Mechanisms of invasion and metastasis in colorectal cancer

We studied the mechanisms driving the metastatic spread in colorectal cancer (CRC), focusing in the MAPK pathway. We developed in vivo a highly metastatic cell line using a KRAS-mutated cell line (SW620) in an ortothopic xenograft mouse model and used both in vivo and in vitro experiments to evaluate the mechanisms of metastasis. We also used data from two large prospective cohorts of incident CRC to evaluate the association of an intronic variant of SMAD7 (rs4939827, 18q21) with the phenotype and molecular characterisitics of CRC. In the first project, we inoculated SW620 luciferase-expressing cells into portal circulation of immunodeficient mice via intrasplenic injection followed by splenectomy, in order to isolate cell populations that target the liver. Comparative transcriptomic analysis identified 194 genes differentially expressed between the parental and the highly metastatic cell line. We found pathways of nitrogen metabolism, cell adhesion molecules and mitogen-activated protein kinases (MAPKs). Downregulation of ERK2 (but not of ERK1) in the highly metastatic cell line reverted its metastatic capacity to the liver, but not to the lung in our mice model. We thus hypothesized that the ability to metastasize the lung by the highly metastatic derivative had to be driven by other mechanism. The expression of parathyroid hormone-like hormone (PTHLH) was upregulated in our highly metastatic derivative and was inversely correlated with the expression of MKK6. Downregulation of PHTLH in the highly metastatic derivative decreased its capacity to colonize the lung without decreasing its capacity to colonize the liver after intra portal inoculation. We evidenced that PTHLH induced apoptosis of human pulmonary endothelial cells via apoptosis-inducing factor mitochondrion-associated 1. In the second project we evaluated the association of the SMAD7 intronic variant with tumor phenotype and several CRC molecular characteristics. We used 1509 CRC cases and 2307 age-matched controls nested within the Nurses Health Study (NHS) and the Health Professionals Follow-up Study (HPFS). We randomly selected between one and three matched controls. Among the 1509 cases with blood or buccal samples in this study, we were able to successfully obtain tissue suitable for molecular analyses in 658 cases. We genotyped rs4939827 (TaqMan) successfully in 98% of the samples in NHS and 99.6% of the samples in HPFS. The phenotipic features evaluated were: TNM stage, grade of differentiation, location of the primary tumor (colon vs. rectum) and age at diagnosis. The evaluated molecular characteristics were DNA methylation of RUNX3 and LINE-1 (long interspersed nucleotide element-1), CpG island methylator phenotype (CIMP), microsatellite instability, TP53 expression by immunohistochemistry and the mutational status of BRAF, KRAS and PIK3CA. We found that the minor allele (G) in rs4939827 was associated with a lower risk of developing tumor stage pT1 or pT2 CRC [multivariate odds ratio (OR), 0.73; 95% confidence interval (CI) 0.62-0.87] but not tumor stage pT3 or pT4 (multivariate OR, 1.07; 95% CI 0.93-1.23, P for heterogeneity = 1.2 x 10-4). The association between rs4939827 and CRC also significantly differed by methylation of RUNX3 (P for heterogeneity = 0.005). Among those with CRC, the minor allele (G) in rs4939827 was significantly associated with poorer overall survival (hazards ratio, 1.20; 95% CI, 1.02-1.42). In conclusion, we provide clinical and molecular evidence showing that ERK2 activation provides colon cancer cells with the ability to seed and colonize the liver and reduced p38 MAPK signalling endows cancer cells with the ability to form lung metastasis from previously established liver lesions. We also show that patients with the rs4939827 CRC-susceptibility locus diagnosed with CRC tend to develop tumors with greater invasiveness (as measured by the pT stage).Parte de esta investigación consiste en explorar los mecanismos involucrados en el patrón metastático de CCR. Asimismo usamos datos epidemiológicos donde evaluamos la asociación entre el polimorfismo intrónico de SMAD7 (rs4939827, 18q21) con el genotipo y características tumorales. En el primer proyecto usamos un modelo murino de metástasis hepáticas para crear un derivado celular con alto tropismo metastático a hígado y pulmón. Mediante análisis de expresión de genes usando chips de transcripción identificamos 194 genes diferencialmente expresados El análisis de muestras clínicas mostró que aquellos pacientes cuyo tumor presentaba bajos niveles de p38 sufrían una mayor frecuencia de metástasis al pulmón, pero no a otros órganos. Al tratar ratones que habían desarrollado metástasis hepáticas derivadas de la línea celular parental con un inhibidor específico de p38, vimos que se incrementaba la afinidad metastática al pulmón. Evidenciamos que p38, a través del silenciamiento de PTHLH, en el derivado celular altamente metastático disminuía su capacidad de colonizar el pulmón. Demostramos que PTHLH induce la apoptosis de células humanas de endotelio pulmonar a través del factor AIFM1, facilitando que las células metastáticas puedan extravasarse al pulmón. En el segundo proyecto evaluamos la asociación de un polimorfismo intrónico del gen SMAD7 con el fenotipo y varias características moleculares del tumor. Para ello empleamos 1509 casos de cáncer de colon y recto y 2307 controles emparejados anidados en las cohortes Nurses Health Study y Health Professionals Follow-up Study. Encontramos que el alelo de menor frecuencia de rs4939827 (G) se asociaba con un menor riesgo de desarrollar un CCR con un estadio pT1 o pT2 [razón de odds (OR) ajustada, 0.73; intervalo de confianza al 95\% (CI) 0.62-0.87] pero no con tumores con estadio pT3 o pT4 (OR ajustada, 1.07; 95\% CI 0.93-1.23, valor p de heterogeneidad = 1.2 x 10-4). La asociación entre el polimorfismo de rs4939827 y CCR también difería significativamente según la metilación de RUNX3 (valor p de heterogeneidad = 0.005). Entre aquellos pacientes diagnosticados con CCR, el alelo de menor frecuencia de rs4939827 (G) estaba significativamente asociado con peor supervivencia (hazards ratio, 1.20; 95\% CI, 1.02-1.42)