Jean M. Winter

DNA methylation within the normal colorectal mucosa is associated with pathway-specific predisposition to cancer

There are two major molecular pathways to sporadic colorectal cancer, the chromosomal instability (CIN) and the CpG island methylator phenotype (CIMP) pathways. This study recruited 166 patients undergoing colonoscopy. Biopsy samples were collected from the cecum, transverse colon, sigmoid colon and rectum. DNA methylation was quantified at ‘type A’ (ESR1, GATA5, HIC1, HPP1, SFRP1) and ‘type C’ markers (MGMT, MLH1, CDKN2A, MINT2, MINT31, IGF2, CACNA1G, NEUROG1, SOCS1, RUNX3), and LINE-1. ‘Type A’ genes are frequently methylated in normal and neoplastic tissues, proportional to tissue age. ‘Type C’ methylation is more specific for neoplasia. The last five ‘type C’ markers comprise a CIMP panel. The mean ‘type A’ and CIMP-panel methylation Z-scores were calculated. In all, 88 patients had adenomatous lesions, 32 had proximal serrated polyps (PSPs) and 50 were normal. Most ‘type A’ genes showed direct correlations between methylation and age (ESR1, ρ=0.66, P<0.0001), with higher methylation distally (ESR1, P<0.0001). On multivariate analysis, ‘type A’ methylation was inversely associated with colorectal adenomas (odds ratio=0.23, P<0.001), the precursor to CIN cancers. CIMP-panel methylation was significantly associated with advanced PSPs (odds ratio=5.1, P=0.009), the precursor to CIMP cancers. DNA methylation in normal mucosa varied with age and region and was associated with pathway-specific pathology. In the future, the colorectal field could yield important information and potentially inform clinical practice.

Butyrylated starch intake can prevent red meat induced O6-methyl-2-deoxyguanosine adducts in human rectal tissue : a randomised clinical trial

Epidemiological studies have identified increased colorectal cancer (CRC) risk with high red meat (HRM) intakes, whereas dietary fibre intake appears to be protective. In the present study, we examined whether a HRM diet increased rectal O6-methyl-2-deoxyguanosine (O6MeG) adduct levels in healthy human subjects, and whether butyrylated high-amylose maize starch (HAMSB) was protective. A group of twenty-three individuals consumed 300 g/d of cooked red meat without (HRM diet) or with 40 g/d of HAMSB (HRM+HAMSB diet) over 4-week periods separated by a 4-week washout in a randomised cross-over design. Stool and rectal biopsy samples were collected for biochemical, microbial and immunohistochemical analyses at baseline and at the end of each 4-week intervention period. The HRM diet increased rectal O6MeG adducts relative to its baseline by 21 % (P< 0·01), whereas the addition of HAMSB to the HRM diet prevented this increase. Epithelial proliferation increased with both the HRM (P< 0·001) and HRM+HAMSB (P< 0·05) diets when compared with their respective baseline levels, but was lower following the HRM+HAMSB diet compared with the HRM diet (P< 0·05). Relative to its baseline, the HRM+HAMSB diet increased the excretion of SCFA by over 20 % (P< 0·05) and increased the absolute abundances of the Clostridium coccoides group (P< 0·05), the Clostridium leptum group (P< 0·05), Lactobacillus spp. (P< 0·01), Parabacteroides distasonis (P< 0·001) and Ruminococcus bromii (P< 0·05), but lowered Ruminococcus torques (P< 0·05) and the proportions of Ruminococcus gnavus, Ruminococcus torques and Escherichia coli (P< 0·01). HRM consumption could increase the risk of CRC through increased formation of colorectal epithelial O6MeG adducts. HAMSB consumption prevented red meat-induced adduct formation, which may be associated with increased stool SCFA levels and/or changes in the microbiota composition.

Inhibition by Resistant Starch of Red Meat-Induced Promutagenic Adducts in Mouse Colon

Population studies have shown that high red meat intake may increase colorectal cancer risk. Our aim was to examine the effect of different amounts and sources of dietary protein on induction of the promutagenic adduct O6-methyl-2-deoxyguanosine (O6MeG) in colonocytes, to relate these to markers of large bowel protein fermentation and ascertain whether increasing colonic carbohydrate fermentation modified these effects. Mice (n = 72) were fed 15% or 30% protein as casein or red meat or 30% protein with 10% high amylose maize starch as the source of resistant starch. Genetic damage in distal colonocytes was detected by immunohistochemical staining for O6MeG and apoptosis. Feces were collected for measurement of pH, ammonia, phenols, p-cresol, and short-chain fatty acids (SCFA). O6MeG and fecal p-cresol concentrations were significantly higher with red meat than with casein (P < 0.018), with adducts accumulating in cells at the crypt apex. DNA adducts (P < 0.01) and apoptosis (P < 0.001) were lower and protein fermentation products (fecal ammonia, P < 0.05; phenol, P < 0.0001) higher in mice fed resistant starch. Fecal SCFA levels were also higher in mice fed resistant starch (P < 0.0001). This is the first demonstration that high protein diets increase promutagenic adducts (O6MeG) in the colon and dietary protein type seems to be the critical factor. The delivery of fermentable carbohydrate to the colon (as resistant starch) seems to switch from fermentation of protein to that of carbohydrate and a reduction in adduct formation, supporting previous observations that dietary resistant starch opposes the mutagenic effects of dietary red meat. Cancer Prev Res; 4(11); 1920–8. ©2011 AACR.

Manipulation of the gut microbiota using resistant starch is associated with protection against colitis-associated colorectal cancer in rats

This study evaluated whether dietary resistant starch (RS) and green tea extract (GTE), which have anti-inflammatory and anticancer properties, protect against colitis-associated colorectal cancer (CAC) using a rat model, also investigated potential mechanisms of action of these agents including their effects on the gut microbiota. Rats were fed a control diet or diets containing 10% RS, 0.5% GTE or a combination of the two (RS + GTE). CAC was initiated with 2 weekly azoxymethane (AOM) injections (10mg/kg) followed by 2% dextran sodium sulphate in drinking water for 7 days after 2 weeks on diets. Rats were killed 20 weeks after the first AOM. Colon tissues and tumours were examined for histopathology by H&E, gene/protein expression by PCR and immunohistochemistry and digesta for analyses of fermentation products and microbiota populations. RS and RS + GTE (but not GTE) diets significantly (P< 0.05) decreased tumour multiplicity and adenocarcinoma formation, relative to the control diet. Effects of RS + GTE were not different from RS alone. RS diet caused significant shifts in microbial composition/diversity, with increases in Parabacteroides, Barnesiella, Ruminococcus, Marvinbryantia and Bifidobacterium as primary contributors to the shift. RS-containing diets increased short chain fatty acids (SCFA) and expression of the SCFA receptor GPR43 mRNA, and reduced inflammation (COX-2, NF-kB, TNF-α and IL-1β mRNA) and cell proliferation P< 0.05. GTE had no effect. This is the first study that demonstrates chemopreventive effects of RS (but not GTE) in a rodent CAC model, suggesting RS might have benefit to patients with ulcerative colitis who are at an increased risk of developing CRC.

Dietary Manipulation of Oncogenic MicroRNA Expression in Human Rectal Mucosa: A Randomized Trial

High red meat (HRM) intake is associated with increased colorectal cancer risk, while resistant starch is probably protective. Resistant starch fermentation produces butyrate, which can alter microRNA (miRNA) levels in colorectal cancer cells in vitro; effects of red meat and resistant starch on miRNA expression in vivo were unknown. This study examined whether a HRM diet altered miRNA expression in rectal mucosa tissue of healthy volunteers, and if supplementation with butyrylated resistant starch (HRM+HAMSB) modified this response. In a randomized cross-over design, 23 volunteers undertook four 4-week dietary interventions; an HRM diet (300 g/day lean red meat) and an HRM+HAMSB diet (HRM with 40 g/day butyrylated high amylose maize starch), preceded by an entry diet and separated by a washout. Fecal butyrate increased with the HRM+HAMSB diet. Levels of oncogenic mature miRNAs, including miR17–92 cluster miRNAs and miR21, increased in the rectal mucosa with the HRM diet, whereas the HRM+HAMSB diet restored miR17–92 miRNAs, but not miR21, to baseline levels. Elevated miR17–92 and miR21 in the HRM diet corresponded with increased cell proliferation, and a decrease in miR17–92 target gene transcript levels, including CDKN1A. The oncogenic miR17–92 cluster is differentially regulated by dietary factors that increase or decrease risk for colorectal cancer, and this may explain, at least in part, the respective risk profiles of HRM and resistant starch. These findings support increased resistant starch consumption as a means of reducing risk associated with an HRM diet. Cancer Prev Res; 7(8); 786–95. ©2014 AACR.

Dietary Red Meat Aggravates Dextran Sulfate Sodium-Induced Colitis in Mice Whereas Resistant Starch Attenuates Inflammation

BackgroundAlthough a genetic component has been identified as a risk factor for developing inflammatory bowel disease, there is evidence that dietary factors also play a role in the development of this disease.AimsThe aim of this study was to determine the effects of feeding a red meat diet with and without resistant starch (RS) to mice with dextran sulfate sodium (DSS)-induced colitis.MethodsColonic experimental colitis was induced in Balb/c mice using DSS. The severity of colitis was evaluated based on a disease activity index (based on bodyweight loss, stool consistency, rectal bleeding, and overall condition of the animal) and a histological score. Estimations were made of numbers of a range of different bacteria in the treatment pools of cecal digesta using quantitative real-time PCR.ResultsConsumption of a diet high in red meat increased DSS-induced colitis as evidenced by higher disease activity and histopathological scores. Addition of RS to the red meat diet exerted a beneficial effect in acute DSS-induced colitis. Subjective analysis of numbers of a range of bacterial targets suggest changes in the gut microbiota abundance were induced by red meat and RS treatments and these changes could contribute to the reported outcomes.ConclusionsA dietary intake of red meat aggravates DSS-induced colitis whereas co-consumption of resistant starch reduces the severity of colitis.

Dietary butyrylated high-amylose starch reduces azoxymethane-induced colonic O6-methylguanine adducts in rats as measured by immunohistochemistry and high-pressure liquid chromatography

O(6)-methyl guanine (O(6)MeG) adducts are major toxic, promutagenic, and procarcinogenic adducts involved in colorectal carcinogenesis. Resistant starch and its colonic metabolite butyrate are known to protect against oncogenesis in the colon. In this study, we hypothesized that a dietary intervention that specifically delivers butyrate to the large bowel (notably butyrylated high-amylose maize starch [HAMSB]) would reduce colonic levels of O(6)MeG in rats shortly after exposure to the deoxyribonucleic acid (DNA) alkylating agent azoxymethane (AOM) when compared with a low-amylose maize starch (LAMS). A further objective was to validate an immunohistochemistry (IHC) method for quantifying O(6)MeG against a high-performance liquid chromatography method using fluorescence and diode array detection. Rats were fed either LAMS or HAMSB diets for 4 weeks followed by a single injection of AOM or saline and killed 6 hours later. After AOM exposure, both IHC and high-performance liquid chromatography method using fluorescence and diode array detection measured a substantially increased quantity of DNA adducts in the colon (P<.001). Both techniques demonstrated equally that consumption of HAMSB provided a protective effect by reducing colonic adduct load compared with the LAMS diet (P<.05). In addition, IHC allowed visualization of the O(6)MeG distribution, where adduct load was reduced in the lower third of the crypt compartment in HAMSB-fed rats (P=.036). The apoptotic response to AOM was higher in the HAMSB-fed rats (P=.002). In conclusion, the reduction in O(6)MeG levels and enhancement of the apoptotic response to DNA damage in the colonic epithelium through consumption of HAMSB provide mechanistic insights into how HAMSB protects against colorectal tumorigenesis.

Anti-mutagenic lichen extract has double-edged effect on azoxymethane-induced colorectal oncogenesis in C57BL/6J mice.

This study compared the effects of three anti-mutagenic lichen extracts on colorectal oncogenesis in azoxymethane (AOM)-treated mice and determined whether the extracts also regulated the homeostatic response to genotoxic damage. C57BL/6J mice (n = 12 per group) were treated with the lichen extracts Antimutagen-He (AMH): AMH-C, AMH-D, or AMH-E dimethyl sulfoxide (DMSO, control) for 2 weeks. At the end of the treatment, mice were given a single AOM injection to induce DNA damage and killed 6 h later for measuring apoptosis and proliferation. Apoptotic and proliferation indexes in mice treated with AMH-C, AMH-D, and AMH-E were 0.61%, 1.41%, and 0.77%; and 30.62%, 21.93%, and 27.27%, respectively, which were significantly lower than those of control mice (5.88% and 38.69%) (p < 0.05). To examine the effects of lichen extracts on colorectal cancer, separate groups of mice (n = 25 per group) treated with AMH-C, AMH-D, AMH-E, or DMSO were given 4-weekly AOM injections to induce oncogenesis. Mice were killed 24 weeks after the last AOM injection for assessing colon tumor formation. Colonic tumor incidences were 47.3%, 13%, and 20%; the tumor volumes were 18.47, 2.75, and 10.78 mm3, respectively, in mice treated with AMH-C (p < 0.05), AMH-D (p < 0.05), and AMH-E (p > 0.05), compared to 24% and 13.28 mm3 in mice of control correspondingly. No lichen extract showed evident toxic effects on mice. No usnic acid was found in these lichen extracts. The regulation of acute apoptosis and cell proliferation in colonic epithelial cells and the anti-mutagenesis do not seem directly related to the cancer protective effect.

Prostate cancer susceptibility gene HIST1H1A is a modulator of androgen receptor signaling and epithelial to mesenchymal transition

In 2018, approximately 165,000 new prostate cancer (PC) cases will be diagnosed, and over 29,000 men will succumb to PC in the U.S. alone. The means of assessing outcome in the clinic are inaccurate, and there is a pressing need to more precisely identify men at risk of aggressive PC. We previously identified HIST1H1A as a susceptibility gene for aggressive PC. HIST1H1A encodes H1.1, a member of the linker histone family that is involved in chromatin organization and compaction. To understand the molecular basis of aggressive PC, we have characterized how germline variation modulates susceptibility to neuroendocrine differentiation, which is a form of aggressive PC. Immunohistochemistry studies revealed that HIST1H1A is over-expressed in normal human prostate tissue compared to prostate adenocarcinoma. Functional characterization of HIST1H1A in prostate LNCaP cells indicated that HIST1HA over-expression increased cell growth, as well as the expression of neuroendocrine and epithelial-to-mesenchymal markers in vitro. Assay for Transposase-Accessible Chromatin (ATAC-seq), which is used to assess chromatin compaction and thus the transcriptional availability of individual genomic regions, demonstrated that H1.1 plays a prominent role in modulating Wnt signaling pathway genes, which are implicated in prostate tumorigenesis. These results demonstrate that HIST1H1A is a modulator of aggressive PC susceptibility.

Acknowledgement to the Reviewers

Chris T. Bolliger, Tygerberg, South Africa Raphael Borie, Paris, France Piera Boschetto, Ferrara, Italy Gabriel T. Bosslet, Indianapolis, USA Louis-Philippe Boulet, Sainte-Foy, Canada A. Bourdin, Montpellier, France John Brannan, Sydney, Australia Joerg Brederlau, Berlin, Germany E.C. Breen, La Jolla, USA Fabienne Bregeon, Marseille, France Pilar Brito-Zeron, Barcelona, Spain Dunja Bruder, Braunschweig, Germany Guy G. Brusselle, Gent, Belgium Martin H. Brutsche, St. Gallen, Switzerland Antonio Bugalho, Lisbon, Portugal Janette Burgess, Camperdown, Australia Umberto Campia, Chicago, USA Giorgio Walter Canonica, Genova, Italy Andre Capderou, Le Plessis Robinson, France Gaetano Caramori, Ferrara, Italy Pierluigi Carratu, Bari, Italy Laura Carrozzi, Pisa, Italy C.R.F. Carvalho, Sao Paulo, Brazil Gian Luca Casoni, Forli, Italy Loris Ceron, Carbonera, Italy Stefania Cerri, Portland, USA Indranil Chakravorty, London, UK Rachel Chambers, London, UK Shi-Chuan Chang, Taipei, Taiwan Ling Chen, Baltimore, USA Alfredo Chetta, Parma, Italy Carlo Chezzi, Parma, Italy Prashant N. Chhajed, Mumbai, India Doo-Sup Choi, Rochester, USA Marco Matteo Ciccone, Bari, Italy Michal Ciurzynski, Warsaw, Poland Donald W. Cockcroft, Saskatoon, Canada Henri Colt, Irvine, USA Alison Condliffe, Cambridge, UK Marco Confalonieri, Trieste, Italy Claudius Conrad, Boston, USA Vittoria Conti, Rome, Italy Robalo Cordeiro, Coimbra, Portugal Massimo Corradi, Parma, Italy Raja Abboud, Vancouver, Canada Yossef Aelony, Rancho Palos Verdes, USA Michalis Agrafiotis, Magoula, Greece Khalid Al Shafi, London, UK Ghada Alsaleh, Illkirch, France Nicolino Ambrosino, Cisanello, Pisa, Italy Kayvan Amjadi, Ottawa, Canada Asha Anandiah, Boston, USA Stephan Andreas, Immenhausen, Germany Jouke T. Annema, Leiden, The Netherlands Katerina Antoniou, Heraklion, Greece Balazs Antus, Budapest, Hungary Juan Carlos Arevalo, Salamanca, Spain Christine Armbruster, Vienna, Austria Laurent Arnaud, Paris, France Hormoz Ashtyani, Hackensack, USA John D. Aubert, Lausanne, Switzerland Najib Ayas, Vancouver, Canada Imran Aziz, Wigan, UK Chung-Xue Bai, Shanghai, China Kristina Bailey, Omaha, USA Petros Bakakos, Athens, Greece Bruno Balbi, Veruno, Italy Ferran Barbe, Lleida, Spain Xavier Basagana Flores, Barcelona, Spain Sandip Basu, Bombay, India Salvatore Battaglia, Palermo, Italy Sevim Bavbek, Ankara, Turkey Gillian Beamer, Columbus, USA H.D. Becker, Heidelberg, Germany Jurgen Behr, Bochum, Germany Carolyn Behrendt, Duarte, USA Maria Belvisi, London, UK Norbert Berend, Glebe, Australia Gidon Berger, Haifa, Israel Robert Berkowitz, Hackensack, USA Nitin Bhatt, Columbus, USA Luca Bianchi, Lumezzane, Italy Andrea Bianco, Campobasso, Italy Semra Bilaceroglu, Izmir, Turkey Jose Blanquer, Valencia, Spain Alexander Blau, Berlin, Germany Konrad E. Bloch, Zurich, Switzerland