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

Chang WC, Wu SL, Huang WC, Hsu JY, Chan SH, Wang JM, Tsai JP, Chen BK - Oncotarget (2015)

Bottom Line: 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 conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacy, Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

EGF induces transcriptional activation of PTX3 gene expression in head and neck squamous cell carcinoma (HNSCC) cell lines(A) HNSCC cell lines were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR and examination in 2% agarose gels. (B) Lysates of cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (C) KB cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR (upper panel) and a real-time quantitative PCR (lower panel). Relative levels of PTX3 were normalized to GADPH. Values represent the mean ± S.E. of three independent experiments. (D) Lysates of EGF-treated KB cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (E) KB cells were treated with 50 ng/ml EGF for a period of time, and then conditioned medium was collected to analyze PTX3 protein by an ELISA. Values represent the mean ± S.E. of three independent experiments. (F) KB cells were transfected with 0.5 μg PTX3 promoter construct by lipofection and then treated with 50 ng/ml EGF for various times as indicated. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
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Figure 1: EGF induces transcriptional activation of PTX3 gene expression in head and neck squamous cell carcinoma (HNSCC) cell lines(A) HNSCC cell lines were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR and examination in 2% agarose gels. (B) Lysates of cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (C) KB cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR (upper panel) and a real-time quantitative PCR (lower panel). Relative levels of PTX3 were normalized to GADPH. Values represent the mean ± S.E. of three independent experiments. (D) Lysates of EGF-treated KB cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (E) KB cells were treated with 50 ng/ml EGF for a period of time, and then conditioned medium was collected to analyze PTX3 protein by an ELISA. Values represent the mean ± S.E. of three independent experiments. (F) KB cells were transfected with 0.5 μg PTX3 promoter construct by lipofection and then treated with 50 ng/ml EGF for various times as indicated. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.

Mentions: To investigate the association of the PTX3 gene expression signature with HNSCC, data mining on the cancer microarray database, Oncomine 4.0 (Oncomine DB at http://www.oncomine.org) [19], was used. PTX3 expressions between normal and malignant or metastatic tissues from HNSCC patients were analyzed using published datasets. The expression level of PTX3 in malignant tissues was higher than that in normal tissues in HNSCC patients (Supplementary Fig. 1A). Significantly, PTX3 expression was obvious in metastatic tissues (Supplementary Fig. 1B). These results suggested that PTX3 mRNA was overexpressed (p < 0.05) in clinical HNSCC tissues. We further studied PTX3 expression in various malignant tumor cells treated with EGF. Interestingly, we found that EGF significantly induced PTX3 gene expression (Fig. 1A) and protein production (Fig. 1B) in time-dependent manners in head and neck cancer cell lines, but a tiny induction was observed in HeLa cells (Supplementary Fig. 2). The RT-PCR and real-time quantitative RT-PCR results showed that the PTX3 mRNA level was substantially elevated and reached a peak after 3 h of EGF treatment (Fig. 1C). These results revealed that PTX3 was significantly induced by EGF in head and neck cancer cells. To further confirm the induction of PTX3 by EGF, the expression and secretion of PTX3 were examined in cell lysates and conditioned media, respectively. As shown in Fig. 1D and 1E, EGF also increased PTX3 protein production and secretion in cultured media in time-dependent manners. To investigate whether the alteration of transcriptional activity was responsible for EGF-induced PTX3 gene expression, we studied the effects of EGF on PTX3 promoter activity using a luciferase reporter assay. As shown in Fig. 1F, EGF induced substantial PTX3 promoter activity in a time-dependent manner. These results revealed that EGF stimulated PTX3 expression through transcriptional activation, resulting in the generation of PTX3.


