Non-neoplastic Colonic Mucosa Research Articles

Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

The Prognostic Roles of PYCR2 and ZBTB18 Expression in Tissues of Colorectal Carcinoma and Non-Neoplastic Tissues: An Immunohistochemical Study

Abstract Background It is important to detect novel biomarkers responsible for the progression and spread of colorectal cancer (CRC) to better evaluate the prognosis of the patients, provide better management, and foster the development of therapeutic targets. In humans, pyrroline-5-carboxylate reductase 2 (PYCR2) is encoded on chromosome 1q42.12, and its metabolic activity has been linked to oncogenesis in many cancers. Zinc finger and broad-complex, tramtrack, and bric-à-brac (BTB) domain-containing protein 18 (ZBTB18), a zinc finger transcriptional repressor, has been found to have a tumor-suppressor role and to be methylated in CRCs. To date, the prognostic roles of PYCR2 and ZBTB18 in CRC patients have not been thoroughly studied. Objective To evaluate the tissue protein expression of PYCR2 and ZBTB18 in CRC and adjacent non-neoplastic intestinal tissues, to detect their roles in CRC carcinogenesis, progression and metastases. Patients and methods After applying the inclusion criteria, 60 CRC patients were included in the study. Tissue samples from the tumor and the adjacent non-neoplastic tissues were stained with PYCR2 and ZBTB18. The patients were followed up for about 30 months (range: 10 to 36 months). We performed a correlation regarding the expression of the markers, and clinicopathological and prognostic parameters. Results Upregulation of PYCR2 and downregulation of ZBTB18 were found to be higher in CRC tissue than in the adjacent non-neoplastic colonic mucosa (p = 0.026 and p &lt; 0.001 respectively). High expression of PYCR2 and low expression of ZBTB18 were positively correlated with large tumor size, higher tumor grade, advanced tumor stage, presence of spread to lymph nodes, and presence of distant metastases (p &lt; 0.001). High PYCR2 and low ZBTB18 expressions were significantly associated with poor response to therapy (p = 0.008 and 0.0.17 respectively), as well as high incidence of progression and recurrence (p = 0.005), and unfavorable overall survival (OS) rates (p = 0.001). Conclusion High expression of PYCR2 and low expression of ZBTB18 were independent predictors of CRC, progression, poor prognosis and unfavorable patient OS and progression-free survival (PFS) rates.

Interpretation of P16 expression as a marker of HPV in colorectal cancer.

Colorectal cancer is one of the most prevalent types of tumors worldwide. P16ᴵᴺᴷ⁴ᵃ is a widely used immunohistochemical marker for high-risk HPV infection. The purpose of this study is to explore the relationship between P16 expression as an indicator of HPV infection and colorectal cancer in Egyptian patients, as well as its association with histopathological characteristics. The study was performed on 59 cases of colorectal carcinoma cases and 30 specimens of normal colonic mucosa. p16 protein was detected in 22% (13 of 59) of patients with colorectal carcinoma. No evidence of P16 expression in all 30 cases of non-neoplastic colonic mucosa was found. More frequent expression of P16 was seen in distal carcinomas. our study demonstrated that P16 protein is expressed in a reasonable percent of colorectal carcinoma cases, suggesting a role of HPV in colorectal carcinogenesis. The present study highlights the role of p16 protein expression which is important in the pathogenesis in colorectal carcinoma, especially regarding distal tumors.

SPATA18 Expression Predicts Favorable Clinical Outcome in Colorectal Cancer.

