Selenobaculum gbiensis gen. nov. sp. nov., a new bacterium isolated from the gut microbiota of a patient with Crohn's disease.

The human gut microbiota is a complex ecology comprising approximately 10 to 100 trillion microbial cells. Most of the bacteria detected by 16s rRNA sequencing have yet to be cultured, but intensive attempts to isolate the novel bacteria have improved our knowledge of the gut microbiome composition and its roles within human host. In our culturomics study, a novel gram-negative, motile, obligately anaerobic, rod-shaped bacteria, designated as strain ICN-92133T, was isolated from a fecal sample of a 26-year-old patient with Crohn's disease. Based on the 16s rRNA sequence of strain ICN-92133T, the phylogeny analysis placed the strain into the family Selenomonadaceae, showing 93.91% similarity with the closely related Massilibacillus massiliensis strain DSM 102838T. Strain ICN-92133T exhibited a genome size of 2,679,003 bp with a GC content of 35.5% which was predicted to contain 26 potential virulence factors and five antimicrobial resistance genes. In comparative genomic analysis, strain ICN-92133T showed digital DNA-DNA Hybridization and OrthoANI values lower than 21.9% and 71.9% with the closest type strains, respectively. In addition, comparing phenotypic, biochemical, and cellular fatty acids with those of closely related strains revealed the distinctiveness of strain ICN-92133T. Based on the taxonogenomic results, strain ICN-92133T is proposed as a novel species belonging to a new genus. Therefore, we suggest the name of the new genus Selenobaculum gen. nov. within the family Selenomonadaceae and strain ICN-92133T (= KCTC 25622T = JCM 36070T) as a type strain of new species Selenobaculum gbiensis sp. nov.

PRRX1 is a master transcription factor of stromal fibroblasts for myofibroblastic lineage progression.

Although stromal fibroblasts play a critical role in cancer progression, their identities remain unclear as they exhibit high heterogeneity and plasticity. Here, a master transcription factor (mTF) constructing core-regulatory circuitry, PRRX1, which determines the fibroblast lineage with a myofibroblastic phenotype, is identified for the fibroblast subgroup. PRRX1 orchestrates the functional drift of fibroblasts into myofibroblastic phenotype via TGF-ß signaling by remodeling a super-enhancer landscape. Such reprogrammed fibroblasts have myofibroblastic functions resulting in markedly enhanced tumorigenicity and aggressiveness of cancer. PRRX1 expression in cancer-associated fibroblast (CAF) has an unfavorable prognosis in multiple cancer types. Fibroblast-specific PRRX1 depletion induces long-term and sustained complete remission of chemotherapy-resistant cancer in genetically engineered mice models. This study reveals CAF subpopulations based on super-enhancer profiles including PRRX1. Therefore, mTFs, including PRRX1, provide another opportunity for establishing a hierarchical classification system of fibroblasts and cancer treatment by targeting fibroblasts.

Validation of ZMYND8 as a new treatment target in hepatocellular carcinoma.

BACKGROUND: ZMYND8 (Zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8) has been known to play an important role in tumor regulation in various types of cancer. However, the results of ZMYND8 expression and their clinical significance in hepatocellular carcinoma (HCC) have not yet been published. In the present study, we investigate the expression of ZMYND8 protein and mRNA in HCC and elucidate its prognostic significance. METHODS: ZMYND8 protein and mRNA expression in 283 and 234 HCCs were investigated using immunohistochemistry and microarray gene expression profiling data. The relationships between ZMYND8 expression with clinicopathologic features and prognosis of HCC patients were evaluated. Furthermore, we performed the invasion, migration, apoptosis, soft agar formation assay and sphere formation assay in HCC cell lines, and evaluated tumorigenicity in a nude mouse model, after ZMYND8 knockdown. RESULTS: Overexpression of ZMYND8 protein and mRNA was observed in 20.5% and 26.9% of HCC cases, respectively. High ZMYND8 expression showed significant correlations with microvascular invasion, high Edmondson grade, advanced American Joint Committee on Cancer, and increased alpha-fetoprotein level. ZMYND8 mRNA overexpression was an independent prognostic factor for predicting early recurrence as well as short recurrence-free survival (RFS). Downregulation of ZMYND8 reduced migration and invasion of HCC cells, and promoted apoptosis of HCC cells in an in vitro model. In a xenograft nude mouse model, knockdown of ZMYND8 significantly reduced tumor growth. CONCLUSION: ZMYND8 mRNA overexpression could be a prognostic marker of shorter RFS in HCC patients after curative resection. ZMYND8 might play an important role in the proliferation and progression of HCC and could be a promising candidate for targeted therapy.

Longitudinal Microbiome Analysis in a Dextran Sulfate Sodium-Induced Colitis Mouse Model.

The role of the gut microbiota in the pathogenesis of inflammatory bowel disease (IBD) has been in focus for decades. Although metagenomic observations in patients/animal colitis models have been attempted, the microbiome results were still indefinite and broad taxonomic presumptions were made due to the cross-sectional studies. Herein, we conducted a longitudinal microbiome analysis in a dextran sulfate sodium (DSS)-induced colitis mouse model with a two-factor design based on serial DSS dose (0, 1, 2, and 3%) and duration for 12 days, and four mice from each group were sacrificed at two-day intervals. During the colitis development, a transition of the cecal microbial diversity from the normal state to dysbiosis and dynamic changes of the populations were observed. We identified genera that significantly induced or depleted depending on DSS exposure, and confirmed the correlations of the individual taxa to the colitis severity indicated by inflammatory biomarkers (intestinal bleeding and neutrophil-derived indicators). Of note, each taxonomic population showed its own susceptibility to the changing colitis status. Our findings suggest that an understanding of the individual susceptibility to colitis conditions may contribute to identifying the role of the gut microbes in the pathogenesis of IBD.

Validation of ORAOV1 as a new treatment target in hepatocellular carcinoma.

PURPOSE: Chromosome 11q13.2, which contains genes cyclin D1 (CCND1), fibroblast growth factor 19 (FGF19), and Oral Cancer Overexpressed 1 (ORAOV1), is the most highly amplified peak in hepatocellular carcinoma (HCC). CCND1 and FGF19 have been already suggested as therapeutic targets of HCC, but the role of ORAOV1 in carcinogenesis of HCCs has not been reported. METHODS: This retrospective study investigated ORAOV1 expression using immunohistochemistry performed on tissue microarray blocks obtained from 259 HCC patients with curative resection, between 2000 and 2006. We assessed the prognostic significance of ORAOV1 expression by Kaplan-Meier method with log-rank test and Cox proportional hazards model. Also, we performed invasion, migration, apoptosis, and cell cycle assays in HCC cell lines, and evaluated the tumorigenicity of HCC xenografts in nude mice, after knockdown of ORAOV1. RESULTS: High expression of ORAOV1 protein was observed in 80% of HCC tissues. The ORAOV1 high expression group showed shorter recurrence free survival (RFS) (p < 0.001) and shorter disease-specific survival (DSS) than the low expression group. It was an independent prognostic factor for predicting early recurrence [Odds ratio 2.74 (95% confidence interval (CI) 1.27-5.93), p = 0.01] as well as short RFS [hazard ratio 2.23 (95% CI 1.40-3.54), p = 0.001] and DSS [hazard ratio 2.30 (95% CI 1.27-4.17), p = 0.006]. Knockdown of ORAOV1 induced significant decreases in migration, invasion, and tumorigenicity of HCC cells in in-vitro model, and inhibited the growth of HCC xenografts in nude mice. CONCLUSION: We demonstrated unfavorable prognostic effect of ORAOV1 expression with supporting experimental data in HCC. ORAOV1 may be used as a biomarker for predicting HCC prognosis and is a potential candidate for targeted therapy.

Anti-Inflammatory and Gut Microbiota Modulatory Effect of Lactobacillus rhamnosus Strain LDTM 7511 in a Dextran Sulfate Sodium-Induced Colitis Murine Model.

Inflammatory bowel disease (IBD) is a group of conditions involving chronic relapsing-remitting inflammation of the gastrointestinal tract with an unknown etiology. Although the cause-effect relationship between gut microbiota and IBD has not been clearly established, emerging evidence from experimental models supports the idea that gut microbes play a fundamental role in the pathogenesis of IBD. As microbiome-based therapeutics for IBD, the beneficial effects of probiotics have been found in animal colitis models and IBD patients. In this study, based on the dextran sulfate sodium (DSS)-induced colitis mouse model, we investigated Lactobacillus rhamnosus strain LDTM 7511 originating from Korean infant feces as a putative probiotic strain for IBD. The strain LDTM 7511 not only alleviated the release of inflammatory mediators, but also induced the transition of gut microbiota from dysbiotic conditions, exhibiting the opposite pattern in the abundance of DSS colitis-associated bacterial taxa to the DSS group. Our findings suggest that the strain LDTM 7511 has the potential to be used as a probiotic treatment for IBD patients in comparison to L. rhamnosus GG (ATCC 53103), which has been frequently used for IBD studies.

New screening system using Twist1 promoter activity identifies dihydrorotenone as a potent drug targeting cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in tumor microenvironments. These cells strongly support tumor progression and are considered to be potent therapeutic targets. Therefore, drugs targeting CAFs have been developed, but most of them have failed in clinical trials. The discovery of additional drugs to inactivate or eliminate CAFs is thus essential. In this study, we developed a high-throughput screening system to find anti-CAF drugs using reporter cells that express Twist1 promoter-GFP. This screening system uses the activity of the Twist1 promoter as an indicator of CAF activation because Twist1 is known to be a central player in CAF activation. Using this screening system, we found that dihydrorotenone (DHR), an inhibitor of electron transfer chain complex 1 in mitochondria, can effectively deactivate CAFs. DHR-treated CAFs exhibited reduced expression of CAF-enriched markers, decreased capability of collagen gel contraction, and impaired ability to engage in tumor-promoting activities, such as facilitating the proliferation and colonization of cancer cells. Furthermore, conditioned media from DHR-treated CAFs attenuated tumor progression in mice grafted with MNK28 cells. In conclusion, DHR can be considered as a candidate drug targeting CAFs.

Ubiquitin-Specific Protease 21 Promotes Colorectal Cancer Metastasis by Acting as a Fra-1 Deubiquitinase.

Fos-related-antigen-1 (Fra-1), a member of the activator protein-1 (AP-1) transcription factor superfamily, has an essential role in cancer progress and metastasis and Fra-1 is considered a therapeutic target in metastatic cancer including metastatic colorectal cancer (mCRC). However, its regulation at protein level has not yet been clearly elucidated. We found that ubiquitin-specific protease 21 (USP21) increases Fra-1 stability by deubiquitinating Fra-1 and enhances the expression of Fra-1 target genes in colon cancer cells. We also showed that USP21 controlled Fra-1-dependent migration and invasion activities. The oncogenic property of USP21 was confirmed by a significant reduction in liver metastasis when USP21-knockdown cancer cells were injected intrasplenically into mice. Consistently, clinicopathological analysis of colorectal cancer patients revealed a correlation of USP21 expression with high-grade carcinoma and life span. These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1. Therefore, USP21 is considered an attractive therapeutic target in mCRC with high Fra-1 expression.

A positive feedback loop bi-stably activates fibroblasts.

Although fibroblasts are dormant in normal tissue, they exhibit explosive activation during wound healing and perpetual activation in pathologic fibrosis and cancer stroma. The key regulatory network controlling these fibroblast dynamics is still unknown. Here, we report that Twist1, a key regulator of cancer-associated fibroblasts, directly upregulates Prrx1, which, in turn, increases the expression of Tenascin-C (TNC). TNC also increases Twist1 expression, consequently forming a Twist1-Prrx1-TNC positive feedback loop (PFL). Systems biology studies reveal that the Twist1-Prrx1-TNC PFL can function as a bistable ON/OFF switch and regulates fibroblast activation. This PFL can be irreversibly activated under pathologic conditions, leading to perpetual fibroblast activation. Sustained activation of the Twist1-Prrx1-TNC PFL reproduces fibrotic nodules similar to idiopathic pulmonary fibrosis in vivo and is implicated in fibrotic disease and cancer stroma. Considering that this PFL is specific to activated fibroblasts, Twist1-Prrx1-TNC PFL may be a fibroblast-specific therapeutic target to deprogram perpetually activated fibroblasts.

Determination of Optimized Growth Medium and Cryoprotective Additives to Enhance the Growth and Survival of Lactobacillus salivarius.

