EGFR and Prion protein promote signaling via FOXO3a-KLF5 resulting in clinical resistance to platinum agents in colorectal cancer

Epidermal growth factor receptor (EGFR) supports colorectal cancer progression via oncogenic signaling. Anti-EGFR therapy is being investigated as a clinical option for colorectal cancer, and an observed interaction between EGFR and Prion protein has been detected in neuronal cells. We hypothesized that PrPC expression levels may regulate EGFR signaling and that detailed understanding of this signaling pathway may enable identification of resistance mechanisms and new actionable targets in colorectal cancer. We performed molecular pathway analysis following knockdown of PrPC or inhibition of EGFR signaling via gefitinib to identify changes in expression of key signaling proteins that determine cellular sensitivity or resistance to cisplatin. Expression of these proteins was examined in matched primary and metastatic patient samples and was correlated for resistance to therapy and progression of disease. Utilizing three colorectal cancer cell lines, we observed a correlation between high expression of PrPC and resistance to cisplatin. Investigation of molecular signaling in a resistant cell line revealed that PrPC contributed to signaling via colocalization with EGFR, which could be overcome by targeting p38 mitogen-activated protein kinases (p38 MAPK). We revealed that the level of Krüppel-like factor 5 (KLF5), a target downstream of p38 MAPK, was predictive for cell line and patient response to platinum agents. Further, high KLF5 expression was observed in BRAF-mutant colorectal cancer. Our study indicates that the EGFR to KLF5 pathway is predictive of patient progression on platinum-based therapy

Analysis of the Association between CIMP and BRAFV600E in Colorectal Cancer by DNA Methylation Profiling

A CpG island methylator phenotype (CIMP) is displayed by a distinct subset of colorectal cancers with a high frequency of DNA hypermethylation in a specific group of CpG islands. Recent studies have shown that an activating mutation of BRAF (BRAFV600E) is tightly associated with CIMP, raising the question of whether BRAFV600E plays a causal role in the development of CIMP or whether CIMP provides a favorable environment for the acquisition of BRAFV600E. We employed Illumina GoldenGate DNA methylation technology, which interrogates 1,505 CpG sites in 807 different genes, to further study this association. We first examined whether expression of BRAFV600E causes DNA hypermethylation by stably expressing BRAFV600E in the CIMP-negative, BRAF wild-type COLO 320DM colorectal cancer cell line. We determined 100 CIMP-associated CpG sites and examined changes in DNA methylation in eight stably transfected clones over multiple passages. We found that BRAFV600E is not sufficient to induce CIMP in our system. Secondly, considering the alternative possibility, we identified genes whose DNA hypermethylation was closely linked to BRAFV600E and CIMP in 235 primary colorectal tumors. Interestingly, genes that showed the most significant link include those that mediate various signaling pathways implicated in colorectal tumorigenesis, such as BMP3 and BMP6 (BMP signaling), EPHA3, KIT, and FLT1 (receptor tyrosine kinases) and SMO (Hedgehog signaling). Furthermore, we identified CIMP-dependent DNA hypermethylation of IGFBP7, which has been shown to mediate BRAFV600E-induced cellular senescence and apoptosis. Promoter DNA hypermethylation of IGFBP7 was associated with silencing of the gene. CIMP-specific inactivation of BRAFV600E-induced senescence and apoptosis pathways by IGFBP7 DNA hypermethylation might create a favorable context for the acquisition of BRAFV600E in CIMP+ colorectal cancer. Our data will be useful for future investigations toward understanding CIMP in colorectal cancer and gaining insights into the role of aberrant DNA hypermethylation in colorectal tumorigenesis

Thrombospondin-4 is a putative tumour-suppressor gene in colorectal cancer that exhibits age-related methylation

<p>Abstract</p> <p>Background</p> <p><it>Thrombospondin-4 </it>(<it>THBS4</it>) is a member of the extracellular calcium-binding protein family and is involved in cell adhesion and migration. The aim of this study was to evaluate the potential role of deregulation of <it>THBS4 </it>expression in colorectal carcinogenesis. Of particular interest was the possible silencing of expression by methylation of the CpG island in the gene promoter.</p> <p>Methods</p> <p>Fifty-five sporadic colorectal tumours stratified for the CpG Island Methylator Phenotype (CIMP) were studied. Immunohistochemical staining of THBS4 protein was assessed in normal and tumour specimens. Relative levels of <it>THBS4 </it>transcript expression in matched tumours and normal mucosa were also determined by quantitative RT-PCR. Colony forming ability was examined in 8 cell lines made to overexpress THBS4. Aberrant promoter hypermethylation was investigated as a possible mechanism of gene disruption using MethyLight. Methylation was also assessed in the normal colonic tissue of 99 patients, with samples biopsied from four regions along the length of the colon.</p> <p>Results</p> <p><it>THBS4 </it>expression was significantly lower in tumour tissue than in matched normal tissue. Immunohistochemical examination demonstrated that THBS4 protein was generally absent from normal epithelial cells and tumours, but was occasionally expressed at low levels in the cytoplasm towards the luminal surface in vesicular structures. Forced THBS4 over-expression caused a 50-60% repression of tumour colony growth in all eight cell lines examined compared to control cell lines. Tumours exhibited significantly higher levels of methylation than matched normal mucosa, and <it>THBS4 </it>methylation correlated with the CpG island methylator phenotype. There was a trend towards decreased gene expression in tumours exhibiting high <it>THBS4 </it>methylation, but the correlation was not significant. <it>THBS4 </it>methylation was detectable in normal mucosal biopsies where it correlated with increasing patient age and negatively with the occurrence of adenomas elsewhere in the colon.</p> <p>Conclusions</p> <p><it>THBS4 </it>shows increased methylation in colorectal cancer, but this is not strongly associated with altered gene expression, either because methylation has not always reached a critical level or because other factors influence <it>THBS4 </it>expression. <it>THBS4 </it>may act as a tumour suppressor gene, demonstrated by its suppression of tumour colony formation <it>in vitro</it>. <it>THBS4 </it>methylation is detectable in normal colonic mucosa and its level may be a biomarker for the occurrence of adenomas and carcinoma.</p

Oncogenic BRAF mutation induces DNA methylation changes in a murine model for human serrated colorectal neoplasia

Colorectal cancer is a major cause of cancer death and approximately 20% arises within serrated polyps, which are under-recognized and poorly understood. Human serrated colorectal polyps frequently exhibit both oncogenic BRAF mutation and widespread DNA methylation changes, which are important in silencing genes restraining neoplastic progression. Here, we investigated whether in vivo induction of mutant Braf is sufficient to result in coordinated promoter methylation changes for multiple cancer-related genes. The BrafV637E mutation was induced in murine intestine on an FVB;C57BL/6J background and assessed for morphological and DNA methylation changes at multiple time points from 10 days to 14 months. Extensive intestinal hyperplasia developed by 10 days post-induction of the mutation. By 8 months, most mice had murine serrated adenomas with dysplasia and invasive cancer developed in 40% of mice by 14 months. From 5 months onwards, Braf mutant mice showed extensive, gene-specific increases in DNA methylation even in hyperplastic mucosa without lesions. This demonstrates that persistent oncogenic Braf signaling is sufficient to induce widespread DNA methylation changes. This occurs over an extended period of time, mimicking the long latency followed by rapid progression of human serrated neoplasia. This study establishes for the first time that DNA methylation arises slowly in direct response to prolonged oncogenic Braf signaling in serrated polyps; this finding has implications both for chemoprevention and for understanding the origin of DNA hypermethylation in cancer generally.Catherine E. Bond, Cheng Liu, Futoshi Kawamata, Diane M. McKeone, Winnie Fernando, Saara Jamieson, Sally-Ann Pearson, Alexandra Kane, Susan L. Woods, Tamsin R.M. Lannagan, Roshini Somashekar, Young Lee, Troy Dumenil, Gunter Hartel, Kevin J. Spring, Jennifer Borowsky, Lochlan Fennell, Mark Bettington, Jason Lee, Daniel L. Worthley, Barbara A. Leggett and Vicki L.J. Whitehal

Copy number profiles of paired primary and metastatic colorectal cancers

This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Liver metastasis is the major cause of death following a diagnosis of colorectal cancer (CRC). In this study, we compared the copy number profiles of paired primary and liver metastatic CRC to better understand how the genomic structure of primary CRC differs from the metastasis. Paired primary and metastatic tumors from 16 patients and their adjacent normal tissue samples were analyzed using single nucleotide polymorphism arrays. Genome-wide chromosomal copy number alterations were assessed, with particular attention to 188 genes known to be somatically altered in CRC and 24 genes that are clinically actionable in CRC. These data were analyzed with respect to the timing of primary and metastatic tissue resection and with exposure to chemotherapy. The genomic differences between the tumor and paired metastases revealed an average copy number discordance of 22.0%. The pairs of tumor samples collected prior to treatment revealed significantly higher copy number differences compared to post-therapy liver metastases (P = 0.014). Loss of heterozygosity acquired in liver metastases was significantly higher in previously treated liver metastasis samples compared to treatment naive liver metastasis samples (P = 0.003). Amplification of the clinically actionable genes ERBB2, FGFR1, PIK3CA or CDK8 was observed in the metastatic tissue of 4 patients but not in the paired primary CRC. These examples highlight the intra-patient genomic discrepancies that can occur between metastases and the primary tumors from which they arose. We propose that precision medicine strategies may therefore identify different actionable targets in metastatic tissue, compared to primary tumors, due to substantial genomic differences

How the BRAF V600E mutation defines a distinct subgroup of colorectal cancer: molecular and clinical implications

The BRAF oncogene is an integral component of the MAP kinase pathway, and an activating V600E mutation occurs in 15% of sporadic colorectal cancer. This is an early event in serrated pathway tumourigenesis, and the BRAF V600E has been commonly associated with the CpG island methylator phenotype, microsatellite instability (MSI), and a consistent clinical presentation including a proximal location and predilection for elderly females. A proportion of the BRAF mutant lesions remain as microsatellite stable (MSS), and in contrast to the MSI cancers, they have an aggressive phenotype and correlate with poor patient outcomes. Recent studies have found that they have clinical and molecular features of both the BRAF mutant/MSI and the conventional BRAF wild-type cancers and comprise a distinct colorectal cancer subgroup. This review highlights the importance of the BRAF mutation occurring in colorectal cancer stratified for molecular background and discusses its prognostic and clinical significance