Dysregulation of mitochondrial quality control has been reported to be associated with cancer and degenerative diseases. SPATA18 (spermatogenesis-associated 18, also known as Mieap) encodes a p53-inducible protein that can induce lysosome-like organelles within mitochondria that eliminate oxidized mitochondrial proteins and has tumor suppressor functions in mitochondrial quality control. In the present study, 268 primary colorectal cancers (CRCs) were evaluated immunohistochemically for SPATA18 expression to assess its predictive utility and its association with cellular proliferation activity. Furthermore, the association with p53 immunoreactivity, a surrogate marker for TP53 mutation, was analyzed. Non-neoplastic colonic mucosa showed cytoplasmic SPATA18 expression. Seventy-two percent of the lesions (193/268) displayed high SPATA18 expression in the cytoplasm of CRC cells. Univariate analyses revealed significant associations between SPATA18 expression and tumor size (p < 0.0001), histological differentiation (p = 0.0017), and lymph node metastasis (p = 0.00039). The log-rank test revealed that patients with SPATA18-high CRCs had significantly better survival than SPATA18-low patients (p < 0.0001). Multivariate Cox hazards regression analysis identified tubular-forming histology (hazard ratio [HR] = 0.25), age < 70 years (HR = 0.50), and SPATA18-high (HR = 0.55) as potential favorable factors. Lymph node metastasis (HR = 1.98) and peritoneal metastasis (HR = 5.45) were cited as potential independent risk factors. Cellular proliferation activity was significantly higher in SPATA18-high tumors. However, no significant correlation was detected between SPATA18 expression and p53 immunoreactivity or KRAS/BRAF mutation status. On the basis of our observations, SPATA18 immunohistochemistry can be used in the prognostication of CRC patients.

High Variability in Cellular Proliferation, Gene Expression, and Cytokine Production in the Nonneoplastic Colonic Epithelium of Young Apc+/Min-FCCC Mice.

An urgent need exists to identify efficacious therapeutic preventive interventions for individuals who are at high risk of developing colorectal cancer. To maximize the benefits of preventive intervention, it is vital to identify the time interval during which the initiation of a preventive intervention will lead to an optimal outcome. The goal of the present study was to determine if oncogenic events can be detected in the nonneoplastic colonic mucosa of Apc+/Min-FCCC mice prior to formation of the first adenoma, thus defining an earlier point of intervention along the cancer continuum. Tissues taken at three potential points of intervention were characterized: prior to Apc mutation (wild type Apc+/+-FCCC mice); after initiation but prior to colon adenoma formation (tumor-free Apc+/Min-FCCC mice); and after formation of the first colon adenoma (tumor-bearing Apc+/Min-FCCC mice). Experimentation focused on molecular processes that are dysregulated in early colon lesions: 1) cellular proliferation (proliferative index and size of the proliferative zone); 2) cellular stemness (expression of Ascl2, Grem1, Lgr5 and Muc2); 3) EGFR signaling (expression of Ereg); and 4) inflammation (expression of Mmp9, Ptsg2, and Reg4, as well as secretion of 18 cytokines involved in immune activation and response). Interestingly, the nonneoplastic colonic mucosa of wild type, tumor-free Apc+/Min-FCCC, and tumor-bearing Apc+/Min-FCCC mice did not display significant differences in average epithelial cell proliferation (fold change 0.8–1.3, p≥0.11), mucosal gene expression (fold change 0.8–1.4, p≥0.22), or secretion of specific cytokines from colonic mucosa (fold change 0.2–1.5, p≥0.06). However, the level of cytokine secretion was highly variable, with many (22% of wild type, 31% of tumor-free Apc+/Min-FCCC, and 31% of tumor-bearing Apc+/Min-FCCC) mice categorized as outliers (> 1.5 x interquartile ranges below the first quartile or above the third quartile) due to elevated expression of at least one cytokine. In summary, no differences were observed in proliferation, stemness, and EGFR signaling in the colonic mucosa of wild type vs Apc+/Min-FCCC mice, with low baseline cytokine expression, prior to the formation of the first colon adenoma. The results of this study provide valuable baseline data to inform the design of future cancer prevention studies.