The beneficial effects of lactic acid bacteria (LAB) have been intensively investigated in recent decades with special focus on modulation of the host intestinal microbiota. Numerous discoveries of effective probiotics are driven by a significantly increasing demand for dietary supplements. Consequently, technological advances in the large-scale production and lyophilization are needed by probiotic-related industries for producing probiotic LAB for commercial use. Our study had a dual objective, to determine the optimum growth medium composition and to investigate appropriate cryoprotective additives (CPAs) for Lactobacillus salivarius, and compare its responses with other Lactobacillus species. The one-factor-at-a-time method and central composite design were applied to determine the optimal medium composition for L. salivarius cultivation. The following composition of the medium was established (per liter): 21.64 g maltose, 85 g yeast extract, 1.21 ml Tween 80, 6 g sodium acetate, 0.2 g MgSO4∙7H2O, 0.02 g MnSO4∙H2O, 1 g K2HPO4, 1.5 g KH2PO4, 0.01 g FeSO4∙7H2O, and 1 g sodium citrate. A cryoprotective additive combination comprising 10% (w/v) skim milk and 10% (w/v) sucrose supplemented with 2.5% (w/v) sodium glutamate was selected for L. salivarius, and its effectiveness was confirmed using culture-independent methods in the freeze-dried cells of the Lactobacillus strains. In conclusion, the optimized medium enhanced the species-specific cultivation of L. salivarius. On the other hand, the cryoprotective effects of the selected CPA mixture may also be dependent on the bacterial strain. This study highlights the necessity for precise and advanced processing techniques for large-scale production of probiotics in the food and feed industries.

Drug repurposing screening identifies bortezomib and panobinostat as drugs targeting cancer associated fibroblasts (CAFs) by synergistic induction of apoptosis.

Cancer associated fibroblasts (CAFs) are the most abundant components of cancer-microenvironment. They play important roles in cancer initiation, progression, and metastasis. In addition, CAFs can confer drug-resistance to cancer cells. Considering their pro-tumorigenic roles, it is recommended to remove CAFs to prevent cancer recurrence after chemotherapy. Despite their clinical significance, few anti-CAF drugs have been developed. The objective of this study was to find a drug that could suppress the viability of patient-derived CAFs through repurposed screening of 51 drugs that were in clinical trials or received FDA approval. As a result, bortezomib (BTZ), carfilzomib (CFZ), and panobinostat (PST) were identified as anti-CAF drug candidates. It was confirmed that BTZ and PST could decrease the viability of various patients derived CAFs through inducing of caspase-3 mediated apoptosis. Interestingly, combination therapy with BTZ and PST showed better efficacy of inhibiting CAFs than single treatment. The synergistic effect between BTZ and PST on viability of CAFs was observed both in vitro CAF culture and in vivo mouse model. Furthermore, combination therapy with BTZ/PST and conventional anticancer compound docetaxel strongly inhibited tumor growth in xenografts of mouse breast cancer cells with mouse CAFs. In conclusion, our present study revealed that BTZ and PST could significantly reduce the viability of CAFs. Therefore, a combination therapy with BTZ/PST and anticancer drugs might be considered as a new rational for the development of anticancer therapy.

Twist1 is highly expressed in cancer-associated fibroblasts of esophageal squamous cell carcinoma with a prognostic significance.

Cancer-associated fibroblasts (CAFs) play important roles in cancer progression. Twist1 was recently reported to be a key regulator of CAFs in gastric cancer, but its role in other types of cancer remains unclear, especially for esophageal squamous cell carcinoma (ESCC). We assessed the Twist1 expression on stromal fibroblasts using immunohistochemistry in 169 tissue specimens from ESCC patients, and performed in vitro and in vivo experiments to confirm the role of Twist1 in CAFs of ESCC. And we investigated the biological pathways that are activated in Twist1-high ESCC using The Cancer Genome Atlas (TCGA) data. The expression of Twist1 in stromal fibroblasts was observed in 89.9% of ESCC patients and positively associated with the increased depth of tumor invasion, lymph node metastasis, and advanced clinical stage, and a significant adverse prognostic factor in overall survival. Twist1-expressing stromal fibroblasts also expressed representative CAF markers, and co-localization of Twist1 and CAF markers were confirmed by confocal immunofluorescence imaging. Bioinformatic analysis of mRNA expression data of esophageal cancer from TCGA revealed that gene sets of CAFs were highly enriched in Twist1-high ESCC. Depletion of Twist1 in ex vivo cultured ESCC CAFs induced significant decrease in migration, invasion, colony formation, sphere formation, and contractibility of ESCC cancer cells compared to control CAFs. Furthermore, Twist1-expressing fibroblasts remarkably enhanced the in vivo tumorigenicity of ESCC in a xenograft model. In conclusion, Twist1 could be a novel CAF marker for the prognostic evaluation of ESCC patients as well as a potent therapeutic target for ESCC.

Autoinducer-2 Quorum Sensing Influences Viability of Escherichia coli O157:H7 under Osmotic and In Vitro Gastrointestinal Stress Conditions.

Bacteria use autoinducer molecules to communicate both at intra-species and inter-species levels by quorum sensing. One such cell density-dependent signaling system is the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2). Virulence of several pathogens is determined by an AI-2 system and is related to colonization and infection of the host. From this concept, numerous papers have suggested that AI-2 inhibition is an important strategy toward designing of new antimicrobial agents. However, recent studies indicate that the AI-2 system is also involved in adaptation and survival under environmental stress conditions. Therefore, we hypothesized that interaction between quorum sensing and environmental conditions may be critical in influencing predicted results in a control and when combating of target pathogens. We investigated the growth of enterohemorrhagic Escherichia coli O157:H7 (EHEC) and its luxS-deficient (non AI-2 producing) mutant strain under various stress conditions, and found significant differences in the growth rate under osmotic stress. Moreover, we could also show the impact of the AI-2 molecule on viability in the gastrointestinal tract model representing a complex environmental condition. Differences in vital responses of the strains suggest that AI-2 quorum sensing has a significant influence on the viability of EHEC under environmental stress conditions.

Development of putative probiotics as feed additives: validation in a porcine-specific gastrointestinal tract model.

Enforced restrictions on the use of antibiotics as growth promoters (AGPs) in animal production have prompted investigations into alternative feed additives in recent decades. Probiotics are currently the main feed additive used in livestock. However, the selection of probiotic candidates relies on human-based methods and little is known about the verification criteria for host-specific selection. We investigated the probiotic potential of Lactobacillus salivarius strains isolated from fed pig feces for their use as porcine feed additives. Two methods were developed that simulated the pig gastrointestinal (GI) tract and the intestinal epithelium, and these were compared with human-based in vitro methods and used for selecting porcine probiotics. Lactobacillus salivarius strain LS6 was identified as a promising probiotic strain for potential use as a porcine feed additive. This strain prevented disruption of the epithelial integrity of pig small intestine (PSI) cells by inhibiting the adherence of enterotoxigenic Escherichia coli K88. It also showed high survival rates in the in vitro pig GI tract model and good adhesion to PSI cells. We propose that host target-specific screening and validation methods are important tools in the development of effective probiotic feed additives, and this approach may support future-oriented agriculture.

Tenascin-C, a Prognostic Determinant of Esophageal Squamous Cell Carcinoma.

BACKGROUND: Tenascin-C, an adhesion modulatory extracellular matrix molecule, is highly expressed in numerous human malignancies; thus, it may contribute to carcinogenesis and tumor progression. We explored the clinicopathological significance of Tenascin-C as a prognostic determinant of esophageal squamous cell carcinoma (ESCC). METHODS: In ESCC patient tissues and cell lines, the presence of isoforms were examined using western blotting. We then investigated Tenascin-C immunohistochemical expression in 136 ESCC tissue samples. The clinical relevance of Tenascin-C expression and the correlation between Tenascin-C expression and expression of other factors related to cancer-associated fibroblasts (CAFs) were also determined. RESULTS: Both 250 and 350 kDa sized isoforms of Tenascin-C were expressed only in esophageal cancer tissue not in normal tissue. Furthermore, both isoforms were also identified in all of four CAFs derived from esophageal cancer tissues. Tenascin-C expression was remarkably higher in ESCC than in adjacent non-tumor esophageal epithelium (p < 0.001). Tenascin-C expression in ESCC stromal fibroblasts was associated with patient's age, tumor (pT) stage, lymph node metastasis, clinical stage, and cancer recurrence. Tenascin-C expression in cancer cells was correlated with an increase in tumor-associated macrophage (TAM) population, cancer recurrence, and hypoxia inducible factor1α (HIF1α) expression. Moreover, Tenascin-C overexpression in cancer cells and stromal fibroblasts was an independent poor prognostic factor for overall survival (OS) and disease-free survival (DFS). In the Cox proportional hazard regression model, Tenascin-C overexpression in cancer cells and stromal fibroblasts was a significant independent hazard factor for OS and DFS in ESCC patients in both univariate and multivariate analyses. Furthermore, Tenascin-C expression in stromal fibroblasts of the ESCC patients was positively correlated with platelet-derived growth factor α (PDGFRα), PDGFRß, and smooth muscle actin (SMA) expression. The 5-year OS and DFS rates were remarkably lower in patients with positive expressions of both Tenascin-C and PDGFRα (p < 0.001), Tenascin-C and PDGFRß (p < 0.001), Tenascin-C and SMA (p < 0.001), Tenascin-C and fibroblast activation protein (FAP) (p < 0.001), and Tenascin-C and fibroblast-stimulating protein-1 (FSP1) (p < 0.001) in ESCC stromal fibroblasts than in patients with negative expressions of both Tenascin-C and one of the abovementioned CAF markers. CONCLUSION: Our results show that Tenascin-C is a reliable and significant prognostic factor in ESCC. Tenascin-C may thus be a potent ESCC therapeutic target.

Influence of gastrointestinal stress on autoinducer-2 activity of two Lactobacillus species.

Quorum sensing is a bacterial communication signalling system that regulates the expression of certain target genes with autoinducers in a cell density-dependent manner. The universal luxS-mediated quorum sensing using the autoinducer-2 (AI-2) signal is present in a wide variety of bacteria with only sparse information on probiotic lactobacilli. Effective probiotics should exhibit tolerance and adaptation to stress conditions typical of the GIT. Adhesion to human intestinal epithelial cells and competitive exclusion of pathogens are also considered important. The AI-2 signal system plays an important role in the response of probiotic lactobacilli to the surrounding environment. Intraspecies-related changes in quorum signalling in the GIT were determined by monitoring the AI-2 activity of two strains each of Lactobacillus rhamnosus and L. plantarum under various stress conditions. Modulation of the AI-2 activity of all the strains was induced by stress responses to pH, bile acid, temperature, osmotic pressure and starvation, and was both species- and strain-specific. AI-2 inhibition correlated with a reduction in the stress-related genes of L. rhamnosus. We therefore suggest that AI-2 quorum signalling of probiotic lactobacilli may represent one way of adapting to the host's ecosystem and of interacting within the intestinal environment.

Twist1 is a key regulator of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAF) are key contributors to malignant progression, but their critical regulators remain largely unknown. In this study, we examined the role of Twist1, a central regulator of epithelial-mesenchymal transition in carcinoma cells, in the transdifferentiation of normal quiescent fibroblasts to CAF and we defined its upstream controls and downstream effectors. Primary human gastric fibroblast and CAF cultures were established from gastrectomy specimens and validated as nontumor cells by somatic mutation analyses. In these cultures, exposure to the proinflammatory cytokine IL6 commonly expressed in tumors was sufficient to induce Twist1 expression in normal fibroblasts and transdifferentiate them into CAFs via STAT3 phosphorylation. In xenograft models, tumor infiltration of Twist1-expressing CAFs was enhanced strongly by ectopic IL6 expression in gastric or breast cancer cells. We found that Twist1 expression was necessary and sufficient for CAF transdifferentiation. Enforced expression of Twist1 in normal fibroblasts was also sufficient to drive CAF marker expression and malignant character in gastric cancer cells both in vitro and in vivo. Conversely, silencing the expression of Twist1 in CAFs abrogated their tumor-promoting properties. Downstream of Twist1, we defined the chemokine CXCL12 as a transcriptional target. Clinically, CXCL12 and Twist1 expression were correlated in CAFs present in gastric tumor specimens. Finally, ectopic expression of Twist1 in normal fibroblasts suppressed premature senescence, whereas Twist1 attenuation accelerated senescence in CAFs. Our findings define Twist1 as a compelling target to deprogram the tumor-supporting features of the cancer microenvironment.

ZNF282 (Zinc finger protein 282), a novel E2F1 co-activator, promotes esophageal squamous cell carcinoma.

