Non-neoplastic Colorectal Tissues Research Articles

HFE variants in colorectal cancer and their clinicopathological correlations

The study aimed to screen mutation of human homeostatic iron regulator (HFE) in colorectal carcinoma (CRC) and detect their associations with clinicopathological parameters. Expression of HFE was determined by quantitative polymerase chain reaction in matched CRC and non neoplastic colorectal mucosal tissue of 76 patients. Genomic DNA extracted were subjected to high high-resolution melt curve analysis and Sanger sequencing to detect mutations in HFE. The associations of the identified mutations with a variety of clinical features were determined. Approximately 60% of CRC showed low HFE expression. Of the ten 10 mutations identified in exons 2 and 4, c.187C>G (H63D), c845G>A (C282Y), c.193A>T (S65C), g.3828T>C, g.5795T>C, and g.5728G>A were known mutations. Four novel mutations were discovered; : c.184G>A, c.220T>G, c.322A>C, and c.324T>C. Heterozygous H63D and C282Y mutations were seen in 71% and 49% of cancer tissue, respectively. Tumour site (p=0.048) and gender (p=0.039) were significantly associated with H63D and C282Y mutation status, respectively. Local spread of cancer was significantly associated with C282Y mutations in CRC cancer and adjacent non-neoplastic tissue (p=0.029 & and p=0.004, respectively). There was a statistically significant association between H63D and C282Y negativity in matched non-neoplastic colorectal mucosa tissue and pathological staging of cancer (p=0.047 & and p=0.001, respectively). Patients with H63D and C282Y mutations in cancer tissue tend to have higher survival rates. Hence HFE mutations are common in CRC and are associated with clinicopathological parameters, implying the potential clinical significance of HFE mutations in colorectal carcinogenesis.

Loss of SDC1 Expression Is Associated with Poor Prognosis of Colorectal Cancer Patients in Northern China

Background Syndecan-1 (SDC1/CD138) is a key cell surface adhesion molecule essential for maintaining cell morphology and the interactions with the surrounding microenvironment. SDC1 tumor immunoexpression may be increased or decreased in epithelial malignant neoplasms compared to that in adjacent non-neoplastic tissue, depending on the type of carcinoma, and it has been correlated with various clinicopathological parameters and patient prognosis. SDC1 expression is decreased in colorectal cancer (CRC) tissue, but the relationship between prognosis and SDC1 expression in CRC patients is controversial. Methods In this study, SDC1 expression was detected in 65 adjacent non-neoplastic colorectal tissues, 477 CRCs, and 79 metastatic lymph nodes using tissue microarray. Results The data show that SDC1 decreased in CRC tissues (p ≤ 0.001) and metastatic lymph node tissues (p ≤ 0.001) compared to that in adjacent non-neoplastic colorectal tissues. Loss of SDC1 protein expression is associated with poor overall (p < 0.0001) and disease-free survival (p < 0.0001), differentiation (p = 0.017), stage (p ≤ 0.001), and lymph node metastasis (p ≤ 0.001) in CRC patients. Conclusions These data suggest that the loss of SDC1 plays an important role in CRC malignant progression. Loss of SDC1 expression indicates poor prognosis in patients from northern China with CRC.

Expression and correlation of phosphorylation extracellular signal-regulated kinase and monopolar spindle 1 in colorectal cancer with wild type or BRAF V600E mutation

