TIPE2 protein restrains invariant NKT activation and protects against immune-mediated hepatitis in mice.

BACKGROUND AND AIMS: Concanavalin A (ConA) administration induces a rapid and severe liver injury in mice, and invariant natural killer T (iNKT) cells are recognized to be the key effector cells in this process. However, the underlying regulatory mechanisms are not well defined. APPROACH AND RESULTS: We found that iNKT cells constitutively expressed TIPE2 (Tumor necrosis factor-α-induced protein 8-like 2, or TNFAIPL2). Genetic TIPE2 ablation strongly sensitized mice to ConA-induced hepatitis, accompanied with hyperactivation of iNKT cells. Moreover, Tipe2-/- mice were also more susceptible to α-galactosylceramide (αGalCer)-induced liver injury, with elevated serum ALT level and enhanced proinflammatory cytokine production. CD1d signaling blockade or iNKT cell elimination through antibodies reduced the effect of TIPE2 deficiency on liver injury. Mechanistic studies revealed that TIPE2 in iNKT cells functioned as a negative regulator, limiting iNKT cell activity and cytokine production through PIP3- AKT/mTOR pathway. TIPE2-mediated protection from liver injury was further validated by the administration of adeno-associated viruses expressing TIPE2, which effectively ameliorated ConA-induced hepatic injury. However, TIPE2 was dispensable in two other liver injury models, including D-GalN/LPS and APAP-induced hepatitis. CONCLUSION: Our findings reveal a new role of TIPE2 in the attenuation of iNKT cell-mediated hepatic injury. We propose that TIPE2 serves as an important regulator of immune homeostasis in the liver, and might be exploited for the therapeutic treatment of autoimmune liver diseases.

Epithelial TIPE1 Protein Guards against Colitis by Inhibiting TNF-α-Mediated Inflammation.

Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response, and IEC dysfunction has been linked to multiple inflammatory diseases, including inflammatory bowel disease. In this study, we found that a member of the TNF-α-induced protein 8 (TNFAIP8 or TIPE) family called TIPE1 is indispensable for maintaining epithelial cell barrier integrity and homeostasis under inflammatory conditions. TIPE1-deficient mice, or chimeric mice that were deficient in TIPE1 in their nonhematopoietic cells, were more sensitive to dextran sulfate sodium-induced experimental colitis; however, TIPE1 deficiency had no impact on the development of inflammation-associated and sporadic colorectal cancers. Mechanistically, TIPE1 prevented experimental colitis through modulation of TNF-α-dependent inflammatory response in IECs. Importantly, genetic deletion of both TIPE1 and its related protein TNFAIP8 in mice led to the development of spontaneous chronic colitis, indicating that both of these two TIPE family members play crucial roles in maintaining intestinal homeostasis. Collectively, our findings highlight an important mechanism by which TIPE family proteins maintain intestinal homeostasis and prevent inflammatory disorders in the gut.

Modified colonic transit test in healthy subjects and constipated patients: A triple-phase, two-center prospective study.

BACKGROUND AND AIM: Classic daily-ingestion single-film protocol using radiopaque markers for colonic transit time (CTT) is unsuitable for Chinese patients because of rapid colonic motility. A new modified method needs to be established. METHODS: The triple-phase study was performed. In Phase I, the classic protocol was assessed to evaluate its feasibility for Chinese subjects. In Phase II, a modified protocol was performed in two centers on 180 healthy subjects and 90 constipated patients to determine optimal conditions. In Phase III, the simplified protocol was validated on 90 constipated patients. RESULTS: All the subjects of the Phase I expelled more than 95% of the markers during the examination period, which means that the classic protocol is unsuitable for Chinese patients. The 20.9-h mean total CTT for healthy Chinese subjects was much faster than that seen in Western countries. As shown by Phase II, the numbers of subjects went beyond the upper limit were 22 in P1TCTT and 10 in P2TCTT (8.14% vs 3.70%, P = 0.029). The percentage of values fall outside of the measurement range of excretion ratio was around half of our study subjects (45-70%), whereas this percentage was only 3.70% using P2TCTT. The simplified protocol had a diagnostic accuracy for constipation of 0.81, with a sensitivity and specificity of 0.46 and 0.97, respectively. CONCLUSIONS: Colon movement in Chinese individuals is significantly faster than that of Western populations. The modified protocol generated in this study is appropriate for diagnosis of constipation in population with rapid colon motility.

RACO-1 modulates Hippo signalling in oesophageal squamous cell carcinoma.

