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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-induced PTX3 regulates the expression of MMP9(A) HONE1 cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3, MMP1, MMP3, MMP7, MMP9, MMP10 and GAPDH mRNA were analyzed by RT-PCR and examined in 2% agarose gel. (B) Parental and shPTX3 HONE1 cells were treated with 50 ng/ml EGF for 6 h. The expression of MMP9 (I), MMP1 (II) and MMP10 (III) mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMPs were normalized by GADPH. Error bars indicate SEM of three independent experiments. shLacZ, negative control. (C) NONE1 cells were treated with 15 μg/ml anti-PTX3 antibodies, 15 μg/ml immunoglobulin G (IgG), 15 μg/ml anti-aryl hydrocarbon receptor nuclear translocator (ARNT) antibodies and 50 ng/ml EGF for 9 h. The expression of MMP9 mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMP9 were normalized by GADPH. Error bars indicate SEM of three independent experiments.
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Figure 7: EGF-induced PTX3 regulates the expression of MMP9(A) HONE1 cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3, MMP1, MMP3, MMP7, MMP9, MMP10 and GAPDH mRNA were analyzed by RT-PCR and examined in 2% agarose gel. (B) Parental and shPTX3 HONE1 cells were treated with 50 ng/ml EGF for 6 h. The expression of MMP9 (I), MMP1 (II) and MMP10 (III) mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMPs were normalized by GADPH. Error bars indicate SEM of three independent experiments. shLacZ, negative control. (C) NONE1 cells were treated with 15 μg/ml anti-PTX3 antibodies, 15 μg/ml immunoglobulin G (IgG), 15 μg/ml anti-aryl hydrocarbon receptor nuclear translocator (ARNT) antibodies and 50 ng/ml EGF for 9 h. The expression of MMP9 mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMP9 were normalized by GADPH. Error bars indicate SEM of three independent experiments.

Mentions: Based on the observation that PTX3 expression is essential for EGF-enhanced cancer metastasis, we next clarified the mechanisms involved in PTX3-regulated cell metastasis. The expressions of fibronectin and integrin β1, and the phosphorylation of Akt increased in EGF-treated cells (Fig. 6A). In addition, EGF inhibited the expression of E-cadherin, but had no effect on vimentin or N-cadherin expressions (Fig. 6A). Although no changes in EGF-induced integrin β1 or phospho-Akt were observed in shPTX3 cells, EGF-regulated expressions of fibronectin and E-cadherin were significantly inhibited in PTX3-knockdown cells (Fig. 6B). EGF-induced expression of fibronectin was also inhibited when PTX3 was neutralized by anti-PTX3 antibodies (Fig. 6C). In addition, repression of fibronectin expression in shPTX3 cells was abolished when cells were treated with EGF and PTX3 (Fig. 6D). To further confirm the function of fibronectin for tumor invasion in EGF-treated cells, we study the transendothelial invasion in EGF- and PTX3-treated fibronectin knockdown cells. As shown in Fig. 6E, even in cotreatment with PTX3, EGF-induced invasion was blocked in loss-of-fibronectin. These results suggested that the induction of PTX3/fibronectin axis was essential for EGF-induced tumor invasion. The activation of the Rac1/cdc42 was a key regulator downstream of fibronectin to mediate cell migration. We examined whether EGF-activated Rac1/cdc42 is regulated by fibronectin expression. We found that EGF-induced phosphorylation of Rac1/cdc42 was significantly inhibited in the depletion of fibronectin (Supplementary Fig. 10A). Again, siRNAs targeted fibronectin also inhibited EGF-induced cell invasion (Supplementary Fig. 10B). To further investigate whether EGF-regulated MMP expressions in HNSCC are due to the induction of PTX3, expressions of MMPs were examined in PTX3-depleted cells. As shown in Fig. 7A and 7B, the expression of EGF-induced MMP9 was repressed in PTX3-knockdown cells. However, no changes in EGF-induced MMP1 or MMP10 were observed in shPTX3 cells. EGF-induced expression of MMP9 was also inhibited when PTX3 was neutralized by anti-PTX3 antibodies (Fig. 7C). Taken together, these results demonstrated that the EGF/PTX3/fibronectin axis is an important pathway in regulating HNSCC migration and invasion.


