Nontransformed Intestinal Epithelial Cells Research Articles

Epithelial aPKC deficiency leads to stem cell loss preceding metaplasia in colorectal cancer initiation

The early mechanisms of spontaneous tumor initiation that precede malignancy are largely unknown. We show that reduced aPKC levels correlate with stem cell loss and the induction of revival and metaplastic programs in serrated- and conventional-initiated premalignant lesions, which is perpetuated in colorectal cancers (CRCs). Acute inactivation of PKCλ/ι invivo and in mouse organoids is sufficient to stimulate JNK in non-transformed intestinal epithelial cells (IECs), which promotes cell death and the rapid loss of the intestinal stem cells (ISCs), including those that are LGR5+. This is followed by the accumulation of revival stem cells (RSCs) at the bottom of the crypt and fetal-metaplastic cells (FMCs) at the top, creating two spatiotemporally distinct cell populations that depend on JNK-induced AP-1 and YAP. These cell lineage changes are maintained during cancer initiation and progression and determine the aggressive phenotype of human CRC, irrespective of their serrated or conventional origin.

Cannabidiol attenuates inflammatory impairment of intestinal cells expanding biomaterial-based therapeutic approaches

Cannabis-based biomaterials have the potential to deliver anti-inflammatory therapeutics specifically to desired cells, tissues, and organs, enhancing drug delivery and the effectiveness of anti-inflammatory treatment while minimizing toxicity. As a major component of Cannabis, Cannabidiol (CBD) has gained major attention in recent years because of its potential therapeutic properties, e.g., for restoring a disturbed barrier resulting from inflammatory conditions. The aim of this study was to test the hypothesis that CBD has beneficial effects under normal and inflammatory conditions in the established non-transformed intestinal epithelial cell model IPEC-J2. CBD induced a significant increase in transepithelial electrical resistance (TER) values and a decrease in the paracellular permeability of [³H]-D-Mannitol, indicating a strengthening effect on the barrier. Under inflammatory conditions induced by tumor necrosis factor alpha (TNFα), CBD stabilized the TER and mitigated the increase in paracellular permeability. Additionally, CBD prevented the barrier-disrupting effects of TNFα on the distribution and localization of sealing TJ proteins. CBD also affected the expression of TNF receptors. These findings demonstrate the potential of CBD as a component of Cannabis-based biomaterials used in the development of novel therapeutic approaches against inflammatory pathogenesis.

Creating a More Perfect Union: Modeling Intestinal Bacteria-Epithelial Interactions Using Organoids.

Intestinal organoids have become indispensable tools for many gastrointestinal researchers, advancing their studies of nontransformed intestinal epithelial cells, and their roles in an array of diseases, including inflammatory bowel disease and colon cancer. In many cases. these diseases, as well as many enteric infections, reflect pathogenic interactions between bacteria and the gut epithelium. The complexity of studying this microbe–epithelial interface in vivo has led to significant focus on modeling this cross-talk using organoid models. Considering how quickly the organoid field is advancing, it can be difficult to keep up to date with the latest techniques, as well as their respective strengths and weaknesses. This review addresses the advantages of using organoids derived from adult stem cells and the recently identified differences that biopsy location and patient age can have on organoids and their interactions with microbes. Several approaches to introducing bacteria in a relevant (apical) manner (ie, microinjecting 3-dimensional spheroids, polarity-reversed organoids, and 2-dimensional monolayers) also are addressed, as are the key readouts that can be obtained using these models. Lastly, the potential for new approaches, such as air–liquid interface, to facilitate studying bacterial interactions with important but understudied epithelial subsets such as goblet cells and their products, is evaluated.

