YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8+ T cells.

Despite tumor-associated macrophages (TAMs) playing a key role in shaping the tumor microenvironment (TME), the mechanisms by which TAMs influence the TME and contribute to cancer progression remain unclear. Here, we show that the N6-methyladenosine reader YTHDF2 regulates the antitumor functions of TAMs. YTHDF2 deficiency in TAMs suppressed tumor growth by reprogramming TAMs toward an antitumoral phenotype and increasing their antigen cross-presentation ability, which in turn enhanced CD8+ T cell-mediated antitumor immunity. YTHDF2 deficiency facilitated the reprogramming of TAMs by targeting interferon-γ-STAT1 signaling. The expression of YTHDF2 in TAMs was regulated by interleukin-10-STAT3 signaling. Selectively targeting YTHDF2 in TAMs using a Toll-like receptor 9 agonist-conjugated small interfering RNA reprogrammed TAMs toward an antitumoral phenotype, restrained tumor growth and enhanced the efficacy of PD-L1 antibody therapy. Collectively, our findings describe the role of YTHDF2 in orchestrating TAMs and suggest that YTHDF2 inhibition is an effective approach to enhance cancer immunotherapy.

Lyso-PAF, a biologically inactive phospholipid, contributes to RAF1 activation.

Phospholipase A2, group VII (PLA2G7) is widely recognized as a secreted, lipoprotein-associated PLA2 in plasma that converts phospholipid platelet-activating factor (PAF) to a biologically inactive product Lyso-PAF during inflammatory response. We report that intracellular PLA2G7 is selectively important for cell proliferation and tumor growth potential of melanoma cells expressing mutant NRAS, but not cells expressing BRAF V600E. Mechanistically, PLA2G7 signals through its product Lyso-PAF to contribute to RAF1 activation by mutant NRAS, which is bypassed by BRAF V600E. Intracellular Lyso-PAF promotes p21-activated kinase 2 (PAK2) activation by binding to its catalytic domain and altering ATP kinetics, while PAK2 significantly contributes to S338-phosphorylation of RAF1 in addition to PAK1. Furthermore, the PLA2G7-PAK2 axis is also required for full activation of RAF1 in cells stimulated by epidermal growth factor (EGF) or cancer cells expressing mutant KRAS. Thus, PLA2G7 and Lyso-PAF exhibit intracellular signaling functions as key elements of RAS-RAF1 signaling.

Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m6A Methylation.

N6-methyladenosine (m6A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m6A methylation in mRNA metabolism has been well documented recently, regulation of the m6A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m6A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m6A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m6A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m6A methylation.

Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.

There is growing evidence that tumour neoantigens have important roles in generating spontaneous antitumour immune responses and predicting clinical responses to immunotherapies1,2. Despite the presence of numerous neoantigens in patients, complete tumour elimination is rare, owing to failures in mounting a sufficient and lasting antitumour immune response3,4. Here we show that durable neoantigen-specific immunity is regulated by mRNA N6-methyadenosine (m6A) methylation through the m6A-binding protein YTHDF15. In contrast to wild-type mice, Ythdf1-deficient mice show an elevated antigen-specific CD8+ T cell antitumour response. Loss of YTHDF1 in classical dendritic cells enhanced the cross-presentation of tumour antigens and the cross-priming of CD8+ T cells in vivo. Mechanistically, transcripts encoding lysosomal proteases are marked by m6A and recognized by YTHDF1. Binding of YTHDF1 to these transcripts increases the translation of lysosomal cathepsins in dendritic cells, and inhibition of cathepsins markedly enhances cross-presentation of wild-type dendritic cells. Furthermore, the therapeutic efficacy of PD-L1 checkpoint blockade is enhanced in Ythdf1-/- mice, implicating YTHDF1 as a potential therapeutic target in anticancer immunotherapy.

Author Correction: Anti-tumour immunity controlled through mRNA m6A methylation and YTHDF1 in dendritic cells.

In this Letter, a citation to 'Fig. 1e' has been corrected to 'Fig. 1d' in the sentence starting "By contrast, the anti-tumour response…". This has been corrected online.

Association of Microsatellite Instability and Gene Expression Profile in Colorectal Carcinoma and Potential Implications for Therapy.

