miR-181b functions as an oncomiR in colorectal cancer by targeting PDCD4

Programmed cell death 4 (PDCD4) is a RNA-binding protein that acts as a tumor suppressor in many cancer types, including colorectal cancer (CRC). During CRC carcinogenesis, PDCD4 protein levels remarkably decrease, but the underlying molecular mechanism for decreased PDCD4 expression is not fully understood. In this study, we performed bioinformatics analysis to identify miRNAs that potentially target PDCD4. We demonstrated miR-181b as a direct regulator of PDCD4. We further showed that activation of IL6/STAT3 signaling pathway increased miR-181b expression and consequently resulted in downregulation of PDCD4 in CRC cells. In addition, we investigated the biological effects of PDCD4 inhibition by miR-181b both in vitro and in vivo and found that miR-181b could promote cell proliferation and migration and suppress apoptosis in CRC cells and accelerate tumor growth in xenograft mice, potentially through targeting PDCD4. Taken together, this study highlights an oncomiR role for miR-181b in regulating PDCD4 in CRC and suggests that miR-181b may be a novel molecular therapeutic target for CRC.     

Genetic Variability in Key Genes in Prostaglandin E2 Pathway (COX-2, HPGD,ABCC4 and SLCO2A1) and Their Involvement in Colorectal Cancer Development

The pro-carcinogenic effects of prostaglandin E2 (PGE₂) in colonic mucosa are not only regulated by the rates between Cyclooxygenase-2 (COX-2) biosynthesis and 15-Hydroxyprostaglandin Dehydrogenase (15-PGDH)-dependent degradation but also the steady-state levels of PGE₂ in extracellular microenvironment, maintained by key specific prostaglandin transporters, the Multidrug Resistance Protein (MRP4) (efflux carrier) and Prostaglandin Transporter (PGT) (influx carrier). To understand the contribution of genetic variability in genes coding for COX-2/15-PGDH/MRP4/PGT proteins in CRC development, we conducted a hospital-based case-control study involving 246 CRC patients and 480 cancer-free controls. A total of 51 tagSNPs were characterized using the Sequenom platform through multiplexed amplification followed by mass-spectrometric product separation or allelic discrimination using real-time PCR. Seven tagSNPs were implicated in CRC development: the rs689466 in COX-2 gene, the rs1346271 and rs1426945 in 15-PGDH, the rs6439448 and rs7616492 in PGT and rs1751051 and rs1751031 in MRP4 coding genes. Upon a stratified analysis a measurable gene-environment interaction was noticed between rs689466 and smoking habits, with individuals ever-smokers carriers of rs689466 GG homozygous genotype having a nearly 6-fold increased susceptibility for CRC onset (95%CI: 1.49–22.42, P = 0.011). Furthermore, the multifactor dimensionality reduction (MDR) analysis identified an overall four-factor best gene-gene interactive model, including the rs1426945, rs6439448, rs1751051 and rs1751031 polymorphisms. This model had the highest cross-validation consistency (10/10, P<0.0001) and an accuracy of 0.6957 and was further associated with a 5-fold increased risk for CRC development (95%CI: 3.89–7.02, P<0.0001). In conclusion, specific low penetrance genes in the pro-carcinogenic PGE₂ pathway appear to modulate the genetic susceptibility for CRC development. A clearer understanding on CRC etiology through the identification of biomarkers of colorectal carcinogenesis might allow a better definition of risk models that are more likely to benefit from targeted preventive strategies to reduce CRC burden.     

Relationship between Circulating and Tissue microRNAs in a Murine Model of Breast Cancer