Objective To determine the correlation between phosphorylation extracellular signal-regulated kinase (p-Erk) and monopolar spindle 1 (Mps1) in colorectal cancer patients with the BRAF V600E mutation or not, and to explore the relationship between the expression of p-Erk and Mps1 with the clinicopathological features and prognosis of colorectal cancer patients. Methods Two hundred and eighty-eight paraffin-embedded tissue sections containing both the carcinoma and its adjacent non-neoplastic colorectal tissue were collected from January 2009 to June 2015 in the First Hospital of Shanxi Medical University. BRAF mutation was detected by Sanger sequencing. Kaplan-Meier survival analysis was used to analyze the correlation between BRAF V600E and prognosis. Immunohistochemistry was used to detect the expression level of p-Erk and Mps1 in colorectal cancer with wild type or BRAF V600E mutation. The correlation between p-ERK and Mps1 expression was analyzed by using linear regression analysis. Results The BRAF V600E mutation rate was 5.2% in colorectal carcinomas. In addition, the poorly differentiated tumours and mucinous tumours had higher incidence of BRAF mutations than well differentiated tumours and non-mucinous tumours respectively [14.3% (9/63) vs. 2.7% (6/225), χ2 = 11.208, P = 0.001; 25.0% (6/24) vs. 3.4% (9/264), χ2 = 16.630, P <0.001). The positive rate of p-Erk and Mps1 in colorectal carcinomas with BRAF V600E was significantly higher than that in colorectal carcinomas with BRAF WT (14/15 vs. 3/15, P < 0.05; 15/15 vs. 3/15, P < 0.05]. It was found that the p-Erk expression correlated positively to the Mps1 expression (R 2 = 0.419, P < 0.001). The expressions of p-Erk protein in poorly differentiated adenocarcinoma was significantly higher and mucinous adenocarcinoma than those in high differentiated adenocarcinoma and non-mucinous adenocarcinoma (χ 2 = 6.679, P = 0.01; χ 2 = 5.735, P = 0.017), as well as in the group with lymph node metastasis than without lymph node metastasis (χ 2 = 5.436, P = 0.02). Positive rate of Mps1 in poorly differentiated carcinoma was higher than that in well differentiated adenocarcinoma of colorectal carcinomas (χ 2 = 7.950, P = 0.009). The Kaplan-Meier survival analysis indicated that patients with BRAF V600E had a worse survival rate than BRAF WT patients. Conclusions BRAF V600E may play an important role in specific pathological kinds of colorectal carcinomas, which is expected to be an independent prognostic factor. The expression of p-Erk is significantly correlated with Mps1 in colorectal carcinomas, suggesting that Mps1 may become a new potential target for targeted therapy. Key words: Colorectal neoplasms; BRAF V600E mutation; Phosphorylation extracellular signal-regulated kinase; Monopolar spindle 1

Clinical and biological significance of miR-193a-3p targeted KRAS in colorectal cancer pathogenesis

This study was to investigate the expression pattern, mechanisms and clinicopathological implications of miR-193a-3p in colorectal cancer. Fresh-frozen tissues from 70 matched colorectal adenocarciomas and the adjacent non-neoplastic mucosae were prospectively collected. Two colorectal cancer cell lines (SW480 and SW48) and a non-neoplastic colon cell line (FHC) were also used. The expression levels of miR193a-3p in the cells and tissues were measured by quantitative real-time polymerase chain reaction. The expression of KRAS protein as a predicted downstream target for miR-193a was studied by immunohistochemistry. Restoration of the miR-193a level in the cell lines by permanent transfection was achieved and multiple functional and immunological assays were performed to analyze the functions of miR-193a in vitro. Down-regulation of miR-193a-3p was noted in 70% of the colorectal cancer tissues when compared to non-neoplastic colorectal tissues. In addition, down-regulation of miR-193a was significantly correlated with carcinoma of early stages (P<.05). Significant inverse correlation between miR-193a-3p and its target KRAS protein was determined (P<.05). Overexpression of miR-193a in colon cancer cells resulted in reduced cell proliferation, increased apoptosis, induced significant changes in cell cycle events and decreased the expression of epithelial-mesenchymal transition marker TWIST. This study confirms the tumor suppressor roles of miR-193a-3p, its downstream target affinity to KRAS and clinical significance in patients with colorectal adenocarcinoma.

MicroRNA-186-5p overexpression modulates colon cancer growth by repressing the expression of the FAM134B tumour inhibitor

