Normal Intestinal Epithelial Cells Research Articles

LKB1 regulates epithelial-mesenchymal transition in Peutz-Jeghers hamartoma and intestinal epithelial cells

To investigate the molecular mechanism by which LKB1 regulates epithelial-mesenchymal transition (EMT) in Peutz-Jeghers hamartoma and intestinal epithelial cells. Immunohistochemistry was used to detect gene expression of LKB1, E-cadherin, and vimentin in 20 hamartoma tissues and 10 normal intestinal tissues, and collagen fiber deposition was analyzed using Masson trichrome staining. Normal intestinal epithelial NCM460 cells were transfected with LKB1 shRNA plasmid or negative control via lentiviral vectors, and the role of LKB1 in cell polarization and migration were determined using CCK8 and Transwell assays. Western blotting, quantitative real-time PCR (qPCR) and immunofluorescence were used to assess the alterations of EMT markers in the cells with LKB1 knockdown. Compared with normal intestinal tissues, hamartoma polyps showed significantly decreased LKB1 and E-cadherin expressions and increased vimentin expression with increased collagen fiber deposition. The cells with LKB1 knockdown exhibited enhanced cell proliferation and migration activities (P<0.01). Western blot analysis, qPCR and immunofluorescence all detected decreased E-cadherin and increased N-cadherin, vimentin, Snail, and Slug expressions in the cells with LKB1 knockdown. s LKB1 deficiency triggers EMT in intestinal epithelial cells and Peutz-Jeghers hamartoma, suggesting that EMT can serve as the therapeutic target for treatment of Peutz-Jeghers syndrome.

Card9-dependent IL-1β regulates IL-22 production from group 3 innate lymphoid cells and promotes colitis-associated cancer.

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9−/− mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9−/− mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.

Role of the inflammasome of tumor cells in modulating the biology of Tumor Infiltrating T lymphocytes (TILs) in colorectal cancer.

e23087 Background: In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the phenotype and biology of Tumor Infiltrating T lymphocytes (TILs) of the tumor microenvironment. One of these mechanisms could be the inflammasome, a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL18 and generate a mucosal Th1/Tc1 (IFNγ) response. However, the inflammasome status of tumor cells in CRC and its potential role on TILs are unknown yet. Methods: Prospective and retrospective cohort studies aimed to determine in CRC patients: the status of the inflammasome in tumor cells (IL18 immunostaining on tissue microarrays (TMA) and in situ detection of active caspase-1 on frozen sections) and the density of TILs (CD8+, T-bet+) in relation with i) the microsatellite stable (MSS) or unstable (MSI) status of CRC, and ii) the levels of cytokines (IL18, IFNγ) secreted in an ex vivo explant culture model of CRC. Finally, we assessed the effect of recombinant human IL18 (rhIL18) on the IFNγ response of isolated TILs. Results: TMA analysis of the retrospective cohort showed that IL18 was significantly expressed (in more than 50% of tumor cells) in 80% of CRC, especially in MSI CRC, and correlated with a high density of T-bet+ and CD8+ intraepithelial TILs (IEL-TILs). Active caspase-1 was detected in tumor cells in 60% of CRC. In the prospective cohort, the presence of active caspase-1 in tumor cells was associated with high levels of mature IL18 secreted in explant cultures, with high density of T-bet+ TILs and with IFNγ release in most cases. In addition, isolated TILs expressing IL18 receptors (IL18Rα), cultured with rhIL18, were able to secrete IFNγ either unstimulated or stimulated with OKT3. Conclusions: The inflammasome of tumor cells, when maintained and active, can contribute to a Th1/Tc1 antitumor immune response elicited by TILs, that can modulate tumor growth. The inflammasome of tumor cells can thus be considered as a potential new therapeutic target to strengthen the antitumor immune response in CRC, in association with other immunotherapies.

Differential cytotoxic activity of Quercetin on colonic cancer cells depends on ROS generation through COX-2 expression

Quercetin is a bioactive compound with anti-inflammatory, antioxidant and anticancer properties. This study exemplifies the differential cytotoxic activity of Quercetin on two human colonic cancer cell lines, HT29 and HCT15. IC50 of Quercetin for HT29 and HCT15 cells were 42.5 μM and 77.4 μM, respectively. Activation of caspase-3, increased level of cytosolic cytochrome c, decreased levels of pAkt, pGSK-3β and cyclin D1 in 40 μM Quercetin treated HT29 cells alone. Though, nuclear translocation of NFkB was increased in 40 μM Quercetin treated HT29 and HCT15 cells, over expression of COX-2 was observed in 40 μM Quercetin treated HT29 cells, whereas, Quercetin treated HCT15 cells did not expressed COX-2. Increased generation of reactive oxygen species (ROS) was observed only in Quercetin treated HT29 cells, which is due to over expression of COX-2, as COX-2 silencing inhibited Quercetin induced apoptosis and ROS generation. Insilico analysis provided evidence that Quercetin could partially inhibit COX-2 enzyme by binding to subunit A which has peroxidase activity and serves as source of ROS. However, Quercetin showed minimal effect on normal intestinal epithelial cells i,e IEC-6. To conclude, differential sensitivity of two cancer cells, HT29 and HCT15, to Quercetin depends on COX-2 dependent ROS generation that induces apoptosis and inhibits cell survival.

Overexpression of the long non-coding RNA, linc-UBC1, is associated with poor prognosis and facilitates cell proliferation, migration, and invasion in colorectal cancer.

Long non-coding RNAs (lncRNAs) serve comprehensive roles in various diseases, including cancer. lncRNA upregulated in bladder cancer 1 (linc-UBC1) is a notable biomarker of prognosis in certain cancer types; however, its involvement in the progression of colorectal cancer (CRC) remains unknown. The present study aimed to investigate the expression of linc-UBC1 in patients with CRC and to investigate its effect on CRC cells. The expression levels of linc-UBC1 were estimated by reverse transcription-quantitative polymerase chain reaction in clinical CRC specimens and matched adjacent non-tumor mucosa from 96 cases of CRC, as well as in a number of CRC cell lines. In addition, the biological roles of linc-UBC1 were examined using a cell counting kit-8 assay, flow cytometry, and migration and invasion assays following the downregulation of linc-UBC1 by small interfering RNA. The results revealed that linc-UBC1 was significantly overexpressed in CRC tissues and the majority of CRC cell lines compared with the matched non-tumor mucosa and normal intestinal epithelial cells. Furthermore, high expression levels of linc-UBC1 were significantly associated with large tumor size, greater tumor depth, lymph node metastasis, and advanced tumor-node-metastasis stages. Patients with abnormal expression of linc-UBC1 had poorer overall survival times according to Kaplan–Meier analyses. Furthermore, multivariate Cox regression analysis indicated that linc-UBC1 was a significant independent prognostic factor. The results also revealed that reducing the expression of linc-UBC1 led to the inhibition of migration, invasion, and proliferation of CRC cells in vitro. Taken together, the results of the present study suggest that overexpression of linc-UBC1 promotes proliferation and metastasis in CRC, and may be considered as a novel diagnostic marker of CRC.

Role of the inflammasome of tumor cells in modulating the biology of Tumor Infiltrating T lymphocytes (TILs) in colorectal cancer.

640 Background: In colorectal cancer (CRC), little is known about mechanisms by which tumor cells can influence the phenotype and biology of Tumor Infiltrating T lymphocytes (TILs) of the tumor microenvironment. One of these mechanisms could be the inflammasome, a molecular platform present in normal intestinal epithelial cells, whose effector protein, caspase-1, can rapidly mature IL18 and generate a mucosal Th1/Tc1 (IFNg) response. However, the inflammasome status of tumor cells in CRC and its potential role on TILs are unknown yet. Methods: Prospective and retrospective cohort studies aimed to determine in CRC patients: the status of the inflammasome in tumor cells (IL18 immunostaining on tissue microarrays (TMA) and in situ detection of active caspase-1 on frozen sections) and the density of TILs (CD8+, T-bet+) in relation with the microsatellite stable (MSS) or unstable (MSI) status of CRC, and the levels of cytokines (IL18, IFNg) secreted in an ex vivo explant culture model of CRC. Finally, we assessed the effect of recombinant human IL18 (rhIL18) on the IFNg response of isolated TILs. Results: TMA analysis of the retrospective cohort showed that IL18 was significantly expressed (in more than 50% of tumor cells) in 80% of CRC, especially in MSI CRC, and correlated with a high density of T-bet+ and CD8+ intraepithelial TILs (IEL-TILs). Active caspase-1 was detected in tumor cells in 60% of CRC. In the prospective cohort, the presence of active caspase-1 in tumor cells was associated with high levels of mature IL18 secreted in explant cultures, with high density of T-bet+ TILs and with IFNg release in most cases. In addition, isolated TILs expressing IL18 receptors (IL18Ra), cultured with rhIL18, were able to secrete IFNg either unstimulated or stimulated with OKT3. Conclusions: The inflammasome of tumor cells, when maintained and active, can contribute to a Th1/Tc1 antitumor immune response elicited by TILs, that can modulate tumor growth. The inflammasome of tumor cells can thus be considered as a potential new therapeutic target to strengthen the antitumor immune response in CRC, in association with other immunotherapies.

Abstract B05: The RNA-binding protein HuR enhances exosome secretion in colorectal cancer

Abstract Enhanced secretion of exosomes by cancer cells is recognized as a means of transferring oncogenic information within the tumor microenvironment. Through their ability to carry specific RNA and protein cargo, tumor-derived exosomes are now being recognized for their ability to impact the tumor microenvironment and as promising cancer biomarkers. However, our knowledge of the cellular factors that promote increased exosome production from tumor cells and how they impact the loading of specific tumor-promoting RNA cargo is limited. Our prior work has established that colorectal cancer (CRC) cells and tumors overexpress the key RNA-binding protein HuR (ELAVL1) early in GI tumor development. When overexpressed and present in the cytoplasm, HuR can promote mRNA stabilization of tumor-promoting genes through binding of 3'UTR AU-rich elements (ARE). These same mRNAs are within tumor-derived exosomes, suggesting a role for HuR. To test this, Tet-regulated HuR-inducible HeLa cells were used to demonstrate that cytoplasmic HuR overexpression promoted a 4-fold increase in exosome production. Furthermore, HuR was detected in exosomes produced only from HuR-overexpressing cells. The presence of HuR in exosomes directly impacted mRNA cargo, as selective uptake of ARE-containing mRNAs were observed in exosomes derived from HuR overexpressing cells. To assess if these effects were seen in CRC cells that endogenously overexpress HuR, exosome levels from CRC cells were compared to normal human intestinal epithelial and myofibroblast cells. CRC cells secrete ~3-fold greater exosome levels than normal cells and enhanced exosome production was dependent upon HuR. siRNA knockdown and CRISPER/Cas9 knockout of HuR in CRC cells showed a &amp;gt;2-fold decrease in secreted exosomes, similar to the levels observed in normal cells. Furthermore, HuR knockout CRC cells showed a ~3-fold reduction in xenograft tumor growth compared to parental CRC cells. These findings were reflected in vivo where GI-tumor bearing APCMin/+ mice produced ~3-fold more serum exosomes, with HuR as exosomal cargo in APCMin/+ mice, whereas limited expression of exosomal HuR was detected in wild-type mice. This work has identified a novel connection between HuR-mediated post-transcriptional regulation and tumor-derived exosome production, along with providing the first preclinical evidence indicating the presence of exosomal HuR as a serum-based CRC biomarker. Citation Format: Ranjan Preet, Wei-Ting Hung, Shufei Zhuang, Lane K. Christenson, Dan A. Dixon. The RNA-binding protein HuR enhances exosome secretion in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B05.

MiR-34c and PlncRNA1 mediated the function of intestinal epithelial barrier by regulating tight junction proteins in inflammatory bowel disease

BackgroundInflammatory bowel disease (IBD) is originated from uncontrolled inflammation, and desired methods for IBD therapy remains the main difficult. The network comprised with miRNA and lncRNA has been verified to play an important role on diverse human diseases. In this study, we demonstrated the role of miR-34c and lncRNA PlncRNA1 on the function of intestinal barrier. MethodsIntestinal epithelial barrier model was constructed based on normal intestinal epithelial cell line Caco-2. 2% DSS was supplemented in the Apical side of the model cells to induce the injury of intestinal epithelial barrier. Real-time PCR or western blot was used to determine mRNA or protein expression of miR-34c, PlncRNA1, Myc-associated zinc finger protein (MAZ), zonula occludens 1 (ZO-1) and occludin. ResultsDSS induced injury of intestinal epithelial barrier, while overexpression of PlncRNA1 seemed to protect intestinal epithelial barrier from injury. Tight junction (TJ) proteins ZO-1 and occludin were regulated by MAZ, while, miR-34c targeted MAZ to regulate its expression, in addition, PlncRNA1 and miR-34c bound together to regulate the expressions of MAZ, ZO-1 and occludin. The protect effects of PlncRNA1 overexpression on intestinal epithelial barrier function was reversed by overexpression of miR-34c. ConclusionMAZ and TJ proteins were involved in the function of intestinal epithelial barrier, while miR-34c and PlncRNA1 regulated the intestinal dysfunction cooperatively.

Sphingosine-1 phosphate promotes intestinal epithelial cell proliferation via S1PR2.

Sphingosine-1 phosphate (S1P) is a potent bioactive lipid mediator that acts both as an intracellular signaling molecule and a natural ligand of five different G protein-coupled receptors (GPCRs), S1PR1-5. The level of S1P in intestinal tissue is abundant. Previous studies have reported that S1P protects intestinal epithelial cell from apoptosis by activating the ERK and Akt signaling pathways. However, the effect of S1P on intestinal epithelial cell proliferation under physiological conditions and the underlying signaling mechanisms remain to be elucidated. Here, we show that, except for S1PR4, all S1PRs are expressed in normal intestinal epithelial cells with S1PR2 being the most abundant. S1P dose-dependently stimulated cell migration and proliferation, which were inhibited by JTE-013, a selective chemical antagonist of S1PR2, and by a S1PR2 shRNA. S1P significantly upregulated the expression of c-Myc, cyclin D1, E-cadherin and zona occluden-1 (ZO-1), which was completely inhibited by downregulation of S1PR2 expression with a shRNA. In total, the results suggest that S1P-mediated activation of the S1PR2 plays an important role in regulating intestinal epithelial cell proliferation and migration.

The Effect of Inflammatory Status on Butyrate and Folate Uptake by Tumoral (Caco-2) and Non-Tumoral (IEC-6) Intestinal Epithelial Cells.

Objective Colorectal cancer (CRC) is the second leading cause of cancer death in occidental countries. Chronic inflammatory bowel disease (crohn’s disease and ulcerative colitis) is associated with an increased risk for CRC development. The aim of this work was to investigate the relationship between inflammatory status and absorption of nutrients with a role in CRC pathogenesis. Materials and Methods In this experimental study, we evaluated the in vitro effect of tumour necrosis factor-alpha (TNF-α), interferon-γ (IF-γ), and acetylsalicylic acid on 14C-butyrate (14C- BT), 3H-folic acid (3H-FA) uptake, and on proliferation, viability and differentiation of Caco-2 and IEC-6 cells in culture. Results The proinflammatory cytokines TNF-α and INF-γ were found to decrease uptake of a low concentration of 14C-BT (10 µM) by Caco-2 (tumoral) and IEC-6 (normal) intestinal epithelial cell lines. However, the effect of TNF-α and INF-γ in IEC-6 cells is most probably related to a cytotoxic and antiproliferative impact. In contrast, INF-γ increases uptake of a high concentration (10 mM) of 14C-BT in Caco-2 cells. The anticarcinogenic effect of BT (10 mM) in these cells is not affected by the presence of this cytokine. On the other hand, acetylsalicylic acid stimulates 14C-BT uptake by Caco-2 cells and potentiates its antiproliferative effect. Finally, both TNF-α and INF-γ cause a significant decrease in 3H-FA uptake by Caco-2 cells. ConclusionThe inflammatory status has an impact upon cellular uptake of BT and FA, two nutrients with a role in CRC pathogenesis. Moreover, the anti-inflammatory acetylsalicylic acid potentiates the anticarcinogenic effect of BT in Caco-2 cells by increasing its cellular uptake.