MiRNAs are key regulators of tumorigenesis that are aberrantly expressed in the circulation and tissue of patients with cancer. The aim of this study was to determine whether miRNA dysregulation in the circulation reflected similar changes in tumour tissue. Athymic nude mice (n = 20) received either a mammary fat pad (n = 8, MFP), or subcutaneous (n = 7, SC) injection of MDA-MB-231 cells. Controls received no tumour cells (n = 5). Tumour volume was monitored weekly and blood sampling performed at weeks 1, 3 and 6 following tumour induction (total n = 60). Animals were sacrificed at week 6 and tumour tissue (n = 15), lungs (n = 20) and enlarged lymph nodes (n = 3) harvested. MicroRNAs were extracted from all samples (n = 98) and relative expression quantified using RQ-PCR. MiR-221 expression was significantly increased in tumour compared to healthy tissue (p<0.001). MiR-10b expression was significantly higher in MFP compared to SC tumours (p<0.05), with the highest levels detected in diseased lymph nodes (p<0.05). MiR-10b was undetectable in the circulation, with no significant change in circulating miR-221 expression detected during disease progression. MiR-195 and miR-497 were significantly decreased in tumour tissue (p<0.05), and also in the circulation of animals 3 weeks following tumour induction (p<0.05). At both tissue and circulating level, a positive correlation was observed between miR-497 and miR-195 (r = 0.61, p<0.001; r = 0.41, p<0.01 respectively). This study highlights the distinct roles of miRNAs in circulation and tissue. It also implicates miRNAs in disease dissemination and progression, which may be important in systemic therapy and biomarker development.     

Concomitant Tumor Expression of EGFR and TATI/SPINK1 Associates with Better Prognosis in Colorectal Cancer

Background

Epidermal growth factor receptor (EGFR) activation plays a role in colorectal cancer (CRC) carcinogenesis, and anti-EGFR drugs are used in treatment of advanced CRC. One of the EGFR ligands is tumor-associated trypsinogen inhibitor TATI, also called serine protease inhibitor Kazal type1 (SPINK 1), which we recently showed to be an independent prognostic marker in CRC.

Methods

We studied the prognostic value of immunohistochemical expression of EGFR and concomitant expression of EGFR and TATI/SPINK1 in a series of 619 colorectal cancer patients.

Results

Of the samples, 92% were positive for EGFR. EGFR+/TATI+ was seen in 62.8%, EGFR+/TATI− in 29.5%, EGFR−/TATI+ in 4.9%, and EGFR−/TATI− in 2.7% of patients. EGFR expression correlated with WHO grade (p = 0.040). In univariate analysis, EGFR expression correlated with favourable survival (p = 0.006). EGFR+/TATI+ patients showed better survival than did those with other combinations (p<0.001). In multivariate analysis, EGFR+/TATI+ was an independent prognostic factor of favourable prognosis (p<0.001).

Conclusion

Concomitant positivity of EGFR and TATI/SPINK1 predicts favourable prognosis in CRC.     

Meta-Analysis of the Association between COX-2 Polymorphisms and Risk of Colorectal Cancer Based on Case–Control Studies

Objective

Cyclooxygenase-2 (COX-2) is an inducible enzyme converting arachidonic acid to prostaglandins and playing important roles in inflammatory diseases as well as tumor development. Previous studies investigating the association between COX-2 polymorphisms and colorectal cancer (CRC) risk reported conflicting results. We performed a meta-analysis of all available studies to explore this association.

Methods

All studies published up to October 2013 on the association between COX-2 polymorphisms and CRC risk were identified by searching electronic databases PubMed, EMBASE, and Cochrane library. The association between COX-2 polymorphisms and CRC risk was assessed by odds ratios (ORs) together with their 95% confidence intervals (CIs).

Results

Ten studies with 6,774 cases and 9,772 controls were included for −1195A>G polymorphism, 13 studies including 6,807 cases and 10,052 controls were available for −765G>C polymorphism, and 8 studies containing 5,121 cases and 7,487 controls were included for 8473T>C polymorphism. With respect to −765G>C polymorphism, we did not find a significant association with CRC risk when all eligible studies were pooled into the meta-analysis. However, in subgroup analyses by ethnicity and cancer location, with a Bonferroni corrected alpha of 0.05/2, statistical significant increased CRC risk was found in the Asian populations (dominant model CC+CG vs. GG: OR = 1.399, 95%CI: 1.113–1.760, P = 0.004) and rectum cancer patients (CC vs. GG: OR = 2.270, 95%CI: 1.295–3.980, P = 0.004; Recessive model CC vs. CG+GG: OR = 2.269, 95%CI: 1.297–3.970, P = 0.004). In subgroup analysis according to source of control, no significant association was detected. With respect to −1195A>G and 8473T>C polymorphisms, no significant association with CRC risk was demonstrated in the overall and subgroup analyses.

Conclusions

The present meta-analysis suggests that the COX-2 −765G>C polymorphism may be a risk factor for CRC in Asians and rectum cancer patients. Further large and well-designed studies are needed to confirm this association.     

The Knowledge of Colorectal Cancer Symptoms and Risk Factors among 10,078 Screening Participants: Are High Risk Individuals More Knowledgeable?

Objectives

We tested the a priori hypothesis that self-perceived and real presences of risks for colorectal cancer (CRC) are associated with better knowledge of the symptoms and risk factors for CRC, respectively.

Methods

One territory-wide invitation for free CRC screening between 2008 to 2012 recruited asymptomatic screening participants aged 50–70 years in Hong Kong. They completed survey items on self-perceived and real presences of risks for CRC (advanced age, male gender, positive family history and smoking) as predictors, and knowledge of CRC symptoms and risk factors as outcome measures, respectively. Their associations were evaluated by binary logistic regression analyses.

Results

From 10,078 eligible participants (average age 59 years), the mean knowledge scores for symptoms and risk factors were 3.23 and 4.06, respectively (both score range 0–9). Male gender (adjusted odds ratio [AOR] = 1.34, 95% C.I. 1.20–1.50, p<0.01), self-perception as not having any risks for CRC (AOR = 1.12, 95% C.I. 1.01–1.24, p = 0.033) or uncertainty about having risks (AOR = 1.94, 95% C.I. 1.55–2.43, p<0.001), smoking (AOR 1.38, 95% C.I. 1.11–1.72, p = 0.004), and the absence of family history (AOR 0.61 to 0.78 for those with positive family history, p<0.001) were associated with poorer knowledge scores (≤4) of CRC symptoms. These factors remained significant for knowledge of risk factors.

Conclusions

Male and smokers were more likely to have poorer knowledge but family history of CRC was associated with better knowledge. Since screening of these higher risk individuals could lead to greater yield of colorectal neoplasm, educational interventions targeted to male smokers were recommended.     

Prognostic Significance of COX-2 Immunohistochemical Expression in Colorectal Cancer: A Meta-Analysis of the Literature

Background

Cyclooxygenase-2 (COX-2) is believed to be an important enzyme in the pathogenesis of colorectal cancer (CRC). Correlations between the expression of COX-2 with tumor growth and distant metastasis have become an issue; thus, attention has been paid to COX-2 as a prognostic factor. Various studies examined the relationship between COX-2 immunohistochemistry (IHC) overexpression with the clinical outcome in patients with colorectal cancer, but yielded conflicting results. The prognostic significance of COX-2 overexpression in colorectal cancer remains controversial.

Methods

Electronic databases updated to October 2012 were searched to find relevant studies. A meta-analysis was conducted with eligible studies which quantitatively evaluated the relationship between COX-2 overexpression and survival of patients with colorectal cancer. Survival data were aggregated and quantitatively analyzed.

Results

We performed a meta-analysis of 23 studies (n  =  4567 patients) that evaluated the correlation between COX-2 overexpression detected by IHC and survival in patients with colorectal cancer. Combined hazard ratios suggested that COX-2 overexpression had an unfavorable impact on overall survival (OS) (HR [hazard ratio]  =  1.193, 95% CI [confidence interval]: 1.02 ∼ 1.37), but not disease free survival (DFS) (HR  =  1.25, 95% CI: 0.99 ∼ 1.50) in patients with colorectal cancer.

Conclusions

Cox-2 overexpression in colorectal cancer detected by IHC appears to have slightly worse overall survival. However, the prognostic value of COX-2 on survival in colorectal cancer still needs further large-scale prospective trials to be clarified.     

