The prognostic impact of TGF-β1, fascin, NF-κB and PKC-ζ expression in soft tissue sarcomas

Aims

Transforming growth factor-β (TGF-β), fascin, nuclear factor-kappa B (NF-κB) p105, protein-kinase C-zeta (PKC-ζ), partioning-defective protein-6 (Par-6), E-cadherin and vimentin are tumor promoting molecules through mechanisms involved in cell dedifferentiation. In soft tissue sarcomas, their expression profile is poorly defined and their significance is uncertain. We aimed to investigate the prognostic impact of TGF-β1, NF-κB p105, PKC-ζ, Par-6α, E-cadherin and vimentin in non-gastrointestinal stromal tumor soft tissue sarcomas (non-GIST STSs).

Patients and Methods

Tumor samples and clinical data from 249 patients with non-GIST STS were obtained, and tissue microarrays (TMAs) were constructed for each specimen. Immunohistochemistry (IHC) was used to evaluate marker expression in tumor cells.

Results

In univariate analysis, the expression levels of TGF-β1 (P = 0.016), fascin (P = 0.006), NF-κB p105 (P = 0.022) and PKC-ζ, (P = 0.042) were significant indicators for disease specific survival (DSS). In the multivariate analysis, high TGF-β1 expression was an independent negative prognostic factor for DSS (HR = 1.6, 95% CI = 1.1–2.4, P = 0.019) in addition to tumor depth, malignancy grade, metastasis at diagnosis, surgery and positive resection margins.

Conclusion

Expression of TGF-β1 was significantly associated with aggressive behavior and shorter DSS in non-GIST STSs.     

Prevalence and Clinical Relevance of T-Helper Cells,Th17 and Th1, in Hepatitis B Virus-Related Hepatocellular Carcinoma

Background and Aims

An immune imbalance in the cytokine profile exerts a profound influence on the progression of hepatitis B virus (HBV) infections and hepatocellular carcinoma (HCC). The present study evaluated the immune status of T helper (Th) 17 and Th1 cells in patients with HBV-related and non-HBV-related HCC.

Methods

We randomly enrolled 150 patients with HCC. Blood samples and tissue samples were obtained. The distributions and phenotypic features of Th17 and Th1 cells were determined by flow cytometry and/or immunohistochemistry.

Results

Compared to corresponding non-tumor regions, the levels of Th17 and Th1 cells were significantly increased in tumors of patients with HCC (P<0.001). The intratumoral densities of IL-17-producing cells and IFN-γ-producing cells were associated with overall survival (OS, P = 0.001) and disease-free survival (DFS, P = 0.001) of patients with HCC. The ratio of Th17 to Th1 in HBV-related HCC was higher than in non-HBV-related HCC. A multivariate Cox analysis revealed that the Th17 to Th1 ratio was an independent prognostic factor for OS (HR = 2.651, P = 0.007) and DFS (HR = 2.456, P = 0.002).

Conclusions

HBV infections can lead to an imbalance in immune status in patients with HCC. An elevated Th17 to Th1 ratio may promote tumor progression. The Th17 to Th1 ratio could serve as a potential prognostic marker for scoring the severity of HCC.     

Involvement of CCR6/CCL20/IL-17 axis in NSCLC disease progression

Objectives

Autocrine and paracrine chemokine/chemokine receptor-based interactions promote non-small-cell-lung-cancer (NSCLC) carcinogenesis. CCL20/CCR6 interactions are involved in prostatic and colonic malignancy pathogenesis. The expression and function of CCL20/CCR6 and its related Th-17 type immune response in NSCLC is not yet defined. We sought to characterize the role of the CCL20/CCR6/IL-17 axis in NSCLC tumor growth.

Methods

A specialized histopathologist blindly assessed CCL20/CCR6 expression levels in 49 tissue samples of NSCLC patients operated in our department. Results were correlated to disease progression. Colony assays, ERK signaling and chemokine production were measured to assess cancer cell responsiveness to CCL20 and IL-17 stimulation.

Results

CCL20 was highly expressed in the majority (38/49, 77.5%) of tumor samples. Only a minority of samples (8/49, 16.5%) showed high CCR6 expression. High CCR6 expression was associated with a shorter disease-free survival (P = 0.008) and conferred a disease stage-independent 4.87-fold increased risk for disease recurrence (P = 0.0076, CI 95% 1.52–15.563). Cancerous cell colony-forming capacity was increased by CCL20 stimulation; this effect was dependent in part on ERK phosphorylation and signaling. IL-17 expression was detected in NSCLC; IL-17 potentiated the production of CCL20 by cancerous cells.

