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Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody.

Kim JG, Kang MJ, Yoon YK, Kim HP, Park J, Song SH, Han SW, Park JW, Kang GH, Kang KW, Oh do Y, Im SA, Bang YJ, Yi EC, Kim TY - PLoS ONE (2012)

Bottom Line: Identification of predictive biomarkers is essential for the successful development of targeted therapy.Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model.We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, Seoul National University, Seoul, South Korea.

ABSTRACT

Background: Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.

Methodology/principal findings: In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.

Conclusion and significance: The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.

Show MeSH

Related in: MedlinePlus

Anti-proliferative effect of figitumumab.A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G0/G1, S, and G2/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *P-values <0.05, **P-values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×107) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm3. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student’s t test. *P-values <0.05; **P-values <0.01 versus control.
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pone-0033322-g001: Anti-proliferative effect of figitumumab.A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G0/G1, S, and G2/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *P-values <0.05, **P-values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×107) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm3. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student’s t test. *P-values <0.05; **P-values <0.01 versus control.

Mentions: As a first step, we assessed the anti-proliferative effect of figitumumab, a monoclonal antibody that prevents ligands from binding to IGF1R [12], on 17 cancer cell lines (Figure 1A). Some cells considered to be sensitive to figitumumab, such as SNU719, HepG2, and SNU368, showed a dose-dependent decrease of cell viability; IC30 value of figitumumab (growth inhibitions of ∼30%) for each cell line were 0.063 µg/ml, 0.062 µg/ml, and 0.047 µg/ml, respectively (Table 1).


Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody.

Kim JG, Kang MJ, Yoon YK, Kim HP, Park J, Song SH, Han SW, Park JW, Kang GH, Kang KW, Oh do Y, Im SA, Bang YJ, Yi EC, Kim TY - PLoS ONE (2012)

Anti-proliferative effect of figitumumab.A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G0/G1, S, and G2/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *P-values <0.05, **P-values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×107) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm3. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student’s t test. *P-values <0.05; **P-values <0.01 versus control.
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Related In: Results  -  Collection

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pone-0033322-g001: Anti-proliferative effect of figitumumab.A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G0/G1, S, and G2/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *P-values <0.05, **P-values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×107) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm3. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student’s t test. *P-values <0.05; **P-values <0.01 versus control.
Mentions: As a first step, we assessed the anti-proliferative effect of figitumumab, a monoclonal antibody that prevents ligands from binding to IGF1R [12], on 17 cancer cell lines (Figure 1A). Some cells considered to be sensitive to figitumumab, such as SNU719, HepG2, and SNU368, showed a dose-dependent decrease of cell viability; IC30 value of figitumumab (growth inhibitions of ∼30%) for each cell line were 0.063 µg/ml, 0.062 µg/ml, and 0.047 µg/ml, respectively (Table 1).

Bottom Line: Identification of predictive biomarkers is essential for the successful development of targeted therapy.Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model.We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Institute, Seoul National University, Seoul, South Korea.

ABSTRACT

Background: Identification of predictive biomarkers is essential for the successful development of targeted therapy. Insulin-like growth factor 1 receptor (IGF1R) has been examined as a potential therapeutic target for various cancers. However, recent clinical trials showed that anti-IGF1R antibody and chemotherapy are not effective for treating lung cancer.

Methodology/principal findings: In order to define biomarkers for predicting successful IGF1R targeted therapy, we evaluated the anti-proliferation effect of figitumumab (CP-751,871), a humanized anti-IGF1R antibody, against nine gastric and eight hepatocellular cancer cell lines. Out of 17 cancer cell lines, figitumumab effectively inhibited the growth of three cell lines (SNU719, HepG2, and SNU368), decreased p-AKT and p-STAT3 levels, and induced G 1 arrest in a dose-dependent manner. Interestingly, these cells showed co-overexpression and altered mobility of the IGF1R and insulin receptor (IR). Immunoprecipitaion (IP) assays and ELISA confirmed the presence of IGF1R/IR heterodimeric receptors in figitumumab-sensitive cells. Treatment with figitumumab led to the dissociation of IGF1-dependent heterodimeric receptors and inhibited tumor growth with decreased levels of heterodimeric receptors in a mouse xenograft model. We next found that both IGF1R and IR were N-linked glyosylated in figitumumab-sensitive cells. In particular, mass spectrometry showed that IGF1R had N-linked glycans at N913 in three figitumumab-sensitive cell lines. We observed that an absence of N-linked glycosylation at N913 led to a lack of membranous localization of IGF1R and figitumumab insensitivity.

Conclusion and significance: The data suggest that the level of N-linked glycosylated IGF1R/IR heterodimeric receptor is highly associated with sensitivity to anti-IGF1R antibody in cancer cells.

Show MeSH
Related in: MedlinePlus