Background: Little is known regarding the fine regulation of the Wnt/?-catenin pathway or its biological functions that might be involved in the pathogenesis of CRC. To identify potential novel CRC biomarkers, the well known azoxymethane (AOM)/ dextran sulfate sodium (DSS) murine model was used to perform a genome-wide expression profiling. The obtained results have been validated in a set of human CRC samples. Material and methods: 30 Balb/c mice were divided in 4 groups: 1) AOM/DSS treated, 2) AOM treated, 3) DSS treated, 4) untreated. 20 weeks from the cancer induction protocol, 6 mice/group were sacrificed. RNA was isolated from each sample using Trizol®. Genome-wide expression microarray was performed using MouseWG-6v2.0 Expression BeadChip (Illumina, Inc, CA). Microarray data were evaluated by state of the art statistics tools. IPA7 (www.ingenuity.com) was used for Gene Ontology (GO) analysis. 6 AOM/DSS treated mice were investigated at 5th week for early colon lesions. Tissues from 10 patients with non-metastatic CRC staged as T3N0 with a G2 grade of cellular differentiation were analysed. Histopathological analysis and immunohistochemistry (IHC) followed standard procedures. Results: Gene expression profiling showed 2036 differentially expressed genes in AOM/DSS induced tumors, including 1092 upregulated and 944 downregulated genes. In addition to the preeminent activation of the Wnt/?-catenin pathway, GO analyses revealed the enhanced expression of genes encoding Wnt antagonists, which might set up a negative feedback response to activated Wnt/?-catenin signaling in CRC. In particular, we found that the NOTUM gene was significantly up-regulated and two heparan sulfate proteoglycans, Glypican-1 (GPC1) and Glypican-3 (GPC3), which are under Notum control and act as competitive inhibitors of Wnt, were up- and down-regulated, respectively. Moreover, the IHC on animal tissues showed a positive correlation of NOTUM overexpression with intracellular ?catenin staining and a correlation with glypicans expression. We revealed an inverse correlation between molecular expression and protein distribution between Notum and GLY3. Conversely, GLY1 showed a very similar expression pattern to Notum in late lesions, although a positive staining was observed in 90% of early lesions. We validated the preclinical results on a set of human colorectal adenocarcinoma. We found a significant alteration of the molecular levels of these Wnt pathway molecular mediators in human CRC samples with respect to normal mucosa. Conclusions: Taken together, our results provide the first demonstration of a perturbed expression in the NOTUM, GPC1 and GPC3 genes in the context of CRC development. Additionally, we show a significant correlation between the expression levels of these molecules and that of ?-catenin, suggesting their role as novel biomarkers in CRC.
Novel insights into Notum and glypicans regulation in colorectal cancer.
Emanuela Signori
2016
Abstract
Background: Little is known regarding the fine regulation of the Wnt/?-catenin pathway or its biological functions that might be involved in the pathogenesis of CRC. To identify potential novel CRC biomarkers, the well known azoxymethane (AOM)/ dextran sulfate sodium (DSS) murine model was used to perform a genome-wide expression profiling. The obtained results have been validated in a set of human CRC samples. Material and methods: 30 Balb/c mice were divided in 4 groups: 1) AOM/DSS treated, 2) AOM treated, 3) DSS treated, 4) untreated. 20 weeks from the cancer induction protocol, 6 mice/group were sacrificed. RNA was isolated from each sample using Trizol®. Genome-wide expression microarray was performed using MouseWG-6v2.0 Expression BeadChip (Illumina, Inc, CA). Microarray data were evaluated by state of the art statistics tools. IPA7 (www.ingenuity.com) was used for Gene Ontology (GO) analysis. 6 AOM/DSS treated mice were investigated at 5th week for early colon lesions. Tissues from 10 patients with non-metastatic CRC staged as T3N0 with a G2 grade of cellular differentiation were analysed. Histopathological analysis and immunohistochemistry (IHC) followed standard procedures. Results: Gene expression profiling showed 2036 differentially expressed genes in AOM/DSS induced tumors, including 1092 upregulated and 944 downregulated genes. In addition to the preeminent activation of the Wnt/?-catenin pathway, GO analyses revealed the enhanced expression of genes encoding Wnt antagonists, which might set up a negative feedback response to activated Wnt/?-catenin signaling in CRC. In particular, we found that the NOTUM gene was significantly up-regulated and two heparan sulfate proteoglycans, Glypican-1 (GPC1) and Glypican-3 (GPC3), which are under Notum control and act as competitive inhibitors of Wnt, were up- and down-regulated, respectively. Moreover, the IHC on animal tissues showed a positive correlation of NOTUM overexpression with intracellular ?catenin staining and a correlation with glypicans expression. We revealed an inverse correlation between molecular expression and protein distribution between Notum and GLY3. Conversely, GLY1 showed a very similar expression pattern to Notum in late lesions, although a positive staining was observed in 90% of early lesions. We validated the preclinical results on a set of human colorectal adenocarcinoma. We found a significant alteration of the molecular levels of these Wnt pathway molecular mediators in human CRC samples with respect to normal mucosa. Conclusions: Taken together, our results provide the first demonstration of a perturbed expression in the NOTUM, GPC1 and GPC3 genes in the context of CRC development. Additionally, we show a significant correlation between the expression levels of these molecules and that of ?-catenin, suggesting their role as novel biomarkers in CRC.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