Abstract 99: Phenotypic characterization of knockdown of CDKs in the intestinal epithelial cells

Abstract Cyclin-dependent kinases (CDKs) are serine/threonine kinases that bind to cyclins and together act as regulators of cell cycle progression. CDK inhibitors have various therapeutic potentials including cancer (e.g. CDK4/6 inhibitors). Intestinal epithelial cells are highly proliferative; however, the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. The IEC6 cell line, a non-transformed rat small intestinal epithelial cell line with characteristics of crypt epithelial cells, was utilized in the current study to understand the role of CDKs in the proliferation and survival of intestinal cells. In the initial experiment CDK4/6 inhibitor palbociclib treatment demonstrated a lack of potent toxicity in the IEC6 in comparison with MCF7 (breast cancer cell line), indicating the absence of intestinal cell reliance on CDK4 or CDK6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, targeted gene knockdown experiments using CDK1, 2, 4, and 6 siRNA and lipofectamine-mediated transfection were conducted. Surprisingly, only CDK1 knockdown causes profound cell death. CDK2, 4, or 6 knockdowns, whether single, double or triple combinations, did not have significant impact on IEC6 cells, as measured by multiple endpoints including impedance, ATP viability, and caspase activity. To further understand the role of CDK1 in cell cycle, various seeding densities of IEC6 cells were used with CDK1 siRNA. Although the impact of CDK1 knockdown on IEC6 cells decreased at the initial proliferation phase with the higher seeding density, there was still a decrease of viability observed at the later time point (when cells reached proliferation plateau), indicating a non-canonical role of CDK1 that is not related to cell cycle. This research supports the concept that CDK1, but not CDKs 2, 4, or 6, is essential for intestinal cell cycle progression and explains the lack of GI toxicity observed with palbociclib. Citation Format: Shuyan Lu, Wenyue Hu, Tae Sung, Brad Hirakawa, Marina Amaro, Bart Jessen. Phenotypic characterization of knockdown of CDKs in the intestinal epithelial cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 99.

Functional impairment triggered by altertoxin II (ATXII) in intestinal cells in vitro: cross-talk between cytotoxicity and mechanotransduction

Intestinal cells are able to continuously integrate response to multiple stimuli/stressors; these include the concomitant activation of “chemically driven” pathways, of paramount importance in the response to toxicants, as well as physical stimulation derived from motility. Altertoxin II (ATXII, 0.1, 1 and 10 µM), a mycotoxin produced by the food contaminant fungus Alternaria alternata was studied in HT-29 intestinal adenocarcinoma cells and in non-transformed intestinal epithelial cells, HCEC. One-hour incubation with ATXII was sufficient to trigger irreversible cytotoxicity in both cell types, as well as to modify cellular responses to concomitant pro-oxidant challenge (H2O2, 100–500 µM, DCF-DA assay) suggesting that even relatively short-time exposure of the intestinal cells could be sufficient to alter their functionality. Combination of ATXII (1 µM) with physical stimulation typical of the intestinal compartment (shear stress) revealed differential response of tumor-derived epithelial cells HT-29 in comparison to HCEC, in particular in the localization of the transcription factor Nrf2 (NF-E2-related factor 2). Moreover, ATXII reduced the migratory potential of HCEC as well as their membrane fluidity, but had no respective impact on HT-29 cells. Taken together, ATXII appeared to alter predominantly membrane functionality in HCEC thus hampering crucial functions for cellular motility/turnover, as well as barrier function of healthy intestinal cells and had very limited activity on the tumor counterparts.

Mitochondria-targeted drugs stimulate mitophagy and abrogate colon cancer cell proliferation

Mutations in the KRAS proto-oncogene are present in 50% of all colorectal cancers and are increasingly associated with chemotherapeutic resistance to frontline biologic drugs. Accumulating evidence indicates key roles for overactive KRAS mutations in the metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis in cancer cells. Here, we sought to exploit the more negative membrane potential of cancer cell mitochondria as an untapped avenue for interfering with energy metabolism in KRAS variant-containing and KRAS WT colorectal cancer cells. Mitochondrial function, intracellular ATP levels, cellular uptake, energy sensor signaling, and functional effects on cancer cell proliferation were assayed. 3-Carboxyl proxyl nitroxide (Mito-CP) and Mito-Metformin, two mitochondria-targeted compounds, depleted intracellular ATP levels and persistently inhibited ATP-linked oxygen consumption in both KRAS WT and KRAS variant-containing colon cancer cells and had only limited effects on nontransformed intestinal epithelial cells. These anti-proliferative effects reflected the activation of AMP-activated protein kinase (AMPK) and the phosphorylation-mediated suppression of the mTOR target ribosomal protein S6 kinase B1 (RPS6KB1 or p70S6K). Moreover, Mito-CP and Mito-Metformin released Unc-51-like autophagy-activating kinase 1 (ULK1) from mTOR-mediated inhibition, affected mitochondrial morphology, and decreased mitochondrial membrane potential, all indicators of mitophagy. Pharmacological inhibition of the AMPK signaling cascade mitigated the anti-proliferative effects of Mito-CP and Mito-Metformin. This is the first demonstration that drugs selectively targeting mitochondria induce mitophagy in cancer cells. Targeting bioenergetic metabolism with mitochondria-targeted drugs to stimulate mitophagy provides an attractive approach for therapeutic intervention in KRAS WT and overactive mutant-expressing colon cancer.

Abstract 3466: Growth inhibitory MEK-ERK signaling in the intestinal epithelium

Abstract Increasing evidence points to members of the PKC family of serine/threonine kinases as important tumor suppressors; however, the signaling pathways involved remain poorly defined. Our previous studies have determined that PKCα, which acts as a tumor suppressor in the epithelium of the intestine/colon, is activated coincident with growth arrest in intestinal crypts in vivo. PKCα activity induces cell cycle withdrawal in intestinal epithelial cells, a process that involves downregulation of cyclin D1 and Id1, upregulation of the cyclin dependent kinase inhibitors p21cip1 and p27kip1, and characteristic changes in pocket proteins and DNA licensing factors. Pharmacological inhibitors of the ERK pathway revealed that these changes are dependent on activation of MEK-ERK signaling, thus identifying a growth inhibitory ERK-mediated signaling axis in the intestinal epithelium. Given the interest in inhibitors of the ERK pathway for treatment of colon and other cancers, characterization of this tumor suppressive pathway will be essential for rational development of this treatment modality and to avoid and/or manage potential adverse effects of ERK inhibition in patients. To this end, the mechanistic basis for the growth suppressive effects of the PKCα→ERK module on cyclin D1, Id1 and p21cip1 are being investigated in non-transformed intestinal epithelial cells and colon cancer cells. Use of selective inhibitors and siRNA-mediated knockdown has determined that PKCα activates multiple components of the ERK pathway, including RAS, RAF, MEK and ERK. Notably, tumor suppressive effects are seen in colon cancer cells that harbor activating mutations in KRAS, indicating that growth inhibitory ERK signaling can override oncogenic signals arising from RAS mutation. PKCα signaling is associated with activation of ARAF, BRAF and CRAF and with induction of RAF heterodimers. A requirement for RAF activity was confirmed by the ability of LY3009120 or sorafenib to block growth inhibitory effects of PKCα. However, knockdown of individual RAF proteins fails to block these effects of PKCα, indicating some redundancy between the RAF proteins in mediating growth inhibitory signals. While the scaffolding protein KSR1 has been shown to be required for oncogenic RAS-ERK signaling, PKCα was still able to elicit growth inhibitory effects in KSR1 knockdown HCT-116 colon cancer cells. These data indicate that differential use of scaffolding proteins may explain both the growth inhibitory effects of PKCα-ERK signaling and their ability to override oncogenic RAS signals. A better understanding of these mechanisms may serve to guide the use of ERK inhibitors for colon cancer therapy. Supported by NIH grants DK60632, CA54807, CA157774 and CA191894. Citation Format: Navneet Kaur, Robert Lewis, Adrian Black, Jennifer Black. Growth inhibitory MEK-ERK signaling in the intestinal epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3466.

