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- Material and methods
- Supporting Information
IGF-1 receptor (IGF-1R) plays a key role in the development of numerous tumors. Blockade of IGF-1R axis using monoclonal antibodies constitutes an interesting approach to inhibit tumor growth. We have previously shown that h7C10, a humanized anti–IGF-1R Mab, exhibited potent antitumor activity in vivo. However, mechanisms of action of h7C10 are still unknown. Here, we showed that h7C10 inhibited IGF-1–induced IGF-1R phosphorylation in a dose-dependent manner. Also, h7C10 abolished IGF-1–induced activation of PI3K/AKT and MAPK pathways. Cell cycle progression and colony formation were affected in the presence of h7C10 probably because of the inhibition of IGF-1–induced cyclin D1 and E expression. In addition, we demonstrated that h7C10 induced a rapid IGF-1R internalization leading to an accumulation into cytoplasm resulting in receptor degradation. Using lysosome and proteasome inhibitors, we observed that the IGF-1R α- and β-chains could follow different degradation routes. Thus, we demonstrated that antitumoral properties of h7C10 are the result of IGF-1–induced cell signaling inhibition and down-regulation of IGF-1R level suggesting that h7C10 could be a candidate for therapeutic applications. © 2008 Wiley-Liss, Inc.
The Type I insulin-like growth factor receptor (IGF-1R) is a tyrosine kinase receptor mediating metabolic, mitogenic and antiapoptotic pathways induced by both insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2).1 It also binds insulin but with 100 to 1,000-fold lower affinity.2 Several in vitro studies showed that inhibition of IGF-1R expression or activation inhibited cancer cell growth and colony formation.3, 4 These findings were confirmed in animal models, where IGF-1R antisense oligonucleotides injected intraperitoneally in nude mice inhibited the growth of human cancers.5, 6 The link between cancer and IGF signaling is also consistent with epidemiological studies showing that elevated levels of IGF-1 are associated with an increased relative risk of developing colon, prostate, breast, lung and bladder cancers.7–9
IGF-1R functions as a heterotetramer composed of 2 extracellular ligand binding α-subunits and 2 β-subunits comprising both the transmembrane and tyrosine kinase domains.1 Once autophosphorylated, IGF-1R binds and phosphorylates on various tyrosine substrates, such as insulin receptor substrates (IRS-1 to 4)10–12 and Shc (Src Homology Collagen protein).13 These substrates serve as docking molecules for other proteins containing SH2 domains including p85 regulatory subunit of PI 3-kinase and Grb2 that lead to the activation of two main signaling pathways, of PI 3-kinase/Akt14 and MAPK pathways.15 MAPK and of PI 3-kinase/Akt pathways are considered to be essential for cell proliferation, anchorage-independent growth and to mediate antiapoptotic signal of IGF-1.
On ligand binding, tyrosine kinase receptors are usually internalized leading to receptor translocation from plasma membrane to lysosomes.16 Because of homologies of IGF-1R with insulin receptor (IR) signaling, it was suggested that IGF-1 binding to IGF-1R leads to receptor internalization into early endosomes via clathrin-coated pits or caveolae17, 18 resulting in a partial receptor degradation through a process requiring receptor ubiquitination.19–23 Recent studies have implicated 2 E3 ligases, Mdm2 and Nedd4, in IGF-1R ubiquitination and degradation.20, 21 Mdm2 requires β-arrestin as an adaptor to interact with IGF-1R.24 The tumor suppressor p53 has also been described as involved in the regulation of IGF-1R degradation through Mdm2. However, its role seems complex and not very well defined.25 In Nedd4 and IGF-1R overexpressing cells, the Grb10/Nedd4/IGF-1R complex has been shown to induce receptor ubiquitination and internalization.20 These studies indicate that ubiquitination of IGF-1R is a well established event whereas the molecular mechanisms (proteasomal and/or lysosomal pathways) involved in the degradation of IGF-1R are dependent on cell type used and are still unclear.22, 25, 26
Strategies targeting IGF pathway constitute a promising tool to inhibit tumor development. Most of the anti–IGF-1 strategies have been directed against the receptor itself. Monoclonal antibodies,27–33 tyrosine kinase inhibitors,34, 35 antisense oligonucleotides directed against IGF-1R mRNA36 or dominant negative approaches have been reported to inhibit in vitro and/or in vivo tumor proliferation. We have previously shown that h7C10, a humanized monoclonal antibody anti–IGF-1R, inhibits both the in vitro and in vivo growth of breast (MCF-7 and MDA-MB231) and NSCLC tumor cells.29, 31 Here, we investigated the effect of h7C10 on IGF-1–induced cell signaling and examined the mechanisms involved by h7C10 to inhibit tumor growth. Our results suggest that h7C10 antitumor activity results from inhibition of IGF-1–induced MAPK and PI3K/AKT activation pathways leading to inhibition of cell cycle progression and anchorage-independent growth. In addition, we showed that h7C10 also induces both internalization and degradation of IGF-1R.
