Down-Regulation of Insulin Receptor by Antibodies Against the Type I

Research Article

Down-regulation of Insulin Receptor by Antibodies against the Type I Insulin-Like Growth Factor Receptor: Implications for Anti–Insulin-Like Growth Factor Therapy in Breast Cancer

Deepali Sachdev,1 Rajeeva Singh,2 Yoko Fujita-Yamaguchi,3 and Douglas Yee1

1University of Minnesota Cancer Center, Minneapolis, Minnesota; 2ImmunoGen, Inc., Cambridge, Massachusetts; and 3Tokai University, Hiratsuka, Japan

Abstract however, to date, there are no reported inhibitors completely specific Insulin-like growth factor-I (IGF-I), IGF-II, and insulin have for IGF1R (3–5). Finally, antibodies directed against IGF1R have been all been implicated in regulating several aspects of the used to inhibit activation of IGF1R. a malignant phenotype via the type I IGF receptor (IGF1R) and The widely studied monoclonal antibody IR3 (6) blocks a insulin receptor (IR). We have previously shown that a binding of IGF-I to IGF1R. IR3 inhibits proliferation of MCF-7 chimeric single-chain antibody against IGF1R (scFv-Fc) and cells in vitro (7). It also inhibits the growth of some (MDA-MB-231 a murine antibody EM164 down-regulate IGF1R, making and T61) but not all (MCF-7) breast cancer xenografts in vivo breast cancer cells unresponsive to IGF-I. To determine if IR (8, 9). Additional monoclonal antibodies directed against IGF1R signaling is affected, we examined regulation of IR in MCF-7 have been reported (10–16). A chimeric antibody directed against cells after exposure to these antibodies. Surprisingly, both IGF1R, scFv-Fc, partially inhibits the xenograft growth of MCF-7 scFv-Fc and EM164 resulted in decreased levels of IR in vitro cells (10). We have previously shown that scFv-Fc is an agonistic and in vivo despite their lack of reactivity against IR. Twenty- antibody but then leads to rapid down-regulation of IGF1R via four-hour pretreatment with EM164 also inhibited insulin- the endocytic pathway, making cells refractory to further mediated phosphorylation of IR and insulin-stimulated mitogenic effects of IGF-I (11). EM164 is a murine antibody proliferation of MCF-7 cells. Neither scFv-Fc nor EM164 against IGF1R and also does not bind IR (12). EM164 inhibits caused down-regulation of IR in cells that express very low activation of IGF1R by IGF-I and IGF-II and also causes down- levels of IGF1R or no IGF1R. Expression of IGF1R was regulation of IGF1R. Other reports confirm that down-regulation required for IR down-regulation, which was specific as of IGF1R levels is a common mechanism of action for all monoclonal antibodies directed against IGF1R studied to date neither antibody caused down-regulation of B1 integrin or epidermal growth factor receptor. Reagents that disrupt lipid (13–15). rafts inhibited IR down-regulation by the antibodies, Several lines of evidence show that insulin also regulates breast suggesting that IR in close physical proximity to IGF1R cancer biology (17–19). IGFs and insulin act via a family of in lipid rafts was being endocytosed. Our data show receptors that includes IGF1R and the insulin receptor (IR). IR that down-regulation of IR by monoclonal antibodies against exists in two isoforms generated by alternative splicing of exon 11; IGF1R requires the coexpression of IGF1R and may be exon 11+ or B isoform (IR-B) and exon 11 or A isoform (IR-A; due to endocytosis of hybrid IR/IGF1R or holo-IR. Thus, ref. 20). Although IR-A and IR-B have similar affinities for insulin, antibodies against IGF1R provide inhibition of both IGF IR-A exhibits higher affinity for IGF-II than IR-B (21). Furthermore, and insulin signaling in cancer cells. (Cancer Res 2006; 66(4): human breast cancer specimens have higher IR content than 2391-402) normal breast tissue and fibroadenoma specimens (22). Breast cancer cells express high levels of IR-A compared with IR-B Introduction (23, 24). Many cells and tissues have hybrid receptors assembled with one chain of the IGF1R and one of IR (25, 26). As IGF1R Abundant data from cell culture, animal, and human studies expression levels are also elevated in primary breast cancer tissues have suggested that insulin-like growth factors (IGF) regulate the (27), increased levels of hybrid IGF1R/IR-A receptors likely exist in malignant phenotype (1). The tyrosine kinase type I IGF receptor breast cancer cells and targeting of both IR and IGF1R may be (IGF1R) has been implicated in several different cancers, although necessary to completely inhibit IGF action. Thus, a strategy that its role has not yet been established in any clinical trial. targets only IGF1R may not be sufficient to block all of the Several approaches have been used to inhibit signaling via IGF1R. receptors important in regulating IGF action. However, due Antisense IGF1R oligodeoxynucleotide-based therapy has been to the importance of IR in glucose homeostasis, an ideal anti- reported in patients with astrocytomas (2). A second way to inhibit IGF strategy would target IGF1R and IR only in tumor cells and IGF1R is the use of small-molecule tyrosine kinase inhibitors. leave host IR in insulin target organs unaffected. Several small-molecule inhibitors of IGF1R have been developed; In this study, we assessed the specificity of scFv-Fc and EM164 by determining their effect on IR. Our results show that both scFv-Fc and EM164 down-regulated IR. Furthermore, long-term

Requests for reprints: Deepali Sachdev or Douglas Yee, University of Minnesota exposure in vitro and in vivo to EM164 inhibited insulin Cancer Center, MMC 806, 420 Delaware Street Southeast, Minneapolis, MN 55455. signaling and insulin-stimulated proliferation of MCF-7 cells. Phone: 612-626-8487; Fax: 612-626-4842; E-mail: [email protected] or yeexx006@ Disruption of membrane lipid rafts inhibited down-regulation of umn.edu. I2006 American Association for Cancer Research. IR by scFv-Fc and EM164. Moreover, antibody-mediated down- doi:10.1158/0008-5472.CAN-05-3126 regulation of IR requires coexpression of IGF1R, suggesting that www.aacrjournals.org 2391 Cancer Res 2006; 66: (4). February 15, 2006

Figure 1. Antibodies directed against IGF1R down-regulated IR in MCF-7 cells. A, MCF-7 cells were grown in 6 cm dishes in regular growth medium. Confluent cells (70%) were washed twice with PBS and serum deprived for 24 hours in serum-free medium (SFM). MCF-7 cells were either untreated (lanes labeled SFM), treated with 60 nmol/L aIR3, 250 nmol/L scFv-Fc, or 120 nmol/L EM164 for various times as indicated below the lane number. Cells were washed thrice with ice-cold PBS on ice and lysed with 300 AL/6 cm dish lysis buffer TNESV. Lysates were clarified by centrifugation at 12,000 Â g for 20 minutes at 4jC and soluble cellular proteins were used for experiments. Forty micrograms of cellular proteins were subjected to reducing SDS-PAGE on 8% polyacrylamide gels. After SDS-PAGE, proteins were transferred to nitrocellulose membrane. Cellular proteins were then immunoblotted (IB) for total levels of IRh and chemiluminescence was done using SuperSignal West Pico substrate (Pierce). The 95 kDa h-subunit of IR is shown in the figure. Cellular proteins were also immunoblotted for total levels of p44/p42 ERK1/2 as loading control. Experiments were repeated thrice with similar results and a representative experiment is shown. B, top, MCF-7 cells were untreated or treated with 5 nmol/L IGF-I or 250 nmol/L scFv-Fc for 24 hours. Cellular proteins (500 Ag) were immunoprecipitated (IP) with an antibody against IGF1R and immunoblotted for IGF1R to assay for holo-IGF1R (lanes 1-3), immunoprecipitated with an antibody against IGF1R, and immunoblotted for IR to assay for hybrid IGF1R/IR (lanes 4-6) or immunoprecipitated with an antibody against IR and immunoblotted for IR to measure holo-IR (lanes 7-9). Lanes 10 to 12, cellular proteins immunoblotted for IR; lanes 13 to 15, total ERK1/2 levels as loading control to indicate equal loading of samples. Bottom, MCF-7 cells were either untreated (lanes 1 and 4) or treated with 10 nmol/L insulin (Ins, lanes 2 and 5) or 120 nmol/L EM164 (lanes 3 and 6) for 15 minutes (lanes 1-3) or 24 hours (lanes 4-6) and assayed for holo-IR as described above. Experiments were repeated thrice with similar results and a representative experiment is shown.

cells with only IR expression may be unaffected by monoclonal Cell lines and culture. MCF-7L cells were obtained from Dr. C. Kent antibody strategies. Osborne (Baylor College of Medicine, Houston, TX) and were maintained as described previously. RÀ cells (28) were obtained from Dr. Renato Baserga (Kimmel Cancer Institute, Thomas Jefferson University, Phila- Materials and Methods delphia, PA) and were maintained in DMEM with 10% fetal bovine serum Reagents. All reagents and chemicals were purchased from Sigma and 50 Ag/mL G418. RÀ cells expressing wild-type human IGF1R (RÀ/ (St. Louis, MO) and cell culture reagents were from Invitrogen/ IGF1R) were made by stably cotransfecting RÀ cells with a cDNA Life Technologies (Rockville, MD). IGF-I and IGF-II were purchased encoding human IGF1R and a plasmid expressing hygromycin resistance from GroPep (Adelaide, Australia) and human insulin was from Eli using Effectene reagent (Qiagen, Inc., Valencia, CA). Lilly (Indianapolis, IN). Antibodies against the h-subunits of IGF1R and Cell stimulation. Seventy percent confluent cells in 6 cm dishes in

IR and the monoclonal antibody against Gai2 were from Santa Cruz regular growth medium were washed twice with PBS and serum deprived Biotechnology, Inc. (Santa Cruz, CA). Antiphosphotyrosine antibody for 24 hours in serum-free medium as described previously (29). For conjugated to horseradish peroxidase (PY20-HRP) was from BD treatment, medium was replaced with serum-free medium containing Transduction Laboratories (Lexington, KY). Antibodies against p44/p42 5 nmol/L IGF-I, 10 nmol/L IGF-II, 10 nmol/L insulin, 60 nmol/L aIR3, extracellular signal-regulated kinase (ERK) 1/2 (phosphospecific and 250 nmol/L scFv-Fc, or 120 nmol/L EM164 for times as indicated total) and Akt (phosphospecific and total) were purchased from Cell in the figure captions. To determine if EM164 inhibited IGF-I, IGF-II, Signaling (Beverly, MA). or insulin-mediated activation of their cognate receptors or other

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Down-regulation of Insulin Receptor in Breast Cancer Therapy downstream pathways, cells were first pretreated with antibodies for 15 Results minutes or 24 hours and then with the various ligands for additional 10 minutes or 5 minutes for insulin. All experiments were repeated a Antibodies against IGF1R down-regulated IR in breast minimum of thrice. cancer cells. We and others have shown that antibodies against Cell lysis. Cells were washed thrice with ice-cold PBS on ice and lysed IGF1R inhibit growth of breast cancer cells and cause efficient down- with 300 AL/6 cm dish lysis buffer TNESV [50 mmol/L Tris-Cl (pH 7.4), regulation of IGF1R (10, 11, 13). To determine if IR was also affected, 1% NP-40, 2 mmol/L EDTA (pH 8.0), 100 mmol/L NaCl, 10 mmol/L Na we examined the effect of three different antibodies on IR levels in orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 20 Ag/mL MCF-7 cells. None of the antibodies caused significant changes in the leupeptin, and 20 Ag/mL aprotinin]. level of IR after 30 minutes of exposure (Fig. 1A). However, scFv-Fc Proliferation assays. Cells were plated in 24-well plates with down-regulation of IR was seen within 6 hours (Fig. 1A, lane 4) and 20,000 cells per well in regular growth medium. Cells were switched to by 24 hours levels of IR were markedly decreased (lane 10) when serum-free medium for 24 hours and then treated as indicated in the a figure captions. All treatments were done in triplicates. Growth was compared with untreated cells. IR3 and EM164 also caused a measured 4 to 5 days after treatment by the 3-(4,5-dimethylthiazol-2-yl)- decrease in IR levels after 6 hours and by 24 hours IR levels were 2,5-diphenyl tetrazolium bromide (MTT) assay as described previously decreased by f50%. The down-regulation of IR by these antibodies (11, 30). was via the endocytic pathway as methylamine, which increases pH Isolation of plasma membrane lipid rafts. Lipid rafts were isolated of the endosomal vesicles, inhibited the down-regulation of IR (data using sequential detergent extraction of membranes as described (31). not shown). We have previously shown that IGF-I given over the Cells were resuspended in ice-cold buffer A [25 mmol/L MES and 150 same time period did not down-regulate IGF1R (11) and it also did mmol/L NaCl (pH 6.5)] and lysed with an equal volume of ice-cold buffer not decrease IR over the same time period (see Fig. 3). Because A with 2% Triton X-100, 2 mmol/L Na orthovanadate, and 2 mmol/L EM164 is an IGF1R antagonist but still causes receptor down- PMSF. Lysates were incubated for 60 minutes on ice with gentle inversion and centrifuged for 30 minutes and the supernatants were saved as regulation, it does not appear that receptor tyrosine phosphoryla- Triton-soluble cytoplasmic and nonraft membrane proteins. Triton- tion is required for these effects. insoluble pellets were extracted with buffer B [10 mmol/L Tris-Cl It is possible that the antibodies down-regulated hybrid receptor (pH 7.6), 500 mmol/L NaCl, 1% Triton X-100, 2 mmol/L Na orthovanadate, and the apparent decrease in total IR levels were a result of 1 mmol/L PMSF, and 60 mmol/L h-octylglucoside] on ice for 30 minutes diminished hybrid levels. To determine if the antibodies down- with frequent mixing. Triton-insoluble and h-octylglucoside–soluble regulated hybrid IGF1R/IR or holo-IR, MCF-7 cell lysates were supernatants containing lipid raft proteins were obtained after centrifu- examined for IR by immunoblot after immunoprecipitation with gation at 15,000 Â g for 20 minutes. either anti-IGF1R (Fig. 1B, top, lanes 4-6) or anti-IR antibodies Disruption of rafts. Lipid rafts were disrupted by treating cells with (Fig. 1B, top, lanes 7-9). Twenty-four-hour treatment with scFv-Fc two different classes of cholesterol-depleting reagents—filipin III and methyl-h-cyclodextrin for 24 hours. caused a down-regulation of the IR within the IGF1R/IR hybrid Immunoblotting. For immunoblotting, 40 Ag of cellular proteins were complex (lane 6 compared with lane 4) and also the IR/IR subjected to reducing SDS-PAGE. Immunoblotting was done as described holoreceptor (lane 9 compared with lane 7). In lanes 10 to 12, total previously (11). To detect levels of receptors, membranes were blotted cell lysates were immunoblotted for IR and a 24-hour treatment with with 1:1,000 dilution of rabbit polyclonal antibody against IRh or IGF1Rh. scFv-Fc caused a down-regulation of IR. As a control, in lanes 1 to Chemiluminescence was done using SuperSignal West Pico substrate 3 lysates were immunoprecipitated with IGF1R and immunoblotted (Pierce, Rockford, IL). For detecting phosphorylated proteins, membranes with IGF1R and as we have previously reported (11), 24-hour were incubated with 1:2,000 dilution of PY20-HRP antiphosphotyrosine treatment with scFv-Fc down-regulated IGF1R. Similar results were antibody in TBST for 1 hour at room temperature. All other antibodies seen with EM164 (Fig. 1B, bottom), which also decreased IR within were used as per instructions of the manufacturer. In all immunoblot analyses, total levels of ERK1/2 were used as loading control to indicate the IR/IR holoreceptor following a 24-hour treatment compared equal loading of samples. with a 15-minute treatment (lane 6 compared with lane 3) and Immunoprecipitations. Five hundred micrograms of total cellular compared with untreated or insulin-treated cells. Thus, antibodies proteins were immunoprecipitated with 2 Ag polyclonal antibody against directed only against IGF1R down-regulate IR in MCF-7 cells. h h IGF1R or IR as described previously (11). These results were specific for IGF1R and IR, as neither h1 Animal studies and xenograft tumor extract analyses. All animal integrin or epidermal growth factor receptor (EGFR) levels were protocols were approved by the University of Minnesota Institutional affected by EM164 and scFv-Fc (data not shown). Moreover, other Animal Care and Use Committee. MCF-7 xenografts were grown in 4- antibodies (rituximab and trastuzumab) did not affect IR or IGF1R week-old female athymic mice implanted with 60-day release pellets of 0.5 mg 17h-estradiol (Innovative Research of America, Sarasota, FL) as (data not shown). Thus, only antibodies directed against IGF1R can described previously (11). Tumor volume was estimated from bidirec- down-regulate IR and IGF1R. tional measurements using the formula length Â breadth2 / 2. When Short-term treatment with EM164 did not inhibit insulin tumors entered the exponential growth phase, mice were treated with signaling in MCF-7 cells. In MCF-7 cells, IRS-1 is the major adaptor 800 Ag EM164 or an isotope-matched control antibody i.p. every 3 days. protein phosphorylated by IGF-I and insulin (29). IGF-I treatment Treatments were continued for 4 weeks and at the end of the resulted in detection of a 185 kilodaltons phosphorylated band in experiment, mice were sacrificed and tumors were harvested. The cells (Fig. 2A, lane 2) and in cells treated with IGF-II (lane 3)or tumors were snap frozen in liquid nitrogen. Frozen tumor samples were insulin (lane 4) but not in untreated cells (lane 1). Pretreatment of homogenized in a tissue pulverizer in a dry ice/ethanol bath as described cells with 120 nmol/L EM164 for 15 minutes before stimulation with previously. IGF-I inhibited IGF-I-induced phosphorylation of IRS-1 (lane 6 To determine the effect of scFv-Fc on receptor levels, mice with MCF-7 xenografts on either side were used. Tumors from the left side of all mice compared with lane 2) and also inhibited phosphorylation of IRS-1 were resected before treatment. Twenty-four hours after resection, mice mediated by IGF-II (lane 7 compared with lane 3), but did not inhibit were injected i.p. with 500 Ag scFv-Fc or PBS as control. Twenty-four phosphorylation of IRS-1 by insulin (lane 8 compared with lane 4). hours after treatment, mice were sacrificed and the remaining tumors Fifteen-minute treatment with EM164 also did not inhibit insulin- were harvested. stimulated phosphorylation of IR (Fig. 2C, top). Because EM164 does www.aacrjournals.org 2393 Cancer Res 2006; 66: (4). February 15, 2006

