MiR-320e is a novel prognostic biomarker in colorectal cancer.

BACKGROUND: Advances in early detection and treatment have improved outcomes in patients with colorectal cancer (CRC). However, there remains a need for robust prognostic and predictive biomarkers. We conducted a systematic discovery and validation of microRNA (miRNA) biomarkers in two clinical trial cohorts of CRC patients. METHODS: We performed an initial 'discovery' phase using Affymetrix miRNA expression arrays to profile stage III CRC patients with and without tumour recurrence (n=50 per group) at 3-years of follow-up. All patients received adjuvant 5-fluorouracil (5-FU) plus oxaliplatin, that is, FOLFOX, treatment. During 'validation', we analysed miRNAs using qRT-PCR in an independent cohort of 237 stage II-IV CRC patients treated with 5-FU-based chemotherapy, as well as in normal colonic mucosa from 20 healthy subjects. Association with disease recurrence, disease-free survival (DFS) and overall survival (OS) was examined using Cox proportional hazard models. RESULTS: In the discovery cohort, miR-320e expression was significantly elevated in stage III colon cancers from patients with vs without recurrence (95% confidence interval (CI)=1.14-1.42; P<0.0001). These results were then independently validated in stage II and III tumours. Specifically, increased miR-320e expression was associated with poorer DFS (hazard ratio (HR)=1.65; 95% CI=1.27-2.13; P=0.0001) and OS (HR=1.78; 95% CI=1.31-2.41; P=0.0003) in stage III CRC patients. CONCLUSIONS: In two clinical trial cohorts, a systematic biomarker discovery and validation approach identified miR-320e to be a novel prognostic biomarker that is associated with adverse clinical outcome in stage III CRC patients treated with 5-FU-based adjuvant chemotherapy. These findings have important implications for the personalised management of CRC patients.

Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

BACKGROUND: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. METHODS: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. RESULTS: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ß-catenin levels. CONCLUSION: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/ß-catenin signaling.

Review of the Lynch syndrome: history, molecular genetics, screening, differential diagnosis, and medicolegal ramifications.

More than one million patients will manifest colorectal cancer (CRC) this year of which, conservatively, approximately 3% (approximately 30,700 cases) will have Lynch syndrome (LS), the most common hereditary CRC predisposing syndrome. Each case belongs to a family with clinical needs that require genetic counseling, DNA testing for mismatch repair genes (most frequently MLH1 or MSH2) and screening for CRC. Colonoscopy is mandated, given CRC's proximal occurrence (70-80% proximal to the splenic flexure). Due to its early age of onset (average 45 years of age), colonoscopy needs to start by age 25, and because of its accelerated carcinogenesis, it should be repeated every 1 to 2 years through age 40 and then annually thereafter. Should CRC occur, subtotal colectomy may be necessary, given the marked frequency of synchronous and metachronous CRC. Because 40-60% of female patients will manifest endometrial cancer, tailored management is essential. Additional extracolonic cancers include ovary, stomach, small bowel, pancreas, hepatobiliary tract, upper uroepithelial tract, brain (Turcot variant) and sebaceous adenomas/carcinomas (Muir-Torre variant). LS explains only 10-25% of familial CRC.

In vivo effects of mesalazine or E. coli Nissle 1917 on microsatellite instability in ulcerative colitis.

BACKGROUND: Microsatellite instability (MSI) occurs in chronically inflamed colorectal tissue and may evolve to colitis-associated cancer. In vitro data suggest that mesalazine (5-ASA) improves MSI. AIM: To analyse the changes in MSI in 156 distal colonic biopsies of 39 patients with ulcerative colitis that had been treated within a randomized, double-blind trial comparing 5-ASA with E. coli Nissle (EcN). METHODS: Two biopsies had been collected before and after 1 year of treatment. MSI testing was performed using a panel of eight markers, including 3 dinucleotide and 5 mononucleotide repeats. RESULTS: No MSI was observed with any of the mono-repeats, and among dinucleotide repeats, only D5S346 (maps to APC) and D17S250 (p53) were consistently informative. Overall, 31/156 (20%) biopsies displayed MSI. After 1 year, 3/11 patients displayed MSI improvement [change to microsatellite stability (MSS); 1 on 5-ASA, 2 on EcN] at D5S346 and 4/11 showed MSI worsening (change from MSS to MSI; all 5-ASA). For D17S250, the corresponding data were for 3/9 patients (2 on 5-ASA, 1 on EcN) and 2/9 (both on 5-ASA), respectively. CONCLUSIONS: In the set of biopsies taken from patients treated with 1.5 g 5-ASA for 1 year, there was no improvement in the prevalence of MSI in the distal colon.

