Runx1 regulates Tff1 expression to expedite viability of retinal microvascular endothelial cells in mice with diabetic retinopathy.

Diabetic retinopathy (DR) represents a major complication of diabetes, and molecular mechanisms related to vascular dysfunction, particularly endothelial dysfunction, in DR remains unclear. In the present work, we generated a DR animal model using mice and a cell model in mouse retinal microvascular endothelial cells (mRMECs) to examine the role of Trefoil factor family 1 (Tff1) in DR. Tff1 was poorly expressed in DR mice and high glucose (HG)-treated mRMECs. Overexpression of Tff1 significantly attenuated streptozotocin-induced retinal proliferation and angiogenesis in DR mice and reduced the secretion of inflammatory factors. In HG-treated mRMECs, overexpression of Tff1 remarkably reduced the proliferation and angiogenesis of mRMECs. In further experiments, we found that Tff1 was transcriptionally repressed by Runt-related transcription factor 1 (Runx1) directly, and Tff1 expression was indirectly modulated by Runx1 via the core-binding factor subunit beta (CBF-ß)/nuclear factor, erythroid 2/microRNA-423-5p axis and the CBF-ß/estrogen receptor 1 (ESR1) axis. Moreover, Tff1 could inhibit the activation of NF-κB signaling pathway, which in turn attenuated retinal endothelial cell proliferation and angiogenesis. It was thus proposed that Runx1/Tff1/NF-κB axis may be a potential target for the treatment strategy of DR, and further studies are needed.

TFF1 gene single nucleotide polymorphism (rs3761376) and colorectal cancer risk.

INTRODUCTION: Trefoil Factor 1 (TFF1) is a secretory peptide with gastrointestinal protective functions. Abnormal TFF1 expression is reported in some cancers and functional promoter polymorphism in TFF1 is believed to be associated with risk of gastric cancer. We evaluated rs3761376 in a sample of Iranian patients with colorectal cancer. METHODS: Peripheral blood samples were taken from pathology confirmed cases of colorectal cancer and healthy volunteers. Genotyping was carried out using Restriction Fragment Length Polymorphism (RFLP) PCR. Any association with clinicopathologic data was assessed by SPSS version 19. RESULTS: A total of 245 participants, including 122 patients with cancer and 123 non-cancer subjects were enrolled. Age, body mass index, and smoking habits were not significantly different between the two groups (P > 0.05). Distribution of TFF1 genotypes was not found to be associated with colorectal cancer. However, distant metastasis was more prevalent in carriers of the mutant allele. CONCLUSION: TFF1 rs3761376 was not associated with colorectal cancer but it may be involved in metastasis. Therefore, further investigation is warranted to determine this relationship.

Association of functional genetic variants in TFF1 and nephrolithiasis risk in a Chinese population.

Trefoil Factor 1 (TFF1) is considered to be able to inhibit the formation of kidney stone. However, genetic variants in TFF1 and corresponding function in kidney stone development are still not well studied. In this study, the discovery set including 230 cases and 250 controls was used to analyze the association between seven tagSNPs of TFF1 gene and the nephrolithiasis risk. Further evaluation was confirmed by the validation set comprising 307 cases and 461 controls. The consequences of the two-stage case-control study indicated that individuals with the rs3761376 A allele have significantly increased nephrolithiasis risk than those with the GG genotypes [adjusted odds ratio (OR) = 1.35, 95% confidence interval (CI) = 1.05-1.73]. Moreover, we also carried out a stratified analysis and found the increased nephrolithiasis risks at A allele among males, overweight individuals, no hypertensive individuals, nondiabetic individuals, smokers, and drinkers. In the following functional experiments, the notably lower expression of TFF1 was exhibited by the vectors carrying A allele compared with those carrying G allele in both luciferase (P = 0.022) and expression vectors (P = 0.041). In addition to tissue detection, we confirmed a significant inverse association of rs3761376 G > A and TFF1 gene expression (P < 0.001). These results suggest that TFF1 rs3761376 may serve as a potential biomarker to predict the risk of nephrolithiasis.

Circ-TFF1 Facilitates Breast Cancer Development via Regulation of miR-338-3p/FGFR1 Axis.