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research not directly bind to IR, insulin-stimulated phosphorylation of IRS-1 ability to be further stimulated by treatment with additional insulin is not inhibited by EM164 following a short-term treatment. (Fig. 2B, lane 10 compared with lane 9). Twenty-four-hour pretreatment with antibodies against To directly assay the effect of EM164 on IR activation, cellular IGF1R inhibited insulin signaling and insulin-stimulated proteins were immunoprecipitated with an antibody against IR proliferation of MCF-7 cells. We hypothesized that if the followed by immunoblotting with an antiphosphotyrosine anti- antibodies down-regulated the holo-IR, then they should more body. Short-term treatment with EM164 did not inhibit phosphor- completely inhibit insulin signaling after a 24-hour exposure. ylation of IR (Fig. 2C, top, lane 8 compared with lane 4), indicating MCF-7 cells were pretreated with EM164 for 24 hours (Fig. 2B, that EM164 does not directly block insulin binding to IR. However, lanes 5-8), or not (lanes 1-4), before exposure to IGF-I, IGF-II, or a 24-hour treatment with EM164 inhibited IR phosphorylation by insulin. Figure 2B (lanes 1-4) shows that IGF-I, IGF-II, and insulin insulin (lane 12 compared with lane 4). Similarly, a 24-hour exposure resulted in phosphorylation of IRS-1 in control cells not treatment with scFv-Fc also decreased phosphorylation of IR pretreated with EM164. When cells were exposed to EM164 for 24 (Fig. 2C, lane 16 compared with lane 4). As a control, the hours followed by treatment with IGF-I (lane 6), IGF-II (lane 7), membrane in Fig. 2C (top) was stripped and reprobed for IR or insulin (lane 8), phosphorylated IRS-1 was not detected. In (bottom) and shows the levels of IR in the immunoprecipitates. contrast, cells treated with insulin for 24 hours still retained the These data show that following a 24-hour treatment with scFv-Fc

Figure 2. EM164 did not inhibit IR activation following a short treatment but inhibited insulin signaling and insulin-stimulated proliferation following a 24-hour treatment. A, short-term treatment with EM164 did not inhibit insulin signaling. Seventy-percent-confluent MCF-7 cells were switched to serum-free medium. Twenty-four hours later, cells were either untreated (lanes 1 and 5), treated with 5 nmol/L IGF-I for 10 minutes (lanes 2 and 6), 10 nmol/L IGF-II for 10 minutes (lanes 3 and 7), or 10 nmol/L insulin for 5 minutes (lanes 4 and 8), without (lanes 1-4) or with a 15-minute preincubation with 120 nmol/L EM164 (lanes 5-8). Cell lysates were immunoblotted with PY20-HRP to assay for phosphorylation of IRS-1. B, 24-hour treatment of cells with EM164 inhibited phosphorylation of IRS-1 induced by insulin. Cells were not pretreated (lanes labeled no pre-Tx), pretreated with EM164 for 24 hours (lanes labeled 24h EM164), or pretreated with insulin (lanes labeled 24h Ins). Following this pretreatment, cells were either left untreated (lanes 1, 5, and 9) or stimulated with 5 nmol/L IGF-I (lanes 2 and 6), 10 nmol/L IGF-II (lanes 3 and 7), or 10 nmol/L insulin (lanes 4, 8, and 10). Cell lysates were immunoblotted with PY20-HRP antibody to assay for phosphorylation of IRS-1. C, 24-hour treatment with EM164 and scFv-Fc also inhibited phosphorylation of IR. MCF-7 cells were untreated (lanes 1, 5, 9, and 13) or treated with IGF-I (lanes 2, 6, 10, and 14), IGF-II (lanes 3, 7, 11, and 15), or insulin (lanes 4, 8, 12, and 16) without EM164 or scFv-Fc exposure (lanes 1-4), following a 15-minute exposure to EM164 (lanes 5-8) after a 24-hour exposure to EM164 (lanes 9-12) or after a 24-hour exposure to scFv-Fc (lanes 13-16). Cellular proteins were immunoprecipitated with IR antibody followed by immunoblotting with an antiphosphotyrosine antibody (top) or immunoprecipitated with IR antibody followed by immunoblotting for IR (bottom).

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Down-regulation of Insulin Receptor in Breast Cancer Therapy and EM164, down-regulation of IR levels resulted in inhibition of cells as shown in Fig. 2E (filled column) and EM164 did not IR activation by insulin. significantly inhibit insulin-stimulated proliferation of MCF-7 cells Furthermore, as shown in Fig. 2D, treatment of cells with when added simultaneously (left). We tested if a 24-hour EM164 for 24 hours also blocked insulin-stimulated phosphory- pretreatment of cells with EM164 before exposure to additional lation of Akt and phosphorylation of p44/p42 ERK1/2 (lane 6) insulin would have any effect on the growth of cells. Cells were compared with the phosphorylation in cells that were not pretreated with either insulin as control or EM164 for 24 hours in pretreated with EM164 (lane 3). These data show that down- Fig. 2E (right). After this pretreatment, cells were incubated with regulation of IR by EM164 partially blocked the ability of insulin or without additional insulin. Pretreatment with insulin followed to initiate downstream signaling. by treatment with subsequent insulin resulted in increased To test the effect of EM164 on insulin-stimulated proliferation, proliferation compared with the control as shown in Fig. 2E.In MCF-7 cells were treated with 10 nmol/L insulin in the absence contrast, pretreatment with EM164 inhibited proliferation in or presence of EM164. Insulin stimulated the growth of MCF-7 response to insulin.

Figure 2 Continued. D, 24-hour treatment with EM164 inhibited downstream signaling pathways activated by insulin. Cells without exposure to EM164 (lanes 1-3) or following exposure to EM164 for 24 hours (lanes 4-6) were untreated (lanes 1 and 4), treated with IGF-I (lanes 2 and 5), or 10 nmol/L insulin for 5 minutes (lanes 3 and 6). Cellular proteins were immunoblotted for phospho-Akt, total Akt, phospho-p44/p42 ERK1/2, and total p44/p42 ERK1/2. All experiments shown in (A-D) were repeated thrice with similar results and a representative experiment is shown. E, pretreatment of cells with EM164 inhibited growth stimulation by insulin. Left, MCF-7 cells in serum-free conditions were treated with insulin, EM164, or insulin and EM164 together for 5 days. Proliferation was then measured by the uptake of the MTT reagent followed by measurement of absorbance at 570 nm. Growth is shown as the absorbance at 570 nm. Columns, mean of triplicate samples; bars, SE. Right, MCF-7 cells in serum-free conditions were pretreated with either insulin (columns labeled 24h pre-Tx: Insulin) or EM164 (columns labeled 24h Tx: EM164) for 24 hours. Medium was removed and cells were treated without (SFM) or with 10 nmol/L insulin for 4 days. Proliferation was then measured by the uptake of the MTT reagent followed by measurement of absorbance at 570 nm. Growth is shown as the absorbance at 570 nm. Columns, mean of triplicate readings; bars, SE. Experiments were repeated thrice with similar results.

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Thus, cells pretreated with insulin still have ample receptor on the of IGF1R (28) and do not express IGF1R (Fig. 3C, top). In these cell surface, allowing for further proliferation in response to insulin. cells, neither scFv-Fc nor EM164 affected IR levels (Fig. 3D, top, In contrast, when cells pretreated with EM164 were exposed to lanes 9 and 10) after 24-hour treatment. insulin, the cells had decreased cell surface IR and did not proliferate Our data from Hs578T and RÀ cells suggested that both IGF1R in response to insulin. and IR need to be expressed on the same cell to see IR down- scFv-Fc and EM164 did not down-regulate IR in Hs578T regulation by anti-IGF1R antibodies. To test this hypothesis, we breast cancer cells with very low levels of IGF1R expression. transfected the cDNA for human IGF1R into RÀ cells to generate Because the antibodies against IGF1R have no immunoreactivity RÀ/IGF1R cells. Two different clones were characterized and the with IR (12), we next examined if expression of IGF1R was required IGF1R stably expressed in these cells was functional as measured for IR down-regulation. Hs578T expressed low levels of IGF1R when by the ability of IGF-I to activate downstream signaling pathways. compared with MCF-7 (Fig. 3A). Hs578T and MCF-7 cells had To test the effect of EM164 and scFv-Fc on IR levels, RÀ/IGF1R equivalent amounts of IR expression (Fig. 3A, top). In Hs578T cells, #11, and RÀ/IGF1R #8 were exposed to IGF-I, insulin, EM164, scFv- 24-hour treatment with scFv-Fc did not result in decreased levels of Fc, or no additional treatment for 15 minutes or 24 hours and IR (Fig. 3A), whereas a similar treatment decreased IR levels in immunoblotted for IGF1R (Fig. 3C) or IR (Fig. 3D). In both clones MCF-7 cells. In both cells, a 15-minute treatment with scFv-Fc did expressing IGF1R, 24-hour treatment with either EM164 (lane 9)or not down-regulate IR. In Hs578T cells, scFv-Fc was able to down- scFv-Fc (lane 10) caused down-regulation of IGF1R as shown in regulate the very low levels of IGF1R in these cells (Fig. 3A, middle, Fig. 3C. Furthermore, in RÀ/IGF1R #11 cells, both EM164 (Fig. 3D, lane 12 compared with lane 10). Figure 3A (bottom) shows the total lane 9) and scFv-Fc (lane 10) also caused down-regulation of IR levels of ERK1/2 as a loading control. Similar results were seen with after a 24-hour treatment compared with untreated cells (lane 6) EM164 (Fig. 3B). or cells treated with the antibodies for only 15 minutes (lanes 4 scFv-Fc- and EM164-mediated down-regulation of IR and 5) as shown in Fig. 3D. Neither IGF-I nor insulin affected required coexpression of both IGF1R and IR on the same cell. IGF1R or IR levels (Fig. 3C and D). Figure 3E shows the levels of RÀ cells are fibroblasts derived from mice with a genetic deletion total ERK1/2 to indicate equal loading of samples in Fig. 3C and D.

Figure 3. Coexpression of IGF1R and IR was required for down-regulation by antibodies. A, scFv-Fc did not down-regulate IR in Hs578T cells with low IGF1R levels. MCF-7 (lanes 1-6) and Hs578T cells (lanes 7-12) were untreated (lanes 1, 4, 7, and 10) or treated with 5 nmol/L IGF-I (lanes 2, 5, 8, and 11)or 250 nmol/L scFv-Fc (lanes 3, 6, 9, and 12) for 15 minutes (lanes 1-3 and 7-9)or 24 hours (lanes 4-6 and 10-12). Cellular proteins were then immunoblotted for total levels of IGF1Rh,IRh, and ERK1/2 (to show equal loading of all samples). Experiments were repeated thrice with similar results and a representative one is shown. B, EM164 did not down-regulate IR in Hs578T cells. Hs578T cells were untreated (lane 1) or treated either with 10 nmol/L insulin (lane 2) or 120 nmol/L EM164 (lane 3) for 24 hours. Cellular proteins were then immunoblotted for total levels of IRh or total ERK1/2 as loading control.

