Antitumor activity of pimasertib, a selective MEK 1/2 inhibitor, in combination with PI3K/mTOR inhibitors or with multi-targeted kinase inhibitors in pimasertib-resistant human lung and colorectal cancer cells.

The RAS/RAF/MEK/MAPK and the PTEN/PI3K/AKT/mTOR pathways are key regulators of proliferation and survival in human cancer cells. Selective inhibitors of different transducer molecules in these pathways have been developed as molecular targeted anti-cancer therapies. The in vitro and in vivo anti-tumor activity of pimasertib, a selective MEK 1/2 inhibitor, alone or in combination with a PI3K inhibitor (PI3Ki), a mTOR inhibitor (everolimus), or with multi-targeted kinase inhibitors (sorafenib and regorafenib), that block also BRAF and CRAF, were tested in a panel of eight human lung and colon cancer cell lines. Following pimasertib treatment, cancer cell lines were classified as pimasertib-sensitive (IC50 for cell growth inhibition of 0.001 µM) or pimasertib-resistant. Evaluation of basal gene expression profiles by microarrays identified several genes that were up-regulated in pimasertib-resistant cancer cells and that were involved in both RAS/RAF/MEK/MAPK and PTEN/PI3K/AKT/mTOR pathways. Therefore, a series of combination experiments with pimasertib and either PI3Ki, everolimus, sorafenib or regorafenib were conducted, demonstrating a synergistic effect in cell growth inhibition and induction of apoptosis with sustained blockade in MAPK- and AKT-dependent signaling pathways in pimasertib-resistant human colon carcinoma (HCT15) and lung adenocarcinoma (H1975) cells. Finally, in nude mice bearing established HCT15 and H1975 subcutaneous tumor xenografts, the combined treatment with pimasertib and BEZ235 (a dual PI3K/mTOR inhibitor) or with sorafenib caused significant tumor growth delays and increase in mice survival as compared to single agent treatment. These results suggest that dual blockade of MAPK and PI3K pathways could overcome intrinsic resistance to MEK inhibition.

DUSP 4 expression identifies a subset of colorectal cancer tumors that differ in MAPK activation, regardless of the genotype.

As dual-specificity phosphatase (DUSP) expression has been correlated to sensitivity to MEK inhibitors, DUSP expression levels may indicate activation of the mitogen-activated protein kinase (MAPK) pathway in many tumor types. In this study, we investigate if DUSP levels can indicate different levels of MAPK activation within colorectal cancer (CRC) patients. In three different CRC patient microarray datasets, we analyzed the expression of DUSP1. DUSP4 and DUSP6 according to mutational status, their correlation with survival and their association with different clinical characteristics. DUSP4 was significantly differentially expressed between all mutational subgroups with the highest expression in BRAF mutated tumors. Moreover, high DUSP4 expression was associated with a worse overall survival and with clinical characteristics typical for BRAF mutant patients. The use of DUSP expression as a predictive biomarker towards MAPK targeted therapy in CRC patients needs further investigation.

KRAS, BRAF, PIK3CA, and PTEN mutations: implications for targeted therapies in metastatic colorectal cancer.

The discovery of mutant KRAS as a predictor of resistance to epidermal growth-factor receptor (EGFR) monoclonal antibodies brought a major change in the treatment of metastatic colorectal cancer. This seminal finding also highlighted our sparse knowledge about key signalling pathways in colorectal tumours. Drugs that inhibit oncogenic alterations such as phospho-MAP2K (also called MEK), phospho-AKT, and mutant B-RAF seem promising as single treatment or when given with EGFR inhibitors. However, our understanding of the precise role these potential drug targets have in colorectal tumours, and the oncogenic dependence that tumours might have on these components, has not progressed at the same rate. As a result, patient selection and prediction of treatment effects remain problematic. We review the role of mutations in genes other than KRAS on the efficacy of anti-EGFR therapy, and discuss strategies to target these oncogenic alterations alone or in combination with receptor tyrosine-kinase inhibition.

Prevalence of Toxoplasma gondii infection in Belgian house cats.