Abstract 3463: Crosstalk between PKCalpha and TGF-beta signaling suppresses inhibitor of DNA binding 1 (Id1) expression in intestinal epithelial cells

Abstract Inhibitor of DNA binding (Id) proteins (Id1-Id4) are dominant negative antagonists of basic helix-loop-helix (bHLH) transcription factors with recognized functions in development, stem cell self-renewal, and cell fate determination. Recent studies have identified Id proteins as master regulators of cancer-initiating cells and tumor aggressiveness, with critical roles in regulation of central hallmarks of cancer, including cell proliferation, survival, senescence, angiogenesis, migration, metastasis, and chemoresistance. In colorectal cancer (CRC), Id1 overexpression is associated with aggressive clinical behavior and poor prognosis, while decreasing sensitivity to chemotherapeutic agents. Protein kinase C α (PKCα) is a serine/threonine kinase that functions as a tumor suppressor in CRC. PKCα signaling induces cell cycle arrest in nontransformed IEC-18 intestinal epithelial cells via an ERK-dependent mechanism. A major target of PKCα negative control in these cells is Id1. To further characterize mechanisms underlying the tumor-suppressive effects of PKCα, we modulated PKCα expression and activation in CRC cells and nontransformed IEC-18 cells and performed RNA-seq analysis to identify changes in gene expression. This analysis identified transforming growth factor beta receptor 1 (TGFβR1) as an mRNA upregulated by PKCα signaling. Subsequent RT-PCR and Western blot analysis confirmed that PKCα upregulates TGFβR1 at both the mRNA and protein levels. The MEK inhibitors U0126 and PD98059 blocked PKCα upregulation of TGFβR1, pointing to an ERK-dependent mechanism. Notably, inhibition of TGFβR1 with GW788388 prevented PKCα-mediated Id1 downregulation, linking TGF-β signaling to the tumor-suppressive effects of PKCα. Further studies indicated that TGFβR1 activates a p38-mediated noncanonical pathway to downregulate Id1 in response to PKCα activity. Collectively, these findings identify a PKCα→Erk→TGFβR1→p38→Id1 signaling axis in intestinal epithelial cells. Both PKCα and TGF-β function as tumor suppressors in the intestinal epithelium and both pathways are inactivated during intestinal tumor progression. By providing a direct link between two tumor-suppressive signaling pathways with established effects on intestinal tumorigenesis, our findings broaden our understanding of CRC and open avenues for potential novel therapeutic approaches to this disease. (Supported by NIH grants DK60632, CA54807, CA036727 and CA191894.) Citation Format: Xinyue Li, Michelle Lum, Adrian Black, Jennifer Black. Crosstalk between PKCalpha and TGF-beta signaling suppresses inhibitor of DNA binding 1 (Id1) expression in intestinal epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3463.

Cellular differentiation of non-transformed intestinal epithelial cells is regulated by Lactobacillus rhamnosus and L. casei strains.

The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.

PWE-350 Resilience and protection against the dose-related toxicity of hydrogen sulphide