Some circular RNAs (circRNAs) have been verified to act as essential regulators in the progression of breast cancer (BC). We aimed to investigate the role of circRNA trefoil factor 1 (circ-TFF1) in BC progression. The expression of circ-TFF1, microRNA-338-3p (miR-338-3p) and fibroblast growth factor receptor 1 (FGFR1) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT), colony formation, and 5-Ethynyl-2'-deoxyuridine (EDU) assays. Cell apoptosis and invasion were assessed by flow cytometry and transwell assay, respectively. Cellular glycolysis, including glucose consumption, lactate production, and ATP/ADP ratio, was detected by commercial kits. All protein levels were measured by western blot assay. The relationship between miR-338-3p and circ-TFF1 or FGFR1 was predicted by online bioinformatics tool and verified by dual-luciferase reporter assay. Xenograft tumor model was established to verify the function of circ-TFF1 in vivo. Circ-TFF1 was overexpressed in BC tissues and cells. Circ-TFF1 knockdown inhibited cell proliferation, invasion and glycolysis and induced apoptosis in BC cells. Circ-TFF1 acted as a sponge of miR-338-3p, and the effects of circ-TFF1 knockdown on BC cell proliferation, apoptosis, invasion, and glycolysis were abolished by miR-338-3p inhibition. FGFR1 was confirmed to be a target gene of miR-338-3p, and miR-338-3p played a tumor-suppressive role in BC by targeting FGFR1. Moreover, circ-TFF1 regulated FGFR1 expression by targeting miR-338-3p. Additionally, circ-TFF1 knockdown hampered tumorigenesis in vivo. Circ-TFF1 knockdown suppressed BC progression by regulating miR-338-3p/FGFR1 axis, providing a promising therapeutic target for BC.

Circ-TFF1 Promotes Breast Cancer Progression Through the miR-129-2-3p/IRAK1 Axis.

Breast cancer (BC) is a common malignant tumor, and circular RNA-trefoil factor 1 (circ-TFF1; hsa_circ_0061825) has been found to be highly expressed in BC tissues and cells and is associated with the poor prognosis of BC patients. However, the interaction between circ-TFF1 and microRNA in BC has not been studied. Quantitative real-time PCR was used to detect the expression of circ-TFF1, miR-129-2-3p, and interleukin (IL)-1 receptor-associated kinase 1 (IRAK1). Through the detection of cell proliferation, migration, invasion, tube formation, and apoptosis, cell function was assessed. The expression levels of angiogenesis-related proteins were detected by western blot. The interaction between miR-129-2-3p and circ-TFF1 or IRAK1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. Xenotransplantation experiments were used to confirm the function of circ-TFF1 in vivo. Circ-TFF1 and IRAK1 were significantly high expressed in BC tissues and cells. Silencing of circ-TFF1 reduced the proliferation, migration, invasion and tube formation, while increased the apoptosis of MDA-MB-361 and SK-Br-3 cells. MiR-129-2-3p was a target of circ-TFF1. Silencing of circ-TFF1 inhibited the malignant behavior of BC cells by releasing miR-129-2-3p. In addition, IRAK1 was a target of miR-129-2-3p. Overexpression of IRAK1 partially restored the inhibitory effect of miR-129-2-3p on cell progression. Animal experiments confirmed the anti-tumor effect of circ-TFF1 knockdown in vivo. Circ-TFF1 regulated the expression of IRAK1 by sponging miR-129-2-3p, thereby, promoting the development of BC. These data provided a novel targeted therapy for BC.

NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1.

BACKGROUND: H. pylori infection is the main risk factor for gastric cancer. In this study, we investigated H. pylori-mediated activation of STAT3 and NF-κB in gastric cancer, using in vitro and in vivo models. METHODS: To investigate the activation of NF-κB and STAT3 by H. pylori strains we used in vitro and in vivo mouse models, western blots, immunofluorescence, ChIP Assay, luciferase and quantitative real-time PCR assays. RESULTS: Following infection with H. pylori in vitro, we found an earlier phosphorylation of NF-kB-p65 (S536), followed by STAT3 (Y705). Immunofluorescence, using in vitro and in vivo models, demonstrated nuclear localization of NF-kB and STAT3, following H. pylori infection. NF-kB and STAT3 luciferase reporter assays confirmed earlier activation of NF-kB followed by STAT3. In vitro and in vivo models demonstrated induction of mRNA expression of IL-6 (p < 0.001), VEGF-α (p < 0.05), IL-17 (p < 0.001), and IL-23 (p < 0.001). Using ChIP, we confirmed co-binding of both NF-kB-p65 and STAT3 on the IL6 promoter. The reconstitution of Trefoil Factor 1 (TFF1) suppressed activation of NF-kB with reduction in IL6 levels and STAT3 activity, in response to H. pylori infection. Using pharmacologic (BAY11-7082) and genetic (IκB super repressor (IκBSR)) inhibitors of NF-kB-p65, we confirmed the requirement of NF-kB-p65 for activation of STAT3, as measured by phosphorylation, transcription activity, and nuclear localization of STAT3 in in vitro and in vivo models. CONCLUSION: Our findings suggest the presence of an early autocrine NF-kB-dependent activation of STAT3 in response to H. pylori infection. TFF1 acts as an anti-inflammatory guard against H. pylori-mediated activation of pro-inflammatory networks.