ObjectivesThe role and underlying mechanism of miR-186-5p in colorectal cancer remain unknown. The present study aims to examine the various cellular effects of miR-186-5p in the carcinogenesis of colorectal cancer. Also, the interacting targets and association of clinicopathological factors with miR-186-5p expression in patients with colorectal cancer were analysed. MethodsThe miR-186-5p expression levels in colorectal cancer tissues (n=126) and colon cancer cell lines (n=3) were analysed by real-time PCR. Matched non-neoplastic colorectal tissues and a non-neoplastic colonic epithelial cell line were used as controls. Various in vitro assays such as cell proliferation, wound healing and colony formation assays were performed to examine the miR-186-5p specific cellular effects. Western blots and immunohistochemistry analysis were performed to examine the modulation of FAM134B, PARP9 and KLF7 proteins expression. ResultsSignificant high expression of miR-186-5p was noted in cancer tissues (p< 0.001) and cell lines (p<0.05) when compared to control tissues and cells. The majority of the patients with colorectal cancer (88/126) had shown overexpression of miR-186-5p. This miR-186-5p overexpression was predominantly noted with in cancer with distant metastasis (p=0.001), lymphovascular permeation (p=0.037), microsatellite instability (MSI) stable (p=0.015), in distal colorectum (p=0.043) and with associated adenomas (p=0.047). Overexpression of miR-186-5p resulted in increased cell proliferation, colony formation, wound healing capacities and induced alteration of cell cycle kinetics in colon cancer cells. On the other hand, inhibition of endogenous miR-186-5p reduced the cancer growth properties. miR-186-5p overexpression reduced FAM134B expression significantly in the cancer cells (p<0.01). Also, FAM134B and miR-186-5p expressions are inversely correlated in colorectal cancer tissues and cells. ConclusionThe miR-186-5p expression promotes colorectal cancer pathogenesis by regulating tumour suppressor FAM134B. Reduced cancer cells growth followed by inhibition of miR-186-5p highlights the potential of miR-186-5p inhibitor as a novel strategy for targeting colorectal cancer initiation and progression.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry.

ABSTRACTObjective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression.Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’s t test.Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03).Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

Role of Raf-kinase inhibitor protein in colorectal cancer and its regulation by hydroxycamptothecine.

BackgroundRecently accumulated evidence suggests that Raf kinase inhibitor protein (RKIP) participates in regulation of many signaling pathways and plays an important role in tumorigenesis and tumor metastasis. However, studies investigating the role of RKIP in colorectal cancer have not been reported. The aim of this study was to investigate the role of RKIP on colorectal cancer cell differentiation, progression and its correlation with chemosensitivity.ResultsImmunohistochemical analysis revealed that RKIP expression was higher in non-neoplastic colorectal tissue (NCRCT) and colorectal cancer tissue (CRCT) than that in metastatic lymph node tissue (MLNT) (P <0.05). P-ERK protein expression was higher in MLNT and CRCT than that in NCRCT (P = 0.02). Immunocytochemical analysis further revealed that RKIP expression was higher in the well differentiated cell line SW1116 as compared to that in the poorly differentiated cell line LoVo. Matrigel invasive assay demonstrated that the inhibition of RKIP by short hairpin RNA (shRNA) 271 transfection significantly increased the number of migrated cells (90.67 ± 4.04 vs. 37.33 ± 2.51, P <0.05), whereas over-expression of RKIP by PEBP-1 plasmid transfection significantly suppressed the number of migrated cells (79.24 ± 5.18 vs. 154.33 ± 7.25, P <0.05). Meanwhile, down-regulation of RKIP induced an increase in the cell survival rate by inhibiting apoptosis induced by hydroxycamptothecine.ConclusionsRKIP was also found to be associated with cell differentiation, with a higher activity in well differentiated colorectal cancer cells than in poorly differentiated ones. The upregulated expression of RKIP in colorectal cancer cells inhibited cell invasion and metastasis, while downregulation of RKIP reduced chemosensitivity by inhibiting apoptosis induced by HCPT.

Immunohistochemical study of the canonical and non-canonical Wnt signaling pathway in colorectal carcinoma and non-neoplastic mucosa

Colorectal cancer is linked to several signaling pathways such as Wnt pathway. Our objective is to detect and verify the integrity of protein members of Wnt signaling pathway in colorectal carcinoma and non-neoplastic colorectal tissue. Sixty-four patients with colorectal carcinoma provided samples of colorectal neoplasia and non-neoplastic tissues, which were prepared in tissue microarray blocks and subjected to immunohistochemical analysis. The primary antibodies used were Wnt-1, Wnt-2, Wnt-5a Frizzled-1, Frizzled-5 and axin. Immunoexpression of Wnt-2 protein was significantly lower in colorectal tumor tissue and axin protein immunoexpression was significantly higher in tumor tissue. There was no significant difference in the expression of Wnt-1, Wnt-5a, Frizzled-1 and Frizzled-5 proteins in both tissues. The higher expression of Wnt-2 protein in non-neoplastic colorectal tissue suggests the participation during the hyperproliferative stage of colorectal mucosa. The increased axin protein immunoexpression in colorectal tumor suggests a decrease in the formation of the β-catenin destructor complex.

