Limits...
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 enhances tumor migration and invasion(A) The migratory properties of KB cells were analyzed by a transwell assay using Boyden chambers. Parental and shPTX3 KB cells were incubated with or without 50 ng/ml EGF and 250 ng/ml PTX3 recombinant protein in the upper chamber of filter inserts. After incubation for 15 h, non-migratory cells were removed, and migratory cells were fixed and stained by a crystal violet procedure. Original magnification, ×200 (left panel). Migrating cells were counted (right panel). Values represent the mean ± S.E. of three determinations. shLacZ, negative control. (B) HONE1 cells were treated with various concentrations of anti-PTX3 antibodies (ab), 15 μg/ml immunoglobulin G (IgG) and 50 ng/ml EGF for 15 h. Migratory cells were fixed and stained by a crystal violet procedure. Migrating cells were counted. Values represent the mean ± S.E. of three determinations. (C) The invasive properties of HONE1 cells were examined using invasion assay as described in “Materials and methods”. Cells were transfected with 30 nM PTX3 siRNA oligonucleotides and scrambled oligonucleotides (SC) by lipofection. After 50 ng/ml EGF treatment for 48 h, the non-invasive cells were removed and invasive cells were fixed and stained by crystal violet procedure. The invasive images were examined using a microscope (upper panel). The number of invasion cells was counted as shown in lower panel. Values represent the mean ± S.E. of three determinations. (D) Endothelial cells were grown to form a monolayer on the bottom of a thick layer of extracellular matrix proteins to mimic intravasation in transendothelial invasion assays. HONE1 cells were transfected with 30 nM PTX3 siRNA oligonucleotides by lipofection. After 50 ng/ml EGF treatment for 6 h and stained with DiI, cells were loaded in the upper chamber of filter inserts. After incubation for 48 h, non-invasive cells were removed. The invasive images were examined using a microscope (left panel). Original magnification, ×200. Invasive cells were counted (right panel). Values represent the mean ± S.E. of three determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4480713&req=5

Figure 4: EGF-induced PTX3 enhances tumor migration and invasion(A) The migratory properties of KB cells were analyzed by a transwell assay using Boyden chambers. Parental and shPTX3 KB cells were incubated with or without 50 ng/ml EGF and 250 ng/ml PTX3 recombinant protein in the upper chamber of filter inserts. After incubation for 15 h, non-migratory cells were removed, and migratory cells were fixed and stained by a crystal violet procedure. Original magnification, ×200 (left panel). Migrating cells were counted (right panel). Values represent the mean ± S.E. of three determinations. shLacZ, negative control. (B) HONE1 cells were treated with various concentrations of anti-PTX3 antibodies (ab), 15 μg/ml immunoglobulin G (IgG) and 50 ng/ml EGF for 15 h. Migratory cells were fixed and stained by a crystal violet procedure. Migrating cells were counted. Values represent the mean ± S.E. of three determinations. (C) The invasive properties of HONE1 cells were examined using invasion assay as described in “Materials and methods”. Cells were transfected with 30 nM PTX3 siRNA oligonucleotides and scrambled oligonucleotides (SC) by lipofection. After 50 ng/ml EGF treatment for 48 h, the non-invasive cells were removed and invasive cells were fixed and stained by crystal violet procedure. The invasive images were examined using a microscope (upper panel). The number of invasion cells was counted as shown in lower panel. Values represent the mean ± S.E. of three determinations. (D) Endothelial cells were grown to form a monolayer on the bottom of a thick layer of extracellular matrix proteins to mimic intravasation in transendothelial invasion assays. HONE1 cells were transfected with 30 nM PTX3 siRNA oligonucleotides by lipofection. After 50 ng/ml EGF treatment for 6 h and stained with DiI, cells were loaded in the upper chamber of filter inserts. After incubation for 48 h, non-invasive cells were removed. The invasive images were examined using a microscope (left panel). Original magnification, ×200. Invasive cells were counted (right panel). Values represent the mean ± S.E. of three determinations.

Mentions: In mice, the PTX3 mutation is associated with a severe defect in female fertility caused by an abnormal location of granulosa cells. An abnormal cumulus oophorus is due to defective organization and stability of the ECM [22]. Thus, we raised the hypothesis that PTX3 may also participate in tumor migration. To investigate the biological significance of EGF-induced PTX3 expression by tumor cells, stable cell lines with PTX3-knockdown via shRNA knockdown of PTX3 (shPTX3) was used. As shown in Supplementary Fig. 7, EGF-induced PTX3 mRNA expression and protein secretion into culture media were dramatically inhibited in shPTX3 cells. These shPTX3 cells were then used to study the effects of EGF on cell migration. As shown in Fig. 4A, EGF induced cancer cell migration in parental but not PTX3-knockdown cells. However, recombinant PTX3 abolished this inhibition in EGF-treated shPTX3 cells. Again, depletion of PTX3 using PTX3 siRNA oligonucleotides significantly blocked EGF-induced cell migration (Supplementary Fig. 8). The involvement of PTX3 in regulating EGF-induced migration was further examined using anti-PTX3 antibodies to neutralize PTX3 in culture media. Anti-PTX3 antibodies blocked EGF-induced cell migration in a dose-dependent manner (Fig. 4B). In addition, we studied whether PTX3 contributed to EGF-induced tumor invasion. Indeed, depletion of PTX3 inhibited EGF-induced HNSCC cell invasion (Fig. 4C). We further studied the effect of PTX3 on the endothelial transmigration of tumor cells in a three-dimensional in vitro model. Briefly, endothelial cells were grown until they formed a monolayer on the bottom of a thick layer of ECM proteins to mimic intravasation in transendothelial assays. As shown in Fig. 4D and Supplementary Fig. 9, EGF-induced transendothelial invasion was significantly reduced in PTX3 knockdown cells. Addition of PTX3 recovered the EGF-induced transeddothelial invasion in PTX3 knockdown cells (Fig. 4D).


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 enhances tumor migration and invasion(A) The migratory properties of KB cells were analyzed by a transwell assay using Boyden chambers. Parental and shPTX3 KB cells were incubated with or without 50 ng/ml EGF and 250 ng/ml PTX3 recombinant protein in the upper chamber of filter inserts. After incubation for 15 h, non-migratory cells were removed, and migratory cells were fixed and stained by a crystal violet procedure. Original magnification, ×200 (left panel). Migrating cells were counted (right panel). Values represent the mean ± S.E. of three determinations. shLacZ, negative control. (B) HONE1 cells were treated with various concentrations of anti-PTX3 antibodies (ab), 15 μg/ml immunoglobulin G (IgG) and 50 ng/ml EGF for 15 h. Migratory cells were fixed and stained by a crystal violet procedure. Migrating cells were counted. Values represent the mean ± S.E. of three determinations. (C) The invasive properties of HONE1 cells were examined using invasion assay as described in “Materials and methods”. Cells were transfected with 30 nM PTX3 siRNA oligonucleotides and scrambled oligonucleotides (SC) by lipofection. After 50 ng/ml EGF treatment for 48 h, the non-invasive cells were removed and invasive cells were fixed and stained by crystal violet procedure. The invasive images were examined using a microscope (upper panel). The number of invasion cells was counted as shown in lower panel. Values represent the mean ± S.E. of three determinations. (D) Endothelial cells were grown to form a monolayer on the bottom of a thick layer of extracellular matrix proteins to mimic intravasation in transendothelial invasion assays. HONE1 cells were transfected with 30 nM PTX3 siRNA oligonucleotides by lipofection. After 50 ng/ml EGF treatment for 6 h and stained with DiI, cells were loaded in the upper chamber of filter inserts. After incubation for 48 h, non-invasive cells were removed. The invasive images were examined using a microscope (left panel). Original magnification, ×200. Invasive cells were counted (right panel). Values represent the mean ± S.E. of three determinations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480713&req=5