Circulating trefoil factors in relation to lung cancer, age and lung function: a cross-sectional study in patients referred for suspected lung cancer.

The trefoil factor family proteins: TFF1, TFF2 and TFF3 are secreted by epithelial cells in the respiratory tract. Here, we explore circulating concentrations of the trefoil factors in relation to lung cancer, age and lung function. We included 751 patients suspected of lung cancer. Lung cancer diagnosis was based on data reported to a national database. Serum TFF1, TFF2 and TFF3 concentrations were measured by ELISA, and spirometry was performed within ±3 days of blood sampling. Forced expiratory volume in the first second (FEV1) in relation to forced vital capacity (FVC), FEV1/FVC (a parameter used to quantify reduced lung function) was recorded. Lung cancer was diagnosed in 163 (22%) patients. Circulating concentrations of TFF3 (p = .021), but not TFF1 and TFF2, were significantly elevated in cancer patients. All three trefoil factors showed an increase in concentration with increasing age (p < .001) and declining lung function (p < .004). In the present cohort, concentrations of all three peptides were elevated compared with previous results published for healthy individuals. In conclusion, we report higher concentrations of TFF3 in patients with lung cancer, while increasing age and reduced lung function are associated with increasing concentrations of all trefoil factors in this specific patient population. The results emphasize that age and lung function should be taken into consideration when evaluating concentrations of trefoil factors in patients. However, the increases in trefoil factor concentrations were relatively small, and consequently, it is unlikely that circulating trefoil factor concentrations may have a role in the diagnosis of lung cancer and lung function impairment.

TFF1 Induces Aggregation and Reduces Motility of Helicobacter pylori.

Gastric cancer is considered one of the most common malignancies in humans and Helicobacter pylori infection is the major environmental risk factor of gastric cancer development. Given the high spread of this bacterium whose infection is mostly asymptomatic, H. pylori colonization persists for a long time, becoming chronic and predisposing to malignant transformation. The first defensive barrier from bacterial infection is constituted by the gastric mucosa that secretes several protective factors, among which is the trefoil factor 1 (TFF1), that, as mucin 5AC, binds the bacterium. Even if the protective role of TFF1 is well-documented, the molecular mechanisms that confer a beneficial function to the interaction among TFF1 and H. pylori remain still unclear. Here we analyze the effects of this interaction on H. pylori at morphological and molecular levels by means of microscopic observation, chemiotaxis and motility assays and real-time PCR analysis. Our results show that TFF1 favors aggregation of H. pylori and significantly slows down the motility of the bacterium across the mucus. Such aggregates significantly reduce both flgE and flaB gene transcription compared with bacteria not incubated with TFF1. Finally, our results suggest that the interaction between TFF1 and the bacterium may explain the frequent persistence of H. pylori in the human host without inducing disease.

Reciprocal expression of trefoil factor-1 and thyroid transcription factor-1 in lung adenocarcinomas.