Conclusion

Our findings suggest that the CCL20/CCR6 axis promotes NSCLC disease progression. CCR6 is identified as a potential new prognostic marker and the CCL20/CCR6/IL-17 axis as a potential new therapeutic target. Larger scale studies are required to consolidate these observations.     

A genetic variant in the promoter region of miR-106b-25 cluster and risk of HBV infection and hepatocellular carcinoma

Background

MiR-106b-25 cluster, hosted in intron 13 of MCM7, may play integral roles in diverse processes including immune response and tumorigenesis. A single nucleotide polymorphism (SNP), rs999885, is located in the promoter region of MCM7.

Methods

We performed a case-control study including 1300 HBV-positive hepatocellular carcinoma (HCC) cases, 1344 HBV persistent carriers and 1344 subjects with HBV natural clearance to test the association between rs999885 and the risk of HBV persistent infection and HCC. We also investigated the genotype-expression correlation between rs999885 and miR-106b-25 cluster in 25 pairs of HCC and adjacent non-tumor liver tissues.

Results

Compared with the HBV natural clearance subjects carrying rs999885 AA genotype, those with AG/GG genotypes had a decreased risk of chronic HBV infection with an adjusted odds ratio (OR) of 0.79 [95% confidence intervals (CIs) = 0.67–0.93]. However, the AG/GG genotypes were significantly associated with an increased HCC risk in HBV persistent carriers (adjusted OR = 1.25, 95% CIs = 1.06–1.47). Expression analysis revealed that the expression level of miR-106b-25 cluster was significantly higher in AG/GG carriers than those in AA carriers in non-tumor liver tissues.

Conclusions

These findings indicate that the A to G base change of rs999885 may provide a protective effect against chronic HBV infection but an increased risk for HCC in HBV persistent carriers by altering the expression of the miR-106b-25 cluster.     

NF-κB induced the donor liver cold preservation related acute lung injury in rat liver transplantation model

Background

We have observed at our clinical work that acute lung injury (ALI) often occurs in patients transplanted with donor livers persevered for long time. So, we conducted this study to investigate the influence of cold preservation time (CPT) of donor liver on ALI induced by liver transplantation (LT), and further study the role of nuclear factor-κB (NF-κB) in the process.

Methods

Wistar rats were used as donors and recipients to establish orthotopic rat liver transplantation models. Donor livers were preserved at 4°C for different lengths of time. The effect of NF-κB inhibitor, ammonium pyrrolidinedithiocarbamate (PDTC), on ALI was detected. All samples were harvested after 3 h reperfusion. The severity of liver injury was evaluated first. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in liver tissue and liver outflow serum were measured respectively. The severity indexes of ALI, the activity of NF-κB and inhibitor-κBα (I-κBα) in lung/liver were measured accordingly.

Results

With the prolonged liver CPT, the liver damage associated indexes and ALI-related indexes all increased significantly. TNF-α and IL-1β in liver outflow serum increased accordingly, and the activity of NF-κB in liver/lung increased correspondingly. All these ALI-associated indexes could be partially reversed by the use of PDTC.

Conclusions

Extended CPT aggravates the damage of donor liver and induces the expressions of TNF-α and IL-1β in liver. These inflammatory factors migrate to lung via liver outflow blood and activate NF-κB in lung, inducing ALI finally. NF-κB may play a critical role in LT-related ALI. Patients with or at risk of ALI may benefit from acute anti-inflammatory treatment with PDTC.     

Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production

Background

Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling.

Methodology/Principal Findings

We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection.

Conclusions/Significance

To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production.     

Nitrosylcobalamin potentiates the anti-neoplastic effects of chemotherapeutic agents via suppression of survival signaling

Background

Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-κB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.

Methodology

Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-κB activation.

Results

Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-κB DNA binding activity, inhibition of IκB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.

Conclusion

The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-κB or AKT.     

MicroRNA profile predicts recurrence after resection in patients with hepatocellular carcinoma within the Milan Criteria

Objective

Hepatocellular carcinoma (HCC) is difficult to manage due to the high frequency of post-surgical recurrence. Early detection of the HCC recurrence after liver resection is important in making further therapeutic options, such as salvage liver transplantation. In this study, we utilized microRNA expression profiling to assess the risk of HCC recurrence after liver resection.

Methods

We examined microRNA expression profiling in paired tumor and non-tumor liver tissues from 73 HCC patients who satisfied the Milan Criteria. We constructed prediction models of recurrence-free survival using the Cox proportional hazard model and principal component analysis. The prediction efficiency was assessed by the leave-one-out cross-validation method, and the time-averaged area under the ROC curve (ta-AUROC).

Results

The univariate Cox analysis identified 13 and 56 recurrence-related microRNAs in the tumor and non-tumor tissues, such as miR-96. The number of recurrence-related microRNAs was significantly larger in the non-tumor-derived microRNAs (N-miRs) than in the tumor-derived microRNAs (T-miRs, P<0.0001). The best ta-AUROC using the whole dataset, T-miRs, N-miRs, and clinicopathological dataset were 0.8281, 0.7530, 0.7152, and 0.6835, respectively. The recurrence-free survival curve of the low-risk group stratified by the best model was significantly better than that of the high-risk group (Log-rank: P = 0.00029). The T-miRs tend to predict early recurrence better than late recurrence, whereas N-miRs tend to predict late recurrence better (P<0.0001). This finding supports the concept of early recurrence by the dissemination of primary tumor cells and multicentric late recurrence by the ‘field effect’.

Conclusion

microRNA profiling can predict HCC recurrence in Milan criteria cases.     

IL-17A Promotes the Migration and Invasiveness of Cervical Cancer Cells by Coordinately Activating MMPs Expression via the p38/NF-κB Signal Pathway

Objective

IL-17A plays an important role in many inflammatory diseases and cancers. We aimed to examine the effect of IL-17A on the invasion of cervical cancer cells and study its related mechanisms.

Methods

Wound healing and matrigel transwell assays were used to examine the effect of IL-17A on cervical cancer cell migration and invasion by a panel of cervical cancer cell lines. The levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) were investigated using western blotting. The activity of p38 and nuclear factor-kappa B (NF-κB) signal pathway was detected too.

Results

Here, we showed that IL-17A could promote the migration and invasion of cervical cancer cells. Further molecular analysis showed that IL-17A could up-regulate the expressions and activities of MMP2 and MMP9, and down-regulate the expressions of TIMP-1 and TIMP-2. Furthermore, IL-17A also activates p38 signal pathway and increased p50 and p65 nuclear expression. In addition, treatment of cervical cancer cells with the pharmacological p38/NF-κB signal pathway inhibitors, SB203580 and PDTC, potently restored the roles of invasion and upregulation of MMPs induced by IL-17A.

Conclusion

IL-17A could promote the migration and invasion of cervical cancer cell via up-regulating MMP2 and MMP9 expression, and down-regulating TIMP-1 and TIMP-2 expression via p38/NF-κB signal pathway. IL-17A may be a potential target to improve the prognosis for patients with cervical cancer.     

Clara cell 10-kDa protein gene transfection inhibits NF-κB activity in airway epithelial cells

Background

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.

Method/Results

To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10''s effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10''s effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.

Conclusion

These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation.     

The accumulation and prognosis value of tumor infiltrating IL-17 producing cells in esophageal squamous cell carcinoma

Background

The role of IL-17 producing cells in tumors is controversial. In the present study, we investigated the prognostic value of measuring tumor-infiltrating IL-17 producing cell levels in human esophageal squamous cell carcinoma (ESCC).

Methodology/Principal Findings

Immunohistochemical staining was performed to investigate the levels of IL-17+ tumor infiltrating lymphocytes (TILs), as well as CD8+ cytotoxic T lymphocytes (CTLs) and CD57+ natural killer (NK) cells from 181 ESCC patients. The prognostic value of measuring the densities of IL-17+TILs and the correlation with CTLs and NK was evaluated. IL-17 producing cells were detected in esophageal squamous cell carcinoma tissues. The IL-17 producing cells were major CD4 positive, but Foxp3 negative. The median level of IL-17+TILs was 3.90 cells/high power microscopic field (HPF). The density of IL-17 producing cells correlated negatively with T stage (P = 0.042). The higher densities of tumor infiltrating IL-17+ lymphocytes were associated with better overall survival (P = 0.031). Furthermore, we found that there were positive correlations between levels of IL-17 producing cells and the densities of CD8+cells, as well as CD57+cells (r = 0.198, P = 0.008 for CD8+ cells and r = 0.261, P<0.001 for CD57+ cells, respectively). The prognosis analysis also showed that the higher levels of CD8+ CTLs and CD57+ NK cells correlated with better overall survival of ESCC patients.

Conclusions

Our study suggests that tumor infiltrating IL-17 producing cells in ESCC patients may have protective roles in the tumor microenvironment and may be treated as a prognostic marker for ESCC patients.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.