Targeting cancer-associated fibroblast-secreted WNT2 restores dendritic cell-mediated antitumour immunity.

OBJECTIVE: Solid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC). DESIGN: Anti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses. RESULTS: A negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades. CONCLUSIONS: CAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

RNA editing of SLC22A3 drives early tumor invasion and metastasis in familial esophageal cancer.

Like many complex human diseases, esophageal squamous cell carcinoma (ESCC) is known to cluster in families. Familial ESCC cases often show early onset and worse prognosis than the sporadic cases. However, the molecular genetic basis underlying the development of familial ESCC is mostly unknown. We reported that SLC22A3 is significantly down-regulated in nontumor esophageal tissues from patients with familial ESCC compared with tissues from patients with sporadic ESCCs. A-to-I RNA editing of the SLC22A3 gene results in its reduced expression in the nontumor esophageal tissues of familial ESCCs and is significantly correlated with lymph node metastasis. The RNA-editing enzyme ADAR2, a familial ESCC susceptibility gene identified by our post hoc genome-wide association study, is positively correlated with the editing level of SLC22A3 Moreover, functional studies showed that SLC22A3 is a metastasis suppressor in ESCC, and deregulation of SLC22A3 facilitates cell invasion and filopodia formation by reducing its direct association with α-actinin-4 (ACTN4), leading to the increased actin-binding activity of ACTN4 in normal esophageal cells. Collectively, we now show that A-to-I RNA editing of SLC22A3 contributes to the early development and progression of familial esophageal cancer in high-risk individuals.

Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis.

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V(H)H) library raised against TcdB, we identified two V(H)H antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD50) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells.

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-ß1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-ß1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

Roles and therapeutic implications of m6A modification in cancer immunotherapy.

Recent studies have demonstrated that N6-methyladenosine (m6A), the most abundant, dynamic, and reversible epigenetic RNA modification in eukaryotes, is regulated by a series of enzymes, including methyltransferases (writers), demethylases (erasers), and m6A recognition proteins (readers). Aberrant regulation of m6A modification is pivotal for tumorigenesis, progression, invasion, metastasis, and apoptosis of malignant tumors. Immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, as recognized by the 2018 Nobel Prize in Medicine and Physiology. However, not all cancer patients response to ICI therapy, which is thought to be the result of intricate immune escape mechanisms. Recently, numerous studies have suggested a novel role for m6A epigenetic modification in the regulation of tumor immune evasion. Herein, we review the relevant mechanisms of m6A regulators in regulating various key signaling pathways in cancer biology and how m6A epigenetic modifications regulate the expression of immune checkpoints, opening a new window to understand the roles and mechanisms of m6A epigenetic modifications in regulating tumor immune evasion. In addition, we highlight the prospects and development directions of future combined immunotherapy strategies based on m6A modification targeting, providing directions for promoting the treatment outcomes of immune checkpoint inhibitors.

Microfluidics-Enabled Nanovesicle Delivers CD47/PD-L1 Antibodies to Enhance Antitumor Immunity and Reduce Immunotoxicity in Lung Adenocarcinoma.

The CD47/PD-L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune-related adverse events (IRAEs) caused by the on-target off-tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics-enabled nanovesicle using ultra-pH-sensitive polymer mannose-poly(carboxybetaine methacrylate)-poly(hydroxyethyl piperidine methacrylate) (Man-PCB-PHEP) is developed to deliver CD47/PD-L1 antibodies (NCPA) for tumor-acidity-activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow-derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD-L1 antibodies accumulation, promoted tumor-associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated.

NOTCH4ΔL12_16 sensitizes lung adenocarcinomas to EGFR-TKIs through transcriptional down-regulation of HES1.

Resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI) remains one of the major challenges in lung adenocarcinoma (LUAD) therapy. Here, we find an increased frequency of the L12_16 amino acid deletion mutation in the signal peptide region of NOTCH4 (NOTCH4ΔL12_16) in EGFR-TKI-sensitive patients. Functionally, exogenous induction of NOTCH4ΔL12_16 in EGFR-TKI -resistant LUAD cells sensitizes them to EGFR-TKIs. This process is mainly mediated by the reduction of the intracellular domain of NOTCH4 (NICD4) caused by the NOTCH4ΔL12_16 mutation, which results in a lower localization of NOTCH4 in the plasma membrane. Mechanistically, NICD4 transcriptionally upregulates the expression of HES1 by competitively binding to the gene promoter relative to p-STAT3. Because p-STAT3 can downregulate the expression of HES1 in EGFR-TKI-resistant LUAD cells, the reduction of NICD4 induced by NOTCH4ΔL12_16 mutation leads to a decrease in HES1. Moreover, inhibition of the NOTCH4-HES1 pathway using inhibitors and siRNAs abolishes the resistance of EGFR-TKI. Overall, we report that the NOTCH4ΔL12_16 mutation sensitizes LUAD patients to EGFR-TKIs through transcriptional down-regulation of HES1 and that targeted blockade of this signaling cohort could reverse EGFR-TKI -resistance in LUAD, providing a potential approach to overcome resistance to EGFR-TKI -therapy.

WNT2-SOX4 positive feedback loop promotes chemoresistance and tumorigenesis by inducing stem-cell like properties in gastric cancer.

Gastric cancer (GC) is characterized by its vigorous chemoresistance to current therapies, which is attributed to the highly heterogeneous and immature phenotype of cancer stem cells (CSCs) during tumor initiation and progression. The secretory WNT2 ligand regulates multiple cancer pathways and has been demonstrated to be a potential therapeutic target for gastrointestinal tumors; however, its role involved in gastric CSCs (GCSCs) remains unclear. Here, we found that overexpression of WNT2 enhanced stemness properties to promote chemoresistance and tumorigenicity in GCSCs. Mechanistically, WNT2 was positively regulated by its transcription factor SOX4, and in turn, SOX4 was upregulated by the canonical WNT2/FZD8/ß-catenin signaling pathway to form an auto-regulatory positive feedback loop, resulting in the maintenance of GCSCs self-renewal and tumorigenicity. Furthermore, simultaneous overexpression of both WNT2 and SOX4 was correlated with poor survival and reduced responsiveness to chemotherapy in clinical GC specimens. Blocking WNT2 using a specific monoclonal antibody significantly disrupted the WNT2-SOX4 positive feedback loop in GCSCs and enhanced the chemotherapeutic efficacy when synergized with the chemo-drugs 5-fluorouracil and oxaliplatin in a GCSC-derived mouse xenograft model. Overall, this study identified a novel WNT2-SOX4 positive feedback loop as a mechanism for GCSCs-induced chemo-drugs resistance and suggested that the WNT2-SOX4 axis may be a potential therapeutic target for gastric cancer treatment.

Retracted: Apoptosis of CT26 colorectal cancer cells induced by Clostridium difficile toxin A stimulates potent anti-tumor immunity.

Clostridium difficile toxin A (TcdA) is one of the main pathogenic factors released by C. difficile. Due to its potent cytotoxic and proinflammatory activities, we investigated the anti-tumor activity of TcdA. CT26 colorectal cancer cells were challenged with recombinant TcdA, and it was found that TcdA could induce apoptosis of CT26 cells. Calreticulin (CRT) exposure to the cell surface during TcdA-induced apoptosis suggested that this apoptosis may correlate with immunogenicity. Moreover, TcdA-treated apoptotic CT26 cells were highly immunogenic since they could stimulate DC activation, T-cell activation, and anti-tumor activity. Furthermore, the anti-tumor immune response generated was specific and long-term. In summary, these studies demonstrate that C. difficile toxin A can induce apoptotic death of CT26 colorectal cancer cells and stimulate potent anti-tumor immunity.

Biochemical characterization of human peroxiredoxin 2, an antioxidative protein.

Human peroxiredoxin 2 (Prx2), which is abundant in erythrocytes, has been shown to play a key role in protecting erythrocytes against oxidative stress by scavenging reactive oxygen species as well as participating in cell signal transduction. Here, human Prx2 gene was successfully cloned into Escherichia coli BL21 (DE3) for Prx2 expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis suggested that the recombinant protein was expressed mainly in a soluble form. The recombinant protein was purified by one-step Ni-nitrilotriacetic acid chelating affinity chromatography to a purity of up to 91.5%. The peroxidase activity of Prx2 to scavenge H(2)O(2) was determined by a ferrithiocyanate assay. The ability of Prx2 to protect plasmid DNA was tested by using a mixed-function oxidation system, and results showed that Prx2 could prevent DNA from undergoing oxidative stress. Ultraviolet (UV)-induced cell apoptosis assay demonstrated that Prx2 is also able to protect NIH/3T3 cells from UV-induced damage, suggesting its possible applications in cosmetics and other areas.

Targeting TROY-mediated P85a/AKT/TBX3 signaling attenuates tumor stemness and elevates treatment response in hepatocellular carcinoma.

BACKGROUND: Previous in vitro hepatocyte differentiation model showed that TROY was specifically expressed in liver progenitor cells and a small proportion of hepatocellular carcinoma cells, suggesting that TROY may participate in hepatocellular carcinoma (HCC) stemness regulation. Here, we aim to investigate the role and mechanism of TROY in HCC pathogenesis. METHOD: Bioinformatics analysis of the TCGA dataset has been used to identify the function and mechanism of TROY. Spheroid, apoptosis, and ALDH assay were performed to evaluate the stemness functions. Validation of the downstream pathway was based on Western blot, co-immunoprecipitation, and double immunofluorescence. RESULTS: HCC tissue microarray study found that a high frequency of TROY-positive cells was detected in 53/130 (40.8%) of HCC cases, which was significantly associated with poor prognosis and tumor metastasis. Functional studies revealed that TROY could promote self-renewal, drug resistance, tumorigenicity, and metastasis of HCC cells. Mechanism study found that TROY could interact with PI3K subunit p85α, inducing its polyubiquitylation and degradation. The degradation of p85α subsequently activate PI3K/AKT/TBX3 signaling and upregulated pluripotent genes expression including SOX2, NANOG, and OCT4, and promoted EMT in HCC cells. Interestingly, immune cell infiltration analysis found that upregulation of TROY in HCC tissues was induced by TGF-ß1 secreted from CAFs. PI3K inhibitor wortmannin could effectively impair tumor stemness to sorafenib. CONCLUSION: We demonstrated that TROY is an HCC CSC marker and plays an important role in HCC stemness regulation. Targeting TROY-positive CSCs with PI3K inhibitor wortmannin combined with chemo- or targeted drugs might be a novel therapeutic strategy for HCC patients.

Downregulation of MTAP promotes Tumor Growth and Metastasis by regulating ODC Activity in Breast Cancer.

5'-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway and has been reported to suppress tumorigenesis. The MTAP gene is located at 9p21, a chromosome region often deleted in breast cancer (BC). However, the clinical and biological significance of MTAP in BC is still unclear. Here, we reported that MTAP was frequently downregulated in 41% (35/85) of primary BCs and 89% (8/9) of BC cell lines. Low expression of MTAP was significantly correlated with a poor survival of BC patients (P=0.0334). Functional studies showed that MTAP was able to suppress both in vitro and in vivo tumorigenic ability of BC cells, including migration, invasion, angiogenesis, tumor growth and metastasis in nude mice with orthotopic xenograft tumor of BC. Mechanistically, we found that downregulation of MTAP could increase the polyamine levels by activating ornithine decarboxylase (ODC). By treating the MTAP-repressing BC cells with specific ODC inhibitor Difluoromethylornithine (DFMO) or treating the MTAP-overexpressing BC cells with additional putrescine, metastasis-promoting or -suppressing phenotype of these MTAP-manipulated cells was significantly reversed, respectively. Taken together, our data suggested that MTAP has a critical metastasis-suppressive role by tightly regulating ODC activity in BC cells, which may serve as a prominent novel therapeutic target for advanced breast cancer treatment.