Influence of MIF polymorphisms on CpG island hyper-methylation of CDKN2A in the patients with ulcerative colitis

BackgroundCDKN2A hypermethylation is among the major events associated with carcinogenesis and is also observed in non-neoplastic colonic mucosa in patients with ulcerative colitis (UC). Macrophage migration inhibitory factor (MIF) plays a crucial role in promoting gastrointestinal inflammation characteristic of UC. The aim of this study is to explore associations between CDKN2A methylation status and MIF polymorphisms (rs755622 and rs5844572).MethodsOne hundred and fifty-nine patients diagnosed with UC were enrolled in this study. The methylation status of p14ARF and p16INK4a was determined by MSP; MIF genotypes were identified by PCR-SSCP.ResultsWe found no differences with respect to mean age, gender, clinical type (chronic continuous or relapse/remitting), or extent of disease among the patients with methylated and unmethylated p14ARF or p16INK4a. Carrying the rs755622 C allele indicated a significantly higher risk for p14ARF methylation (odds ratio (OR), 2.16; 95% confidence interval (CI), 1.08–4.32; p = 0.030); similarly, carrying the rs5844572 7-repeat allele indicated a significantly higher risk for p16INK4a methylation (OR, 2.57; 95% CI, 1.26–5.24; p = 0.0094) after an adjusted regression analysis. The carriers of the rs755662 C allele or the rs5844572 7-repeat allele were both at a significantly higher risk for methylation of both p14ARF and p16INK4a when compared to the cohort in which neither of the genes were methylated (OR, 2.70; 95% CI, 1.22–6.01; p = 0.015 and OR, 2.87; 95% CI, 1.25–6.62; p = 0.013, respectively). Additionally, carrying rs755622 C allele was significantly associated with CIHM in chronic continuous of clinical type and total colitis (OR, 25.9; 95% CI, 2.55–262.6; p = 0.0059 and OR, 4.38; 95% CI, 1.12–17.2; p = 0.034, respectively), and carrying 7-repeat allele of rs5844572 was significantly associated in chronic continuous type (OR, 14.5; 95%CI, 1.46–144.3; p = 0.022).ConclusionsTaken together, our findings suggest that MIF genotypes associated with inflammation may also be involved in promoting carcinogenesis via CDKN2A hypermethylation in patients diagnosed with UC.

S1616 A Nonspecific Gastrointestinal Symptom Leads to a Rare Colonic Metastasis in a Patient With Remote History of Breast Cancer

INTRODUCTION: Breast cancer is the second most common malignancy in women and can metastasize to the lungs, bone, and liver; colonic metastases are exceedingly rare. We discuss a breast cancer survivor who presented with non-specific GI symptoms and was found to have metastatic breast cancer in the colon. CASE DESCRIPTION/METHODS: This is a 74-year-old female with a remote history of localized right breast cancer initially managed with breast-conserving surgery and radiation in the late 1980s, then maintained on an estrogen receptor (ER) antagonist before receiving a mastectomy in the late 1990s. She adhered to mammogram surveillance that confirmed her remission for the following two decades. At our clinic, she complained of decreased stool caliber and constipation refractory to over-the-counter laxatives. Symptoms were present for four weeks, associated with nausea, and without weight loss, abdominal pain, or blood in stool. She was up-to-date with colonoscopies. Exam revealed a soft abdomen with a firm focus near the umbilicus without tenderness on palpation. Colonoscopy revealed a region of erythematous friable mucosa with ulcerations at the transverse colon measuring 3 cm wide (Figure 1A). Pathology revealed a poorly differentiated carcinoma and GATA3, a breast carcinoma marker, was strongly positive (Figure 1B/D). E-cadherin was negative indicating this is most likely lobular carcinoma and ER, progesterone receptor, and HER-2 were all negative. Subsequent imaging confirmed metastases to the colon and retroperitoneum. She established care with oncology and has since been initiated on chemotherapy. DISCUSSION: Breast cancer metastases to the colon are a rare finding. The frequency at which metastases occur and the time at which they present depends on histologic subtype and hormone receptor status, respectively. Lobular carcinomas, as in our patient, tend to metastasize more than other subtypes. Also, hormone receptor-negative disease tends to metastasize earlier than hormone receptor-positive disease, with the former typically appearing within five years after initial diagnosis and the latter presenting well beyond that. Although our patient has lobular carcinoma, she also has triple-negative disease confirming exceptions to these rules. We present this case not only to highlight an atypical presentation of a common malignancy but to emphasize that providers should maintain a high index of suspicion for cancer recurrence when evaluating nonspecific gastrointestinal complaints in cancer survivors.Figure 1.: A. Endoscopic view of transverse colon demonstrating erythematous friable mucosa with ulceration. Ulcer measures three centimeters wide. B. Hematoxylin and Eosin stain: Ulcerated mucosa with underlying dense infiltrate of poorly differentiated neoplastic cells in the lamina propria. Of note, non-neoplastic colonic mucosa with unremarkable colonic glands/crypts are seen on the left of the dotted red line (arrow). C. Immunohistochemistry for CDX2. The neoplastic cells are negative for CDX-2, ruling out the possibility of adenocarcinoma of the GI tract. D. Immunohistochemistry for GATA 3. The neoplastic cells are positive for GATA3, suggesting breast origin of these neoplastic cells.

