Background: IGF-IR signaling in rhabdomyosarcoma (RMS) and neuroblastoma (NB) cell lines is thought to be dysregulated by overexpression of the IGF-II ligand. However, functional linkage between IGF-IR signaling and activation of a critical node in signaling, Akt, has not been defined.
Methods: RMS and NB cell lines were grown in vitro under normoxic (21% O2) or hypoxic (1% O2) conditions. Cells were treated for 24h with CP751871 (1μg/ml), a human antibody targeting IGF-IR. IGF-IR, Akt, GSK-3β, S6 and their phosphorylated derivatives were determined by Western blot. In vivo, the same proteins were monitored 24h after administration of CP751871 (0.25 mg) to mice bearing tumor xenografts.
Results: Under serum-free conditions CP751871 completely blocked IGF-I stimulation of Akt phosphorylation in all cell lines. Under normoxic conditions in serum-containing media, CP751871 caused downregulation of IGF-IR in 3 of 4 RMS lines but only slightly reduced pAkt levels. Under hypoxic conditions CP751871 markedly inhibited pAkt in 3 of 4 RMS lines. Of note, whereas total IGF-IR levels did not increase under hypoxia, pIGF-IR dramatically increased, and this increase was completely suppressed by CP751871. Similar results were obtained for 3 of 4 NB cell lines, suggesting feedback activation of IGF-IR in some RMS and NB cell lines. Rapamycin (106nM) completely or partially suppressed hypoxia-induced increases in pIGF-IR, indicating its dependence on mTOR signaling. In vivo, CP751871 dramatically downregulated IGF-IR in 4 of 4 RMS xenografts, but had little effect on pAkt. In contrast, CP751871 downregulated IGF-IR and suppressed pAkt in 1/1 Ewing sarcoma (EWS) and 1/1 NB xenografts.
Conclusions: Our results suggest that CP751871 potently inhibits IGF-IR signaling in vitro. Under normoxia, inhibition of IGF-IR has relatively small effect on pAkt levels, suggesting that alternate receptor-mediated pathways signal to Akt, or that there is upregulation of these pathways when IGF-IR signaling is inhibited. Under conditions of hypoxia, the signaling through IGF-IR becomes more important. Phosphorylation of IGF-IR (Tyr1131) is increased in most cell lines, and CP751871 suppressed pAkt, indicating ′linkage′ between the receptor and Akt under hypoxia. Further, increased pIGF-IR is in part mTOR dependent. In vivo, CP751871 dramatically downregulates IGF-IR in RMS tumors, and suppressed pAkt in EWS and NB xenografts, but failed to suppress pAkt in RMS xenografts, due in part to upregulation of insulin receptor signaling. These results indicate that in vitro hypoxia induces IGF-IR dependence, whereas in vivo inhibition of IGF-IR leads to accommodation through alternate receptors.
Supported by USPHS grants CA77776 and CA96696 and by ALSAC.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA