Loss of miR-200 family in 5-fluorouracil resistant colon cancer drives lymphendothelial invasiveness in vitro.

Invasive colorectal cancer is associated with poor prognosis requiring treatment with systemic chemotherapies usually including 5-fluorouracil. A consequence of prolonged treatment is the acquisition of resistance eventually resulting in the recurrence of highly metastatic cancer cells. To address the relationship between drug resistance and increased lymphatic metastatic potential, we used a 3D co-culture model of colon tumour cell spheroids of parent CCL227 cells and subclones with gradually increasing resistance against 5-fluorouracil. From each investigated cell line, homogeneous tumour spheroids were generated in the presence of methylcellulose yielding emboli of ∼700 µm diameter. When invasive, tumour spheroids disrupt the continuous lymphendothelial cell (LEC) layer and generate a 'circular chemorepellent-induced defect' (CCID), reminiscent of the entry gates through which tumour emboli intravasate lymphatic vasculature. Here we provide evidence that increasingly chemoresistant colon cancer spheroids were strongly associated with enhanced intravasative properties. In naïve CCL227 spheroids, miR-200 family members were released into exosomes thereby repressing the epithelial to mesenchymal transition-regulating transcription factors ZEB1 and SLUG in LEC. As a consequence of attenuated plasticity and migration of LEC, CCID formation was impaired. Loss of exosomal transferred miR-200c in resistant colon cells rendered LEC more susceptible to pro-migratory signals that were generated and directly transmitted by colon cancer spheroids. This observation indicates a common molecular axis in colon cancer and LEC where miR-200 family members act as regulators of ZEB proteins. The data support the notion that horizontal miR-200 signalling prevents the permeation of cells into adjacent epithelia and contributes to organ integrity.

The influence of methotrexate on the gene expression of the pro-inflammatory cytokine IL-12A in the therapy of rheumatoid arthritis.

OBJECTIVES: Methotrexate (MTX) is a cornerstone in the treatment of rheumatoid arthritis (RA). Among its anti-proliferative activity, the anti-inflammatory mechanisms of MTX seem to play a major role in the treatment of RA. MTX reduces the production of pro-inflammatory cytokines such as interleukin (IL)-1, IL-2, IL-6 and interferon (INF)-γ, while the gene expression of anti-inflammatory Th2 cytokines like IL-4 and IL-10 is increased - altogether resulting in the anti-inflammatory effect. As little is known about the impact of MTX on other cytokines involved in the pathogenesis of RA, the present trial investigated the effect of MTX on IL-12A and IL-18 gene expression by peripheral blood mononuclear cells (PBMCs). For comparison, the effect on IL-6 and tumour necrosis factor (TNF) was analysed. METHODS: Using real-time PCR, mRNA concentrations of pro-inflammatory cytokines were determined in PBMCs from 17 patients before and during MTX therapy. Furthermore, gene expression was correlated with clinical and pharmacokinetic parameters such as methotrexate polyglutamate concentrations (Spearman's correlation coefficient). To eliminate concomitant corticosteroids as confounding factor, a subgroup analysis for methotrexate without corticosteroids was performed in 6 patients. RESULTS: MTX statistically significantly reduced the mRNA expression of IL-12A by PBMCs in rheumatoid arthritis patients (Wilcoxon-test for paired samples, p<0.046). Consistent with other reports, IL-6 was reduced under MTX treatment. Although the combination of MTX and corticosteroids significantly reduced the gene expression of IL-18, this key molecule was unaffected by MTX without corticosteroids. Our results were further supported by a negative correlation of methotrexate polyglutamate concentrations and the mRNA expression of the pro-inflammatory cytokines IL-6 and IL-12A. CONCLUSIONS: We describe a novel effect of MTX reducing the gene expression of IL-12A independently of corticosteroid application in patients. This impact was further enhanced by a reduction of IL-12A-producing lymphocytes and neutrophils under MTX treatment. These results expand the understanding of the mechanism of action of the most widely used drug in RA.

Osteoclast-like giant cell tumor in mucinous cystadenocarcinoma of the pancreas: an immunohistochemical and molecular analysis.

Osteoclast-like giant cell tumors (OLGT) are rare neoplasms of the pancreas and mostly associated with ductal adenocarcinomas. In this report, we present the rare case of OLGT associated with mucinous cystadenocarcinoma (MCC). We investigated the expression profile of both tumors by methods of molecular biology and immunohistochemistry. The panel of markers included osteopontin, her2/neu, mismatch repair genes, K-ras, p53, E-cadherin, VEGF-C, and podoplanin. Osteopontin was expressed by the osteoclast-like giant cells but not by the mononuclear tumor cells of the OLGT. We detected an amplification and overexpression of her2/neu in the MCC but not in the OLGT. Although we observed an immunohistochemical expression of hMSH2 and hMLH1 in the OLGT, we were not able to confirm this result by western blot analysis. We also did not find any microsatellite instability (D2S123, BAT26). While mutation of K-ras codon 12 was found in both tumor components, there was wild-type DNA of p53. E-cadherin was expressed in MCC but not in OLGT. VEGF-C was only positive in osteoclast-like giant cells and some of the mononuclear cells of OLGT. The vessel-rich stroma of OLGT did not present any podoplanin-positive lymphatic vessel. The observation of our case and others in the published literature may indicate separating OLGT with undifferentiated carcinoma from OLGT with MCC for the better clinical outcome of the latter.

Dissecting progressive stages of 5-fluorouracil resistance in vitro using RNA expression profiling.

Resistance to anticancer drugs such as the widely used antimetabolite 5-fluorouracil (FU) is one of the most important obstacles to cancer chemotherapy. Using GeneChip arrays, we compared the expression profile of different stages of FU resistance in colon cancer cells after in vitro selection of low-, intermediate- and high-resistance phenotypes. Drug resistance was associated with significant changes in expression of 330 genes, mainly during early or intermediate stage. Functional annotation revealed a majority of genes involved in signal transduction, cell adhesion and cytoskeleton with subsequent alterations in apoptotic response, cell cycle control, drug transport, fluoropyrimidine metabolism and DNA repair. A set of 33 genes distinguished all resistant subclones from sensitive progenitor cells. In the early stage, downregulation of collagens and keratins, together with upregulation of profilin 2 and ICAM-2, suggested cytoskeletal changes and cell adhesion remodeling. Interestingly, 6 members of the S100 calcium-binding protein family were suppressed. Acquisition of the intermediate-resistance phenotype included upregulation of the well-known drug resistance gene ABCC6 (ATP-binding cassette subfamily C member 6). The very small number of genes affected during transition to high resistance included the primary FU target thymidylate synthase. Although limited to an in vitro model, our data suggest that resistance to FU cannot be explained by known mechanisms alone and substantially involves a wide molecular repertoire. This study emphasizes the understanding of resistance as a time-depending process: the cell is particularly challenged at the beginning of this process, while acquisition of the high-resistance phenotype seems to be less demanding.