Functional Role of DUSP6 in the EGFR-mediated Progression of Head and Neck Squamous Cell Carcinoma.

BACKGROUND/AIM: Epidermal growth factor receptor (EGFR) over-expression is commonly observed in advanced head and neck squamous cell carcinoma (HNSCC) and is correlated with poor patient outcomes. However, the role of dual-specificity phosphatase 6 (DUSP6) in EGFR-associated HNSCC progression remains poorly understood. This study aimed to investigate the correlation between DUSP6 expression and EGFR signaling in malignant HNSCC tissues. MATERIALS AND METHODS: Data mining and in vitro assays were employed to assess DUSP6 expression levels in HNSCC tissues compared to normal tissues. Additionally, the correlation between DUSP6 and EGFR expression was examined. Functional assays were conducted to investigate the modulation of DUSP6 expression by EGFR signaling and its involvement in EGF-induced cell migration and anoikis resistance. RESULTS: Our analysis revealed a significant elevation in DUSP6 expression in HNSCC tissues compared to normal tissues and a strong correlation between DUSP6 and EGFR expression. EGFR signaling modulated DUSP6 expression in a dose- and time-dependent manner, primarily through the extracellular signal-regulated kinase (ERK) pathway. Knockdown experiments demonstrated the functional role of DUSP6 in EGF-induced cell migration and anoikis resistance. CONCLUSION: The findings of this study elucidate the intricate signaling networks governing DUSP6 expression and its interplay with EGFR signaling in HNSCC. Moreover, the results provide insights into the potential role of DUSP6 as a therapeutic target and highlight the importance of personalized treatment strategies in HNSCC management.

RNA-binding protein-regulated fibronectin is essential for EGFR-activated metastasis of head and neck squamous cell carcinoma.

There is a higher expression level of epidermal growth factor receptor (EGFR) in up to 90% of advanced head and neck squamous cell carcinoma (HNSCC) tissue than in normal surrounding tissues. However, the role of RNA-binding proteins (RBPs) in EGFR-associated metastasis of HNSCC remains unclear. In this study, we reveal that RBPs, specifically nucleolin (NCL) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1), correlated with the mesenchymal phenotype of HNSCC. The depletion of RBPs significantly attenuated EGF-induced HNSCC metastasis. Intriguingly, the EGF-induced EMT markers, such as fibronectin, were regulated by RBPs through the ERK and NF-κB pathway, followed by the enhancement of mRNA stability of fibronectin through the 5' untranslated region (5'-UTR) of the gene. The upregulation of fibronectin triggered the integrin signaling activation to enhance tumor cells' attachment to endothelial cells and increase endothelial permeability. In addition, the concurrence of EGFR and RBPs or EGFR and fibronectin was associated with overall survival and disease-free survival of HNSCC. The in vivo study showed that depletion of NCL, hnRNPA2B1, and fibronectin significantly inhibited EGF-promoted extravasation of tumor cells into lung tissues. The depletion of fibronectin or treatment with integrin inhibitors dramatically attenuated EGF-induced HNSCC metastatic nodules in the lung. Our data suggest that the RBPs/fibronectin axis is essential for EGF-induced tumor-endothelial cell interactions to enhance HNSCC cell metastasis.

Oleic acid-induced metastasis of KRAS/p53-mutant colorectal cancer relies on concurrent KRAS activation and IL-8 expression bypassing EGFR activation.

