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. 2008 Oct 1;68(19):8039-48.
doi: 10.1158/0008-5472.CAN-08-1712.

Addiction to elevated insulin-like growth factor I receptor and initial modulation of the AKT pathway define the responsiveness of rhabdomyosarcoma to the targeting antibody

Liang Cao  1 , Yunkai Yu, Isaac Darko, Duane Currier, Linnia H Mayeenuddin, Xiaolin Wan, Chand Khanna, Lee J Helman
Affiliations

Addiction to elevated insulin-like growth factor I receptor and initial modulation of the AKT pathway define the responsiveness of rhabdomyosarcoma to the targeting antibody

Liang Cao et al. Cancer Res. .

Abstract

Insulin-like growth factor I receptor (IGF-IR) and its ligands are overexpressed by tumors, mediating proliferation and protecting against stress-induced apoptosis. Accordingly, there has been a considerable amount of interest in developing therapeutic agents against IGF-IR. IGF-IR is believed to be ubiquitously expressed without detectable mutation or amplification in cancer. We explored the determinants of cellular response to a humanized anti-IGF-IR antibody. Our results showed a large variation in IGF-IR levels in rhabdomyosarcoma tumor specimens that were comparable with those in rhabdomyosarcoma cell lines. In vitro analysis revealed a direct and very significant correlation between elevated IGF-IR levels and antiproliferative effects of the antibody and defined a receptor number that would predict sensitivity. Our data further suggested a strong dependence on IGF-IR for AKT signaling in cells with elevated IGF-IR. The sensitivity of the high IGF-IR-expressing cells was blocked with a constitutively active AKT. The extracellular signal-regulated kinase pathway was not affected by the antibody. In vivo studies showed that anti-IGF-IR had single-agent antitumor activity; furthermore, predictions of responses based on IGF-IR levels were accurate. In vivo biomarker analysis suggested that h7C10 down-regulated both IGF-IR and p-AKT initially, concordant with antitumor activity. Subsequent progression of tumors was associated with reactivation of p-AKT despite sustained suppression of IGF-IR. These results identified the first predictive biomarker for anti-IGF-IR therapies in cancer.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
Determination of IGF-IR in rhabdomyosarcoma tumors and cell lines and validation of ECL-based IGF-IR sandwich immunoassay assay. A, levels of IGF-IR in rhabdomyosarcoma (RMS) tumors and cell lines. IGF-IR was determined for tumor tissue from rhabdomyosarcoma patients. Ctrl, skeletal muscle from a donor; P1-P9, rhabdomyosarcoma tumors from patients at the NCI. IGF-IR was also determined for a panel of rhabdomyosarcoma cell lines. Data are shown as nanograms of IGF-IR per milligram of lysate. B, standard curve was generated with purified recombinant IGF-IR with ECL assay (see Materials and Methods). Points, mean (n = 3), with coefficient of variation of ~10%; bars, SE. Cutoff was defined from negative controls (1% BSA) as mean + 5 × SD. The assay has a sensitivity of 1 pg IGF-IR. C, IGF-IR mRNA in rhabdomyosarcoma cell lines was determined with normalized qPCR analysis. D, IGF-IR protein was detected with immunoblot with a different IGF-IR antibody. The results of IGF-IR mRNA and protein analysis were summarized to show high degrees of agreement between ECL, immunoblot, and qPCR results.
Figure 2
Figure 2
In vitro antiproliferative activity of h7C10 is correlated with the levels of IGF-IR in rhabdomyosarcoma cells A, rhabdomyosarcoma cell lines were treated with 10 μg/mL anti–IGF-IR antibody h7C10 or a mock antibody (MOPC 21) for 5 d. The cell proliferation was determined using ATP assay. The percentage of inhibition was defined as (V ctrl − V treated)/V ctrl. The data were obtained from a representative proliferation experiment and analyzed against their IGF-IR levels using GraphPad. Points, mean (n = 3); bars, SE. Pearson correlation was performed with GraphPad to determine the correlation between antiproliferative activity of h7C10 and IGF-IR levels of the rhabdomyosarcoma cell lines. B, dose curve of h7C10 in antiproliferative assay against sensitive Rh4 (IC50, 7 ng/mL) and resistant Rh18 cells (IC50 >10,000 ng/mL). Points, mean (n = 3); bars, SE. C, effects of h7C10 on rhabdomyosarcoma in clonogenic assay with representative rhabdomyosarcoma cell lines of low (Rh18, 1.0 ng/mg), medium (Rh30, 7.2 ng/mg), and high (Rh4, 15.1 ng/mg) levels of IGF-IR.