Class III β-tubulin Expression in Colorectal Neoplasms Is a Potential Predictive Biomarker for Paclitaxel Response.

The challenges of cololorectal cancer (CRC) management include prediction of outcome and drug response or chemoresistance. This study aimed at examining whether βIII-tubulin (TUBB3), present in various types of normal tissues and cancer, is a biomarker for the response of colorectal neoplasms to paclitaxel. Six tissue microarrays (TMAs) including 14 colon mucosa, 78 polyps and 202 CRCs were constructed. Assessment of TUBB3 expression was performed by immunohistochemistry, and it was scored as negative, focal and positive. In the HCT116 cell line, TUBB3 expression was silenced with siRNA. Paclitaxel toxicity was evaluated in TUBB3-silenced and control HCT116 cell lines. The non-neoplastic colon mucosa was negative for TUBB3, while some of colon adenomas and CRCs expressed TUBB3 in various levels from focal to diffuse. TUBB3-expressing CRCs tended to have poor prognosis and silencing of TUBB3 sensitized the cells to paclitaxel. TUBB3 was expressed in a subgroup of colorectal neoplasms. Suppression of TUBB3 potentialy sensitizes neoplastic cells to taxanes.

Combined activity of COX-1 and COX-2 is increased in non-neoplastic colonic mucosa from colorectal neoplasia patients

BackgroundCyclooxygenase (COX) activity is increased in endoscopic normal colonic mucosa from patients with colorectal neoplasia (CRN). COX-2 is thought to be the predominant COX isozyme involved in neoplasia. Meanwhile, relative contributions of COX-1 and COX-2 isoforms are unknown. Knowledge about their mutual activity in colonic mucosa is important for diagnostics and targeted therapy for CRN. The aim of this study was to assess the relative function, expression and localization of COX-1 and COX-2 enzymes in colonic non-neoplastic human mucosa and thereby to potentially reveal a mucosal disease predisposition for better treatment.MethodsBiopsies were pinched from normal appearing colonic mucosa in patients undergoing endoscopy. Ussing chamber technique was applied for an indirect assessment of epithelial activity, RT-qPCR for expression and immunohistochemistry for localization of COX-1 and COX-2 enzymes in patients without (ctrls) and with a history of CRN (CRN-pts).ResultsCombined COX-1 and COX-2 activity was higher in CRN-pts, p = 0.036. COX-2 was primarily localized in absorptive cells, while COX-1 appeared to be restricted to nonenteroendocrine tuft cells of the colonic epithelium.ConclusionsIn biopsies from endoscopic normal appearing colonic mucosa, combined activity of COX-1 and COX-2 enzymes is increased in CRN-pts compared with ctrls. This indicates that COX-1 and COX-2 together contribute to an increased proliferation process. Of note, in colonic epithelial cell lining, the COX-1 enzyme seems localized in tuft cells.

