Clinical studies indicate that Herceptin (trastuzumab), a recombinant humanized monoclonal antibody directed against the human epidermal growth factor receptor-2 (HER-2) tyrosine kinase growth factor receptor, provides a significant but transient survival advantage to a subset of patients with HER-2-overexpressing metastatic breast cancer when given as a first-line agent. Increased insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling has recently been identified as a potential factor adversely influencing the response to Herceptin. We examined the effect of recombinant human IGF binding protein 3 (rhIGFBP-3), an antagonist of IGF-IR signaling, in Herceptin-resistant breast cells in vitro and in tumors in vivo. Consistent with results obtained using HER-2- or IGF-IR-transfected cells (MCF-7/HER2-18 and SKBR3/IGF-IR, respectively), we found that rhIGFBP-3 significantly reduced IGF-I-induced IGF-IR phosphorylation and displayed a synergistic interaction with Herceptin against cultured HER-2-overexpressing breast cancer cells in vitro. We show, for the first time, the antitumor activity of rhIGFBP-3 against advanced-stage MCF-7/HER2-18–transfected human breast cancer xenografts and its potentiation of Herceptin activity. We also provide evidence that IGF-IR activation counters the early suppressive effect of Herceptin on HER-2 signaling via Akt and p44/p42 mitogen-activated protein kinase (MAPK), and that inhibition of HER-2-overexpressing human breast tumor growth by rhIGFBP-3 is associated with restored down-regulation of Akt and p44/p42 MAPK phosphorylation in vitro and in vivo. These results emphasize the merit of evaluating simultaneous blockade of the HER-2 and IGF-IR pathways using combination therapy with rhIGFBP-3 plus Herceptin in human clinical trials of patients with HER-2-positive breast cancer. (Cancer Res 2006; 66(14): 7245-52)

Human epidermal growth factor receptor-2 (HER-2/erbB2) belongs to the type I family of tyrosine kinase receptors involved in signal transduction pathways that regulate epithelial cell growth, differentiation, apoptosis, and metastasis (1). HER-2 is activated on heterodimerization with other HER receptors, leading to simultaneous induction of multiple signaling networks, including the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3′-kinase (PI3K) pathways (2). Enhanced MAPK signaling couples HER-2 to cell cycle progression by mediating degradation of the cyclin-dependent kinase inhibitor p27Kip1. Increased recruitment and phosphorylation of PI3K by HER-2 promotes cell survival through activation of the antiapoptotic serine/threonine kinase Akt. HER-2 is overexpressed and constitutively activated in 20% to 30% of human breast cancers and is associated with enhanced tumor aggressiveness and a high risk of relapse and death (3). Herceptin (trastuzumab; Genentech, San Francisco, CA) is a humanized monoclonal antibody that targets the extracellular domain of HER-2 (4) and has shown clinical activity against HER-2-overexpressing breast tumors (5, 6). However, the response rate to Herceptin is <40% as a single agent in first-line treatment of HER-2-positive metastatic breast cancer and the median duration of response is between 9 and 12 months (6, 7). Thus, the efficacy of Herceptin is limited by intrinsic and acquired resistance.

The mechanism(s) by which Herceptin inhibits growth of HER-2-overexpressing breast tumors is complex and not completely understood. Current data (reviewed in refs. 8, 9) suggest that Herceptin acts via down-regulation of HER-2 from the cell surface, prevention of HER-2-containing heterodimer formation, induction of G1 arrest through up-regulation of p27Kip1, down-regulation of PI3K signaling through activation of the phosphatase and tensin homologue (10), inhibition of production of angiogenic factors such as vascular endothelial growth factor, and, possibly, induction of a host tumor response by way of antibody-dependent cellular cytotoxicity and complement activation. Hence, there are several potential mechanisms through which resistance to Herceptin may occur. Among those documented are a loss of p27Kip1 expression (11) and nuclear localization (12) and impaired phosphatase and tensin homologue function (10).

The insulin-like growth factor (IGF) system is also critically involved in the development and maintenance of breast cancer (13, 14). The IGF-I receptor (IGF-IR) is a type II transmembrane tyrosine kinase receptor that is activated upon binding of its ligands, IGF-I and IGF-II. Subsequent recruitment and transphosphorylation of IGF-IR adaptor/effector molecules activates multiple downstream signaling networks also targeted by HER-2. The PI3K survival pathway is activated by IGF-IR-mediated tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), which relays the signal to Akt via PI3K. The Ras/MAPK cascade, linked to cell growth and proliferation, is activated by the IGF-IR-phosphorylated substrates, IRS-1 and Src-collagen homology protein, via interaction with Grb-2/Sos. IGF bioactivity is regulated by a family of high affinity binding proteins (IGFBPs), predominantly IGFBP-3, which limits IGF bioavailability and distribution to target tissues (15). IGFBP-3 also exerts antiproliferative and proapoptotic activities independent of IGFs (16, 17). The IGF-IR and its ligands are frequently overexpressed in human breast tumors (13, 14) and epidemiologic studies indicate that increased levels of IGF-I, reduced levels of IGFBP-3, or an increased ratio of IGF-I to IGFBP-3 in the circulation is associated with increased breast cancer risk (18, 19). In a recent study, Lu et al. (20) showed that increased IGF-IR signaling also plays a role in resistance to Herceptin. These authors showed that the in vitro growth-suppressive effect of Herceptin was significantly reduced in human breast cancer cells (MCF-7/HER2-18 and SKBR3/IGF-IR) co-overexpressing HER-2 and IGF-I receptors when compared with those overexpressing HER-2 alone (SKBR3). Herceptin resistance was overcome by blocking IGF-IR signaling through cotreatment with a monoclonal antibody directed against the IGF-IR (α-IR3) or with recombinant IGFBP-3. The results of a subsequent study suggest that IGF-I-induced antagonism of Herceptin activity involves up-regulation of ubiquitin-related p27Kip degradation that is dependent on the PI3K pathway (21).

Increased expression of natural IGFBP-3 or treatment with recombinant human IGFBP-3 (rhIGFBP-3) has been shown not only to inhibit cancer cell growth in a variety of experimental systems but also to enhance the efficacy of radiation, proapoptotic, and chemotherapeutic agents (reviewed in ref. 14). The present study was designed to further explore the ability of rhIGFBP-3 to inhibit the growth of HER-2-positive breast tumors and to sensitize their response to Herceptin. We confirm that reduced Herceptin activity against HER-2-overexpressing breast cancer cells correlates with elevated IGF-IR levels and show that growth inhibition by rhIGFBP-3 is associated with down-regulation of IGF-IR phosphorylation and downstream signaling events in vitro and in vivo. Furthermore, we show that rhIGFBP-3 has single-agent antitumor activity and potentiates Herceptin activity in a xenograft model of Herceptin-resistant breast cancer. These results support the clinical development of rhIGFBP-3 for the treatment of HER-2-positive breast cancer.

Human breast cancer cell lines. Parental BT474 and BT474/HerR, a Herceptin-resistant derivative obtained by serial passage in the presence of increasing concentrations of Herceptin (100 μg/mL final maintenance dose), were provided by Dr. Mark Pegram (Division of Hematology/Oncology, Department of Medicine, UCLA School of Medicine, Los Angeles, CA; ref. 22). Parental SKBR3 and IGF-IR-transfected lines, SKBR3/IGF-IR and MCF-7/HER2-18 cells (23), were provided by Dr. Michael Pollak (Lady Davis Institute for Medical Research, Department of Oncology, McGill University, Montreal, Quebec, Canada; ref. 20). MCF-7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Characteristics of cell lines used in this study are given in Table 1.

Table 1.

Characteristics of human breast cancer cell lines used in this study

Cell lineIGF-IR status*HER-2 status*Origin
MCF-7 (parental) High, endogenous Low, endogenous Mammary adenocarcinoma; pleural effusion (ATCC) 
MCF-7/HER2-18 High, endogenous High, transfected DNA 23 
SKBR3 (parental) Low, endogenous High, endogenous Mammary adenocarcinoma; pleural effusion (ATCC) 
SKBR3/IGF-IR High, transfected DNA High, endogenous 20 
BT474 (parental) Low, endogenous High, endogenous Mammary gland ductal carcinoma (ATCC) 
BT474/HerR Moderate, endogenous High, endogenous 22 
Cell lineIGF-IR status*HER-2 status*Origin
MCF-7 (parental) High, endogenous Low, endogenous Mammary adenocarcinoma; pleural effusion (ATCC) 
MCF-7/HER2-18 High, endogenous High, transfected DNA 23 
SKBR3 (parental) Low, endogenous High, endogenous Mammary adenocarcinoma; pleural effusion (ATCC) 
SKBR3/IGF-IR High, transfected DNA High, endogenous 20 
BT474 (parental) Low, endogenous High, endogenous Mammary gland ductal carcinoma (ATCC) 
BT474/HerR Moderate, endogenous High, endogenous 22 
*

Relative levels of IGF-I and HER-2 receptors were determined by Western blot analysis and are shown in Fig. 1A.

Cell culture. Unless otherwise stated, all cell culture reagents were from Gibco, Inc. (Grand Island, NY). Cells were maintained in RPMI (BT474, BT474/HerR, MCF-7, and MCF-7/HER2-18) or McCoy's 5A Medium Modified (SKBR3 and SKBR3/IGF-IR) supplemented with 10% fetal bovine serum (FBS), 2 mmol/L l-glutamine, 10 mmol/L HEPES buffer, and antibiotic-antimycotic (penicillin, streptomycin, amphotericin B). MCF-7/HER2-18 and SKBR3/IGF-IR were maintained in 700 μg/mL and 800 μg/mL Geneticin (G418), respectively. BT474/HerR was maintained in 100 μg/mL Herceptin (trastuzumab; Hoffmann-La Roche, Mississauga, Ontario, Canada). All cells were grown at 37°C and 5% CO2.

Cell survival assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. Human breast cancer cells (3 × 103) were seeded in 200 μL complete medium (RPMI or McCoy's) in 96-well microdilution plates. Following cell adherence (24 hours later), experimental medium containing serial dilutions of Herceptin (0.01-100 μg/mL), rhIGFBP-3 (0.1-100 μg/mL; Insmed, Inc., Richmond, VA), or a combination of a fixed dose of Herceptin (2.5 μg/mL) and increasing doses of rhIGFBP-3 was added to appropriate octuplet wells and plates were incubated at 37°C for 72 hours. Cells were washed with drug-free medium and cell survival was measured by the ability of viable cells to reduce the pale yellow tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), to a dark blue formazan product (24). Plates were incubated for 4 hours at 37°C following addition of 25 μL of substrate (5 mg/mL MTT; Sigma, St. Louis, MO) to 100 μL cells in complete medium. Solubilization of the formazan product was accomplished by the addition of 100 μL of solubilization buffer (35% dimethylformamide, 15% acetic acid, 15% SDS) and further incubation at 37°C for 24 hours. The absorbances of the samples were read at 540 nm on a Bio-Rad Model 3550 microplate reader (Bio-Rad Laboratories, Hercules, CA). Cell survival in the presence of test agents was expressed as a percentage of that obtained in drug-free medium (control). All experiments were done in triplicate.

For the combination experiments, using the CalcuSyn Software (Biosoft, Cambridge, United Kingdom), analysis of dose-effect relationships was done according to the median-effect method of Chou and Talalay and mean combination index values were determined, where combination index <1, combination index = 1, and combination index >1 represent synergism, additivity, and antagonism of both drugs, respectively (25).

IGF-I/rhIGFBP-3 treatment and preparation of total cell lysates. Approximately 5 × 105 cells (BT474/HerR, MCF-7/HER2-18, and SKBR3/IGF-IR) were cultured in 3 mL of medium (described above) in six-well plates. After 24 hours of incubation at 37°C, cells were washed twice with the appropriate serum-free medium and then incubated in serum-free medium for 24 hours. During the last 30 minutes of culture, the cells were either untreated or treated with 40 ng/mL recombinant human IGF-I (Sigma), 0.25 μg/mL rhIGFBP-3 (Insmed), and 2.5 μg/mL Herceptin, alone or in combination. Cell monolayers were washed twice with ice-cold PBS solution and then lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mmol/L Tris-Cl (pH 8), 150 mmol/L NaCl, 0.1% SDS, 0.5% deoxycholic acid, 1% NP40, 2 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 2 μg/mL leupeptin, 2 μg/mL pepstatin, 1 mmol/L sodium orthovanadate] at 4°C for 30 minutes. Lysates were clarified by microcentrifugation at 14,000 rpm for 30 minutes at 4°C. Protein concentration was determined by the method of Bradford using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories).

Western blot analysis. Proteins (20-75 μg) were resolved on denaturing SDS-polyacrylamide gels (10%), transferred to Immuno-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories), and probed with the following primary antibodies: anti–phospho-IGF-IR [Tyr113, 1/500 in 5% bovine serum albumin (BSA)], anti-p44/42 MAPK (1/1,500 in 5% BSA), and anti-Akt (1/1,000 in 5% BSA; Cell Signaling Technology, Beverly, MA); anti–protein-tyrosine phosphatase 1D (PTP1D)/SHP2 (1/2,500 in 5% skim milk; BD Transduction Laboratories, Mississauga, Ontario, Canada); anti–phospho-ErbB2/HER-2 (Y1248, 1/4,000 in 3% skim milk) and anti-erbB2/HER-2 (1/2,500 in 4% skim milk; Upstate, Lake Placid, NY); and anti-IGF-IRβ (C-20, 1/400 in 4% skim milk; Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were revealed using peroxidase-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) and visualized with the ECL Plus detection system (Amersham Biosciences, Piscataway, NJ). Membranes were stripped with Re-Blot Plus (Chemicon International, Temecula, CA) and reprobed as required. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D using the formula (A / B) × (C / D), where A is the PTP1D densitometry of sample 1, B is the PTP1D densitometry of sample 2, C is the specific antibody densitometry of sample 2, and D is the specific antibody densitometry of sample 1.

HER-2-overexpressing human breast carcinoma xenografts. Six- to eight-week-old female CD1 (nu/nu) athymic mice (Charles River, Wilmington, MA) were injected s.c., bilaterally on the flank, with 8 × 106 MCF-7/HER2-18 cells, resuspended in 150 μL PBS containing 45% Matrigel (BD Biosciences, Bedford, MA). One day before tumor cell injection and once weekly thereafter for the duration of the experiment, all mice received s.c. injection of 1.5 mg/kg estradiol cypionate (Pharmacia & Upjohn Company, Kalamazoo, MI) to promote tumor cell growth. Animals were monitored daily for general health and body weights were measured twice-weekly. Tumors were measured twice-weekly with slide calipers and volumes (mm3) were calculated as (L × W2) / 2, where L and W are the major and minor diameters (in millimeters), respectively. Once tumor volumes reached 400 to 500 mm3 (∼3 weeks later), seven to eight mice were randomized to each treatment or control group. Mice were primed with an initial loading dose of Herceptin at 6.0 mg/kg (diluted in sterile PBS) by i.p. injection and then received a maintenance dose of 3 mg/kg twice-weekly. Control mice received sterile PBS or human myeloma IgG1 antibody (6 mg/kg loading, then 3 mg/kg maintenance; Calbiochem-Novabiochem, La Jolla, CA) on the same schedule as Herceptin. rhIGFBP-3 (dissolved in sterile water) was administered b.i.d., s.c. at 10 mg/kg. Mice received treatment with single or combined agents for 3 weeks. Relative body weights (%) were calculated as (Wt / Wi) × 100, where Wt is the body weight at any given time and Wi is the body weight at treatment initiation. Net tumor growth was calculated as VtVi, where Vt is the tumor volume at any given time and Vi is the tumor volume at treatment initiation. At the termination of the study, tumors from three representative mice in each group were harvested and homogenized using a Polytron System PT 2100 homogenizer (Brinkmann Instruments, Mississauga, Ontario, Canada) in RIPA lysis buffer, minus detergents. After homogenization, 0.1% SDS, 0.5% deoxycholic acid, and 1% NP40 were added and tumor cell lysates were processed as described for cell monolayers. Equal amounts of protein were then subject to Western blot analysis.

Statistical methods. Comparisons in net tumor growth between mouse treatment groups, and cell survival in the presence of test agents versus control conditions in vitro, were done using statistical tests (F test for determination of variance, followed by Student's t test). Differences were considered to be statistically significant when P < 0.05.

Characterization of IGF-I and HER-2 receptor levels in human breast cancer cells. The goal of the first part of this study was to compare the in vitro growth inhibitory effects of rhIGFBP-3 and Herceptin, alone and in combination, on Herceptin-resistant human breast tumor cell lines (Table 1). Figure 1A is a representative Western blot showing the relative amounts of IGF-I and HER-2 receptors in the parental and Herceptin-resistant cells. The antibody used to measure total IGF-IR levels (C-20, Santa Cruz Biotechnology) is directed at the IGF-IRβ subunit, which becomes phosphorylated upon activation. Thus, measurements of phospho-specific and total IGF-IR levels are based on detection of the same receptor subunit. IGF-IR expression was highest in IGF-IR-transfected SKBR3 cells, followed by MCF-7 and MCF-7/HER2-18, both of which overexpress the receptor endogenously. Of particular interest to this study, BT474 cells with Herceptin resistance acquired through serial passage in the presence of Herceptin (BT474/HerR) displayed elevated IGF-IR levels (3- to 5-fold) when compared with their Herceptin-sensitive counterparts (BT474). HER-2 was constitutively activated at high levels in all cell lines overexpressing the receptor (Fig. 1A).

Figure 1.

Reduced growth inhibition by Herceptin correlates with elevated IGF-IR levels. A, comparison of total IGF-IR and HER-2 levels in parental and Herceptin-resistant human breast cancer cell lines. Cell lysates were prepared from monolayers grown in complete medium containing 10% FBS. Equal amounts (75 μg) of protein from each cell line were subjected to Western blot analysis using total IGF-IR and HER-2 antibodies. PTP1D was used for assessment of loading equivalence between samples. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. B to D, cells were exposed to a concentration range of Herceptin (0.01-100 μg/mL) for 72 hours in the presence of 10% FBS and cell survival was measured using the MTT assay with growth in drug-free medium serving as control. All results are based on octuplet samples and experiments were repeated in triplicate. Columns, mean; bars, SE.

Figure 1.

Reduced growth inhibition by Herceptin correlates with elevated IGF-IR levels. A, comparison of total IGF-IR and HER-2 levels in parental and Herceptin-resistant human breast cancer cell lines. Cell lysates were prepared from monolayers grown in complete medium containing 10% FBS. Equal amounts (75 μg) of protein from each cell line were subjected to Western blot analysis using total IGF-IR and HER-2 antibodies. PTP1D was used for assessment of loading equivalence between samples. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. B to D, cells were exposed to a concentration range of Herceptin (0.01-100 μg/mL) for 72 hours in the presence of 10% FBS and cell survival was measured using the MTT assay with growth in drug-free medium serving as control. All results are based on octuplet samples and experiments were repeated in triplicate. Columns, mean; bars, SE.

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Reduced growth inhibition of HER-2-overexpressing breast cancer cells by herceptin correlates with elevated IGF-IR levels. The relative responsiveness of human breast cancer cell line pairs to a concentration range of Herceptin was determined in vitro using the MTT assay. Because parental MCF-7 cells do not overexpress the HER-2 receptor (Fig. 1A), they are insensitive to Herceptin (20) and were thus omitted from survival studies. In the cell lines with elevated levels of the IGF-I receptor, survival was reduced by only 15% (MCF-7/HER2-18; Fig. 1B), 17% (SKBR3/IGF-IR; Fig. 1C), and 18% (BT474/HerR; Fig. 1D) at the same Herceptin concentration. Maximal growth inhibition of parental SKBR3 (33%) and BT474 (40%) cells was seen at 2.5 μg/mL Herceptin (Fig. 1C and D) in the presence of 10% FBS. Thus, we confirmed previous reports that Herceptin sensitivity correlates inversely with IGF-IR levels in receptor-transfected cell lines (20) and extended these observations to a more clinically relevant cell line (BT474/HerR) with drug resistance and up-regulation of IGF-IR levels acquired by prolonged exposure to Herceptin.

Dose-dependent growth inhibition of herceptin-resistant cells and enhancement of herceptin sensitivity by rhIGFBP-3. Next, we evaluated the efficacy of rhIGFBP-3 to reduce growth of the Herceptin-resistant breast tumor cell lines. In the two sublines (MCF-7/HER2-18 and SKBR3/IGF-IR) expressing high levels of both receptors, treatment with rhIGFBP-3 resulted in dose-dependent growth inhibition (15-55% and 12-40%, respectively), superior to the most effective dose of Herceptin (Fig. 2A and B). The rhIGFBP-3 single-agent effect was much less pronounced in BT474/HerR (10-26% growth inhibition; Fig. 2C), expressing intermediate levels of the IGF-IR. rhIGFBP-3 elicited a strong statistically significant (P < 0.05) dose-dependent increase in Herceptin sensitivity in all three resistant cell lines (compare hatched columns to solid black columns in Fig. 2A-C).