Figure 4: EGF-induced PTX3 enhances tumor migration and invasion(A) The migratory properties of KB cells were analyzed by a transwell assay using Boyden chambers. Parental and shPTX3 KB cells were incubated with or without 50 ng/ml EGF and 250 ng/ml PTX3 recombinant protein in the upper chamber of filter inserts. After incubation for 15 h, non-migratory cells were removed, and migratory cells were fixed and stained by a crystal violet procedure. Original magnification, ×200 (left panel). Migrating cells were counted (right panel). Values represent the mean ± S.E. of three determinations. shLacZ, negative control. (B) HONE1 cells were treated with various concentrations of anti-PTX3 antibodies (ab), 15 μg/ml immunoglobulin G (IgG) and 50 ng/ml EGF for 15 h. Migratory cells were fixed and stained by a crystal violet procedure. Migrating cells were counted. Values represent the mean ± S.E. of three determinations. (C) The invasive properties of HONE1 cells were examined using invasion assay as described in “Materials and methods”. Cells were transfected with 30 nM PTX3 siRNA oligonucleotides and scrambled oligonucleotides (SC) by lipofection. After 50 ng/ml EGF treatment for 48 h, the non-invasive cells were removed and invasive cells were fixed and stained by crystal violet procedure. The invasive images were examined using a microscope (upper panel). The number of invasion cells was counted as shown in lower panel. Values represent the mean ± S.E. of three determinations. (D) Endothelial cells were grown to form a monolayer on the bottom of a thick layer of extracellular matrix proteins to mimic intravasation in transendothelial invasion assays. HONE1 cells were transfected with 30 nM PTX3 siRNA oligonucleotides by lipofection. After 50 ng/ml EGF treatment for 6 h and stained with DiI, cells were loaded in the upper chamber of filter inserts. After incubation for 48 h, non-invasive cells were removed. The invasive images were examined using a microscope (left panel). Original magnification, ×200. Invasive cells were counted (right panel). Values represent the mean ± S.E. of three determinations.
Mentions: In mice, the PTX3 mutation is associated with a severe defect in female fertility caused by an abnormal location of granulosa cells. An abnormal cumulus oophorus is due to defective organization and stability of the ECM [22]. Thus, we raised the hypothesis that PTX3 may also participate in tumor migration. To investigate the biological significance of EGF-induced PTX3 expression by tumor cells, stable cell lines with PTX3-knockdown via shRNA knockdown of PTX3 (shPTX3) was used. As shown in Supplementary Fig. 7, EGF-induced PTX3 mRNA expression and protein secretion into culture media were dramatically inhibited in shPTX3 cells. These shPTX3 cells were then used to study the effects of EGF on cell migration. As shown in Fig. 4A, EGF induced cancer cell migration in parental but not PTX3-knockdown cells. However, recombinant PTX3 abolished this inhibition in EGF-treated shPTX3 cells. Again, depletion of PTX3 using PTX3 siRNA oligonucleotides significantly blocked EGF-induced cell migration (Supplementary Fig. 8). The involvement of PTX3 in regulating EGF-induced migration was further examined using anti-PTX3 antibodies to neutralize PTX3 in culture media. Anti-PTX3 antibodies blocked EGF-induced cell migration in a dose-dependent manner (Fig. 4B). In addition, we studied whether PTX3 contributed to EGF-induced tumor invasion. Indeed, depletion of PTX3 inhibited EGF-induced HNSCC cell invasion (Fig. 4C). We further studied the effect of PTX3 on the endothelial transmigration of tumor cells in a three-dimensional in vitro model. Briefly, endothelial cells were grown until they formed a monolayer on the bottom of a thick layer of ECM proteins to mimic intravasation in transendothelial assays. As shown in Fig. 4D and Supplementary Fig. 9, EGF-induced transendothelial invasion was significantly reduced in PTX3 knockdown cells. Addition of PTX3 recovered the EGF-induced transeddothelial invasion in PTX3 knockdown cells (Fig. 4D).

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