Uninvolved Colonic Mucosa Research Articles

Race‐dependent Association of Sulfidogenic Bacteria with Colorectal Cancer

In the US, there is a higher incidence of colorectal cancer (CRC) in African Americans (AAs) compared to non‐Hispanic whites (NHWs). Recent evidence links consumption of a diet high in animal protein and fat as an environmental risk factor for CRC development, and the intestinal microbiota modulates the tumor promoting or protective effects of diet. Hydrogen sulfide, produced by resident sulfidogenic bacteria, triggers pro‐inflammatory pathways and hyper‐proliferation, and is genotoxic. We hypothesized that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHWs, and may be correlated with differences in dietary composition. Colonic biopsies from uninvolved or healthy mucosa from CRC cases and controls were collected from five medical centers through the Chicago Colorectal Cancer Consortium. Using quantitative PCR, sulfidogenic bacterial abundance was measured in uninvolved colonic mucosa of 97 AA and 56 NHW CRC cases, and 100 AA and 76 NHW controls. In addition, 16S rRNA sequencing was performed in AA cases and AA controls. A Block Brief 2000 Food Frequency Questionnaire was collected from a subset of subjects of 50 AA and 31 NHW CRC cases and 30 AA and 24 NHW controls. Differences were examined among bacterial targets, race, disease status, and dietary intake. AAs harbored a greater abundance (p<0.001) of sulfidogenic bacteria compared to NHWs regardless of disease status, including the functional gene for H2S production in sulfate reducing bacteria, dissimilatory sulfate reductase (pan‐dsrA), as well as Bilophila wadsworthia‐specific dsrA, and 16S rRNA genes for Desulfobacter spp., Desulfovibrio spp., and Desulfotomaculum spp. Bilophila wadsworthia‐specific dsrA was significantly more abundant in AA cases compared to AA controls (p<0.001). Linear discriminant analysis of 16S rRNA gene sequences highlighted the sulfidogenic Bilophila, Lactococcus, Odoribacter, Porphyromonas and Pyramidobacter genera as significant in AA cases. Fat intake and daily servings of meat were significantly higher (p<0.01) in AAs compared with NHWs, and dietary fat intake correlated positively with pan‐dsrA abundance (p=0.011). Additionally, intake of dairy and calcium was lower (p<0.001) in AA, and servings of dairy correlated negatively with pan‐dsrA abundance (p=0.007). Together, these results implicate sulfidogenic bacteria as an environmental risk factor contributing to CRC development in AAs.Support or Funding InformationThis work was supported by grants from the National Cancer Institute (U01 CA153060 and P30 CA023074, NAE; RO1 CA204808, HRG, EM, LTH; RO1 CA141057, BJ) and the American Cancer Society Illinois Division (223187, XL). PGW was supported by a predoctoral fellowship from Mayo Clinic and University of Illinois Alliance for Technology‐Based Healthcare. CY was supported by a training grant from National Institute of Health (5T32DK007788‐15). GJA was supported by a Cancer Biology Training Grant (T32CA009213).

Race-dependent association of sulfidogenic bacteria with colorectal cancer

ObjectiveColorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC...

Dysregulation of MicroRNAs in Colonic Field Carcinogenesis: Implications for Screening

Colorectal cancer (CRC) screening tests often have a trade-off between efficacy and patient acceptability/cost. Fecal tests (occult blood, methylation) engender excellent patient compliance but lack requisite performance underscoring the need for better population screening tests. We assessed the utility of microRNAs (miRNAs) as markers of field carcinogenesis and their potential role for CRC screening using the azoxymethane (AOM)-treated rat model. We found that 63 miRNAs were upregulated and miR-122, miR-296-5p and miR-503# were downregulated in the uninvolved colonic mucosa of AOM rats. We monitored the expression of selected miRNAs in colonic biopsies of AOM rats at 16 weeks and correlated it with tumor development. We noted that the tumor bearing rats had significantly greater miRNA modulation compared to those without tumors. The miRNAs showed good diagnostic performance with an area under the receiver operator curve (AUROC) of >0.7. We also noted that the miRNA induction in the colonic mucosa was mirrorred in the mucus layer fecal colonocytes isolated from AOM rat stool and the degree of miRNA induction was greater in the tumor bearing rats compared to those without tumors. Lastly, we also noted significant miRNA modulation in the Pirc rats- the genetic model of colon carcinogenesis, both in the uninvolved colonic mucosa and the fecal colonocytes. We thus demonstrate that miRNAs are excellent markers of field carcinogenesis and could accurately predict future neoplasia. Based on our results, we propose an accurate, inexpensive, non-invasive miRNA test for CRC risk stratification based on rectal brushings or from abraded fecal colonocytes.

