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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

Activation of NF-κB is essential for EGF-induced PTX3 expression(A) RelA-, MEK1-, and JNK2-deficient cell lines were selected by infecting KB cells with a lentivirus containing an expression vector encoding short hairpin (sh)RNA against RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2). Expressions of RelA, MEK1, JNK2, and GAPDH mRNAs were analyzed by an RT-PCR and examined in 2% agarose gels. shLacZ, negative control. (B) shRNA containing cells was treated with 50 ng/ml EGF for 3 h, and expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and Western blotting (WB). shLacZ, negative control. (C) KB cells were treated with 25 μM LY294002, 10 μM parthenolide, or 0.1% DMSO for 1 h, followed by treatment with 50 ng/ml EGF for 3 h. Expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and WB. (D) The construct of the pTK promoter with five repeated NF-κB-binding sites bearing the luciferase gene is presented (upper panel). KB cells were transfected with 0.5 μg pTK-NF-κB promoter, 1 μg dominant negative IκB (DN-IκB) expression vector, and 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized (lower panel). (E) KB cells were transfected with 1 μg DN-IκB expression vector or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h before extraction of RNA or lysates. Expressions of PTX3, IκB, GAPDH, and α-tubulin mRNAs and proteins were respectively analyzed by an RT-PCR (PCR) and Western blotting (WB). (F) KB cells were transfected with 0.5 μg PTX3 promoter construct, 1 μg DN-IκB expression vector, or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
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Figure 2: Activation of NF-κB is essential for EGF-induced PTX3 expression(A) RelA-, MEK1-, and JNK2-deficient cell lines were selected by infecting KB cells with a lentivirus containing an expression vector encoding short hairpin (sh)RNA against RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2). Expressions of RelA, MEK1, JNK2, and GAPDH mRNAs were analyzed by an RT-PCR and examined in 2% agarose gels. shLacZ, negative control. (B) shRNA containing cells was treated with 50 ng/ml EGF for 3 h, and expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and Western blotting (WB). shLacZ, negative control. (C) KB cells were treated with 25 μM LY294002, 10 μM parthenolide, or 0.1% DMSO for 1 h, followed by treatment with 50 ng/ml EGF for 3 h. Expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and WB. (D) The construct of the pTK promoter with five repeated NF-κB-binding sites bearing the luciferase gene is presented (upper panel). KB cells were transfected with 0.5 μg pTK-NF-κB promoter, 1 μg dominant negative IκB (DN-IκB) expression vector, and 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized (lower panel). (E) KB cells were transfected with 1 μg DN-IκB expression vector or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h before extraction of RNA or lysates. Expressions of PTX3, IκB, GAPDH, and α-tubulin mRNAs and proteins were respectively analyzed by an RT-PCR (PCR) and Western blotting (WB). (F) KB cells were transfected with 0.5 μg PTX3 promoter construct, 1 μg DN-IκB expression vector, or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.

Mentions: To clarify the signaling pathways involved in the regulation of EGF-induced PTX3 expression, downstream targets regulated by EGF were examined. As shown in Supplementary Fig. 3A, EGF stimulated activation of ERK1/2 and Akt by enhancing kinase phosphorylation in head and neck cancer cells. In addition, EGF also increased the phosphorylation IκBα, resulting in a decrease in the IκBα level (Supplementary Fig. 3A). Subsequently, we investigated whether the decrease in IκBα led to an increase in the nuclear translocation of NF-κB in EGF-treated cells. Indeed, EGF significantly induced the nuclear translocation of NF-κB (Supplementary Fig. 3B). These results suggest that EGF activates the ERK and Akt/NF-κB signaling pathways in HNSCC. To clarify which EGF-activated signaling pathways are involved in regulating PTX3 expression, stable cell lines with RelA-, MEK1-, and JNK2-knockdown via short hairpin (sh)RNA knockdown of RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2), respectively, were confirmed and used (Fig. 2A). As shown in Fig. 2B, EGF-induced expression of PTX3 was inhibited in shRelA cells, but not in shMEK1 or shJNK2 cells. In addition, LY294002 and parthenolide, inhibitors of phosphoinositide 3-kinases (PI3Ks) and NF-κB, respectively inhibited EGF-induced PTX3 mRNA and protein expressions (Fig. 2C). To further study the effect of permanently preventing NF-κB activation on EGF-induced PTX3 expression, we utilized a dominant negative form of IκB (DN-IκB) that lacked all N-terminal phosphorylation sites, thus it is resistant to degradation but still had the ability to bind to NF-κB [20, 21]. We found that the overexpression of DN-IκB inhibited the basal activity of NF-κB, and also significantly reduced EGF-induced NF-κB activity (Fig. 2D). Consistently, EGF-induced PTX3 mRNA and protein expressions were dramatically reduced in DN-IκB-expressing cells (Fig. 2E). In addition, DN-IκB also inhibited EGF-induced PTX3 promoter activity (Fig. 2F). These results indicated that EGF-induced PTX3 expression was, at least in part, through activation of the PI3K/Akt and NF-κB pathways.


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)

Activation of NF-κB is essential for EGF-induced PTX3 expression(A) RelA-, MEK1-, and JNK2-deficient cell lines were selected by infecting KB cells with a lentivirus containing an expression vector encoding short hairpin (sh)RNA against RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2). Expressions of RelA, MEK1, JNK2, and GAPDH mRNAs were analyzed by an RT-PCR and examined in 2% agarose gels. shLacZ, negative control. (B) shRNA containing cells was treated with 50 ng/ml EGF for 3 h, and expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and Western blotting (WB). shLacZ, negative control. (C) KB cells were treated with 25 μM LY294002, 10 μM parthenolide, or 0.1% DMSO for 1 h, followed by treatment with 50 ng/ml EGF for 3 h. Expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and WB. (D) The construct of the pTK promoter with five repeated NF-κB-binding sites bearing the luciferase gene is presented (upper panel). KB cells were transfected with 0.5 μg pTK-NF-κB promoter, 1 μg dominant negative IκB (DN-IκB) expression vector, and 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized (lower panel). (E) KB cells were transfected with 1 μg DN-IκB expression vector or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h before extraction of RNA or lysates. Expressions of PTX3, IκB, GAPDH, and α-tubulin mRNAs and proteins were respectively analyzed by an RT-PCR (PCR) and Western blotting (WB). (F) KB cells were transfected with 0.5 μg PTX3 promoter construct, 1 μg DN-IκB expression vector, or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
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Figure 2: Activation of NF-κB is essential for EGF-induced PTX3 expression(A) RelA-, MEK1-, and JNK2-deficient cell lines were selected by infecting KB cells with a lentivirus containing an expression vector encoding short hairpin (sh)RNA against RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2). Expressions of RelA, MEK1, JNK2, and GAPDH mRNAs were analyzed by an RT-PCR and examined in 2% agarose gels. shLacZ, negative control. (B) shRNA containing cells was treated with 50 ng/ml EGF for 3 h, and expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and Western blotting (WB). shLacZ, negative control. (C) KB cells were treated with 25 μM LY294002, 10 μM parthenolide, or 0.1% DMSO for 1 h, followed by treatment with 50 ng/ml EGF for 3 h. Expressions of PTX3 mRNA and protein were respectively analyzed by an RT-PCR and WB. (D) The construct of the pTK promoter with five repeated NF-κB-binding sites bearing the luciferase gene is presented (upper panel). KB cells were transfected with 0.5 μg pTK-NF-κB promoter, 1 μg dominant negative IκB (DN-IκB) expression vector, and 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized (lower panel). (E) KB cells were transfected with 1 μg DN-IκB expression vector or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h before extraction of RNA or lysates. Expressions of PTX3, IκB, GAPDH, and α-tubulin mRNAs and proteins were respectively analyzed by an RT-PCR (PCR) and Western blotting (WB). (F) KB cells were transfected with 0.5 μg PTX3 promoter construct, 1 μg DN-IκB expression vector, or 1 μg control vector by lipofection and then treated with 50 ng/ml EGF for 6 h. Luciferase activities and protein concentrations were then determined and normalized. Values represent the mean ± S.E. of three determinations.
Mentions: To clarify the signaling pathways involved in the regulation of EGF-induced PTX3 expression, downstream targets regulated by EGF were examined. As shown in Supplementary Fig. 3A, EGF stimulated activation of ERK1/2 and Akt by enhancing kinase phosphorylation in head and neck cancer cells. In addition, EGF also increased the phosphorylation IκBα, resulting in a decrease in the IκBα level (Supplementary Fig. 3A). Subsequently, we investigated whether the decrease in IκBα led to an increase in the nuclear translocation of NF-κB in EGF-treated cells. Indeed, EGF significantly induced the nuclear translocation of NF-κB (Supplementary Fig. 3B). These results suggest that EGF activates the ERK and Akt/NF-κB signaling pathways in HNSCC. To clarify which EGF-activated signaling pathways are involved in regulating PTX3 expression, stable cell lines with RelA-, MEK1-, and JNK2-knockdown via short hairpin (sh)RNA knockdown of RelA (shRelA), MEK1 (shMEK1), and JNK2 (shJNK2), respectively, were confirmed and used (Fig. 2A). As shown in Fig. 2B, EGF-induced expression of PTX3 was inhibited in shRelA cells, but not in shMEK1 or shJNK2 cells. In addition, LY294002 and parthenolide, inhibitors of phosphoinositide 3-kinases (PI3Ks) and NF-κB, respectively inhibited EGF-induced PTX3 mRNA and protein expressions (Fig. 2C). To further study the effect of permanently preventing NF-κB activation on EGF-induced PTX3 expression, we utilized a dominant negative form of IκB (DN-IκB) that lacked all N-terminal phosphorylation sites, thus it is resistant to degradation but still had the ability to bind to NF-κB [20, 21]. We found that the overexpression of DN-IκB inhibited the basal activity of NF-κB, and also significantly reduced EGF-induced NF-κB activity (Fig. 2D). Consistently, EGF-induced PTX3 mRNA and protein expressions were dramatically reduced in DN-IκB-expressing cells (Fig. 2E). In addition, DN-IκB also inhibited EGF-induced PTX3 promoter activity (Fig. 2F). These results indicated that EGF-induced PTX3 expression was, at least in part, through activation of the PI3K/Akt and NF-κB pathways.

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