William M. Grady

Transforming Growth Factor β Receptor Type II Inactivation Induces the Malignant Transformation of Intestinal Neoplasms Initiated by Apc Mutation

The transforming growth factor-beta (TGF-beta) signaling pathway is a tumor-suppressor pathway that is commonly inactivated in colon cancer. TGF-beta is a secreted ligand that mediates its effects through a transmembrane heteromeric receptor complex, which consists of type I (TGFBR1) and type II subunits (TGFBR2). Approximately 30% of colon cancers carry TGFBR2 mutations, demonstrating that it is a common target for mutational inactivation in this cancer. To assess the functional role of TGFBR2 inactivation in the multistep progression sequence of colon cancer, we generated a mouse model that recapitulates two common genetic events observed in human colon cancer by mating Apc(1638N/wt) mice with mice that are null for Tgfbr2 in the intestinal epithelium, Villin-Cre;Tgfbr2(E2flx/E2flx) mice. In this model, we observed a dramatic increase in the number of intestinal adenocarcinomas in the Apc(1638N/wt);Villin-Cre;Tgfbr2(E2flx/E2flx) mice (called Apc(1638N/wt);Tgfbr2(IEKO)) compared with those mice with intact Tgfbr2 (Apc(1638N/wt);Tgfbr2(E2flx/E2flx)). Additionally, in vitro analyses of epithelial tumor cells derived from the Apc(1638N/wt);Tgfbr2(IEKO) mice showed enhanced expression and activity of matrix metalloproteinase MMP-2 and MMP-9, as well as increased TGF-beta1 secretion in the conditioned medium. Similarly, primary tumor tissues from the Apc(1638N/wt);Tgfbr2(IEKO) mice also showed elevated amounts of TGF-beta1 as well as higher MMP-2 activity in comparison with Apc(1638N/wt);Tgfbr2(E2flx/E2flx)-derived tumors. Thus, loss of TGFBR2 in intestinal epithelial cells promotes the invasion and malignant transformation of tumors initiated by Apc mutation, providing evidence that Wnt signaling deregulation and TGF-beta signaling inactivation cooperate to drive the initiation and progression, respectively, of intestinal cancers in vivo.

TGF-β receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-β

TGF-beta is a pluripotent cytokine that mediates its effects through a receptor composed of TGF-beta receptor type II (TGFBR2) and type I (TGFBR1). The TGF-beta receptor can regulate Smad and nonSmad signaling pathways, which then ultimately dictate TGF-betas biological effects. We postulated that control of the level of TGFBR2 is a mechanism for regulating the specificity of TGF-beta signaling pathway activation and TGF-betas biological effects. We used a precisely regulatable TGFBR2 expression system to assess the effects of TGFBR2 expression levels on signaling and TGF-beta mediated apoptosis. We found Smad signaling and MAPK-ERK signaling activation levels correlate directly with TGFBR2 expression levels. Furthermore, p21 levels and TGF-beta induced apoptosis appear to depend on relatively high TGFBR2 expression and on the activation of the MAPK-ERK and Smad pathways. Thus, control of TGFBR2 expression and the differential activation of TGF-beta signaling pathways appears to be a mechanism for regulating the specificity of the biological effects of TGF-beta.

The aberrant methylation of TSP1 suppresses TGF-β1 activation in colorectal cancer

Colorectal cancer arises from the progressive accumulation of mutations and epigenetic alterations in colon epithelial cells. Such alterations often deregulate signaling pathways that affect the formation of colon cancer, such as the Wnt, RAS‐MAPK and TGF‐β pathways. The tumor promoting effects of mutations in genes, such as APC, have been demonstrated in cancer cell lines and in mouse models of intestinal cancer; however, the biological effects of most epigenetic events identified in colorectal cancer remain unknown. Consequently, we assessed whether the aberrant methylation of TSP1, the gene for thrombospondin 1, a regulator of TGF‐β ligand activation, is an epigenetic mechanism for inhibiting the TGF‐β signaling pathway. We found methylated TSP1 occurs in colon cancer cell lines (33%), colon adenomas (14%) and colon adenocarcinomas (21%). In primary colorectal cancers, loss of TSP1 expression correlated with impaired TGF‐β signaling as indicated by decreased Smad2 phosphorylation and nuclear localization. Furthermore, methylation‐induced silencing of TSP1 expression reduced the concentration of secreted active TGF‐β1 and attenuated TGF‐β signaling. Reversal of TSP1 methylation resulted in increased TSP1 mediated activation of the latent LAP:TGF‐β complex and subsequent TGF‐β receptor activation. Our results demonstrate that the aberrant methylation of TSP1 has biological consequences and provide evidence that the aberrant methylation of TSP1 is a novel epigenetic mechanism for suppressing TGF‐β signaling in colorectal cancer.