Background: Multigene mutations in colorectal cancer (CRC), including KRAS, BRAF, and p53, afford high metastatic ability and resistance to EGFR-targeting therapy. Understanding the molecular mechanisms regulating anti-EGFR-resistant CRC metastasis can improve CRC therapy. This study aimed to investigate the effects of IL-8 and the activation of KRAS on reactive oxygen species (ROS) production and metastasis of hyperlipidemia-associated CRC harboring mutations of KRAS and p53. Methods: The cytokine array analysis determined the up-expression of secreted factors, including IL-8. The clinical relevance of the relationship between IL-8 and angiopoietin-like 4 (ANGPTL4) was examined in CRC patients from National Cheng Kung University Hospital and TCGA dataset. Expressions of IL-8, ANGPTL4, NADPH oxidase 4 (NOX4), and epithelial-mesenchymal transition (EMT) markers in free fatty acids (FFAs)-treated KRAS/p53 mutant CRC cells were determined. The hyperlipidemia-triggered metastatic ability of CRC cells under treatments of antioxidants, statin, and cetuximab or knockdown of IL-8, KRAS, and EGFR was evaluated in vitro and in vivo. In addition, the effects of antioxidants and depletion of IL-8 and KRAS on the correlation between ROS production and hyperlipidemia-promoted CRC metastasis were also clarified. Results: In this study, we found that free fatty acids promoted KRAS/p53-mutant but not single-mutant or non-mutant CRC cell metastasis. IL-8, the most abundant secreted factor in KRAS/p53-mutant cells, was correlated with the upregulation of NOX4 expression and ROS production under oleic acid (OA)-treated conditions. In addition, the metastasis of KRAS/p53-mutant CRC relies on the ANGPTL4/IL-8/NOX4 axis and the activation of KRAS. The antioxidants and inactivation of KRAS also inhibited OA-induced EMT and metastasis. Although KRAS mediated EGF- and OA-promoted CRC cell invasion, the inhibition of EGFR did not affect OA-induced ANGPTL4/IL-8/NOX4 axis and CRC metastasis. The high-fat diet mice fed with vitamin E and statin or in IL-8-depleted cells significantly inhibited tumor extravasation and metastatic lung growth of CRC. Conclusion: The antioxidants, statins, and targeting IL-8 may provide better outcomes for treating metastatic CRC that harbors multigene mutations and anti-EGFR resistance.

ARNT deficiency represses pyruvate dehydrogenase kinase 1 to trigger ROS production and melanoma metastasis.

The metabolic changes in melanoma cells that are required for tumor metastasis have not been fully elucidated. In this study, we show that the increase in glucose uptake and mitochondrial oxidative phosphorylation confers metastatic ability as a result of aryl hydrocarbon receptor nuclear translocator (ARNT) deficiency. In clinical tissue specimens, increased ARNT, pyruvate dehydrogenase kinase 1 (PDK1), and NAD(P)H quinine oxidoreductase-1 (NQO1) was observed in benign nevi, whereas lower expression was observed in melanoma. The depletion of ARNT dramatically repressed PDK1 and NQO1 expression, which resulted in an increase of ROS levels. The elimination of ROS using N-acetylcysteine (NAC) and inhibition of oxidative phosphorylation using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and rotenone inhibited the ARNT and PDK1 deficiency-induced cell migration and invasion. In addition, ARNT deficiency in tumor cells manipulated the glycolytic pathway through enhancement of the glucose uptake rate, which reduced glucose dependence. Intriguingly, CCCP and NAC dramatically inhibited ARNT and PDK1 deficiency-induced tumor cell extravasation in mouse models. Our work demonstrates that downregulation of ARNT and PDK1 expression serves as a prognosticator, which confers metastatic potential as the metastasizing cells depend on metabolic changes.

Evaluation for the Genetic Association between Store-Operated Calcium Influx Pathway (STIM1 and ORAI1) and Human Hepatocellular Carcinoma in Patients with Chronic Hepatitis B Infection.

Hepatocellular carcinoma (HCC) often develops from chronic hepatitis B (CHB) through replication of hepatitis B virus (HBV) infection. Calcium (Ca2+) signaling plays an essential role in HBV replication. Store-operated calcium (SOC) channels are a major pathway of Ca2+ entry into non-excitable cells such as immune cells and cancer cells. The basic components of SOC signaling include the STIM1 and ORAI1 genes. However, the roles of STIM1 and ORAI1 in HBV-mediated HCC are still unclear. Thus, long-term follow-up of HBV cohort was carried out in this study. This study recruited 3631 patients with chronic hepatitis (345 patients with HCC, 3286 patients without HCC) in a Taiwanese population. Genetic variants of the STIM1 and ORAI1 genes were detected using an Axiom CHB1 genome-wide array. Clinical associations of 40 polymorphisms were analyzed. Three of the STIM1 single-nucleotide polymorphisms (SNPs) (rs6578418, rs7116520, and rs11030472) and one SNP of ORAI1 (rs6486795) showed a trend of being associated with HCC disease (p < 0.05). However, after correction for multiple testing, none of the SNPs reached a significant level (q > 0.05); in contrast, neither STIM1 nor ORAI1 showed a significant association with HCC progression in CHB patients. Functional studies by both total internal reflection fluorescence images and transwell migration assay indicated the critical roles of SOC-mediated signaling in HCC migration. In conclusion, we reported a weak correlation between STIM1/ORAI1 polymorphisms and the risk of HCC progression in CHB patients.

Hinokitiol induces cell death and inhibits epidermal growth factor-induced cell migration and signaling pathways in human cervical adenocarcinoma.

OBJECTIVE: The aim of this study was to examine the antitumor activity of hinokitiol for its clinical application in the treatment of human cervical carcinoma. MATERIALS AND METHODS: Cervical carcinoma HeLa cells were treated by different concentrations of hinokitiol. Flow cytometry was used to analyze cell cycle. Senescence-associated ß-galactosidase (SA-ß-gal) assay was used to identify senescent cells. The effects of hinokitiol on EGF-induced cell migration were determined by wound healing and transwell migration assays. Western blot was used to detect proteins involved in cell cycle progression, apoptosis, autophagy, and EGF-induced signaling pathways. RESULTS: Hinokitiol suppressed cell viability in a dose-dependent manner. Flow cytometric analysis indicated that hinokitiol treatment resulted in cell cycle arrest at G1 phase, with reduced number of cells in the G2/M phase. Western blot analysis further demonstrated that hinokitiol treatment increased the levels of p53 and p21, and concomitantly reduced the expression of cell cycle regulatory proteins, including cyclin D and cyclin E. SA-ß-gal assay showed that hinokitiol treatment significantly induced ß-galactosidase activity. In addition, treatment with hinokitiol increased the accumulation of the autophagy regulators, beclin 1 and microtubule-associated protein 1 light chain 3 (LC3-II), in a dose-dependent manner; however, it did not induce caspase-3 activation and poly ADP ribose polymerase (PARP) cleavage. In addition, epidermal growth factor-induced cell migration and c-Jun N-terminal kinase (JNK) and focal adhesion kinase (FAK) phosphorylation were significantly inhibited by hinokitiol. CONCLUSION: Our findings revealed that hinokitiol might serve as a potential therapeutic agent for cervical carcinoma therapy.

Oleic acid-induced NOX4 is dependent on ANGPTL4 expression to promote human colorectal cancer metastasis.

Background: Colorectal cancer (CRC) progression and related mortality are highly associated with metabolic disorders. However, the molecular mechanism involved in the regulation of hyperlipidemia-associated CRC metastasis remains unclear. This study aimed to investigate the effects of angiopoietin-like 4 (ANGPTL4) on NADPH oxidase 4 (NOX4) expression and reactive oxygen species (ROS) production, which might provide new targets for improving outcomes in patients with hyperlipidemia-associated CRC metastasis. Methods: The clinical relevance of relationship between NOX4 expression and ANGPTL4 was examined in CRC patients by the Oncomine and TCGA data set. Expressions of NOX4, epithelial-mesenchymal transition (EMT) markers, and gene regulation of NOX4 in free fatty acids (FFAs)-treated CRC cells were determined. The FFAs-triggered metastatic ability of CRC cells under treatments of antioxidants or knockdown of NOX4, ANGPTL4, and MMPs was evaluated in vitro and in vivo. In addition, effects of antioxidants and depletion of metastasis-associated molecules on the correlation between ROS production and FFAs-promoted CRC metastasis were also clarified. Results: In this study, we found that the induction of NOX4, followed by the increased ROS was essential for oleic acid (OA)-promoted CRC cell metastasis. The depletion of ANGPTL4 significantly inhibited c-Jun-mediated transactivation of NOX4 expression, accompanied with reduced levels of ROS, MMP-1, and MMP-9, resulting in the disruption of OA-promoted CRC cell metastasis. Moreover, knockdown of ANGPTL4, NOX4, MMP-1, and MMP-9 or the treatment of antioxidants dramatically inhibited circulating OA-enhanced tumor cell extravasation and metastatic seeding of tumor cells in lungs, indicating that the ANGPTL4/NOX4 axis was critical for dyslipidemia-associated tumor metastasis. Conclusion: The coincident expression of NOX4 and ANGPTL4 in CRC tumor specimens provides the insight into the potential therapeutic targets for the treatment of dyslipidemia-associated CRC metastasis.

Epidermal growth factor-induced COX-2 regulates metastasis of head and neck squamous cell carcinoma through upregulation of angiopoietin-like 4.

Epidermal growth factor receptor (EGFR) expression and activation are the major causes of metastasis in cancers such as head and neck squamous cell carcinoma (HNSCC). However, the reciprocal effect of EGF-induced COX-2 and angiopoietin-like 4 (ANGPTL4) on HNSCC metastasis remains unclear. In this study, we revealed that the expression of ANGPTL4 is essential for COX-2-derived prostaglandin E2 (PGE2 )-induced tumor cell metastasis. We showed that EGF-induced ANGPTL4 expression was dramatically inhibited with the depletion and inactivation of COX-2 by knockdown of COX-2 and celecoxib treatment, respectively. Prostaglandin E2 induced ANGPTL4 expression in a time- and dose-dependent manners in various HNSCC cell lines through the ERK pathway. In addition, the depletion of ANGPTL4 and MMP1 significantly impeded the PGE2 -induced transendothelial invasion ability of HNSCC cells and the binding of tumor cells to endothelial cells. The induction of molecules involved in the regulation of epithelial-mesenchymal transition was also dependent on ANGPTL4 expression in PGE2 -treated cells. The depletion of ANGPTL4 further blocked PGE2 -primed tumor cell metastatic seeding of lungs. These results indicate that the EGF-activated PGE2 /ANGPTL4 axis enhanced HNSCC metastasis. The concurrent expression of COX-2 and ANGPTL4 in HNSCC tumor specimens provides insight into potential therapeutic targets for the treatment of EGFR-associated HNSCC metastasis.

Functional Effects of let-7g Expression in Colon Cancer Metastasis.

MicroRNA regulation is crucial for gene expression and cell functions. It has been linked to tumorigenesis, development and metastasis in colorectal cancer (CRC). Recently, the let-7 family has been identified as a tumor suppressor in different types of cancers. However, the function of the let-7 family in CRC metastasis has not been fully investigated. Here, we focused on analyzing the role of let-7g in CRC. The Cancer Genome Atlas (TCGA) genomic datasets of CRC and detailed data from a Taiwanese CRC cohort were applied to study the expression pattern of let-7g. In addition, in vitro as well as in vivo studies have been performed to uncover the effects of let-7g on CRC. We found that the expression of let-7g was significantly lower in CRC specimens. Our results further supported the inhibitory effects of let-7g on CRC cell migration, invasion and extracellular calcium influx through store-operated calcium channels. We report a critical role for let-7g in the pathogenesis of CRC and suggest let-7g as a potential therapeutic target for CRC treatment.

Integrative genomic analysis for the functional roles of ITPKC in bone mineral density.

Osteoporosis is defined by low bone mineral density (BMD), which is mainly due to the imbalances in osteoclast and osteoblast activity. Previous studies indicated that early activation of osteoclasts relies on calcium entry through store-operated calcium (SOC) entry, and several genes, including STIM1, ORAI1, and ITPKC, are known as key regulators of SOC entry. However, the relationships between STIM1, ORAI1, ITPKC, and human BMD are still unclear. In order to investigate the plausible associations between these genes and BMD, we conducted a meta-analysis of genes expression and BMD using the publicly available GEO database. We further recruited 1044 subjects and tested associations between polymorphisms in these genes and BMD. Clinical information (including age, sex, and BMI) was collected and used for the analysis. Our results indicated that ITPKC gene expression was significantly associated with BMD. Furthermore, we found that one ITPKC SNP (rs2607420) was significantly associated with lumbar spine BMD. Through bioinformatics analysis, rs2607420 was found to be very likely to participate in the regulation of ITPKC expression. Our findings suggest that ITPKC is a susceptibility gene for BMD, and rs2607420 may play an important role in the regulation of this gene.

Prediction for Intravenous Immunoglobulin Resistance by Using Weighted Genetic Risk Score Identified From Genome-Wide Association Study in Kawasaki Disease.

BACKGROUND: Intravenous immunoglobulin (IVIG) is the treatment of choice in Kawasaki disease (KD). IVIG is used to prevent cardiovascular complications related to KD. However, a proportion of KD patients have persistent fever after IVIG treatment and are defined as IVIG resistant. METHODS AND RESULTS: To develop a risk scoring system based on genetic markers to predict IVIG responsiveness in KD patients, a total of 150 KD patients (126 IVIG responders and 24 IVIG nonresponders) were recruited for this study. A genome-wide association analysis was performed to compare the 2 groups and identified risk alleles for IVIG resistance. A weighted genetic risk score was calculated by the natural log of the odds ratio multiplied by the number of risk alleles. Eleven single-nucleotide polymorphisms were identified by genome-wide association study. The KD patients were categorized into 3 groups based on their calculated weighted genetic risk score. Results indicated a significant association between weighted genetic risk score (groups 3 and 4 versus group 1) and the response to IVIG (Fisher's exact P value 4.518×10-03 and 8.224×10-10, respectively). CONCLUSIONS: This is the first weighted genetic risk score study based on a genome-wide association study in KD. The predictive model integrated the additive effects of all 11 single-nucleotide polymorphisms to provide a prediction of the responsiveness to IVIG.

Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 expression enhances head and neck squamous cell carcinoma metastasis via up-regulation of fibronectin.

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in such cancers as head and neck squamous cell carcinoma (HNSCC); however, whether the metabolic enzyme, pyruvate dehydrogenase kinase 1 (PDK1), mediates EGF-enhanced HNSCC metastasis remains unclear. Of interest, we found that EGF induced PDK1 expression in HNSCC. Tumor cell transformation induced by EGF was repressed by PDK1 knockdown, and the down-regulation of PDK1 expression or inhibition of its activity significantly blocked EGF-enhanced cell migration and invasion. In addition, depletion of PDK1 impeded EGF-enhanced binding of HNSCC cells to endothelial cells as well as the metastatic seeding of tumor cells in lungs. PDK1 depletion inhibited EGF-induced matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Furthermore, PDK1 overexpression induced MMP-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. Of interest, depletion of fibronectin inhibited PDK1-enhanced MMP-1-3 and MMP-9 expression as well as Rac1/cdc42 activation and tumor invasion. These results demonstrate that EGF-induced PDK1 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. Inhibition of PDK1 may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.-Hsu, J.-Y., Chang, J.-Y., Chang, K.-Y., Chang, W.-C., Chen B.-K. Epidermal growth factor-induced pyruvate dehydrogenase kinase 1 expression enhances head and neck squamous cell carcinoma metastasis via up-regulation of fibronectin.

Oleate-induced PTX3 promotes head and neck squamous cell carcinoma metastasis through the up-regulation of vimentin.

The association between metabolic diseases and the risk of developing cancer is emerging. However, the impact of long pentraxin-3 (PTX3) on dyslipidemia-associated tumor metastasis remains unknown. In this study, we found that oleate induced PTX3 expression and secretion through the activation of Akt/NF-κB pathway in head and neck squamous cell carcinomas (HNSCCs). The activation of NF-κB was essential for the oleate-induced stabilization of PTX3 mRNA. In addition, both the depletion of PTX3 and the inhibition of NF-κB significantly inhibited oleate-induced tumor cell migration and invasion. The enhancement of binding between tumor and endothelial cells was observed in oleate-treated cells but not in the depletion and neutralization of PTX3 with siPTX3 and anti-PTX3 antibodies, respectively. The levels of oleate-induced epithelial-mesenchymal transition (EMT) markers, such as vimentin and MMP-3, were significantly reduced in PTX3-depleted cells. Knocking down vimentin also repressed oleate-induced HNSCC invasion. Furthermore, the depletion of PTX3 blocked the oleate-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that oleate enhances HNSCC metastasis through the PTX3/vimentin signaling axes. The inhibition of PTX3 could be a potential strategy for the treatment of dyslipidemia-mediated HNSCC metastasis.

Oleic acid-induced ANGPTL4 enhances head and neck squamous cell carcinoma anoikis resistance and metastasis via up-regulation of fibronectin.

Obese patients have higher levels of free fatty acids (FFAs) in their plasma and a higher risk of cancer than their non-obese counterparts. However, the mechanisms involved in the regulation of cancer metastasis by FFAs remain unclear. In this study, we found that oleic acid (OA) induced angiopoietin-like 4 (ANGPTL4) protein expression and secretion and conferred anoikis resistance to head and neck squamous cell carcinomas (HNSCCs). The autocrine production of OA-induced ANGPTL4 further promoted HNSCC migration and invasion. In addition, the expression of peroxisome proliferator-activated receptor (PPAR) was essential for the OA-induced ANGPTL4 expression and invasion. The levels of OA-induced epithelial-mesenchymal transition markers, such as vimentin, MMP-9, and fibronectin and its downstream effectors Rac1/Cdc42, were significantly reduced in ANGPTL4-depleted cells. Knocking down fibronectin inhibited the expression of MMP-9 and repressed OA- and recombinant ANGPTL4-induced HNSCC invasion. On the other hand, ANGPTL4 siRNA inhibited OA-induced MMP-9 expression, which was reversed in fibronectin-overexpressing cells. Furthermore, the depletion of ANGPTL4 impeded the OA-primed metastatic seeding of tumor cells in the lungs. These results demonstrate that OA enhances HNSCC metastasis through the ANGPTL4/fibronectin/Rac1/Cdc42 and ANGPTL4/fibronectin/MMP-9 signaling axes.

Involvement of L-type Ca²âº channel and toll-like receptor-4 in nickel-induced interleukin-8 gene expression.

The metal nickel (Ni(2+)) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP-1 cell line as a model. Interleukin (IL)-8 promoter activity as well as gene expression were tested by luciferase assay and real-time polymerase chain reaction. The underlying mechanisms of nickel-induced IL-8 were investigated. We found that nickel induced IL-8 gene expression via the L-type Ca(2+) channel, Toll-like receptor-4 (TRL-4) and nuclear factor NF-κB signal transduction pathways. Nickel activated NF-κB expression through extracellular signal-regulated kinase 1/2 phosphorylation and then increased IL-8 expression. Thus, the L-type Ca(2+) channel and TRL-4 play important roles in nickel-induced inflammatory gene expressions.

TNF up-regulates Pentraxin3 expression in human airway smooth muscle cells via JNK and ERK1/2 MAPK pathways.

BACKGROUND: Long pentraxin 3 (PTX3) is a novel candidate marker for inflammation in many chronic diseases. As a soluble pattern recognition receptor, PTX3 is involved in amplification of inflammatory reactions and regulation of innate immunity. Previously, we demonstrate that human airway smooth muscle cells (HASMC) express constitutively PTX3 and upon TNF stimulation. However, very little is known about the mechanism governing its expression in HASMC. We sought to investigate the mechanism governing TNF induced PTX3 expression in primary HASMC. METHODS: HASMC were stimulated with TNF in the presence of transcriptional inhibitor actinomycin D (ActD) or MAPKs pharmacological inhibitors. PTX3 mRNA and protein expression were analyzed by Real-time RT-PCR and ELISA, respectively. PTX3 promoter activity was determined using luciferase assay. RESULTS: PTX3 mRNA and protein are expressed constitutively by HASMC and significantly up-regulated by TNF. TNF-induced PTX3 mRNA and protein release in HASMC were inhibited by transcriptional inhibitor actinomycin D. TNF induced significantly PTX3 promoter activation in HASMC. MAPK JNK and ERK1/2 specific inhibitors (SP600125 and UO126), but not p38, significantly down regulates TNF induced PTX3 promoter activity and protein release in HASMC. Finally, TNF mediated PTX3 promoter activity in HASMC was abolished upon mutation of NF-κß and AP1 binding sites. CONCLUSIONS: Our data suggest that TNF induced PTX3 in HASMC at least via a transcriptional mechanism that involved MAPK (JNK and ERK1/2), NF-κß and AP1 pathways. These results rise the possibility that HASMC derived PTX3 may participate in immune regulation in the airways.

Targeting chemotherapy-induced PTX3 in tumor stroma to prevent the progression of drug-resistant cancers.

The tumor microenvironment has been suggested to participate in tumorigenesis, but the nature of the communication between cancer cells and the microenvironment, especially in response to anticancer drugs, remains obscure. We determined that activation of the CCAAT/enhancer binding protein delta (CEBPD) response to Cisplatin and 5-Fluorouracil in cancer-associated macrophages and fibroblasts contributed to the metastasis, invasion, acquired chemoresistance and stemness of cancer cells by in vitro and in vivo assays. Specifically, reporter and in vivo DNA binding assays were used to determine that Pentraxin 3 (PTX3) is a CEBPD responsive gene and serves a protumor role upon anticancer drug treatment. Finally, a PTX3 peptide inhibitor RI37 was developed and assessed the antitumor effects by in vivo assays. RI37 could function as a promising inhibitor for preventing cancer progression and the metastasis, invasion and progression of drug-resistant cancers. The identification of PTX3 provided a new insight in the interaction between host and tumor and the RI37 peptide showed a great opportunity to largely reduce the risk of invasion and metastasis of cancer and drug-resistant cancers.

Down-regulation of ARNT promotes cancer metastasis by activating the fibronectin/integrin ß1/FAK axis.

The aryl hydrocarbon receptor nuclear translocator (ARNT) is broadly involved in regulating tumorigenesis by inducing genes that are involved in tumor growth and angiogenesis. Tumorigenesis usually involves normoxic conditions. However, the role of ARNT in tumor metastasis during normoxia remains unclear. Here, we demonstrate that ARNT protein levels were decreased in late-stage human colorectal cancer using immunohistochemical analysis. Down-regulation of ARNT protein promoted cancer cell migration and invasion, which was mediated by activation of the fibronectin/integrin ß1/FAK signaling axis. In addition, the enhancement of migration and invasion in ANRT knockdown cells was blocked when ARNT was restored in the cells. In xenografts in severe combined immunodeficiency mice, tumor growth was significantly inhibited in the ARNT-knockdown condition. However, the tail-vein injection animal model revealed that the depletion of ARNT-induced metastatic lung colonies was further enhanced when ARNT expression was recovered post-injection. Interestingly, chemotherapeutic drugs inhibited ARNT expression and promoted the invasion of residual tumor cells. These results suggest that ARNT may play a positive role during tumor growth (either in early-stage tumor growth or in organ metastases), but plays a negative role in tumor migration and invasion. Therefore, the efficiency of ARNT-targeted therapy during different cancer stages should be carefully evaluated.

PTX3 gene activation in EGF-induced head and neck cancer cell metastasis.

Overexpression of the epidermal growth factor (EGF) receptor (EGFR) is associated with enhanced invasion and metastasis in head and neck squamous cell carcinoma (HNSCC). Long Pentraxin PTX3 is involved in immune escape in cancer cells. Here, we identified PTX3 as a promoting factor that mediates EGF-induced HNSCC metastasis. EGF-induced PTX3 transcriptional activation is via the binding of c-Jun to the activator protein (AP)-1 binding site of the PTX3 promoter. PI3K/Akt and NF-κB were essential for the PTX3 activation. EGF-induced PTX3 expression was blocked in c-Jun- and NF-κB-knockdown cells. EGF-mediated PTX3 secretion resulted in the enhancement of cell migration and invasion, and interactions between cancer and endothelial cells. The tail-vein injection animal model revealed that depletion of PTX3 decreased EGF-primed tumor cell metastatic seeding of the lungs. In addition, fibronectin, matrix metalloproteinase-9 (MMP9) and E-cadherin were essential components in EGFR/PTX3-mediated cancer metastasis. In conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF. Autocrine production of EGF-induced PTX3 in turn induces metastatic molecules, activating inflammatory cascades and metastasis.

Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation.

Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE2 compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE2. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis.