A Diagnostic Dilemma: Metastatic Neuroendocrine Tumor Mimicking Hepatocellular Carcinoma.

Carcinoid syndrome arises from neuroendocrine tumors, characterized by the presence of neurosecretory granules. The diagnosis of carcinoid syndrome involves biochemical testing and various imaging techniques. We report the case of a 62-year-old man with Parkinson's Disease who was found to have new-onset cirrhosis and multiple hepatic lesions with necrosis on CT imaging. These findings were concerning for metastatic malignancy of unknown primary origin. Subsequent MRI characterization of the liver lesions indicated hepatocellular carcinoma as the most likely diagnosis. However, a transthoracic echocardiogram, performed for anasarca and dyspnea on exertion, revealed a thickened tricuspid leaflet, highly suspicious for carcinoid valvulitis. A biopsy of one of the hepatic lesions was consistent with neuroendocrine tumor, confirming the diagnosis of carcinoid syndrome. This case highlights the limitations of diagnostic imaging approaches in distinguishing hepatocellular carcinoma from neuroendocrine tumors.

Betulinic acid alleviates neuropathic pain induced by chronic constriction injury of the sciatic nerve in mice.

Neuropathic pain refers to a type of pain that arises from primary damage and dysfunction within the nervous system. Addressing this condition presents significant challenges and complexities. Betulinic acid (BA), known for its potent antioxidative and anti-inflammatory properties, has garnered extensive attention; nevertheless, the impact upon neuropathic pain induced by CCI is still uncertain. This paper explores the analgesic effects concerning BA on mice experiencing neuropathic pain due to sciatic nerve injury. Throughout the experiment, mice with CCI received oral gavage of BA at dosages of 3, 10, and 30 mg/kg for consecutively 8 days from the 7th day post-surgery. To assess their responses, behavioral tests and sciatic functional index (SFI) evaluations were conducted on zeroth, seventh, eighth, tenth, twelveth and fourteenth day post-CCI. On day 14, histopathological examinations and measurements of biochemical markers were performed. Immunofluorescence techniques were employed to detect Nrf2 and glial cell activation, while the Western blot method was utilized to evaluate Nrf2/HO-1 protein levels and pro-inflammatory cytokine expression. The results elucidated that BA significantly alleviated hyperalgesia and allodynia, demonstrating a dose-dependent enhancement in sciatic nerve function and facilitating the recovery of sciatic nerve injury. Furthermore, BA prominently augmented the entire antioxidative capacity (T-AOC) and T-SOD levels, concomitantly reducing MDA concentrations. Notably, BA activated the Nrf2/HO-1 signaling pathway, inhibited glial cell activation, and downregulation of the expression levels of pro-inflammatory cytokines, specifically, TNF-α, IL-1ß, and IL-6 were observed. As such, this study provides a basis to support BA as a candidate drug for the treatment of neuropathic pain, attributing its analgesic effects to its anti-inflammatory, antioxidative, and neuroprotective properties.

Downregulation of SODD mediates carnosol-induced reduction in cell proliferation in esophageal adenocarcinoma cells.

Esophageal adenocarcinoma carries a poor prognosis associated with a 5-year survival rate of 12.5-20%. Therefore, a new therapeutic modality is needed for this lethal tumor. Carnosol is a phenolic diterpene purified from the herbs such as rosemary and Mountain desert sage and has been shown to have anticancer activities in multiple cancers. In this study we examined the effect of carnosol on cell proliferation in esophageal adenocarcinoma cells. We found that carnosol dose-dependently decreased cell proliferation in FLO-1 esophageal adenocarcinoma cells and significantly increased caspase-3 protein, indicating that carnosol decreases cell proliferation and increases cell apoptosis in FLO-1 cells. Carnosol significantly increased H2O2 production and N-acetyl cysteine, a reactive oxygen species (ROS) scavenger, significantly inhibited carnosol-induced decrease in cell proliferation, indicating that ROS may mediate carnosol-induced decrease in cell proliferation. Carnosol-induced decrease in cell proliferation was partially reversed by NADPH oxidase inhibitor apocynin, suggesting that NADPH oxidases may be partially involved in carnosol's effect. In addition, carnosol significantly downregulated SODD protein and mRNA expression and knockdown of SODD significantly inhibited the carnosol-induced reduction in cell proliferation, suggesting that downregulation of SODD may contribute to carnosol-induced reduction in cell proliferation. We conclude that carnosol dose-dependently decreased cell proliferation and significantly increased caspase-3 protein. Carnosol's effect may be through the overproduction of ROS and the downregulation of SODD. Carnosol might be useful for the treatment of esophageal adenocarcinoma.

Pulmonary tumor embolism: A retrospective study over a 30-year period.

BACKGROUND: Pulmonary tumor embolism (PTE) is difficult to detect before death, and it is unclear whether the discrepancy between antemortem clinical and postmortem diagnosis improves with the advance of the diagnostic technologies. In this study we determined the incidence of PTE and analyzed the discrepancy between antemortem clinical and postmortem diagnosis. METHODS: We performed a retrospective autopsy study on patients with the history of malignant solid tumors from 1990 to 2020 and reviewed all the slides of the patients with PTE. We also analyzed the discrepancies between antemortem clinical and postmortem diagnosis in 1999, 2009 and 2019 by using the Goldman criteria. Goldman category major 1 refers to cases in which an autopsy diagnosis was the direct cause of death and was not recognized clinically, but if it had been recognized, it may have changed treatment or prolonged survival. RESULTS: We found 20 (3%) cases with PTE out of the 658 autopsy cases with solid malignancies. Out of these 20 cases, urothelial carcinoma (30%, 6/20) and invasive ductal carcinoma of the breast (4/20, 20%) were the most common primary malignancies. Seven patients with shortness of breath died within 3-17 days (average 8.4±2.2 days) after onset of the symptoms. Pulmonary embolism was clinically suspected in seven out of twenty (35%, 7/20) patients before death, but only two patients (10, 2/20) were diagnosed by imaging studies before death. The rate of Goldman category major 1 was 13.2% (10/76) in 1999, 7.3% (4/55) in 2009 and 6.9% (8/116) in 2019. Although the rate of Goldman category major 1 appeared decreasing, the difference was not statistically significant. The autopsy rate was significantly higher in 2019 (8.4%, 116/1386) than in 2009 (4.4%, 55/1240). CONCLUSIONS: The incidence of PTE is uncommon. Despite the advances of the radiological techniques, radiological imaging studies did not detect the majority of PTEs. The discrepancy between the antemortem clinical and the postmortem diagnosis has not improved significantly over the past 30 years, emphasizing the value of autopsy.

Immune Microenvironment in Gallbladder Adenocarcinomas.

Programmed death-1 (PD1) expression has not been reported in gallbladder adenocarcinoma. In this study we examined PD1 expression in gallbladder cancer to explore the correlation between PD1 expression and the clinicopathologic parameters. We found that 98% (46/47) cases expressed programmed death-ligand 1 (PD-L1) with 85% cases being PD-L1 3+. PD1+ tumor-infiltrating lymphocytes (TILs) were present in 78.7% cases (37/47). The tumor size was significantly smaller and the stromal CD3+ TILs were significantly higher in tumors with PD1+ TILs than those with PD1- TILs. In the tumors with size of <3 cm, stromal CD3+ TILs >115/HPF or stromal CD8+ TILs >45/HPF were associated with much better survival than those with stromal CD3+ TILs ≤115/HPF or stromal CD8+ TILs ≤45/HPF. In tumors with the size of 3 cm or larger, PD1+ TILs or stromal CD8+ TILs >45/HPF carried a significantly poorer survival than PD1- tumors or stromal CD8+ TILs <=45/HPF. No correlation was identified between PD1 expression and lymphovascular invasion, distant metastasis, pathologic tumor stage or prognostic stage. Multivariate survival analysis showed that tumor TNM stage and age were independent prognostic factors in gallbladder adenocarcinomas. We conclude that gallbladder adenocarcinomas may have high PD-L1 expression and PD1+ TILs. Smaller tumor size and greater amount of stromal CD3+ T cells were found in tumors with PD1+ TILs. In small tumors (<3 cm), high stromal CD3+ TILs or high stromal CD8+ TILs were associated with better survival. However, in large tumors (≥3 cm), PD1+ TILs or high stromal CD8+ TILs carried a poorer survival. Our study implied that immune-based therapy including PD1/PD-L1 checkpoint blockade might be useful in gallbladder adenocarcinomas.

SHP2 regulates the development of intestinal epithelium by modifying OSTERIX+ crypt stem cell self-renewal and proliferation.

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is ubiquitously expressed and essential for the development and/or maintenance of multiple tissues and organs. SHP2 is involved in gastrointestinal (GI) epithelium development and homeostasis, but the underlying mechanisms remain elusive. While studying SHP2's role in skeletal development, we made osteoblast-specific SHP2 deficient mice using Osterix (Osx)-Cre as a driver to excise Ptpn11 floxed alleles. Phenotypic characterization of these SHP2 mutants unexpectedly revealed a critical role of SHP2 in GI biology. Mice lacking SHP2 in Osx+ cells developed a fatal GI pathology with dramatic villus hypoplasia. OSTERIX, an OB-specific zinc finger-containing transcription factor is for the first time found to be expressed in GI crypt cells, and SHP2 expression in the crypt Osx+ cells is critical for self-renewal and proliferation. Further, immunostaining revealed the colocalization of OSTERIX with OLFM4 and LGR5, two bona fide GI stem cell markers, at the crypt cells. Furthermore, OSTERIX expression is found to be associated with GI malignancies. Knockdown of SHP2 expression had no apparent influence on the relative numbers of enterocytes, goblet cells or Paneth cells. Given SHP2's key regulatory role in OB differentiation, our studies suggest that OSTERIX and SHP2 are indispensable for gut homeostasis, analogous to SOX9's dual role as a master regulator of cartilage and an important regulator of crypt stem cell biology. Our findings also provide a foundation for new avenues of inquiry into GI stem cell biology and of OSTERIX's therapeutic and diagnostic potential.

Fatal Mucormycosis and Aspergillosis in an Atypical Host: What Do We Know about Mixed Invasive Mold Infections?

Mixed invasive mold infections (MIMIs) are considered rare. We present a case of fatal aspergillosis and mucormycosis in an elderly host with history of chronic lymphocytic leukemia (CLL) and potential mold exposures. Notably, he had no classic risk factors for IMI other than high-dose corticosteroids, which may be an important risk factor for (M)IMI, based on the current and previous reports. There is an urgent need for studies on the "net state of immunosuppression," environmental exposure as risk factors for (M)IMIs, and noninvasive fungal diagnostics.

Effect of Proton Pump Inhibitor Therapy on NOX5, mPGES1 and iNOS expression in Barrett's Esophagus.

Acid reflux may contribute to the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). However, it is not clear whether the molecular changes present in BE patients are reversible after proton pump inhibitor (PPI) treatment. In this study we examined whether PPI treatment affects NOX5, microsomal prostaglandin E synthase (mPGES)-1 and inducible nitric oxide synthase (iNOS) expression. We found that NADPH oxidase 5 (NOX5), mPGES-1 and iNOS were significantly increased in BE mucosa. One-month PPI treatment significantly decreased NOX5, mPGES1 and iNOS. In BAR-T cells, NOX5 mRNA and p16 promoter methylation increased after pulsed acid treatment in a time-dependent manner. Four or eight-week-acid induced increase in NOX5 mRNA, NOX5 protein and p16 methylation may be reversible. Twelve-week acid treatment also significantly increased NOX5, mPGES1 and iNOS mRNA expression. However, twelve-week-acid-induced changes only partially restored or did not recover at all after the cells were cultured at pH 7.2 for 8 weeks. We conclude that NOX5, mPGES1 and iNOS may be reversible after PPI treatment. Short-term acid-induced increase in NOX5 expression and p16 methylation might be reversible, whereas long-term acid-induced changes only partially recovered 8 weeks after removal of acid treatment.

Utility of 15(S)-HETE as a Serological Marker for Eosinophilic Esophagitis.

The pathogenesis of eosinophilic esophagitis (EoE) involves Th2-mediated eosinophil recruitment and degranulation into the esophagus. However, measuring serum Th2 cytokines, eosinophils, and eosinophil-derived products does not reliably distinguish EoE from control populations. Non-invasive methods to diagnose EoE are lacking. We evaluated the diagnostic value of a novel candidate biomarker of EoE: 15(S)-hydroxyeicosatetraenoic acid (HETE). We used immunoassay to measure 15(S)-HETE and cytokine profiles in patients undergoing endoscopy with known or suspected EoE. 31 subjects were enrolled, 16 with EoE, and 15 with an alternate diagnosis. 15(S)-HETE was elevated in the EoE group compared to non-EoE group. The sensitivity and specificity of 15(S)-HETE to be used as a non-invasive marker is 50% and 80%, respectively. 15(S)-HETE may aid in the diagnosis of EoE.

TGR5 expression in normal kidney and renal neoplasms.

BACKGROUND: The G protein-coupled bile acid receptor (TGR5) is a cell surface receptor which induces the production of intracellular cAMP and promotes epithelial-mesenchymal transition in gastric cancer cell lines. TGR5 is found in a wide variety of tissues including the kidney. However, the patterns of TGR5 expression have not been well characterized in physiologic kidney or renal neoplasms. We explore the expression of TGR5 in benign renal tissue and renal neoplasms and assess its utility as a diagnostic marker. METHODS: Sixty-one renal cortical neoplasms from 2000 to 2014 were retrieved. TGR5 protein expression was examined by immunohistochemistry. TGR5 mRNA was also measured by real-time PCR. RESULTS: In normal renal tissue, TGR5 was strongly positive in collecting ducts, distal convoluted tubules and thin loop of Henle. Proximal convoluted tubules showed absent or focal weak staining. In clear cell renal cell carcinomas (RCCs), 25 of 27 cases (92%) were negative for TGR5 (p < 0.001). TGR5 mRNA was also significantly decreased in clear cell RCCs, suggesting that decreased TGR5 protein expression may be attributable to the downregulation of TGR5 mRNA in these tumors. All 11 papillary RCCs expressed TGR5 with 45% (5/11) exhibiting moderate to strong staining. All chromophobe RCCs and oncocytomas were positive for TGR5 with weak to moderate staining. TGR5 mRNA expression in these tumors was similar to normal kidney. All urothelial carcinomas of the renal pelvis strongly expressed TGR5 including a poorly differentiated urothelial carcinoma with sarcomatoid features. CONCLUSION: TGR5 is strongly expressed in collecting ducts, distal convoluted tubules and thin loop of Henle. TGR5 protein and mRNA expression were notably decreased in clear cell RCCs and may be helpful in differentiating these tumors from other RCCs.

TGR5 expression in benign, preneoplastic and neoplastic lesions of Barrett's esophagus: Case series and findings.

AIM: To examined the bile acid receptor TGR5 expression in squamous mucosa, Barrett's mucosa, dysplasia and esophageal adenocarcinoma (EA). METHODS: Slides were stained with TGR5 antibody. The staining intensity was scored as 1+, 2+ and 3+. The extent of staining (percentage of cells staining) was scored as follows: 1+, 1%-10%, 2+, 11%-50%, 3+, 51%-100%. A combined score of intensity and extent was calculated and categorized as negative, weak, moderate and strong staining. TGR5 mRNA was measured by real time PCR. RESULTS: We found that levels of TGR5 mRNA were significantly increased in Barrett's dysplastic cell line CP-D and EA cell line SK-GT-4, when compared with Barrett's cell line CP-A. Moderate to strong TGR5 staining was significantly higher in high-grade dysplasia and EA cases than in Barrett's esophagus (BE) or in low-grade dysplasia. Moderate to strong staining was slightly higher in low-grade dysplasia than in BE mucosa, but there is no statistical significance. TGR5 staining had no significant difference between high-grade dysplasia and EA. In addition, TGR5 staining intensity was not associated with the clinical stage, the pathological stage and the status of lymph node metastasis. CONCLUSION: We conclude that TGR5 immunostaining was much stronger in high-grade dysplasia and EA than in BE mucosa or low-grade dysplasia and that its staining intensity was not associated with the clinical stage, the pathological stage and the status of lymph node metastasis. TGR5 might be a potential marker for the progression from BE to high-grade dysplasia and EA.

Silencer-of-Death Domain Mediates Acid-Induced Decrease in Cell Apoptosis in Barrett's Associated Esophageal Adenocarcinoma Cells.

