Majority Of Sporadic Colorectal Cancers Research Articles

Adenomatous Polyposis Coli as a Major Regulator of Human Embryonic Stem Cells Self-Renewal.

Human embryonic stem cells (hESCs) provide an essential tool to investigate early human development, study disease pathogenesis, and examine therapeutic interventions. Adenomatous polyposis coli (APC) is a negative regulator of Wnt/β-catenin signaling, implicated in the majority of sporadic colorectal cancers and in the autosomal dominant inherited syndrome familial adenomatous polyposis (FAP). Studies into the role of Wnt/β-catenin signaling in hESCs arrived at conflicting results, due at least in part to variations in culture conditions and the use of external inhibitors and agonists. Here, we directly targeted APC in hESCs carrying a germline APC mutation, derived from affected blastocysts following preimplantation genetic diagnosis (PGD) for FAP, in order to answer open questions regarding the role of APC in regulating pluripotency and differentiation potential of hESCs. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), we generated second hit APC mutations in FAP-hESCs. Despite high CRISPR/Cas9 targeting efficiency and the successful isolation of many clones, none of the isolated clones carried a loss of function mutation in the wild-type (WT) APC allele. Using a fluorescent β-catenin reporter and analysis of mutated-allele frequencies in the APC locus, we show that APC double mutant hESCs robustly activate Wnt/β-catenin signaling that results in rapid differentiation to endodermal and mesodermal lineages. Here, we provide direct evidence for a strict requirement for constant β-catenin degradation through the APC destruction complex in order to maintain pluripotency, highlighting a fundamental role for APC in self-renewal of hESCs. Stem Cells 2019;37:1505-1515.

Infection with genotoxin-producing Salmonella enterica synergises with loss of the tumour suppressor APC in promoting genomic instability via the PI3K pathway in colonic epithelial cells.

Several commensal and pathogenic Gram‐negative bacteria produce DNA‐damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin‐producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin‐producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC‐deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3‐kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin‐producing bacteria in promoting a microenvironment conducive to malignant transformation.

TALEN-mediated apc mutation in Xenopus tropicalis phenocopies familial adenomatous polyposis.

Truncating mutations in the tumor suppressor gene adenomatous polyposis coli (APC) are the initiating step in the vast majority of sporadic colorectal cancers, and they underlie familial adenomatous polyposis (FAP) syndromes. Modeling of APC- driven tumor formation in the mouse has contributed substantially to our mechanistic understanding of the associated disease, but additional models are needed to explore therapeutic opportunities and overcome current limitations of mouse models. We report on a novel and penetrant genetic cancer model in Xenopus tropicalis, an aquatic tetrapod vertebrate with external development, diploid genome and short life cycle. Tadpoles and froglets derived from embryos injected with TAL effector nucleases targeting the apc gene rapidly developed intestinal hyperplasia and other neoplasms observed in FAP patients, including desmoid tumors and medulloblastomas. Bi-allelic apc mutations causing frame shifts were detected in the tumors, which displayed activation of the Wnt/β-catenin pathway and showed increased cellular proliferation. We further demonstrate that simultaneous double bi-allelic mutation of apc and a non-relevant gene is possible in the neoplasias, opening the door for identification and characterization of effector or modifier genes in tumors expressing truncated apc. Our results demonstrate the power of modeling human cancer in Xenopus tropicalis using mosaic TALEN-mediated bi-allelic gene disruption.

Identification of driver genes in microsatellite-unstable colorectal cancers

SUMMARY Somatic mutations in key driver genes are necessary for colorectal tumor formation. Whereas the majority of sporadic colorectal cancer (CRC) cases have chromosomal instability, a sixth of cases generate somatic mutations by a deficiency in the mismatch repair system. This choice of mutational mechanism has a strong impact on which genes in CRC pathways are the most likely to be mutated in an individual tumor. A specific challenge in understanding CRC with microsatellite instability is to discriminate driver from passenger genes given the large number of nucleotide repeats that are frequently found mutated in patient tumors. This review, therefore, discusses strategies for the identification and validation of cancer genes in microsatellite-unstable colorectal cancers.

Colorectal Cancer: Sailing with a T-Cell EMAST

During the past two decades, considerable progress has been made in understanding and defining the molecular basis of human colorectal cancer (CRC). The genetic heterogeneity of this disease is now well-established, and it has become clear that a distinct subgroup of CRC possesses a unique form of genetic instability, defined as microsatellite instability (MSI). The MSI phenotype is a hallmark of defective DNA mismatch repair (MMR), and inactivation of MMR genes permits increased instability and accumulation of genomic alterations in the sequences containing mono[e.g.(A)n] and di-nucleotide [e.g. (CA)n] microsatellite repeats. Approximately 12–15% of CRC exhibit high frequency MSI (MSI-H), which includes the majority of Lynch syndrome cancers with germline mutation of a MMR gene and about 12% of sporadic tumors due to methylation-induced transcriptional silencing of the MLH1 gene [1]. The remaining 85% of CRCs either possess very low levels of MSI (MSI-L) or are microsatellite stable (MSS); however, the molecular basis of these tumors remains poorly understood. Almost a decade ago, Sidransky and colleagues described a distinct form of genetic instability present in respiratory tumors, which was termed as elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) [2]. In contrast to instability at the monoand di-nucleotide repeats seen in MSI CRCs, EMAST tumors are identified when the frameshift mutations occur at certain tetranucleotide repeat sequences [e.g. (AAAG)n or (ATAG)n]. Since its initial discovery in non-small cell lung cancers [2, 3], EMAST has subsequently been observed in several other human cancers, including skin [4], ovarian [5], endometrial [6], bladder [4, 7], prostate [8, 9] and colorectal [10–13]. Most microsatellite sequences (either mono-, dior tetra-nucleotide repeats) are present in the non-coding regions of DNA; however, small minorities of these tandem repeats exist within the coding sequences of growth regulatory genes that play a critical role in driving CRC pathogenesis. Frameshift mutations within this latter class of genes, such as observed with EMAST, have direct functional consequences for the growth and proliferation of tumor cells. The underlying molecular mechanism(s) causing EMAST remain unclear. The earliest reports in this regard proposed an association of EMAST with mutations in the p53 gene [3, 4]. It was suggested that exposure to environmental carcinogens may exacerbate this phenotype [14]. With the limited evidence at hand, EMAST appears to be distinct from the classic MSI signature that is frequently observed in Lynch syndrome and sporadic CRC. Whether EMAST contributes to tumor progression by specific geneinactivating mutations within repetitive coding sequences, as is the case with MSI-positive CRC, is a matter of ongoing investigation. Recently, it was shown that EMAST is present in the majority of sporadic CRCs [10]. Haugen et al. demonstrated that EMAST is associated with MSI-L and a deficiency in MSH3 protein expression in colorectal cell lines and tumor tissues [10]. Building upon this evidence, later on it was demonstrated that EMAST can be acquired during the adenoma to carcinoma transformation, as well as upon transformation of well-differentiated carcinomas to moderately and poorly differentiated CRCs [12]. Interestingly in this study, EMAST best correlated in CRCs with ulcerated features, and the authors suggested that inflammation may help drive EMAST [12]. A. Goel (&) Gastrointestinal Cancer Research Laboratory, Sammons Cancer Center and Baylor Research Institute, Baylor University Medical Center, 3500 Gaston Avenue, Suite H-250, Dallas, TX 75246, USA e-mail: ajay.goel@baylorhealth.edu

A Targeted Constitutive Mutation in the Apc Tumor Suppressor Gene Underlies Mammary But Not Intestinal Tumorigenesis

Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/β-catenin signaling. Notably, genotype–phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/β-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/β-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc +/1572T mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc +/1572T mice suggests that specific dosages of Wnt/β-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.

Editorial: The Influence of Timing of Colonoscopy on Adenoma Detection: Is Timing Everything?

Colorectal cancer is a common disease in the United States. The majority of sporadic colorectal cancers develop from adenomatous polyps. In the United States, the incidence of colorectal cancer is declining, most likely because of colonoscopic polypectomy. Adenoma detection rate is affected by the quality of prep, careful mucosal inspection, and withdrawal time. In this issue of the Journal, there is intriguing evidence that the timing of colonoscopy, morning vs. afternoon, affects adenoma detection rate. Physician fatigue is postulated as a factor leading to lower polyp detection rates in the afternoon compared with the morning. Further, prospective studies are needed to confirm these findings and determine the effect on clinical practice.