Stool Microbiome and Metabolome Differences between Colorectal Cancer Patients and Healthy Adults

In this study we used stool profiling to identify intestinal bacteria and metabolites that are differentially represented in humans with colorectal cancer (CRC) compared to healthy controls to identify how microbial functions may influence CRC development. Stool samples were collected from healthy adults (n = 10) and colorectal cancer patients (n = 11) prior to colon resection surgery at the University of Colorado Health-Poudre Valley Hospital in Fort Collins, CO. The V4 region of the 16s rRNA gene was pyrosequenced and both short chain fatty acids and global stool metabolites were extracted and analyzed utilizing Gas Chromatography-Mass Spectrometry (GC-MS). There were no significant differences in the overall microbial community structure associated with the disease state, but several bacterial genera, particularly butyrate-producing species, were under-represented in the CRC samples, while a mucin-degrading species, Akkermansia muciniphila, was about 4-fold higher in CRC (p<0.01). Proportionately higher amounts of butyrate were seen in stool of healthy individuals while relative concentrations of acetate were higher in stools of CRC patients. GC-MS profiling revealed higher concentrations of amino acids in stool samples from CRC patients and higher poly and monounsaturated fatty acids and ursodeoxycholic acid, a conjugated bile acid in stool samples from healthy adults (p<0.01). Correlative analysis between the combined datasets revealed some potential relationships between stool metabolites and certain bacterial species. These associations could provide insight into microbial functions occurring in a cancer environment and will help direct future mechanistic studies. Using integrated “omics” approaches may prove a useful tool in identifying functional groups of gastrointestinal bacteria and their associated metabolites as novel therapeutic and chemopreventive targets.     

LncRNA HAND2-AS1 inhibits 5-fluorouracil resistance by modulating miR-20a/PDCD4 axis in colorectal cancer

BackgroundChemoresistance is one of the main obstacles in the therapy of human cancers, including colorectal cancer (CRC). Long non-coding RNA heart and neural crest derivatives expressed 2-antisense RNA 1 (lncRNA HAND2-AS1) has been demonstrated to be associated with CRC. However, the function of HAND2-AS1 in 5-Fluorouracil (5-FU) resistance of CRC remains unclear.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression of HAND2-AS1, miR-20a and programmed cell death factor 4 (PDCD4) mRNA. 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate IC50 of 5-FU and cell proliferation. Flow cytometry analysis was used to determine cell apoptosis. Transwell assay was carried out to measure cell migration and invasion. Western blot assay was conducted to examine the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-Associated X (Bax), matrix metalloprotein 2 (MMP2), MMP9 and PDCD4. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay were utilized to verify the combination between miR-20a and HAND2-AS1. Dual-luciferase reporter assay was used to analyze the association between miR-20a and PDCD4. Murine xenograft assay was used to confirm the function of HAND2-AS1 in vivo.ResultsHAND2-AS1 and PDCD4 were downregulated and miR-20a was upregulated in 5-FU-resistant CRC tissues and cells. HAND2-AS1 suppressed 5-FU resistance, cell proliferation, migration and invasion and promoted cell apoptosis in 5-FU-resistant CRC cells. HAND2-AS1 acted as a sponge of miR-20a to regulate PDCD4 expression. Moreover, HAND2-AS1 suppressed cell progression and 5-FU resistance by upregulating PDCD4 via sponging miR-20a in 5-FU-resistant CRC cells. Besides, HAND2-AS1 inhibited tumor growth in vivo.ConclusionHAND2-AS1/miR-20a/PDCD4 axis inhibited cell progression and 5-FU resistance in 5-FU-resistant CRC cells.     

Circulating MicroRNAs and Aerobic Fitness – The HUNT-Study

Aerobic fitness, measured as maximal oxygen uptake (VO_2max), is a good indicator of cardiovascular health, and a strong predictor of cardiovascular mortality. Biomarkers associated with low VO_2max may therefore represent potential early markers of future cardiovascular disease (CVD). The aim of this study was to assess whether circulating microRNAs (miRs) are associated with VO_2max-level in healthy individuals. In a screening study, 720 miRs were measured in serum samples from healthy individuals (40–45 yrs) with high (n = 12) or low (n = 12) VO_2max matched for gender, age and physical activity. Candiate miRs were validated in a second cohort of subjects with high (n = 38) or low (n = 38) VO_2max. miR-210 and miR-222 were found to be higher in the low VO_2max-group (p<0.05). In addition, miR-21 was increased in male participants with low VO_2max (p<0.05). There were no correlations between traditional risk factors for CVD (blood pressure, cholesterol, smoking habit, or obesity) and miR-21, miR-210 and miR-222. DIANA-mirPath identified 611 potential gene-targets of miR-21, miR-210 and miR-222, and pathway analysis indicated alterations in several important signaling systems in subjects with low VO_2max. Potential bias involve that blood was collected from non-fasting individuals, and that 8 performed exercise within 24 h before sampling. In conclusion, we found that miR-210, miR-21, and miR-222 were increased in healthy subjects with low VO_2max. The lack of association between these three miRs, and other fitness related variables as well as traditional CVD risk factors, suggests that these miRs may have a potential as new independent biomarkers of fitness level and future CVD.     

