Murine Colonic Cells Research Articles

Abstract 1465: Phase specific microRNA deregulation of oncogenesis and stemness in a mouse model of sporadic CRC

Abstract Background: The definition of cancer stem cells (CSCs) as “tumor cells that contain self-renewal and multilineage differentiation capacity” still lacks conclusive experimental evidence. This is partly because the gold standard assay to identify CSCs, serial transplantation in animal models, is an operation that can undervalue total CSC numbers. Recently, the combination of mouse models that spontaneously develop tumors with genetic tracing provided exciting support for the CSC theory. Here, we took advantage of the use of cell-surface biomarkers, to purify, to some extent, murine cell populations with CSC phenotype by cell sorting on fresh spontaneous colorectal cancer (CRC). The aim of our study was to define a panel of miRNAs pathways with a potential function in the orchestration of selfrenewal and cell fate during cancer development using tissue of colorectal cancer from both mouse and human origin. Methods: Different populations of murine colon cells derived from AOM/DSS-induced carcinoma were isolated with cell sorting performed on a BD FACSAria system, by the expression of cell surface biomarkers (CD44, EphB2), then stem/progenitor cells-like property was analysed by the expression of stemness/differentiation genes (gene expression and IHC analysis). Specific regions of AOM/DSS treated colon mucosa containing dysplastic ACFs, microadenoma, adenoma, carcinoma compared to normal mucosa were isolated with Laser Capture Microdissection for RNA extraction. miRNAs expression profiling were performed using TaqMan Low Density Arrays (LifeTechnologies) both on cells and tissue samples. Microarray data were evaluated by state of the art statistics and bioinformatics tools. Single Real-Time PCR for selected miRNAs were performed on a training set of 60 FFPE human samples. Results: Global miRNAs expression profiles showed a total of ∼100 miRNAs with a significantly (p<0,05) up/down regulation in a gradual or punctual way in 4 different stages of murine CRC. Numerous miRNAs were already demonstrated to be deregulated in hCRC, confirming the high reliability of the murine model to mimic the human pathology. A set of ∼100 miRNAs distinguished the 4 steps of CRC progression in a unsupervised hierarchical clustering analysis (p<0,05). The analysis of miRNAs expression pattern in the sorted stem-like cell populations in comparison to miRNAs altered in the murine CRC initiation/progression steps revealed a significant cancer stemness signature, composed by miRNAs related to pluripotency and differentiation. Some of these changes, including miR-200b, mir-429, mir-365, mir-215, mir-26b, mir-135, were studied as well in the human samples, showing a similar pattern of that observed in the murine model. Conclusions: These data provide a comprehensive miRNAs signature implicated in the regulation of tumorigenesis, stemness, and cell fate determination that could be exploited for diagnosis and therapeutic design. Citation Format: Mariangela De Robertis, Eva Bandrés, Maria Luana Poeta, Luisa Loiacono, Giuseppe Lamorte, Massimo Sanchez, Luigi Marchionni, Maria Grazia Diodoro, Edoardo Pescarmona, Angelo Luigi Vescovi, Jesus Garcia-Foncillas, Vito Michele Fazio. Phase specific microRNA deregulation of oncogenesis and stemness in a mouse model of sporadic CRC. [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 1465. doi:10.1158/1538-7445.AM2014-1465

Upregulation of heme oxygenase-1 in colorectal cancer patients with increased circulation carbon monoxide levels, potentially affects chemotherapeutic sensitivity.

BackgroundHeme oxygenase-1 (HO-1) and its major product carbon monoxide (CO) are known to be involved in the development and progression of many tumors. The present study was to elucidate the expression and function of HO-1 in colorectal cancer (CRC), specially focusing on the circulation CO levels in CRC patients and the possible roles of HO-1 in chemoresistance of colon cancer cells.MethodsOne hundred and eighteen patients received resection for colorectal cancer and polyps at China Medical University Sheng Jing Hospital, were collected in this study. HO-1 expression in CRC tissues was analyzed by immnuohistochemical staining; circulation CO levels as carboxyhemoglobin (COHb) in CRC patients were analyzed by an ABL800 FLEX blood gas analyzer. HO-1 expression in murine colon cells C26 and human colon cancer cells HT29 and DLD1 under HO-1 inducer hemin and anticancer drug pirarubicin (THP) treatment was examined by RT-PCR, and the cell viability after each treatment was investigated by MTT assay. Data were analyzed by student’s t-test or one-way ANOVA followed by Bonferroni t-test or Fisher's exact test.ResultsHO-1 expression in tumor tissues of CRC (61.0%) was significantly higher than in normal colorectal tissues and polyps tissues (29.7%, P < 0.01); well-differentiated CRC seemed to express more HO-1 (81.5%) than moderately/poorly-differentiated cancers (59.5%, P < 0.05). However, the nuclear HO-1 expression is apparently higher in moderately/poorly differentiated CRC than well-differentiated CRC probably suggesting a new mechanism of function involved in HO-1 in cancer. In parallel with HO-1 expression, circulation CO levels in CRC patients also significantly accelerated. Moreover, HO-1 expression/induction also related to the chemosensitivity of colon cells; HO inhibitor zinc protoporphyrin significantly increased cytotoxicities of THP (i.e., 2.6 – 5.3 folds compared to cells without zinc protoporphyrin treatment).ConclusionsThese findings strongly suggested HO-1/COHb is a useful diagnostic and prognostic indicator for CRC, and inhibition of HO-1 may be a option to enhance the chemotherapeutic effects of conventional anticancer drugs toward CRC.