Immunohistochemistry expression of TCF4 protein on carcinoma, adenoma and non neoplastic colorectal mucosa

PurposeTo detect and quantify the immunoreactivity of TCF4 protein in colorectal carci- noma, colorectal adenoma and non-neoplasic colorectal epithelium. MethodsWe studied 129 individuals: 40 with colorectal cancer, 52 with colorectal ad- enoma and 37 with non-neoplastic colorectal epithelium. The colorectal adenoma and carcinoma samples were obtained from patients who underwent surgical procedures, and colonoscopies and samples of non-neoplastic colorectal epithelium were taken from patients who died from cardiovascular diseases, without diseases of the large intestine. Samples of different tissues were included in paraffin blocks, and the immunohistochem- ical expression of protein TCF4 was analyzed using the technique of tissue microarray (TMA) with polyclonal antibody TCF4. The immunoreactivity was analyzed and classified as positive and negative. ResultsThe immunohistochemical expression of TCF4 protein was significantly higher (p < 0.01) in colorectal carcinoma than in the non-neoplastic colorectal epithelium and adenoma. There was no difference (p = 0.76) between TCF4 protein immunohistochemical expression in colorectal adenoma and non-neoplastic colorectal tissue. ConclusionsTCF4 protein showed a more intense expression in colorectal carcinoma than in non-neoplastic colorectal epithelium and adenoma, indicating that this protein is in- volved in colorectal carcinogenesis.

A panel of genes methylated with high frequency in colorectal cancer.

BackgroundThe development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests.MethodsCombined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP).ResultsCombined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas.ConclusionsThis study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes (SOX21, SLC6A15, NPY, GRASP, ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes (BCAT1, COL4A2, DLX5, FGF5, FOXF1, FOXI2, GRASP, IKZF1, IRF4, SDC2 and SOX21) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Abstract 4134: miR1288 in colorectal cancers: Altered expression and its clinicopathological significance

Abstract Introduction: MicroRNA-1288 (miR-1288) is a non-coding RNA located in 17p11.2 and not been studied solely in cancer. We aim to examine the miR-1288 expression in a large cohort of patients with colorectal carcinoma, adenoma and non-neoplastic colorectal tissues. The implications of miR-1288 expression and clinicopathological features were also studied. Methods: Tissues from 102 patients with surgical resection of colorectum (82 adenocarcinomas, 10 adenomas and 10 non-neoplastic tissues), two colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. miRNA was extracted from all these samples and were converted in to cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for the detection of miR-1288 expression. The results were correlated with the clinical and pathological data. Results: miR-1288 expression was decreased in majority of colorectal adenocarcinoma when compared to colorectal adenoma and non-neoplastic tissues. Reduced or absence expression of miR-1288 was noted in 78% (n=64) of the cancers. The colon cancer cell lines showed reduced expression of miR-1288 compared to normal colonic epithelial cell line. Distal colorectal cancers showed higher expression levels of miR-1288 than proximal cancers (p= 0.007). The expression levels of miR-1288 were significantly altered in different levels of cancer invasion (T staging) (p= 0.023). Conclusions: Altered expression pattern of miR-1288 in colorectal cancers compare to non-neoplastic tissues was identified. Also, differential regulation of miR-1288 was found to be related to location and pathological staging of colorectal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4134. doi:1538-7445.AM2012-4134

Overexpression of EIF5A2 promotes colorectal carcinoma cell aggressiveness by upregulating MTA1 through C-myc to induce epithelial–mesenchymaltransition

Background and AimsThe authors have previously isolated a putative oncogene, eukaryotic initiation factor 5A2 (EIF5A2) from 3q26. In this study, EIF5A2 was characterised for its role in colorectal carcinoma (CRC)...