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- Material and methods
- Supporting Information
IGF-1R antibodies demonstrated in vitro and in vivo activities against numerous tumors. h7C10 is a humanized monoclonal antibody that induced tumor regression in subcutaneous xenograft and orthotopic models. We have already shown that h7C10 specifically inhibited IGF-1 binding to IGF-1R, receptor autophosphorylation and proliferation. To better characterize mechanisms by which h7C10 inhibits tumor growth, we first studied the h7C10 effect on IGF-1R signaling in MCF-7 cells. Here, we have shown that h7C10 inhibited specifically IGF-1–induced and IGF-2–induced IGF-1R autophosphorylation. No effect of this antibody was observed on insulin-induced or on IGF-2–induced activation of insulin receptor. Likewise, no interference was observed with EGF-R phosphorylation evaluated as a negative control. The dramatic inhibition of IGF-1R phosphorylation by h7C10 could probably explain the complete abrogation of tyrosine phosphorylation of IRS-1, IRS-2 and Shc, but also the total inactivation of the PI3K/Akt and MAPK signaling pathways in response to IGF-1. These two latter signaling pathways are survival signals able to protect cancer cells from apoptosis and it has been described that simultaneous inactivation of both PI3K/Akt and MAPK Erk1/2 is required to inhibit IGF-1R capacity of protecting cells from apoptotic injuries.39 The concomitant inactivation of PI3K/Akt and MAPK Erk1/2 pathways by h7C10 is probably one of the mechanisms directly involved in the successful in vivo inhibition of cell proliferation and tumor growth. Signaling impairment could also lead to cell cycle perturbations. Indeed, it has been well described that defective function of cell cycle regulators is a main cause for tumor development and progression. For example, the cell cycle promoter cyclin D1 is frequently overexpressed in cancer cells.40 IGF-1 has been shown to regulate both the expression and the activity of various molecules involved in cell cycle progression. In MCF-7 cells, IGF-1 induces cyclin D1 expression and Rb hyperphosphorylation through PI3K but not MAPK pathways.41 In this study, we showed that h7C10 treatment decreased expression of both IGF-1–induced cyclins D1 and E and inhibited Rb phosphorylation. Thus, we can speculate that these data are related to the inhibition of the PI-3-kinase/Akt signaling pathway in response to h7C10.
In another set of experiment, we have observed that h7C10-induced both in vivo and in vitro down-regulation of IGF-1R. Down-regulation of IGF1-R is a mechanism of action that has already been described for most of the monoclonal antibodies directed against IGF-1R.27, 30, 42 However, molecular mechanisms involved in this process are still unclear. Degradation of cell surface receptor can be due to either receptor internalization or receptor shedding. In this study, we have shown that h7C10 induces IGF-1R internalization and degradation, suggesting that IGF-1R degradation did not involve receptor shedding. On the other hand, IGF-1 ligand did not induce apparent IGF-1R degradation in MCF-7 as already reported.27, 29, 43 To further investigate the mechanism of down-regulation, both lysosome (methylamine, chloroquine, bafilomycin A1) and proteasome (epoxomycin, bortezomib, MG115, MG132) inhibitors were used and either α-chain or β-chain degradation were monitored. Uncorrelated results between degradation of these two chains were noticed suggesting that they followed different degradation pathways. Indeed, we have demonstrated that all lysosome inhibitors significantly reversed IGF-1R α-chain degradation whereas bafilomycin A1 and in a lesser extend methylamine reversed β-chain degradation. Results obtained with MG115 and MG132 suggested that proteasomes could also participate in both IGF-1R α- and β-chains degradation. However, only one minor effect was observed with either bortezomib or epoxomycin. This apparent discrepancy could be explained by the lack of specificity described for some proteasome inhibitors. Although specificity of bortezomib and epoxomycin on proteasome activity was clearly demonstrated,44, 45 the specificity of MG115 and MG132 is less evident. Several reports clearly described that peptide aldehydes could inhibit lysosomal enzymes.46–48 For example, MG132 can also inhibit cathepsin B, which is a lysosomal proteinase of the papain family involved in protein degradation.46 Based on this observation, cathepsin B could be responsible for α-chain degradation. However, the inefficiency of chloroquine and NH4Cl, 2 lysosome inhibitors, on β-chain-degradation lead us to speculate that degradation of α- and β-chains occur in different compartments.