Expression of trefoil factors and TWIST1 in colorectal cancer and their correlation with metastatic potential and prognosis

To detect the expression of trefoil factors (TFFs) and TWIST1 in colorectal cancer (CRC) and analyze their correlation with metastasis and survival. This study examined the expression of TFF1, TFF3 and TWIST1 in a total of 75 tumor samples, 47 matched normal samples (15 cm from the lesion margin), 30 metastatic lymph nodes, and 10 liver metastatic cancer samples from patients with CRC. The relationship was then analyzed between the protein expression and different clinical records. TFF1, TFF3, TWIST1,E-cadherin, vimentin and β-catenin mRNA and protein expression levels were measured in colon cancer cell lines with different metastatic potentials (HIEC, HT29, SW620, and LoVo cells), and the correlation of the expression levels with epithelial-mesenchymal transition (EMT) was discussed. It was found that 66.7% (50/75), 78.7% (59/75) and 54.7% (41/75) of tumor tissue samples exhibited positive staining for TFF1, TFF3 and TWIST1 and so did 27.3% (13/47), 100% (47/47) and 17% (8/47) of adjacent normal colorectal tissues. Compared with adjacent normal tissues, significant differences were found in the expression of all three proteins in different cancerous tissues (P < 0.05). Higher expression of TFF3 and TWIST1 was significantly correlated with lymph node metastasis (P = 0.034, P = 0.000), advanced stage (P = 0.031, P = 0.003), and poorer survival (P = 0.042 for the TFF3 group, P = 0.003 for the TWIST1 group). The expression of TFF3 and TWIST1 in cancer cell lines was higher than that in HIEC (a normal human intestinal epithelial cell line)(P < 0.05), and the expression intensity demonstrated a tendency to rise with increased metastatic potential both at the protein and mRNA levels. However, TFF1 expression demonstrated the opposite tendency. It was also observed that the expression of E-cadherin and β-catenin tended to decrease while that of vimentin, TWIST1 and Snail tended to rise with the increase in metastatic potential. The expression of TFF3 and TWIST1 might be associated with the survival of patients with CRC after curative resection and might be pivotal predictors of disease progression. TFF3 may be correlated to the invasiveness of CRC.

Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways.

Lactoferrin (Lf) is an iron-binding glycoprotein that is present at high concentrations in milk. Bovine lactoferricin (LfcinB) is a peptide fragment generated by pepsin proteolysis of bovine lactoferrin (bLf). LfcinB consists of amino acid residues 17-41 proximal to the N-terminus of bLf and a disulfide bond between residues 19 and 36, forming a loop. Both bLf and LfcinB have been demonstrated to have antitumor activities. Colorectal cancer is the second most common cause of cancer death in developed countries. We hypothesized that bLf and LfcinB exert antitumor activities on colon cancer cells (HT-29) by triggering various signaling pathways. bLf and LfcinB significantly induced apoptosis in HT-29 cells but not in normal human intestinal epithelial cells, as revealed by the ApoTox-Glo Triplex Assay. The LIVE/DEAD cell viability assay showed that both bLf and LfcinB reduced the viability of HT-29 cells. Transcriptome analysis indicated that bLf, cyclic LfcinB, and linear LfcinB exerted antitumor activities by differentially activating diverse signaling pathways, including p53, apoptosis, and angiopoietin signaling. Immunoblotting results confirmed that both bLf and LfcinBs increased expression of caspase-8, p53, and p21, critical proteins in tumor suppression. These results provide valuable information regarding bLf and LfcinB for potential clinical applications in colon cancer therapy.

Subcellular proteomics analysis of different stages of colorectal cancer cell lines.

Studying cell differentiation and transformation allows a better understanding of the mechanisms involved in the initiation and the evolution of cancer. The role of proteins which participate in these processes is dependent on their location within the cell. Determining the subcellular localization of proteins or the changes in localization is, therefore, paramount in elucidating their role. Using quantitative mass spectrometry, we characterized the protein expression and subcellular localization of nearly 5000 proteins from seven different colorectal cancer (CRC) cell lines, as well as normal colon fibroblasts and intestinal epithelial cells. This cellular characterization allowed the identification of colon cancer-associated proteins with differential expression patterns as well as deregulated protein networks and pathways. Indeed, our results demonstrate differential expression of proteins involved in cell adhesion, cytoskeleton, and transcription in colon cancer cells compared to normal colon-derived cells. Pathway analyses identified different cellular functions, including endocytosis and eIF2 signaling, whose deregulation correlates with mutations found in the different CRC phenotypes. Our results provide an unbiased, quantitative and high-throughput approach to measure changes in protein expression and subcellular protein locations in different CRC cell lines.

Abrogation of MUC5AC Expression Contributes to the Apoptosis and Cell Cycle Arrest of Colon Cancer Cells.

Deregulated expressions of mucins have been found in various malignancies and play a pivotal role in carcinogenesis. MUC5AC, as a secreted mucin, is reported to be aberrantly expressed during epithelial cancer progression, including colon cancer. However, the mechanisms of the oncoprotein MUC5AC in the initiation of colon cancer requires further investigation. Here, we collected colon cancer tissues (n = 20) and corresponding paracancerous tissues (n = 20) and found that the expression of MUC5AC was significantly elevated in colon cancer tissues when compared with the corresponding paracancerous tissues. Immunofluorescence indicated that all colon cancer cell lines, including HT29, SW620, and the normal human intestinal epithelial cells FHC, showed the positive expression of MUC5AC, and SW620 exhibited the highest expression. Moreover, knockdown of MUC5AC in SW620 cells remarkably suppressed cell vitality and promoted apoptosis and G1 cell cycle arrest, resulting in the impaired ability of colony formation. Furthermore, the inhibition of MUC5AC in SW620 cells dramatically repressed the cell migration and invasion. These results demonstrated that MUC5AC as an oncogene could be a promising target in the treatment of colon cancer.

MiR-29a Regulates Radiosensitivity in Human Intestinal Cells by Targeting PTEN Gene.

Two major challenges encountered during radiotherapy for colorectal cancer (CRC) are radioresistance of tumor cells and damage to normal cells. An understanding of the mechanisms of radioresistance in CRC may lead to new strategies for overcoming obstacles to affective clinical therapy. In this study, the miR-29a expression was compared among four cell lines: the normal human intestinal epithelial crypt cell line, HIEC and three CRC cell lines, HT29, DLD-1 and HCT116. The roles of miR-29a in regulating cellular radiosensitivity were then investigated. The findings from this study showed that miR-29a mimic enhanced radioresistance in HIEC, HT29 and DLD-1 cells with low levels of intrinsic miR-29a. On the other hand, a miR-29a inhibitor significantly sensitized HCT116 cells with high levels of miR-29a after irradiation. Further studies indicated that PTEN was the direct functional target of miR-29a and was involved in radiosensitivity. MiR-29a could activate the PI3K/Akt signaling pathway through negatively regulated PTEN expression. In conclusion, miR-29a may regulate the radiosensitivity of intestinal cell lines by targeting the PTEN gene, which indicates miR-29a might serve as a novel approach to enhance radiosensitivity in CRC.

Polaprezinc protects normal intestinal epithelium against exposure to ionizing radiation in mice.

Polaprezinc (PZ), an antiulcer drug, has been reported to have antioxidant effects. The purpose of the present study was to assess the radioprotective effects of PZ in the normal intestine of C57BL/6J mice. PZ was orally administered at 100 mg/kg body weight in the drinking water. Firstly, the present study compared the survival of normal intestinal crypt epithelial cells with mice that received PZ prior to or following irradiation. Next, the present study examined the sequential changes of the incidence of apoptosis in the normal intestine of mice that received irradiation. The mice that received PZ prior to irradiation demonstrated a stronger protective effect on the normal intestine compared with those that received PZ after irradiation. The present study therefore administrated PZ 2 h before irradiation in the subsequent experiments. The mice receiving PZ developed fewer apoptotic cells in the duodenum, jejunum and ileum. Radiation-induced cell death occurred with a peak at position 10 or lower from the base of the crypt axis, and was subsequently reduced by PZ treatment. Pretreatment with PZ protected the normal intestinal tissues from radiation-induced apoptosis.