We have shown that NADPH oxidase (NOX)5-S may mediate the acid-induced decrease in cell apoptosis. However, mechanisms of NOX5-S-dependent decrease in cell apoptosis are not fully understood. In this study, we found that silencer-of-death domain (SODD) was significantly increased in esophageal adenocarcinoma (EA) tissues, EA cell lines FLO and OE33, and a dysplastic cell line CP-B. Strong SODD immunostaining was significantly higher in low-grade dysplasia (66.7%), high-grade dysplasia (81.2%), and EA (71.2%) than in Barrett's mucosa (10.5%). Acid treatment significantly increased SODD protein and mRNA expression and promoter activity in FLO cells, an increase that was significantly decreased by the knockdown of NOX5-S and nuclear factor κB (NF-κB)1 p50 with their small interfering RNAs. Similarly, acid-induced increase of SODD mRNA was blocked by knockdown of NOX5-S and p50 in a BE cell line CP-A. Overexpression of NOX5-S significantly increased SODD protein expression in FLO cells. Moreover, overexpression of NOX5-S or p50 significantly increased the SODD promoter activity and decreased the caspase 9 activity or apoptosis. NOX5-S overexpression-induced increase in SODD promoter activity was significantly decreased by knockdown of p50. In addition, acid treatment significantly decreased the caspase 9 activity, a decrease that was significantly inhibited by knockdown of SODD. Furthermore, chromatin immunoprecipitation assay showed that NF-κB1 p50 bound to SODD genomic DNA containing a NF-κB-binding element GGGGACACCCT. This binding element was further confirmed by a gel mobility shift assay. We conclude that acid-induced increase in SODD expression and decrease in cell apoptosis may depend on the activation of NOX5-S and NF-κB1 p50 in FLO cells.

Bile acid receptor TGR5, NADPH Oxidase NOX5-S and CREB Mediate Bile Acid-Induced DNA Damage In Barrett's Esophageal Adenocarcinoma Cells.

The mechanisms whereby bile acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. In this study we found that bile acid taurodeoxycholic acid (TDCA) significantly increased the tail moment (TM) and histone H2AX phosphorylation in FLO-1 EA cells, an increase which was significantly decreased by knockdown of TGR5. Overexpression of TGR5 significantly increased TDCA-induced TM increase and H2AX phosphorylation. In addition, NADPH oxidase inhibitor diphenylene iodonium significantly inhibited the TDCA-induced increase in TM and H2AX phosphorylation. TDCA-induced increase in TM and H2AX phosphorylation was significantly decreased by knockdown of NOX5-S and overexpression of NOX5-S significantly increased TDCA-induced increase in the tail moment and H2AX phosphorylation. Furthermore, TDCA significantly increased cAMP response element binding protein (CREB) phosphorylation in FLO-1 cells. Knockdown of CREB significantly decreased TDCA-induced increase in NOX5-S mRNA and the tail moment. Conversely, overexpression of CREB significantly increased TDCA-induced TM increase. We conclude that TDCA-induced DNA damage may depend on the activation of TGR5, CREB and NOX5-S. It is possible that in Barrett's patients bile acids may activate NOX5-S and increase reactive oxygen species (ROS) production via activation of TGR5 and CREB. NOX5-S-derived ROS may cause DNA damage, thereby contributing to the progression from BE to EA.

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

Mechanisms of the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

TMEFF2 deregulation contributes to gastric carcinogenesis and indicates poor survival outcome.

PURPOSE: The role and clinical implication of the transmembrane protein with EGF and two follistatin motifs 2 (TMEFF2) in gastric cancer is poorly understood. EXPERIMENTAL DESIGN: Gene expression profile analyses were performed and Gene Set Enrichment Analysis (GSEA) was used to explore its gene signatures. AGS and MKN45 cells were transfected with TMEFF2 or control plasmids and analyzed for gene expression patterns, proliferation, and apoptosis. TMEFF2 expression was knocked down with shRNAs, and the effects on genome stability were assessed. Interactions between TMEFF2 and SHP-1 were determined by mass spectrometry and immunoprecipitation assays. RESULTS: Integrated analysis revealed that TMEFF2 expression was significantly decreased in gastric cancer cases and its expression was negatively correlated with the poor pathologic stage, large tumor size, and poor prognosis. GSEA in The Cancer Genome Atlas (TCGA) and Jilin datasets revealed that cell proliferation, apoptosis, and DNA damage-related genes were enriched in TMEFF2 lower expression patients. Gain of TMEFF2 function decreased cell proliferation by increasing of apoptosis and blocking of cell cycle in gastric cancer cells. The protein tyrosine phosphatase SHP-1 was identified as a binding partner of TMEEF2 and mediator of TMEFF2 function. TMEFF2 expression positively correlated with SHP-1, and a favorable prognosis was more likely in patients with gastric cancer with higher levels of both TMEFF2 and SHP-1. CONCLUSION: TMEFF2 acts as a tumor suppressor in gastric cancer through direct interaction with SHP-1 and can be a potential biomarker of carcinogenesis.

Role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in Barrett's cells and Barrett's esophageal adenocarcinoma cells.

Mechanisms whereby acid reflux may accelerate the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. Acid and reactive oxygen species (ROS) have been reported to cause DNA damage in Barrett's cells. We have previously shown that NADPH oxidase NOX5-S is responsible for acid-induced H2O2 production in Barrett's cells and in EA cells. In this study we examined the role of intracellular calcium and NADPH oxidase NOX5-S in acid-induced DNA damage in a Barrett's EA cell line FLO and a Barrett's cell line CP-A. We found that pulsed acid treatment significantly increased tail moment in FLO and CP-A cells and histone H2AX phosphorylation in FLO cells. In addition, acid treatment significantly increased intracellular Ca(2+) in FLO cells, an increase that is blocked by Ca(2+)-free medium with EGTA and thapsigargin. Acid-induced increase in tail moment was significantly decreased by NADPH oxidase inhibitor diphenylene iodonium in FLO cells, and by blockade of intracellular Ca(2+) increase or knockdown of NOX5-S with NOX5 small-interfering RNA (siRNA) in FLO and CP-A cells. Acid-induced increase in histone H2AX phosphorylation was significantly decreased by NOX5 siRNA in FLO cells. Conversely, overexpression of NOX5-S significantly increased tail moment and histone H2AX phosphorylation in FLO cells. We conclude that pulsed acid treatment causes DNA damage via increase of intracellular calcium and activation of NOX5-S. It is possible that in BE acid reflux increases intracellular calcium, activates NOX5-S, and increases ROS production, which causes DNA damage, thereby contributing to the progression from BE to EA.

Role of C9orf140 in the promotion of colorectal cancer progression and mechanisms of its upregulation via activation of STAT5, ß-catenin and EZH2.

C9orf140 is a newly identified and characterized gene which is associated with cell proliferation and tumorigenicity. Expression of C9orf140 is upregulated in human gastric cancer and colorectal cancer (CRC); however, little is known about its role in CRC progression. We have investigated the clinical significance, biological effects and mechanisms of C9orf140 signaling. We found that the expression of C9orf140 is dramatically increased in a subset of CRC and correlates significantly with vascular invasion and lymph node metastasis. Our finding showed that knockdown of C9orf140 significantly reduced cell proliferation and invasion in vitro and dramatically increased overall survival and decreased lung metastasis in vivo. Conversely, overexpression of C9orf140 significantly increased lung metastasis and shortened overall survival when compared with control tumors. C9orf140-induced CRC cell invasion may depend on promoting the epithelial-mesenchymal transition progression. STAT5 may directly interact with the enhancer of zeste homolog 2 (EZH2) and ß-catenin to enhance C9orf140 gene transactivation. Furthermore, C9orf140 may participate in cell invasion which is induced by STAT5, EZH2 or ß-catenin activation. We describe the role of C9orf140 in CRC progression and find that C9orf140 overexpression may be regulated by STAT5, EZH2 and ß-catenin interaction.

Role of NADPH oxidase NOX5-S, NF-κB, and DNMT1 in acid-induced p16 hypermethylation in Barrett's cells.

