The Low-density Lipoprotein Receptor-related Protein 6 Pathway in the Treatment of Intestinal Barrier Dysfunction Induced by Hypoxia and Intestinal Microbiota through the Wnt/ß-catenin Pathway.

Our study is to explore the key molecular of Low-density lipoprotein receptor-related protein 6 (LRP6) and the related Wnt/ß-catenin pathway regulated by LRP6 during the intestinal barrier dysfunction. Colorectal protein profile analysis showed that LRP6 expression was decreased in dextran sulfate sodium (DSS)-induced colitis mice, and mice received fecal bacteria transplantation from stroke patients. Mice with intestinal hypoxia and intestinal epithelial cells cultured in hypoxia showed decreased expression of LRP6. Overexpression of LPR6 or its N-terminus rescued the Wnt/ß-catenin signaling pathway which was inhibited by hypoxia and endoplasmic reticulum stress. In mice overexpressing of LRP6, the expression of ß-catenin and DKK1 increased, Bcl2 decreased, and Bax increased. Mice with LRP6 knockout showed an opposite trend, and the expression of Claudin2, Occludin and ZO-1 decreased. Two drugs, curcumin and auranofin could alleviate intestinal barrier damage in DSS-induced colitis mice by targeting LRP-6. Therefore, gut microbiota dysbiosis and hypoxia can inhibit the LRP6 and Wnt/ß-catenin pathway, and drugs targeting LRP6 can protect the intestinal barrier.

Suppression of Colon Tumorigenesis in Mutant Apc Mice by a Novel PDE10 Inhibitor that Reduces Oncogenic ß-Catenin.

Previous studies have reported that phosphodiesterase 10A (PDE10) is overexpressed in colon epithelium during early stages of colon tumorigenesis and essential for colon cancer cell growth. Here we describe a novel non-COX inhibitory derivative of the anti-inflammatory drug, sulindac, with selective PDE10 inhibitory activity, ADT 061. ADT 061 potently inhibited the growth of colon cancer cells expressing high levels of PDE10, but not normal colonocytes that do not express PDE10. The concentration range by which ADT 061 inhibited colon cancer cell growth was identical to concentrations that inhibit recombinant PDE10. ADT 061 inhibited PDE10 by a competitive mechanism and did not affect the activity of other PDE isozymes at concentrations that inhibit colon cancer cell growth. Treatment of colon cancer cells with ADT 061 activated cGMP/PKG signaling, induced phosphorylation of oncogenic ß-catenin, inhibited Wnt-induced nuclear translocation of ß-catenin, and suppressed TCF/LEF transcription at concentrations that inhibit cancer cell growth. Oral administration of ADT 061 resulted in high concentrations in the colon mucosa and significantly suppressed the formation of colon adenomas in the Apc+/min-FCCC mouse model of colorectal cancer without discernable toxicity. These results support the development of ADT 061 for the treatment or prevention of adenomas in individuals at risk of developing colorectal cancer. PREVENTION RELEVANCE: PDE10 is overexpressed in colon tumors whereby inhibition activates cGMP/PKG signaling and suppresses Wnt/ß-catenin transcription to selectively induce apoptosis of colon cancer cells. ADT 061 is a novel PDE10 inhibitor that shows promising cancer chemopreventive activity and tolerance in a mouse model of colon cancer.

Proteomic Characterization of Colorectal Cancer Cells versus Normal-Derived Colon Mucosa Cells: Approaching Identification of Novel Diagnostic Protein Biomarkers in Colorectal Cancer.

In the western world, colorectal cancer (CRC) is the third most common cause of cancer-related deaths. Survival is closely related to the stage of cancer at diagnosis striking the clinical need for biomarkers capable of early detection. To search for possible biological parameters for early diagnosis of CRC we evaluated protein expression for three CREC (acronym: Cab45, reticulocalbin, ERC-55, calumenin) proteins: reticulocalbin, calumenin, and ERC-55 in a cellular model consisting of a normal derived colon mucosa cell line, NCM460, and a primary adenocarcinoma cell line of the colon, SW480. Furthermore, this cellular model was analyzed by a top-down proteomic approach, 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for novel putative diagnostic markers by identification of differentially expressed proteins between the two cell lines. A different colorectal carcinoma cell line, HCT 116, was used in a bottom-up proteomic approach with label-free quantification (LFQ) LC-MS/MS. The two cellular models gave sets of putative diagnostic CRC biomarkers. Various of these novel putative markers were verified with increased expression in CRC patient neoplastic tissue compared to the expression in a non-involved part of the colon, including reticulocalbin, calumenin, S100A6 and protein SET. Characterization of these novel identified biological features for CRC patients may have diagnostic potential and therapeutic relevance in this malignancy characterized by a still unmet clinical need.