MAPK10 Expression as a Prognostic Marker of the Immunosuppressive Tumor Microenvironment in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains a devastating malignancy worldwide due to lack of effective therapy. The immune-rich contexture of HCC tumor microenvironment (TME) makes this tumor an appealing target for immune-based therapies; however, the immunosuppressive TME is still a major challenge for more efficient immunotherapy in HCC. Using bioinformatics analysis based on the TCGA database, here we found that MAPK10 is frequently down-regulated in HCC tumors and significantly correlates with poor survival of HCC patients. HCC patients with low MAPK10 expression have lower expression scores of tumor infiltration lymphocytes (TILs) and stromal cells in the TME and increased scores of tumor cells than those with high MAPK10 expression. Further transcriptomic analyses revealed that the immune activity in the TME of HCC was markedly reduced in the low-MAPK10 group of HCC patients compared to the high-MAPK10 group. Additionally, we identified 495 differentially expressed immune-associated genes (DIGs), with 482 genes down-regulated and 13 genes up-regulated in parallel with the decrease of MAPK10 expression. GO enrichment and KEGG pathway analyses indicated that the biological functions of these DIGs included cell chemotaxis, leukocyte migration and positive regulation of the response to cytokine-cytokine receptor interaction, T cell receptor activation and MAPK signaling pathway. Protein-protein interaction (PPI) analyses of the 495 DIGs revealed five potential downstream hub genes of MAPK10, including SYK, CBL, VAV1, LCK, and CD3G. Several hub genes such as SYK, LCK, and VAV1 could respond to the immunological costimulatory signaling mediated by the transmembrane protein ICAM1, which was identified as a down-regulated DIG associated with low-MAPK10 expression. Moreover, ectopic overexpression or knock-down of MAPK10 could up-regulate or down-regulate ICAM1 expression via phosphorylation of c-jun at Ser63 in HCC cell lines, respectively. Collectively, our results demonstrated that MAPK10 down-regulation likely contributes to the immunosuppressive TME of HCC, and this gene might serve as a potential immunotherapeutic target and a prognostic factor for HCC patients.

WNT2-Mediated FZD2 Stabilization Regulates Esophageal Cancer Metastasis via STAT3 Signaling.

Esophageal cancer micro environment factor WNT2 was critical in cancer metastasis. However, very little is known about WNT2 receptors and their role in the malignant progression of ESCC. The clinical significance and underlying molecular mechanisms of FZD2, one of the receptors of WNT2, was further investigated in ESCC. We found that FZD2 expression was positively correlated with WNT2 levels in clinical ESCC specimens through database analysis. Upregulated FZD2 expression was detected in 69% (69/100) of the primary ESCC cases examined, and increased FZD2 expression was significantly correlated with poor prognosis (P < 0.05). Mechanistically, FZD2 induced the migration and invasion of ESCC cells by regulating the FZD2/STAT3 signaling. In vivo xenograft experiments further revealed the metastasis-promoting role of FZD2 in ESCC. Moreover, we found that the WNT2 ligand could stabilize and phosphorylate the FZD2 receptor by attenuating FZD2 ubiquitination, leading to the activation of STAT3 signaling and the initiation of ESCC cell metastasis. Collectively, our data revealed that a novel non-canonical WNT2/FZD2/STAT3 signaling axis is critical for ESCC progression. Strategies targeting this specific signaling axis might be developed to treat patients with ESCC.

The immune landscape of esophageal cancer.

Esophageal cancer (EC) seriously threatens human health, and a promising new avenue for EC treatment involves cancer immunotherapy. To improve the efficacy of EC immunotherapy and to develop novel strategies for EC prognosis prediction or clinical treatment, understanding the immune landscapes in EC is required. EC cells harbor abundant tumor antigens, including tumor-associated antigens and neoantigens, which have the ability to initiate dendritic cell-mediated tumor-killing cytotoxic T lymphocytes in the early stage of cancer development. As EC cells battle the immune system, they obtain an ability to suppress antitumor immunity through immune checkpoints, secreted factors, and negative regulatory immune cells. Cancer-associated fibroblasts also contribute to the immune evasion of EC cells. Some factors of the immune landscape in EC tumor microenvironment are associated with cancer development, patient survival, or treatment response. Based on the immune landscape, peptide vaccines, adoptive T cell therapy, and immune checkpoint blockade can be used for EC immunotherapy. Combined strategies are required for better clinical outcome in EC. This review provides directions to design novel and effective strategies for prognosis prediction and immunotherapy in EC.

Therapeutic targeting of the crosstalk between cancer-associated fibroblasts and cancer stem cells.