JC virus infects the enteric glia of patients with chronic idiopathic intestinal pseudo-obstruction.

BACKGROUND AND AIMS: Chronic idiopathic intestinal pseudo-obstruction (CIIP) is characterised by severe impairment of intestinal propulsive motility that mimics bowel obstruction. JC virus (JCV) is a polyomavirus that can infect brain glial cells causing a fatal disease, but may also be found throughout the normal gastrointestinal tract. The hypothesis that JCV infects the myenteric plexuses of patients with CIIP was tested. METHODS: 10 patients with CIIP and 61 normal specimens (30 ascending colon and 31 ileum) from patients with uncomplicated colon cancer were studied. DNA was extracted from the myenteric plexuses, and JCV T antigen (TAg) DNA and the viral regulatory region were detected by PCR and sequencing. Immunohistochemistry was performed to detect JCV viral protein expression, neuronal and glial markers. Fluorescence in situ hybridisation was performed for cellular localisation of the JCV infection. RESULTS: Clinical studies demonstrated neurogenic impairment, and pathological analyses showed neuropathy in each patient with CIIP. JCV TAg DNA was found in the myenteric plexuses of 8/10 (80%) of the patients with CIIP and 3/31 (9.7%) of the control patients (p<0.001). All samples were JCV Mad-1 strains. Seven of the 10 CIIP specimens expressed both JCV TAg and the JCV viral protein VP1, while none of the controls expressed either. JCV infection co-localised with glial fibrillary acidic protein expression, a marker of enteric glial cells. CONCLUSION: JCV infection occurs in the myenteric plexuses of patients with CIIP. The JCV localisation in enteroglial cells suggests a possible pathological role for this virus in enteric neuropathy.

Zoledronic acid induces antiproliferative and apoptotic effects in human pancreatic cancer cells in vitro.

Bisphosphonates (BPs) are an emerging class of drugs mostly used in the palliative care of cancer patients. We investigated the in vitro activity of the most potent antiresorptive BP, zoledronic acid (ZOL), on the growth and survival of three human pancreatic cancer (PC) cell lines (BxPC-3, CFPAC-1 and PANC-1). Pancreatic cancer frequently has a dysregulated p21(ras) pathway and therefore appears to be a suitable target for BPs that interfere with the prenylation of small GTP-binding proteins such as p21(ras). We found that ZOL induces growth inhibition (IC(50):10-50 micro M) and apoptotic death of PC cells. The proapoptotic effect was correlated to cleavage/activation of caspase-9 and poly(ADP)-ribose polymerase, but not of caspase-3. Moreover, we studied the p21(ras) signalling in cells exposed to ZOL and detected a reduction of p21(ras) and Raf-1 content and functional downregulation of the terminal enzyme ERK/MAPkinase and of the pKB/Akt survival pathway. Finally, we observed that ZOL induces significant cytoskeletal rearrangements. In conclusion, we demonstrated that ZOL induces growth inhibition and apoptosis on PC cells and interferes with growth and survival pathways downstream to p21(ras). These findings might be relevant for expanding application of BPs in cancer treatment.

BRCA1 expression modulates chemosensitivity of BRCA1-defective HCC1937 human breast cancer cells.

Germline mutations of the tumour suppressor gene BRCA1 are involved in the predisposition and development of breast cancer and account for 20-45% of all hereditary cases. There is an increasing evidence that these tumours are characterised by a specific phenotype and pattern of gene expression. We have hypothesised that differences in chemosensitivity might parallel molecular heterogeneity of hereditary and sporadic breast tumours. To this end, we have investigated the chemosensitivity of the BRCA1-defective HCC1937 breast cancer cell line, and the BRCA1-competent MCF-7 (hormone-sensitive) and MDA-MB231 (hormone-insensitive) breast cancer cell lines using the MTT assay. The 50% inhibitory concentration (IC(50)) for the individual compounds were derived by interpolate plot analysis of the logarithmic scalar concentration curve after a 48 h exposure. HCC1937 cells were significantly (P<0.005) more sensitive to cisplatin (CDDP) (IC(50) : 30-40 microM) compared with MCF-7 (IC(50) : 60-70 microM) and MDA-MB231 (IC(50) : 90-100 microM) cells. On the other hand, BRCA1-defective breast cancer cells were significantly less sensitive to doxorubicin (Dox) (IC(50) : 45-50 microM) compared with MCF-7 (IC(50) : 1-5 microM) and MDA-MB231 (IC(50) : 5-10 microM) (P<0.02), as well as to paclitaxel (Tax) (IC(50) : >2 microM for HCC1937, 0.1-0.2 microM for MCF-7 and 0.01-0.02 microM for MDA-MB231) (P<0.001). Full-length BRCA1 cDNA transfection of BRCA1-defective HCC1937 cells led to the reconstituted expression of BRCA1 protein in HCC1937/(WT)BRCA1-derived cell clone, but did not reduce tumour cell growth in soft agar. BRCA1 reconstitution reverted the hypersensitivity to CDDP (P<0.02), and restored the sensitivity to Dox (P<0.05) and Tax (P<0.001), compared with parental HCC1937 cells. Taken together, our findings suggest a specific chemosensitivity profile of BRCA1-defective cells in vitro, which is dependent on BRCA1 protein expression, and suggest prospective preclinical and clinical investigation for the development of tailored therapeutical approaches in this setting.

JC virus: a biomarker for colorectal cancer?

Chromosomal instability (CIN) is present in most colorectal cancers, though the mechanism for these genetic aberrations is unclear. An explanation may lie in the possible link between JC virus (JCV) Mad-1 strain, found in colorectal cancers, and aneuploid neoplasia. It is proposed here to test the hypothesis that detection of JCV in colorectal cancer patients may serve as a clinically useful biomarker for the presence of colorectal tumors. This may be tested by looking for any correlation that may exist between JCV DNA, viral proteins, and anti-JCV anti-sera detected in samples of stool, blood, and urine obtained from patients with colorectal neoplasm compared with normal age-matched controls.

Oxidative stress increases frameshift mutations in human colorectal cancer cells.

Chronic inflammation in the gastrointestinal tract increases the risk for development of cancer by an incompletely understood pathway, which may involve microsatellite instability (MSI). Low frequency of MSI referred to as "MSI-L" occurs frequently in chronically inflamed nonneoplastic tissue. In this work, we have tested the hypothesis that oxidative stress may induce the accumulation of frameshift mutations in human microsatellite DNA. Mismatch repair (MMR)-proficient HCT116+chr3 and MMR-deficient HCT116 cells were transfected with pCMV-(CA)13-EGFP, a plasmid that contains a (CA)13 dinucleotide repeat, which disrupts the reading frame of the downstream enhanced green fluorescent protein gene. A dose-dependent increase in frameshift mutations restoring the enhanced green fluorescent protein reading frame was detected in HCT116 by flow cytometry. At 1 mM H2O2, the mutant fraction was 9-fold higher than that in mock-treated control cells. Although demonstrating stability at lower H2O2 concentrations, MMR-proficient HCT116+chr3 cells accumulated mutations at the 1 mM H2O2 level (4.1-fold above mock-treated control). No significant mutations were detected when HCT116 cells were transfected with the pCMV-(N)26-EGFP construct that contains 26 nucleotides in a random sequence. These data indicate that oxidative stress is a potential mutagen leading to accumulation of frameshift mutations and may contribute to MSI in the setting of chronic inflammation.

Specific inhibition of cyclooxygenase-2 (COX-2) expression by dietary curcumin in HT-29 human colon cancer cells.

Curcumin, a major yellow pigment and active component of turmeric, has been shown to possess anti-inflammatory and anti-cancer activities. Cyclooxygenase (COX)-2 plays an important role in colon carcinogenesis. To investigate the effect of curcumin on COX-2 expression, we treated HT-29 human colon cancer cells with various concentrations of curcumin. Curcumin inhibited the cell growth of HT-29 cells in a concentration- and time-dependent manner. Curcumin markedly inhibited the mRNA and protein expression of COX-2, but not COX-1. These data suggest that a non-toxic concentration of curcumin has a significant effect on the in vitro growth of HT-29 cells, specifically inhibits COX-2 expression, and may have value as a safe chemopreventive agent for colon cancer.

Colonic mucosal prostaglandin E2 and cyclooxygenase expression before and after low aspirin doses in subjects at high risk or at normal risk for colorectal cancer.

UNLABELLED: Development of potential cancer chemopreventive drugs involves the systematic evaluation of these drugs in preliminary Phase I and II studies in human beings to identify the optimal drug dose, drug toxicity, and surrogate end point biomarker modulation. OBJECTIVES: We tested the hypothesis that aspirin, at a single, once-daily 81-mg dose, will reduce colonic mucosal concentration of prostaglandin estradiol (E2) in individuals at high risk for colorectal cancer development similar to our prior observations in a young normal-risk population. METHODS: Aspirin was administered at a dose of 81 mg once daily for 28 days in a cohort of 92 matched high-risk and normal-risk colorectal cancer subjects. Prostaglandin E2 and cyclooxygenase expression were assayed from distal sigmoid biopsies from all of the subjects before and after treatment. RESULTS: The mean prostaglandin E2 for normal-risk subjects before aspirin treatment was 11.3 +/- 1.7 pg/microg (mean +/- SE) tissue protein and after aspirin treatment was 4.9 +/- 0.91 pg/microg tissue protein (P < 0.0001). In high-risk subjects, mean pretreatment prostaglandin E2 was 14.4 +/- 1.7 pg/microg tissue protein and after aspirin treatment was 4.7 +/- 0.70 pg/microg tissue protein (P < 0.0001). Aspirin treatment did not alter cyclooxygenase-1 protein expression. CONCLUSIONS: Aspirin treatment at a dose of 81 mg reduces colorectal mucosal prostaglandin E2 concentration after 28 daily doses. Risk for colorectal carcinoma did not modify colorectal mucosal baseline or post-aspirin prostaglandin E2, or cyclooxygenase expression. Colorectal mucosal prostaglandin concentration may be used as a "drug-effect surrogate biomarker," that is, a surrogate to assess sufficient delivery and tissue effect of a chemopreventive agent.

Germline characterization of early-aged onset of hereditary non-polyposis colorectal cancer.

OBJECTIVES: Hereditary non-polyposis colorectal cancer (HNPCC) is characterized by the early onset of colorectal cancer (approximately 40 years). Adolescent colorectal cancer is unusual in HNPCC families. We speculated that some DNA mismatch repair germline mutations might be associated with early onset of disease. STUDY DESIGN: Genomic DNA was extracted from members of a kindred with virulent HNPCC fitting the Amsterdam Criteria for HNPCC and sequenced for 2 DNA mismatch repair genes, hMSH2 and hMLH1. A sigmoid adenocarcinoma from the 14-year-old proband was analyzed for highfrequency microsatellite instability and immunostained for DNA mismatch repair gene expression. RESULTS: A germline mutation was identified at nucleotide 676 (codon 226) of the hMLH1 gene. The C to T transition created a nonsense mutation, truncating the hMLH1 protein. This mutation also alters the splice donor sequence, because nucleotide 676 is 2 base pairs from the 3' end of the exon 8. The proband's tumor demonstrated high-frequency microsatellite instability and displayed loss of hMLH1 expression, indicating bi-allelic inactivation of hMLH1. CONCLUSIONS: A complex mutation of hMLH1 at codon 226 is associated with adolescent onset of colorectal cancer in an HNPCC family. Genetic screening of other suspected HNPCC families with unusually young members with cancer might reveal certain genotypes with particularly virulent forms of this disease.

Genetic pathways in the evolution of morphologically distinct colorectal neoplasms.

Colorectal adenomas can be morphologically classified as exophytic or flat. Polypoid cancers and cancers arising de novo (ie., without any adenomatous component) might be the results of genetic progression from exophytic and flat adenomas, respectively. In this study, we examined 94 morphologically distinct neoplastic specimens for mutations in K-RAS and analyzed 10 microsatellite loci tightly linked to the tumor suppressor genes APC, p53, DCC/SMAD4, hMSH2, and hMLH1. K-RAS mutations were significantly associated with exophytic adenomas [11 of 21 (52%)] compared to flat adenomas [2 of 13(15%), P < 0.03] and polypoid cancers [17 of 25 (68%)] compared to cancers arising de novo [7 of 25 (28%), P < 0.01]. Two polypoid cancer cases demonstrated three and four different K-RAS mutations, respectively, suggesting multiple areas of clonal expansion. Cancers arising de novo were significantly associated with loss of heterozygosity (LOH) at chromosome 3p compared to pol ypoid cancers [6 of 18(33%) versus 1 of 20(5%), P < 0.03], whereas the prevalence of LOH at chromosomes 2p, 5q, 17p, and 18q and microsatellite instability were not different between the groups. For all cancers, LOH at chromosomes 17p and 18q occurred in 47 and 51%, respectively. However, LOH at 17p and 18q occurred in 0 and 16% of benign lesions, respectively, suggesting their role in malignant transformation. There was no difference in LOH at chromosomes 17p and 18q between exophytic and flat lesions. These findings suggest that (a) mutant K-RAS is associated with the exophytic growth of colonic neoplasms, and that (b) some colorectal cancers arising de novo lose chromosome 3p during their evolution, which is not seen in polypoid cancers. Half of all cancers lose chromosomes 17p and 18q at or near the malignant transition of benign lesions as reported previously, irrespective of morphology. There may be more than one genetic avenue for colorectal cancer formation, and this correlates with the morphological characteristics.

Mad-1 is the exclusive JC virus strain present in the human colon, and its transcriptional control region has a deleted 98-base-pair sequence in colon cancer tissues.

JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23- and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [chi(2) = 23.6; P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.