Center for Cell Based Therapy and National Institute of Science and Technology in Stem Cell and Cell Therapy, Ribeirão Preto, SP, Brazil. 7. Center for Integrative Systems Biology - CISBi, NAP/USP, Ribeirão Preto, SP, Brazil. 8. Laboratory of Applied Science and Technology in Health (LASTH), Instituto Carlos Chagas, Fundação Oswaldo Cruz, Curitiba, PR, Brazil. 9. Laboratory of Immunofluorescence and Electron Microscopy (LIME), Presidente Dutra University Hospital (HUUFMA), São Luís, MA, Brazil. 10. Departament of Medical Clinic, Medical School of Ribeirão Preto, University of São Paulo, USP, Ribeirão Preto, SP, Brazil. 11. Department of Surgery and Anatomy, School of Medicine of Ribeirão Preto, University of São Paulo, Sao Paulo, Brazil. 12. Department of Genetics, Ribeirão Preto Medical School, University of São Paulo, Av Bandeirantes, 3900, CEP: 14049-900, Monte Alegre, Ribeirão Preto, SP, Brazil. wilsonjr@usp.br. 13. Center for Cell Based Therapy and National Institute of Science and Technology in Stem Cell and Cell Therapy, Ribeirão Preto, SP, Brazil. wilsonjr@usp.br. 14. Center for Integrative Systems Biology - CISBi, NAP/USP, Ribeirão Preto, SP, Brazil. wilsonjr@usp.br.
Abstract
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.
BACKGROUND:Colorectal cancer (CRC) is one of the most common cancers worldwide; it is the fourth leading cause of death in the world and the third in Brazil. Mutations in the APC, DCC, KRAS and TP53 genes have been associated with the progression of sporadic CRC, occurring at defined pathological stages of the tumor progression and consequently modulating several genes in the corresponding signaling pathways. Therefore, the identification of gene signatures that occur at each stage during the CRC progression is critical and can present an impact on the diagnosis and prognosis of the patient. In this study, our main goal was to determine these signatures, by evaluating the gene expression of paired colorectal adenoma and adenocarcinoma samples to identify novel genetic markers in association to the adenoma-adenocarcinoma stage transition. METHODS: Ten paired adenoma and adenocarcinoma colorectal samples were subjected to microarray gene expression analysis. In addition, mutations in APC, KRAS and TP53 genes were investigated by DNA sequencing in paired samples of adenoma, adenocarcinoma, normal tissue, and peripheral blood from ten patients. RESULTS: Gene expression analysis revealed a signature of 689 differentially expressed genes (DEG) (fold-change> 2, p< 0.05), between the adenoma and adenocarcinoma paired samples analyzed. Gene pathway analysis using the 689 DEG identified important cancer pathways such as remodeling of the extracellular matrix and epithelial-mesenchymal transition. Among these DEG, the ETV4 stood out as one of the most expressed in the adenocarcinoma samples, further confirmed in the adenocarcinoma set of samples from the TCGA database. Subsequent in vitro siRNA assays against ETV4 resulted in the decrease of cell proliferation, colony formation and cell migration in the HT29 and SW480 colorectal cell lines. DNA sequencing analysis revealed KRAS and TP53 gene pathogenic mutations, exclusively in the adenocarcinomas samples. CONCLUSION: Our study identified a set of genes with high potential to be used as biomarkers in CRC, with a special emphasis on the ETV4 gene, which demonstrated involvement in proliferation and migration.
Authors: Alvaro Aytes; Antonina Mitrofanova; Carolyn Waugh Kinkade; Celine Lefebvre; Ming Lei; Vanessa Phelan; H Carl LeKaye; Jason A Koutcher; Robert D Cardiff; Andrea Califano; Michael M Shen; Cory Abate-Shen Journal: Proc Natl Acad Sci U S A Date: 2013-08-05 Impact factor: 11.205