Ibuprofen Inhibits Overexpression of Tumor-Related Rac1b through SRSF1

Abstract: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen. Purpose: The objective of our study is to understand the molecular regulation of Rac1b alternative splicing event and how it contributes to tumorigenesis. Experimental description: HT29 colorectal cell line was used as model to test several signaling pathways after 48h of treatment with ibuprofen. For this we analyzed the proteins of interest by Western Blot and the transcript levels by RT-PCR. Results: Mechanistic studies in cultured HT29 colorectal tumor cells revealed that ibuprofen inhibited Rac1b expression in a cyclooxygenase inhibition–independent manner and targets directly the alternative splicing event. Here, we provide evidence that ibuprofen leads to a decrease in expression of SRSF1, a splicing factor that we previously identified to promote Rac1b alternative splicing. Together, our results suggest that stromal cues, namely, inflammation, can trigger changes in Rac1b expression in the colon and identify ibuprofen as a highly specific and efficient inhibitor of Rac1b overexpression in colorectal tumors. Conclusions: Our data identify an additional cyclooxygenase–independent action of ibuprofen and suggest it may be beneficial in the treatment of patients with the subtype of BRAF-mutated serrated colorectal tumors.

Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

Ibuprofen inhibits overexpression of tumor-related Rac1b through SRSF1

Introduction: The serrated pathway to colorectal tumor formation involves oncogenic mutations in the BRAF gene, which are sufficient for initiation of hyperplastic growth but not for tumor progression. A previous analysis of colorectal tumors revealed that overexpression of splice variant Rac1b occurs in around 80% of tumors with mutant BRAF and both events proved to cooperate in tumor cell survival. Patients with inflamed human colonic mucosa also have increased expression of Rac1b as well as mice with experimentally induced colitis. The increase of Rac1b in the mouse model was specifically prevented by the nonsteroidal anti-inflammatory drug ibuprofen.

Roles of the Immune Cytokine Environment on Clonal Growth of Murine and Human Tet2 Mutant Bone Marrow Progenitors: Implications for Myelodysplastic Syndromes

TET2 is a tumor suppressor gene that has been implicated in the epigenetic regulation of gene expression. Inactivating TET2 mutations are common in MDS. These mutations may contribute to early clonal dominance and myeloid transformation, although the exact mechanisms remain to be elucidated. Common to the environment of MDS are elevations in cytokines, such as TNFα and IFN-γ. It was hypothesized that inflammatory cytokines TNF-α and IFN-γ may promote clonal expansion of TET2 mutant progenitors. Adult (10-14 weeks-old) Tet2 wild type (+/+) and Tet2 mutant (-/-) C57BL/6 mice strains were chosen as a model system. Lineage negative cells (Lin-), enriched for hematopoietic stem and progenitor cells, were isolated from Tet2 +/+ and -/- bone marrow and cultured in the absence or presence of varying concentrations of TNFα or IFN-γ in methylcellulose colony formation assays and long term cell culture assays, over a period of 12 and 30 days respectively, and their colony growth, cell count, immunophenotype and resistance to apoptosis were examined. Where indicated, serial re-plating was performed. Expression of apoptotic regulators was assessed by qRT-PCR. In the triplicate experiments, starting with equal densities of Tet2 +/+ and -/- Lin- cells, Tet2 -/- Lin- cells displayed increased resistance to cytokine-induced growth suppression and superior colony forming ability over +/+ in the serial re-plating assays under stress of increasing TNFα or IFN γ. Tet2 -/- progenitors also displayed a lower apoptotic index compared to +/+ under stress of increasing TNFα, suggesting increased resistance to TNFα induced apoptosis. Transcriptional data showed low expression of Tnfr1, Fas and caspase 8, as well as a high expression of Bcl-2 and Iap1 in Tet2 -/- compared to +/+ under stress of TNFα. Tet2-/- also showed increased basal expression of endogenous TNFα mRNA compared to +/+. In the human colony growth assay, the clonal growth of TET2 mutant CFU-GM progenitors was enhanced at low TNFα concentrations. Conclusion: Mutations that promote resistance to environmental stem cell stressors are a known mechanism of clonal selection in aplastic anaemia and JAK2-mutant MPN and our findings suggest that this mechanism may be critical to clonal selection and dominance in MDS.

Growth of boehmite nanofibers by assembling nanoparticles with surfactant micelles

It is known that boehmite (AlOOH) nanofibers formed in the presence of nonionic poly(ethylene oxide) (PEO) surfactant at 373 K. A novel approach is proposed in this study for the growth of the boehmite nanofibers: when fresh aluminum hydrate precipitate was added at regular interval to initial mixture of boehmite and PEO surfactant at 373 K, the nanofibers grow from 40 to 50 nm long to over 100 nm. It is believed that the surfactant micelles play an important role in the nanofiber growth: directing the assembly of aluminum hydrate particles through hydrogen bonding with the hydroxyls on the surface of aluminum hydrate particles. Meanwhile a gradual improvement in the crystallinity of the fibers during growth is observed and attributed to the Ostwald ripening process. This approach allows us to precisely control the size and morphology of boehmite nanofibers using soft chemical methods and could be useful for low temperature, aqueous syntheses of other oxide nanomaterials with tailorable structural specificity such as size, dimension and morphology.

New particle formation and growth at a remote, sub-tropical coastal location

A month-long intensive measurement campaign was conducted in March/April 2007 at Agnes Water, a remote coastal site just south of the Great Barrier Reef on the east coast of Australia. Particle and ion size distributions were continuously measured during the campaign. Coastal nucleation events were observed in clean, marine air masses coming from the south-east on 65% of the days. The events usually began at ~10:00 local time and lasted for 1-4 hrs. They were characterised by the appearance of a nucleation mode with a peak diameter of ~10 nm. The freshly nucleated particles grew within 1-4 hrs up to sizes of 20-50 nm. The events occurred when solar intensity was high (~1000 W m-2) and RH was low (~60%). Interestingly, the events were not related to tide height. The volatile and hygroscopic properties of freshly nucleated particles (17-22.5 nm), simultaneously measured with a volatility-hygroscopicity-tandem differential mobility analyser (VH-TDMA), were used to infer chemical composition. The majority of the volume of these particles was attributed to internally mixed sulphate and organic components. After ruling out coagulation as a source of significant particle growth, we conclude that the condensation of sulphate and/or organic vapours was most likely responsible for driving particle growth during the nucleation events. We cannot make any direct conclusions regarding the chemical species that participated in the initial particle nucleation. However, we suggest that nucleation may have resulted from the photo-oxidation products of unknown sulphur or organic vapours emitted from the waters of Hervey Bay, or from the formation of DMS-derived sulphate clusters over the open ocean that were activated to observable particles by condensable vapours emitted from the nutrient rich waters around Fraser Island or Hervey Bay. Furthermore, a unique and particularly strong nucleation event was observed during northerly wind. The event began early one morning (08:00) and lasted almost the entire day resulting in the production of a large number of ~80 nm particles (average modal concentration during the event was 3200 cm-3). The Great Barrier Reef was the most likely source of precursor vapours responsible for this event.