TGEV infection up-regulates FcRn expression via activation of NF-κB signaling.

It has been well characterized that the neonatal Fc receptor (FcRn) transports maternal IgG to a fetus or newborn and protects IgG from degradation. We previously reported that FcRn is expressed in a model of normal porcine intestinal epithelial cells (IPEC-J2). Transmissible gastroenteritis is an acute enteric disease of swine that is caused by transmissible gastroenteritis virus (TGEV). How porcine FcRn (pFcRn) expression is regulated by pathogenic infection remains unknown. Our research shows that IPEC-J2 cells infected with TGEV had up-regulated pFcRn expression. In addition, the NF-κB signaling pathway was activated in IPEC-J2 cells by TGEV infection. Furthermore, treatment of TGEV-infected IPEC-J2 cells with the NF-κB-specific inhibitor BAY 11-7082 resulted in down-regulation of pFcRn expression. Transient transfection of pFcRn promoter luciferase report plasmids with overexpression of NF-κB p65 transcription factor enhanced the activation of the luciferase report plasmids. We identified four NF-κB transcription factor binding sites in the promoter region of this gene using luciferase reporter system, chromatin immunoprecipitation, electromobility shift assay, and supershift analysis. Together, the data provide the first evidence that TGEV infection up-regulates pFcRn expression via activation of NF-κB signaling.

Abstract A41: The RNA binding protein HuR enhances exosome secretion in colorectal cancer

Abstract Enhanced secretion of extracellular vesicles such as microvesicles and exosomes by cancer cells has been recently recognized as a new means of transferring oncogenic information within the tumor microenvironment. Through their ability to carry specific RNA and protein cargo, tumor-derived exosomes can directly impact neighboring cells to support tumor progression. However, it remains unclear what regulates exosome production in tumor cells and the factors influencing loading of tumor-promoting RNA cargo. Our prior work has established that colorectal cells (CRC) and tumors overexpress the key RNA-binding protein HuR early in GI tumor development. When overexpressed and present in the cytoplasm, HuR can promote mRNA stabilization of tumor-promoting genes through binding of 3'UTR AU-rich elements (ARE). While the ability of HuR overexpression to promote gene expression is well recognized, our understanding of how HuR communicates this information within the tumor microenvironment is limited. To test if HuR overexpression could impact exosomes, Tet-regulated HuR-inducible HeLa cells were used to demonstrate that cytoplasmic HuR overexpression promoted a 4-fold increase in exosomes produced. Furthermore, HuR was detected in exosomes produced only from HuR-overexpressing cells. The presence of HuR in exosomes directly impacted mRNA cargo, as selective uptake of ARE-containing mRNAs were observed in exosomes derived from HuR overexpressing cells. To test if these effects were seen in CRC cells that endogenously overexpress HuR, exosome levels from CRC cells were compared to normal primary human intestinal epithelial and myofibroblast cells. When normalized to total cell numbers, CRC cells secrete ~3-fold greater exosome levels than normal cells and enhanced exosome production was dependent upon HuR. Knockdown of HuR in CRC cells directly impacted exosome secretion to levels observed in normal cells. HuR was also only detected in exosomes produced from CRC cells. Using an inducible model of RasV12-mediated transformation of intestinal epithelial cells, endogenous HuR overexpression was observed concomitant with 4-fold greater levels of exosomes containing HuR as cargo. These findings were reflected in vivo where GI-tumor bearing APCMin/+ mice produced ~3-fold more serum exosomes, with HuR as exosomal cargo in APCMin/+ mice whereas no expression of exosomal HuR was detected in wild-type mice. This work has identified a novel connection between HuR-mediated post-transcriptional regulation and tumor-derived exosome production, along with providing the first evidence indicating the presence of exosomal HuR as a serum-based CRC biomarker. Citation Format: Ranjan Preet, Shufei Zhuang, Wei-Ting Hung, Lane K. Christenson, Dan A. Dixon. The RNA binding protein HuR enhances exosome secretion in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A41.