Background and Objective: In sporadic colorectal carcinomas (CRC), microsatellite instability (MSI) pathways play important roles. Previously, we showed differences in DNA methylation patterns in microsatellite stable (MSS) colorectal carcinomas and MSI-CRC. In the current study, we explore the similarities and differences in gene expression profiles in MSS and MSI at the gene level and at the pathway level to better understand CRC pathogenesis and/or the potential for therapeutic opportunities. Material and Methods: Seventy-one CRC patients (MSI = 18, MSS = 53) were studied. Paired tumor and adjacent normal tissues were used for genome-wide gene expression assays. Result: At the gene level, we compared the list of differentially expressed genes (fold change (FC) ≥ 3 and FDR < 0.05) in tumor tissues compared to corresponding normal tissue in CRC patients with MSI tumors (190 genes) and MSS tumors (129 genes). Of these, 107 genes overlapped. The list of genes that were differentially expressed in MSI tumors only showed enrichment predominantly in two broad categories of pathways-(a) Inflammation-related pathways including the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, chemokine signaling, nuclear factor kappa B (NFκB) signaling, and cytokine-cytokine interactions, and (b) metabolism-related pathways, including retinol metabolism, steroid hormone biosynthesis, drug metabolism, pentose and glucoronate interconversions, and ascorbate and aldarate metabolism. The genes in inflammation-related pathways were up-regulated whereas genes in metabolism-related pathways were down-regulated in MSI tumor tissue. Pathway-level analysis also revealed similar results confirming the gene enrichment findings. For example, the 150 genes involved in the IL-17 signaling pathway were on average up-regulated by 1.19 fold (CI 1.16-1.21) in MSI compared to 1.14 fold (CI 1.13-1.16) in MSS patients (interaction p = 0.0009). Conclusions: We document an association between MSI status and differential gene expression that broadens our understanding of CRC pathogenesis. Furthermore, targeting one or more of these dysregulated pathways could provide the basis for improved therapies for MSI and MSS CRC.

Western Diet Promotes Intestinal Colonization by Collagenolytic Microbes and Promotes Tumor Formation After Colorectal Surgery.

BACKGROUND & AIMS: The Western diet, which is high in fat, is a modifiable risk factor for colorectal recurrence after curative resection. We investigated the mechanisms by which the Western diet promotes tumor recurrence, including changes in the microbiome, in mice that underwent colorectal resection. METHODS: BALB/c male mice were fed either standard chow diet or Western-type diet (characterized by high fat, no fiber, and decreased minerals and vitamins) for 4 weeks; some mice were given antibiotics or ABA-PEG20k-Pi20 (Pi-PEG), which inhibits collagenase production by bacteria, but not bacterial growth, in drinking water. Colorectal resections and anastomoses were then performed. The first day after surgery, mice were given enemas containing a collagenolytic rodent-derived strain of Enterococcus faecalis (strain E2), and on the second day they were given mouse colon carcinoma cells (CT26). Twenty-one days later, distal colons were removed, and colon contents (feces, distal colon, and tumor) were collected. Colon tissues were analyzed by histology for the presence of collagenolytic colonies and by 16S ribosomal RNA sequencing, which determined the anatomic distribution of E faecalis at the site of the anastomosis and within tumors using in situ hybridization. Mouse imaging analyses were used to identify metastases. RESULTS: Colorectal tumors were found in 88% of mice fed the Western diet and given antibiotics, surgery, and E faecalis compared with only 30% of mice fed the standard diet followed by the same procedures. Colon tumor formation correlated with the presence of collagenolytic E faecalis and Proteus mirabilis. Antibiotics eliminated collagenolytic E faecalis and P mirabilis but did not reduce tumor formation. However, antibiotics promoted emergence of Candida parapsilosis, a collagenase-producing microorganism. Administration of a Pi-PEG reduced tumor formation and maintained diversity of the colon microbiome. CONCLUSIONS: We identified a mechanisms by which diet and antibiotic use can promote tumorigenesis by colon cancer cells at the anastomosis after colorectal surgery. Strategies to prevent emergence of these microbe communities or their enzymatic activities might be used to reduce the risk of tumor recurrence in patients undergoing colorectal cancer surgery.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12-CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12-CXCR4/CXCR7 axis as a treatment strategy for CRC.

A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase.

Although valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene - either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model's small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors - unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.

Impact of Angiotensin II Signaling Blockade on Clinical Outcomes in Patients with Inflammatory Bowel Disease.

BACKGROUND: Preclinical data demonstrate that activation of the renin-angiotensin system (RAS) contributes to mucosal inflammation, and RAS inhibition by angiotensin-converting-enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) improves colitis in animal models. Less is known regarding the effects of RAS inhibition on clinical outcomes in inflammatory bowel disease (IBD) patients. AIM: Evaluate the impact of ACEI and ARB on clinical outcomes in IBD. METHODS: Rates of IBD-related hospitalizations, operations, and corticosteroid use were evaluated retrospectively in two groups. First, 111 IBD patients taking an ACEI or ARB were compared to nonusers matched 1:1 based on sex, age, diagnosis, disease location, and hypertension diagnosis. Second, outcomes in a cohort of 130 IBD patients were compared prior to and during ACEI/ARB exposure. RESULTS: Compared to matched controls, all IBD patients together with ACEI/ARB exposure had fewer hospitalizations (OR 0.26, p < 0.01), operations (OR 0.08, p = 0.02), and corticosteroid prescriptions (OR 0.5, p = 0.01). Comparing outcomes before and during ACEI/ARB use, there were no differences in hospitalizations, operations, or corticosteroid use for all IBD patients together, but patients with UC had increased hospitalizations (0.08 pre- vs. 0.16 during ACEI/ARB exposure, p = 0.03) and decreased corticosteroid use (0.24 pre-ACEI/ARB vs. 0.12 during ACEI/ARB exposure, p < 0.01) during ACEI/ARB use. CONCLUSIONS: IBD patients with ACEI/ARB exposure had fewer hospitalizations, operations, and corticosteroid use compared to matched controls. No differences in outcomes were observed in individuals on ACEI/ARB therapy when compared to a period of time prior to medication exposure.

Early increase in blood supply (EIBS) is associated with tumor risk in the Azoxymethane model of colon cancer.

BACKGROUND: The present study aimed to investigate the role of blood supply in early tumorigenesis in colorectal cancer. We leveraged the renin angiotensin system (RAS) to alter colonic blood supply and determine the effect on tumor initiation and progression. METHODS: To test the effect of blood supply on tumorigenesis, 53 male A/J mice were randomly assigned to one of three RAS modulation groups and one of two AOM treatments. The RAS modulation groups were I) water (RAS-unmodulated) as a control group, II) angiotensin-II and III) the angiotensin receptor blocker, Losartan. The mice in each group were then randomly split into either the saline control condition or the AOM-treated condition in which tumors were induced with a standard protocol of serial azoxymethane (AOM) injections. To monitor microvascular changes in the rectal mucosa during the study, we used confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal hemoglobin concentration ([Hb]) and blood vessel radius (BVR). RESULTS: At 12 weeks post-AOM injections and before tumor formation, CLE images revealed many traditional hallmarks of angiogenesis including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting in the pericryptal capillaries of the rectal mucosa in AOM-Water tumor bearing mice. PGS measurements at the same time-point showed increased rectal [Hb] and decreased BVR. At later time points, CLE images showed pronounced angiogenic features including irregular networks throughout the colon. Notably, the AOM-Losartan mice had significantly lower tumor multiplicity and did not exhibit the same angiogenic features observed with CLE, or the increase in [Hb] or decrease in BVR measured with PGS. The AOM-AngII mice did not have any significant trends. CONCLUSION: In-vivo PGS measurements of rectal colonic blood supply as well as CLE imaging revealed angiogenic disruptions to the capillary network prior to tumor formation. Losartan demonstrated an effective way to mitigate the changes to blood supply during tumorigenesis and reduce tumor multiplicity. These effects can be used in future studies to understand the early vessel changes observed.

Lectin-functionalized mesoporous silica nanoparticles for endoscopic detection of premalignant colonic lesions.

Colorectal cancer (CRC) is one of the leading causes of cancer-deaths worldwide. Methods for the early in situ detection of colorectal adenomatous polyps and their precursors - prior to their malignancy transformation into CRC - are urgently needed. Unfortunately at present, the primary diagnostic method, colonoscopy, can only detect polyps and carcinomas by shape/morphology; with sessile polyps more likely to go unnoticed than polypoid lesions. Here we describe our development of polyp-targeting, fluorescently-labeled mesoporous silica nanoparticles (MSNs) that serve as targeted endoscopic contrast agents for the early detection of colorectal polyps and cancer. In vitro cell studies, ex vivo histopathological analysis, and in vivo colonoscopy and endoscopy of murine colorectal cancer models, demonstrate significant binding specificity of our nanoconstructs to pathological lesions via targeting aberrant α-L-fucose expression. Our findings strongly suggest that lectin-functionalized fluorescent MSNs could serve as a promising endoscopic contrast agent for in situ diagnostic imaging of premalignant colonic lesions.

Significant difference in active metabolite levels of ginseng in humans consuming Asian or Western diet: The link with enteric microbiota.

After ingestion of ginseng, the bioavailability of its parent compounds is low and enteric microbiota plays an important role in parent compound biotransformation to their metabolites. Diet type can influence the enteric microbiota profile. When human subjects on different diets ingest ginseng, their different gut microbiota profiles may influence the metabolism of ginseng parent compounds. In this study, the effects of different diet type on gut microbiota metabolism of American ginseng saponins were investigated. We recruited six healthy adults who regularly consumed different diet types. These subjects received 7 days' oral American ginseng, and their biological samples were collected for LC-Q-TOF-MS analysis. We observed significant ginsenoside Rb1 (a major parent compound) and compound K (a major active metabolite) level differences in the samples from the subjects consuming different diets. Subjects on an Asian diet had much higher Rb1 levels but much lower compound K levels compared with those on a Western diet. Since compound K possesses much better cancer chemoprevention potential, our data suggested that consumers on a Western diet should obtain better cancer prevention effects with American ginseng intake compared with those on an Asian diet. Ginseng compound levels could be enhanced or reduced via gut microbiota manipulation for clinical utility.

Daikenchuto (TU-100) Suppresses Tumor Development in the Azoxymethane and APCmin/+ Mouse Models of Experimental Colon Cancer.

Chemopreventative properties of traditional medicines and underlying mechanisms of action are incompletely investigated. This study demonstrates that dietary daikenchuto (TU-100), comprised of ginger, ginseng, and Japanese pepper effectively suppresses intestinal tumor development and progression in the azoxymethane (AOM) and APCmin/+ mouse models. For the AOM model, TU-100 was provided after the first of six biweekly AOM injections. Mice were sacrificed at 30 weeks. APCmin/+ mice were fed diet without or with TU-100 starting at 6 weeks, and sacrificed at 24 weeks. In both models, dietary TU-100 decreased tumor size. In APC min/+ mice, the number of small intestinal tumors was significantly decreased. In the AOM model, both TU-100 and Japanese ginseng decreased colon tumor numbers. Decreased Ki-67 and ß-catenin immunostaining and activation of numerous transduction pathways involved in tumor initiation and progression were observed. EGF receptor expression and stimulation/phosphorylation in vitro were investigated in C2BBe1 cells. TU-100, ginger, and 6-gingerol suppressed EGF receptor induced Akt activation. TU-100 and ginseng and to a lesser extent ginger or 6-gingerol inhibited EGF ERK1/2 activation. TU-100 and some of its components and metabolites of these components inhibit tumor progression in two mouse models of colon cancer by blocking downstream pathways of EGF receptor activation. Copyright © 2016 John Wiley & Sons, Ltd.

The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon.

OBJECTIVE: ATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohn's disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells. DESIGN: We genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant. RESULTS: We found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway. CONCLUSIONS: The CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.

Hsp70 exerts oncogenic activity in the Apc mutant Min mouse model.

Colorectal cancer (CRC) develops from colonic epithelial cells that lose expression of key tumor suppressor genes and/or gain expression of proproliferative and antiapoptotic genes like heat shock protein 70 (Hsp70). Heat shock protein 70 is overexpressed in CRC, but it is not known whether this is in response to the proteotoxic stress induced by transformation, or if it contributes to the process of transformation itself. Here, using the Apc (Min/+) mouse model of CRC, we show that Hsp70 regulates mitogenic signaling in intestinal epithelial cells through stabilization of proteins involved in the receptor tyrosine kinase (RTK) and WNT signaling pathways. Loss of Hsp70 reduced tumor size with decreased proliferation and increased tumor cell death. Hsp70 loss also led to decreased expression of ErbB2, Akt, ERK and ß-catenin along with decreased ß-catenin transcriptional activity as measured by c-myc and axin2 expression. Upregulation of RTK or WNT signals are frequent oncogenic events in CRC and many other cancers. Thus, in addition to the role of Hsp70 in cell-survival after transformation, Hsp70 stabilization of ß-catenin, Akt, ERK and ErbB2 are predicted to contribute to transformation. This has important implications not only for understanding the pathophysiology of these cancers, but also for treatment since anti-EGFR antibodies are in clinical use for CRC and EGFR is a major ErbB2 heterodimeric partner. Targeting Hsp70, therefore, might provide an alternative or complementary strategy for achieving better outcomes for CRC and other related cancer types.

X-ray fluorescence microscopy demonstrates preferential accumulation of a vanadium-based magnetic resonance imaging contrast agent in murine colonic tumors.

Contrast agents that specifically enhance cancers on magnetic resonance imaging (MRI) will allow earlier detection. Vanadium-based chelates (VCs) selectively enhance rodent cancers on MRI, suggesting selective uptake of VCs by cancers. Here we report x-ray fluorescence microscopy (XFM) of VC uptake by murine colon cancer. Colonic tumors in mice treated with azoxymethane/dextran sulfate sodium were identified by MRI. Then a gadolinium-based contrast agent and a VC were injected intravenously; mice were sacrificed and colons sectioned. VC distribution was sampled at 120 minutes after injection to evaluate the long-term accumulation. Gadolinium distribution was sampled at 10 minutes after injection due to its rapid washout. XFM was performed on 72 regions of normal and cancerous colon from five normal mice and four cancer-bearing mice. XFM showed that all gadolinium was extracellular, with similar concentrations in colon cancers and normal colon. In contrast, the average VC concentration was twofold higher in cancers versus normal tissue (p < .002). Cancers also contained numerous "hot spots" with intracellular VC concentrations sixfold higher than the concentration in normal colon (p < .0001). No hot spots were detected in normal colon. This is the first direct demonstration that VCs selectively accumulate in cancer cells and thus may improve cancer detection.

Metformin for primary colorectal cancer prevention in patients with diabetes: a case-control study in a US population.

BACKGROUND: Emerging evidence from observational studies has suggested that metformin may be beneficial in the primary prevention of colorectal cancer (CRC). However, to the authors' knowledge, none of these studies was conducted in a US population. Because environmental factors such as Western diet and obesity are implicated in the causation of CRC, a large case-control study was performed to assess the effects of metformin on the incidence of CRC in a US population. METHODS: MarketScan databases were used to identify diabetic patients with CRC. A case was defined as having an incident diagnosis of CRC. Up to 2 controls matched for age, sex, and geographical region were selected for each case. Metformin exposure was assessed by prescription tracking within the 12-month period before the index date. Conditional logistic regression was used to adjust for multiple potential confounders and to calculate adjusted odds ratios (AORs). RESULTS: The mean age of the study participants was 55 years and 57 years, respectively, in the control and case groups (P = 1.0). Approximately 60% of the study participants were male and 40% were female in each group. In the multivariable model, any metformin use was associated with a 15% reduction in the odds of CRC (AOR, 0.85; 95% confidence interval, 0.76-0.95 [P = .007]). After adjusting for health care use, the beneficial effect of metformin was reduced to 12% (AOR, 0.88; 95% confidence interval, 0.77-1.00 [P = .05]). The dose-response analyses demonstrated no significant association with metformin dose, duration, or total exposure. CONCLUSIONS: Metformin use appears to be associated with a reduced risk of developing CRC among diabetic patients in the United States.

Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

T-oligo as an anticancer agent in colorectal cancer.

In the United States, there will be an estimated 96,830 new cases of colorectal cancer (CRC) and 50,310 deaths in 2014. CRC is often detected at late stages of the disease, at which point there is no effective chemotherapy. Thus, there is an urgent need for effective novel therapies that have minimal effects on normal cells. T-oligo, an oligonucleotide homologous to the 3'-telomere overhang, induces potent DNA damage responses in multiple malignant cell types, however, its efficacy in CRC has not been studied. This is the first investigation demonstrating T-oligo-induced anticancer effects in two CRC cell lines, HT-29 and LoVo, which are highly resistant to conventional chemotherapies. In this investigation, we show that T-oligo may mediate its DNA damage responses through the p53/p73 pathway, thereby inhibiting cellular proliferation and inducing apoptosis or senescence. Additionally, upregulation of downstream DNA damage response proteins, including E2F1, p53 or p73, was observed. In LoVo cells, T-oligo induced senescence, decreased clonogenicity, and increased expression of senescence associated proteins p21, p27, and p53. In addition, downregulation of POT1 and TRF2, two components of the shelterin protein complex which protects telomeric ends, was observed. Moreover, we studied the antiproliferative effects of T-oligo in combination with an EGFR tyrosine kinase inhibitor, Gefitinib, which resulted in an additive inhibitory effect on cellular proliferation. Collectively, these data provide evidence that T-oligo alone, or in combination with other molecularly targeted therapies, has potential as an anti-cancer agent in CRC.