Ishfaq Ahmed

Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigenesis.

DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial–mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20–34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-β (TGFβ), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8u2009h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Critical Roles of Notch and Wnt/β-Catenin Pathways in the Regulation of Hyperplasia and/or Colitis in Response to Bacterial Infection

ABSTRACT Notch and Wnt/β-catenin signals play essential roles in intestinal development and homeostasis. Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and various degrees of inflammation, depending upon the genetic background. We aimed at delineating the role of the Notch and Wnt/β-catenin pathways in the regulation of colonic crypt hyperplasia and/or colitis following C. rodentium infection. During TMCH, relative levels of the Notch intracellular domain (NICD) increased significantly, along with increases in Jagged-1 and Hes-1 coinciding with the progression and regression phases of hyperplasia. Blocking of Notch signaling with dibenzazepine (DBZ) for 5 days before the onset of hyperplasia also blocked Wnt/β-catenin signaling. Targeting the Notch pathway for 5 days after the onset of hyperplasia failed to inhibit Wnt/β-catenin-regulated crypt hyperplasia. Chronic DBZ administration for 10 days blocked both Notch and Wnt signaling, disrupted the intestinal barrier, and induced colitis. Core-3−/− mice, which are defective in mucin secretion and are susceptible to experimental triggers of colitis, also exhibited significant colitis in response to C. rodentium plus DBZ. Chronic DBZ administration in these mice did not result in depletion of the putative stem cell marker doublecortin-like kinase-1 (DCLK1) in the crypts. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored signaling via the Notch and Wnt/β-catenin pathways, thereby promoting crypt regeneration, and also replenished the mucus layer, leading to amelioration of C. rodentium- and DBZ-induced colitis in NIH:Swiss mice. Thus, the balancing act between cell proliferation and mucus production to restore barrier integrity seems to depend upon the interplay between the Wnt/β-catenin and Notch pathways in the TMCH model.

Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2.

The enhancer of zeste homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and ApcMin/+ mice were infected with CR (108u2009CFU); BLT1−/−ApcMin/+ mice, azoxymethane (AOM)/dextran sodium sulfate (DSS)-treated mice and de-identified human adenocarcinoma samples were the models of colon cancer. Following infection with wild-type but not mutant CR, elevated EZH2 levels in the crypt at days 6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3’s occupancy on WIF1 (Wnt inhibitory factor 1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via small interfering RNA or EZH2-inhibitor deazaneplanocin A (Dznep) either alone or in combination with histone deacetylase inhibitor suberoylanilide hydroxamic acid, WIF1 promoter activity increased significantly while the overexpression of EZH2 attenuated WIF1 reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels, whereas H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, although β-catenin levels were lower in EZH2-knocked down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days 6 and 12 post infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected ApcMin/+ mice paralleled changes recorded in BLT1−/−ApcMin/+, AOM/DSS and human adenocarcinomas. Thus, EZH2-induced downregulation of WIF1 expression may partially regulate Wnt/β-catenin-dependent crypt hyperplasia in response to CR infection.

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation, Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.

Evidence of functional cross talk between the Notch and NF-κB pathways in nonneoplastic hyperproliferating colonic epithelium

The Notch and NF-κB signaling pathways regulate stem cell function and inflammation in the gut, respectively. We investigate whether a functional cross talk exists between the two pathways during transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium (CR). During TMCH, NF-κB activity and subunit phosphorylation in colonic crypts of NIH Swiss mice at days 6 and 12 were associated with increases in downstream target CXC chemokine ligand (CXCL)-1/keratinocyte-derived chemokine (KC) expression. Blocking Notch signaling acutely for 5 days with the Notch blocker dibenzazepine (DBZ) failed to inhibit crypt NF-κB activity or CXCL-1/KC expression. Chronic DBZ administration for 10 days, however, blocked Notch and NF-κB signaling in the crypts and abrogated hyperplasia. Intriguingly, chronic Notch inhibition was associated with significant increases in IL-1α, granulocyte colony-stimulating factor, monocyte chemoattractant protein 1, macrophage inflammatory protein 2, and KC in the crypt-denuded lamina propria or whole distal colon, with concomitant increases in myeloperoxidase activity. In core-3(-/-) mice, which are defective in intestinal mucin, DBZ administration replicated the results of NIH Swiss mice; in Apc(Min/+) mice, which are associated with CR-induced elevation of NF-κB-p65(276) expression, DBZ reversed the increase in NF-κB-p65(276), which may have blocked rapid proliferation of the mutated crypts. DBZ further blocked reporter activities involving the NF-κB-luciferase reporter plasmid or the Toll-like receptor 4/NF-κB/SEAPorter HEK-293 reporter cell line, while ectopic expression of Notch-N(ICD) reversed the inhibitory effect. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored Notch and NF-κB cross talk in NIH Swiss mice, inhibited CR/DBZ-induced apoptosis in the crypts, and promoted crypt regeneration. Thus functional cross talk between the Notch and NF-κB pathways during TMCH regulates hyperplasia and/or inflammation in response to CR infection.

An ornamental plant targets epigenetic signaling to block cancer stem cell-driven colon carcinogenesis

Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea(AR; Hollyhock) is an ornamental plant with known anti-inflammatory properties. This study explored its role as an anticancer agent. The AR seed extract (AR extract) inhibited proliferation and colony formation in a dose- and time-dependent manner and promoted apoptosis as was evidenced by cleavage of PARP and increased expression of Bax accompanying reduced levels of BCL-xl protein in HCT116 and SW480 cells, respectively. In addition, AR extract-arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. AR extract-treated cells exhibited reduced number and size of colonospheres in a dose-dependent manner concomitant with decreases in cancer stem cell (CSC) markers ALDH1A1 and Dclk1. Relative levels of β-catenin, Notch-ICD, Hes1 and EZH2 were also attenuated by AR extract. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown.In vivo, AR extract attenuated tumor growth due probably to reduced levels of EZH2, β-catenin, CyclinD1 and Ki-67 along with reduced levels of CSC markers. Since partial purification via HPLC yielded a prominent peak, efforts are underway to identify the active ingredient(s). Taken together, the results clearly suggest that AR extract/active component(s) can be an effective preventative/therapeutic agent to target colon cancer.

Abstract LB-317: Energy distruptor 2-deoxyglucose mitigates bacterial infection responsive hyperplasia and/or colitis

Transmissible murine crypt hyperplasia (TMCH) is a pathological outcome of Citrobacter rodentium (CR) infection, accompanied by intestinal inflammation during colitis though excessive induction of epithelial regeneration and repair mechanisms. The TMCH model, provides an excellent template to determine how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection. CR is a valuable infection model for the study of pathogenesis of the clinically significant human pathogens, enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC). During TMCH, there is a complex interplay between the Notch and Wnt/β-catenin pathways in colonic crypt hyperplasia. Blocking Notch signaling via chronic dibenzazepine (DBZ) administration inhibits both Notch and Wnt signaling, and disrupts the intestinal barrier during CR induced colitis. 2-deoxyglucose (2DG) is a glycolytic inhibitor has been used for cancer therapy exploiting the cancer cell9s high metabolic state. Added, the similarity of 2DG to mannose produces improper N-linked glycosylation resulting in an unfolded protein response (UPR) slowing growth of tumors. Here, using CR infection model, we show that dietary treatment of 2DG (0.4%) mitigates C. rodentium- and Notch signaling blocker dibenzazepine (DBZ)-induced colitis in NIH:Swiss mice. The survival curve shows no loss in mice administered with 2DG, compared to 40% loss in mice with vehicle alone during CR infection together with DBZ treatment. At tissue level, 2DG dietary intervention resulted in restoration of Hes-1, and β-catenin protein levels as observed from the western analyses of the C. rodentium-infected and DBZ-treated mice crypts. Histological staining show reduced dysbiosis upon 2DG intervention, as seen from reduced infiltration of inflammatory response cells during CR infection and the therapeutic effects are further substantiated with the Ki-67 staining. Thus, the energy distruptor 2-deoxyglucose (2DG) is described for the first time here to mitigate bacterial infection and its responsive hyperplasia/colitis. The current model in this novel mechanism of action will be presented. Citation Format: Ishfaq Ahmed, Shahid Umar, Rao V. Papineni. Energy distruptor 2-deoxyglucose mitigates bacterial infection responsive hyperplasia and/or colitis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-317.

Abstract 3297: Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LAnnBackground. Intestinal mucus layer disruption and gut microflora modification in conjunction with tight junction (TJ) changes can increase colonic permeability that allows bacterial dissemination and intestinal and systemic disease. Employing Citrobacter rodentium (CR) as a model system to mimic the pathogenic mechanisms of Enteropathogenic and Enterohaemorrhagic E. coli, we showed previously that a functional cross-talk between the Notch and Wnt/β-catenin pathways regulates hyperplasia and/or colitis following CR infection. Aim: To test the hypothesis that CR infection combined with chronic Notch inhibition will alter the composition of the colonic mucus to promote dysbiosis, tight junction disruption, loss of multipotent intestinal stem cells (ISCs) and colitis. Material/methods: Immune-competent NIH:Swiss outbred mice were infected with CR (108 CFUs) and treated with Notch blocker Dibenzazepine [DBZ, i.p. @ 10μmol/Kg body weight]. Mucus composition, fecal 16S rDNA analysis and components of TJ integrity were measured and correlated with changes in components of the Notch pathway. 6% Pectin diet served as a SCFA-delivery system to the colon. Results: In the colons of CR-infected/DBZ-treated mice, mucus analysis revealed dramatic alterations in the composition of trace O-glycans and complex type and hybrid N-glycans, compared to CR-infected alone that preceded alterations in 16S rDNA microbial community structure. Indeed, mucin degrading and colitogenic bacterium, Akkermansia muciniphila exhibited dramatic increases in the feces of CR-infected and DBZ-treated mice. At the same time, colons from CR-infected/DBZ-treated mice had decreased expression of antimicrobial peptides Angiogenin-4, Retnlβ, Intelectin-1 and Intelectin-2, respectively. Expression levels of both TJ and adherens junction proteins (e.g., Claudin-5, ZO-1, ZO-2, E-Cadherin, β-catenin) also decreased significantly in the colonic crypts of CR-infected/DBZ-treated mice that paralleled loss of Notch signaling. Both C3H/HeN mice that exhibit exaggerated response to CR infection and T- and B-cell deficient Rag-1 mice following CR-infection and DBZ-treatment, exhibited dramatic increases in paracellular permeability, goblet cell metaplasia and exacerbation of colitis. Chronic blockade of the Notch pathway depleted colonic epithelium of ISC markers Dclk1 and Lgr5 concomitant with exacerbation of colitis. 6% Pectin diet ameliorated colitis by restoring barrier integrity via recruitment of Bacteroides vulgatus, Streptococcus gordonii and Lactococcus lactis, bacteria with potential anti-inflammatory functions. Conclusions: 1. These studies help us delineate the mechanistic basis of colitis development in the aftermath of chronic Notch inhibition. 2. Our findings also caution against the use of Notch inhibitors for patients suffering from colitis-associated colon cancer.nnCitation Format: Ishfaq Ahmed, Badal Roy, Rita-marie McFadden, Shrikant Anant, Seth Septer, Shahid Umar. Altered mucus composition and bacterial dysbiosis promote development of colitis following chronic Notch inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3297.

Abstract 4656: Phytochemicals target epigenetic signaling to block cancer stem cell-driven colon carcinogenesis

Background: Phytochemicals modulate key cellular signaling pathways and have proven anticancer effects. Alcea rosea (AR; Hollyhock) is an ornamental plant in the Malvaceae family with known anti-inflammatory properties. Its role as an anti-cancer agent however, has not yet been confirmed. The present investigation is focused on the mechanistic insight of AR seed extract as an anti-cancer agent for colon cancer. Methodology: Colon cancer cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell proliferation, cell cycle and stemness were determined by hexoseaminidase, flow cytometry and spheroid assays, respectively. TOP-Flash reporter and WIF1 promoter activities were performed in HEK-293T cells. For in vivo effects, we used the HCT116-tumor xenograft model for colon tumorigenesis. Real Time PCR, Western blot and immunohistochemistry studies were performed for determining gene expression. Results: The AR seed extract inhibited proliferation and colony formation in a dose and time dependent manner in colon cancer cell lines. The anti-proliferative activity appeared to be mediated by apoptotic mechanisms, as cleavage of PARP and increased expression of Bax accompanied decreased levels of BCL-xl protein. In addition, AR seed extract arrested cells at Go/G1 phase of cell cycle and exhibited decreases in Cyclin D1. Treatment of cells with AR seed extract also reduced the number and size of colonospheres in a dose-dependent manner which coincided with decreases in the expression of ALDH1A1 and Dclk1, markers of CSCs (Cancer Stem Cells). During analyses of pathways critical for CSC maintenance and colon cancer progression, AR seed extract attenuated levels of β-catenin, Jagged1, Notch-ICD, Hes1 and EZH2. TOP-flash reporter activity, a measure of Wnt signaling, decreased significantly in response to treatment while overexpression of wild type but not mutant EZH2, reversed the inhibitory effects. Moreover, WIF1 (a Wnt antagonist) promoter activity increased dramatically following treatment with AR seed extract which phenocopied increases in WIF1 reporter activity following EZH2 knockdown. To determine the effect of AR seed extract on tumor growth in vivo, athymic nude mice bearing tumor xenografts were administered AR seed extract intraperitoneally at 200 mg/Kg daily for 21 days. The AR seed extract significantly inhibited tumor xenograft growth. In addition, there were reduced levels of EZH2, β-catenin, CyclinD1 and Ki-67 along with attenuated levels of Dclk1 in the xenograft tissues. Finally, preliminary run through HPLC yielded three prominent peaks and further identification via NMR spectroscopy is underway to identify the active ingredient(s). Conclusion: These results clearly suggest that AR seed extracts can be an effective preventative/therapeutic agent for colon cancer. Citation Format: Shahid Umar, Ishfaq Ahmed Ahmed, Dharmalingam Subramaniam. Phytochemicals target epigenetic signaling to block cancer stem cell-driven colon carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2015-4656

Abstract 4901: Clostridium cluster XIVa species an early biomarker in radiation injury

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnRadiation therapy is being used in the treatment of nearly 60% of the current cancer patients. In spite of the best efforts to provide special care in minimizing radiation exposure, significant radiation injury remains a common side-effect particularly to the gastro intestinal tract. Severe injury to GI tract may lead to multiple organ dysfunction syndrome. The status of fecal microbiota has been proposed as a rapid diagnostic platform in radiation injury. Gut microbes play a role in immunity, health, and disease. Imbalances in gut microbiota are related to numerous disorders, such as inflammatory bowel disease, colorectal cancer (CRC), and atopy. In patients with immune suppression due to high-dose chemotherapy or that undergoing radiation therapy, disruption of the microbiota through antibiotics and impairment of host immunity gives rise to perturbations favoring intestinal domination by pathogenic species, resulting in increased bacterial translocation and susceptibility to systemic infection. Here, using real-time PCR assays, we assessed the immediate changes and quantitative alterations to fecal microbiota in response to radiation injury. Swiss-albino Mice were subjected to 10 Gy X-ray whole body irradiation doses using commercially available X-RAD 320 irradiator (1Gy/min; F2 beam hardening filter 1.5mm Al, 0.25mm Cu, 0.75mm Sn; Precision X-ray inc, USA). Fecal samples collected immediately after the radiation exposure were analyzed. We show that Clostridium cluster XIVa that accounts for almost 60% of the mucin-adhered microbiota is significantly increased (11.71 +/- 0.29 fold n=6) in the fecal samples, compared to the total bacteria and other bacteria that tend to enrich in the luminal region. Along with the Bacteroidetes and Proteobacteria from the luminal region, minimal changes in the levels of Firmicutes that colonize the mucin layer were observed. Further, these changes varied considerably between the subjects and were inconsistent compared to the Clostridium cluster species. Clostridium cluster XIVa has been proposed to be the mucosal butyrate producers producing butyrate close to the epithelium. This enhances butyrate bioavailability required in treating diseases such as inflammatory bowel disease. These findings provide opportunities to evaluate the potential use of intestinal microbiota as biomarkers in radiation injury. Also, provides insights into the microbial changes tantamount to the radiation induced GI injury.nnCitation Format: Ishfaq Ahmed, William McLauglin, Shrikant Anant, Shahid Umar, Rao V. Papineni. Clostridium cluster XIVa species an early biomarker in radiation injury. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4901. doi:10.1158/1538-7445.AM2014-4901