Genome-Wide Search for Gene-Gene Interactions in Colorectal Cancer

Genome-wide association studies (GWAS) have successfully identified a number of single-nucleotide polymorphisms (SNPs) associated with colorectal cancer (CRC) risk. However, these susceptibility loci known today explain only a small fraction of the genetic risk. Gene-gene interaction (GxG) is considered to be one source of the missing heritability. To address this, we performed a genome-wide search for pair-wise GxG associated with CRC risk using 8,380 cases and 10,558 controls in the discovery phase and 2,527 cases and 2,658 controls in the replication phase. We developed a simple, but powerful method for testing interaction, which we term the Average Risk Due to Interaction (ARDI). With this method, we conducted a genome-wide search to identify SNPs showing evidence for GxG with previously identified CRC susceptibility loci from 14 independent regions. We also conducted a genome-wide search for GxG using the marginal association screening and examining interaction among SNPs that pass the screening threshold (p<10⁻⁴). For the known locus rs10795668 (10p14), we found an interacting SNP rs367615 (5q21) with replication p = 0.01 and combined p = 4.19×10⁻⁸. Among the top marginal SNPs after LD pruning (n = 163), we identified an interaction between rs1571218 (20p12.3) and rs10879357 (12q21.1) (nominal combined p = 2.51×10⁻⁶; Bonferroni adjusted p = 0.03). Our study represents the first comprehensive search for GxG in CRC, and our results may provide new insight into the genetic etiology of CRC.     

A High Degree of LINE-1 Hypomethylation Is a Unique Feature of Early-Onset Colorectal Cancer

Objective

Early-onset colorectal cancer (CRC) represents a clinically distinct form of CRC that is often associated with a poor prognosis. Methylation levels of genomic repeats such as LINE-1 elements have been recognized as independent factors for increased cancer-related mortality. The methylation status of LINE-1 elements in early-onset CRC has not been analyzed previously.

Design

We analyzed 343 CRC tissues and 32 normal colonic mucosa samples, including 2 independent cohorts of CRC diagnosed ≤50 years old (n = 188), a group of sporadic CRC >50 years (MSS n = 89; MSI n = 46), and a group of Lynch syndrome CRCs (n = 20). Tumor mismatch repair protein expression, microsatellite instability status, LINE-1 and MLH1 methylation, somatic BRAF V600E mutation, and germline MUTYH mutations were evaluated.

Results

Mean LINE-1 methylation levels (±SD) in the five study groups were early-onset CRC, 56.6% (8.6); sporadic MSI, 67.1% (5.5); sporadic MSS, 65.1% (6.3); Lynch syndrome, 66.3% (4.5) and normal mucosa, 76.5% (1.5). Early-onset CRC had significantly lower LINE-1 methylation than any other group (p<0.0001). Compared to patients with <65% LINE-1 methylation in tumors, those with ≥65% LINE-1 methylation had significantly better overall survival (p = 0.026, log rank test).

Conclusions

LINE-1 hypomethylation constitutes a potentially important feature of early-onset CRC, and suggests a distinct molecular subtype. Further studies are needed to assess the potential of LINE-1 methylation status as a prognostic biomarker for young people with CRC.     

Stromal Expression of MiR-21 Predicts Biochemical Failure in Prostate Cancer Patients with Gleason Score 6

Aim

microRNAs (miRNAs) are involved in various neoplastic diseases, including prostate cancer (PCs). The aim of this study was to investigate the miRNA profile in PC tissue, to assess their association with clinicopathologic data, and to evaluate the potential of miRNAs as diagnostic and prognostic markers.

Materials and Methods

From a cohort of 535 patients submitted to radical prostatectomy (RP), a sample of 30 patients (14 patients with rapid biochemical failure (BF) and 16 patients without BF) with Gleason score 7 were analyzed. A total of 1435 miRNAs were quantified by microarray hybridization, and selected miRNAs with the highest Standard deviation (n = 50) were validated by real-time quantitative PCR (qRT-PCR). In situ hybridization (ISH) was used to evaluate the expression of miR-21.