Figure 2.

rhIGFBP-3 significantly enhances Herceptin activity in resistant human breast cancer cells. Cells were cultured with rhIGFBP-3 (0.1-100 μg/mL) alone or together with Herceptin (2.5 μg/mL) for 72 hours in the presence of 10% FBS and cell survival was measured using the MTT assay with growth in drug-free medium serving as control. A, MCF-7/HER2-18; B, SKBR3/IGF-IR; C, BT474/HerR. *, P < 0.05, combined agents versus Herceptin alone. **, P < 0.05, combined agents versus Herceptin alone and rhIGFBP-3 alone. All results are based on octuplet samples and experiments were repeated in triplicate. Columns, mean; bars, SE.

Figure 2.

rhIGFBP-3 significantly enhances Herceptin activity in resistant human breast cancer cells. Cells were cultured with rhIGFBP-3 (0.1-100 μg/mL) alone or together with Herceptin (2.5 μg/mL) for 72 hours in the presence of 10% FBS and cell survival was measured using the MTT assay with growth in drug-free medium serving as control. A, MCF-7/HER2-18; B, SKBR3/IGF-IR; C, BT474/HerR. *, P < 0.05, combined agents versus Herceptin alone. **, P < 0.05, combined agents versus Herceptin alone and rhIGFBP-3 alone. All results are based on octuplet samples and experiments were repeated in triplicate. Columns, mean; bars, SE.

Close modal

The synergistic activity of rhIGFBP-3 plus Herceptin in all resistant lines was corroborated by analysis of the combination effect according to the median effect method of Chou and Talalay. Combination index values significantly lower than 1 in MCF-7/HER2-18 (ranging from 0.26 to 0.75) and lower than 0.1 in SKBR3/IGF-IR and BT474/HerR were observed, indicating clear synergism (Table 2).

Table 2.

Combination indexes for MCF-7/HER2-18, SKBR3/IGF-IR, and BT474/HerR

Agents
Combination index value
rhIGFBP-3 (μg/mL)Herceptin (μg/mL)MCF-7/HER2-18SKBR3/IGF-IRBT474/HerR
0.1 2.5 0.264 0.025 0.054 
2.5 0.284 0.011 0.048 
2.5 0.624 0.01 0.032 
10 2.5 0.758 0.023 0.04 
100 2.5 0.413 0.056 0.017 
Agents
Combination index value
rhIGFBP-3 (μg/mL)Herceptin (μg/mL)MCF-7/HER2-18SKBR3/IGF-IRBT474/HerR
0.1 2.5 0.264 0.025 0.054 
2.5 0.284 0.011 0.048 
2.5 0.624 0.01 0.032 
10 2.5 0.758 0.023 0.04 
100 2.5 0.413 0.056 0.017 

The combination of agents did not enhance Herceptin sensitivity of parental BT474 and had a modest effect in SKBR3 (data not shown). Thus, rhIGFBP-3 displayed single-agent (MCF-7/HER2-18 and SKBR3/IGF-IR) and combinatorial antitumor activities with Herceptin (MCF-7/HER2-18, SKBR3/IGF-IR, BT474/HerR) in Herceptin-resistant breast carcinoma cells.

rhIGFBP-3 inhibits IGF-IR phosphorylation and downstream signaling events in HER-2-overexpressing breast cancer cells. In an attempt to correlate the observed in vitro cell survival effects by rhIGFBP-3 with its ability to reduce IGF-IR signaling, Western blot analyses were conducted using lysates of cells cultured under various growth conditions. Basal and activated levels of the IGF-IR were determined for cells grown in serum-free medium for 24 hours, followed by treatment with IGF-I and/or rhIGFBP-3 for 30 minutes. As seen in Fig. 3A, to C (lanes 1), IGF-I-induced activation of the IGF-IR was seen in the Herceptin-resistant cells. Cotreatment with rhIGFBP-3 suppressed IGF-I-induced IGF-IR activation (compare Fig. 3A-C, lanes 2 and 3) without changing total receptor levels.

Figure 3.

rhIGFBP-3 reduces IGF-IR phosphorylation and downstream signaling events in HER-2-overexpressing breast cancer cells. MCF-7/HER2-18 (A), SKBR3/IGF-IR (B), and BT474/HerR (C) cells were grown to 50% confluence in complete medium, washed twice, and incubated in serum-free conditions for 24 hours. During the last 30 minutes of culture, cells were untreated or treated with IGF-I (40 ng/mL), rhIGFBP-3 (0.25 μg/mL), and Herceptin (2.5 μg/mL), alone or in combination. Cell lysates were prepared and Western blot analysis (50 μg protein) was done. PTP1D served as the loading control for all blots. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies.

Figure 3.

rhIGFBP-3 reduces IGF-IR phosphorylation and downstream signaling events in HER-2-overexpressing breast cancer cells. MCF-7/HER2-18 (A), SKBR3/IGF-IR (B), and BT474/HerR (C) cells were grown to 50% confluence in complete medium, washed twice, and incubated in serum-free conditions for 24 hours. During the last 30 minutes of culture, cells were untreated or treated with IGF-I (40 ng/mL), rhIGFBP-3 (0.25 μg/mL), and Herceptin (2.5 μg/mL), alone or in combination. Cell lysates were prepared and Western blot analysis (50 μg protein) was done. PTP1D served as the loading control for all blots. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies.

