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.

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.

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.

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.

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.

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.

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.

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.

Phosphorylation of SRSF1 by SRPK1 regulates alternative splicing of tumor-related Rac1b in colorectal cells.

The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3ß, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3ß or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.

Validation of NCM460 cell model as control in antitumor strategies targeting colon adenocarcinoma metabolic reprogramming: trichostatin A as a case study.

BACKGROUND: Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected. METHODS: Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-(13)C2]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism. RESULTS: We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells. CONCLUSIONS: We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming. GENERAL SIGNIFICANCE: Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.

DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism.

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.

A SMYD3 Small-Molecule Inhibitor Impairing Cancer Cell Growth.

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

Effect of Negative Pressure Therapy on the Inflammatory Response of the Intestinal Microenvironment in a Porcine Septic Model.

In a swine model of ischemia/reperfusion injury coupled with sepsis, we have previously shown attenuation of secondary organ injury and decreased mortality with negative pressure therapy (NPT). We hypothesized that NPT modulates the intestinal microenvironment by mediating the innate immune system. Sepsis was induced in 12 anesthetized female pigs. Group 1 (n = 6) was decompressed at 12 hrs after injury (T 12) and treated with standard of care (SOC), and group 2 (n = 6) with NPT for up to T 48. Immunoparalysis was evident as lymphocytopenia at T 24 in both groups; however, survival was improved in the NPT group versus SOC (Odds ratio = 4.0). The SOC group showed significant reduction in lymphocyte numbers compared to NPT group by T 48 (p < 0.05). The capacity of peritoneal fluid to stimulate a robust reactive oxygen species response in vitro was greater for the NPT group, peaking at T 24 for both M1 (p = 0.0197) and M2 macrophages (p = 0.085). Plasma elicited little if any effect which was confirmed by microarray analysis. In this septic swine model NPT appeared to modulate the intestinal microenvironment, facilitating an early robust, yet transient, host defense mediated by M1 and M2 macrophages. NPT may help overcome immunoparalysis that occurs during inflammatory response to septic injury.

Antioxidant capacity of broccoli sprouts subjected to gastrointestinal digestion.

BACKGROUND: Broccoli is a common vegetable recognized as a rich source of antioxidants. To date, research on the antioxidant properties of broccoli, predominantly conducted on extracts, has not considered the lesions of composition and this activity after gastrointestinal digestion. Here the stability of antioxidants during gastrointestinal digestion was evaluated in conjunction with the protective effects of broccoli sprouts (BS) against oxidative stress in human colon cells. RESULTS: The obtained data suggest that, among the biocompounds identified in BS, glucosinolates were mainly degraded under gastrointestinal digestion, while phenolics, particularly hydroxycinnamic acid derivatives, were the most resistant constituents. The antioxidant capacity of BS extract subjected to gastrointestinal digestion was similar to or higher than that determined for non-digested BS. Gastrointestinal digested BS extract exhibited reactive oxygen species (ROS)-inhibitory capacity in NCM460 human colon cells, with 1 mg mL(-1) showing an ROS clearance of 76.59%. A 57.33% reduction in oxidative DNA damage in NCM460 cells due to treatment with digested BS extract was observed. CONCLUSION: The results lend support to the possible application of BS as a rich source of antioxidants to improve the defensive system against oxidative stress in the human colon mucosa.

Knockout of MIMP protein in lactobacillus plantarum lost its regulation of intestinal permeability on NCM460 epithelial cells through the zonulin pathway.

BACKGROUND: Previous studies indicated that the micro integral membrane protein located within the media place of the integral membrane protein of Lactobacillus plantarum CGMCC 1258 had protective effects against the intestinal epithelial injury. In our study, we mean to establish micro integral membrane protein -knockout Lactobacillus plantarum (LPKM) to investigate the change of its protective effects and verify the role of micro integral membrane protein on protection of normal intestinal barrier function. METHODS: Binding assay and intestinal permeability were performed to verify the protective effects of micro integral membrane protein on intestinal permeability in vitro and in vivo. Molecular mechanism was also determined as the zonulin pathway. Clinical data were also collected for further verification of relationship between zonulin level and postoperative septicemia. RESULTS: LPKM got decreased inhibition of EPEC adhesion to NCM460 cells. LPKM had lower ability to alleviate the decrease of intestinal permeability induced by enteropathogenic-e.coli, and prevent enteropathogenic-e.coli -induced increase of zonulin expression. Overexpression of zonulin lowered the intestinal permeability regulated by Lactobacillus plantarum. There was a positive correlation between zonulin level and postoperative septicemia. Therefore, micro integral membrane protein could be necessary for the protective effects of Lactobacillus plantarum on intestinal barrier. CONCLUSION: MIMP might be a positive factor for Lactobacillus plantarum to protect the intestinal epithelial cells from injury, which could be related to the zonulin pathway.

SP1 mediates the link between methylation of the tumour suppressor miR-149 and outcome in colorectal cancer.

Although recent studies indicate that DNA methylation contributes to the down-regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation-silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5-aza-2'-deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation-specific PCR. We found that miRNA-149 (miR-149) was epigenetically silenced in CRC and down-regulation of miR-149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT-PCR analysis demonstrated that the miR-149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non-cancerous tissues. In addition, low expression of miR-149 was associated with a greater depth of invasion (p = 0.012), lower 5-year survival rate (p = 0.025), and was found to be an independent prognostic factor for overall survival (p = 0.016) in a multivariate analysis. Moreover, transfection of miR-149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR-149. Our data suggest that, as a methylation-sensitive miRNA, miR-149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications.

Invasive colon cancer, but not non-invasive adenomas induce a gradient effect of Wnt pathway receptor frizzled 1 (Fz1) expression in the tumor microenvironment.

BACKGROUND: Wnt signaling in the colon cancer tumor microenvironment (TME) may affect cancer biologic properties including invasion and metastatic dissemination. Prior reports have suggested that the expression of select frizzled (Fz) receptors may be altered in cancers and in the TME. METHODS: Colon cancer, colonic adenoma and normal colonic mucosal specimens were obtained under institutional review board approval and analyzed for the expression of Fz1 and Fz2 by confocal fluorescent immunohistochemistry and Wnt-specific membrane array. In vitro, the effect of Wnt3a on Fz1 expression was examined in normal-derived NCM460 cells by qRT-PCR and immunohistochemistry. RESULTS: Fz1 was expressed in colon cancer and villous adenomas but not in more benign tubular adenomas. Fz1 expression was seen in normal colonic mucosa in close proximity to colon cancer, but not villous or tubular adenomas. Normal colonic mucosa distant from colon cancer did not express Fz1. Fz2 was expressed ubiquitously in cancer, adenomas and normal colonic mucosa. Fz1 expression was induced by Wnt3a in a normal colon mucosa-derived cell line in vitro. CONCLUSIONS: Fz1 is a Wnt responsive gene in colon-derived tissues. Fz1 expression exhibited increased expression in normal mucosa only in close proximity to colon cancer. This field effect was not seen with pre-malignant adenomas and may be due to Wnt/ß-catenin signaling within the TME. Fz1 may represent a new TME-directed therapeutic target for patients with colon cancer.

Termination and activation of store-operated cyclic AMP production.

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca(2+) ] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this 'store-operated' pathway requires the ER Ca(2+) sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca(2+) entry via the plasma membrane Ca(2+) channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca(2+) /cAMP crosstalk system.