Bafilomycin A1 which blocked both α- and β-chains degradation is a specific inhibitor of the vacuolar proton pump V-type ATPase that prevents from acidification of endosome compartments required for lysosomal proteases maturation.49, 50 Since it does not interfere with localization of surface molecules to early endosome or late multivesicular bodies (MVBs) but inhibits their transport to lysosome,50, 51 our data suggest that h7C10 binding leads to IGF-1R endocytosis of both α- and β-chains into endosomes and then into MVBs. Colocalization of α-chain with either early endosomes and lysosomes and protective effect of lysosome inhibitors on α-chain suggests that IGF-1R α-chain is mainly degraded into lysosomes, whereas the degradation route of β-chain remains to be elucidated. The involvement of proteasome pathway in β-chain degradation was already suggested.27, 52 Since proteasome inhibitors slightly protect both IGF-1R α- and β-chains from degradation, we can not exclude a role of proteasome in IGF-1R process degradation. For example, it has been reported that treatment with proteasome inhibitors decreased the translocation efficiency of the EGFR from outer limiting membrane to internal vesicles of MVB,46 but proteasome inhibitors failed to protect EGFR from degradation.48 The fact that IGF-1–induced a rapid and transient ubiquitination, whereas h7C10 effect was sustained which lead us to hypothesize that ubiquitination is required for h7C10-induced IGF-1R degradation but not absolutely necessary.
IGF-1R ubiquitination on IGF-1 binding was described to play a role in IGF-1R degradation. In melanoma cells, Mdm2-mediated ubiquitination of IGF-1R.21 In this study, they suggested that p53/mdm2 could decide the fate of IGF-1R by controlling receptor internalization and endosomal sorting process. Mdm2-mediated IGF-1R ubiquitination under both basal and IGF-1–stimulated conditions required β-arrestins.21 However, in our study, neither IGF-1 nor h7C10 increased association of either Mdm2 or β-arrestin with IGF-1R. This discrepancy could be explained by the fact that IGF-1R signaling and degradation are different according to the cell type.22, 26 Although Grb10/nedd4 were also shown to participate to IGF-1R ubiquitination,20 h7C10 did not increase basal association of Gbr10 or Nedd4 with IGF-1R. Further investigations using knockdown and/or overexpression of Mdm2 and Nedd4 are needed to confirm that these E3 ubiquitin ligases (Mdm2 and Nedd4) are not involved in the h7C10-induced IGF-1R ubiquitination and degradation. Recently, c-Cbl was also shown to participate in IGF-1R ubiquitination leading to IGF-1R degradation through the caveolin/lipid raft pathway.53 The role of c-Cbl in h7C10-induced IGF-1R ubiquitination and degradation remains to be determined but could represent an alternative E3 ligase to Mdm2 in ubiquitination of IGF-1R. Unexpectedly, h7C10 significantly increased p53 association with IGF-1R. The regulation of p53 activity occurs through a variety of mechanisms but interaction of p53 directly with other proteins seems to be the most important factor in regulating p53 function and stability. Mdm2 is required to maintain p53 at low levels and to suppress the ability of p53 to induce cell death. In this study, p53 level started to increase after 30 min in the presence of h7C10. At this time point, most of the receptors are internalized suggesting that p53 associates with internalized IGF-1R. On the basis of our preliminary results, it is tempting to suggest that this p53 up-regulation in response to h7C10 is responsible to cell cycle arrest and anchorage-independent growth inhibition. The data described herein also suggest that p53 pathway may play a central role in the regulation of IGF-1R expression but the exact role of p53 in IGF-1R degradation remains to be elucidated.
Several neutralizing antibodies anti–IGF-1R were described and their binding induced receptor internalization and degradation. In this study, we have shown that anti–IGF-1R antibody h7C10 inhibited tumor growth through IGF-1R internalization and degradation. Degradation of IGF-1R α-chain occurs through lysosomes, but ubiquitination of IGF-1R could also play a role in facilitating IGF-1R degradation. Our preliminary results suggest that receptor internalization could induce p53 up-regulation and consequently contribute to cell cycle arrest and induction of apoptosis as suggested by Baserga et al.54 The effect of h7C10 on cell cycle arrest and anchorage-independent growth probably constitutes an important property to target tumor growth. The effect of h7C10 on cell cycle is due to inhibition of IGF-1–induced cyclinD1 and cyclin E expression and Rb phosphorylation. In conclusion, h7C10 could block IGF-1R signaling and inhibit tumor growth via antagonization of ligand binding and via a rapid IGF-1R internalization and degradation.