Abstract 5611: Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting.

Abstract Photochemical internalization (PCI) is a modality for intracellular delivery of drugs which lack an effective transport mechanism into the cell. Such drugs are taken up by means of endocytosis and are transported to the lysosomes where they are degraded before they have exerted their biological effect. PCI utilizes photosensitizers which accumulate in the membranes of these endo/lysosomal compartments. Light exposure causes rupture of these membranes and subsequent cytosolic release of the drug which freely can interact with its target. PCI of the conventional cytostatic drug Bleomycin has been shown to be highly effective in vivo and is currently evaluated in clinical trials with promising results. Bleomycin is, however, probably not optimal for PCI mediated delivery, and therefore cannot fully take use of the technology. Recombinant targeted toxins may be designed to possess all characteristics for an optimal drug to be delivered by PCI: (1) Exclusive uptake in tumor cells, (2) Intracellular target, (3) Not able to penetrate the plasma membrane, (4) Suboptimal escape from endocytic compartments without PCI, (5) Able to diffuse into the tumor tissue. We here present PCI of a recombinant fusion toxin composted of VEGF121, and gelonin, a type I ribosome inactivating protein toxin. VEGF121/rGel administration has previously been shown to be effective in suppressing tumor xenografts and metastasis. Severe adverse effects may, however, limit the possibilities to obtain complete responses with VEGF121/rGel monotherapy. PCI was shown to increase the selectivity of VEGF121/rGel in VEGFR1 and VEGFR2 transfected porcine endothelial cells (PAE) in vitro. The PCI treatment also resulted in a dramatic reduction in the dosage of VEGF121/rGel to 1/100 as measured by a LD90 of 10 nM for the fusion toxin alone compared to 100 pM with the PCI treatment in VEGFR2 expressing PAE cells. For in vivo experiments the murine colon cell line CT26.CL25 was injected subcutaneously in the left hip of BALB c mice and PCI of VEGF121/rGel was administered one week after implantation. By day 24 after PCI all animals in the non-treatment control group were sacrificed due to tumor size (&amp;gt;1000m3),while a 50% complete response (CR) was found with PCI of VEGF121/rGel. The animals were followed up to day 60 when the experiment was terminated. No tumor regression was observed in CR animals in this time-frame. About 50%CR was also detected in PCI of bleomycin-treated animals. Animals receiving PCI of bleomycin showed an average weight loss of 15% compared to no significant weight loss in the PCI of the VEGF121/rGel treated group. This is the first report on PCI of a tumor vascular targeting drug. In conclusion, the present results indicate PCI of VEGF121/rGel as a highly selective method for destroying tumor vasculature. The project is supported by the Norwegian Cancer Society. Research was conducted, in part, by the Clayton Foundation for Research Citation Format: Anette Weyergang, Lawrence H. Cheung, Michael Rosenblum, Khalid Mohamedali, Johannes Waltenberger, Kristian Berg. Photochemical internalization of VEGF121/rGel: a strategy for optimizing tumor-vasculature targeting. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5611. doi:10.1158/1538-7445.AM2013-5611

EGFR Signals Downregulate Tumor Suppressors miR-143 and miR-145 in Western Diet–Promoted Murine Colon Cancer: Role of G1 Regulators