Folate, genomic stability and colon cancer: The use of single cell gel electrophoresis in assessing the impact of folate in vitro, in vivo and in human biomonitoring.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

MicroRNA-21 increases the expression level of occludin through regulating ROCK1 in prevention of intestinal barrier dysfunction.

OBJECTIVE: The aim of this study is to investigate the role of molecular mechanism of microRNA (miR)-21 on tight junction (TJ)-proteins and its protective effects on the intestinal barrier. METHODS: TJ proteins and target genes expression were analyzed in miR-21 inhibition and overexpression NCM460 cell lines. To further verify the role of miR-21, the mmu-miR-21 intestinal epithelial conditional knockout (IKO) mice model was established. MiR-21 expression was detected in clinical specimens of acute stercoral obstruction patients. RESULTS: Rho-associated protein kinase 1 (ROCK1) were identified as target genes of miR-21. There is a negative correlation between miR-21 expression level and TJ proteins levels. TJ protein and ROCK1 were significantly decreased in miR-21 IKO mice, which presented intestinal inflammation response and intestinal barrier dysfunction (both P < 0.05). Determination of clinical samples showed consistent results with NCM460 cell line and miR-21 IKO mice. CONCLUSIONS: MiR-21 could be a protective factor of intestinal barrier dysfunction, which promoting the expression of TJ protein by targeting ROCK1 in vivo and in vitro.

Protective Effects of Let-7b on the Expression of Occludin by Targeting P38 MAPK in Preventing Intestinal Barrier Dysfunction.

BACKGROUND/AIMS: Let-7b was dramatically reduced after a dicer knockout of mice with intestinal barrier function injuries. This paper aims to investigate the molecular mechanism of let-7b by targeting p38 MAPK in preventing intestinal barrier dysfunction. METHODS: A total of 186 patients were enrolled, with 93 in the control group and 93 in the PRO group. Only 158 patients completed the entire study, whereas the others either did not meet the inclusion criteria or refused to participate. To further verify the role of let-7b, intestinal epithelial conditional knockout (IKO) mice of mmu-let-7b model were established. Serum let-7b, zonulin, IL-6, and TNF-α concentrations were measured by ELISA or quantitative RT-PCR. Permeability assay was done by ussing chamber. The apoptotic cells were identified using an In Situ Cell Death Detection Kit. Protein was detected by western blot. RESULTS: Probiotics can lower infection-related complications, as well as increase the serum and tissue let-7b levels. P38 MAPK was identified as the target of let-7b, as verified by NCM460 cells. P38 MAPK expression was increased, whereas tight-junction (TJ) proteins were significantly decreased in let-7b IKO mice (both P<0.05). Negative regulation of p38 MAPK molecular signaling pathways was involved in the protective effects of let-7b on intestinal barrier function. CONCLUSION: Let-7b was identified as a novel diagnosis biomarker or a potential treatment target for preventing intestinal barrier dysfunction.

Beta-catenin cleavage enhances transcriptional activation.

Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

Epithelial TNF Receptor Signaling Promotes Mucosal Repair in Inflammatory Bowel Disease.

TNF plays an integral role in inflammatory bowel disease (IBD), as evidenced by the dramatic therapeutic responses in Crohn's disease (CD) patients induced by chimeric anti-TNF mAbs. However, treatment of CD patients with etanercept, a decoy receptor that binds soluble TNF, fails to improve disease. To explore this discrepancy, we investigated the role of TNF signaling in Wnt/ß-catenin-mediated intestinal stem cell and progenitor cell expansion in CD patients, human cells, and preclinical mouse models. We hypothesized that TNF exerts beneficial effects on intestinal epithelial cell (IEC) responses to injury. In CD patients, intestinal stem cell and progenitor cell Wnt/ß-catenin signaling correlates with inflammation status. TNF-deficient (Tnf-/-) mice exhibited increased apoptosis, less IEC proliferation, and less Wnt signaling when stimulated with anti-CD3 mAb. Bone marrow (BM) chimera mice revealed that mucosal repair depended on TNF production by BM-derived cells and TNFR expression by radioresistant IECs. Wild-type→Tnfr1/2-/- BM chimera mice with chronic dextran sodium sulfate colitis exhibited delayed ulcer healing, more mucosal inflammation, and impaired Wnt/ß-catenin signaling, consistent with the hypothesis that epithelial TNFR signaling participates in mucosal healing. The direct effect of TNF on stem cells was demonstrated by studies of TNF-induced Wnt/ß-catenin target gene expression in murine enteroids and colonoid cultures and TNF-induced ß-catenin activation in nontransformed human NCM460 cells (TOPFlash) and mice (TOP-GAL). Together, these data support the hypothesis that TNF plays a beneficial role in enhancing Wnt/ß-catenin signaling during ulcer healing in IBD. These novel findings will inform clinicians and therapeutic chemists alike as they strive to develop novel therapies for IBD patients.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

Fusobacterium nucleatum Increases Proliferation of Colorectal Cancer Cells and Tumor Development in Mice by Activating Toll-Like Receptor 4 Signaling to Nuclear Factor-κB, and Up-regulating Expression of MicroRNA-21.

BACKGROUND & AIMS: Nearly 20% of the global cancer burden can be linked to infectious agents. Fusobacterium nucleatum promotes tumor formation by epithelial cells via unclear mechanisms. We aimed to identify microRNAs (miRNAs) induced by F nucleatum and evaluate their ability to promote colorectal carcinogenesis in mice. METHODS: Colorectal cancer (CRC) cell lines were incubated with F nucleatum or control reagents and analyzed in proliferation and would healing assays. HCT116, HT29, LoVo, and SW480 CRC cell lines were incubated with F nucleatum or phosphate-buffered saline (PBS [control]) and analyzed for miRNA expression patterns and in chromatin immunoprecipitation assays. Cells were incubated with miRNAs mimics, control sequences, or small interfering RNAs; expression of reporter constructs was measured in luciferase assays. CRC cells were incubated with F nucleatum or PBS and injected into BALB/C nude mice; growth of xenograft tumors was measured. C57BL adenomatous polyposis colimin/+, C57BL miR21a-/-, and C57BL mice with full-length miR21a (controls) were given F nucleatum by gavage; some mice were given azoxymethane and dextran sodium sulfate to induce colitis and colon tumors. Intestinal tissues were collected and tumors were counted. Serum samples from mice were analyzed for cytokine levels by enzyme-linked immunosorbent assay. We performed in situ hybridization analyses to detect enrichment of F nucleatum in CRC cells. Fusobacterium nucleatum DNA in 90 tumor and matched nontumor tissues from patients in China were explored for the expression correlation analysis; levels in 125 tumor tissues from patients in Japan were compared with their survival times. RESULTS: Fusobacterium nucleatum increased proliferation and invasive activities of CRC cell lines compared with control cells. CRC cell lines infected with F nucleatum formed larger tumors, more rapidly, in nude mice than uninfected cells. Adenomatous polyposis colimin/+ mice gavaged with F nucleatum developed significantly more colorectal tumors than mice given PBS and had shorter survival times. We found several inflammatory factors to be significantly increased in serum from mice given F nucleatum (interleukin 17F, interleukin 21, and interleukin 22, and MIP3A). We found 50 miRNAs to be significantly up-regulated and 52 miRNAs to be significantly down-regulated in CRCs incubated with F nucleatum vs PBS; levels of miR21 increased by the greatest amount (>4-fold). Inhibitors of miR21 prevented F nucleatum from inducing cell proliferation and invasion in culture. miR21a-/- mice had a later appearance of fecal blood and diarrhea after administration of azoxymethane and dextran sodium sulfate, and had longer survival times compared with control mice. The colorectum of miR21a-/- mice had fewer tumors, of smaller size, and the miR21a-/- mice survived longer than control mice. We found RASA1, which encodes an RAS GTPase, to be one of the target genes consistently down-regulated in cells that overexpressed miR21 and up-regulated in cells exposed to miR21 inhibitors. Infection of cells with F nucleatum increased expression of miR21 by activating Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB. Levels of F nucleatum DNA and miR21 were increased in tumor tissues (and even more so in advanced tumor tissues) compared with non-tumor colon tissues from patients. Patients whose tumors had high amounts of F nucleatum DNA and miR21 had shorter survival times than patients whose tumors had lower amounts. CONCLUSIONS: We found infection of CRC cells with F nucleatum to increase their proliferation, invasive activity, and ability to form xenograft tumors in mice. Fusobacterium nucleatum activates Toll-like receptor 4 signaling to MYD88, leading to activation of the nuclear factor-κB and increased expression of miR21; this miRNA reduces levels of the RAS GTPase RASA1. Patients with both high amount of tissue F nucleatum DNA and miR21 demonstrated a higher risk for poor outcomes.