Abstract 5104: The RNA binding protein HuR enhances exosome secretion in colorectal cancer

Abstract Enhanced secretion of extracellular vesicles such as microvesicles and exosomes by cancer cells has been recently recognized as a new means of transferring oncogenic information within the tumor microenvironment. Through their ability to carry specific RNA and protein cargo, tumor-derived exosomes can directly impact neighboring cells to support tumor progression. However, it remains unclear what regulates exosome production in tumor cells and the factors influencing loading of tumor-promoting RNA cargo. Our prior work has established that colorectal cancer (CRC) cells and tumors overexpress the key RNA-binding protein HuR early in GI tumor development. When overexpressed and present in the cytoplasm, HuR can promote mRNA stabilization of tumor-promoting genes through binding of 3’UTR AU-rich elements (ARE). While the ability of HuR overexpression to promote gene expression is well recognized, our understanding of how HuR communicates this information within the tumor microenvironment is limited. To test if HuR overexpression could impact secreted exosomes, Tet-regulated HuR-inducible HeLa cells were used to demonstrate that cytoplasmic HuR overexpression promoted a 4-fold increase in exosomes produced. Furthermore, HuR was detected in exosomes produced only from HuR-overexpressing cells. The presence of HuR in exosomes directly impacted mRNA cargo, as selective uptake of ARE-containing mRNAs was observed in exosomes derived from HuR overexpressing cells. To test if these effects were seen in CRC cells that endogenously overexpress HuR, exosome levels from CRC cells were compared to normal primary human intestinal epithelial and myofibroblast cells. When normalized to total cell numbers, CRC cells secrete ∼3-fold greater exosome levels than normal cells and enhanced exosome production was dependent upon HuR. Knockdown of HuR in CRC cells directly impacted exosome secretion to levels observed in normal cells. HuR was also only detected in exosomes produced from CRC cells. Using an inducible model of RasV12-mediated transformation of intestinal epithelial cells, endogenous HuR overexpression was observed associated with 4-fold greater levels of exosomes containing HuR as cargo. These findings were reflected in vivo where GI-tumor bearing APCMin/+ mice produced ∼3-fold more serum exosomes, with HuR as exosomal cargo in APCMin/+ mice where as no expression of exosomal HuR was detected in wild-type mice. This work has identified a novel connection between HuR-mediated post-transcriptional regulation and tumor-derived exosome production, along with providing the first evidence indicating the presence of exosomal HuR as a serum-based CRC biomarker. Citation Format: Ranjan Preet, Shufei Zhuang, Wei-ting Hung, Lane K. Christenson, Dan A. Dixon. The RNA binding protein HuR enhances exosome secretion in colorectal cancer. [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 5104.

Estrogen receptor beta reduces colon cancer metastasis through a novel miR-205 - PROX1 mechanism.

Colon cancer is a common cause of cancer death in the Western world. Accumulating evidence supports a protective role of estrogen via estrogen receptor beta (ERβ) but the mechanism of action is not known. Here, we elucidate a molecular mechanism whereby ERβ represses the oncogenic prospero homebox 1 (PROX1) through the upregulation of miR-205. We show that PROX1 is a potential target of miR-205 and that in clinical specimens from The Cancer Genome Atlas data, ERβ and miR-205 are decreased in colorectal cancer tissue compared to non-tumorous colon, while PROX1 levels are increased. Through mechanistic studies in multiple colorectal cancer cell lines, we show that ERβ upregulates miR-205, and that miR-205 targets and represses PROX1 through direct interaction with its 3′UTR. Through the generation of intestine-specific ERβ knockout mice, we establish that this pathway is correspondingly regulated in normal intestinal epithelial cells in vivo. Functionally, we demonstrate that miR-205 decreases cell proliferation and decreases migratory and invasive potential of colon cancer cells, leading to a reduction of micrometastasis in vivo. In conclusion, ERβ in both normal and cancerous colon epithelial cells upregulates miRNA-205, which subsequently reduces PROX1 through direct interaction with its 3′UTR. This results in reduced proliferative and metastatic potential of the cells. Our study proposes a novel pathway that may be exploited using ERβ-selective agonists and/or miR-205-replacement therapy in order to improve preventive and therapeutic approaches against colon cancer.