Giant filiform polyposis in a patient with inflammatory bowel disease presenting with intestinal obstruction

A 50-year-old known patient with ulcerative colitis with relapses and remissions presented with clinical features suggestive of bowel obstruction. Radiological studies confirmed an obstructive mass lesion in the hepatic flexure of the colon. Due to bowel obstruction and suspicion of malignancy an emergency laporatomy and partial ileo-colectomy was performed. A complex polypoid mass comprising numerous slender straight finger-like villi lined by non-neoplastic colonic mucosa with serrations at the base and prominent lamina proprial smooth muscle splaying was identified and a diagnosis of filiform polyposis was made. We report this unusual manifestation of an inflammatory reaction presenting as a colonic mass clinically and radiologically mimicking malignancy.

Identifying DCN and HSPD1 as Potential Biomarkers in Colon Cancer Using 2D-LC-MS/MS Combined with iTRAQ Technology.

Colon cancer is one of the most common types of gastrointestinal cancers and the fourth cause of cancer death worldwide. To discover novel diagnostic biomarkers for colon cancer and investigate potential mechanisms of oncogenesis, quantitative proteomic approach using iTRAQ-tagging and 2D-LC-MS/MS was performed to characterize proteins alterations in colon cancer and non-neoplastic colonic mucosa (NNCM) using laser capture microdissection-harvested from the two types of tissues, respectively. As a result, 188 DEPs were identified, and the differential expression of two DEPs (DCN and HSPD1) was further verified by Western blotting and immunohistochemistry. KEGG pathway analysis disclosed that the DEPs were related to signaling pathways associated with cancer; furthermore, DCN and HSPD1 are in the relative central hub position among protein-protein interaction subnetwork of the DEPs. The results not only shed light on the mechanism by the DEPs contributed to colonic carcinogenesis, but also showed that DCN and HSPD1 are novel potential biomarkers for the diagnosis of colon cancer.

Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC.

Background Promoterhypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC.ResultsThe expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC.MethodsColonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR.ConclusionsMethylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance.

Abstract 1053: Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC)

Abstract Background: Patients with Ulcerative colitis (UC) have more increased risk for the development of colorectal cancers compared with the average-risk population. Methods or strategies other than intensive colonoscopic surveillance to diagnose ulcerative colitis-associated colorectal carcinoma (UCAC) at early stage are limited. DNA methylation is deeply involved in carcinogenesis in many tumor types and its potential utility as a biomarker has been suggested. Aim: To evaluate potential utility of DNA methylation as the risk marker for UCAC, we quantified DNA methylation of inflamed rectal mucosa, neoplastic tissue and adjacent colonic mucosa of UC patients. Method: Inflamed rectal mucosa (n = 84) and normal appealing proximal mucosa (n = 10) of 84 cancer-free UC patients were examined. Neoplastic tissue (n = 33) and adjacent colonic mucosa (n = 26) from 16 UC patients with neoplasia (UCAC or DALM) were also studied. Bisulfite pyro sequencing was performed for the quantification of 45 cancer or age-related candidate panels and LINE1 repetitive element. Illumina Infinium Human Methylation 450 platform was also used to determine 470,000 CpG sites distributed across the genome. Results: Inflamed mucosa in UC was characterized as hyper methylation in candidate panels using the helical unsupervised clustering. In several panels, hyper methylation was associated with severe clinical phenotypes including steroid dependent, refractory, longstanding severe inflammation etc. (DPYS and miR-1247: p&amp;lt;0.05. GSTP1: p&amp;lt;0.01. mean of three: p&amp;lt;0.0001). Notably, in many panels, hyper methylation was significantly associated with longer duration of disease in cancer-free patients. Among them, some panels showed hyper methyhlation in both neoplastic and adjacent tissue compared to rectal mucosa of cancer-free cases (DPYS and N33, both p values &amp;lt;0.0001). Genome scale analysis revealed that UC with severe inflammation or neoplasia was characterized as hyper methylation at CpG sites rather than outside CpG sites. Gene ontology analysis demonstrated frequently methylated promoter CpG sites in these cases frequently encoded protein related to biosynthetic process (Fold enrichment: 2.07. p value using Benjamini correction: 5.69E-05). Conclusion: Hyper methylation at CpG sites of non-neoplastic colonic mucosa in UC with severe inflammation and neoplasia suggested the potential utility of DNA methytlation as a biomarker for prediction of UCAC. Moreover, enrichment of specific pathway in methylated genes suggests the possibility of targeting therapy for treating UCAC. Citation Format: Tomomitsu Tahara, Naoko Nakano, Mitsuo Nagasaka, Yoshihito Nakagawa, Tomoyuki Shibata, Naoki Ohmiya, Ichiro Hirata. Potential utility of DNA methytlation as a biomarker for prediction of ulcerative colitis associated colitic cancer (UCAC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1053. doi:10.1158/1538-7445.AM2015-1053

Telomere length in non-neoplastic colonic mucosa in ulcerative colitis (UC) and its relationship to the severe clinical phenotypes.

Telomere shortening occurs with human aging in many organs and tissues and is accelerated by rapid cell turnover and oxidative injury. To clarify the clinical importance of telomere shortening in colonic mucosa in ulcerative colitis (UC), we measured average telomere length using quantitative real-time PCR in non-neoplastic colonic mucosa in UC patients and assessed its relationship to various clinical subtypes. Relative telomere length in genomic DNA was measured in colonic biopsies obtained from rectal inflammatory mucosa from 86 UC patients as well as paired non-inflammatory proximal colonic mucosae from 10 patients. Data were correlated with various clinical phenotypes. In paired samples, average relative telomere length of rectal inflammatory mucosa was shortened compared to normal appearing proximal colon in eight out of ten cases (p=0.01). Telomere length shortening was significantly associated with more severe Mayo endoscopic subscore (p<0.0001) and cases needing surgery due to toxic megacolon or cancer occurrence (p=0.043). When the severe clinical phenotype was defined as having at least one of following phenotypes, more than two times of hospitalization, highest Mayo endoscopic subscore, steroid dependent, refractory, or needing operation, average relative telomere length was significantly shortened in the same phenotypes than the others (p=0.003). Telomere shortening is associated with more severe clinical phenotypes of UC, reflecting severe inflammatory state in the colonic mucosa.

Src Plays a Key Role in ADAM28 Expression in v-src–Transformed Epithelial Cells and Human Carcinoma Cells

ADAM28, a disintegrin and metalloproteinase 28, is overexpressed by carcinoma cells with direct correlations with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, the molecular mechanisms of ADAM28 gene expression in carcinoma cells remain elusive. Herein, we investigated the expression of ADAM28 in Madin-Darby canine kidney epithelial cells transformed by oncogenes, including v-src, LMP1, ErbB2, Ha-Ras, and c-Fos, and found that v-src transformants selectively induce ADAM28. Implantation of the v-src transformants showed a progressively growing tumor, which was significantly suppressed by local injections of anti-ADAM28 antibody. ADAM28 expression in v-src transformants was partially inhibited by treatment with inhibitors to Src kinase, mitogen-activated protein kinase kinase (MEK), phosphatidylinositol 3-kinase (PI3K), or mammalian target of rapamycin, and abrogated by v-Src kinase inhibitor, radicicol, or a mixture of MEK and PI3K inhibitors. Human carcinoma cell lines of the lung, breast, ovary, kidney, and colon showed ADAM28 expression, which was correlated with phosphorylation of c-Src and suppressed by the inhibitors in a similar way to v-src transformants. IHC of the human tumor tissues demonstrated co-expression of ADAM28 and phosphorylated Src in neoplastic cells of the breast, lung, and colon carcinomas and some adenomas of the colon, but not in nonneoplastic colon mucosa. Our data provide, to the best of our knowledge, the first evidence that Src is an inducer of ADAM28 gene expression through the MEK/extracellular signal-regulated kinase and PI3K/mammalian target of rapamycin pathways.