Oesophageal cancer is one of the most lethal malignancies worldwide, whereas the 5-year survival is less than 20%. Although the detailed carcinogenic mechanisms are not totally clear, recent genomic sequencing data showed dysregulation of Hippo signalling could be a critical factor for oesophageal squamous cell carcinoma (ESCC) progression. Therefore, understanding of the molecular mechanisms that control Hippo signalling activity is of great importance to improve ESCC diagnostics and therapeutics. Our current study revealed RACO-1 as an inhibitory protein for YAP/TEAD axis. Depletion of RACO-1 increases the protein level of YAP and expression of YAP/TEAD target gene. Besides, RACO-1 silencing could promote ESCC cell invasion and migration, which effect could be rescued by YAP depletion in ESCC cells. Immunoprecipitation showed that RACO-1 associated with YAP and promote ubiquitination and degradation of YAP at k48 poly-ubiquitination site. Our research discovered a new regulator of Hippo signalling via modulating YAP stability. RACO-1 could be a promising factor, which serves cancer diagnostics and therapeutics in ESCC patients.

RNF168 facilitates proliferation and invasion of esophageal carcinoma, possibly via stabilizing STAT1.

Oesophageal cancer ranks as one of the most common malignancy in China and worldwide. Although genome-wide association studies and molecular biology studies aim to elucidate the driver molecules in oesophageal cancer progression, the detailed mechanisms remain to be identified. Interestingly, RNF168 (RING finger protein 168) shows a high frequency of gene amplification in oesophageal cancer from TCGA database. Here, we report an important function for RNF168 protein in supporting oesophageal cancer growth and invasion by stabilizing STAT1 protein. RNF168 gene is amplified in oesophageal cancer samples, which tends to correlate with poor prognosis. Depletion RNF168 causes decreased cell proliferation and invasion in oesophageal cancer cells. Through unbiased RNA sequencing in RNF168 depleted oesophageal cancer cell, we identifies JAK-STAT pathway is dramatically decreased. Depletion RNF168 reduced JAK-STAT target genes, such as IRF1, IRF9 and IFITM1. Immuno-precipitation reveals that RNF168 associates with STAT1 in the nucleus, stabilizing STAT1 protein and inhibiting its poly-ubiquitination and degradation. Our study provides a novel mechanism that RNF168 promoting JAK-STAT signalling in supporting oesophageal cancer progression. It could be a promising strategy to target RNF168 for oesophageal cancer treatment.

Maslinic acid activates mitochondria-dependent apoptotic pathway in cardiac carcinoma.

PURPOSE: Cardiac carcinoma is the most common subtype of gastric cancer and its incidence has increased in recent years. The current chemotherapeutic drugs exhibit limited effectiveness and significant side effects in patients. Maslinic acid (MA) exerts an anti-tumor activity on a wide range of cancers and has no significant side effect; however, the anti-tumor effect of MA on cardiac carcinoma has not yet been explored. METHODS: MTT assays, tumor xenograft animal model, immunoblotting, MMP assessment and flow cytometry were performed in this study. RESULTS: MA was able to suppress the viability of cardiac carcinoma cells in both a time- and dose-dependent manner. This natural compound exhibited no cytotoxicity in normal cells. Its inhibitory effect on tumor growth was further confirmed in a mouse model. Mechanistically, MA induced the activation of p38 MAPK in cardiac carcinoma cells and, in turn, changed their mitochondrial membrane potential (MMP). Finally, caspase cascades were activated by a series of cleavages, leading to apoptosis in cardiac cancer cells. Inhibition of p38 MAPK signaling was able to rescue the effect of MA on cardiac carcinoma cells. CONCLUSION: Our data demonstrated that natural compound, MA, suppressed the growth of cardiac carcinoma by inducing apoptosis via the p38 MAPK/mitochondria/caspase pathway. MA and its derivatives may be promising anti-tumor agents for cardiac carcinoma treatment in the future. (Supplemental Figures available here.).

Prediction of methylation status using WGS data of plasma cfDNA for multi-cancer early detection (MCED).

BACKGROUND: Cell-free DNA (cfDNA) contains a large amount of molecular information that can be used for multi-cancer early detection (MCED), including changes in epigenetic status of cfDNA, such as cfDNA fragmentation profile. The fragmentation of cfDNA is non-random and may be related to cfDNA methylation. This study provides clinical evidence for the feasibility of inferring cfDNA methylation levels based on cfDNA fragmentation patterns. We performed whole-genome bisulfite sequencing and whole-genome sequencing (WGS) on both healthy individuals and cancer patients. Using the information of whole-genome methylation levels, we investigated cytosine-phosphate-guanine (CpG) cleavage profile and validated the method of predicting the methylation level of individual CpG sites using WGS data. RESULTS: We conducted CpG cleavage profile biomarker analysis on data from both healthy individuals and cancer patients. We obtained unique or shared potential biomarkers for each group and built models accordingly. The modeling results proved the feasibility to predict the methylation status of single CpG sites in cfDNA using cleavage profile model from WGS data. CONCLUSION: By combining cfDNA cleavage profile of CpG sites with machine learning algorithms, we have identified specific CpG cleavage profile as biomarkers to predict the methylation status of individual CpG sites. Therefore, methylation profile, a widely used epigenetic biomarker, can be obtained from a single WGS assay for MCED.