Zinc finger protein 282 (ZNF282) is a newly identified transcription factor and little is known about its expression and function. Originally, ZNF282 is known to bind U5RE (U5 repressive element) of HLTV-1 (human T cell leukemia virus type 1) with a repressive effect. Recently we reported that ZNF282 functions as an estrogen receptor co-activator and plays an essential role in breast tumorigenesis. Although these results suggest the possible role of ZNF282 in cancers, clinical significance and function of ZNF282 are completely unknown in most of cancers. Here we found that ZNF282 was frequently overexpressed in esophageal squamous cell carcinoma (ESCC) (n=165) compared with normal esophageal epithelium and its overexpression was correlated with adverse clinical outcome. Multivariate survival analysis indicated that ZNF282 expression was an independent prognostic predictor for poor survival in ESCC (HR: 2.56 (95% CI 1.54-4.26), p<0.001). In addition, depletion of ZNF282 inhibited the cell cycle progression, migration, and invasion of ESCC cells and reduced the tumorigenicity of ESCC xenograft in nude mouse. We further showed that ZNF282 is required for E2F1-mediated gene expression in ESCC cells. Thus, ZNF282 is E2F1 co-activator involved in ESCC and elevated expression of ZNF282 is an independent adverse prognostic factor in ESCC.

Selection of functional lactic acid bacteria as starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler ex Sprengel.).

The purpose of this research was to find safe and suitable starter cultures for the fermentation of Korean leek (Allium tuberosum Rottler), also known as garlic chives or Oriental garlic. This traditional herb has several functional properties and a strong flavour; its leaves are used as food material. Eighteen strains of lactic acid bacteria (LAB) were isolated from well-fermented leek kimchi. Controlled fermentation of the leek leaves was conducted with 2 strains (Weissella confusa LK4 and Lactobacillus plantarum LK8), selected as potential starter cultures on the basis of their safety properties, and on the pH, total titratable acidity (TTA), and viable cell numbers [colony forming units (CFUml(-1))] achieved during the fermentation. Microbial dynamics was also followed during fermentation by using PCR-DGGE (Denaturing Gradient Gel Electrophoresis) on DNA level. To analyse bioactive compounds such as thiols and allicin (diallyl thiosulfinates), the total flavonoid and polyphenolic contents were determined by colorimetric methods. Functional properties were assessed on the basis of anti-oxidative capacities by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect, and ferric reducing antioxidant power (FRAP). W. confusa LK4 rapidly increased during the first stage of leek fermentation, and was mainly responsible for accelerated fermentation during the early period in contrast to L. plantarum LK8, a stronger acid producer during the later stages of fermentation. After 48 h fermentation, leeks fermented with W. confusa LK4 showed the highest radical scavenging effects and reducing ability. The detectable amount of allicin of fermented leeks decreased relative to the change in pH, whereas the concentration of thiols significantly increased. Total flavonoid and poly-phenolic contents changed during fermentation and showed correlation with anti-oxidant effects. We therefore suggest the suitability of W. confusa LK4 as a potential starter culture for fermentation of leeks.

The prognostic significance of cancer-associated fibroblasts in esophageal squamous cell carcinoma.

BACKGROUND: Cancer-associated fibroblasts (CAF) are activated fibroblasts in the cancer stroma and play an important role in cancer progression. Some reports have indicated the correlation between the expression of CAF markers and adverse prognosis in several cancers. However, no reports have studied CAF phenotype and its clinical relevance in esophageal squamous cell carcinoma (ESCC). METHODS: We investigated CAF phenotype of ESCC based on histology and immunohistochemical expressions of five CAF markers such as fibroblast activation protein (FAP), smooth muscle actin (SMA), fibroblast-specific protein-1 (FSP1), platelet-derived growth factor receptor (PDGFRα), and PDGFRß in 116 ESCC tissue samples. Besides, we also examined the correlation of the CAF phenotype with clinical relevance as well as other cancer-microenvironment related factors. RESULTS: Histologically immature CAF phenotype was correlated with poor prognosis (p<0.001) and associated with increased microvessel density, increased tumor associated macrophages, and epithelial to mesenchymal transition. CAF markers were characteristically expressed in stromal fibroblast close to tumor cells and the expression pattern of 5 CAF markers was highly heterogeneous in every individual cases. Of five CAF markers, SMA, FSP1, and PDGFRα were unfavorable prognostic indicators of ESCC. The number of positive CAF markers was greater in ESCC with immature CAFs than in those with mature ones. CONCLUSIONS: Our results demonstrate that histologic classification of CAF phenotype is a reliable and significant prognostic predictor in ESCC. CAF markers have the potential to be diagnostic and therapeutic targets in ESCC.