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-induced PTX3 regulates the expression of MMP9(A) HONE1 cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3, MMP1, MMP3, MMP7, MMP9, MMP10 and GAPDH mRNA were analyzed by RT-PCR and examined in 2% agarose gel. (B) Parental and shPTX3 HONE1 cells were treated with 50 ng/ml EGF for 6 h. The expression of MMP9 (I), MMP1 (II) and MMP10 (III) mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMPs were normalized by GADPH. Error bars indicate SEM of three independent experiments. shLacZ, negative control. (C) NONE1 cells were treated with 15 μg/ml anti-PTX3 antibodies, 15 μg/ml immunoglobulin G (IgG), 15 μg/ml anti-aryl hydrocarbon receptor nuclear translocator (ARNT) antibodies and 50 ng/ml EGF for 9 h. The expression of MMP9 mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMP9 were normalized by GADPH. Error bars indicate SEM of three independent experiments.
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Related In: Results  -  Collection

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Figure 7: EGF-induced PTX3 regulates the expression of MMP9(A) HONE1 cells were treated with 50 ng/ml EGF for a period of time as indicated. Expressions of PTX3, MMP1, MMP3, MMP7, MMP9, MMP10 and GAPDH mRNA were analyzed by RT-PCR and examined in 2% agarose gel. (B) Parental and shPTX3 HONE1 cells were treated with 50 ng/ml EGF for 6 h. The expression of MMP9 (I), MMP1 (II) and MMP10 (III) mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMPs were normalized by GADPH. Error bars indicate SEM of three independent experiments. shLacZ, negative control. (C) NONE1 cells were treated with 15 μg/ml anti-PTX3 antibodies, 15 μg/ml immunoglobulin G (IgG), 15 μg/ml anti-aryl hydrocarbon receptor nuclear translocator (ARNT) antibodies and 50 ng/ml EGF for 9 h. The expression of MMP9 mRNA was also analyzed by Real-time quantitative PCR. Relative levels of MMP9 were normalized by GADPH. Error bars indicate SEM of three independent experiments.
Mentions: Based on the observation that PTX3 expression is essential for EGF-enhanced cancer metastasis, we next clarified the mechanisms involved in PTX3-regulated cell metastasis. The expressions of fibronectin and integrin β1, and the phosphorylation of Akt increased in EGF-treated cells (Fig. 6A). In addition, EGF inhibited the expression of E-cadherin, but had no effect on vimentin or N-cadherin expressions (Fig. 6A). Although no changes in EGF-induced integrin β1 or phospho-Akt were observed in shPTX3 cells, EGF-regulated expressions of fibronectin and E-cadherin were significantly inhibited in PTX3-knockdown cells (Fig. 6B). EGF-induced expression of fibronectin was also inhibited when PTX3 was neutralized by anti-PTX3 antibodies (Fig. 6C). In addition, repression of fibronectin expression in shPTX3 cells was abolished when cells were treated with EGF and PTX3 (Fig. 6D). To further confirm the function of fibronectin for tumor invasion in EGF-treated cells, we study the transendothelial invasion in EGF- and PTX3-treated fibronectin knockdown cells. As shown in Fig. 6E, even in cotreatment with PTX3, EGF-induced invasion was blocked in loss-of-fibronectin. These results suggested that the induction of PTX3/fibronectin axis was essential for EGF-induced tumor invasion. The activation of the Rac1/cdc42 was a key regulator downstream of fibronectin to mediate cell migration. We examined whether EGF-activated Rac1/cdc42 is regulated by fibronectin expression. We found that EGF-induced phosphorylation of Rac1/cdc42 was significantly inhibited in the depletion of fibronectin (Supplementary Fig. 10A). Again, siRNAs targeted fibronectin also inhibited EGF-induced cell invasion (Supplementary Fig. 10B). To further investigate whether EGF-regulated MMP expressions in HNSCC are due to the induction of PTX3, expressions of MMPs were examined in PTX3-depleted cells. As shown in Fig. 7A and 7B, the expression of EGF-induced MMP9 was repressed in PTX3-knockdown cells. However, no changes in EGF-induced MMP1 or MMP10 were observed in shPTX3 cells. EGF-induced expression of MMP9 was also inhibited when PTX3 was neutralized by anti-PTX3 antibodies (Fig. 7C). Taken together, these results demonstrated that the EGF/PTX3/fibronectin axis is an important pathway in regulating HNSCC migration and invasion.

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