Identification of stromal differentially expressed proteins in the colon carcinoma by quantitative proteomics

Tumor microenvironment plays very important roles in the carcinogenesis. A variety of stromal cells in the microenvironment have been modified to support the unique needs of the malignant state. This study was to discover stromal differentially expressed proteins (DEPs) that were involved in colon carcinoma carcinogenesis. Laser capture microdissection (LCM) was captured and isolated the stromal cells from colon adenocarcinoma (CAC) and non-neoplastic colon mucosa (NNCM) tissues, respectively. Seventy DEPs were identified between the pooled LCM-enriched CAC and NNCM stroma samples by iTRAQ-based quantitative proteomics. Gene Ontology (GO) relationship analysis revealed that DEPs were hierarchically grouped into 10 clusters, and were involved in multiple biological functions that were altered during carcinogenesis, including extracellular matrix organization, cytoskeleton, transport, metabolism, inflammatory response, protein polymerization, and cell motility. Pathway network analysis revealed 6 networks and 56 network eligible proteins with Ingenuity pathway analysis. Four significant networks functioned in digestive system development and its function, inflammatory disease, and developmental disorder. Eight DEPs (DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ) were validated by Western blotting, and four DEPs (DCN, FN1, PKM2, and HSP90B1) were validated by immunohistochemical analysis. It is the first report of stromal DEPs between CAC and NNCM tissues. It will be helpful to recognize the roles of stromas in the colon carcinoma microenvironment, and improve the understanding of carcinogenesis in colon carcinoma. The present data suggest that DCN, FN1, PKM2, HSP90B1, S100A9, MYH9, TUBB, and YWHAZ might be the potential targets for colon cancer prevention and therapy.

Abstract 182: Differential response of epithelial cells from colorectal adenomas and adjacent nonneoplastic colonic mucosa to aspirin and atorvastatin in Apc+/Min-FCCC mice.

Abstract Although various agents have been identified that are efficacious in inhibiting colorectal adenomas in humans, the precise mechanisms responsible for their antitumor activity remain ill-defined. Significant insight into the use of agents for the prevention and treatment of colorectal cancer can be gained by examining the biological response of nonneoplastic colonic mucosa and colorectal adenomas to drug exposure. The present study compared the gene expression profile of neoplastic and nonneoplastic colonic epithelial cells following short-term administration of aspirin and atorvastatin to a strain of mice that uniquely develop multiple colorectal adenomas (Apc+/Min-FCCC). Tumor-bearing male Apc+/Min-FCCC mice (70 days of age) received either control Teklad 2018SX diet or diet supplemented with aspirin (300 ppm) or atorvastatin (100 ppm) for 7 days. At euthanasia, the colon was examined for gross lesions and matched pairs of colorectal adenomas and adjacent normal mucosa were frozen in OCT for gene expression analyses. Colonic epithelial cells were laser microdissected from pairs of pathologically-confirmed colorectal adenomas and nonneoplastic mucosa (4/treatment group). Amplified RNA was subjected to gene expression profiling using Agilent Mouse Whole Genome 4 X 44K microarrays. Genes differentially expressed in colon tissue from untreated vs. drug-treated mice were identified using LIMMA methodology and filtered (p = 0.01). The number of unique genes differentially expressed in neoplastic epithelial cells exposed to aspirin was 1052, while a much smaller set of genes (N=74) was altered in the nonneoplastic epithelium. Functional enrichment analyses revealed that the pathways modulated in nonneoplastic cells (i.e. protein folding, posttranscriptional regulation, DNA damage response, vascular permeability) were distinct from those altered in adenomas (i.e. cell adhesion, DNA repair, TOR signaling, apoptosis, glucose transport). In contrast, fewer genes were differentially expressed in neoplastic cells (N = 286) than in the adjacent nonneoplastic colonic mucosa (N = 749) following exposure to atorvastatin. The majority of the cellular processes modified in nonneoplastic cells by atorvastatin (i.e. DNA damage, cell death, immune function, cilium assembly) differed from those affected in neoplastic cells (i.e. lipid metabolism, apoptosis, cell cycle control, DNA repair). These data suggest that the mechanisms responsible for the protection conferred to nonneoplastic cells by aspirin or atorvastatin vary from those that lead to inhibition of tumor growth. Further mining of these expression data when combined with endoscopic monitoring of individual lesions during drug exposure should significantly enhance our understanding of the cellular processes responsible for their activity. (Supported by NIH N01 CN43309) Citation Format: Lisa Vanderveer, Suresh Paramasivam, Kara Williams, Harry Cooper, Karthik Devarajan, Suraj Peri, Yan Zhou, Laura Henik, Wen-Chi Chang, Ronald Lubet, Margie Clapper. Differential response of epithelial cells from colorectal adenomas and adjacent nonneoplastic colonic mucosa to aspirin and atorvastatin in Apc+/Min-FCCC mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 182. doi:10.1158/1538-7445.AM2013-182

The topography of DNA methylation in the non‐neoplastic colonic mucosa surrounding colorectal cancers

The degree of gene hypermethylation in non-neoplastic colonic mucosa (NNCM) is a potentially important event in the development of colorectal cancer (CRC), particularly for the subgroup with a CpG island methylator phenotype (CIMP). In this study, we aimed to use an unbiased and high-throughput approach to evaluate the topography of DNA methylation in the non-neoplastic colonic mucosa (NNCM) surrounding colorectal cancer (CRC). A total of 61 tissue samples comprising 53 NNCM and 8 tumor samples were obtained from hemicolectomy specimens of two CRC patients (Cases 1 and 2). NNCM was stripped from the underlying colonic wall and samples taken at varying distances from the tumor. The level of DNA methylation in NNCM and tumor tissues was assessed at 1,505 CpG sites in 807 cancer-related genes using Illumina GoldenGate® methylation arrays. Case 1 tumor showed significantly higher levels of methylation compared to surrounding NNCM samples (P < 0.001). The average level of methylation in NNCM decreased with increasing distance from the tumor (r = -0.418; P = 0.017), however this was not continuous and "patches" with higher levels of methylation were observed. Case 2 tumor was less methylated than Case 1 tumor (average β-value 0.181 vs. 0.415) and no significant difference in the level of methylation was observed in comparison to the surrounding NNCM. No evidence was found for a diminishing gradient of methylation in the NNCM surrounding CRC with a high level of methylation. Further work is required to determine whether CIMP+ CRC develop from within "patches" of NCCM that display high levels of methylation.

Tu1324 Telomere Length in Non-Neoplastic Colonic Mucosa in Ulcerative Colitis and Its Relationship to the Severe Clinical Phenotypes

Tu1324 Telomere Length in Non-Neoplastic Colonic Mucosa in Ulcerative Colitis and Its Relationship to the Severe Clinical Phenotypes

Characterization and significance of ACE2 and Mas receptor in human colon adenocarcinoma

A new arm of the renin-angiotensin system (RAS) has been recently characterized; this includes angiotensin converting enzyme (ACE)2 and angiotensin (Ang)1-7, a heptapeptide acting through the Mas receptor (MasR). Recent studies show that Ang1-7 has an antiproliferative action on lung adenocarcinoma cells. The aim of this study was to characterize RAS expression in human colon adenocarcinoma and to investigate whether Ang1-7 exerts an antiproliferative effect on human colon adenocarcinoma cells. Gene, protein expression and enzymatic activity of the main components of the RAS were determined on non-neoplastic colon mucosa as well as on the tumor mass and the mucosa taken 5 cm distant from it, both collected from patients with colon adenocarcinoma. Two different human colon cancer cell lines were treated with AngII and Ang1-7. The novel finding of this study was that MasR was significantly upregulated in colon adenocarcinoma compared with non-neoplastic colon mucosa, which showed little or no expression of it. ACE gene expression and enzymatic activity were also increased in the tumors. However, AngII and Ang1-7 did not have any pro-/antiproliferative effects in the cell lines studied. The data suggest that upregulation of the MasR could be used as a diagnostic marker of colon adenocarcinoma.