Cancer-associated fibroblasts (CAFs) play critical roles in cancer progression and treatment failure. CAFs display extreme phenotypic heterogeneity and functional diversity. Some subpopulations of CAFs have the ability to reconstitute cancer stemness by promoting the expansion of cancer stem cells (CSCs) or by inducing the generation of CSCs from differentiated cancer cells. CAFs regulate cancer stemness in different types of solid tumors by activating a wide array of CSC-related signaling by secreting proteins and exosomes. As feedback, the CSCs can also induce the proliferation and further activation of CAFs to promote their CSC-supporting activities, thus completing the loop of CAF-CSC crosstalk. Current research on targeting CAF-CSC crosstalk could be classified into (i) specific depletion of CAF subpopulations that have CSC-supporting activities and (ii) targeting molecular signaling in CAF-CSC crosstalk, such as the IL6/STAT3, TGF-ß/SDF-1/PI3K, WNT/ß-catenin, HGF/cMET and SHH/Hh pathways. Strategies targeting CAF-CSC crosstalk may open new avenues for overcoming cancer progression and therapeutic resistance.

Epigenetic alterations of a novel antioxidant gene SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

Esophageal squamous cell carcinoma (ESCC) occurs with the highest frequency in China, especially in the high-risk Northern Chinese. Recent studies have reported that SLC22A3 is significantly downregulated in non-tumor (NT) esophageal tissues from familial ESCC patients compared with those from sporadic ESCC. However, the mechanism of how SLC22A3 regulates familial ESCC remains unknown. In this study, post hoc genome-wide association studies (GWAS) in 496 cases with a family history of upper gastrointestinal tract cancers and 1056 controls were performed and the results revealed that SLC22A3 is a novel susceptibility gene for familial ESCC. Reduced expression of SLC22A3 in NT esophageal tissues from familial ESCC patients significantly correlates with its promoter hypermethylation. Moreover, case-control study of Chinese descendants from different risk areas of China revealed that the methylation of the SLC22A3 gene in peripheral blood leukocyte (PBL) DNA samples could be a risk factor for developing ESCC in this high-risk population. Functional studies showed that SLC22A3 is a novel antioxidant gene, and deregulation of SLC22A3 facilitates heat stress-induced oxidative DNA damage and formation of γ-H2AX foci in normal esophageal epithelial cells. Collectively, we show that epigenetic alterations of SLC22A3 predispose susceptible individuals to increased risk of esophageal cancer.

FZD7 is a novel prognostic marker and promotes tumor metastasis via WNT and EMT signaling pathways in esophageal squamous cell carcinoma.

Frizzled (FZD) proteins are receptors for secreted WNT proteins and play a critical role in the malignant progression of various cancers. However, the role of human FZD family members in esophageal squamous cell carcinoma (ESCC) was rarely investigated. In this study, we found that the FZD7 gene was the most commonly up-regulated FZD member in ESCC cell lines compared with other FZDs. TMA studies further validated that FZD7 protein was up-regulated in 165 of 252 (65.5%) informative ESCC patients and significantly correlated with poor overall survival (P=0.001). Additionally, multivariate Cox regression analysis showed that FZD7 overexpression was an independent prognostic factor for ESCC patients. Ectopic expression of FZD7 could promote ESCC cell metastasis both in vitro and in vivo. Under WNT3A stimulation, FZD7 was able to induce the nuclear translocation of ß-catenin and activate the downstream targets of WNT/ß-catenin signaling, as well as promote epithelial-mesenchymal transition (EMT) potential in ESCC cells. Our study demonstrated for the first time that FZD7 contributes to the malignant progression of ESCC and represents a novel prognostic marker and a potential therapeutic target for ESCC patients.

Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis.

Clostridium difficile TcdB harbors a glucosyltransferase that targets host Rho GTPases. However, the role of the enzyme activity in the induction of host intestinal disease has not been demonstrated. In this study, we established a mouse acute intestinal disease model by cecum injection of wild type and glucosyltransferase-deficient TcdB and a chronic model by delivering toxin intraluminally via engineered surrogate host Bacillus megaterium. We demonstrated, for the first time, that the glucosyltransferase activity of TcdB is essential for inducing disease symptoms and intestinal pathological responses that resemble human disease, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.