Epidermal growth factor receptors (EGFR) contribute to colonic tumorigenesis in experimental models of colon cancer. We previously showed that EGFR was also required for colonic tumor promotion by Western diet. The goal of this study was to identify EGFR-regulated microRNAs that contribute to diet-promoted colonic tumorigenesis. Murine colonic tumors from Egfr(wt) and hypomorphic Egfr(wa2) mice were screened using micro RNA (miRNA) arrays and miR-143 and miR-145 changes confirmed by Northern, real-time PCR, and in situ analysis. Rodent and human sporadic and ulcerative colitis (UC)-associated colon cancers were examined for miR-143 and miR-145. Effects of EGFR on miR-143 and miR-145 expression were assessed in murine and human colonic cells and their putative targets examined in vitro and in vivo. miR-143 and miR-145 were readily detected in normal colonocytes and comparable in Egfr(wt) and Egfr(wa2) mice. These miRNAs were downregulated in azoxymethane and inflammation-associated colonic tumors from Egfr(wt) mice but upregulated in Egfr(wa2) tumors. They were also reduced in human sporadic and UC colon cancers. EGFR signals suppressed miR-143 and miR-145 in human and murine colonic cells. Transfected miR-143 and miR-145 inhibited HCT116 cell growth in vitro and in vivo and downregulated G(1) regulators, K-Ras, MYC, CCND2, cdk6, and E2F3, putative or established targets of these miRNAs. miRNA targets Ras and MYC were increased in colonic tumors from Egfr(wt) but not Egfr(wa2) mice fed a Western diet. EGFR suppresses miR-143 and miR-145 in murine models of colon cancer. Furthermore, Western diet unmasks the tumor suppressor roles of these EGFR-regulated miRNAs.

Downregulation of cytoprotective intestinal heat shock proteins in inflamed mucosa and by inflammatory cytokines

Background & Aims: Inducd)/e heat shock proteins (hsp) protect viability, and function of intestinal epithelial cells (IEC) against inflammation-associated injury. Unlike small intestine, they are expressed in coloinc IEC under pfiysiological conditions, an eft~:ct dependent on colonic flora as well as mucosal immune cells Because increased hsp expression in inflamed umcosa would be predicted as a host response to limit the extent and severity of mucosal injury, we invrstigated hsp expression in mucosal biopsies from IBD patients, DSS-induced colitis, and in vitro. Methods: Protein expression of inducfble hsps, hsp25/27 and hsp72, and'the constitutively expressed hsc73 were determined by Western blots and immnnostaining (IH) m colonic tnucosa d-ore 1BD patients and DSS-induced colitis. Proqnflannnatory cytokines IL-1~, TNFoc 1FNy were used to elucidate potential factors ahering hsp expression in murine colon-cells (YAMC) and in vivo. Results: Hsp27 and hsp72, but not hsc73, were significantly decreased (66% and 75% respectively) in areas of active inflammation in IBD patients vs uninvolved or healthy' subject samples. When normalized to two epithelial cell markers (cytokeratm 18 and villin), the decreased expression was highly significant (p<0.01), These changes were paralleled by diminished expression of colonic epithelial cell hsps, assessed by IH. In DSS-induced colitis, a rune-dependent decrease in hsp25 (95%) and hsp72 (53%) protein expression and IH (nadir at day 7) were observed and inversely related to increased myelopenaxidase activuy and histologic grading. By contrast, no changes in mucosal hsc73, villin, or cytokemtin expression were observed. The treatment of YAMC cell lines by I IA~ (5ng/ml) or combined TNFmqFNy (100ng/ml; 200U/relY completely inhibited induced hsp25 and hsp72 expression. In addition, If-l!3 and TNBeFIFNy administered IP to healthy mice resuked in marked depletion of hsp25 (80%) and hsp72 (91%), but not hse73, expression in colon mucosa Conclusion: Expression of cytoprotective hsps, hsp25 and hap72, are decreased in the areas of active inflammation in colon, an effect mediated in part by pro-inflammatory c3aokines, Such an effect would render the colonic mucosa more susceptible to inflammation-associated injury. Conversely, augmentation of the cytopmtective hsp response by pharmacological, dietary, or probiotic treatment could benefit patients with IBD and other conditions of injured mucosa.

APC protein expression in murine colonic cells

APC protein expression in murine colonic cells

CONVERSION OF AN ANTIPROLIFERATIVE TO A PROLIFERATIVE RESPONSE IN INTESTINAL EPITHELIAL CELLS BY SWITCHING TNFα SIGNALING FROM SUSTAINED TO TRANSIENT MAPK ACTIVATION

4 Our lab has demonstrated that tumor necrosis factor (TNF) α (100 ng/mL) inhibits proliferation, stimulates the sustained activation of mitogen activated protein kinase (MAPK) ERK1/ERK2 through TNFα receptor R1 (55 kDa) and that 5-amino salicylic acid blocks TNFα-stimulated MAPK activation in intestinal epithelial cells. The kinetics of ERK1/ERK2 activation are believed important in determining whether neural cells enter a proliferation or differentiation pathway in response to growth factors. Rapid, pulsed activation of ERK1/ERK2 is postulated as proliferative, while sustained activation is theorized to stimulate differentiation (growth arrest). The purpose of this study was to determine if the disruption of TNFα's sustained activation of ERK1/ERK2 would alter TNFα's inhibition of proliferation and to determine the intracellular localization of active-ERK1/ERK2. The conditionally immortalized murine colon cell line (YAMC) was studied in a non-immortalized state. The effect of murine EGF (10 ng/mL), TNFα (100 ng/mL) and PD 98059 (MAPK kinase inhibitor) was studied on intestinal epithelial cell proliferation by hemocytometry and signal transduction by immunodetection. After treatment with EGF, TNFα and/or PD 98059 for various times, Triton-soluble lysates were prepared and proteins separated by SDS-PAGE. The activity of MAPK was determined by Western blot analysis with antibody to the dually phosphorylated ERK1/ERK2 while the intracellular localization of activated ERK1/ERK2 was studied by immunofluorescence. A concentration curve demonstrated that PD 98059 (100 nM) inhibited TNFα-, but not EGF-mediated MAPK activation. When added 30 min prior to TNFα (100 ng/mL), PD 98059 completely reversed TNFα's antiproliferative effect. Interestingly, when PD 98059 was added 30 min after TNFα, to mimic a pulsed activation of ERK1/ERK2 similar to EGF, MAPK activity was transiently increased for only 30 min and TNFα (100 ng/mL) stimulated proliferation (p < 0.001). By confocal immunofluorescent microscopy, TNFα-stimulated MAPK activity was localized in the nucleus within 15 min and sustained for at least 2 hr. EGF-stimulated MAPK activity, however, appeared only cytoplasmic. These findings demonstrate that the kinetics ERK1/ERK2 activation are important in TNFα's regulation of intestinal epithelial cell proliferation and that an antiproliferative signal can be converted to a proliferative signal by switching from sustained to transient MAPK activation.

TUMOR NECROSIS FACTOR ALPHA ACTIVATES MAP KINASES THROUGH TNFα RECEPTOR R1 IN INTESTINAL EPITHELIAL CELLS

Our lab has recently shown that high dose TNFα (100 ng/mL) inhibits both intestinal cell proliferation and growth factor-mediated mitogenesis through TNFα receptor R1 (55 kDa), while low dose TNFα (1 ng/mL) stimulates intestinal cell proliferation through TNFα receptor R2 (75 kDa). One mechanism of cytokine and growth factor regulation of cellular function is determined by the kinetics of MAP kinase activation. The purpose of this study was to determine the effects of EGF and TNFα on the kinetics of MAP kinase phosphorylation in intestinal epithelial cells and to determine the independent roles of TNFα receptors R1 and R2 on the signaling events of MAP kinase family members Erk1/Erk2 (p42/p44), JNK/SAPK (p46/p54) and p38. The conditionally immortalized murine colon cell line (YAMC) was studied in a non-immortalized state. Following 24 h incubation at 37°C in media with 0.5% serum, the effects of murine EGF (10 ng/mL) and TNFα (1 and 100 ng/mL) were studied at exposures of 2, 15, 30, 60 and 120 min. Anti-TNFα receptor antibodies were utilized to elucidate TNFα signaling pathways. After treatment with EGF, TNFα or anti-TNFα receptor antibodies, Triton-soluble lysates were obtained and proteins separated by SDS-PAGE. Activated Erk1/Erk2, JNK/SAPK and p38 were determined by Western blot analysis. EGF activates Erk1/Erk2 maximally at 2 min with a return to baseline by 60 min. High dose TNFα (100 ng/mL) activates Erk1/Erk2 maximally at 15 min with sustained activation through 120 min. Similarly, high dose TNFα phosphorylates JNK/SAPK maximally at 15 min. Anti-TNFα receptor R1 agonist antibody activated Erk1/Erk2 and JNK/SAPK. TNFα's activation of Erk1/Erk2 and JNK/SAPK was blocked by anti-TNFα receptor R1 antagonist antibody, while anti-TNFα receptor R2 antagonist antibody had no effect. When low dose TNFα (1 ng/mL) was studied, neither Erk1/Erk2 nor JNK/SAPK were activated. In addition, neither high dose TNFα, low dose TNFα nor EGF appear to activate p38. In summary, TNFα (100 ng/mL) activates both Erk1/Erk2 and JNK/SAPK pathways. Phosphorylation of Erk1/Erk2 by TNFα receptor R1 and EGF showed different patterns of maximal activation and duration. These findings, combined with our previously reported data, suggest that TNFα alters EGF-mediated signaling through TNFα receptor R1 by disrupting MAP kinase activation.