Introduction Hydrogen sulphide (H2S) is thought to have a biphasic effect on cells leading to cellular proliferation at “low” doses (micromolar) and cell death in “high” doses (millimolar). It is unclear how this is related to colonic physiology and in-vivo concentrations. H2S is cytotoxic by inhibiting cytochrome c, while genotoxicity may be mediated by free radicals of uncertain origin. We tested the effect of sub-physiological and physiological concentration of H2S on epithelial cell lines to identify proliferative/cytotoxic effects and whether a free radical scavenger could be protective. Method Cell lines were seeded at a concentration of 2.5 × 10 4 per well and cultured until 70–80% confluency. NaHS was dissolved in distilled water at 0, 100 μM, 500 μM, 1 mM, 2.5 mM and 5 mM and added to the culture. The number of surviving cells was counted after 24 h. In the protection experiments 1mM Tiron, a free radical scavenger, dissolved in distilled water was added concurrently. Results H2S proved considerably cytotoxic to CACO2 cells at even physiological doses (100 μM, 76.7% death), while at the higher end of the claimed physiological range (2.5 mM) 98.8% cell death was noted). Similar findings occurred with HCT116, though increased survival was noted with HT29 cells compared to the other colorectal cell lines (100 µM 43.9% death). Tiron was protective in all cell lines, especially in the CACO2 cells, with 100 μM + 1 mM Tiron mitigating cell death at 20% and 2.5 mM at 68.8%. P-values were significant at p Conclusion H2S is cytotoxic to cell lines, especially colonic lines, at estimated physiological doses. Tiron, a free-radical scavenger, increases cell survival after H2S exposure. This implicates excess free radicals in cytotoxicity. HT29 cells, with intact mismatch repair, were more resilient than other colorectal lines with mismatch deficiency. Contrary to some published research, this experiment suggests that H2S is cytotoxic, even at physiological levels, via a free radical mechanism, rather than protective against such insults. Colonic H2S may potentiate the risk of colorectal cancer. Steps to reduce its production or inhibit its action may reduce the risk of progression to cancer and reduce the chance of recurrence after surgery. Disclosure of interest None Declared. References Szabo C, et al . Tumor-derived hydrogen sulfide, produced by cystathionine-beta-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Proc Natl Acad Sci USA. 2013;110(30):12474–9 Deplancke B, Gaskins HR. Hydrogen sulfide induces serum-independent cell cycle entry in nontransformed rat intestinal epithelial cells. FASEB J. 2003;17(10):1310–2

Novel curcumin analogs to overcome EGFR–TKI lung adenocarcinoma drug resistance and reduce EGFR–TKI-induced GI adverse effects

Curcumin (1) down-regulates the expression as well as phosphorylation of epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells expressing gefitinib-resistant EGFR. Thirty-seven newly synthesized curcumin analogues including dimethoxycurcumin (2, DMC) were evaluated for their effects on EGFR expression as well as phosphorylation in two gefitinib-resistant lung adenocarcinoma cell lines, CL1-5 (EGFRwt) and H1975 (EGFRL858R+T790M). Based on the identified structure–activity relationships, methoxy substitution at C-3′, C-4′, or both positions favored inhibitory activity (compounds 1, 2, 5, 8–15, 17, 36), while compounds with more polar substituents were generally less active in both cell lines. Compound 36 with a fluorine substituent at C-6′ and its protonated counterpart 2 did not lose activity, suggesting halogen tolerance. In addition, a conjugated linker was essential for activity. Among all evaluated curcumin derivatives, compound 2 showed the best inhibitory effects on both wild-type and mutant EGFR by efficiently inducing gefitinib-insensitive EGFR degradation. Compound 23 also reduced gefitinib-induced gastrointestinal damage in the non-transformed intestinal epithelial cell line IEC-18.

Polymethoxyflavone Apigenin-Trimethylether Suppresses LPS-Induced Inflammatory Response in Nontransformed Porcine Intestinal Cell Line IPEC-J2.

The in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 μM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.