Molecular targeted therapies against EGFR and ALK have improved the quality of life of lung adenocarcinoma patients. However, targetable driver mutations are mainly found in thyroid transcription factor-1 (TTF-1)/NK2 homeobox 1 (NKX2-1)-positive terminal respiratory unit (TRU) types and rarely in non-TRU types. To elucidate the molecular characteristics of the major subtypes of non-TRU-type adenocarcinomas, we analyzed 19 lung adenocarcinoma cell lines (11 TRU types and 8 non-TRU types). A characteristic of non-TRU-type cell lines was the strong expression of TFF-1 (trefoil factor-1), a gastric mucosal protective factor. An immunohistochemical analysis of 238 primary lung adenocarcinomas resected at Jichi Medical University Hospital revealed that TFF-1 was positive in 31 cases (13%). Expression of TFF-1 was frequently detected in invasive mucinous (14/15, 93%), enteric (2/2, 100%), and colloid (1/1, 100%) adenocarcinomas, less frequent in acinar (5/24, 21%), papillary (7/120, 6%), and solid (2/43, 5%) adenocarcinomas, and negative in micropapillary (0/1, 0%), lepidic (0/23, 0%), and microinvasive adenocarcinomas or adenocarcinoma in situ (0/9, 0%). Expression of TFF-1 correlated with the expression of HNF4-α and MUC5AC (P < .0001, P < .0001, respectively) and inversely correlated with that of TTF-1/NKX2-1 (P < .0001). These results indicate that TFF-1 is characteristically expressed in non-TRU-type adenocarcinomas with gastrointestinal features. The TFF-1-positive cases harbored KRAS mutations at a high frequency, but no EGFR or ALK mutations. Expression of TFF-1 correlated with tumor spread through air spaces, and a poor prognosis in advanced stages. Moreover, the knockdown of TFF-1 inhibited cell proliferation and soft-agar colony formation and induced apoptosis in a TFF-1-high and KRAS-mutated lung adenocarcinoma cell line. These results indicate that TFF-1 is not only a biomarker, but also a potential molecular target for non-TRU-type lung adenocarcinomas.

Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. Aberrant expression of microRNAs is strongly implicated in gastric tumorigenesis; however, their contribution in response to H. pylori infection has not been fully elucidated. In this study, we evaluated the expression of miR-135b-5p and its role in gastric cancer. We describe the overexpression of miR-135b-5p in human gastric cancer tissue samples compared with normal tissue samples. Furthermore, we found that miR-135b-5p is also up-regulated in gastric tumors from the trefoil factor 1-knockout mouse model. Infection with H. pylori induced the expression of miR-135b-5p in the in vitro and in vivo models. miR-135b-5p induction was mediated by NF-κB. Treatment of gastric cancer cells with TNF-α induced miR-135b-5p in a NF-κB-dependent manner. Mechanistically, we found that miR-135b-5p targets Krüppel-like factor 4 (KLF4) and binds to its 3' UTR, leading to reduced KLF4 expression. Functionally, high levels of miR-135b-5p suppress apoptosis and induce cisplatin resistance. Our results uncovered a mechanistic link between H. pylori infection and miR-135b-5p-KLF4, suggesting that targeting miR-135b-5p could be a potential therapeutic approach to circumvent resistance to cisplatin.-Shao, L., Chen, Z., Soutto, M., Zhu, S., Lu, H., Romero-Gallo, J., Peek, R., Zhang, S., El-Rifai, W. Helicobacter pylori-induced miR-135b-5p promotes cisplatin resistance in gastric cancer.

Molecular Alterations in the Stomach of Tff1-Deficient Mice: Early Steps in Antral Carcinogenesis.

TFF1 is a peptide of the gastric mucosa co-secreted with the mucin MUC5AC. It plays a key role in gastric mucosal protection and repair. Tff1-deficient (Tff1KO) mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas. Thus, these mice represent a model for gastric tumorigenesis. Here, we compared the expression of selected genes in Tff1KO mice and the corresponding wild-type animals (RT-PCR analyses). Furthermore, we systematically investigated the different molecular forms of Tff1 and its heterodimer partner gastrokine-2 (Gkn2) in the stomach (Western blot analyses). As a hallmark, a large portion of murine Tff1 occurs in a monomeric form. This is unexpected because of its odd number of seven cysteine residues. Probably the three conserved acid amino acid residues (EEE) flanking the 7th cysteine residue allow monomeric secretion. As a consequence, the free thiol of monomeric Tff1 could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. Furthermore, a minor subset of Tff1 forms a disulfide-linked heterodimer with IgG Fc binding protein (Fcgbp). Of special note, in Tff1KO animals a homodimeric form of Gkn2 was observed. In addition, Tff1KO animals showed strongly reduced Tff2 transcript and protein levels, which might explain their increased sensitivity to Helicobacter pylori infection.

The Tumor Suppressor TFF1 Occurs in Different Forms and Interacts with Multiple Partners in the Human Gastric Mucus Barrier: Indications for Diverse Protective Functions.