Aberrant DNA methylation occurs in colon neoplasms arising in the azoxymethane colon cancer model

Mouse models of intestinal tumors have advanced our understanding of the role of gene mutations in colorectal malignancy. However, the utility of these systems for studying the role of epigenetic alterations in intestinal neoplasms remains to be defined. Consequently, we assessed the role of aberrant DNA methylation in the azoxymethane (AOM) rodent model of colon cancer. AOM induced tumors display global DNA hypomethylation, which is similar to human colorectal cancer. We next assessed the methylation status of a panel of candidate genes previously shown to be aberrantly methylated in human cancer or in mouse models of malignant neoplasms. This analysis revealed different patterns of DNA methylation that were gene specific. Zik1 and Gja9 demonstrated cancer‐specific aberrant DNA methylation, whereas, Cdkn2a/p16, Igfbp3, Mgmt, Id4, and Cxcr4 were methylated in both the AOM tumors and normal colon mucosa. No aberrant methylation of Dapk1 or Mlt1 was detected in the neoplasms, but normal colon mucosa samples displayed methylation of these genes. Finally, p19Arf, Tslc1, Hltf, and Mlh1 were unmethylated in both the AOM tumors and normal colon mucosa. Thus, aberrant DNA methylation does occur in AOM tumors, although the frequency of aberrantly methylated genes appears to be less common than in human colorectal cancer. Additional studies are necessary to further characterize the patterns of aberrantly methylated genes in AOM tumors.

Evidence for the role of aberrant DNA methylation in the pathogenesis of Lynch syndrome adenomas

Colorectal cancer (CRC) forms through a series of histologic steps that are accompanied by mutations and epigenetic alterations, which is called the polyp‐cancer sequence. The role of epigenetic alterations, such as aberrant DNA methylation, in the polyp‐cancer sequence in sporadic CRC and particularly in hereditary colon cancer is not well understood. Consequently, we assessed the methylation status of CDKN2A/p16, MGMT, MLH1 and p14ARF in adenomas arising in the Lynch syndrome, a familial colon cancer syndrome caused by MLH1 and MSH2 mutations, to determine if DNA methylation is a “second hit” mechanism in CRC and to characterize the role of DNA methylation in the polyp phase of the Lynch syndrome. We found MLH1 and p14ARF are methylated in 53 and 60% of the Lynch syndrome adenomas and in 4 and 20% of sporadic adenomas, whereas CDKN2A/p16 and MGMT are methylated in 6 and 14% of the Lynch syndrome adenomas versus 50 and 64% of sporadic adenomas. Therefore, the frequency and pattern of gene methylation varies between the Lynch syndrome and sporadic colon adenomas, implying differences in the molecular pathogenesis of the tumors. MLH1 methylation in the Lynch syndrome adenomas suggests gene methylation might have a role in the initiation of these neoplasms.

Insights into the role of DNA methylation in disease through the use of mouse models.

Epigenetics was originally defined as the interaction of genes with their environment that brings the phenotype into being. It now refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. To date, the best understood epigenetic mechanisms are CpG DNA methylation and histone modifications. DNA methylation in particular has been the subject of intense interest because of its recently recognized role in disease, as well as in the development and normal function of organisms. Much of the focus of disease-related research has been on cancer because of the recognition that epigenetic alterations are common in cancer and probably cooperate with genetic alterations to drive cancer formation. Our understanding of epigenetic mechanisms in controlling gene expression has resulted from the study of cell line systems and simple model systems, such as Arabidopsis thaliana. We are now moving into an era of more complex model systems, such as transgenic and knockout mouse models, which will lead to further insight into epigenetics in development and human disease. The current models have revealed complex, tissue-specific effects of epigenetic mechanisms and have further informed our understanding of the role of DNA methylation and histone modifications on disease and development. The current state of these models is the subject of this Commentary.

TGF-beta has paradoxical and context dependent effects on proliferation and anoikis in human colorectal cancer cell lines.

Transforming growth factor-β (TGF-β) is a pluripotent cytokine that can have both tumor suppressing and tumor promoting effects on epithelial cells. It is unclear what determines when TGF-β and its signaling pathway act predominantly as a tumor suppressor pathway or as a tumor-promoter pathway and whether TGF-β can have both classes of effects concurrently on a cell. We investigated the effect of TGF-β on anoikis in colorectal cancer cell lines sensitive to TGF-β-mediated growth inhibition to determine if the context of the cells could be one of the factors that would affect whether TGF-β exerts tumor suppressor or oncogene activity on colon cancer cells. We observed variable effects of TGF-β on anoikis in these cell lines, even though they all are growth-inhibited by TGF-β. Thus, we show that TGF-β has variable effects on anoikis in colon cancer cell lines that likely reflects the effects of concurrent gene mutations in the cancer cells and the activation state of the signaling pathways controlled by these genes.