Abstract 98: A novel mouse model for intestinal serrated polyposis

Abstract Serrated polyps have recently been proposed to comprise at least 30% of the origins of human colorectal cancer (CRC). Unlike adenomatous polyps, which are initiated by APC/β-catenin mutations, the genetic and molecular mechanisms underlying the serrated pathway that leads to serrated polyp initiation and progression remain poorly understood. Here we describe a novel genetically modified mouse model of serrated polyposis via conditional activation of SMAD7, an inhibitory SMAD protein, in the intestinal epithelia. We show that SMAD7 activation is sufficient to drive serrated polyp initiation and progression to aggressive adenocarcinoma in both mouse intestine and colon. Mechanistically, we find that SMAD7-induced foci formation in non-transformed intestinal epithelial cells in vitro and serrated polyposis in vivo are Wnt/β-catenin independent. Furthermore, we find that, in addition to its regulation of TGFβ/BMP pathway, SMAD7 interacts with BRAF/MAPK signaling to regulate serrated polyposis. Taken together, our studies implicate for the first time the involvement of SMAD7 in serrated polyposis and reveal a potential novel molecular mechanism underlying the serrated polyp pathway. Citation Format: Jennifer Cotton, He Huang, Mihir Rajurkar, Junhao Mao. A novel mouse model for intestinal serrated polyposis. [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 98. doi:10.1158/1538-7445.AM2014-98

Oxysterol-binding Protein (OSBP)-related Protein 4 (ORP4) Is Essential for Cell Proliferation and Survival

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) comprise a large gene family with sterol/lipid transport and regulatory activities. ORP4 (OSBP2) is a closely related paralogue of OSBP, but its function is unknown. Here we show that ORP4 binds similar sterol and lipid ligands as OSBP and other ORPs but is uniquely required for the proliferation and survival of cultured cells. Recombinant ORP4L and a variant without a pleckstrin homology (PH) domain (ORP4S) bind 25-hydroxycholesterol and extract and transfer cholesterol between liposomes. Two conserved histidine residues in the OSBP homology domain ORP4 are essential for binding phosphatidylinositol 4-phosphate but not sterols. The PH domain of ORP4L also binds phosphatidylinositol 4-phosphate in the Golgi apparatus. However, in the context of ORP4L, the PH domain is required for normal organization of the vimentin network. Unlike OSBP, RNAi silencing of all ORP4 variants (including a partial PH domain truncation termed ORP4M) in HEK293 and HeLa cells resulted in growth arrest but not cell death. ORP4 silencing in non-transformed intestinal epithelial cells (IEC)-18 caused apoptosis characterized by caspase 3 and poly(ADP-ribose) polymerase processing, DNA cleavage, and JNK phosphorylation. IEC-18 transformed with oncogenic H-Ras have increased expression of ORP4L and ORP4S proteins and are resistant to the growth-inhibitory effects of ORP4 silencing. Results suggest that ORP4 promotes the survival of rapidly proliferating cells.

Transforming Growth Factor β Regulates P-Body Formation through Induction of the mRNA Decay Factor Tristetraprolin

Transforming growth factor β (TGF-β) is a potent growth regulator and tumor suppressor in normal intestinal epithelium. Likewise, epithelial cell growth is controlled by rapid decay of growth-related mRNAs mediated through 3' untranslated region (UTR) AU-rich element (ARE) motifs. We demonstrate that treatment of nontransformed intestinal epithelial cells with TGF-β inhibited ARE-mRNA expression. This effect of TGF-β was promoted through increased assembly of cytoplasmic RNA processing (P) bodies where ARE-mRNA localization was observed. P-body formation was dependent on TGF-β/Smad signaling, as Smad3 deletion abrogated P-body formation. In concert with increased P-body formation, TGF-β induced expression of the ARE-binding protein tristetraprolin (TTP), which colocalized to P bodies. TTP expression was necessary for TGF-β-dependent P-body formation and promoted growth inhibition by TGF-β. The significance of this was observed in vivo, where colonic epithelium deficient in TGF-β/Smad signaling or TTP expression showed attenuated P-body levels. These results provide new insight into TGF-β's antiproliferative properties and identify TGF-β as a novel mRNA stability regulator in intestinal epithelium through its ability to promote TTP expression and subsequent P-body formation.