TFF1 is a protective peptide of the Trefoil Factor Family (TFF), which is co-secreted with the mucin MUC5AC, gastrokine 2 (GKN2), and IgG Fc binding protein (FCGBP) from gastric surface mucous cells. Tff1-deficient mice obligatorily develop antropyloric adenoma and about 30% progress to carcinomas, indicating that Tff1 is a tumor suppressor. As a hallmark, TFF1 contains seven cysteine residues with three disulfide bonds stabilizing the conserved TFF domain. Here, we systematically investigated the molecular forms of TFF1 in the human gastric mucosa. TFF1 mainly occurs in an unusual monomeric form, but also as a homodimer. Furthermore, minor amounts of TFF1 form heterodimers with GKN2, FCGBP, and an unknown partner protein, respectively. TFF1 also binds to the mucin MUC6 in vitro, as shown by overlay assays with synthetic 125I-labeled TFF1 homodimer. The dominant presence of a monomeric form with a free thiol group at Cys-58 is in agreement with previous studies in Xenopus laevis and mouse. Cys-58 is likely highly reactive due to flanking acid residues (PPEEEC58EF) and might act as a scavenger for extracellular reactive oxygen/nitrogen species protecting the gastric mucosa from damage by oxidative stress, e.g., H2O2 generated by dual oxidase (DUOX).

Subcellular Localization of the TFF Peptides xP1 and xP4 in the Xenopus laevis Gastric/Esophageal Mucosa: Different Secretion Modes Reflecting Diverse Protective Functions.

The TFF peptides xP1 and xP4 from Xenopus laevis are orthologs of TFF1 and TFF2, respectively. xP1 is secreted as a monomer from gastric surface mucous cells and is generally not associated with mucins, whereas xP4 is a typical secretory peptide from esophageal goblet cells, and gastric mucous neck and antral gland cells tightly associated as a lectin with the ortholog of mucin MUC6. Both TFF peptides have diverse protective functions, xP1 as a scavenger for reactive oxygen species preventing oxidative damage and xP4 as a constituent of the water-insoluble adherent inner mucus barrier. Here, we present localization studies using immunofluorescence and immunoelectron microscopy. xP1 is concentrated in dense cores of secretory granules of surface mucous cells, whereas xP4 mixes with MUC6 in esophageal goblet cells. Of note, we observe two different types of goblet cells, which differ in their xP4 synthesis, and this is even visible morphologically at the electron microscopic level. xP4-negative granules are recognized by their halo, which is probably the result of shrinkage during the processing of samples for electron microscopy. Probably, the tight lectin binding of xP4 and MUC6 creates a crosslinked mucous network forming a stabile granule matrix, which prevents shrinkage.

miR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

Tamoxifen (TAM) is a major adjuvant therapy for patients who are diagnosed with estrogen receptor-α (ER)-positive breast cancer; however, TAM resistance occurs often during treatment and the underlying mechanism is unclear. Here, we report that miR-125a-3p inhibits ERα transcriptional activity and, thus, ER+ breast cancer cell proliferation, which causes cell-cycle arrest at the G1/S stage, inducing apoptosis and suppressing tumor growth by targeting cyclin-dependent kinase 3 (CDK3) in vitro and in vivo. In addition, CDK3 and miR-125a-3p expression levels were measured in 37 cancerous tissues paired with noncancerous samples, and their expression levels were negatively associated with miR-125a-3p level. Of interest, miR-125a-3p level is down-regulated in MCF-7 TAM-resistant (TamR) cells. Of more importance, up-regulation of miR-125a-3p resensitizes MCF-7 TamR cells to TAM, which is dependent on CDK3 expression. These results suggest that miR-125a-3p can function as a novel tumor suppressor in ER+ breast cancer by targeting CDK3, which may be a potential therapeutic approach for TamR breast cancer therapy.-Zheng, L., Meng, X., Li, X., Zhang, Y., Li, C., Xiang, C., Xing, Y., Xia, Y., Xi, T. miR-125a-3p inhibits ERα transactivation and overrides tamoxifen resistance by targeting CDK3 in estrogen receptor-positive breast cancer.

Deoxynivalenol inhibits the expression of trefoil factors (TFF) by intestinal human and porcine goblet cells.

