Glycolytic Activation of CD14+ Intestinal Macrophages Contributes to the Inflammatory Responses via Exosomal Membrane Tumor Necrosis Factor in Crohn's Disease.

BACKGROUND: Macrophage (Mφ) activation plays a critical role in the inflammatory response. Activated Mφ go through profound reprogramming of cellular metabolism. However, changes in their intracellular energy metabolism and its effect on inflammatory responses in Crohn's disease (CD) remain currently unclear. The aim of this study is to explore metabolic signatures of CD14+ Mφ and their potential role in CD pathogenesis as well as the underlying mechanisms. METHODS: CD14+ Mφ were isolated from peripheral blood or intestinal tissues of CD patients and control subjects. Real-time flux measurements and enzyme-linked immunosorbent assay were used to determine the inflammatory states of Mφ and metabolic signatures. Multiple metabolic routes were suppressed to determine their relevance to cytokine production. RESULTS: Intestinal CD14+ Mφ in CD patients exhibited activated glycolysis compared with those in control patients. Specifically, macrophagic glycolysis in CD largely induced inflammatory cytokine release. The intestinal inflammatory microenvironment in CD elicited abnormal glycolysis in Mφ. Mechanistically, CD14+ Mφ derived exosomes expressed membrane tumor necrosis factor (TNF), which engaged TNFR2 and triggered glycolytic activation via TNF/nuclear factor κB autocrine and paracrine signaling. Importantly, clinically applicable anti-TNF antibodies effectively prevented exosomal membrane TNF-induced glycolytic activation in CD14+ Mφ. CONCLUSIONS: CD14+ Mφ take part in CD pathogenesis by inducing glycolytic activation via membrane TNF-mediated exosomal autocrine and paracrine signaling. These results provide novel insights into pathogenesis of CD and enhance understanding of the mechanisms of anti-TNF agents.

TIMP1 down-regulation enhances gemcitabine sensitivity and reverses chemoresistance in pancreatic cancer.

The therapeutic effect of gemcitabine (GEM) in pancreatic ductal adenocarcinoma (PDAC) is limited due to low drug sensitivity and high drug resistance. Tissue inhibitor of matrix metalloprotease 1 (TIMP1) is reportedly associated with GEM resistance in PDAC. However, the effect of TIMP1 down-regulation in combination with GEM treatment is unknown. We analyzed the expression of TIMP1 in human PDAC tissue using western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry. TIMP1 was highly expressed in PDAC specimens. Kaplan-Meier survival analysis suggested that a higher level of TIMP1 was correlated with poorer overall survival in 103 PDAC patients. The mRNA and protein expression profiles of TIMP1 were explored in the HTERT-HPNE human pancreatic ductal epithelium cell line, five PDAC cell lines (MIA PaCa-2, PANC-1, BxPC-3, Capan2, and SW1990), and two GEM-resistant PDAC cell lines (MIA PaCa-2R and PANC-1R). Compared with HTERT-HPNE, TIMP1 was highly expressed in the PDAC cell lines. In addition, TIMP1 was upregulated in GEM-resistant PDAC cell lines compared with their parental cells. When TIMP1 was knocked-down using short hairpin RNA, GEM-induced cytotoxicity and apoptosis were increased, while colony formation was repressed in MIA PaCa-2, PANC-1, and their GEM-resistant cells. When Bax was activated by BAM7 or Bcl-2 was inhibited by venetoclax, CCK-8 assays demonstrated that GEM sensitivity was restored in GEM-resistant cells. When Bax was down-regulated by siRNA, CCK-8 assays verified that GEM sensitivity was decreased in PDAC cells. The observations that TIMP1 knockdown enhanced GEM sensitivity and reversed chemoresistance by inducing cells apoptosis indicated cooperative antitumor effects of shTIMP1 and GEM therapy on PDAC cells. The combination may be a potential strategy for PDAC therapy.

Bmi-1-induced miR-27a and miR-155 promote tumor metastasis and chemoresistance by targeting RKIP in gastric cancer.

BACKGROUND: We previously reported an inverse relationship between B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) and Raf kinase inhibitory protein (RKIP), which is associated with the prognosis of gastric cancer (GC). In this study, we further explored the microRNA (miRNA) regulatory mechanism between Bmi-1 and RKIP. METHODS: Microarray analysis was first carried out to identify miRNA profiles that were differentially expressed in cells overexpressing Bmi-1. Then, miRNAs that could regulate RKIP were identified. Quantitative real-time PCR (qRT-PCR) and Western blotting were performed to measure the expression of Bmi-1, miR-155, miR-27a and RKIP. RKIP was confirmed as a target of miR-27a and miR-155 through luciferase reporter assays, qRT-PCR and Western blotting. The effects of the Bmi-1/miR-27a/RKIP and Bmi-1/miR-155/RKIP axes on tumor growth, proliferation, migration, invasion, colony-formation ability, metastasis and chemoresistance were investigated both in vitro and in vivo. RESULTS: The downregulation of RKIP by Bmi-1 occurred at the protein but not mRNA level. This indicates probable posttranscriptional regulation. miRNA expression profiles of cells with ectopic expression of Bmi-1 were analyzed and compared to those of control cells by microarray analysis. A total of 51 upregulated and 72 downregulated miRNAs were identified. Based on publicly available algorithms, miR-27a and miR-155 were predicted, selected and demonstrated to target RKIP. Bmi-1, miR-27a and miR-155 are elevated in human GC and associated with poor prognosis of GC, while RKIP is expressed at lower levels in GC and correlated with good prognosis. Then, in vitro tests shown that in addition to regulating RKIP expression via miR-27a and miR-155, Bmi-1 was also able to regulate the migration, invasion, proliferation, colony-formation ability and chemosensitivity of GC cells through the same pathway. Finally, the in vivo test showed similar results, whereby the knockdown of the Bmi-1 gene led to the inhibition of tumor growth, metastasis and chemoresistance through miR-27a and miR-155. CONCLUSIONS: Bmi-1 was proven to induce the expression of miR-27a and miR-155 and thus promote tumor metastasis and chemoresistance by targeting RKIP in GC. Overall, miR-27a and miR-155 might be promising targets for the screening, diagnosis, prognosis, treatment and disease monitoring of GC.

Development and validation of a novel nomogram for pretreatment prediction of liver metastasis in pancreatic cancer.

PURPOSE: The diagnostic value of nomogram in pancreatic cancer (PC) with liver metastasis (PCLM) is still largely unknown. We sought to develop and validate a novel nomogram for the prediction of liver metastasis in patients with PC. METHOD: About 604 pathologically confirmed PC patients from the Sun Yat-sen University Cancer Center (SYSUCC) between July, 2001 and December, 2013 were retrospectively studied. The SYSUCC cohort was randomly assigned to as the training set and internal validation set. Using these two sets, we derived and validated a prognostic model by using concordance index and calibration curves. Another two independent cohorts between August, 2002 and December, 2013 from the Sun Yat-sen Memorial Hospital (SYSMH, n = 335) and Guangdong General Hospital (GDGH, n = 503) was used for external validation. RESULT: Computed tomography (CT) reported liver metastasis status, carcinoembryonic antigen (CEA) level and differentiation type were identified as risk factors for PCLM in the training set. The final diagnostic model demonstrated good calibration and discrimination with a concordance index of 0.97 and had a robust internal validation. The score ability to diagnose PCLM was further externally validated in SYSMH and GDGH with a concordance index of 0.93. The model showed better calibration and discrimination than CT, CEA and differentiation in each cohort. CONCLUSION: Based on a large multi-institution database and on the routinely observed CT-reported status, CEA level and tumor differentiation in clinical practice, we developed and validated a novel nomogram to predict PLCM.

Expression of the PTEN/FOXO3a/PLZF signalling pathway in pancreatic cancer and its significance in tumourigenesis and progression.

Pancreatic cancer (PC) is one of the most lethal gastrointestinal malignancies. The PTEN/AKT signalling pathway is closely related to the tumourigenesis and progression of PC. The downstream effectors, FOXO3a, PLZF and VEGF, are reported to be involved in angiogenesis, lymph node metastasis and poor survival in PC. By using tissue microarrays and immunohistochemistry, we found, that PTEN, FOXO3a and PLZF expression was significantly decreased in PC specimens compared with that in chronic pancreatitis (CP) specimens, while VEGF expression was significantly increased. Furthermore, the expression of PTEN was positively correlated with that of FOXO3a and PLZF but negatively correlated with that of VEGF. Our results suggest that the PTEN/FOXO3a/PLZF signalling pathway may negatively regulate VEGF expression in PC. Through clinical analysis of 69 PC patients, PTEN, FOXO3a and PLZF expression was found to be significantly decreased in specimens from PC patients with lymph node metastasis and poor prognosis, while VEGF expression was significantly increased. Taken together, these reaults suggest that the PTEN/FOXO3a/PLZF signalling pathway may be capable of inhibiting growth and metastasis in PC by regulating VEGF-mediated angiogenesis, which requires further in vivo and in vitro studies and can potentially be a therapeutic target for PC.