GAEC1 and colorectal cancer: a study of the relationships between a novel oncogene and clinicopathologic features

GAEC1 is a novel gene located at 7q22.1 that was detected in our previous work in esophageal cancer. The aims of the present study are to identify the copy number of GAEC1 in different colorectal tissues including carcinomas, adenomas, and nonneoplastic tissues and characterize any links to pathologic factors. The copy number of GAEC1 was studied by evaluating the quantitative amplification of GAEC1 DNA in 259 colorectal tissues (144 adenocarcinomas, 31 adenomas, and 84 nonneoplastic tissues) using real-time polymerase chain reaction. Copy number of GAEC1 DNA in colorectal adenocarcinomas was higher in comparison with nonneoplastic colorectum. Seventy-nine percent of the colorectal adenocarcinomas showed amplification and 15% showed deletion of GAEC1 (P < .0001). Of the adenomas, 90% showed deletion of GAEC1, with the remaining 10% showing normal copy number. The differences in GAEC1 copy number between colorectal adenocarcinoma, colorectal adenoma, and nonneoplastic colorectal tissue are significant (P < .0001). GAEC1 copy number was significantly higher in adenocarcinomas located in distal colorectum compared with proximal colon (P = .03). In conclusion, GAEC1 copy number was significantly different between colorectal adenocarcinomas, adenomas, and nonneoplastic colorectal tissues. The copy number was also related to the site of the cancer. These findings along with previous work in esophageal cancer imply that GAEC1 is commonly involved in the pathogenesis of colorectal adenocarcinoma.

Expression of melanoma antigen-encoding genes (MAGE) by common primers for MAGE-A1 to -A6 in colorectal carcinomas among Koreans.

This study was to investigate Melanoma-antigen gene (MAGE) expression by reverse transcription-nested polymerase chain reaction (RT-nested PCR) with the original common primers of MAGE-A1 to -A6 and analysis of correlation between its expression and the well-known clinical parameters in addition to evaluate the clinical feasibility of the common primers. Surgical tumor and corresponding nonneoplastic tissue samples from 38 patients with colorectal cancer were studied. To confirm the identities of RT-PCR products, direct sequencing was done after in vitro subcloning. No expression of MAGE was observed in the non-neoplastic colorectal mucosal tissues. Sixteen (42.1%) of 38 carcinomas expressed at least one of MAGE A-1 to -6. The expression of the MAGE genes was not related to age, sex, histological grades, the depth of invasion, metastasis to lymph nodes, vessel, neural, or perineural invasion. The identities with the corresponding mRNAs were confirmed in 6 cases for MAGE-A2 (15.8%), 6 cases for MAGE-A4 (15.8%), 2 cases for MAGE-A3 (5.3%), and one case for MAGE A-6 (2.6%). These results suggest that MAGE expressions, except those of MAGE-A2 and -A4, seem to have a limited role in the molecular pathogenesis of colon cancer. However, the common primer sets to detect of expressions for MAGE-A1 to -A6 simultaneously appear to be feasible to differentiate malignant from benign lesions in colorectal diseases.

Biochemistry and pathological importance of mucin-associated antigens in gastrointestinal neoplasia

Publisher Summary This chapter focuses on mechanisms of alterations of Mucin-Associated Antigens in gastrointestinal neoplasia and their implications in clinical diagnostic and therapeutic oncology. The chapter focuses on real mucins (MUC), taking MUC1 and MUC2 as models. In addition, the chapter defines mucins and discusses the biochemistry and molecular biology of mucins, such as structural aspects of mucins, biosynthesis of mucins, glycosylation site specificity and biosynthetic routes of O-glycosylation, and antigenicity of mucins. During carcinogenesis, alterations of mucin peptide and mucin glycosylation occur. Although glycosylation of proteins should be dependent on the rate of synthesis, and overexpression of mucin species in certain cancers is associated with reduced glycosylation, there is no clear-cut evidence for a causal relationship between these events. The chapter provides an account for the expression of mucin-asssociated antigens in gastric tissues (in non-neoplastic and neoplastic gastric tissues); and expression in colorectal tissues (in non-neoplastic colorectal tissues and colorectal adenoma-carcinoma sequence). Mucin-associated peptide and carbohydrate antigens play an important role in different aspects of proliferation, local invasion, and metastasis of cancer cells. Thus, the carbohydrate antigens gained clinical importance as prognostic markers in a great variety of human carcinomas. Serum tumor markers (circulating antigens) also help in clinical tumor diagnostics. The chapter concludes with the perspectives in tumor immunology and cancer therapy. Mucin peptide cores, in addition to substituting carbohydrate structures, are involved in different types of cellular and humoral immune response in cancer. Biosynthesized glycopeptides containing clustered saccharide domains and peptides that mimic carbohydrates may be applied in future studies on therapy of gastrointestinal carcinomas.