N52 monodeamidated Bcl­xL shows impaired oncogenic properties in vivo and in vitro.

Bcl-xL is a member of the Bcl-2 family, playing a critical role in the survival of tumor cells. Here, we show that Bcl-xL oncogenic function can be uncoupled from its anti-apoptotic activity when it is regulated by the post-translational deamidation of its Asn52.Bcl-xL activity can be regulated by post-translational modifications: deamidation of Asn52 and 66 into Asp residues was reported to occur exclusively in response to DNA damage, and to cripple its anti-apoptotic activity. Our work reports for the first time the spontaneous occurrence of monodeamidated Asp52Bcl-xL in control conditions, in vivo and in vitro. In the normal and cancer cell lines tested, no less than 30% and up to 56% of Bcl-xL was singly deamidated on Asn52. Functional analyses revealed that singly deamidated Bcl-xL retains anti-apoptotic functions, and exhibits enhanced autophagic activity while harboring impaired clonogenic and tumorigenic properties compared to native Bcl-xL. Additionally, Asp52Bcl-xL remains phosphorylatable, and thus is still an eligible target of anti-neoplasic agents. Altogether our results complement the existing data on Bcl-xL deamidation: they challenge the common acceptance that Asn52 and Asn66 are equally eligible for deamidation, and provide a valuable improvement of our knowledge on the regulation of Bcl-xLoncogenic functions by deamidation.

A Cytosolic Multiprotein Complex Containing p85α Is Required for ß-Catenin Activation in Colitis and Colitis-associated Cancer.

Wnt/ß-catenin signaling is required for crypt structure maintenance. We previously observed nuclear accumulation of Ser-552 phosphorylated ß-catenin (pß-Cat(Ser-552)) in intestinal epithelial cells (IEC) during colitis and colitis-associated cancer. Data here delineate a novel multiprotein cytosolic complex (MCC) involved in ß-catenin signaling in the intestine. The MCC contains p85α, the class IA subunit of PI3K, along with ß-catenin, 14-3-3ζ, Akt, and p110α. MCC levels in IEC increase in colitis and colitis-associated cancer patients. IEC-specific p85α-deficient (p85(ΔIEC)) mice develop more severe dextran sodium sulfate colitis due to delayed ulcer healing and reduced epithelial ß-catenin activation. In colonic IEC, p85α deficiency did not alter PI3K signaling. In vitro shRNA depletion of individual complex members disrupts the MCC and reduces ß-catenin signaling. Despite worse colitis, p85(ΔIEC) mice have reduced tumor burden after azoxymethane/dextran sodium sulfate treatment. Together the data indicate that the ß-catenin MCC is needed for mucosal repair and carcinogenesis. This novel MCC may be an attractive therapeutic target in preventing cancer in colitis patients.

Gastrointestinal digested Sambucus nigra L. fruit extract protects in vitro cultured human colon cells against oxidative stress.

Elderberry (EDB) Sambucus nigra L. is one of the oldest medicinal plants which is useful for therapeutic and nutritional purposes due to a large amount of biologically active constituents, including compounds with a high antioxidant capacity. The present study focused on the antioxidant potential of the colon-available EDB fruit extract, derived from the artificial gastrointestinal tract, with regard to human colonic mucosa cells cultured in vitro. Despite the significant loss of EDB bioactive compounds due to the digestion process, the colon-digested extract was able to reduce the excessive intracellular ROS production (22%) and oxidative DNA damage (46%) in the colon cells at a dose of 1 mg of freeze-dried EDB powder/ml. Moreover, the colon-digested EDB extract inhibited oxidant-induced mutagenicity (26%) in the Salmonella typhimurium TA102 strain, as determined by the Ames test. In conclusion, the current in vitro study confirmed that the fruits of S. nigra are capable of protecting colonic cells against the detrimental effects of oxidative stress.