Abstract C38: Small molecule targeting of the mRNA stability factor HuR impacts COX-2 expression in colon cancer

Abstract Background: Colorectal cancer (CRC) is the third most common cancer among American adults and a leading cause of cancer-related mortality. The initiation and progression of colorectal tumorigenesis is characterized by genetic alterations that promote expression of tumor-promoting genes such as COX-2. In non-transformed cells, the mRNAs of these genes are often targeted for rapid decay through AU-rich elements (ARE) present in their 3'UTR. However, ARE-mediated mRNA decay is compromised during colorectal tumorigenesis, allowing for pathogenic gene overexpression. Our prior work identified the RNA-binding protein HuR to be overexpressed during colon tumorigenesis. In this capacity, HuR promotes COX-2 expression by interfering with rapid mRNA degradation and is associated with poor clinical prognosis. In the present study, we examined the therapeutic potential of inhibiting HuR using the small molecule MS-444. Methods: HCT116, HCA-7, SW480, RKO, and HT-29 colon cancer cells along with non-transformed intestinal epithelial (RIE-1) and colonocytes (YAMC) were used. MS-444 was obtained from Novartis. Cell proliferation and apoptosis were assayed by MTT assay and flow cytometry. HuR trafficking was visualized by immunofluorescence. qPCR, western blot, and immunohistochemistry were performed to assess changes in gene expression. In vivo studies were performed to xenograft tumor models of colon cancer cells using IVIS-based imaging for COX-2. Results: Treatment of the CRC cells lines HCT116, HCA-7, SW480, RKO, and HT-29 with MS-444 resulted in growth inhibition with IC50 values ranging between 5.6-12.8 μM, and this observed growth inhibition resulted from increased cell apoptosis. Importantly, treatment of non-transformed intestinal and colonic epithelial cells with MS-444 exhibited IC50 values of 40.7μM and 28.1 μM, respectively, and were resistant to MS-444-induced apoptosis. The expression of HuR was not impacted by MS-444, whereas MS-444 altered HuR's presence in the cytoplasm. HuR performs its mRNA stabilization function when present in the cytoplasm, and CRC cells treated with MS-444 displayed nuclear HuR as a result inhibition of HuR cytoplasmic trafficking. Consistent with growth-inhibitory IC50 values, MS-444 inhibited COX-2 mRNA and protein expression in CRC cells. This inhibition required the COX-2 ARE-containing 3'UTR and occurred on a post-transcriptional level. In vivo, delivery of MS-444 resulted in xenograft CRC tumor growth inhibition and prevented HuR cytoplasmic localization. Furthermore, treatment of MS-444 inhibited tumor COX-2 overexpression. Conclusions: These findings indicate that small molecule-based inhibition of HuR results in selective CRC cell death in part by promoting COX-2 mRNA degradation, while also indicating that HuR is a necessary component for colorectal tumor cell survival. Citation Format: Fernando F. Blanco, Yuan Li, Nicole Meisner-Kober, Shrikant Anant, Dan A. Dixon. Small molecule targeting of the mRNA stability factor HuR impacts COX-2 expression in colon cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr C38.

Deoxynivalenol as a New Factor in the Persistence of Intestinal Inflammatory Diseases: An Emerging Hypothesis through Possible Modulation of Th17-Mediated Response

Background/AimsDeoxynivalenol (DON) is a mycotoxin produced by Fusarium species which is commonly found in temperate regions worldwide as a natural contaminant of cereals. It is of great concern not only in terms of economic losses but also in terms of animal and public health. The digestive tract is the first and main target of this food contaminant and it represents a major site of immune tolerance. A finely tuned cross-talk between the innate and the adaptive immune systems ensures the homeostatic equilibrium between the mucosal immune system and commensal microorganisms. The aim of this study was to analyze the impact of DON on the intestinal immune response.MethodologyNon-transformed intestinal porcine epithelial cells IPEC-1 and porcine jejunal explants were used to investigate the effect of DON on the intestinal immune response and the modulation of naive T cells differentiation. Transcriptomic proteomic and flow cytometry analysis were performed.Results DON induced a pro-inflammatory response with a significant increase of expression of mRNA encoding for IL-8, IL-1α and IL-1β, TNF-α in all used models. Additionally, DON significantly induced the expression of genes involved in the differentiation of Th17 cells (STAT3, IL–17A, IL-6, IL-1β) at the expenses of the pathway of regulatory T cells (Treg) (FoxP3, RALDH1). DON also induced genes related to the pathogenic Th17 cells subset such as IL–23A, IL-22 and IL-21 and not genes related to the regulatory Th17 cells (rTh17) such as TGF-β and IL-10.Conclusion DON triggered multiple immune modulatory effects which could be associated with an increased susceptibility to intestinal inflammatory diseases.