The Hippo-YAP signaling pathway drives CD24-mediated immune evasion in esophageal squamous cell carcinoma via macrophage phagocytosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies in the world with poor prognosis. Despite the promising applications of immunotherapy, the objective response rate is still unsatisfactory. We have previously shown that Hippo/YAP signaling acts as a powerful tumor promoter in ESCC. However, whether Hippo/YAP signaling is involved in tumor immune escape in ESCC remains largely unknown. Here, we show that YAP directly activates transcription of the "don't eat me" signal CD24, and plays a crucial role in driving tumor cells to avoid phagocytosis by macrophages. Mechanistically, YAP regulates CD24 expression by interacting with TEAD and binding the CD24 promoter to initiate transcription, which facilitates tumor cell escape from macrophage-mediated immune attack. Our animal model data and clinical data show that YAP combined with CD24 in tumor microenvironment redefines the impact of TAMs on the prognosis of ESCC patients which will provide a valuable basis for precision medicine. Moreover, treatment with YAP inhibitor altered the distribution of macrophages and suppressed tumorigenesis and progression of ESCC in vivo. Together, our study provides a novel link between Hippo/YAP signaling and macrophage-mediated immune escape, which suggests that the Hippo-YAP-CD24 axis may act as a promising target to improve the prognosis of ESCC patients. A proposed model for the regulatory mechanism of Hippo-YAP-CD24-signaling axis in the tumor-associated macrophages mediated immune escape.

Effect of Xeroderma pigmentosum complementation group F polymorphisms and H.pylori infection on the risk of gastric cancer.

OBJECTIVE: We conducted a case-control study by genotyping three potential functional SNPs to assess the association of Xeroderma pigmentosum complementation group F (XPF) polymorphisms with gastric cancer susceptibility, and role of XPF polymorphisms in combination with H.pylori infection in the risk of gastric cancer. METHODOLOGY: A hospital case-control study was conducted. A total of 331 patients with gastric cancer and 355 controls were collected. Three SNPs of XPF, XPF rs180067, rs1799801 and rs2276466, were genotyped by Taqman real-time PCR method with a 7900 HT sequence detector system. RESULTS: The gastric cancer patients were more likely to have smoking habit, a family history of cancer and H.pylori infection. We did not find the significant difference in the genotype distributions of XPF rs180067, rs1799801 and rs2276466 between cases and controls. Multivariate logistic analysis showed a non-significant decreased risk in patients carrying rs180067 G allele, rs1799801 T allele or rs2276466 T allele genotypes. The stratification by H.pylori infection was not significantly different in polymorphisms of XPF rs180067, rs1799801 and rs2276466. CONCLUSION: There was no evidence that polymorphisms in rs180067, rs1799801 and rs2276466 significantly affect the risk of gastric cancer. Further large sample size studies are strongly needed to validate their association.

Relationship between various Chinese medicine types and T-cell subsets in patients with ulcerative colitis.

OBJECTIVE: To investigate the relationship between various Chinese medicine (CM) types and T-cell subsets (CD4(+) and CD8(+)) in the colonic mucous membranes of patients with ulcerative colitis (UC). METHODS: Fifty UC patients were enrolled, after differentiation into four types by CM syndromes, i.e., the internal heat-damp accumulation type (IHDA), the qi-stagnancy with blood stasis type (QSBS), the Pi-Shen yang-deficiency type (PSYD) and the yin-blood deficiency type (YBD). From every patient, 3-5 pieces of intestinal mucous membrane tissues were taken through colonoscopy to determine the levels of the T-cell subsets (CD4(+) and CD8(+)) using immunohistochemical method. The results were compared with those in the normal control. RESULTS: The level of CD8(+)increased and the ratio of CD4(+)/CD8(+)decreased mainly in colonic mucous membranous tissues in UC patients. The level of CD4(+)decreased significantly in IHDA types (P<0.01), but decreased only slightly in the PSYD, QSBS and YBD types. CD8(+)increased significantly in the IHDA types (P<0.01), but only slightly in the other three types. CONCLUSION: The IHDA types of UC are closely related with T-cell subsets. The difference of T-cell subsets in various IHDA types of UC patients has provided a theoretical basis for syndrome differentiation in the CM typing of UC.

PLCE1 Promotes Esophageal Cancer Cell Progression by Maintaining the Transcriptional Activity of Snail.