Close modal

rhIGFBP-3 also attenuated signaling events downstream of the IGF-IR in MCF-7/HER2-18 and SKBR3/IGF-IR. Specifically, IGF-I-induced activation (but not total receptor levels) of Akt and p44/p42 MAPK was dampened or suppressed by rhIGFBP-3 in these Herceptin-resistant cells (compare Fig. 3A, and B, lanes 2 and 3). By contrast, Akt was constitutively activated and insensitive to rhIGFBP-3 treatment in parental BT474 (data not shown) and BT474/HerR cells (Fig. 3C). Similarly, p44/p42 MAPK was induced by IGF-I in BT474/HerR but was unaffected by rhIGFBP-3 treatment.

Herceptin has been reported to inhibit p44/p42 MAPK and Akt activity in BT474 and SKBR3 cells in vitro (10, 26). We measured the effect of short exposure (30 minutes) to Herceptin alone, Herceptin in combination with IGF-I, and Herceptin together with IGF-I plus rhIGFBP-3 on p44/p42 MAPK and Akt activation (Fig. 3). We found that treatment with Herceptin alone reduced basal (lane 4), but not IGF-I-induced (lane 5), phosphorylation of Akt in MCF-7/HER2-18 and SKBR3/IGF-IR and of p44/p42 MAPK in MCF-7/HER2-18. The results for combination treatment with rhIGFBP-3 plus Herceptin and IGF-I (lane 6) were similar to rhIGFBP-3 plus IGF-I alone (lane 3) in these two cell lines. Thus, increased IGF-IR activity antagonized Herceptin blockade of HER-2 signaling through Akt and p44/p42 MAPK, an effect which was overcome by treatment with rhIGFBP-3. Unlike the effect seen in MCF-7/HER2-18 and SKBR3/IGF-IR, p44/p42 MAPK was rapidly activated in response to 30-minute treatment with Herceptin in BT474/HerR cells and was unresponsive to IGF-I and rhIGFBP-3 (Fig. 3C, lanes 4-6). The results were identical for parental BT474 and SKBR3 (data not shown) expressing low IGF-IR levels. Yakes et al. (26) reported a similar transient increase in phospho-MAPK in BT474 cells following 1 hour of Herceptin treatment, before inhibition at 8 hours. Collectively, our results suggest a link between down-regulation of IGF-IR signaling and the antiproliferative effect of rhIGFBP-3 against MCF-7/HER2-18 and SKBR3/IGF-IR Herceptin-resistant breast cancer cells (Fig. 2). rhIGFBP-3-enhanced suppression of BT474/HerR growth by Herceptin may be related to interference with IGF-IR targets not measured in this study and/or IGF-independent effects.

rhIGFBP-3 inhibits growth of established MCF-7/HER2-18 xenografts and sensitizes tumors to herceptin. We recently showed the ability of rhIGFBP-3 to inhibit the in vivo growth of breast, lung, and colon carcinomas, alone or in combination with standard chemotherapeutic agents (refs. 27, 28).5

5

Manuscripts submitted.

To further explore the potential therapeutic utility of rhIGFBP-3 for the treatment of HER-2-positive breast cancer, we conducted a complementary study in vivo. Rapidly growing MCF-7/HER2-18 xenografts were chosen for evaluation in this portion of the study because SKBR3/IGF-IR cells, like parental SKBR3 (29), did not form tumors in immunocompromised nude mice and BT474/HerR cells formed heterogeneously-sized tumors with a long latency, which were very slow-growing. Estrogen-supplemented nude mice bearing established HER-2-transfected (MCF-7/HER2-18) human breast carcinomas (∼400 mm3) were treated for 3 weeks with either Herceptin (6 mg/kg loading dose and then 3 mg/kg, twice weekly), rhIGFBP-3 (10 mg/kg, twice daily), or a combination of the two agents. Control mice received vehicle alone or human myeloma IgG1 (Herceptin isotype-matched human IgG, both containing κ light chains), following the same dose and schedule as Herceptin. No unacceptable toxicity was seen in any of the treatment regimens as determined by physical examination and body weight measurements (Fig. 4A). As a single agent, Herceptin treatment did not yield a statistically significant reduction in net tumor growth at any point during the 3-week study when compared with the control groups (Fig. 4B). Mice receiving rhIGFBP-3 alone showed a consistent trend toward net tumor growth inhibition (32-74%) throughout the study. Combination therapy with rhIGFBP-3 plus Herceptin resulted in a statistically significant reduction (76-106%) in xenograft volume throughout the study when compared with either Herceptin (P < 0.006) or rhIGFBP-3 (P < 0.05) alone. Fifty-six percent of tumors from mice receiving the combined agents showed either no growth or a reduction in volume by the end of the study as compared with only 6% of those tumors treated with Herceptin monotherapy. These in vivo results are in agreement with the single-agent rhIGFBP-3 growth inhibitory activity against cultured MCF-7/HER2-18 cells and exceeded the level of Herceptin sensitization afforded by rhIGFBP-3 in vitro (Fig. 2A).