Trefoil factors (TFFs) are bioactive peptides expressed by several epithelia, including the intestine, where they regulate key functions such as tissue regeneration, barrier function and inflammation. Although food-associated mycotoxins, including deoxynivalenol (DON), are known to impact many intestinal functions, modulation of TFFs during mycotoxicosis has never been investigated. Here, we analyzed the effect of DON on TFFs expression using both human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that very low doses of DON (nanomolar range) inhibit the secretion of TFFs by human goblet cells (IC50 of 361, 387 and 243 nM for TFF1, 2 and 3, respectively) and prevent wound healing. RT-qPCR analysis demonstrated that the inhibitory effect of DON is related to a suppression of TFFs mRNA expression. Experiments conducted on porcine intestinal explants confirmed the results obtained on cells. Finally, the use of specific inhibitors of signal pathways demonstrated that DON-mediated suppression of TFFs expression mainly involved Protein Kinase R and the MAP kinases (MAPK) p38 and ERK1/2. Taken together, our results show for the first time that at very low doses, DON suppresses the expression and production of intestinal TFFs and alters wound healing. Given the critical role of TFFs in tissue repair, our results suggest that DON-mediated suppression of TFFs contributes to the alterations of intestinal integrity the caused by this toxin.

The Interaction of Helicobacter pylori with TFF1 and Its Role in Mediating the Tropism of the Bacteria Within the Stomach.

Helicobacter pylori colonises the human stomach and has tropism for the gastric mucin, MUC5AC. The majority of organisms live in the adherent mucus layer within their preferred location, close to the epithelial surface where the pH is near neutral. Trefoil factor 1 (TFF1) is a small trefoil protein co-expressed with the gastric mucin MUC5AC in surface foveolar cells and co-secreted with MUC5AC into gastric mucus. Helicobacter pylori binds with greater avidity to TFF1 dimer, which is present in gastric mucus, than to TFF1 monomer. Binding of H. pylori to TFF1 is mediated by the core oligosaccharide subunit of H. pylori lipopolysaccharide at pH 5.0-6.0. Treatment of H. pylori lipopolysaccharide with mannosidase or glucosidase inhibits its interaction with TFF1. Both TFF1 and H. pylori have a propensity for binding to mucins with terminal non-reducing α- or ß-linked N-acetyl-d-glucosamine or α-(2,3) linked sialic acid or Gal-3-SO42-. These findings are strong evidence that TFF1 has carbohydrate-binding properties that may involve a conserved patch of aromatic hydrophobic residues on the surface of its trefoil domain. The pH-dependent lectin properties of TFF1 may serve to locate H. pylori deep in the gastric mucus layer close to the epithelium rather than at the epithelial surface. This restricted localisation could limit the interaction of H. pylori with epithelial cells and the subsequent host signalling events that promote inflammation.

The TFF Peptides xP1 and xP4 Appear in Distinctive Forms in the Xenopus laevis Gastric Mucosa: Indications for Different Protective Functions.

The gastric secretory trefoil factor family (TFF) peptides xP1 and xP4 are the Xenopus laevis orthologs of mammalian TFF1 and TFF2, respectively. The aim of this study was to analyze the molecular forms of xP1 and xP4 in the X. laevis gastric mucosa by FPLC. xP1 mainly occurred in a monomeric low-molecular-mass form and only a minor subset is associated with the mucus fraction. The occurrence of monomeric xP1 is unexpected because of its odd number of cysteine residues. Probably a conserved acidic residue flanking Cys55 allows monomeric secretion. Furthermore, Cys55 is probably post-translationally modified. For the first time, we hypothesize that the free thiol of monomeric xP1-and probably also its mammalian ortholog TFF1-could have a protective scavenger function, e.g., for reactive oxygen/nitrogen species. In contrast, xP4 mainly occurs in a high-molecular-mass form and is non-covalently bound to a mucin similarly as TFF2. In vitro binding studies with radioactively labeled porcine TFF2 even showed binding to X. laevis gastric mucin. Thus, xP4 is expected to bind as a lectin to an evolutionary conserved sugar epitope of the X. laevis ortholog of mucin MUC6 creating a tight mucus barrier. Taken together, xP1 and xP4 appear to have different gastric protective functions.

A functional polymorphism in TFF1 promoter is associated with the risk and prognosis of gastric cancer.

