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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

Involvement of GRK 2 and GRK 6 in CP induced signaling and receptor down-regulation.(a) Cells were plated, and transiently transfected with plasmids expressing GRK2 or GRK6 or with siRNAs for GRK2 or GRK6 for 48 hours. Cells were lysed and protein samples were analyzed by WB for GRK2, GRK6 and GAPDH. (b) Involvement of GRKs in CP induced signaling. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (c) Involvement of GRKs in CP induced receptor down-regulation. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for IGF-1R and GAPDH. 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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f6: Involvement of GRK 2 and GRK 6 in CP induced signaling and receptor down-regulation.(a) Cells were plated, and transiently transfected with plasmids expressing GRK2 or GRK6 or with siRNAs for GRK2 or GRK6 for 48 hours. Cells were lysed and protein samples were analyzed by WB for GRK2, GRK6 and GAPDH. (b) Involvement of GRKs in CP induced signaling. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (c) Involvement of GRKs in CP induced receptor down-regulation. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for IGF-1R and GAPDH. 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: IGF-1 induced IGF-1R activation can not only utilize β-arrestin1 but also GRKs of GPCRs signaling machinery as regulators222330. So, the next step was to investigate whether GRKs were involved in the effects of CP. It was indicated that GRK2 decreased the ERK1/2 activation, and prevented receptor degradation, whereas GRK6 acted on the contrary. For these series of experiments, we used HEK-293T cells, which expressed relatively very higher level of GRK2 compared to GRK6. HEK 293T cells, transfected either with GRK2/GRK6 plasmids for overexpression or respective siRNA for down-regulation, were used in subsequent experiments (Figure 6a). Figure 6b showed that with overexpression of GRK6 and lower expression of GRK2, there was an increase in ERK1/2 activation when the cells were treated with 1 ug/ml CP, whereas lower expression of GRK6 and overexpression of GRK2 decreased the ERK1/2 activation slightly. As for degradation of IGF-1R, GRK2 could prevent the receptor to be down-regulated, and GRK6 accelerated the down-regulation process greatly. The above results demonstrated the regulatory roles of GRKs in CP induced IGF-1R signaling and down-regulation.


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)

Involvement of GRK 2 and GRK 6 in CP induced signaling and receptor down-regulation.(a) Cells were plated, and transiently transfected with plasmids expressing GRK2 or GRK6 or with siRNAs for GRK2 or GRK6 for 48 hours. Cells were lysed and protein samples were analyzed by WB for GRK2, GRK6 and GAPDH. (b) Involvement of GRKs in CP induced signaling. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (c) Involvement of GRKs in CP induced receptor down-regulation. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for IGF-1R and GAPDH. 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

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

f6: Involvement of GRK 2 and GRK 6 in CP induced signaling and receptor down-regulation.(a) Cells were plated, and transiently transfected with plasmids expressing GRK2 or GRK6 or with siRNAs for GRK2 or GRK6 for 48 hours. Cells were lysed and protein samples were analyzed by WB for GRK2, GRK6 and GAPDH. (b) Involvement of GRKs in CP induced signaling. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for p-ERK1/2 and t-ERK1/2. (c) Involvement of GRKs in CP induced receptor down-regulation. Transfected cells were splitted into 24 well-plates, allowed to attach and serum starved for 12 h. Cells were then stimulated with 1 ug/ml CP for indicated times, and were lysed then. Protein samples were analyzed by WB for IGF-1R and GAPDH. 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: IGF-1 induced IGF-1R activation can not only utilize β-arrestin1 but also GRKs of GPCRs signaling machinery as regulators222330. So, the next step was to investigate whether GRKs were involved in the effects of CP. It was indicated that GRK2 decreased the ERK1/2 activation, and prevented receptor degradation, whereas GRK6 acted on the contrary. For these series of experiments, we used HEK-293T cells, which expressed relatively very higher level of GRK2 compared to GRK6. HEK 293T cells, transfected either with GRK2/GRK6 plasmids for overexpression or respective siRNA for down-regulation, were used in subsequent experiments (Figure 6a). Figure 6b showed that with overexpression of GRK6 and lower expression of GRK2, there was an increase in ERK1/2 activation when the cells were treated with 1 ug/ml CP, whereas lower expression of GRK6 and overexpression of GRK2 decreased the ERK1/2 activation slightly. As for degradation of IGF-1R, GRK2 could prevent the receptor to be down-regulated, and GRK6 accelerated the down-regulation process greatly. The above results demonstrated the regulatory roles of GRKs in CP induced IGF-1R signaling and down-regulation.

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