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Thus, expression of IGF1R in RÀ cells leads to the down-regulation expressed as percent of the respective receptor levels in untreated of IR by both EM164 and scFv-Fc. These results support the cells. G protein a inhibitory subunit (Gai2) was used as a loading observation that expression of both receptors on the surface of the control and marker for lipid rafts (36) and was present only in lanes same cell is required for down-regulation of IR by anti-IGF1R 4 to 6, which represent the raft fraction (Fig. 4C). antibodies. Disruption of rafts partially rescued down-regulation of IR scFv-Fc down-regulated IGF1R and IR in lipid rafts. Several by antibodies directed against IGF1R. Because both IGF1R and reports have described the presence of both IGF1R and IR in IR were also found in rafts, we hypothesized that IGF1R antibodies plasma membrane lipid rafts in adipocytes and fibroblasts (32–35). down-regulated IR due to receptor colocalization in rafts. In this Therefore, we determined if IGF1R and IR are found in lipid rafts in model, initiation of endocytosis or transcytosis of IGF1R in the MCF-7 cells. Both IGF1R and IR were present in the Triton-soluble rafts also down-regulated IR in this membrane compartment. cytosolic and membrane fraction (Fig. 4A and B, lanes 1-3) and Therefore, we disrupted lipid rafts with either filipin III or methyl- scFv-Fc down-regulated both receptors. In addition, IGF1R and IR h-cyclodextrin and determined the effect on down-regulation of IR were also found in the Triton-insoluble/octylglucoside-soluble levels by the antibodies. In Fig. 5A, both EM164 (lane 4) and fraction in lanes 4 to 6, which represents the lipid raft proteins. scFv-Fc (lane 5) down-regulated IR after a 24-hour exposure when About 20% to 25% of the total IGF1R and IR found in MCF-7 cells rafts were intact. When cells were treated with 2 Ag/mL filipin III was in lipid rafts. Furthermore, 24-hour treatment with scFv-Fc (Fig. 5B) or 2 mmol/L methyl-h-cyclodextrin (Fig. 5C) for 24 hours, down-regulated both IR and IGF1R in the lipid raft (lane 6) in Fig. down-regulation of IR by EM164 (lane 4) and scFv-Fc (lane 5) was 4A and B, respectively. Figure 4D shows the quantitation of the partially rescued. Immunoblotting for ERK1/2 was used as loading levels of IGF1R and IR in the Triton-insoluble/octylglucoside- control and total ERK1/2 levels were equal in all samples (data not soluble fraction in Fig. 4A and B with the relative levels of either shown). Figure 5D represents densitometric quantitation of the

Figure 3 Continued. C, D, and E, scFv-Fc and EM164 down-regulated IR in RÀ cells expressing IGF1R. RÀ,RÀ/IGF1R #11, and RÀ/IGF1R #8 cells were untreated (lanes 1 and 6), treated with 5 nmol/L IGF-I (lanes 2 and 7), 10 nmol/L insulin (lanes 3 and 8), 120 nmol/L EM164 (lanes 4 and 9), or 250 nmol/L scFv-Fc (lanes 5 and 10) for 15 minutes (lanes 1-5)or 24 hours (lanes 6-10). Cellular lysates were immunoblotted for IGF1Rh (C), IRh (D), and total ERK1/2 to show equal loading of all samples (E). Experiments were repeated four times and a representative experiment is shown.

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regulation in xenograft tumors. Figure 6A shows the levels of IR in the tumors before treatment (before PBS and before scFv-Fc lanes). Twenty-four hours after treatment, tumors taken from animals treated with scFv-Fc had significant down-regulation of IR (bottom, after scFv-Fc lanes). No change in IR was observed in PBS- treated animals (top, after PBS lanes). Densitometric analysis showed that after treatment with scFv-Fc, IR levels in xenograft tumor extracts were f38% (n = 3) of that in tumor extracts before scFv-Fc treatment. In contrast, in mice that received control PBS injections, the IR levels were f99% of that before treatment. EM164 inhibited xenograft growth of MCF-7 cells and down- regulated IGF1R and IR in vivo. Maloney et al. (12) have previously shown that EM164 inhibits IGF-I-mediated proliferation of MCF-7 cells in vitro and the xenograft growth of BxPC-3 human pancreatic cancer cells. Figure 6B shows that EM164 inhibited xenograft growth of MCF-7 cells compared with control antibody. Animals treated with EM164 showed significant decrease in tumor volume (P = 0.0033 at day 39, t test; n = 5). Tumor volumes were significantly lower in EM164-treated animals over the period of treatment. After 4 weeks of treatment, mice were sacrificed, tumors were harvested, and tumor extracts from mice treated with EM164 or control antibody were assayed for levels of IGF1R and IR. As shown in Fig. 6C, EM164 caused down-regulation of both IGF1R and IR, whereas the control antibody did not affect levels of either. Figure 6C shows the quantitation of IR and IGF1R levels in the xenograft tumors. In EM164-treated tumors, IR levels were decreased by 32% and IGF1R levels by 75% compared with levels in control antibody-treated tumors. Furthermore, the inhibition of xenograft tumor growth by EM164 (Fig. 6B) and decrease in receptor levels (Fig. 6C and D) was also paralleled by the inhibition of IGF-I-mediated activation of IGF1R and phosphatidylinositol-3 kinase pathway in MCF-7 xenograft tumors in vivo by EM164 (Fig. 6D). Treatment with IGF- I for 30 minutes before sacrifice led to phosphorylation of IRS-1 and Akt in MCF-7 xenograft tumors (Fig. 6D, lane 5) compared with mice injected with PBS (lane 4). Treatment with EM164 24 hours before treatment with IGF-I caused inhibition of IRS-1 Figure 4. IGF1R and IR were found in lipid rafts and scFv-Fc down-regulated phosphorylation (lane 6). Similar inhibition of phosphorylation of both receptor levels in lipid rafts. A to C, MCF-7 cells were untreated (lanes 1 Akt was seen in the xenograft tumor following a 24-hour and 4), or treated with either 5 nmol/L IGF-I (lanes 2 and 5) or 250 nmol/L scFv-Fc (lanes 3 and 6) for 24 hours. Triton-soluble (TS) proteins (lanes 1-3) treatment with EM164. Total levels of Akt were unchanged by and Triton-insoluble/octylglucoside-soluble (TI) lipid raft proteins (lanes 4-6) treatment with EM164. were immunoblotted for IRh (A), IGF1Rh (B), and Gai2 (C). D, densitometric quantitation of the levels of IR and IGF1R in the Triton-insoluble/ octylglucoside-soluble fraction in (A and B). The relative levels of IR and IGF1R following treatment with IGF-I or scFv-Fc are expressed as percent of the Discussion level of the respective receptor in untreated cells. Experiment was repeated thrice and similar results were obtained. Perhaps the best example of a well-validated target for cancer therapy is estrogen receptor (ER) a in breast cancer. By disrupting receptor function with selective ER modulators or by lowering relative levels of IR and IGF1R in Fig. 5A to C expressed as serum estradiol levels via ovarian suppression or aromatase percentage of the respective receptor level in untreated cells in lane inhibition, essentially all stages of breast cancer are effectively 1 of Fig. 5A. As shown in Fig. 5D, disruption of lipid rafts by filipin treated. Although the idea that ovarian-derived factors stimulated increased IR levels from 16.5% to 38.9% following 24-hour breast cancer growth preceded the molecular identification of treatment with EM164. Furthermore, methyl-h-cyclodextrin was ERa, we now understand that only tumors that express this target even more efficient in reversing IR down-regulation by EM164 or benefit from its inhibition, although other ERs have been scFv-Fc. In contrast, IGF1R was down-regulated by EM164 (lane 4) identified. Thus, a firm understanding of the effector of estrogen and scFv-Fc (lane 5) even when rafts were disrupted by filipin III or action has provided substantial clinical benefit for breast cancer methyl-h-cyclodextrin, indicating that down-regulation of the patients. IGF1R was partially due to direct binding of the antibodies to There are potentially many other growth regulatory pathways surface IGF1R followed by endocytosis. that affect cancer cell biology. It is evident that IGF system regulates scFv-Fc caused in vivo down-regulation of IR in xenograft various aspects of the malignant phenotype. All of the IGF ligands tumors. BecausewehavepreviouslyshownthatscFv-Fc (insulin, IGF-I, and IGF-II) have been implicated in cancer cell down-regulates IGF1R in vivo (11), we examined IR down- biology. Thus, strategies to inhibit IGF1R have emerged as potential

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Down-regulation of Insulin Receptor in Breast Cancer Therapy cancer treatments. However, in addition to IGF1R, preclinical data These unexpected results raise the question, how did scFv-Fc and support an important role for IR regulating IGF action, either as a EM164 decrease levels of holo-IR? hybrid or holoreceptor (37). An optimal anti-IGF therapy in cancer Our data show that scFv-Fc and EM164 down-regulate IR by would block all of the receptors responsible for IGF signaling, endocytosis of IR present in lipid rafts along with IGF1R. Indeed, including IR. Certainly, IGF1R selectivity is desirable in host tissues lipid rafts have gained prominence as specialized platforms that but this may not be the case in cancer cells. can bring together components of a signaling cascade in a spatial Antibodies that target IGF1R have been developed for clinical manner in the plasma membrane (38). Many reports have shown use. We have previously shown that scFv-Fc and EM164 inhibit that IGF1R and IR can be found in lipid rafts (32–34). In this IGF-I-mediated effects and down-regulate IGF1R efficiently (12), study, we show that IGF1R was found in lipid rafts in MCF-7 cells suggesting that down-regulation of IGF1R is an important (Fig. 5). Furthermore, because MCF-7 cells lack caveolin-1 (39), mechanism of action for antibodies against IGF1R. Therefore, we these cells should only have noncaveolin flat lipid rafts and not sought to determine the effects of scFv-Fc and EM164 on IR and the caveolin-containing invaginated lipid rafts or caveolae. It is insulin signaling. In this study, we show that both these antibodies possible that the binding of antibodies to IGF1R may result in the also down-regulate IR, inhibit signaling via IR, and inhibit insulin- sequestration of some of the antibody-IGF1R complexes in lipid stimulated proliferation of breast cancer cells in vitro. This was rafts from where they undergo endocytosis or transcytosis. In surprising because neither antibody binds IR. It is possible that addition to the classic clathrin-dependent endocytic pathways the decrease in IR levels reflects the down-regulation of IR, which (40), endocytosis can also occur via clathrin-independent caveolar is present as part of the hybrid IGF1R/IR via direct binding of pathways (41, 42). Thus, although internalization of receptors via the antibodies to the hybrid receptor. However, we show that both caveolae has been documented (41, 43–45), endocytosis of cell scFv-Fc and EM164 also down-regulate holo-IR in MCF-7 cells. surface receptors from noncaveolin flat lipid rafts has not yet been

Figure 5. Disruption of lipid rafts reversed down-regulation of IR by antibodies. A to C, MCF-7 cells were untreated (lane 1), or treated with either 5 nmol/L IGF-I (lane 2), 10 nmol/L insulin (lane 3), 120 nmol/L EM164 (lane 4), or 250 nmol/L scFv-Fc (lane 5) for 24 hours in the absence (A)or presence of either 2 Ag/mL filipin III (B)or 2 mmol/L methyl-h-cyclodextrin (C) for 24 hours. Cellular proteins were separated by SDS-PAGE and immunoblotted for IRh (left) or IGF1Rh (right). Equal loading of samples was ensured by immunoblotting for total levels of ERK1/2 (data not shown). D, densitometric quantitation of the levels of IRh (left) and IGF1Rh (right)in(A-C). Columns, percentage of the level of the respective receptor in untreated cells in (A). Experiments disrupting lipid rafts were repeated four times with similar results and a representative experiment is shown.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research reported. However, it has recently been shown that SV40 can enter kinase receptors can also be internalized from noncaveolin lipid cells via an endocytic pathway that involves neither clathrin nor rafts. caveolae (46), suggesting that receptors can be internalized from We were able to show that scFv-Fc decreases the amount of flat lipid rafts. We have shown for the first time that tyrosine IGF1R in lipid rafts (Fig. 4). Furthermore, in MCF-7 cells, IR was also

Figure 6. Antibodies down-regulated IR in vivo and inhibited xenograft growth. A, scFv-Fc down-regulated IR in vivo. Female athymic mice bearing MCF-7 xenograft tumors on each side were studied. Tumors from the left side were resected from each mouse before treatment. The next day, three mice were injected i.p. with 500 Ag scFv-Fc and three with PBS. Twenty-four hours after treatment, tumors were collected. Tumor samples were frozen in liquid nitrogen and homogenized in TNESV buffer using a pulverizer as described in Materials and Methods. Tumor extracts (100 Ag) were immunoblotted for total IRh levels. Lanes 1 to 3 (top and bottom), total IR levels in the tumor extracts in the six mice before treatment. IR levels in the mice that were injected with scFv-Fc is shown in lanes labeled ‘‘after scFv-Fc’’ (lanes 4 to 6, bottom). Lanes 4 to 6 (top) show IR levels in tumor extracts from mice after treatment with PBS. Experiments were repeated thrice and a representative one is shown. B, EM164 inhibited xenograft growth of MCF-7 cells in athymic mice. Sixty-day-release E2 pellets were implanted in 4- to 5-week-old female mice. The next day, 5 Â 106 MCF-7 cells were injected into the second mammary fat pad on the left side of each mouse. Twelve days after cells were injected, when the tumors were in the exponential phase of growth, five mice were injected with 800 Ag EM164 and five were injected with 800 Ag of an isotype-matched control antibody. Treatments were done every 3 days. Tumors were measured every 3 days and tumor volumes were calculated using the formula length Â width2 /2. Treatments were continued for 4 weeks. Tumor growth is shown as the tumor volume over time. Points, average volume (n = 5 for each group); bars, SE. Statistical significance of inhibition of tumor growth in EM164-treated animals compared with control antibody–treated animals was assessed using a t test. **, P < 0.005; *, P < 0.03, significant inhibition of tumor growth. Tumor growth experiments were repeated thrice and a representative experiment is shown. C, EM164 down-regulated IR in xenograft tumors. At the end of the experiment in (B), mice were sacrificed and tumors were harvested. Tumor extracts (100 Ag) from three mice treated with the control antibody (lanes 2-4) and three mice treated with EM164 (lanes 5-7) were subjected to SDS-PAGE and immunoblotted for IGF1Rh and IRh. MCF-7 cell lysate (40 Ag) was run as a control (lane 1). Bottom graph, quantitation of the average levels of IRh and IGF1Rh in the tumors expressed as percentage of the level in control antibody (C-Ab)–treated tumors. D, EM164 inhibited IGF-I-mediated activation of IGF1R in MCF-7 xenograft tumors. Female athymic mice bearing MCF-7 xenograft tumors were treated i.p. either with 800 Ag EM164 (lane 6) or PBS (lanes 4 and 5). Twenty-four hours later, mice were injected i.p. with 200 Ag IGF-I in a volume of 200 AL(lanes 5 and 6) or PBS (lane 4). Thirty minutes later, mice were sacrificed and tumors were harvested. The tumors were snap frozen, homogenized, and 100 Ag extracts were immunoblotted for phosphotyrosine (top), phospho-Akt (middle), and total Akt (bottom).

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Down-regulation of Insulin Receptor in Breast Cancer Therapy found in lipid rafts. Thus, the ability of antibodies against IGF1R to targeting IGF1R are unknown. However, the existence of human down-regulate holo-IR may be due to the localization of IR in the populations with low levels of IGF-I (49) provides hope that same membrane compartment as some of the antibody-IGF1R inhibition of IGF effects may be tolerated without morbidity. complexes undergoing endocytosis. This might represent only a Furthermore, humans with IGF1R mutations have been de- small fraction of the total IR and explains why the antibodies down- scribed (50), suggesting that disruption of IGF1R can be regulate IGF1R more efficiently than IR as IGF1R antibodies can tolerated. If it is true that maximum benefit would be derived down-regulate IGF1R in and out of the lipid rafts. This mechanism is from targeting all receptors responsible for the action of IGF-I also supported by our finding that reagents that disrupt lipid rafts and IGF-II, and that would include IR-A, then unbalancing inhibit down-regulation of IR by EM164 and scFv-Fc. Both filipin III glucose homeostasis would not be a desirable effect of anti- and methyl-h-cyclodextrin reversed down-regulation of IR by IGF1R therapy especially if it has to be given over a long period EM164 and scFv-Fc. In contrast, these agents did not have a of time. In that respect, we have shown that antibodies against significant effect on down-regulation of IGF1R by EM164 and scFv- IGF1R may be efficient at inhibiting mitogenic effects mediated Fc as these antibodies should bind IGF1R in any cell surface by insulin and IGF-II via the IR only in tumor cells without membrane compartment. Other growth factor receptors have been affecting IR in target organs such as the liver. Currently, an shown to be internalized by multiple pathways. Thus, transforming antibody against IGF1R is in clinical trial for multiple myeloma growth factor-h receptors can be internalized via the clathrin- (http://www.mayoclinic.org/multiple-myeloma/clintrials.html or dependent endocytic and lipid raft caveolar pathways and reside in http://www.moffitt.usf.edu/about_moffitt/publications/clinical_- both lipid raft and nonraft membrane domains (47). Similarly, IFN-g trials_update/nbcmonths/2004s4.pdf) and others will soon be in receptors have been reported to be internalized by both clathrin- clinical trials for solid tumors. In conclusion, it is a certainty dependent and caveolar endocytic pathways (48). that anti-IGF1R reagents will be tested in human cancer. We have also shown that expression of both IGF1R and IR is Understanding the key targets, mechanism of action, and toxicities required for scFv-Fc or EM164 to down-regulate IR and thus inhibit associated with the different types of reagents will be required to insulin signaling in breast cancer cells. This characteristic of these most appropriately identify patients who could benefit from such a antibodies may be highly advantageous as insulin target organs, therapy. Identifying the role of IR in solid tumors needs to be more such as liver, which lack IGF1R, may be unaffected by antibodies completely defined. directed against IGF1R. Furthermore, in HepG2 cells, which are a hepatoma cell line widely used to study insulin effects, scFv-Fc and Acknowledgments EM164 did not down-regulate IR (data not shown). This further Received 9/23/2005; revised 11/21/2005; accepted 12/15/2005. supports the notion that in liver cells anti-IGF1R antibodies should Grant support: NIH grant R01 CA74285 and USPHS Cancer Center Support grant not disrupt insulin function. P30 CA77398 from the National Cancer Institute (D. Yee). The costs of publication of this article were defrayed in part by the payment of page Unlike HER2, which is amplified in cancers, IGF1R and IR are charges. This article must therefore be hereby marked advertisement in accordance ubiquitously expressed and the effects of systemic therapy with 18 U.S.C. Section 1734 solely to indicate this fact.

References insulin-like growth factor II (IGF-II). Eur J Cancer with the fully human anti-type 1 insulin-like growth 1993;4:562–9. factor receptor monoclonal antibody CP-751,871. Clin 1. Sachdev D, Yee D. The IGF system and breast cancer. 9. Arteaga CL, Kitten LJ, Coronado EB, et al. Blockade of Cancer Res 2005;11:2063–73. Endocr Relat Cancer 2001;8:197–209. the type I somatomedin receptor inhibits growth of 16. Wang Y, Hailey J, Williams D, et al. Inhibition of 2. Andrews DW, Resnicoff M, Flanders AE, et al. Results human breast cancer cells in athymic mice. J Clin Invest insulin-like growth factor-I receptor (IGF-IR) signaling of a pilot study involving the use of an antisense 1989;84:1418–23. and tumor cell growth by a fully human neutralizing oligodeoxynucleotide directed against the insulin-like 10. Li SL, Liang SJ, Guo N, Wu AM, Fujita-Yamaguchi Y. anti-IGF-IR antibody. Mol Cancer Ther 2005;4:1214–21. growth factor type I receptor in malignant astrocyto- Single-chain antibodies against human insulin-like 17. Osborne CK, Bolan G, Monaco ME, Lippman ME. mas. J Clin Oncol 2001;19:2189–200. growth factor I receptor: expression, purification, and Hormone responsive human breast cancer in long-term 3. Parrizas M, Gazit A, Levitzki A, Wertheimer E, effect on tumor growth. Cancer Immunol Immunother tissue culture: effect of insulin. Proc Natl Acad Sci U S A LeRoith D. Specific inhibition of insulin-like growth 2000;49:243–52. 1976;73:4536–40. factor-1 and insulin receptor tyrosine kinase activity 11. Sachdev D, Li SL, Hartell JS, Fujita-Yamaguchi Y, 18. Papa V, Belfiore A. Insulin receptors in breast cancer: and biological function by tyrphostins. Endocrinology Miller JS, Yee D. A chimeric humanized single-chain biological and clinical role. J Endocrinol Invest 1997;138:1427–33. antibody against the type I insulin-like growth factor 1996;19:324–33. 4. Blum G, Gazit A, Levitzki A. Development of new (IGF) receptor renders breast cancer cells refractory 19. Frittitta L, Cerrato A, Sacco MG, Weidner N, Goldfine insulin-like growth factor-1 receptor kinase inhib- to the mitogenic effects of IGF-I. Cancer Res ID, Vigneri R. The insulin receptor content is increased itors using catechol mimics. J Biol Chem 2003;278: 2003;63:627–35. in breast cancers initiated by three different oncogenes 40442–54. 12. Maloney EK, McLaughlin JL, Dagdigian NE, et al. An in transgenic mice. Breast Cancer Res Treat 1997;45: 5. Garcia-Echeverria C, Pearson MA, Marti A, et al. anti-insulin-like growth factor I receptor antibody that 141–7. In vivo antitumor activity of NVP-AEW541—a novel, is a potent inhibitor of cancer cell proliferation. Cancer 20. Seino S, Bell GI. Alternative splicing of human insulin potent, and selective inhibitor of the IGF-IR kinase. Res 2003;63:5073–83. receptor messenger RNA. Biochem Biophys Res Com- Cancer Cell 2004;5:231–9. 13. Burtrum D, Zhu Z, Lu D, et al. A fully human mun 1989;159:312–6. 6. Kull FC, Jr., Jacobs S, Su YF, Svoboda ME, Van Wyk monoclonal antibody to the insulin-like growth factor 21. Pandini G, Frasca F, Mineo R, Sciacca L, Vigneri R, JJ, Cuatrecasas P. Monoclonal antibodies to receptors I receptor blocks ligand-dependent signaling and Belfiore A. Insulin/insulin-like growth factor I hybrid for insulin and somatomedin-C. J Biol Chem 1983;258: inhibits human tumor growth in vivo. Cancer Res receptors have different biological characteristics 6561–6. 2003;63:8912–21. depending on the insulin receptor isoform involved. 7. Arteaga CL, Osborne CK. Growth inhibition of 14. Jackson-Booth PG, Terry C, Lackey B, Lopaczynska J Biol Chem 2002;277:39684–95. human breast cancer cells in vitro with an antibody M, Nissley P. Inhibition of the biologic response to 22. Papa V, Pezzino V, Costantino A, et al. Elevated against the type I somatomedin receptor. Cancer Res insulin-like growth factor I in MCF-7 breast cancer cells insulin receptor content in human breast cancer. J Clin 1989;49:6237–41. by a new monoclonal antibody to the insulin-like Invest 1990;86:1503–10. 8. Brunner N, Yee D, Kern FG, Spang-Thomsen M, growth factor-I receptor. The importance of receptor 23. Sciacca L, Costantino A, Pandini G, et al. Insulin Lippman ME, Cullen KJ. Effect of endocrine therapy down-regulation. Horm Metab Res 2003;35:850–6. receptor activation by IGF-II in breast cancers: evidence on growth of T61 human breast cancer xenografts is 15. Cohen BD, Baker DA, Soderstrom C, et al. Combina- for a new autocrine/paracrine mechanism. Oncogene directly correlated to a specific down-regulation of tion therapy enhances the inhibition of tumor growth 1999;18:2471–9. www.aacrjournals.org 2401 Cancer Res 2006; 66: (4). February 15, 2006

24. Frasca F, Pandini G, Scalia P, et al. Insulin receptor survival in prostate cancer cells. Cancer Res 2002;62: 42. Helms JB, Zurzolo C. Lipids as targeting signals: isoform A, a newly recognized, high-affinity insulin-like 2227–31. lipid rafts and intracellular trafficking. Traffic 2004;5: growth factor II receptor in fetal and cancer cells. Mol 32. Vainio S, Heino S, Mansson JE, et al. Dynamic 247–54. Cell Biol 1999;19:3278–88. association of human insulin receptor with lipid rafts 43. Lobie PE, Sadir R, Graichen R, Mertani HC, Morel G. 25. Pandini G, Vigneri R, Costantino A, et al. Insulin in cells lacking caveolae. EMBO Rep 2002;3:95–100. Caveolar internalization of growth hormone. Exp Cell and insulin-like growth factor-I (IGF-I) receptor over- 33. Huo H, Guo X, Hong S, Jiang M, Liu X, Liao K. Res 1999;246:47–55. expression in breast cancers leads to insulin/IGF-I Lipid rafts/caveolae are essential for insulin-like 44. Pelkmans L, Burli T, Zerial M, Helenius A. Caveolin- hybrid receptor overexpression: evidence for a second growth factor-1 receptor signaling during 3T3-1 pre- stabilized membrane domains as multifunctional trans- mechanism of IGF-I signaling. Clin Cancer Res 1999;5: adipocyte differentiation induction. J Biol Chem 2003; port and sorting devices in endocytic membrane traffic. 1935–44. 278:11561–9. Cell 2004;118:767–80. 26. Frasca F, Pandini G, Vigneri R, Goldfine ID. 34. Foti M, Moukil MA, Dudognon P, Carpentier JL. 45. D’Alessio A, Al-Lamki RS, Bradley JR, Pober JS. Insulin and hybrid insulin/IGF receptors are major Insulin and IGF-1 receptor trafficking and signalling. Caveolae participate in tumor necrosis factor receptor regulators of breast cancer cells. Breast Dis 2003;17: Novartis Found Symp 2004;262:125–41; discussion 41–7, 1 signaling and internalization in a human endothelial 73–89. 265–8. cell line. Am J Pathol 2005;166:1273–82. 27. Pezzino V, Papa V, Milazzo G, Gliozzo B, Russo P, 35. Hong S, Huo H, Xu J, Liao K. Insulin-like growth 46. Damm EM, Pelkmans L, Kartenbeck J, Mezzacasa Scalia PL. Insulin-like growth factor-I (IGF-I) recep- factor-1 receptor signaling in 3T3-1 adipocyte differen- A, Kurzchalia T, Helenius A. Clathrin- and caveolin- tors in breast cancer. Ann N Y Acad Sci 1996;784: tiation requires lipid rafts but not caveolae. Cell Death 1-independent endocytosis: entry of simian virus 40 189–201. Differ 2004;11:714–23. into cells devoid of caveolae. J Cell Biol 2005;168: 28. Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, 36. Zhuang L, Kim J, Adam RM, Solomon KR, Freeman 477–88. Baserga R. Simian virus 40 large tumor antigen is unable MR. Cholesterol targeting alters lipid raft composition 47. Di Guglielmo GM, Le Roy C, Goodfellow AF, Wrana to transform mouse embryonic fibroblasts lacking type and cell survival in prostate cancer cells and xenografts. JL. Distinct endocytic pathways regulate TGF-h 1 insulin-like growth factor receptor. Proc Natl Acad Sci J Clin Invest 2005;115:959–68. receptor signalling and turnover. Nat Cell Biol 2003; U S A 1993;90:11217–21. 37. Morrione A, Valentinis B, Xu SQ, et al. Insulin-like 5:410–21. 29. Jackson JG, White MF, Yee D. Insulin receptor growth factor II stimulates cell proliferation through 48. Sadir R, Lambert A, Lortat-Jacob H, Morel G. substrate-1 is the predominant signaling molecule the insulin receptor. Proc Natl Acad Sci U S A 1997; Caveolae and clathrin-coated vesicles: two possible activated by insulin-like growth factor-I, insulin, 94:3777–82. internalization pathways for IFN-g and IFN-g receptor. and interleukin-4 in estrogen receptor-positive 38. Simons K, Toomre D. Lipid rafts and signal Cytokine 2001;14:19–26. human breast cancer cells. J Biol Chem 1998;273: transduction. Nat Rev Mol Cell Biol 2000;1:31–9. 49. Laron Z. The essential role of IGF-I: lessons from the 9994–10003. 39. Lavie Y, Fiucci G, Liscovitch M. Up-regulation of long-term study and treatment of children and adults 30. Twentyman PR, Luscombe M. A study of some caveolae and caveolar constituents in multidrug- with Laron syndrome. J Clin Endocrinol Metab 1999;84: variables in a tetrazolium dye (MTT) based assay for cell resistant cancer cells. J Biol Chem 1998;273:32380–3. 4397–404. growth and chemosensitivity. Br J Cancer 1987;56:279– 40. Mellman I. Endocytosis and molecular sorting. Annu 50. Abuzzahab MJ, Schneider A, Goddard A, et al. IGF- 85. Rev Cell Dev Biol 1996;12:575–625. I receptor mutations resulting in intrauterine and 31. Zhuang L, Lin J, Lu ML, Solomon KR, Freeman 41. Nichols B. Caveosomes and endocytosis of lipid rafts. postnatal growth retardation. N Engl J Med 2003;349: MR. Cholesterol-rich lipid rafts mediate akt-regulated J Cell Sci 2003;116:4707–14. 2211–22.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Down-regulation of Insulin Receptor by Antibodies against the Type I Insulin-Like Growth Factor Receptor: Implications for Anti −Insulin-Like Growth Factor Therapy in Breast Cancer

Deepali Sachdev, Rajeeva Singh, Yoko Fujita-Yamaguchi, et al.

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