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Antitumor effects and molecular mechanisms of figitumumab, a humanized monoclonal antibody to IGF-1 receptor, in esophageal carcinoma.

Zhang T, Shen H, Dong W, Qu X, Liu Q, Du J - Sci Rep (2014)

Bottom Line: Our results showed that CP not only inhibited IGF-1 induced receptor autophosphorylation and downstream signaling, but also triggered β-arrestin1 and G protein-coupled receptor kinases (GRKs) mediated ERK1/2 activation, indicating CP as a biased agonist for IGF-1R.Inhibition of ERK1/2 enhanced the antitumor activity of CP.Furthermore, CP was a more powerful agonist for IGF-1R down-regulation than IGF-1, and dysregulation of β-arrestin1 and GRKs affected this down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, P.R. China.

ABSTRACT
The insulin-like growth factor type 1 receptor (IGF-1R) plays an essential role in the development of numerous cancers. Figitumumab (CP) is not only a monoclonal antibody, it also has agonist activity on IGF-1R. The antitumor activity of CP in esophageal squamous cell carcinoma (ESCC) is still unclear. In our study, we identified IGF-1R as an independent prognostic factor in ESCC patients, and investigated the antitumor effects of CP in ESCC cell lines. CP suppressed tumor growth and sensitized cells to chemotherapeutic drugs. In addition, CP inhibited cell proliferation, migration, colony forming activity and anti-apoptosis induced by IGF-1. Our results showed that CP not only inhibited IGF-1 induced receptor autophosphorylation and downstream signaling, but also triggered β-arrestin1 and G protein-coupled receptor kinases (GRKs) mediated ERK1/2 activation, indicating CP as a biased agonist for IGF-1R. Inhibition of ERK1/2 enhanced the antitumor activity of CP. Furthermore, CP was a more powerful agonist for IGF-1R down-regulation than IGF-1, and dysregulation of β-arrestin1 and GRKs affected this down-regulation. Thus, we demonstrated antitumor activities of CP on ESCC, and as a biased agonist, CP induced ERK1/2 activation and receptor down-regulation required β-arrestin1 and GRKs, suggesting a promising role for targeting IGF-1R in ESCC.

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Related in: MedlinePlus

β-arrestin1 dependent ERK1/2 signaling activation indicates CP as a biased agonist for IGF-1R. (a) CP induced ERK1/2 activation.Cells were plated, allowed to adhere and serum starved for 12 hours. Cells were treated with IGF-1 or CP for indicated times and were lysed. Protein samples were collected and analyzed by western blot for p-IGF-1R, p-Akt, p-ERK1/2, t-IGF-1R and GAPDH. (b) and (c) β-arrestin1 involvement of CP induced ERK1/2 activation. Cells were plated and transfected with β-arrestin1 siRNA for 48 h. Transfection efficiency was detected by WB for β-arrestin1 and GAPDH. Cells were starved and treated with IGF-1 or CP for 10 min, and were then lysed. Protein samples were analyzed by WB for p-ERK1/2 and total-ERK1/2. CTL, control; si, β-arrestin1 siRNA transfected; (d) Cells were plated, allowed to attach, and serum starved for 12 h. Then cells were pretreated with MEK inhibitor (U0126) or PBS for 1 h, and treated with CP or not for 10 min. Cells were lysed and protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (e) Combinational effects between CP and U0126 treatment. Cells were plated, allowed to adhere, and serum starved for 12 h. Cells were treated with 1 ng/ml CP with indicated concentrations of U0126 for 48 h. Number of cells are displayed as percentage of untreated cells. One-way ANOVA were used for statistical analysis properly. *p<0.05; **p<0.01. Cropped blots were used in the figures and the gels were run under the same experimental conditions. Representative full length blots were shown in Supplementary information.
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f5: β-arrestin1 dependent ERK1/2 signaling activation indicates CP as a biased agonist for IGF-1R. (a) CP induced ERK1/2 activation.Cells were plated, allowed to adhere and serum starved for 12 hours. Cells were treated with IGF-1 or CP for indicated times and were lysed. Protein samples were collected and analyzed by western blot for p-IGF-1R, p-Akt, p-ERK1/2, t-IGF-1R and GAPDH. (b) and (c) β-arrestin1 involvement of CP induced ERK1/2 activation. Cells were plated and transfected with β-arrestin1 siRNA for 48 h. Transfection efficiency was detected by WB for β-arrestin1 and GAPDH. Cells were starved and treated with IGF-1 or CP for 10 min, and were then lysed. Protein samples were analyzed by WB for p-ERK1/2 and total-ERK1/2. CTL, control; si, β-arrestin1 siRNA transfected; (d) Cells were plated, allowed to attach, and serum starved for 12 h. Then cells were pretreated with MEK inhibitor (U0126) or PBS for 1 h, and treated with CP or not for 10 min. Cells were lysed and protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (e) Combinational effects between CP and U0126 treatment. Cells were plated, allowed to adhere, and serum starved for 12 h. Cells were treated with 1 ng/ml CP with indicated concentrations of U0126 for 48 h. Number of cells are displayed as percentage of untreated cells. One-way ANOVA were used for statistical analysis properly. *p<0.05; **p<0.01. Cropped blots were used in the figures and the gels were run under the same experimental conditions. Representative full length blots were shown in Supplementary information.