Figure 4.

rhIGFBP-3 treatment inhibits Herceptin-resistant breast tumor growth and reduces activated Akt and p44/p42 MAPK levels in vivo. Female CD1 (nu/nu) athymic mice (randomized into groups of seven to eight each) bearing HER-2-transfected (MCF-7/HER2-18) human breast carcinomas (∼400 mm3) were treated with vehicle (▪; sterile PBS), Herceptin (♦; 6 mg/kg loading dose, then 3 mg/kg, twice weekly), IgG1 (□; same dose and schedule as Herceptin), rhIGFBP-3 (○; 10 mg/kg, twice daily), or rhIGFBP-3 + Herceptin (•) for 3 weeks. A, no treatment regimen showed unacceptable toxicity based on body weight measurements. B, rhIGFBP-3 treatment yielded a consistent growth inhibitory effect throughout the study. As a single agent, Herceptin did not significantly reduce net tumor growth. *, mice receiving combination therapy with rhIGFBP-3 plus Herceptin had significant reductions in net tumor growth when compared with Herceptin (P < 0.006) or rhIGFBP-3 (P < 0.05) alone. A and B, points, mean; bars, SE. C, at the termination of the study, tumors from mice receiving rhIGFBP-3, alone (lane 4) and in combination with Herceptin (lane 5), showed reductions in the phosphorylated forms of Akt and p44/p42 MAPK when compared with control tumors and those treated with Herceptin. Each lane contains 100 μg of protein from tumor cell lysates. PTP1D served as the loading control. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies.

Figure 4.

rhIGFBP-3 treatment inhibits Herceptin-resistant breast tumor growth and reduces activated Akt and p44/p42 MAPK levels in vivo. Female CD1 (nu/nu) athymic mice (randomized into groups of seven to eight each) bearing HER-2-transfected (MCF-7/HER2-18) human breast carcinomas (∼400 mm3) were treated with vehicle (▪; sterile PBS), Herceptin (♦; 6 mg/kg loading dose, then 3 mg/kg, twice weekly), IgG1 (□; same dose and schedule as Herceptin), rhIGFBP-3 (○; 10 mg/kg, twice daily), or rhIGFBP-3 + Herceptin (•) for 3 weeks. A, no treatment regimen showed unacceptable toxicity based on body weight measurements. B, rhIGFBP-3 treatment yielded a consistent growth inhibitory effect throughout the study. As a single agent, Herceptin did not significantly reduce net tumor growth. *, mice receiving combination therapy with rhIGFBP-3 plus Herceptin had significant reductions in net tumor growth when compared with Herceptin (P < 0.006) or rhIGFBP-3 (P < 0.05) alone. A and B, points, mean; bars, SE. C, at the termination of the study, tumors from mice receiving rhIGFBP-3, alone (lane 4) and in combination with Herceptin (lane 5), showed reductions in the phosphorylated forms of Akt and p44/p42 MAPK when compared with control tumors and those treated with Herceptin. Each lane contains 100 μg of protein from tumor cell lysates. PTP1D served as the loading control. Densitometric quantitation of each band was normalized in comparison with the relevant expression of PTP1D and the values are indicated at the bottom. Membranes were sequentially stripped and reprobed with the indicated total and phospho-specific antibodies.

Close modal

Activated Akt and p44/p42 MAPK levels are reduced only in tumors from mice receiving rhIGFBP-3 treatment. To correlate the observed tumor growth reduction by rhIGFBP-3 with its antagonistic effect on IGF-IR signaling, Western blot analysis was done at the termination of the study using tumor lysates obtained from mice belonging to each group. Results from representative tumors are shown in Fig. 4C. Although the phosphorylated IGF-IR/total IGF-IRβ ratio was low in all groups, differential activation of its downstream effectors was observed, as seen in vitro. In the vehicle and IgG control groups, total and activated forms of Akt and p44/p42 MAPK were present at high levels (lanes 1 and 2). Herceptin has previously been shown to inhibit HER-2 signaling via down-regulation of Akt and MAPK activity in HER-2-positive BT474 xenografts (30). Our results show that MCF-7/HER2-18 tumors from mice receiving monotherapy with Herceptin (lane 3) had levels of activated Akt and p44/p42 MAPK indistinguishable from IgG1-treated controls. Conversely, those groups treated with rhIGFBP-3, alone (lane 4) and in combination with Herceptin (lane 5), exhibited reductions in the phosphorylated forms of Akt and p44/p42 MAPK. Thus, the in vivo growth inhibitory effect of rhIGFBP-3 on Herceptin-resistant MCF-7/HER2-18 tumors correlates with its ability to suppress growth and survival signals mediated by the Ras/MAPK and PI3K/Akt pathways, presumably by its ability to sequester IGF-I and prevent its interaction with the IGF-IR.

The convergence of multiple growth factor receptor signaling networks on common downstream effector molecules poses a limitation to the efficacy of receptor-specific antineoplastic therapies and justifies the use of combining strategies that simultaneously target overlapping signal transduction pathways. Our results confirmed that events downstream of IGF-IR activation interfere with Herceptin activity in cultured HER-2-overexpressing MCF-7/HER2-18 and SKBR3/IGF-IR cells (Fig. 3A and B). Specifically, induction with IGF-I prevented the early suppressive effect of Herceptin on HER-2 signaling via Akt (MCF-7/HER2-18 and SKBR3/IGF-IR) and p44/p42 MAPK (MCF-7/HER2-18). rhIGFBP-3 treatment reduced survival of these cells (Fig. 2A and B) in association with decreased IGF-I-induced activation of Akt and p44/p42 MAPK (Fig. 3A and B). Concurrent blockade of HER-2 and IGF-IR signaling pathways via cotreatment with rhIGFBP-3 plus Herceptin (Fig. 2) was synergistic for both MCF-7/HER2-18 and SKBR3/IGF-IR cell lines in vitro.

The results with Herceptin-resistant BT474 cells were more complex. These cells retained high levels of HER-2 expression and activation and had acquired up-regulation of the IGF-I receptor relative to parental BT474 (Fig. 1A). The IGF-IR was phosphorylated in response to IGF-I and subject to repression by rhIGFBP-3 (Fig. 3C). Akt was constitutively activated and unresponsive to Herceptin, IGF-I, and rhIGFBP-3 (Fig. 3C), suggesting a role in the Herceptin resistance of these cells. This is in agreement with the finding that expression of constitutively active Akt prevents Herceptin-induced growth inhibition of BT474 cells and apoptosis of SKBR3 cells (26). The phosphorylation status of p44/p42 MAPK was IGF-I independent in BT474/HerR cells whereas it was rapidly increased in response to Herceptin and immune to down-regulation by rhIGFBP-3. Nonetheless, the combination of rhIGFBP-3 and Herceptin in BT474/HerR cells was highly synergistic (Fig. 2C; Table 2), suggesting that increased expression of the IGF-IR contributes, in part, to their reduced responsiveness to Herceptin. It is possible that rhIGFBP-3 influenced IGF-IR adaptor/effector proteins downstream of Akt and/or p44/p42 MAPK not evaluated in our study. Other groups have generated, by continuous in vitro drug exposure, Herceptin-resistant BT474 or SKBR3 cells that have been characterized as having reduced p27Kip1 expression (11) or nuclear localization (12). Lu et al. (21) showed that elevated IGF-IR signaling via the PI3K pathway antagonized the Herceptin-induced increase in p27Kip1 levels in SKBR3/IGF-IR cells. Growth suppression of BT474/HerR cells by rhIGFBP-3 may also be independent of IGF signaling (31). IGFBP-3 has been reported to directly induce apoptosis in cells lacking the IGF-IR (17, 32), an effect which is at least partially attributable to its ability to induce alterations in the ratio of proapoptotic (Bax) to antiapoptotic (Bcl-2) proteins (33). Future studies involving measurement of p27Kip1 and other adaptor/effector molecules downstream of the IGF-IR and HER-2 signaling pathways will increase our understanding of the factors imparting Herceptin resistance to BT474/HerR cells.

Targeting of the IGF-IR through IGFBP-3 represents a novel strategy for inhibition of human cancer cell growth and enhancement of conventional chemotherapeutic drugs in vivo. Endogenous overexpression of IGFBP-3 has been shown to significantly reduce tumor formation against human non–small-cell lung cancer and prostate xenografts (34, 35). Delivery of IGFBP-3 into H1299 non–small-cell lung cancer xenografts via a recombinant adenovirus reduced tumor volume (36). We have observed significant tumor growth inhibition following rhIGFBP-3 treatment in mice bearing 3LL Lewis lung and LoVo colorectal carcinomas and combinatorial antitumor activity with CPT-11 and paclitaxel against LoVo colorectal and MCF-7 breast carcinoma xenografts, respectively (27, 28). The current study extends the spectrum of IGFBP-3-responsive tumors to Herceptin-resistant human breast cancer xenografts co-overexpressing the HER-2 and IGF-I receptors (MCF-7/HER2-18). Consistent with our findings in vitro, monotherapy with rhIGFBP-3 was superior to Herceptin against established HER-2-transfected human breast carcinomas (Fig. 4B). Furthermore, rhIGFBP-3 significantly sensitized advanced tumors (≥400 mm3) to Herceptin, reducing tumor volumes by 76% to 106%.

Inhibition of Akt activity is known to be necessary for the antitumor effect of Herceptin (26). Herceptin has been shown to down-regulate HER-2 signaling via both Akt and p44/p42 MAPK in vitro (26) and in vivo (30). We found that Akt and p44/p42 MAPK were highly phosphorylated in tumors from control mice and those receiving treatment with Herceptin (Fig. 4C). By contrast, treatment with rhIGFBP-3, alone or in combination with Herceptin, led to reduced Akt and p44/p42 MAPK phosphorylation in parallel with its antitumor effect. These results are congruent with the hypothesis that increased IGF-IR signaling via Akt and p44/p42 MAPK circumvents the growth-suppressive effect of Herceptin in MCF-7/HER2-18 cells. The findings of this study also suggest that the antitumor activities of rhIGFBP-3 are dependent on its ability to block IGF-induced Akt and p42/p44 MAPK activity.

In summary, we have presented data to support the use of rhIGFBP-3 against Herceptin-resistant HER-2-overexpressing human breast tumor cells. In agreement with the proposed inhibitory effect of IGF-IR signaling on Herceptin activity in receptor-transfected human breast cancer cells (MCF-7/HER2-18 and SKBR3/IGF-IR), we found elevated IGF-IR levels in Herceptin-resistant cells (BT474/HerR) generated by continuous drug exposure. rhIGFBP-3, an antiproliferative and proapoptotic agent with IGF-dependent and -independent activity, displayed significant dose-dependent growth inhibition of HER-2-overexpressing, Herceptin-resistant breast cancer cells with elevated IGF-IR levels and synergized with Herceptin in vitro (MCF-7/HER2-18, SKBR3/IGF-IR, and BT474/HerR) and enhanced Herceptin activity against established (MCF-7/HER2-18) tumors in vivo. These effects were associated with the ability of rhIGFBP-3 to reduce HER-2 and IGF-IR activation of the Ras/MAPK and PI3K/Akt pathways. The findings of our study have significant clinical relevance and suggest that combining HER-2 and IGF-IR targeting agents may be an effective therapeutic strategy for Herceptin-resistant breast cancer.

†Q. Yu is deceased as of September 2004.

Grant support: Insmed, Inc. (Richmond, VA).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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