Cross-Species Comparison of Human and Mouse Intestinal Polyps Reveals Conserved Mechanisms in Adenomatous Polyposis Coli ( APC)-Driven Tumorigenesis

Expression profiling is a well established tool for the genome-wide analysis of human cancers. However, the high sensitivity of this approach combined with the well known cellular and molecular heterogeneity of cancer often result in extremely complex expression signatures that are difficult to interpret functionally. The majority of sporadic colorectal cancers are triggered by mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, leading to the constitutive activation of the Wnt/beta-catenin signaling pathway and formation of adenomas. Despite this common genetic basis, colorectal cancers are very heterogeneous in their degree of differentiation, growth rate, and malignancy potential. Here, we applied a cross-species comparison of expression profiles of intestinal polyps derived from hereditary colorectal cancer patients carrying APC germline mutations and from mice carrying a targeted inactivating mutation in the mouse homologue Apc. This comparative approach resulted in the establishment of a conserved signature of 166 genes that were differentially expressed between adenomas and normal intestinal mucosa in both species. Functional analyses of the conserved genes revealed a general increase in cell proliferation and the activation of the Wnt/beta-catenin signaling pathway. Moreover, the conserved signature was able to resolve expression profiles from hereditary polyposis patients carrying APC germline mutations from those with bi-allelic inactivation of the MYH gene, supporting the usefulness of such comparisons to discriminate among patients with distinct genetic defects.

The Bone Morphogenetic Protein Pathway Is Inactivated in the Majority of Sporadic Colorectal Cancers

The finding of bone morphogenetic protein (BMP) receptor 1a mutations in juvenile polyposis suggests that BMPs are important in colorectal cancer (CRC). We investigated the BMP pathway in sporadic CRC. We investigated BMP receptor (BMPR) expression using immunoblotting and sequenced BMPR2 in CRC cell lines. We assessed the expression of BMPRs, SMAD4, and pSMAD1/5/8 in 72 sporadic CRCs using a tissue microarray and immunohistochemistry. We assessed the effect of reintroduction of wild-type BMPR2 on BMP pathway activity and the effect of wild-type or mutated BMPR2 3' untranslated region (UTR) sequences on protein expression by attachment to pCMV-Luc. BMPR2 and SMAD4 protein expression is abrogated in microsatellite unstable (MSI) and microsatellite stable (MSS) cell lines, respectively. BMPR2 3'UTR is mutated in all MSI and in none of the MSS cell lines. Mutant BMPR2 3'UTR sequences reduced luciferase expression 10-fold compared with wild-type BMPR2 3'UTR. BMPR2 expression is impaired more frequently in MSI CRCs than MSS (85% vs 29%; P < .0001) and shows a mutually exclusive pattern of impaired expression compared with SMAD4. Nine of 11 MSI cancers with impaired expression of BMPR2 have microsatellite mutations. The BMP pathway is inactivated, as judged by nuclear pSMAD1/5/8 expression, in 70% of CRCs, and this correlates with BMPR and SMAD4 loss. Our data suggest that the BMP pathway is inactivated in the majority of sporadic CRCs. In MSI CRC this is associated predominantly with impaired BMPR2 expression and in MSS CRC with impaired SMAD4 expression.

Epigenetic Inactivation of a Cluster of Genes Flanking MLH1 in Microsatellite-Unstable Colorectal Cancer

Biallelic promoter methylation and transcriptional silencing of the MLH1 gene occurs in the majority of sporadic colorectal cancers exhibiting microsatellite instability due to defective DNA mismatch repair. Long-range epigenetic silencing of contiguous genes has been found on chromosome 2q14 in colorectal cancer. We hypothesized that epigenetic silencing of MLH1 could occur on a regional scale affecting additional genes within 3p22, rather than as a focal event. We studied the levels of CpG island methylation and expression of multiple contiguous genes across a 4 Mb segment of 3p22 including MLH1 in microsatellite-unstable and -stable cancers, and their paired normal colonic mucosa. We found concordant CpG island hypermethylation, H3-K9 dimethylation and transcriptional silencing of MLH1 and multiple flanking genes spanning up to 2.4 Mb in microsatellite-unstable colorectal cancers. This region was interspersed with unmethylated genes, which were also transcriptionally repressed. Expression of both methylated and unmethylated genes was reactivated by methyltransferase and histone deacetylase inhibitors in a microsatellite-unstable colorectal carcinoma cell line. Two genes at the telomeric end of the region were also hypermethylated in microsatellite-stable cancers, adenomas, and at low levels in normal colonic mucosa from older individuals. Thus, the cluster of genes flanking MLH1 that was specifically methylated in the microsatellite-unstable group of cancers extended across 1.1 Mb. Our results show that coordinate epigenetic silencing extends across a large chromosomal region encompassing MLH1 in microsatellite-unstable colorectal cancers. Simultaneous epigenetic silencing of this cluster of 3p22 genes may contribute to the development or progression of this type of cancer.