Mentions: It is reported that CP directly activates ERK1/2 signaling in a β-arrestin1 biased mechanism in Ewing's sarcoma cell lines25. We therefore investigated the direct effects of CP on IGF-1R signaling in ESCC cells by detection of the kinetics of IGF-1- or CP-mediated activation of the ERK1/2 pathway and Akt pathway downstream of IGF-1R signaling. Upon IGF-1 stimulation, the IGF-1R was gradually phosphorylated for up to 60 minutes, demonstrating an increase in its kinase activity. Kinetic of Akt phosphorylation was in accordance with phosphorylation of IGF-1R, whereby ERK1/2 phosphorylation decreased markedly after 10 minutes (Figures 3b and 5a). In the case of CP direct stimulation, IGF-1R and Akt phosphorylation were undetectable; however, clear ERK1/2 phosphorylation induced by CP were indicated in both of the ESCC cell lines (Figure 5a). ERK1/2 activation levels were lower and postponed compared with that of IGF-1 stimulated activation, suggesting that the majority of ERK1/2 phosphorylation was dependent on the IGF-1R kinase activity, and part of ERK1/2 activation was independent of IGF-1R kinase activation, possibly through a β-arrestin1 mediated mechanism. Eca-109 and TE-1 cells were then transfected with β-arrestin1 siRNA to down-regulate β-arrestin1 expression (Figure 5b). As demonstrated in figure 5c, CP induced ERK1/2 phosphorylation was greatly decreased in the absence of β-arrestin1, whereas IGF-1 induced ERK1/2 phosphorylation was slightly affected. These results indicated a CP-induced and β-arrestin1–mediated ERK1/2 signaling activation downstream of IGF-1R signaling transduction in ESCC cells.


Antitumor effects and molecular mechanisms of figitumumab, a humanized monoclonal antibody to IGF-1 receptor, in esophageal carcinoma.

Zhang T, Shen H, Dong W, Qu X, Liu Q, Du J - Sci Rep (2014)

β-arrestin1 dependent ERK1/2 signaling activation indicates CP as a biased agonist for IGF-1R. (a) CP induced ERK1/2 activation.Cells were plated, allowed to adhere and serum starved for 12 hours. Cells were treated with IGF-1 or CP for indicated times and were lysed. Protein samples were collected and analyzed by western blot for p-IGF-1R, p-Akt, p-ERK1/2, t-IGF-1R and GAPDH. (b) and (c) β-arrestin1 involvement of CP induced ERK1/2 activation. Cells were plated and transfected with β-arrestin1 siRNA for 48 h. Transfection efficiency was detected by WB for β-arrestin1 and GAPDH. Cells were starved and treated with IGF-1 or CP for 10 min, and were then lysed. Protein samples were analyzed by WB for p-ERK1/2 and total-ERK1/2. CTL, control; si, β-arrestin1 siRNA transfected; (d) Cells were plated, allowed to attach, and serum starved for 12 h. Then cells were pretreated with MEK inhibitor (U0126) or PBS for 1 h, and treated with CP or not for 10 min. Cells were lysed and protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (e) Combinational effects between CP and U0126 treatment. Cells were plated, allowed to adhere, and serum starved for 12 h. Cells were treated with 1 ng/ml CP with indicated concentrations of U0126 for 48 h. Number of cells are displayed as percentage of untreated cells. One-way ANOVA were used for statistical analysis properly. *p<0.05; **p<0.01. Cropped blots were used in the figures and the gels were run under the same experimental conditions. Representative full length blots were shown in Supplementary information.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215295&req=5

f5: β-arrestin1 dependent ERK1/2 signaling activation indicates CP as a biased agonist for IGF-1R. (a) CP induced ERK1/2 activation.Cells were plated, allowed to adhere and serum starved for 12 hours. Cells were treated with IGF-1 or CP for indicated times and were lysed. Protein samples were collected and analyzed by western blot for p-IGF-1R, p-Akt, p-ERK1/2, t-IGF-1R and GAPDH. (b) and (c) β-arrestin1 involvement of CP induced ERK1/2 activation. Cells were plated and transfected with β-arrestin1 siRNA for 48 h. Transfection efficiency was detected by WB for β-arrestin1 and GAPDH. Cells were starved and treated with IGF-1 or CP for 10 min, and were then lysed. Protein samples were analyzed by WB for p-ERK1/2 and total-ERK1/2. CTL, control; si, β-arrestin1 siRNA transfected; (d) Cells were plated, allowed to attach, and serum starved for 12 h. Then cells were pretreated with MEK inhibitor (U0126) or PBS for 1 h, and treated with CP or not for 10 min. Cells were lysed and protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (e) Combinational effects between CP and U0126 treatment. Cells were plated, allowed to adhere, and serum starved for 12 h. Cells were treated with 1 ng/ml CP with indicated concentrations of U0126 for 48 h. Number of cells are displayed as percentage of untreated cells. One-way ANOVA were used for statistical analysis properly. *p<0.05; **p<0.01. Cropped blots were used in the figures and the gels were run under the same experimental conditions. Representative full length blots were shown in Supplementary information.
Mentions: It is reported that CP directly activates ERK1/2 signaling in a β-arrestin1 biased mechanism in Ewing's sarcoma cell lines25. We therefore investigated the direct effects of CP on IGF-1R signaling in ESCC cells by detection of the kinetics of IGF-1- or CP-mediated activation of the ERK1/2 pathway and Akt pathway downstream of IGF-1R signaling. Upon IGF-1 stimulation, the IGF-1R was gradually phosphorylated for up to 60 minutes, demonstrating an increase in its kinase activity. Kinetic of Akt phosphorylation was in accordance with phosphorylation of IGF-1R, whereby ERK1/2 phosphorylation decreased markedly after 10 minutes (Figures 3b and 5a). In the case of CP direct stimulation, IGF-1R and Akt phosphorylation were undetectable; however, clear ERK1/2 phosphorylation induced by CP were indicated in both of the ESCC cell lines (Figure 5a). ERK1/2 activation levels were lower and postponed compared with that of IGF-1 stimulated activation, suggesting that the majority of ERK1/2 phosphorylation was dependent on the IGF-1R kinase activity, and part of ERK1/2 activation was independent of IGF-1R kinase activation, possibly through a β-arrestin1 mediated mechanism. Eca-109 and TE-1 cells were then transfected with β-arrestin1 siRNA to down-regulate β-arrestin1 expression (Figure 5b). As demonstrated in figure 5c, CP induced ERK1/2 phosphorylation was greatly decreased in the absence of β-arrestin1, whereas IGF-1 induced ERK1/2 phosphorylation was slightly affected. These results indicated a CP-induced and β-arrestin1–mediated ERK1/2 signaling activation downstream of IGF-1R signaling transduction in ESCC cells.

Bottom Line: Our results showed that CP not only inhibited IGF-1 induced receptor autophosphorylation and downstream signaling, but also triggered β-arrestin1 and G protein-coupled receptor kinases (GRKs) mediated ERK1/2 activation, indicating CP as a biased agonist for IGF-1R.Inhibition of ERK1/2 enhanced the antitumor activity of CP.Furthermore, CP was a more powerful agonist for IGF-1R down-regulation than IGF-1, and dysregulation of β-arrestin1 and GRKs affected this down-regulation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Shandong University, Jinan, P.R. China.

ABSTRACT
The insulin-like growth factor type 1 receptor (IGF-1R) plays an essential role in the development of numerous cancers. Figitumumab (CP) is not only a monoclonal antibody, it also has agonist activity on IGF-1R. The antitumor activity of CP in esophageal squamous cell carcinoma (ESCC) is still unclear. In our study, we identified IGF-1R as an independent prognostic factor in ESCC patients, and investigated the antitumor effects of CP in ESCC cell lines. CP suppressed tumor growth and sensitized cells to chemotherapeutic drugs. In addition, CP inhibited cell proliferation, migration, colony forming activity and anti-apoptosis induced by IGF-1. Our results showed that CP not only inhibited IGF-1 induced receptor autophosphorylation and downstream signaling, but also triggered β-arrestin1 and G protein-coupled receptor kinases (GRKs) mediated ERK1/2 activation, indicating CP as a biased agonist for IGF-1R. Inhibition of ERK1/2 enhanced the antitumor activity of CP. Furthermore, CP was a more powerful agonist for IGF-1R down-regulation than IGF-1, and dysregulation of β-arrestin1 and GRKs affected this down-regulation. Thus, we demonstrated antitumor activities of CP on ESCC, and as a biased agonist, CP induced ERK1/2 activation and receptor down-regulation required β-arrestin1 and GRKs, suggesting a promising role for targeting IGF-1R in ESCC.

Show MeSH
Related in: MedlinePlus