Wnt/β-Catenin Signaling Regulates Yes-associated Protein (YAP) Gene Expression in Colorectal Carcinoma Cells

Mutations in the Wnt/β-catenin pathway occur in most colorectal cancers (CRCs), and these mutations lead to increased nuclear accumulation of the β-catenin transcriptional co-activator. In the nucleus, β-catenin associates with TCF/LEF sequence specific transcription factors to activate target gene expression. The Hippo pathway restricts cellular growth by preventing nuclear accumulation of the Yes-associated protein (YAP) transcriptional co-activator. YAP expression is elevated in CRCs suggesting that, like Wnt/β-catenin signaling, the Hippo pathway may contribute to colorectal carcinogenesis. Regulation of YAP at the post-translational level has been well studied but the transcription factors that control YAP gene expression are unknown. Here we demonstrate that β-catenin/TCF4 complexes bind a DNA enhancer element within the first intron of the YAP gene to drive YAP expression in CRC cells. As such, reducing β-catenin expression in CRC cells using shRNAs leads to decreased YAP mRNA and protein levels. YAP is abundantly expressed in the cytoplasm and nuclei of several established human colon cancer cell lines and this localization pattern is insensitive to plating density. Finally, we show that YAP expression is elevated in the majority of a panel of primary human colorectal tumors compared with its expression in uninvolved colonic mucosa, and that YAP and β-catenin localize to the nuclear compartment of tumor cells. Together, these results implicate YAP as an oncogene whose expression is driven by aberrant Wnt/β-catenin signaling in human CRC cells.

Similarity in the metabolic profile in macroscopically involved and un-involved colonic mucosa in patients with inflammatory bowel disease: an in vitro proton ( 1H) MR spectroscopy study

Background The histological extent of inflammatory bowel disease (IBD) is greater than that evident by colonoscopic evaluation. We hypothesized that metabolic profile in macroscopically un-involved colonic mucosa in IBD is similar to that of controls with healthy colon. We thus assessed the differences in metabolic profile in macroscopically involved and un-involved colonic mucosa of IBD patients to further substantiate the extent of disease. Patients and Methods Colonic mucosal biopsies were obtained and snap frozen from both the macroscopically un-involved and involved colonic mucosa of IBD patients and macroscopically normal colonic mucosa of controls and were subjected to in-vitro high-resolution proton ( 1H) magnetic resonance (MR) spectroscopy and the concentrations of metabolites were determined. Results Thirty-two metabolites were assigned in the proton MR spectrum of colonic mucosa of IBD patients. The concentrations of amino acids (isoleucine, leucine, valine, arginine, lysine, glutamine/glutamate, alanine), membrane metabolites (choline, glycerophosphorylcholine/phosphorylcholine), glycolytic product (lactate) and short chain fatty acid (formate) were significantly lower while significantly high level of glucose were observed in the macroscopically un-involved colonic mucosa of IBD patients compared to the macroscopically normal mucosa of controls. There was no significant difference in the concentrations of metabolites in macroscopically involved and un-involved colonic mucosa of IBD patients. Conclusions The metabolic profile in macroscopically un-involved colonic mucosa of IBD patients is similar to that of macroscopically involved mucosa but different from colonic mucosa of controls. This suggests that even macroscopically un-involved colonic mucosa is metabolically abnormal and may explain the increase in extent of disease with time.

Risk Stratification of Colon Carcinogenesis through Enhanced Backscattering Spectroscopy Analysis of the Uninvolved Colonic Mucosa

Our group has been interested in applying advances in biomedical optics to colorectal cancer risk stratification. Through a recent technological breakthrough, we have been able to harness information from enhanced backscattering spectroscopy, an optics phenomenon that allows quantitative, depth-selective analysis of the epithelial microscale/nanoscale architecture. In the present study, we investigated the ability of enhanced backscattering analysis of the preneoplastic mucosa to predict risk of colon carcinogenesis. Enhanced backscattering analysis was done on intestinal mucosa at preneoplastic time points from two experimental models of colorectal cancer: the azoxymethane-treated rat and the multiple intestinal neoplasia (MIN) mouse. Data were analyzed using two previously validated spectral markers: spectral slope and principle components. We then did a pilot study on mucosal biopsies from 63 subjects undergoing screening colonoscopy. In the azoxymethane-treated rat, when compared with saline-treated controls, significant changes in the enhanced backscattering markers were observed as early as 2 weeks after azoxymethane treatment (before the development of aberrant crypt foci and adenomas). Enhanced backscattering markers continued to progress over time in a manner consonant with future neoplasia. These data were replicated in the preneoplastic MIN mouse mucosa. In humans, spectral slopes in the endoscopically normal cecum, midtransverse colon, and rectum were markedly reduced in patients harboring adenomas when compared with those who were neoplasia free. We show, for the first time, that enhanced backscattering analysis of an aliquot of uninvolved mucosa has the potential for predicting neoplastic risk throughout the colon in both experimental colorectal cancer models and humans.

Multiple Lymphomatous Polyposis Presenting with Lower Gastointestinal Bleeding

Multiple lymphomatous polyposis is an uncommon disease, which is considered to be an intestinal form of mantle cell lymphoma. We present a case of a 66-year-old African American female who was admitted with a 1-week history of painless hematochezia. She has a history of mantle cell lymphoma that was diagnosed from an axillary lymph node biopsy on a prior admission. She refused chemotherapy at that time and was lost to follow up. On this admission she was initially orthostatic and had a hematocrit of 19. Physical examination was pertinent for mandibular, axillary and inguinal lymphadenopathy and rectal examination revealed dark red blood. NG lavage was clear. After resuscitation, she underwent a colonoscopy, which showed multiple polyps throughout the colon, ranging in size from 3 mm to 5cm. There were no rectal polyps. Some of the larger polyps were friable and oozing blood. There was a moderate amount of fresh blood mixed with clots in the lumen up to the transverse colon. Pathology showed diffuse infiltration of the lamnia propria by a monotonous population of atypical lymphocytes that was consistent with mantle cell lymphoma. In addition to the colonic polyps, CT abdomen also showed enlarged mesenteric and retroperitoneal lymph nodes. The liver, spleen and stomach appeared normal. The patient subsequently underwent a subtotal colectomy with ileo-rectal anastomosis for ongoing bleeding. There were innumerable colonic polyps many of which were hemorrhagic. The small bowel was inspected intra-operatively and found to have no lesions. The uninvolved colonic mucosa was unremarkable. The patient recovered uneventfully and was discharged with follow up in oncology clinic after she agreed to chemotherapy. [figure 1]Figure

Down-regulation of PLK3 gene expression by types and amount of dietary fat in rat colon tumors

Epidemiological studies suggest that high intake of dietary fat rich in saturated fatty acids increases the colon cancer risk whereas dietary fish oil high in omega-3 fatty acids reduces the colon cancer risk. Previously, we reported that consumption of omega-6 fatty acid rich diets such as corn oil strongly promotes azoxymethane (AOM)-induced colon carcinogenesis in rats as compared to ingestion of a diet with equivalent amount of fat containing fish oil (HFFO) or low-fat diet (LFCO). Expression of PLK3 (Polo-like kinase-3, previously named Prk) is negatively correlated with the development of certain cancers. Ectopic expression of human PLK3 results in cell cycle arrest or induces apoptosis. To understand the role of PLK3 in colon carcinogenesis and to study the effect of types and amount of dietary fat on the expression levels of PLK3 in colon tumors, we analyzed the colon tumors and mucosa of rats administered the diets containing fish oil and corn oil for PLK mRNA expression. Here we report that expression of PLK3 was down-regulated in rat colon tumors. Quantitative polymerase chain reaction demonstrated that PLK3 mRNA levels were significantly lower in carcinogen (azoxymethane)-induced rat colon tumors than their uninvolved normal colonic mucosa. Among the normal mucosa isolated from rats fed on diets with various levels of fat (LFCO, or high fat diet with corn oil, HFCO, or supplemented with fish oil, HFFO), no significant changes in PLK3 mRNA expression was detected. Tumors isolated from rats fed with HFCO diet contained a very low level of PLK3 mRNA expression. Interestingly, tumors from rats fed the HFFO diet did not exhibit as dramatic down-regulation of PLK3 as the tumors of animals fed the HFCO diet. Furthermore, our results also indicate that the ectopic expression of a kinase active PLK3 construct induced apoptosis in HT-29 colon carcinoma cells. These observations suggest for the first time that a decreased activity of PLK3 may play a key role in colon tumor development as well as in HFCO-induced colon tumorigenesis.

The expression of IL-12 p40 and its homologue, Epstein-Barr virus-induced gene 3, in inflammatory bowel disease.