The APC gene in colorectal cancer

Mutations in the adenomatous polyposis coli (APC) gene are not only responsible for familial adenomatous polyposis (FAP), but also play a rate-limiting role in the majority of sporadic colorectal cancers. Colorectal tumours are known to arise through a gradual series of histological changes, the so-called ‘adenoma–carcinoma’ sequence, each accompanied by a genetic alteration in a specific oncogene or tumour suppressor gene. Loss of APC function triggers this chain of molecular and histological changes. In general, an intestinal cell needs to comply with two essential requirements to develop into a cancer: it must acquire selective advantage to allow for the initial clonal expansion, and genetic instability to allow for multiple hits at other genes responsible for tumour progression and malignant transformation. Inactivation of APC seems to fulfill both requirements. In this short review, I will discuss the role played by APC in providing, when mutated, selective advantage, through constitutional activation of the Wnt signal transduction pathway, and chromosomal instability to the nascent intestinal tumor cell.

DNA methylation and gastrointestinal malignancies: functional consequences and clinical implications.

Evidence now suggests that the transcriptional silencing of selected genes by DNA methylation plays a crucial role in the development and progression of human gastrointestinal malignancies. To date, genes involved in the regulation of the cell cycle, DNA repair, angiogenesis, and apoptosis have all been shown to be inactivated by the hypermethylation of their respective 5' CpG islands. The specific causes and effects of the changes in DNA methylation that occur in cancer remain unknown, however. Nevertheless, recently developed techniques for detecting changes in DNA methylation have dramatically increased the amount of information available on the patterns of methylation that occur as cancers progress. One key process involved in aberrant methylation is related to aging, and because it affects a large number of CpG islands, age-related methylation may be a primary cause of the increased incidence of cancer seen among older individuals. Other patterns of methylation are cancer-specific and are detected only in a subset of tumors exhibiting the CpG island methylator phenotype (CIMP). In this regard, the majority of sporadic colorectal cancers, exhibiting microsatellite instability (MSI) appear to be associated with CIMP, which leads to aberrant methylation of human MutL homologue (hMLH1) and the loss of its expression. We anticipate that as the various components of the molecular machinery involved in aberrant DNA methylation become better understood, they will prove to be useful targets, serving as the basis for the development of new methods for the diagnosis and treatment of gastrointestinal malignancies.

CpG island methylator phenotypes in aging and cancer

CpG islands are short stretches of CpG rich regions that are frequently associated with the promoter region of genes. Aberrant methylation of CpG islands is one mechanism of inactivating tumor suppressor genes (TSGs) in neoplasia, and there is growing evidence that altered cytosine methylabtion play important roles in cancer development. However, the differences in global CpG island methylation patterns between normal and cancer cells remain poorly understood. By examining a large number of loci in a series of cancers, global methylation profiles can be constructed. Such studies revealed that in colorectal cancer, there appears to be two types of methylation that are associated with cancer progression: type A (for age-related) methylation, and type C (for cancer-specific) methylation. Initially, type A methylation arises as a function of age in normal colorectal epithelial cells. By affecting genes that regulate the growth and/or differentiation of these cells, such methylation may result in a predisposition state that precedes tumor formation in the colon. Type C methylation, by contrast, was found exclusively in a subset of cancers, which display a CpG island methylator phenotype (CIMP). CIMP is a novel molecular instability pathway that appears to be responsible for most cases of aberrant TSG methylation in colorectal cancer, and which has important interactions with genetic pathways as well. In fact, CIMP+ tumors account for the majority of sporadic colorectal cancers with microsatellite instability, through methylation of the mismatch repair gene hMLH1. This model whereby age-related methylation increases cell-susceptibility to transformation and cancer-specific methylabtion results in neoplastic progression in a subset of cases may be applicable to many human neoplasms.

CpG island methylator phenotype in colorectal cancer.

Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP+ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.