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Colorectal cancer (CRC) is one of the most common malignancies worldwide. Inflammation contributes to cancer development and inflammatory bowel disease is an important risk factor for CRC. The aim of this study is to assess whether a widely used probiotic Enterococcus faecalis can modulate the NLRP3 inflammasome and protect against colitis and colitis-associated CRC. We studied the effect of heat-killed cells of E. faecalis on NLRP3 inflammasome activation in THP-1-derived macrophages. Pretreatment of E. faecalis or NLRP3 siRNA can inhibit NLRP3 inflammasome activation in macrophages in response to fecal content or commensal microbes, P. mirabilis or E. coli, according to the reduction of caspase-1 activation and IL-1β maturation. Mechanistically, E. faecalis attenuates the phagocytosis that is required for the full activation of the NLRP3 inflammasome. In in vivo mouse experiments, E. faecalis can ameliorate the severity of intestinal inflammation and thereby protect mice from dextran sodium sulfate (DSS)-induced colitis and the formation of CRC in wild type mice. On the other hand, E. faecalis cannot prevent DSS-induced colitis in NLRP3 knockout mice. Our findings indicate that application of the inactivated probiotic, E. faecalis, may be a useful and safe strategy for attenuation of NLRP3-mediated colitis and inflammation-associated colon carcinogenesis.

Activation of the nod-like receptor 3 (NLRP3) inflammasomes is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Cbl plays a pivotal role in suppressing NLRP3 inflammasome activation by inhibiting Pyk2-mediated apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization. Here, we showed that Cbl dampened NLRP3 inflammasome activation by inhibiting glycolysis, as demonstrated with Cbl knockout cells and treatment with the Cbl inhibitor hydrocotarnine. We revealed that the inhibition of Cbl promoted caspase-1 cleavage and interleukin (IL)-1β secretion through a glycolysis-dependent mechanism. Inhibiting Cbl increased cellular glucose uptake, glycolytic capacity, and mitochondrial oxidative phosphorylation capacity. Upon NLRP3 inflammasome activation, inhibiting Cbl increased glycolysis-dependent activation of mitochondrial respiration and increased the production of reactive oxygen species, which contributes to NLRP3 inflammasome activation and IL-1β secretion. Mechanistically, inhibiting Cbl increased surface expression of glucose transporter 1 (GLUT1) protein through post-transcriptional regulation, which increased cellular glucose uptake and consequently raised glycolytic capacity, and in turn enhanced NLRP3 inflammasome activation. Together, our findings provide new insights into the role of Cbl in NLRP3 inflammasome regulation through GLUT1 downregulation. We also show that a novel Cbl inhibitor, hydrocortanine, increased NLRP3 inflammasome activity via its effect on glycolysis.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). Methods In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. Results Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-β upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. Conclusions These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Activation of the NLRP3 inflammasome is crucial for immune defense, but improper and excessive activation causes inflammatory diseases. We previously reported that Pyk2 is essential for NLRP3 inflammasome activation. Here we show that the Src-family kinases (SFKs)-Cbl axis plays a pivotal role in suppressing NLRP3 inflammasome activation in response to stimulation by nigericin or ATP, as assessed using gene knockout and gene knockdown cells, dominant active/negative mutants, and pharmacological inhibition. We reveal that the phosphorylation of Cbl is regulated by SFKs, and that phosphorylation of Cbl at Tyr371 suppresses NLRP3 inflammasome activation. Mechanistically, Cbl decreases the level of phosphorylated Pyk2 (p-Pyk2) through ubiquitination-mediated proteasomal degradation and reduces mitochondrial ROS (mtROS) production by contributing to the maintenance of mitochondrial size. The lower levels of p-Pyk2 and mtROS dampen NLRP3 inflammasome activation. In vivo, inhibition of Cbl with an analgesic drug, hydrocotarnine, increases inflammasome-mediated IL-18 secretion in the colon, and protects mice from dextran sulphate sodium-induced colitis. Together, our novel findings provide new insights into the role of the SFK-Cbl axis in suppressing NLRP3 inflammasome activation and identify a novel clinical utility of hydrocortanine for disease treatment.

The inflammasome adaptor protein, ASC, contributes to both innate immune responses and inflammatory diseases via self-oligomerization, which leads to the activation of the protease, caspase-1. Here, we report that the cytosolic tyrosine kinases, FAK and Pyk2, are differentially involved in NLRP3 and AIM2 inflammasome activation. The inhibition of FAK and Pyk2 with RNA interference or chemical inhibitors dramatically abolished ASC oligomerization, caspase-1 activation, and IL-1β secretion in response to NLRP3 or AIM2 stimulation. Pyk2 is phosphorylated by the kinase Syk and relocalizes to the ASC specks upon NLRP3 inflammasome activation. Pyk2, but not FAK, could directly phosphorylate ASC at Tyr146, and only the phosphorylated ASC could participate in speck formation and trigger IL-1β secretion. Moreover, the clinical-trial-tested Pyk2/FAK dual inhibitor PF-562271 reduced monosodium urate-mediated peritonitis, a disease model used for studying the consequences of NLRP3 activation. Our results suggest that although Pyk2 and FAK are involved in inflammasome activation, only Pyk2 directly phosphorylates ASC and brings ASC into an oligomerization-competent state by allowing Tyr146 phosphorylation to participate ASC speck formation and subsequent NLRP3 inflammation.

Approximately, 25% of nasopharyngeal carcinoma (NPC) patients develop recurrent disease. NPC may involve relatively few genomic alterations compared to other cancers due to its association with Epstein‐Barr virus (EBV). We envisioned that in‐depth sequencing of tumor tissues might provide new insights into the genetic alterations of this cancer. Thirty‐three NPC paired tumor/adjacent normal or peripheral blood mononuclear cell samples were deep‐sequenced (>1000×) with respect to a panel of 409 cancer‐related genes. Newly identified mutations and its correlation with clinical outcomes were evaluated. Profiling of somatic mutations and copy number variations (CNV) in NPC tumors identified alterations in RTK/RAS/PI3K, NOTCH, DNA repair, chromatin remodeling, cell cycle, NF‐κB, and TGF‐β pathways. In addition, patients harbored CNV among 409 cancer‐related genes and missense mutations in TGF‐β/SMAD signaling were associated with poor overall survival and poor recurrence‐free survival, respectively. The CNV events were correlated with plasma EBV copies, while mutations in TGFBR2 and SMAD4 abrogate SMAD‐dependent TGF‐β signaling. Functional analysis revealed that the new TGFBR2 kinase domain mutants were incapable of transducing the signal, leading to failure of phosphorylation of SMAD2/3 and activation of downstream TGF‐β‐mediated cell growth arrest. This study provides evidence supporting CNV and dysregulated TGF‐β signaling contributes to exacerbating the NPC pathogenesis. Profiling of somatic mutations and copy number variations (CNV) in NPC tumors identified alterations in RTK/RAS/PI3K, NOTCH, DNA repair, chromatin remodeling, cell cycle, NF‐κB, and TGF‐β pathways. Among them, TGFBR2 loss‐of‐function mutations are resistant to TGF‐β‐induced growth inhibition. This study provides evidence supporting CNV and dysregulated TGF‐β signaling contributes to exacerbating the NPC pathogenesis.

The involvement of enolase-1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1-targeting antibody has demonstrated the pathological roles of extracellular ENO1 (surface or secreted forms). However, although ENO1 was first identified as a glycolytic enzyme in the cytosol, to the best of our knowledge, extracellular ENO1 has not been implicated in glycolysis thus far. In the present study, the effects of extracellular ENO1 on glycolysis and other related pro-cancer activities were investigated in multiple myeloma (MM) cells in vitro and in vivo. Knockdown of ENO1 expression reduced lactate production, cell viability, cell migration and surface ENO1 expression in MM cells. Notably, addition of extracellular ENO1 protein in cancer cell culture enhanced glycolytic activity, hypoxia-inducible factor 1-α (HIF-1α) expression, glycolysis-related gene (GRG) expression and pro-cancer activities, such as cell migration, cell viability and tumor-promoting cytokine secretion. Consistently, these extracellular ENO1-induced cellular effects were inhibited by an ENO1-specific monoclonal antibody (mAb). In addition, extracellular ENO1-mediated glycolysis, GRG expression and pro-cancer activities were also reduced by HIF-1α silencing. Lastly, administration of an ENO1 mAb reduced tumor growth and serum lactate levels in an MM xenograft model. These results suggested that extracellular ENO1 (surface or secreted forms) enhanced a HIF-1α-mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1-specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