Five hundred and sixty seven sera of healthy house cats aged 3 months to 7 years, were examined for the presence of anti-toxoplasma antibodies by indirect immunofluorescence assay and compared to SAG1 and TLA enzyme linked immunosorbent assays as alternative test. Twenty-five percent of cats tested positive for IgG and/or IgM. Seroprevalence increased with age from 2% below 12 months of age up to 44% at age 7. Sensitivities of SAG1 and TLA ELISA were 84.1% and 88.6%, respectively. Peak levels in seroprevalence were correlated to increased IgG titers in TLA ELISA. Our results suggest that T. gondii infections are common in house cats and that there is a high chance for a negative cat to seroconvert in its second life-year.

The tyrosine phosphatase PTPRO sensitizes colon cancer cells to anti-EGFR therapy through activation of SRC-mediated EGFR signaling.

Inappropriate activation of epidermal growth factor receptor (EGFR) plays a causal role in many cancers including colon cancer. The activation of EGFR by phosphorylation is balanced by receptor kinase and protein tyrosine phosphatase activities. However, the mechanisms of negative EGFR regulation by tyrosine phosphatases remain largely unexplored. Our previous results indicate that protein tyrosine phosphatase receptor type O (PTPRO) is down-regulated in a subset of colorectal cancer (CRC) patients with a poor prognosis. Here we identified PTPRO as a phosphatase that negatively regulates SRC by directly dephosphorylating Y416 phosphorylation site. SRC activation triggered by PTPRO down-regulation induces phosphorylation of both EGFR at Y845 and the c-CBL ubiquitin ligase at Y731. Increased EGFR phosphorylation at Y845 promotes its receptor activity, whereas enhanced phosphorylation of c-CBL triggers its degradation promoting EGFR stability. Importantly, hyperactivation of SRC/EGFR signaling triggered by loss of PTPRO leads to high resistance of colon cancer to EGFR inhibitors. Our results not only highlight the PTPRO contribution in negative regulation of SRC/EGFR signaling but also suggest that tumors with low PTPRO expression may be therapeutically targetable by anti-SRC therapies.

Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.

PURPOSE: BRAF(V600E) mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAF(V600E) CRC. EXPERIMENTAL DESIGN: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAF(V600E) CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAF(V600E) CRC. RESULTS: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAF(V600E) GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression. CONCLUSIONS: We have established a novel GEMM to interrogate BRAF(V600E) CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials.

Repeated 24-hour recalls versus dietary records for estimating nutrient intakes in a national food consumption survey.

BACKGROUND: The methodology used in the first Belgian food consumption survey followed to a large extent the instructions of the European Food Consumption (EFCOSUM) reports, where repeated 24-hour recalls (24HR) using EPIC-SOFT were recommended. OBJECTIVES: To evaluate the relative validity of two non-consecutive 24HR using EPIC-SOFT by comparison with 5-day estimated dietary records (EDR). To assess misreporting in energy for both methods by comparing energy intake with energy expenditure from accelerometery in a subsample. DESIGN: A total of 175 subjects (aged 15 and over) were recruited to participate in the study. Repeated 24HR were performed with an interval of 2-8 weeks. After completion of the second interview, subjects were instructed to keep an EDR. Dietary intakes were adjusted for within-person variability to reflect usual intakes. A Student's t-test was calculated to assess differences between both methods. Spearman and Kappa correlation coefficients were used to investigate agreement. RESULTS: In total, 127 subjects completed the required repeated 24HR, as well as the five record days. From 76 participants, accelerometer data were available. In both methods, about 35% of participants had ratios of Energy Intake/Total Energy Expenditure (EI/TEE) above or below 95% confidence intervals for EI/TEE, suggesting misreporting of energy. Significant differences between the two dietary intake methods were found for total energy, total fat, fatty acids, cholesterol, alcohol, vitamin C, thiamine, riboflavin and iron. In general, intakes from 24HR were higher compared to EDR. Correlation coefficients for all nutrients ranged from 0.16 for thiamine to 0.70 for water. CONCLUSIONS: The results from this study show that in the context of nutritional surveillance, duplicate 24HR can be used to asses intakes of protein, carbohydrates, starch, sugar, water, potassium and calcium.