Activation of the NF-κB pathway as a mechanism of alcohol enhanced progression and metastasis of human hepatocellular carcinoma

Background

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is the third leading cause of cancer-related death in human. Alcohol is a known risk factor for HCC. However it is still unclear whether and how alcohol enhances the progression and metastasis of existing HCC.

Methods and results

We first retrospectively investigated 52 HCC patients (24 alcohol-drinkers and 28 non-drinkers), and found a positive correlation between alcohol consumption and advanced Tumor-Node-Metastasis (TNM) stages, higher vessel invasion and poorer prognosis. In vitro and in vivo experiments further indicated that alcohol promoted the progression and migration/invasion of HCC. Specifically, in a 3-D tumor/endothelial co-culture system, we found that alcohol enhanced the migration/invasion of HepG2 cells and increased tumor angiogenesis. Consistently, higher expression of VEGF, MCP-1 and NF-κB was observed in HCC tissues of alcohol-drinkers. Alcohol induced the accumulation of intracellular reactive oxygen species (ROS) and the activation of NF-κB signaling in HepG2 cells. Conversely, blockage of alcohol-mediated ROS accumulation and NF-κB signaling inhibited alcohol-induced expression of VEGF and MCP-1, the tumor growth, angiogenesis and metastasis.

Conclusion

This study suggested that chronic moderate alcohol consumption may promote the progression and metastasis of HCC; the oncogenic effect may be at least partially mediated by the ROS accumulation and NF-ĸB-dependent VEGF and MCP-1 up-regulation.     

Pim-2 Activates API-5 to Inhibit the Apoptosis of Hepatocellular Carcinoma Cells Through NF-κB Pathway

Pim-2 is proved to be relevant to the tumorigenesis of hepatocellular carcinoma (HCC), but the mechanism is unclear. We studied the relationship among Pim-2, NF-κB and API-5. In our experiment, expression level of the three factors and phosphorylation level of API-5, as well as NF-κB activity, were detected in HCC tissues and the nontumorous controls. Then Pim-2 gene was transfected into nontumorous liver cells L02, and Pim-2 SiRNA was transfected into hepatoblastoma cell line HepG2. Parthenolide was added as NF-κB inhibitor. The same detections as above were repeated in the cells, along with the apoptosis analysis. We found the levels of Pim-2, NF-κB and API-5, as well as NF-κB activity, were significantly higher in HCC tissues. Pim-2 level was increased in L02 cells after the transfection of Pim-2 gene, but decreased in HepG2 cells after the transfection of Pim-2 SiRNA. The levels of NF-κB and API-5, as well as NF-κB activity and API-5 phosphorylation level, were in accordance with Pim-2 level, but could be reversed by Parthenolide. Cell apoptosis rates were negatively correlated with API-5 phosphorylation level. Therefore, we infer that Pim-2 could activate API-5 to inhibit the apoptosis of liver cells, and NF-κB is the key regulator.     

The Two Faces of NF-κB Signaling in Cancer Development and Therapy

Constitutive activation of NF-κB signaling can promote oncogenesis, providing a rationale for anticancer strategies that inhibit NF-κB signaling. Two recent publications in Genes & Development provide evidence that, in contexts where prosurvival signals derive from other oncogenes, NF-κB activity instead enhances sensitivity to cytotoxic chemotherapy, thereby exerting a tumor-suppressor function.     

The Plasmodium Circumsporozoite Protein, a Novel NF-κB Inhibitor, Suppresses the Growth of SW480

The blocking of NF-κB activation is a promising strategy for the treatment of colorectal cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was recently reported to block NF-κB activation in hepatocytes. Thus, we investigated the effect of the CSP on the growth of the human colon cancer cell line, SW480. We demonstrated that transfection with a recombinant plasmid expressing CSP inhibited the proliferation of SW480 in a dose-dependent manner and induced the apoptosis of SW480. A NF-κB gene reporter assay showed that both the CSP and its nuclear localization signal (NLS) motif could equally suppress the activation of NF-κB following the stimulation with human recombinant TNF-α in the SW480. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but could sharply inhibit the nuclear translocation of NF-κB in TNF-α stimulated SW480. Hence, our data suggest that the CSP might be explored as a new NF-κB inhibitor for the treatment of colorectal cancer.     

HIF-α/MIF and NF-κB/IL-6 Axes Contribute to the Recruitment of CD11b+Gr-1+ Myeloid Cells in Hypoxic Microenvironment of HNSCC

CD11b+Gr-1+ myeloid cells have gained much attention due to their roles in tumor immunity suppression as well as promotion of angiogenesis, invasion, and metastases. However, the mechanisms by which CD11b+Gr-1+ myeloid cells recruit to the tumor site have not been well clarified. In the present study, we showed that hypoxia could stimulate the migration of CD11b+Gr-1+ myeloid cells through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Hypoxia-inducible factor-1α (HIF-1α)- and HIF-2α-dependent MIF regulated chemotaxis, differentiation, and pro-angiogenic function of CD11b+Gr-1+ myeloid cells through binding to CD74/CXCR2, and CD74/CXCR4 complexes, and then activating p38/mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases (PI3K)/AKT signaling pathways. Knockdown (KD) of HIF-1α and HIF-2α in HNSCC cells decreased MIF level but failed to inhibit the CD11b+Gr-1+ myeloid cell migration, because HIF-1α/2α KD enhanced nuclear factor κB (NF-κB) activity that increased IL-6 secretion. Simultaneously blocking NF-κB and HIF-1α/HIF-2α had better inhibitory effect on CD11b+Gr-1+ myeloid cell recruitment in the hypoxic zone than individually silencing HIF-1α/2α or NF-κB. In conclusion, the interaction between HIF-α/MIF and NF-κB/IL-6 axes plays an important role in the hypoxia-induced accumulation of CD11b+Gr-1+ myeloid cells and tumor growth in HNSCC.     

4-hexylresorcinol inhibits NF-κB phosphorylation and has a synergistic effect with cisplatin in KB cells

Cisplatin is a representative anti-cancer drug and 4-hexylresorcinol (4-HR) is known as an antiparasitic and antiseptic agent. The aims of this study were to evaluate the effect of 4-HR on the activation of nuclear factor-κB (NF-κB) in cell cultures, to evaluate the antitumor effect of 4-HR plus cisplatin combination therapy in a xenograft model, and to evaluate transglutaminase-2 (TG-2) and phosphorylated NF-κB (pNF-κB) expression in the xenograft model. To determine the effect of 4-HR on NF-κB phosphorylation, co-immunoprecipitation and Western blot analysis were done in KB cells. To examine the in?vivo effect of the cisplatin plus 4-HR combination therapy, KB cells were grafted into nude mice. Drugs were injected into the peritoneal cavity daily. Tumor size, body weight, and duration of survival were checked daily. Specimens from main mass were used in immunohistochemical staining for the analysis of TG-2 and pNF-κB expression. In the in?vitro test, as the 4-HR concentrations increased, the fraction of the bound complex NF-κB-inhibitory-κB (IκB) increased. Consequently, the level of free IκB decreased. In the xenograft model, the cisplatin plus 4-HR group exhibited a significantly decreased tumor growth rate than in the saline group (P=0.039). The mean survival time of the cisplatin plus 4-HR group was 51.20±3.96 days and was significantly prolonged compared with the other groups (P<0.05). The body weight of the cisplatin plus 4-HR group had significantly less weight loss than the cisplatin only group (P=0.045). In the immunohistochemical analysis, the cisplatin plus 4-HR group had a significantly lower expression of TG-2 and pNF-κB compared to the saline group (P<0.05). In conclusion, cisplatin plus 4-HR combination therapy had clear advantages over the cisplatin only treatment such as similar tumor growth inhibition compared to the cisplatin only treatment despite the reduced dosage of cisplatin, less body weight loss, and prolonged survival time.     

Activation of Akt involves resistance to NF-κB inhibition and abrogation of both triggers synergistic apoptosis in lung adenocarcinoma cells

Objectives

Although nuclear factor (NF)-κB and phosphoinositide 3-kinase (PI3K)-Akt-mTOR comprise key pathways, their interrelationship in lung cancer cell survival is poorly understood and needs further analyses.

Materials and methods

We examined the activation of the NF-κB and Akt-mTORC1-p70 S6 kinase (S6K) pathways and the effect of inhibitors for NF-κB, mTORC1, and Akt using fresh lung adenocarcinoma cells.

Results

The cases used for this study showed constitutive NF-κB activity; however, all cases but one showed resistance to NF-κB inhibition. Further examination revealed that the resistant cases were also active in the Akt-mTORC1-S6K pathway. These cases were insensitive to mTORC1 inhibition but sensitive to Akt inhibition. Akt inhibition recovered sensitivity to NF-κB inhibition and dual inhibition showed a synergistic effect on apoptosis induction.

Conclusion

These results indicate that the activation of Akt involves resistance to NF-κB inhibition and both pathways synergistically support the survival of lung adenocarcinoma cells. The results also indicate that inhibition of the mTORC1-S6K pathway does not inhibit the survival of these cells.     

Concurrent blockade of the NF-κB and Akt pathways potently sensitizes cancer cells to chemotherapeutic-induced cytotoxicity

Nuclear factor-κB (NF-κB) and Akt are two major cell survival pathways that are often constitutively activated and can be further stimulated by chemotherpeutics in cancer cells. Although individually targeting the NF-κB or Akt has been reported to sensitize caner therapy, the effectiveness of concurrent blocking these two pathways for chemosensitizing of cancer cells to genotoxic therapeutics has not been investigated. In the present study, we investigate the activation of the NF-κB and Akt pathways by two frontline anticancer drugs cisplatin and etopside in a variety of cancer cell lines. The effects of blocking these two survival pathways individually or concurrently on cisplatin- or etopside-induced cytotoxicity were detected. The results show that cisplatin and etopside activate both NF-κB and Akt in cancer cells. Blockade of either of these pathways with chemical inhibitors or siRNA moderately sensitized cancer cells to cisplatin- or etopside-induced cytotoxicity. Strikingly, much more effective potentiation of cytotoxicity to these anticancer drugs was achieved when NF-κB and Akt were concurrently blocked. These data suggest that NF-κB and Akt cooperatively attenuate therapeutic-induced cytotoxicity and concurrently blocking these pathways is an effective strategy for improving the anticancer efficacy of therapeutics.     

Response and resistance to NF-κB inhibitors in mouse models of lung adenocarcinoma

Lung adenocarcinoma is a leading cause of cancer death worldwide. We recently showed that genetic inhibition of the NF-κB pathway affects both the initiation and the maintenance of lung cancer, identifying this pathway as a promising therapeutic target. In this investigation, we tested the efficacy of small-molecule NF-κB inhibitors in mouse models of lung cancer. In murine lung adenocarcinoma cell lines with high NF-κB activity, the proteasome inhibitor bortezomib efficiently reduced nuclear p65, repressed NF-κB target genes, and rapidly induced apoptosis. Bortezomib also induced lung tumor regression and prolonged survival in tumor-bearing Kras(LSL-G12D/wt);p53(flox/flox) mice but not in Kras(LSL-G12D/wt) mice. After repeated treatment, initially sensitive lung tumors became resistant to bortezomib. A second NF-κB inhibitor, Bay-117082, showed similar therapeutic efficacy and acquired resistance in mice. Our results using preclinical mouse models support the NF-κB pathway as a potential therapeutic target for a defined subset of lung adenocarcinoma. SIGNIFICANCE: Using small-molecule compounds that inhibit NF-κB activity, we provide evidence that NF-κB inhibition has therapeutic efficacy in the treatment of lung cancer. Our results also illustrate the value of mouse models in validating new drug targets in vivo and indicate that acquired chemoresistance may later influence bortezomib treatment in lung cancer.     

Inhibition of NF-κB improves sensitivity to irradiation and EGFR-TKIs and decreases irradiation-induced lung toxicity

Resistance to radiotherapy and to EGFR tyrosine kinase inhibitors (EGFR-TKIs), as well as therapy-related lung toxicity, are serious problems in the treatment of lung cancer. NF-κB has been reported to be associated with radioresistance. Therefore, we evaluated its effects on sensitivity to irradiation and to EGFR-TKIs; irradiation-induced lung toxicity; and the effects of irradiation on sensitivity to EGFR-TKIs. We used IKKβ inhibitor IMD 0354 or p65 depletion to explore their effects on sensitivity to irradiation and to EGFR-TKIs in vitro and in vivo. We evaluated the efficacy of IMD 0354 in a radiation-induced pulmonary-fibrosis mouse model. Irradiation enhanced activation and expression of MET and therefore suppressed the sensitivity of lung cancer cells to irradiation or EGFR-TKIs. Inhibition of NF-κB by IMD 0354 or by p65 depletion reversed irradiation-induced MET activation and increased the sensitivity of lung cancer cells to irradiation, to EGFR-TKIs and to the combination thereof in vitro and in vivo. In addition, IMD 0354 significantly reduced lung toxicity in a murine model of irradiation-induced pneumonia and lung fibrosis. These findings indicated that NF-κB inhibition can improve sensitivity to irradiation and to EGFR-TKIs and can decrease irradiation-induced lung toxicity in lung cancer.