Background Overexpression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a DNA/RNA binding protein, is associated with metastasis in nasopharyngeal carcinoma (NPC). However, the mechanisms underlying hnRNP K-mediated metastasis is unclear. The aim of the present study was to determine the role of matrix metalloproteinase (MMP) in hnRNP K-mediated metastasis in NPC. Methods We studied hnRNP K-regulated MMPs by analyzing the expression profiles of MMP family genes in NPC tissues and hnRNP K-knockdown NPC cells using Affymetrix microarray analysis and quantitative RT-PCR. The association of hnRNP K and MMP12 expression in 82 clinically proven NPC cases was determined by immunohistochemical analysis. The hnRNP K-mediated MMP12 regulation was determined by zymography and Western blot, as well as by promoter, DNA pull-down and chromatin immunoprecipitation (ChIP) assays. The functional role of MMP12 in cell migration and invasion was demonstrated by MMP12-knockdown and the treatment of MMP12-specific inhibitor, PF-356231. Results MMP12 was overexpressed in NPC tissues, and this high level of expression was significantly correlated with high-level expression of hnRNP K (P = 0.026). The levels of mRNA, protein and enzyme activity of MMP12 were reduced in hnRNP K-knockdown NPC cells. HnRNP K interacting with the region spanning −42 to −33 bp of the transcription start site triggered transcriptional activation of the MMP12 promoter. Furthermore, inhibiting MMP12 by MMP12 knockdown and MMP12-specific inhibitor, PF-356231, significantly reduced the migration and invasion of NPC cells. Conclusions Overexpression of MMP12 was significantly correlated with hnRNP K in NPC tissues. HnRNP K can induce MMP12 expression and enzyme activity through activating MMP12 promoter, which promotes cell migration and invasion in NPC cells. In vitro experiments suggest that NPC metastasis with high MMP12 expression may be treated with PF-356231. HnRNP K and MMP12 may be potential therapeutic markers for NPC, but additional validation studies are warranted.

The involvement of enolase-1 (ENO1), intracellularly or extracellularly, has been implicated in cancer development. Moreover, anticancer activities of an ENO1-targeting antibody has demonstrated the pathological roles of extracellular ENO1 (surface or secreted forms). However, although ENO1 was first identified as a glycolytic enzyme in the cytosol, to the best of our knowledge, extracellular ENO1 has not been implicated in glycolysis thus far. In the present study, the effects of extracellular ENO1 on glycolysis and other related pro-cancer activities were investigated in multiple myeloma (MM) cells in vitro and in vivo. Knockdown of ENO1 expression reduced lactate production, cell viability, cell migration and surface ENO1 expression in MM cells. Notably, addition of extracellular ENO1 protein in cancer cell culture enhanced glycolytic activity, hypoxia-inducible factor 1-[alpha] (HIF-1[alpha]) expression, glycolysis-related gene (GRG) expression and pro-cancer activities, such as cell migration, cell viability and tumor-promoting cytokine secretion. Consistently, these extracellular ENO1-induced cellular effects were inhibited by an ENO1-specific monoclonal antibody (mAb). In addition, extracellular ENO1-mediated glycolysis, GRG expression and pro-cancer activities were also reduced by HIF-1[alpha] silencing. Lastly, administration of an ENO1 mAb reduced tumor growth and serum lactate levels in an MM xenograft model. These results suggested that extracellular ENO1 (surface or secreted forms) enhanced a HIF-1[alpha]-mediated glycolytic pathway, in addition to its already identified roles. Therefore, the results of the present study highlighted the therapeutic potential of ENO1-specific antibodies in treating MM, possibly via glycolysis inhibition, and warrant further studies in other types of cancer.

Patients with chronic liver disease (CLD) have a higher risk of mortality when infected with severe acute respiratory syndrome coronavirus 2. Although the fibrosis-4 (FIB-4) index, aspartate aminotransferase-to-platelet ratio index (APRI), and albumin-bilirubin grade (ALBI) score can predict mortality in CLD, their correlation with the clinical outcomes of CLD patients with coronavirus disease 2019 (COVID-19) is unclear. This study aimed to investigate the association between the liver severity and the mortality in hospitalized patients with non-cirrhotic CLD and COVID-19. This retrospective study analyzed 231 patients with non-cirrhotic CLD and COVID-19. Clinical characteristics, laboratory data, including liver status indices, and clinical outcomes were assessed to determine the correlation between liver status indices and the mortality among patients with non-cirrhotic CLD and COVID-19. Non-survivors had higher levels of prothrombin time-international normalized ratio (PT-INR), alanine aminotransferase, aspartate aminotransferase, and high-sensitivity C-reactive protein (hs-CRP) and lower albumin levels. Multivariable analysis showed that ALBI grade 3 (odds ratio (OR): 22.80, 95% confidence interval (CI) [1.70-305.38], = 0.018), FIB-4 index ≥ 3.25 (OR: 10.62, 95% CI [1.12-100.31], = 0.039), PT-INR (OR: 19.81, 95% CI [1.31-299.49], = 0.031), hs-CRP (OR: 1.02, 95% CI [1.01-1.02], = 0.001), albumin level (OR: 0.08, 95% CI [0.02-0.39], = 0.002), and use of vasopressors (OR: 4.98, 95% CI [1.27-19.46], = 0.021) were associated with the mortality. The ALBI grade 3 and FIB-4 index ≥ 3.25, higher PT-INR, hsCRP levels and lower albumin levels could be associated with mortality in non-cirrhotic CLD patients with COVID-19. Clinicians could assess the ALBI grade, FIB-4 index, PT-INR, hs-CRP, and albumin levels of patients with non-cirrhotic CLD upon admission.