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

Chang WC, Wu SL, Huang WC, Hsu JY, Chan SH, Wang JM, Tsai JP, Chen BK - Oncotarget (2015)

EGF induces transcriptional activation of PTX3 gene expression in head and neck squamous cell carcinoma (HNSCC) cell lines(A) HNSCC cell lines were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR and examination in 2% agarose gels. (B) Lysates of cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (C) KB cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR (upper panel) and a real-time quantitative PCR (lower panel). Relative levels of PTX3 were normalized to GADPH. Values represent the mean ± S.E. of three independent experiments. (D) Lysates of EGF-treated KB cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (E) KB cells were treated with 50 ng/ml EGF for a period of time, and then conditioned medium was collected to analyze PTX3 protein by an ELISA. Values represent the mean ± S.E. of three independent experiments. (F) KB cells were transfected with 0.5 μg PTX3 promoter construct by lipofection and then treated with 50 ng/ml EGF for various times as indicated. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
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Related In: Results  -  Collection

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Figure 1: EGF induces transcriptional activation of PTX3 gene expression in head and neck squamous cell carcinoma (HNSCC) cell lines(A) HNSCC cell lines were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR and examination in 2% agarose gels. (B) Lysates of cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (C) KB cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3 and GAPDH mRNA were analyzed by an RT-PCR (upper panel) and a real-time quantitative PCR (lower panel). Relative levels of PTX3 were normalized to GADPH. Values represent the mean ± S.E. of three independent experiments. (D) Lysates of EGF-treated KB cells were prepared and subjected to SDS-PAGE and analyzed by Western blotting with antibodies against PTX3 and α-tubulin. (E) KB cells were treated with 50 ng/ml EGF for a period of time, and then conditioned medium was collected to analyze PTX3 protein by an ELISA. Values represent the mean ± S.E. of three independent experiments. (F) KB cells were transfected with 0.5 μg PTX3 promoter construct by lipofection and then treated with 50 ng/ml EGF for various times as indicated. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
Mentions: To investigate the association of the PTX3 gene expression signature with HNSCC, data mining on the cancer microarray database, Oncomine 4.0 (Oncomine DB at http://www.oncomine.org) [19], was used. PTX3 expressions between normal and malignant or metastatic tissues from HNSCC patients were analyzed using published datasets. The expression level of PTX3 in malignant tissues was higher than that in normal tissues in HNSCC patients (Supplementary Fig. 1A). Significantly, PTX3 expression was obvious in metastatic tissues (Supplementary Fig. 1B). These results suggested that PTX3 mRNA was overexpressed (p < 0.05) in clinical HNSCC tissues. We further studied PTX3 expression in various malignant tumor cells treated with EGF. Interestingly, we found that EGF significantly induced PTX3 gene expression (Fig. 1A) and protein production (Fig. 1B) in time-dependent manners in head and neck cancer cell lines, but a tiny induction was observed in HeLa cells (Supplementary Fig. 2). The RT-PCR and real-time quantitative RT-PCR results showed that the PTX3 mRNA level was substantially elevated and reached a peak after 3 h of EGF treatment (Fig. 1C). These results revealed that PTX3 was significantly induced by EGF in head and neck cancer cells. To further confirm the induction of PTX3 by EGF, the expression and secretion of PTX3 were examined in cell lysates and conditioned media, respectively. As shown in Fig. 1D and 1E, EGF also increased PTX3 protein production and secretion in cultured media in time-dependent manners. To investigate whether the alteration of transcriptional activity was responsible for EGF-induced PTX3 gene expression, we studied the effects of EGF on PTX3 promoter activity using a luciferase reporter assay. As shown in Fig. 1F, EGF induced substantial PTX3 promoter activity in a time-dependent manner. These results revealed that EGF stimulated PTX3 expression through transcriptional activation, resulting in the generation of PTX3.

Bottom Line: 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 conclusion, PI3K/Akt and NF-κB-dependent regulation of AP-1 mediates PTX3 transcriptional responses to EGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacy, Master Program for Clinical Pharmacogenomics and Pharmacoproteomics, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus