NQO1 expression correlates inversely with NFκB activation in human breast cancer

NQO1 participates in cellular defense against oxidative stress and regulates apoptosis via p53- and NF??B-mediated pathways. We have previously found that homozygous missense variant NQO1*2 (rs1800566) predicts poor survival among breast cancer patients, particularly after anthracycline-based adjuvant chemotherapy. Here, we investigated NQO1 and NF??B protein expression and global gene expression profiles in breast tumors with correlation to tumor characteristics and survival after adjuvant chemotherapy. We used immunohistochemical analysis of tissue microarrays to study NQO1 and NF??B expression in two series of tumors: 1000 breast tumors unselected for treatment and 113 from a clinical trial comparing chemotherapy regimens after anthracycline treatment in advanced breast cancer. We used gene expression arrays to define genes co-expressed with NQO1 and NF??B. NQO1 and nuclear NF??B were expressed in 83% and 11% of breast tumors, and correlated inversely (P?=?0.012). NQO1 protein expression was associated with estrogen receptor (ER) expression (P?=?0.011), whereas 34.5% of NF??B-nuclear/activated tumors were ER negative (P?=?0.001). NQO1 protein expression and NF??B activation showed only trends, but no statistical significance for patient survival or outcome after anthracycline treatment. Gene expression analysis highlighted 193 genes that significantly correlated with both NQO1 and NF??B in opposite directions, consistent with the expression patterns of the two proteins. Inverse correlation was found with genes related to oxidation/reduction, lipid biosynthesis and steroid metabolism, immune response, lymphocyte activation, Jak-STAT signaling and apoptosis. The inverse relationship between NQO1 protein expression and NF??B activation, underlined also by inverse patterns of association with ER and gene expression profiles of tumors, suggests that NQO1?CNF??B interaction in breast cancer is different from several other tissue types, possibly due to estrogen receptor signaling in breast cancer. Neither NQO1 nor NF??B protein expression appear as significant prognostic or predictive markers in breast cancer.     

NFκB signalling is upregulated in a subset of castrate-resistant prostate cancer patients and correlates with disease progression

Background:

Cell line models suggest that activation of NFκB is associated with progression of prostate cancer. This pathway may be a therapeutic target if these observations translate to clinical specimens.

Methods:

Immunohistochemistry measured NFκBp65 (p65), NFκBp65 nuclear localisation signal (NLS), NFκBp65 phosphorylated at ser 276 (p65^ser276), NFκBp65 phosphorylated at ser 536 (p65^ser536), IκBα phosphorylated at ser 32/36 (pIκBα^ser32/36) and MMP-9 protein expression in 61 matched hormone naive prostate cancer (HNPC) and castrate-resistant prostate cancer (CRPC) tumours. Animal and cell models were used to investigate the role of NFκB inhibition in prostate carcinogenesis.

Results:

In HNPC tumours, NLS expression significantly associated with a shorter time to disease recurrence and disease-specific death. In CRPC tumours p65, pIκBα^ser32/36 and MMP-9 expression significantly associated with shorter time to death from disease recurrence and shorter disease-specific death. MMP-9 and pIκBα^ser32/36 expression significantly associated with metastases at recurrence and were independent of Gleason sum and prostate-specific antigen at recurrence. Expression of phosphorylated Akt was associated with increased p65 activation in mouse models and inhibition of NFκB in LNCaP cells significantly reduced cellular proliferation and induced apoptosis.

Conclusion:

These results provide further evidence that the NFκB pathway could be exploited as a target for CRPC.     

Gambogic acid synergistically potentiates cisplatin-induced apoptosis in non-small-cell lung cancer through suppressing NF-κB and MAPK/HO-1 signalling

Background:

Gambogic acid (GA) has been reported to have potent anticancer activity and is authorised to be tested in phase II clinical trials for treatment of non-small-cell lung cancer (NSCLC). The present study aims to investigate whether GA would be synergistic with cisplatin (CDDP) against the NSCLC.

Methods:

1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT), combination index (CI) isobologram, western blot, quantitative PCR, flow cytometry, electrophoretic mobility shift assay, xenograft tumour models and terminal deoxynucleotide transferase-mediated dUTP nick-end labelling analysis were used in this study.