6 TUMOR NECROSIS FACTOR ALPHA REGULATES TYROSINE PHOSPHORYLATION AND EXPRESSION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR IN INTESTINAL EPITHELIAL CELLS

While the mitogenic effect of epidermal growth factor (EGF) on intestinal cells is well documented, our lab has recently shown that TNFα inhibits EGF-mediated proliferation. The purpose of this study was to determine the effects of TNFα on EGF receptor signaling in intestinal cells. The conditionally immortalized murine colon cell line (YAMC) was studied in a non-immortalized state. Following 24 h incubation at 37°C in media supplemented with 0.5% serum, the effects of murine EGF and TNFα on EGF receptor signaling were analyzed. EGF (10 ng/mL) was studied at exposures of 1, 15 and 30 minutes. TNFα (100 ng/mL) was added 30 min prior to the addition of EGF when studied in combination or incubated with cells for 30 min when studied alone. After treatment with EGF and/or TNFα, the EGF receptor was isolated by immunoprecipitation from Triton-soluble lysates. Proteins were separated by SDS-PAGE and the EGF receptor and its tyrosine phosphorylation state determined by Western blot analysis. TNFα rapidly decreased EGF-stimulated tyrosine phosphorylation of the EGF receptor two-fold within 1 min. Increased recovery of the EGF receptor was detected within 30 min of exposure to TNFα. This was further studied and confirmed by ligand binding assay using [125l]-EGF, where the EGF receptor number was seen to increase from 54,000 receptors/cell to 96,000 receptors/cell after 30 min of exposure to TNFα. In summary, TNFα not only decreases early EGF-stimulated tyrosine phosphorylation of the EGF receptor, but also alters the available pool of EGF receptors. These findings, combined with our previously reported data, suggest that TNFα-mediated signals disrupt EGF receptor signaling pathways required for EGF-mediated intestinal cell proliferation.

Effect of different doses of dietary calcium on murine colonic cell proliferation

One-hundred and twenty 6-week-old 257BL/65 mice were fed for 2 or 7 weeks with diets supplemented with different combinations of bile acids and calcium. The effect of calcium, bile acids and the duration of these treatments on proliferative indices of the colonic mucosa was studied with a multiway analysis of variance. In mice not treated with bile acids, a low level (0.1%) of calcium in the diet was related to a significantly higher number of cells in each compartment of the crypt, compared with diets supplemented with 0.5 and 1% calcium (P < 0.01). There was no difference between the groups fed with normal and high calcium diets. Bile acids significantly increased proliferative indices in all animal groups whatever the duration of the treatment; however, this effect was significantly lower in the mice fed with 0.5% and 1% calcium than in those fed with 0.1% calcium (P < 0.01). There was a significant interaction between the effect of bile acids and the effect of calcium regarding the number of labeled cells and the labeling indices. Duration of the treatment had little effect on these indices. The effect of bile acids on colonic proliferative activity could be significantly reduced by calcium supplementation, and this effect was stable with time. Although there was no toxic effect of the highest calcium diet, there was no advantage in increasing the calcium dose beyond 0.5%.

P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines: Evidence for a saturation of active daunorubicin transport

We studied the resistance of colon tumors to anticancer agents in vitro. Using daunorubicin (DN), a number of cellular parameters which normally indicate acquired or multidrug resistance (MDR), were compared for several human wild-type colon cell lines, i.e. HT29, SW1116 and COLO 320, and the murine colon cell line C-26. The sensitive/MDR human ovarian cancer cell line couple A2780/2780 AD was used as a reference. The amount of P-glycoprotein (P-gp) was in the order HT29, A2780 ⩽ SW1116 < C26 ⩽ COLO320 < 2780 AD. The MDR modifiers verapamil, Cremophor EL, cyclosporin A and Ro 11-2933/001 had significant effects on DN cytotoxicity, total DN accumulation and efflux, only if P-gp was present. A flow-through system was used to study the mechanism of DN transport. For the first time, evidence for saturation of an active transport of DN from the cells is reported. We discussed the possible presence of cooperative activity between at least two binding sites on the protein responsible for DN efflux, likely to be P-gp.