It has recently been suggested that Crohn's Disease (CD) is associated with an exaggerated T helper 1 cytokine response as manifest by increased production of interleukin-12 (IL-12) and interferon-gamma (IFN-gamma). Epstein-Barr virus-induced gene 3 (EBI3) encodes a 34-kDa glycoprotein that is 27% identical to the p40 unit of IL-12 and has recently been reported to be up-regulated in ulcerative colitis (UC). To determine whether mucosal expression of IL-12 p40 or EBI3 correlates with inflammatory bowel disease (IBD). mRNA expression in colonic mucosa from patients with UC, Crohn's disease (CD) and non-IBD controls was measured by reverse-transcribed real-time polymerase chain reaction (PCR). EBI3 was significantly increased in both involved and uninvolved colonic mucosa in patients with UC. Although IL-12 p40 was increased in some patients with CD relative to non-IBD controls, the increase was not statistically significant. However, 5-aminosalicylic acid (5-ASA) use was significantly correlated with reduced IL-12 p40 levels in the patients with CD, but not in UC cases. A similar reduction was not seen in 5-ASA-treated UC patients. IL-12 p40 expression in CD is heterogeneous. In contrast, expression of the IL-12 p40 homologue, EBI3, is up-regulated in nearly all UC cases and in a subset of CD.

Transforming Growth Factor-α and Epidermal Growth Factor Receptor in Colonic Mucosa in Active and Inactive Inflammatory Bowel Disease

Transforming growth factor-α (TGF-α) is overexpressed in colonic carcinomas and promotes mucosal wound healing. It may be implicated in chronic inflammatory bowel disease (IBD). We analyzed the expression of TGF-α and its receptor, epidermal growth factor receptor (EGF-r), in the colonic mucosa of patients with Crohn's disease (CD) or ulcerative colitis (UC), in active or inactive stages, as compared with controls. Proteins and mRNA were detected in biopsies from the right and left colon and in surgical colonic specimens. Immunoblot analysis revealed TGF-α protein as a 29 k Da band. This band was normally expressed in uninvolved colonic mucosa of patients with CD or UC whether in active or inactive stages, but decreased or absent in involved mucosa of active IBD, even when TGF-α mRNA and EGF-r protein were detected. In the unaffected mucosa of CD, the intensity of TGF-α immunoreactivity was similar to that of controls in the right colon but stronger (P 0.05) in the left colon. There was no TGF-α overexpression in dysplastic regions. In conclusion, in active IBD disease, the decreased TGF-α protein amount seems not only related to epithelial cell loss but reflects a down-regulation at least at the protein level. We speculate that TGF-α does not play a role within the active stage but may be implicated later in the repair process.

MRNA Differential Display of Colonic Mucosa Cells in Ulcerative Colitis

Involvement of mucosal cells in inflammatory bowel disease (IBD) may be a sequenced process and the molecular difference between involved and uninvolved cells might implicate a possible mechanism in the disease process. The aim of this study was to compare gene expression between involved and uninvolved colonic mucosa cells in an individual with ulcerative colitis (UC) and to clone, sequence, and identify those differentially expressed genes. mRNA differential display was used to identify the gene expression in the mucosa of the UC patient. Anchored oligo(dT) primers and random 5′ oligonucleotide 10-mer were used to carry out polymerase chain reaction on reverse-transcribed RNA (RT–;PCR). The amplified cDNAs were displayed on a standard sequencing gel and comparisons were drawn between each two lanes representing either involved or uninvolved cells from a specific combination of two primers. Wherever differences were noted between lanes, the bands were reamplified using PCR, cloned into specialized vectors for positive selection, and then confirmed by dot blot. The cloned genes were then sequenced and compared with the GenBank database. About 1200 mRNA species were displayed in the sequencing gel. Among them, 106 fragments were differentially expressed between the two groups. Twenty-five of those differentially displayed gene fragments have been isolated, reamplified, and cloned for sequencing and dot blot analysis. Seventeen of the fragments were differentially expressed using the dot blot technique. Among those 25 gene fragments, 14 have homology to known genes and 11 have no match to any reported genes. Those matched known genes included genes for parathyroid tumor, T cell receptor-β, α-nascent polypeptide-associated complex, ovarian cancer, and myeloblast. This is the first study using mRNA differential display to observe differential gene expression between involved and uninvolved mucosa cells in UC and it shows that differential display is a rapid method for characterizing gene changesin vivoin ulcerative colitis. The results from this study may provide useful information and facilitate further gene studies in this disease.

Immunohistochemical changes in morphologically involved and uninvolved colonic mucosa of patients with idiopathic proctitis.

Alterations in secretory component, IgA, IgG, and IgM were studied by immunofluorescent techniques in mucosal biopsy specimens obtained at colonoscopy from inflamed and grossly uninvolved colonic mucosa from 12 patients with idiopathic proctitis. Parotid-salivary secretory component and IgA and serum immunoglobulins were also investigated. Decreased secretory IgA was observed in the epithelium of all grossly involved rectal mucosa and in 40% of proximal normal mucosa. Salivary secretory IgA was not diminished. These observations suggest that a local immune defect may be pathogenetically related to idiopathic proctitis.