Results:

The cell viability results showed that sequential CDDP-GA treatment resulted in a strong synergistic action in A549, NCI-H460, and NCI-H1299 cell lines, whereas the reverse sequence and simultaneous treatments led to a slight synergistic or additive action. Increased sub-G1 phase cells and enhanced PARP cleavage demonstrated that the sequence of CDDP-GA treatment markedly increased apoptosis in comparison with other treatments. Furthermore, the sequential combination could enhance the activation of caspase-3, -8, and 9, increase the expression of Fas and Bax, and decrease the expression of Bcl-2, survivin and X-inhibitor of apoptosis protein (X-IAP) in A549 and NCI-H460 cell lines. In addition, increased apoptosis was correlated with enhanced reactive oxygen species generation. Importantly, it was found that, followed by CDDP treatment, GA could inhibit NF-κB and mitogen-activated protein kinase (MAPK)/heme oxygenase-1 (HO-1) signalling pathways, which have been validated to reduce ROS release and confer CDDP resistance. The roles of NF-κB and MAPK pathways were further confirmed by using specific inhibitors, which significantly increased ROS release and apoptosis induced by the sequential combination of CDDP and GA. Moreover, our results indicated that the combination of CDDP and GA exerted increased antitumour effects on A549 xenograft models through inhibiting NF-κB, HO-1, and subsequently inducing apoptosis.

Conclusion:

Gambogic acid sensitises lung cancer cells to CDDP in vitro and in vivo in NSCLC through inactivation of NF-κB and MAPK/HO-1 signalling pathways, providing a rationale for the combined use of CDDP and GA in lung cancer chemotherapy.     

Id-1 regulates Bcl-2 and Bax expression through p53 and NF-κB in MCF-7 breast cancer cells

Although increasing evidence supports the protective role of inhibitor of differentiation and DNA binding-1 (Id-1) against anticancer drug-induced apoptosis, the underlying molecular mechanisms seem to vary depending on the tumor system. Here, we examined the direct role of Id-1 in MCF-7 breast cancer cells by ectopically overexpressing Id-1 under serum-free condition, where the endogenous Id-1 expression was suppressed. Id-1 expression resulted in increased number of viable cells, reduced Bax expression, enhanced Bcl-2 expression, but no change in Bcl-xL expression. The expression of nuclear factor-κB (NF-κB) was augmented, while those of p53 and IκB were reduced. Such changes in p53 and NF-κB pathways were also functional, as assessed by real-time polymerase chain reactions and reporter assays of their known downstream targets, p21 and Il-6, as well as Bax and Bcl-2 genes. Finally, Id-1 played a protective role against taxol-induced apoptosis in breast cancer cells as assessed by MTT assay and apoptotic cell count upon taxol treatment (0–200 nM). Reduced Bax expression and enhanced Bcl-2 expression by Id-1 were also noted in the presence of taxol. Taken together, we present a molecular mechanism where Id-1 regulates p53 and NF-κB pathways, which in turn regulates Bax and Bcl-2 genes, thus providing a survival advantage under exogenous stress such as serum-free or taxol treatment in MCF-7 breast cancer cells. In this regard, inactivation of Id-1 may provide a potential therapeutic strategy leading to inhibition of breast cancer progression and anti-cancer drug resistance. Hwan Kim and Heekyoung Chung equally contributed to this work.     

HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 by activation of p38 MAPK and NF-κB through receptors for advanced glycation end-products in macrophages

The high mobility group box-1 (HMGB1) protein and NALP3 inflammasome have been identified to play important roles in inflammation and cancer pathogenesis, but the relationships between the two and cancer remain unclear. The current study investigated the relationship between HMGB1 and the NALP3 inflammasome in THP-1 macrophages. HMGB1 was found unable to activate the NALP3 inflammasome and failed to induce the release of the IL-1β and IL-18 in THP-1 macrophages. HMGB1 was also found significantly enhanced the activity of ATP to induce IL-1β and IL-18 by the induction of increased expression of pro-IL-1β and pro-IL-18. This process was dependent on activation of RAGE, MAPK p38 and NF-κB signaling pathway. These results demonstrate that HMGB1 promotes the synthesis of pro-IL-1β and pro-IL-18 in THP-1 macrophages by the activation of p38 MAPK and NF-κB through RAGE. HMGB1 likely plays an important role in the first step of the release of the IL-1β and IL-18, preparing for other cytokines to induce excessive release of IL-1β and IL-18 which promote inflammation and cancer progression.     

TGF-β1 modulates the homeostasis between MMPs and MMP inhibitors through p38 MAPK and ERK1/2 in highly invasive breast cancer cells

Background  

Metastasis is the main factor responsible for death in breast cancer patients. Matrix metalloproteinases (MMPs) and their inhibitors, known as tissue inhibitors of MMPs (TIMPs), and the membrane-associated MMP inhibitor (RECK), are essential for the metastatic process. We have previously shown a positive correlation between MMPs and their inhibitors expression during breast cancer progression; however, the molecular mechanisms underlying this coordinate regulation remain unknown. In this report, we investigated whether TGF-β1 could be a common regulator for MMPs, TIMPs and RECK in human breast cancer cell models.     

Guggulsterone sensitizes glioblastoma cells to Sonic hedgehog inhibitor SANT-1 induced apoptosis in a Ras/NFκB dependent manner

Since Shh pathway effector, Gli1, is overexpressed in gliomas, we investigated the effect of novel Shh inhibitor SANT-1 on glioma cell viability. Though SANT-1 failed to induce apoptosis, it reduced proliferation of glioma stem-like cells. Apart from canonical Shh cascade, Gli1 is also induced by non-canonical pathways including NFκB. Therefore, a combinatorial strategy with Ras/NFκB inhibitor, Guggulsterone, was employed to enhance effectiveness of SANT-1. Guggulsterone inhibited Ras and NFκB activity and sensitized cells to SANT-1 induced apoptosis via intrinsic apoptotic mechanism. Inhibition of either Ras or NFκB activity was sufficient to sensitize cells to SANT-1. Guggulsterone induced ERK activation also contributed to Caspase-9 activation. Since SANT-1 and Guggulsterone differentially target stem-like and non-stem glioma cells respectively, this combination warrants investigation as an effective anti-glioma therapy.     

Troglitazone, a PPAR agonist, inhibits human prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression

PPARγ ligands have been reported to reduce proliferation of human prostate cancer cells. However, the molecular mechanism of PPARγ agonist-induced cell growth inhibition of prostate cancer cells is not clear. GSK-3β expression and NFκB activity have important roles in prostate cancer development. To investigate the mechanisms of the PPARγ agonist-induced prostate cancer cell growth inhibition, we examined the effect of troglitazone on the expression of PPARγ, GSK-3β and activity of NFκB as well as on the prostate cancer cell growth. Troglitazone induced the expression of PPARγ in the nuclear of PC-3 cells, but not in LNCaP cells. Troglitazone (0-16 uM) inhibited cancer cell growth in a similar extend between both cells accompanied by the induction of cell cycle arrest in G(0)/G(1) phase and an increased in the similar extent of apoptotic cell death in concentration dependent manner. Troglitazone inhibited the constitutive expression of GSK-3β and activation of NFκB. Co-treatment of troglitazone with a GSK-3β inhibitor (AR-a014418) or GSK-3β siRNA significantly augmented the inhibitory effect of troglitazone on the NFκB activity and on prostate cancer cell growth inhibition and apoptotic cell death. However, overexpression of GSK-3β hindered troglitazone-induced cell growth inhibition and NFκB inactivation. These results suggest that PPARγ agonist, troglitazone, inhibits prostate cancer cell growth through inactivation of NFκB via suppression of GSK-3β expression.     

p38γ promotes breast cancer cell motility and metastasis through regulation of RhoC GTPase, cytoskeletal architecture, and a novel leading edge behavior

Understanding the molecular alterations that confer cancer cells with motile, metastatic properties is needed to improve patient survival. Here, we report that p38γ motogen-activated protein kinase regulates breast cancer cell motility and metastasis, in part, by controlling expression of the metastasis-associated small GTPase RhoC. This p38γ-RhoC regulatory connection was mediated by a novel mechanism of modulating RhoC ubiquitination. This relationship persisted across multiple cell lines and in clinical breast cancer specimens. Using a computational mechanical model based on the finite element method, we showed that p38γ-mediated cytoskeletal changes are sufficient to control cell motility. This model predicted novel dynamics of leading edge actin protrusions, which were experimentally verified and established to be closely related to cell shape and cytoskeletal morphology. Clinical relevance was supported by evidence that elevated expression of p38γ is associated with lower overall survival of patients with breast cancer. Taken together, our results offer a detailed characterization of how p38γ contributes to breast cancer progression. Herein we present a new mechanics-based analysis of cell motility, and report on the discovery of a leading edge behavior in motile cells to accommodate modified cytoskeletal architecture. In summary, these findings not only identify a novel mechanism for regulating RhoC expression but also advance p38γ as a candidate therapeutic target.     

Expression of Frat1 correlates with expression of β-catenin and is associated with a poor clinical outcome in human SCC and AC

Overexpression of frequently rearranged in advanced T-cell lymphomas 1 (Frat1) has been reported in several human malignant tumors, but the relationship between Frat1 and ??-catenin in lung cancer is still unclear. Our goal was to investigate the correlation between Frat1 and ??-catenin in patients with lung cancers. Immunohistochemistry was performed in 110 cases of non-small cell lung cancer (NSCLC) with clinical follow-up. Results showed that both Frat1 and ??-catenin were overexpressed in NSCLC. The expression of Frat1 and ??-catenin was significantly correlated with tumor differentiation, TNM stage, and lymph node metastasis. Interestingly, the overexpression of ??-catenin was positively correlated with the overexpression of Frat1 (correlation coefficient?=?0.285; P?=?0.003). In addition, overexpression of Frat1 and abnormal expression of ??-catenin were found to represent a poor prognosis for the patients. Furthermore, based on the transfection of Frat1 and ??-catenin, we found that Frat1 can upregulate the expression of ??-catenin in BE1 cells.     

TGF-Δ isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity

The literature on TGF-Δ in cancer including data on the expression or activation of TGF-Δ pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-Δ pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-Δ isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-Δ1, TGF-Δ2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-Δ isoform directed therapies. Of note, TGF-Δ antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-β isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-Δ directed antisense therapies based upon TGF-β immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.     

Polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma A375 cells through a mitochondria-mediated ROS–p38–p53 pathway

Polygonatum cyrtonema lectin (PCL), a mannose-binding lectin, has been reported to induce cytotoxicity and apoptosis. Herein, we demonstrated that PCL-induced apoptosis and autophagy in A375 cells. The apoptotic mechanism was that PCL treatment regulated Bax, Bcl-xL and Bcl-2 proteins, leading to mitochondrial depolarization, cytochrome c release and caspase activation. Subsequently, we found that PCL treatment abrogated glutathione antioxidant system and induced mitochondria to generate ROS accumulation, resulting in p38–p53 activation. Moreover, we confirmed that the ROS–p38–p53 pathway was involved in PCL-induced autophagy. In conclusion, these results indicate that PCL induces apoptosis and autophagy via a mitochondrial-mediated ROS–p38–p53 pathway.     

p38 MAPK and NF-κB pathways are involved in naphtho[1,2-b] furan-4,5-dione induced anti-proliferation and apoptosis of human hepatoma cells

Naphtho[1,2-b] furan-4,5-dione (NFD) was investigated for its anti-proliferation effect on human hepatocellular carcinoma (HCC), Hep3B, HepG₂, and Huh-7 cells. The effect of NFD on inhibiting proliferation and apoptosis was correlated with up-regulation of pro-apoptotic protein and down-regulation of pro-survival proteins. Remarkably, we found that NFD inhibited the nuclear translocation of NF-κB, likely accounting for the down-regulation of pro-survival Bcl-2 family. Furthermore, suppression of p38 MAPK activity by a specific inhibitor significantly rescued the cell proliferation inhibited by NFD. These findings suggest that signaling imbalance between p38 MAPK and NF-κB by NFD results in the proliferative inhibition and apoptosis of HCC tumor cells.     

Zipper-interacting protein kinase promotes epithelial-mesenchymal transition,invasion and metastasis through AKT and NF-κB signaling and is associated with metastasis and poor prognosis in gastric cancer patients

Zipper-interacting Protein Kinase (ZIPK) belongs to the death-associated protein kinase family. ZIPK has been characterized as a tumor suppressor in various tumors, including gastric cancer. On the other hand, ZIPK also promotes cell survival. In this study, both in vitro and in vivo assays indicated that ZIPK promoted cell growth, proliferation, migration, invasion, tumor formation and metastasis in nude mice. ZIPK induced epithelial-mesenchymal transition (EMT) with increasing expression of β-catenin, mesenchymal markers, Snail and Slug, and with decreasing expression of E-cadherin. Furthermore, ZIPK activated the AKT/IκB/NF-κB pathway, which can promote EMT and metastasis. Additionally, ZIPK expression was detected in human primary gastric cancer and their matched metastatic lymph node samples by immunohistochemistry. Increased expression of ZIPK in lymph node metastases was significantly associated with stage VI and abdominal organ invasion. Survival analysis revealed that patients with increased ZIPK expression in metastatic lymph nodes had poor disease-specific survival. Taken together, our study reveals that ZIPK is a pro-oncogenic factor, which promotes cancer metastasis.