Esophageal cancer is among the most deadly malignant diseases. However, the genetic factors contributing to its occurrence are poorly understood. Multiple studies with large clinic-based cohorts revealed that variations of the phospholipase C epsilon (PLCE1) gene were associated with esophageal cancer susceptibility. However, the causative role of PLCE1 in esophageal cancer is not clear. We inactivated the functional alleles of PLCE1 by CRISPR/Cas9 genome editing technology. The resultant PLCE1 inactivated cells were analyzed both in vitro and in vivo. Our results showed that loss of PLCE1 dramatically decreased the invasion and proliferation capacity of esophageal carcinoma cells in vitro. Moreover, such PLCE1 inactivated tumor grafts exhibited significantly decreased tumor size in mice. We found that PLCE1 was required to maintain protein level of snail a key transcription factor responsible for invasion. Our further transcriptomic data revealed that deficient cells were significantly decreased in expression of genes enriched as targets of Snail. Strikingly, recovery of Snail protein at least partially rescued the invasion and proliferation capacity in PLCE1 inactivated cells. In ESCC clinical specimens, PLCE1 was correlated with tumor stage (P<.0001). Interestingly, PLCE1 expression was positively correlated Snail by immunohistochemistry in such specimens (P<.0001). Therefore, our functional experiments showed the essential roles of PLCE1 in esophageal carcinoma cells and provided evidences that targeting PLCE1 and its downstream molecules could be effective therapies for esophageal cancer.

Downregulation of microRNA-382 is associated with poor outcome of esophageal squamous cell carcinoma.

AIM: To study the potential prognostic role of microRNA-382 (miR-382) in esophageal squamous cell carcinoma (ESCC). METHODS: Forty six patients were divided into 2 groups according to postoperative survival time: the poor outcome group (28 patients), who showed early metastasis but no recurrence, and died within 1 year after surgery, 12 patients of the group received postoperative chemotherapy treatment that was given after early metastasis happening; the good outcome group (18 patients), who had no clinical metastasis and recurrence, and survived 5 years or more after surgery, all patients did not receive any postoperative treatment. Total RNA was extracted from the patients' formalin-fixed and paraffin-embedded esophageal cancer tissues. miR-382 level was evaluated using high-throughput real-time quantitative polymerase chain reaction analysis. The correlation between miR-382 level and clinicopathologic features was analyzed through COX regression model, and Kaplan-Meier analysis was used to analyze the relationship between miR-382 level and patient survival time. RESULTS: miR-382 was differentially expressed in the two groups. Overall the average miR-382 level in the ESCC patients with good outcome was 9.8 ± 3.8, while miR-382 level in the ESCC patients with poor outcome was 3.0 ± 0.8. The differences of miR-382 levels between two groups were significant (P < 0.05). Kaplan-Meier analysis results showed that miR-382 expression level generally had a significant reverse-correlation with ESCC patient survival time (P < 0.001), in which the patients with higher expressions of miR-382 had a longer survival time either among individuals with the same tumor stage or among the overall patients. CONCLUSION: miR-382 levels are reverse-correlated with ESCC poor outcomes, suggesting that miR-382 could be a potential predictive biomarker for both prognosis and treatment of ESCC.

microRNA-195-Cdc42 axis acts as a prognostic factor of esophageal squamous cell carcinoma.

BACKGROUND AND PURPOSE: Previous studies observed the downregulation of microRNA (miR)-195 in esophageal squamous cell carcinoma (ESCC) tissues, confirmed cell division cycle 42 (Cdc42) as one target gene of miR-195, and demonstrated that miR-195 may act as a tumor suppressor in ESCC by regulating Cdc42 expression. This study aimed to explore the association of miR-195 and Cdc42 combined expression with clinicopathologic factors and prognosis. METHODS: Expression of miR-195 and Cdc42 mRNA in 98 pairs of ESCC and paracancerous tissues were detected using real-time quantitative RT-PCR. RESULTS: miR-195 downregulation and Cdc42 upregulation were both prevalent in ESCC tissues, and negatively correlated with each other. In addition, miR-195 expression negatively correlated with TNM stage (P=0.008) and lymphatic metastasis (P=0.022), while Cdc42 expression positively correlated with TNM stage (P=0.011) and tumor differentiation (P=0.024). Moreover, combined expression of miR-195 and Cdc42 (miR-195/Cdc42) was found to be prognostic indicators for progression-free survival and overall survival of ESCC patients both in univariate and multivariate analyses. CONCLUSION: The main findings of this study indicate the involvement of miR-195-Cdc42 axis in the progression of ESCC and suggest that the combined aberrant expression of miR-195 and Cdc42 mRNA can serve as a promising unfavorable prognostic biomarker in ESCC.