Inactivation of tumor suppressor genes via promoter hypermethylation may play an important role in the progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA). We have previously shown that acid-induced p16 gene promoter hypermethylation may depend on activation of NADPH oxidase NOX5-S in BAR-T cells and OE33 EA cells. DNA methyltransferase 1 (DNMT1) is known to participate in maintaining established patterns of DNA methylation in dividing cells and may play an important role in the development of cancer. Therefore, we examined whether DNMT1 is involved in acid-induced p16 gene promoter hypermethylation in BAR-T cells. We found that the acid significantly increased p16 gene promoter methylation, decreased p16 mRNA, and increased cell proliferation, effects that may depend on activation of DNMT1 in BAR-T cells. DNMT1 is overexpressed in EA cells FLO and OE33 and EA tissues. Acid treatment upregulated DNMT1 mRNA expression and increased DNMT1 promoter activity. Acid-induced increases in DNMT1 mRNA expression and promoter activity were significantly decreased by knockdown of NOX5-S and NF-κB1 p50. Conversely, overexpression of NOX5-S, p50, or p65 significantly increased DNMT1 promoter activity. Knockdown of NOX5-S significantly decreased the acid-induced increase in luciferase activity in cells transfected with pNFκB-Luc. An NF-κB binding element GGGGTATCCC was identified in the DNMT1 gene promoter. We conclude that the acid-induced increase in p16 gene promoter methylation, downregulation of p16 mRNA, and increase in cell proliferation may depend on activation of DNMT1 in BAR-T cells. Acid-induced DNMT1 expression may depend on sequential activation of NOX5-S and NF-κB1 p50.

NADPH oxidase NOX5-S and nuclear factor κB1 mediate acid-induced microsomal prostaglandin E synthase-1 expression in Barrett's esophageal adenocarcinoma cells.

The mechanisms of progression from Barrett's esophagus (BE) to esophageal adenocarcinoma (EA) are not known. Cycloxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) has been shown to be important in esophageal tumorigenesis. We have shown that COX-2 mediates acid-induced PGE2 production. The prostaglandin E synthase (PGES) responsible for acid-induced PGE2 production in BE, however, is not known. We found that microsomal PGES1 (mPGES1), mPGES2, and cytosolic PGES (cPGES) were present in FLO EA cells. Pulsed acid treatment significantly increased mPGES1 mRNA and protein levels but had little or no effect on mPGES2 or cPGES mRNA. Knockdown of mPGES1 by mPGES1 small interfering RNA (siRNA) blocked acid-induced increase in PGE2 production and thymidine incorporation. Knockdown of NADPH oxidase, NOX5-S, a variant lacking calcium-binding domains, by NOX5 siRNA significantly inhibited acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Overexpression of NOX5-S significantly increased the luciferase activity in FLO cells transfected with a nuclear factor κB (NF-κB) in vivo activation reporter plasmid pNF-κB-Luc. Knockdown of NF-κB1 p50 by p50 siRNA significantly decreased acid-induced increase in mPGES1 expression, thymidine incorporation, and PGE2 production. Two novel NF-κB binding elements, GGAGTCTCCC and CGGGACACCC, were identified in the mPGES1 gene promoter. We conclude that mPGES1 mediates acid-induced increase in PGE2 production and cell proliferation. Acid-induced mPGES1 expression depends on activation of NOX5-S and NF-κB1 p50. Microsomal PGES1 may be a potential target to prevent or treat EA.

Role of STAT3 and vitamin D receptor in EZH2-mediated invasion of human colorectal cancer.

The polycomb group protein enhancer of zeste homologue 2 (EZH2), which has histone methyltransferase (HMT) activity, is overexpressed in malignant tumours. However, the role of EZH2 in colorectal cancer (CRC) invasion is little known. Here we investigated the clinical significance, biological effects, and mechanisms of EZH2 signalling. Knockdown of EZH2 significantly reduced cell invasion and secretion of matrix metalloproteinases 2/9 (MMP2/9) in in vitro studies. Knockdown of EZH2 dramatically increased overall survival and decreased metastasis of lung in in vivo studies. Conversely, overexpression of EZH2 significantly increased lung metastasis and shortened overall survival when compared with control tumours. EZH2-induced CRC cell invasion may depend on down-regulation of vitamin D receptor (VDR), which is considered to be a marker of CRC invasion. EZH2 regulates the histone trimethylation of lysine 27 (H3K27me3) in the VDR promoter. Moreover, we found that STAT3 directly binds to the EZH2 promoter and regulates VDR down-regulation in CRC cells. Significant inverse correlations were observed between the expression of EZH2 and pSTAT3 and that of VDR in CRC tissues compared with normal tissue in patients. We show the role of EZH2 in CRC metastasis and identify VDR as a target gene of EZH2. EZH2 expression may be directly regulated by STAT3, and STAT3 may play an important role in EZH2-mediated VDR down-regulation in CRC. This pathway may provide potential targets in aggressive CRC.