Regulation of Cysteinyl Leukotriene Receptor 2 Expression—A Potential Anti-Tumor Mechanism

BackgroundThe cysteinyl leukotrienes receptors (CysLTRs) are implicated in many different pathological conditions, such as inflammation and cancer. We have previously shown that colon cancer patients with high CysLT1R and low CysLT2R expression demonstrate poor prognosis. Therefore, we wanted to investigate ways for the transcriptional regulation of CysLT2R, which still remains to be poorly understood.Methodology/Principal FindingsWe investigated the potential role of the anti-tumorigenic interferon α (IFN-α) and the mitogenic epidermal growth factor (EGF) on CysLT2R regulation using non-transformed intestinal epithelial cell lines and colon cancer cells to elucidate the effects on the CysLT2R expression and regulation. This was done using Western blot, qPCR, luciferase reporter assay and a colon cancer patient array. We found a binding site for the transcription factor IRF-7 in the putative promoter region of CysLT2R. This site was involved in the IFN-α induced activity of the CysLT2R luciferase reporter assay. In addition, IFN-α induced the activity of the differentiation marker alkaline phosphatase along with the expression of mucin-2, which protects the epithelial layer from damage. Interestingly, EGF suppressed both the expression and promoter activity of the CysLT2R. E-boxes present in the CysLT2R putative promoter region were involved in the suppressing effect. CysLT2R signaling was able to suppress cell migration that was induced by EGF signaling.Conclusions/SignificanceThe patient array showed that aggressive tumors generally expressed less IFN-α receptor and more EGFR. Interestingly, there was a negative correlation between CysLT2R and EGFR expression. Our data strengthens the idea that there is a protective role against tumor progression for CysLT2R and that it highlights new possibilities to regulate the CysLT2R.

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

Selective Glucocorticoid Receptor Agonists for Treatment of Inflammatory Bowel Disease – in vitro studies

UDCA slows down intestinal cell proliferation by inducing high and sustained ERK phosphorylation

Ursodeoxycholic acid (UDCA) attenuates colon carcinogenesis in humans and in animal models by an unknown mechanism. We investigated UDCA effects on normal intestinal epithelium in vivo and in vitro to identify the potential chemopreventive mechanism. Feeding of mice with 0.4% UDCA reduced cell proliferation to 50% and suppressed several potential proproliferatory genes including insulin receptor substrate 1 (Irs-1). A similar transcriptional response was observed in the rat intestinal cell line IEC-6 which was then used as an in vitro model. UDCA slowed down the proliferation of IEC-6 cells and induced sustained hyperphosphorylation of ERK1/ERK2 kinases which completely inhibited the proproliferatory effects of EGF and IGF-1. The hyperphosphorylation of ERK1 led to a transcriptional suppression of the Irs-1 gene. Both, the hyperphosphorylation of ERK as well as the suppression of Irs-1 were sufficient to inhibit proliferation of IEC-6 cells. ERK1/ERK2 inhibition in vitro or ERK1 elimination in vitro or in vivo abrogated the antiproliferatory effects of UDCA. We show that UDCA inhibits proliferation of nontransformed intestinal epithelial cells by inducing a sustained hyperphosphorylation of ERK1 kinase which slows down the cell cycle and reduces expression of Irs-1 protein. These data extend our understanding of the physiological and potentially chemopreventive effects of UDCA and identify new targets for chemoprevention.