Results

miR-21 was the only miR that was significantly up-regulated in the BF group (p = 0.045) miR-21 was up-regulated in patients with BF compared with non-BF group (p = 0.05). In univariate analyses, high stromal expression of miR-21 had predictive impact on biochemical failure-free survival (BFFS) and clinical failure-free survival (CFFS) (p = 0.006 and p = 0.04, respectively). In the multivariate analysis, high stromal expression of miR-21 expression was found to be an independent prognostic factor for BFFS in patients with Gleason score 6 (HR 2.41, CI 95% 1.06–5.49, p = 0.037).

Conclusion

High stromal expression of miR-21 was associated with poor biochemical recurrence-free survival after RP. For patients with Gleason score 6, miR-21 may help predict the risk of future disease progression and thereby help select patients for potential adjuvant treatment or a more stringent follow-up.     

Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.     

Sporadic Colorectal Cancer Development Shows Rejuvenescence Regarding Epithelial Proliferation and Apoptosis

Background and Aims

Sporadic colorectal cancer (CRC) development is a sequential process showing age-dependency, uncontrolled epithelial proliferation and decreased apoptosis. During juvenile growth cellular proliferation and apoptosis are well balanced, which may be perturbed upon aging. Our aim was to correlate proliferative and apoptotic activities in aging human colonic epithelium and colorectal cancer. We also tested the underlying molecular biology concerning the proliferation- and apoptosis-regulating gene expression alterations.

Materials and Methods

Colorectal biopsies from healthy children (n₁ = 14), healthy adults (n₂ = 10), adult adenomas (n₃ = 10) and CRCs (n₄ = 10) in adults were tested for Ki-67 immunohistochemistry and TUNEL apoptosis assay. Mitosis- and apoptosis-related gene expression was also studied in healthy children (n₁ = 6), adult (n₂ = 41) samples and in CRC (n₃ = 34) in HGU133plus2.0 microarray platform. Measured alterations were confirmed with RT-PCR both on dependent and independent sample sets (n₁ = 6, n₂ = 6, n₃ = 6).

Results

Mitotic index (MI) was significantly higher (p<0.05) in intact juvenile (MI = 0.33±0.06) and CRC samples (MI = 0.42±0.10) compared to healthy adult samples (MI = 0.15±0.06). In contrast, apoptotic index (AI) was decreased in children (0.13±0.06) and significantly lower in cancer (0.06±0.03) compared to healthy adult samples (0.17±0.05). Eight proliferation- (e.g. MKI67, CCNE1) and 11 apoptosis-associated genes (e.g. TNFSF10, IFI6) had altered mRNA expression both in the course of normal aging and carcinogenesis, mainly inducing proliferation and reducing apoptosis compared to healthy adults. Eight proliferation-associated genes including CCND1, CDK1, CDK6 and 26 apoptosis-regulating genes (e.g. SOCS3) were differently expressed between juvenile and cancer groups mostly supporting the pronounced cell growth in CRC.

Conclusion

Colorectal samples from children and CRC patients can be characterized by similarly increased proliferative and decreased apoptotic activities compared to healthy colonic samples from adults. Therefore, cell kinetic alterations during colorectal cancer development show uncontrolled rejuvenescence as opposed to the controlled cell growth in juvenile colonic epithelium.     

Intestinal PTGS2 mRNA Levels,PTGS2 Gene Polymorphisms,and Colorectal Carcinogenesis

Background & Aims

Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the PTGS2 gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.

Methods

PTGS2 mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional PTGS2 polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.

Results

PTGS2 mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, PTGS2 mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). PTGS2 A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele (PTGS2 AGC) were at increased risk of CRC as compared to homozygous carriers of the PTGS2 AGT (A-1195G, G-765C, T8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and PTGS2 mRNA levels could be detected.

Conclusion

High intestinal PTGS2 mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. PTGS2 polymorphisms that have been associated with altered PTGS2 mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both PTGS2 polymorphisms and PTGS2 mRNA levels may provide information regarding CRC risk.