Antioxidant effects of gastrointestinal digested purple carrot extract on the human cells of colonic mucosa.

Purple carrot (PC) is a potential dietary constituent, which represents a valuable source of antioxidants and can modulate the reactive oxygen species (ROS) level in the gastrointestinal tract. Antioxidant capacity of a PC extract subjected to digestion process simulated in the artificial alimentary tract, including the stomach, small intestine and colon, was analyzed in normal human cells of colon mucosa. Results indicated that the extract obtained upon passage through the gastrointestinal tract, which could come into contact with the colonic cells in situ, was less potent than the extract, which was not subjected to digestion process. Digested PC extract exhibited intracellular ROS-inhibitory capacity, with 1mg/mL showing the ROS clearance of 18.4%. A 20.7% reduction in oxidative DNA damage due to colon mucosa cells' treatment with digested PC extract was observed. These findings indicate that PC extract is capable of colonic cells' protection against the adverse effects of oxidative stress.

Long non-coding RNA CCAL regulates colorectal cancer progression by activating Wnt/ß-catenin signalling pathway via suppression of activator protein 2α.

OBJECTIVE: Long non-coding RNAs (lncRNAs) are emerging as key molecules in cancers, yet their potential molecular mechanisms are not well understood. The objective of this study is to examine the expression and functions of lncRNAs in the development of colorectal cancer (CRC). METHODS: LncRNA expression profiling of CRC, adenoma and normal colorectal tissues was performed to identify tumour-related lncRNAs involved in colorectal malignant transformation. Then, we used quantitative reverse transcription PCR assays to measure the tumour-related lncRNA and to assess its association with survival and response to adjuvant chemotherapy in 252 patients with CRC. The mechanisms of CCAL function and regulation in CRC were examined using molecular biological methods. RESULTS: We identified colorectal cancer-associated lncRNA (CCAL) as a key regulator of CRC progression. Patients whose tumours had high CCAL expression had a shorter overall survival and a worse response to adjuvant chemotherapy than patients whose tumours had low CCAL expression. CCAL promoted CRC progression by targeting activator protein 2α (AP-2α), which in turn activated Wnt/ß-catenin pathway. CCAL induced multidrug resistance (MDR) through activating Wnt/ß-catenin signalling by suppressing AP-2α and further upregulating MDR1/P-gp expression. In addition, we found that histone H3 methylation and deacetylases contributed to the upregulation of CCAL in CRC. CONCLUSIONS: Our results suggest that CCAL is a crucial oncogenic regulator involved in CRC tumorigenesis and progression.

Loss of PTEN Facilitates Rosiglitazone-Mediated Enhancement of Platinum(IV) Complex LA-12-Induced Apoptosis in Colon Cancer Cells.

We demonstrated for the first time an outstanding ability of rosiglitazone to mediate a profound enhancement of LA-12-induced apoptosis associated with activation of mitochondrial pathway in human colon cancer cells. This effect was preferentially observed in the G1 cell cycle phase, independent on p53 and PPARγ proteins, and accompanied with significant changes of selected Bcl-2 family protein levels. Further stimulation of cooperative synergic cytotoxic action of rosiglitazone and LA-12 was demonstrated in the cells deficient for PTEN, where mitochondrial apoptotic pathway was more stimulated and G1-phase-associated dying was reinforced. Our results suggest that combined treatment with rosiglitazone and LA-12 might be promising anticancer strategy in colon-derived tumours regardless of their p53 status, and also favourable in those defective in PTEN function.

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 around 80% of colorectal tumors carrying a mutation in BRAF also overexpress splice variant Rac1b. We used normal NCM460 colonocytes as a model to express oncogenic B-Raf-V600E in the presence or absence of co-transfected Rac1b and then analyzed the effect on expression of senescence markers. When oncogenic B-Raf-V600E was expressed we observed the induction of the senescence-associated ß-galactosidase and of the cell-cycle inhibitors p14, p15 and p21 whereas proliferation marker Ki67 was suppressed. Upon co-expression of splice variant Rac1b, but not of Rac1, the B-Raf-induced senescence phenotype was reverted and expression of the cell-cycle inhibitors downregulated in a reactive oxygen-species dependent manner. We thus provide evidence that co-expression of splice variant Rac1b counteracts B-Raf-induced senescence, indicating the selection for increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.