Rare loss of function variants in candidate genes and risk of colorectal cancer

Although ~ 25% of colorectal cancer or polyp (CRC/P) cases show familial aggregation, current germline genetic testing identifies a causal genotype in the 16 major genes associated with high penetrance CRC/P in only 20% of these cases. As there are likely other genes underlying heritable CRC/P, we evaluated the association of variation at novel loci with CRC/P. We evaluated 158 a priori selected candidate genes by comparing the number of rare potentially disruptive variants (PDVs) found in 84 CRC/P cases without an identified CRC/P risk-associated variant and 2440 controls. We repeated this analysis using an additional 73 CRC/P cases. We also compared the frequency of PDVs in select genes among CRC/P cases with two publicly available data sets. We found a significant enrichment of PDVs in cases vs. controls: 20% of cases vs. 11.5% of controls with ≥ 1 PDV (OR = 1.9, p = 0.01) in the original set of cases. Among the second cohort of CRC/P cases, 18% had a PDV, significantly different from 11.5% (p = 0.02). Logistic regression, adjusting for ancestry and multiple testing, indicated association between CRC/P and PDVs in NTHL1 (p = 0.0001), BRCA2 (p = 0.01) and BRIP1 (p = 0.04). However, there was no significant difference in the frequency of PDVs at each of these genes between all 157 CRC/P cases and two publicly available data sets. These results suggest an increased presence of PDVs in CRC/P cases and support further investigation of the association of NTHL1, BRCA2 and BRIP1 variation with CRC/P.

Case Report of Molecular Signature in Poorly Differentiated Esophageal Adenocarcinoma.

Esophageal adenocarcinoma (EAC) arises from Barrett’s metaplasia (BE) in a process involving discrete genetic mutations and epigenetic alterations that remain poorly defined. An intriguing model underlying BE and EAC is that epithelial progenitor cells persist at the gastroesophageal junction, implicating dysregulation of differentiation in the pathogenesis of BE and EAC. Herein, we chose to elucidate the molecular signatures of primary poorly differentiated EAC cases. Using RNA-Seq to analyze aberrant gene expression, we found that expression of genes involved in morphogenesis and keratinocyte differentiation were decreased, whereas expression of genes involved in cell death and proliferation were increased in patients with poorly differentiated EAC. In addition, the levels of two histone methyltransferases were increased in these poorly differentiated EAC cases, including the “suppressor of variegation 39 homolog” (SUV39H) and the “enhancer of zeste homolog 2” (EZH2). SUV39H and EZH2 are responsible for trimethylation of histone 3 lysine 9 (H3K9me3) and lysine 27 (H3K27me3), respectively. Using chromatin immunoprecipitation and deep-sequencing (ChIP-Seq) to decipher abnormal epigenetic regulation, we identified putative targets aberrantly enriched with H3K9me3 or H3K27me3 in these poorly differentiated EAC cases. Moreover, comparative analysis of DNA methylation, ChIP-Seq, and RNASeq data suggests that dysregulation of histone 3 lysine 27 tri-methylation (H3K27me3) was the most common epigenetic alterations associated with gene expression change in a collection of poorly differentiated EAC. Importantly, our result implicates that the abundance and colocalization of H3K27me3 and primitive epithelial marker KRT-7 would present a unique signature of the poorly differentiated EAC cases. The significance of this *Corresponding author: Chin-Hsing Annie Lin, Department of Biology, University of Texas at San Antonio, San Antonio, TX 78249. Email: annie.lin@utsa.edu Citation: Andrew M. Kaz, et al. Case Report of Molecular Signature in Poorly Differentiated Esophageal Adenocarcinoma. Cancer Research Frontiers. 2017; 3(1): 170-191. doi: 10.17980/2017.170 Copyright: @ 2017 Andrew M. Kaz, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Competing Interests: The authors declare no competing financial interests. Received Feb 22, 2018; Revised Apr 17, 2018; Accepted Apr 23, 2018. Published May 21, 2018 Cancer Research Frontiers. 2017; 3(1): 170-191. doi: 10.17980/2017.170 Case Report 171 case report highlights that defects in epithelial differentiation paradigm underscore the epigenetic dysregulation via H3K27me3, also presents potential biomarkers for detection of the poorly differentiated EAC cases.

DCC and RET pathway analysis to identify factors associated with advanced colorectal cancer.

457 Background: DCC (deleted in colon cancer; 18q21.3) is frequently lost in colorectal cancers (CRC), but few mutations in DCC have been discovered, even in tumors with 18q loss of heterozygosity. DCC has been shown to be a dependence receptor, with differential signaling depending on the presence (proliferative) or absence (pro-apoptotic) of the netrin-1 ligand (NTN1). DCC-mutated CRC tend to present at advanced stage and have a poor prognosis. RET, another dependence receptor, is a possible tumor suppressor gene in CRC and associates with CRC progression. Given the apparent role of DCC and RET in CRC progression, we carried out a genetic association study to determine if specific genetic variants in these pathways associate with advanced vs. early CRC. Methods: Imputed HapMap genome-wide association study (GWAS) from the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), a collection of 19 international case-control and cohort studies, was used to identify single nucleotide polymorphism...