Figure 3
Figure 3
Correlation between IGF-IR and p-AKT levels in rhabdomyosarcoma cell lines and a cellular dependence on IGF-IR for endogenous AKT signaling in the presence of 10% serum. A, immunoblot analysis of AKT, p-AKT, ERK, and p-ERK in rhabdomyosarcoma cell lines. B, quantitative analysis of EGFR and IGF-IR in the rhabdomyosarcoma cell lines. The number of EGFR molecules per cell was determined in reference to that in A431 at 2 × 106 EGFR per cell. The number of IGF-IR molecules per cell was determined with a recombinant IGF-IR standard. C, down-regulation of IGF-IR by h7C10. Rhabdomyosarcoma cell lines were treated with 10 μg/mL anti–IGF-IR antibody h7C10 for 24 h. The cell lysates were analyzed for total IGF-IR via an ECL immunoassay. D, the cell lysates from C were analyzed with immunoblots against t/p-EKR and t/p-AKT. Further quantitative ECL analysis of p-AKT following h7C10 treatment was performed and the relative levels of p-AKT were shown in a bar chart. Resistant Rh1 cells: ctrl, 1; h7C10, 0.75. Sensitive Rh30 cells: ctrl, 26; Rh30, 4.2.
Figure 4
Figure 4
Evaluation of the role of AKT pathway in h7C10-mediated growth suppression with a constitutively active (CA) AKT gene. A, immunoblot analysis of Rh30-v and Rh30-CA-AKT cells treated with anti–IGF-IR antibody h7C10. Cells were infected with retroviruses containing the vector or CA-AKT gene, selected with puromycin, and pooled. The stable cells were treated with h7C10 for 24 h and analyzed for myc-tag, total and p-AKT, and a downstream target p-GSK3β. CA-AKT migrates at a slower rate when compared with the endogenous AKT. B, examination of the antiproliferative activity of h7C10 in Rh30-CA-AKT cells. Cells infected with vector and CA-AKT viruses were treated with mock or h7C10 for 3 and 5 d. The results are from a representative experiment. Points, mean (n = 3); bars, SE. C, statistical analysis of Rh30.v and Rh30-CA-Akt cells treated with h7C10 for 5 d, followed by ATP measurement in triplicate. Columns, mean (n = 3); bars, SE. Student’s t test was performed and P values of relevant pairs are shown. D, effects of h7C10 on Rh30-vec and Rh30-CA-AKT in a clonogenic assay. CTR is another rhabdomyosarcoma cell line with very high IGF-IR level and is very sensitive to h7C10. Cells were treated with h7C10 in triplicates for 2 wk before fixing and staining.
Figure 5
Figure 5
In vivo antitumor effects of h7C10 against rhabdomyosarcoma and biomarker analysisA, mice with RD xenografts were treated with saline or h7C10 starting from day 8. Data shown represent the average size of the tumor in their maximal diameter. Points, mean (n = 10); bars, SE. B, mice with Rh18 xenografts (low IGF-IR) were treated with saline or h7C10 from day 8. Columns, mean maximal diameter (n = 10); bars, SE. C, pharmacodynamic marker analysis of IGF-IR, p-AKT, and t-AKT following 1 to 5 d of h7C10 treatment with RD xenografts (n = 3). D, end point analysis of IGF-IR, p-AKT, and t-AKT with RD tumors collected at the end of the experiment (n = 4). Columns, mean relative ECL units; bars, SE. P values for relevant pairs are shown.
Figure 6
Figure 6
In vivo antitumor effects of h7C10 and rapamycinA, a four-arm study with h7C10 and rapamycin against mice with rhabdomyosarcoma cell line Rh30. Mice were treated with DMSO, h7C10, rapamycin, or both from day 8. Points, mean maximal diameter (n = 10); bars, SE. B, after 2 wk of drug treatment, in vivo tumor imaging was performed with a Xenogen imager. Columns, mean luminescence units (n = 4); bars, SE. C, PD marker analysis of IGF-IR and p-AKT following 5 d of h7C10 treatment (n = 3). D, end point analysis of IGF-IR and p-AKT with tumors collected at the end of the experiment (n = 4). Columns, mean relative ECL units; bars, SE. P values for the relevant pairs are shown.
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