MicroRNA-224 is associated with colorectal cancer progression and response to 5-fluorouracil-based chemotherapy by KRAS-dependent and -independent mechanisms.

BACKGROUND: Colorectal cancers arise from benign adenomas, although not all adenomas progress to cancer and there are marked interpatient differences in disease progression. We have previously associated KRAS mutations with disease progression and reduced survival in colorectal cancer patients. METHODS: We used TaqMan low-density array (TLDA) qRT-PCR analysis to identify miRNAs differentially expressed in normal colorectal mucosa, adenomas and cancers and in isogeneic KRAS WT and mutant HCT116 cells, and used a variety of phenotypic assays to assess the influence of miRNA expression on KRAS activity, chemosensitivity, proliferation and invasion. RESULTS: MicroRNA-224 was differentially expressed in dysplastic colorectal disease and in isogeneic KRAS WT and mutant HCT116 cells. Antagomir-mediated miR-224 silencing in HCT116 KRAS WT cells phenocopied KRAS mutation, increased KRAS activity and ERK and AKT phosphorylation. 5-FU chemosensitivity was significantly increased in miR-224 knockdown cells, and in NIH3T3 cells expressing KRAS and BRAF mutant proteins. Bioinformatics analysis of predicted miR-224 target genes predicted altered cell proliferation, invasion and epithelial-mesenchymal transition (EMT) phenotypes that were experimentally confirmed in miR-224 knockdown cells. CONCLUSIONS: We describe a novel mechanism of KRAS regulation, and highlight the clinical utility of colorectal cancer-specific miRNAs as disease progression or clinical response biomarkers.

Environmental control of trypsin secretion in the midgut of the two-spotted field cricket, Gryllus bimaculatus.

The two most important environmental factors controlling the release of trypsin in Gryllus bimaculatus are temperature and food consumption. Food consumption is in turn controlled by food availability (quantity), food quality (contained nutrients, inhibitors), developmental stage, age, sex and the daily light-dark cycle. The secretion of trypsin was higher at an acclimation temperature (AT) of 22°C than at 32°C, although the weight of caecal tissue and body weight were lower. The trypsin secretion at both experimental temperatures (25°C and 35°C) was almost 2 times greater in crickets maintained at 22°C AT since egg hatch than those maintained only since the last larval stage, but not at 32°C AT. Acclimation became increasingly rotational with increased exposure time at different rearing temperatures. The more food consumed the higher the trypsin secretion. Secretion was highest on day 3 in adult females and day 2 in males, corresponding to the day of maximal food consumption. Secretion was less than 20% in starved or cellulose fed females compared to those fed a control diet. Food reached the caeca in starved crickets within 30min and induced an increased trypsin secretion. Crickets started feeding at the onset of darkness, and trypsin secretion was significantly elevated near the end of the scotophase. The in vivo response to 0.4% soybean trypsin inhibitor (SBTI) fed throughout the last larval stage resulted in reduced growth and a 50% decrease in trypsin secretion in 2day old adult females. An adaptation to the reduction of trypsin secretion occurred when G. bimaculatus was fed 0.1% and 0.2% SBTI, but not when fed with 0.4%.

Glutathione S-transferase genotype is associated with sensitivity to psoralen-ultraviolet A photochemotherapy.

BACKGROUND: There is marked interpatient variation in responses to psoralen-ultraviolet A (PUVA) photochemotherapy. Identification of molecular biomarkers of PUVA sensitivity may facilitate treatment predictability.The glutathione S-transferases (GSTs) influence cutaneous defence against UV radiation-induced oxidative stress and are therefore candidate biomarkers of PUVA sensitivity. Several human GSTs, including GSTM1 and GSTT1, are polymorphic, and null polymorphisms have been associated with increased UVB erythemal sensitivity and skin cancer risk. PUVA also increases skin cancer risk. OBJECTIVES: To investigate the effect of GST genotype on PUVA sensitivity. METHODS: We investigated GST genotype in patients starting PUVA (n=111) and the effects of 8-methoxypsoralen (8-MOP) on antioxidant response element (ARE)-regulated gene expression in mammalian cells. RESULTS: Lower minimal phototoxic doses (MPD) (P=0·022) and higher serum 8-MOP concentrations (P=0·052) were seen in GSTM1-null allele homozygotes compared with patients with one or two active alleles. In a subset of patients with psoriasis (n=50), the GSTM1 genotype was not associated with PUVA outcomes, although MPD [hazard ratio (HR) 1·37; 95% confidence interval (CI) for HR 1·15-1·64] and GSTT1-null (HR 2·39; 95% CI for HR 1·31-4·35) and GSTP1b (HR 1·96; 95% CI for HR 1·10-3·51) genotypes were associated with clearance of psoriasis in this patient group. Exposure of mammalian cells to 8-MOP induced gene expression via the ARE, a regulatory sequence in promoters of cytoprotective genes including GSTs, suggesting that these genes may be implicated in 8-MOP metabolism. CONCLUSION: The polymorphic human GSTs are associated with PUVA sensitivity. Further studies are required to examine the clinical relevance of these preliminary findings.

Pyrosequencing-based methods reveal marked inter-individual differences in oncogene mutation burden in human colorectal tumours.

BACKGROUND: The epidermal growth factor receptor-targeted monoclonal antibody cetuximab (Erbitux) was recently introduced for the treatment of metastatic colorectal cancer. Treatment response is dependent on Kirsten-Ras (K-Ras) mutation status, in which the majority of patients with tumour-specific K-Ras mutations fail to respond to treatment. Mutations in the oncogenes B-Raf and PIK3CA (phosphoinositide-3-kinase) may also influence cetuximab response, highlighting the need for a sensitive, accurate and quantitative assessment of tumour mutation burden. METHODS: Mutations in K-Ras, B-Raf and PIK3CA were identified by both dideoxy and quantitative pyrosequencing-based methods in a cohort of unselected colorectal tumours (n=102), and pyrosequencing-based mutation calls correlated with various clinico-pathological parameters. RESULTS: The use of quantitative pyrosequencing-based methods allowed us to report a 13.7% increase in mutation burden, and to identify low-frequency (<30% mutation burden) mutations not routinely detected by dideoxy sequencing. K-Ras and B-Raf mutations were mutually exclusive and independently associated with a more advanced tumour phenotype. CONCLUSION: Pyrosequencing-based methods facilitate the identification of low-frequency tumour mutations and allow more accurate assessment of tumour mutation burden. Quantitative assessment of mutation burden may permit a more detailed evaluation of the role of specific tumour mutations in the pathogenesis and progression of colorectal cancer and may improve future patient selection for targeted drug therapies.