Trefoil Factor 1 (TFF1, also named pS2), which serves as the gastrointestinal mucosal protector, is known as gastric-specific tumor suppressor gene. However, the genetic variants of TFF1 are still not well studied. In our study, we aim to explore the effects of tagging single nucleotide polymorphisms (tagSNPs) of TFF1 on risk and prognosis of gastric cancer. Seven tagSNPs of TFF1 gene were first analyzed in the discovery set, which was consisted of 753 cases and 950 cancer-free controls. Then, the validation set (940 cases and 1,042 controls) was used for further evaluation. Moreover, we also tested the relation between these tagSNPs and prognosis of gastric cancer (GC). A series of experiments were performed to investigate the underlying mechanisms. We found that rs3761376 AA in the promoter region of TFF1, could reduce the expression of TFF1 by affecting the binding affinity of estrogen receptor 1 (ESR1, ERα), and thereby increased the risk of GC (1.29, 1.08-1.53). Moreover, the rs3761376 AA genotype was also found associated with worse prognosis among patients receiving 5-FU based chemotherapy after surgery (1.71, 1.18-2.48). Further functional assays demonstrated that TFF1 could increase the chemosensitivity of 5-FU by modulating NF-κB targeted genes. These results identified the effect of rs3761376 on TFF1 expression, which accounted for the correlation with susceptibility and prognosis of GC; and this genetic variant may be a potential biomarker to predict the risk and survival of GC.

TFF1 antagonizes TIMP-1 mediated proliferative functions in gastric cancer.

Tissue inhibitor matrix metalloproteinase-1 (TIMP1) is one of four identified members of the TIMP family. We evaluated the role of TIMP1 in gastric cancer using human and mouse tissues along with gastric organoids and in vitro cell models. Using quantitative real-time RT-PCR, we detected significant overexpression of TIMP1 in the human gastric cancer samples, as compared to normal stomach samples (P < 0.01). We also detected overexpression of Timp1 in neoplastic gastric lesions of the Tff1-knockout (KO) mice, as compared to normal stomach tissues. Reconstitution of TFF1 in human gastric cancer cell lines led to a significant decrease in the mRNA expression level of TIMP1 (P < 0.05). In vitro analysis demonstrated that TIMP1 mRNA expression is induced by TNF-α and activation of NF-κB whereas inhibition of NF-κB using BAY11-7082 led to inhibition of NF-κB and downregulation of TIMP1. Western blot analysis confirmed the decrease in TIMP1 protein level following reconstitution of TFF1. By using immunofluorescence, we showed nuclear localization of NF-κB and expression of TIMP1 in gastric organoids established from the Tff1-KO stomach where reconstitution of Tff1 using recombinant protein led to a notable reduction in the expression of both NF-κB and TIMP1. Using EDU assay, as a measure of proliferating cells, we found that TIMP1 promotes cellular proliferation whereas TFF1 reconstitution leads to a significant decrease in cellular proliferation (P < 0.05). In summary, our findings demonstrate overexpression of TIMP1 in mouse and human gastric cancers through NF-kB-dependent mechanism. We also show that TFF1 suppresses NF-κB and inhibits TIMP1-mediated proliferative potential in gastric cancer.

Transcriptomic and histopathological analysis of cholangiolocellular differentiation trait in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous entity with diverse aetiologies, morphologies and clinical outcomes. Recently, histopathological distinction of cholangiolocellular differentiation (CD) of iCCA has been suggested. However, its genome-wide molecular features and clinical significance remain unclear. METHODS: Based on CD status, we stratified iCCAs into iCCA with CD (n=20) and iCCA without CD (n=102), and performed an integrative analysis using transcriptomic and clinicopathological profiles. RESULTS: iCCA with CD revealed less aggressive histopathological features compared to iCCA without CD, and iCCA with CD showed favourable clinical outcomes of overall survival and time to recurrence than iCCA without CD (P<.05 for all). Transcriptomic profiling revealed that iCCA with CD resembled an inflammation-related subtype, while iCCA without CD resembled a proliferation subtype. In addition, we identified a CD signature that can predict prognostic outcomes of iCCA (CD_UP, n=486 and CD_DOWN, n=308). iCCAs were subgrouped into G1 (positivity for CRP and CDH2, 7%), G3 (positivity for S100P and TFF1, 32%) and G2 (the others, 61%). Prognostic outcomes for overall survival (P=.001) and time to recurrence (P=.017) were the most favourable in G1-iCCAs, intermediate in G2-iCCAs and the worst in G3-iCCAs. Similar result was confirmed in the iCCA set from GSE26566 (n=68). CONCLUSIONS: CD signature was identified to predict the prognosis of iCCA. The combined evaluation of histology of CD and protein expression status of CRP, CDH2, TFF1 and S100P might help subtyping and predicting clinical outcomes of iCCA.