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IGF1R-directed targeted therapy enhances the cytotoxic effect of chemotherapy in endometrial cancer
Cancer Letters 335 (2013) 153–159
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
IGF1R-directed targeted therapy enhances the cytotoxic effect of chemotherapy in endometrial cancer Connie Bitelman a,1, Rive Sarfstein a,1, Menahem Sarig b,1, Zohar Attias-Geva a, Ami Fishman b, Haim Werner a, Ilan Bruchim b,⇑ a b
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba 44281, Israel
a r t i c l e
i n f o
Article history: Received 21 November 2012 Received in revised form 3 February 2013 Accepted 4 February 2013
Keywords: Endometrial cancer Uterine serous carcinoma IGF1 IGF1 receptor MK-0646
a b s t r a c t This study evaluated the potential ability of MK-0646 to inhibit IGF1-mediated biological actions and cell signaling events in Type 1 and Type 2 endometrial cancer. We found that MK-0646 treatment significantly decreased IGF1R expression. In addition, pretreatment with MK-0646 decreased the IGF1-induced phosphorylation of IGF1R, AKT and ERK. Apoptosis analyses showed that MK-0646 abolished the antiapoptotic effect of IGF1. Furthermore, MK-0646 treatment abolished the IGF1-stimulatory effect on proliferation and enhanced the cytotoxic effect of cisplatin. These findings indicate that specific inhibition of IGF1R could be a useful therapeutic approach for Type 1 and Type 2 endometrial cancer. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endometrial cancer is the most widespread gynecologic cancer in Western countries. Women have a 2–3% lifetime risk of developing the disease, and more than 40,000 new cases were diagnosed in 2009 in the United States alone [1]. The incidence of the disease has been increasing in recent years, presumably because of the growing obesity epidemic. Differences in molecular pathways, epidemiology and prognosis account for the two major groups of endometrial cancer, Type 1 and Type 2, with Type 1, the estrogen-related form, being the most frequent (more than 80% of cases) [2,3]. Type 1 tumors exhibit an endometrioid, well-differentiated morphology, and are usually associated with a relatively good prognosis, as opposed to Type 2 tumors which display a less differentiated phenotype, and have a worse prognosis. Uterine serous carcinoma (USC) constitutes the predominant histological class among Type 2 tumors [4,5]. USC represents 10% of all endometrial carcinomas, is considered to be a high grade tumor, and has the worst prognosis among endometrioid tumors [6]. A number of different genetic alterations have been detected in Type 1 endome-
⇑ Corresponding author. Address: Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Meir Medical Center, 59 Tschernichovsky Street, Kfar Saba, Israel. Tel.: +972 97472561; fax: +972 97471620. E-mail address: [email protected] (I. Bruchim). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.009
trial cancers, including microsatellite instability and mutations of the pTEN, KRAS2, and ß-catenin genes and near-diploid karyotype. In contrast, Type 2 tumors are associated with p53 and ERBB-2 (HER-2/neu) mutations and loss of heterozygosity on several chromosomes, and in most cases are not diploid [7–9]. Systemic hormonal and chemotherapeutic treatments represent the cornerstones of advanced and recurrent endometrial cancer management, although it is still characterized by a poor prognosis. Therefore, there is an urgent need to identify new therapeutic regimens that will prolong survival and transform response to treatment into cure. Recent research has focused on optimizing chemotherapy regimens, the development of alternative hormonal therapy strategies, and the introduction of targeted therapies. Moreover, ongoing trials are exploring multimodal therapies incorporating chemotherapy with novel molecular agents for advanced primary and recurrent disease. The insulin-like growth factors 1 and 2 (IGF1 and IGF2) are a family of mitogenic polypeptides with important roles in growth and differentiation. The biological actions of the IGFs are mediated by the IGF1 receptor (IGF1R), a transmembrane tyrosine kinase structurally related to the insulin receptor [10–12]. In addition to its central role in normal cell cycle progression, the IGF1R has a pivotal role in tumorigenesis [13,14]. Thus, most tumors and cancer-derived cell lines express high levels of IGF1R mRNA and protein, while suppression of IGF1R was shown to block IGF1stimulated cellular proliferation [15]. Several studies underline
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the crucial role of IGF1R action in endometrial cancer and the importance of altered IGF1R gene expression in the development of a malignant phenotype [16–18]. MK-0646 (Dalotuzumab, h7C10; Merck, Sharp and Dohme) is a recombinant, humanized IgG1 monoclonal antibody that binds with high affinity to IGF1R, leading to receptor internalization and subsequent degradation; hence, preventing its ligand-mediated activation. Recent in vivo and in vitro studies showed that MK-0646 exhibits potent antitumor activity in a number of malignancies. Preliminary data from recent Phase 1 clinical trials suggest that MK-0646 is safe and well-tolerated and showed clinical activity against advanced solid tumors [19]. The current study was designed to obtain preclinical data regarding the use of MK-0646 for treating endometrial cancer.
2.4. Cell cycle analysis Cells were serum-starved for 24 h and incubated in the presence or absence of MK-0646 and/or IGF1 for 72 or 120 h. After incubation, cells were washed with phosphate buffered saline, trypsinized, permeabilized with Triton X-100 (4%) and stained with propidium iodide (50 mg/ml). Stained cells were analyzed using a FACSort Flow Cytometer (Beckton-Dickinson, CA, USA).
3. Results The IGF1R gene has emerged in recent years as a promising therapeutic target. MK-0646 is a monoclonal anti-IGF1R antibody, currently in preclinical and clinical evaluations. In this study, we focused on three uterine carcinoma cell lines, which constitute validated models for Type 1 (ECC-1) and Type 2 (USPC-1 and USPC-2) endometrial cancers.
2. Materials and methods
3.1. MK-0646 down-regulates IGF1R expression 2.1. Cell cultures and treatments Human endometrioid endometrial cancer (Type 1) and USC (Type 2) cell lines were used in this study. For endometrioid cancer, the ECC-1 cell line was used, and for USC, the USPC-1 and USPC-2 cell lines were used. ECC-1 cells were kindly provided by Dr. Y. Sharoni (Ben Gurion University, Beer Sheba, Israel) and USC cells by Dr. A. Santin (Yale University School of Medicine, New Haven, CT, USA). ECC-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l D-glucose (Gibco BRL, Paisley, Scotland) and USPC-1 and USPC2 cells were maintained in RPMI-1640 medium (Biological Industries Ltd., Beit Haemek, Israel). Cells were supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 mg/ml gentamicin sulfate, and 5.6 mg/l amphotericin B. All reagents were purchased from Biological Industries Ltd. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Human monoclonal IGF1R antibody MK-0646 (Dalotuzumab; Merck, Sharp and Dohme, Whitehouse Station, NJ, USA) was diluted in 20 mM histidine and 150 mM NaCl (20.95 g/l) and stored at 4 °C. In all experiments, cells were serum-starved for 24 h, after which they were treated with 10 lg/ml MK0646 in the presence or absence of IGF1 (50 ng/ml) (PeproTech Ltd., Rocky Hill, NJ, USA). In some experiments, cells were treated with 10 lg/ml MK-0646 for 5 h, or 24–144 h, separately and in combination with varying concentrations of cisplatin (1 lM, 5 lM or 10 lM) (Sigma–Aldrich Ltd., St. Louis, MO, USA). 2.2. Western immunoblots Cells were starved overnight and then incubated with MK-0646 in the presence or absence of IGF1 for the indicated periods. After incubation, cells were harvested and lysed in a buffer containing protease inhibitors (#9803, Cell Signaling Technology Beverly, MA, USA). Protein content was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA) as a standard. Samples were electrophoresed through 10% or 5% SDS-PAGE gels, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with either 5% skim milk and/or 3% BSA, the blots were incubated overnight with the antibodies listed below, washed and incubated with the appropriate horseradish peroxidase (HRP)conjugated secondary antibody. Antibodies against phospho-IGF1R (3024), IGF1R b-subunit (3027), phospho-AKT (9271), AKT (9272), phospho-ERK1/2 (9106), poly ADP ribose polymerase (PARP; 9542), and caspase-3 (9661), were obtained from Cell Signaling Technology. An antibody against ERK1 (K-23) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-tubulin (T5168) was purchased from Sigma–Aldrich Ltd. The secondary antibodies were HRP-conjugated goat anti-rabbit IgG (1:50,000) and donkey anti-mouse IgG (1:25,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Proteins were detected using the SuperSignal West PicoChemiluminescent Substrate (Pierce, Rockford, IL, USA). Tubulin expression was used as a loading control. 2.3. Proliferation measurements To determine the effect of MK-0646 on ECC-1 and USPC-1 cell proliferation, cells were seeded onto 24-well plates (2 104 ECC-1 cells/well and 5 104 USPC-2 cells/well). After 24 h, the cells were treated with either MK-0646 (10 lg/ml), IGF1 (80 ng/ml), or a combination of both for 24 and 48 h, in triplicate wells. Cell viability was assessed using a standard thiazolyl blue tetrazolium bromide (MTT) method [20]. The color developed was quantitated by measuring absorbance at a wavelength of 530 nm and reference wavelength of 630 nm on a Microplate Reader (Molecular Devices, SpectraMax 190, GMI, Inc., Minnesota, USA). Cell viability was expressed as a percentage of the optical density values obtained upon treatment relative to controls. In addition, we investigated a potential synergy between MK-0646 and cisplatin. To this end, cells were treated with MK-0646, cisplatin (1 lM, 5 lM or 10 lM), or a combination of MK-0646 with each dose of cisplatin for 120 h.
We first explored the effect of MK-0646 on IGF1R expression levels. To this end, ECC-1 and USPC-1 cells were seeded in full medium and upon reaching 100% confluence, were treated with MK0646 (10 lg/ml) for 24 or 48 h. The cells were then lysed and analyzed by Western blotting using an IGF1R b subunit antibody. Results obtained showed that treatment with MK-0646 caused a marked decrease in IGF1R levels in both cell lines at both time points (Fig. 1A). Of note, the levels of both IGF1R precursor (250kDa) and mature (97-kDa) forms were reduced. Specifically, MK0646 decreased IGF1R levels by 63% (24 h) and 48% (48 h) in ECC-1 cells and by 26% (24 h) and 34% (48 h) in USPC-1 cells versus untreated cells (100%; Fig. 1B). No changes in tubulin levels were observed in MK-0646-treated cells. In addition, we recently showed that IGF1R mRNA levels were correlated with the protein levels in both cell lines [21]. 3.2. MK-0646 decreases phosphorylation of the IGF1R and associated downstream signaling mediators In order to determine the ability of MK-0646 to block activation of the various cellular pathways normally activated by the IGF1R, phosphorylation levels of the receptor were tested using an antiphospho-IGF1R antibody. Results obtained showed that MK-0646 attenuated the IGF1-induced IGF1R phosphorylation in both ECC-1 [68% versus IGF1-treated cells (100%)] and USPC-1 cells [53% versus IGF1-treated cells (100%) (Fig. 2A and B)]. Next, the effects of MK-0646 on downstream signaling pathways induced by IGF1R were examined. Phosphorylation levels of AKT and ERK1/2 were measured as representatives of the PI3K/AKT/PKB and MAPK pathways, respectively. As expected, treatment with IGF1 for 10 min stimulated AKT phosphorylation in both cell types, while pretreatment with MK-0646 for 5 h attenuated this effect. No significant effect was seen on the total AKT level after treatment with MK-0646 and IGF1 or with IGF1 alone, after these short exposures (Fig. 2A). A similar trend was seen in both phosphorylation and expression of ERK1/2 after treatment with MK-0646 and/or IGF1. 3.3. MK-0646 abrogates the anti-apoptotic effect of IGF1 in endometrial cancer cells To investigate the effect of MK-0646 on apoptosis, cleaved PARP and caspase-3 were measured using Western blots. Cleavage of the 116-kDa PARP molecule into an 85-kDa band is considered a typical hallmark of early apoptosis [22]. Caspase-3 has been shown to be a key component of the apoptotic machinery. It is activated in apoptotic cells and cleaves several cellular proteins, including PARP. Caspase-3 is a protease that promotes the splitting of PARP
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Fig. 1. IGF1R levels. ECC-1 and USPC-1 cells were treated with MK-0646 for 24 h and 48 h. At the end of the incubation period, cells were lysed and whole-cell lysates (100 lg) were resolved on SDS-PAGE and immunoblotted with antibodies against IGF1R b-subunit and tubulin as described in Materials and methods. (A) Scanning densitometry analysis represents the IGF1R levels in response to treatment with MK-0646. MK-0646 decreased IGF1R after 24 h and 48 h. Bars represent IGF1R values normalized to the corresponding tubulin levels, (B) the figure shows the result of a typical experiment, repeated three times with similar results.
Fig. 2. IGF1-mediated signaling pathways. ECC-1 and USPC-1 cell were treated for 5 h with MK-0646 (or left untreated) and/or IGF1 during the last 10 min of the incubation period. Whole-cell lysates (100 lg) were resolved on SDS-PAGE and immunoblotted with antibodies against pIGF1R, TIGF1R, pAKT, TAKT, pERK1/2 and TERK1/2. (A) Optical density was expressed as phospho-IGF-IR values normalized to the corresponding total IGF1R levels and (B) the figure shows the result of a typical experiment, repeated three times with similar results.
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Fig. 3. IGF1-stimulated protection from apoptosis. Western blot analysis of the effect of MK-0646 on PARP (A) and caspase-3 (B) on ECC-1 and USPC-1 cells. Serum-starved cells were treated with IGF1 for the indicated times in the presence or absence of MK-0646. After incubation, cell lysates were prepared and electrophoresed (100 lg). Results are representative of three independent experiments.
after it itself undergoes cleavage into 17- and 19-kDa fragments. Evaluation of cleaved PARP and caspase-3 constitute bona fide markers of apoptosis. Results obtained revealed that after 24 h and 48 h, IGF1 treatment led to a reduction in cleaved PARP levels in both cell types (Fig. 3A). Treatment with MK-0646 for 24 h and 48 h abolished the anti-apoptotic effect of IGF1 in both cell lines, as indicated by the increased levels of cleaved PARP compared to IGF1-treated cells (Fig. 3A). In addition, IGF1 significantly decreased the levels of cleaved caspase-3, while MK-0646 attenuated this decrease. In ECC-1 cells, however, the ability of MK-0646 to reverse the effect of IGF1 on caspase-3 cleavage was seen only at 24 h (Fig. 3B).
Table 1 Effect of MK-0646 on the cell cycle in endometrial cancer cells. ECC-1 and USPC-1 cells were seeded in quadruplicate dishes, serum-starved for 24 h, and treated with MK-0646 (or left untreated, controls) for 120 h (ECC-1) and 72 h (USPC-1). Cell cycle distribution was assessed by FACS analysis. The values in the table denote mean ± SEM. Cell type/stage ECC1 G0/G1 S+G2/M USPC1 G0/G1 S+G2/M * **
DMSO (%) 86 ± 0.05 14 ± 0.4 89.4 ± 0.8 10.6 ± 0.8
IGF1 (%) 78 ± 0.04* 22 ± 0.09* 79 ± 0.7* 21 ± 0.6*
IGF1 + MK-0646 (%) 84 ± 0.05** 16 ± 0.02** 90.8 ± 0.8** 9.2 ± 0.7**
p < 0.01 versus control cells (DMSO). p 6 0.01 versus IGF1-treated cells.
3.4. MK-0646 inhibits the IGF1-stimulated cell cycle progression The effect of MK-0646 on cell cycle progression was examined in ECC-1 and USPC-1 cells using FACS analysis. In ECC-1 cells, IGF1 increased the proportion of cells at the S+G2/M phase from 14 ± 0.4% to 22 ± 0.09% (Table 1). The addition of MK-0646 on top of IGF1 caused a decrease in the proportion of cells at the S+G2/ M phase from 22 ± 0.09% to 16 ± 0.02%, and increased the proportion of cells at the G0/G1 phase from 78 ± 0.04% to 84 ± 0.5%. Similarly, in USPC-1 cells IGF1 treatment led to an increase in the number of cells at S+G2/M from 10.6 ± 0.8% to 21 ± 0.6% and decreased the proportion of cells at the G0/G1 phase from
89.4 ± 0.8% to 79 ± 0.7% (Table 1). Addition of MK-0646 on top of IGF1 reduced the proportion of cells at S+G2/M from 21 ± 0.6% to 9.2 ± 0.7% and increased the proportion of cells at the G0/G1 phase from 79.4 ± 0.7% to 90.8 ± 0.8%.
3.5. Effect of IGF1R inhibition on proliferation The effect of MK-0646 on proliferation rate was determined using the MTT method. Proliferation rates were examined
Fig. 4. Endometrial cancer cell proliferation. Cells were placed in 24-well plates at a density of 2 104 cells/well for ECC-1 (A) and 5 104 cells/well for USPC-2 cells (B). Cells were treated with IGF1, or IGF1 in combination with MK-0646 (or left untreated) for 24 h and 48 h, after which the proliferation rate was measured by MTT assays. The number of cells at time 0 was assigned a value of 100% and they were used as control. The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate samples. p 6 0.01 versus untreated cells and p 6 0.01 versus IGF1-treated cells.
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Fig. 5. Endometrial cancer cell proliferation with MK-0646 and varying doses of cisplatin. Cells were plated in 24-well plates at a density of 2 104 cells/well for ECC-1 (A) and 5 104 cells/well for USPC-1 cells (B). The number of cells at time 0 was assigned a value of 100% and they were used as control. The bars represent the mean ± SEM of three independent experiments, each performed in triplicate samples. p 6 0.05 versus untreated cells and p 6 0.05 versus cisplatin-treated cells.
following treatment of ECC-1 and USPC-2 cells with IGF1, MK-0646, or both, compared to untreated cells. Treatment with IGF1 stimulated proliferation in both cell lines after 24 h and 48 h (Fig. 4). Co-treatment with IGF1 and MK-0646 inhibited proliferation in both cell lines after 24 h and 48 h, compared to cells treated with IGF1 alone. Specifically, in ECC-1 cells (Fig. 4A), MK-0646 inhibited proliferation by 59.2% and 73.7% at 24 h and 48 h, respectively and in USPC-2 cells by 58.5% and 72.7%, respectively (Fig. 4B). Similar results were obtained in preliminary assays using trypan blue staining followed by hemocytometer counting. 3.6. MK-0646 potentiates the effect of cisplatin Finally, the effects of MK-0646 on the resistance of endometrial cancer cells against chemotherapeutic agents, as well as possible synergistic effects of co-treatment with MK-0646 and cisplatin were studied. ECC-1 and USPC-1 were treated with cisplatin in the presence or absence of MK-0646. Proliferation rates were measured after 120 h using the MTT method. Treatment with cisplatin had a major cytotoxic effect on both cell types (Fig. 5A). Thus, after treatment with 10 lM cisplatin alone, there was a 49.8% decrease in proliferation rate in ECC-1 cells (Fig. 5A) and a 39.4% decrease in USPC-1 cells (Fig. 5B) compared to control. Next, co-incubation experiments using MK-0646 and increasing doses of cisplatin were performed in ECC-1 cells. At every dose of cisplatin, the addition of MK-0646 decreased the number of surviving cells in a dose-dependent fashion (Fig. 5A). Treatment of both ECC-1 and USPC-1 cells with MK-0646 and 10 lM of cisplatin caused decreases in proliferation of 20.4% and 10.3%, respectively, compared to treatment with 10 lM of cisplatin alone (Fig. 5A and B). In summary, addition of MK-0646 to cisplatin in endometrial cancer cells enhanced the cytotoxic effects of the chemotherapeutic treatment.
4. Discussion The IGF pathway has been implicated in the etiology of several epithelial malignancies, including breast, colon, prostate and gynecologic cancers [23]. Various technologies are currently being employed to down-regulate IGF1R expression and signaling with the purpose of achieving improved response to cancer therapy. Among the different IGF1R targeting strategies, the most promising approaches to date are the antagonistic antibodies and the small molecule kinase inhibitors. A number of reports showed a correlation between components of the IGF system and endometrial cancer risk [24–26]. Moreover, several studies reported significant increases in IGF1R expression in endometrial cancer [16,27] and correlation to disease grade and stage [17,28]. We have recently demonstrated that a specific IGF1R tyrosine kinase inhibitor (NVP-AEW541, Novartis Pharma, Basel, Switzerland) and a fully human monoclonal antibody against the IGF1R (IMC-A12, ImClone Systems, New York, USA), effectively blocked IGF1R activity in endometrial cancer cell lines and abolished the IGF1-mediated signaling cascades [21,29]. IGF1R targeting for cancer therapy is currently being evaluated in over 100 clinical trials and by more than 30 drugs in clinical and laboratory studies [30]. In recent years, several IGF1R inhibitors have been developed, some of which were evaluated in Phase I-III clinical trials as monotherapy, as well as in combination with chemotherapy, radiotherapy, and/or antibodybased therapy. Results of these clinical trials in solid tumors have shown variable responses to IGF1R-directed therapies and recent Phase III trials in unselected patients were discontinued due to futility. MK-0646 is a recombinant, humanized IgG1 monoclonal antibody that binds to the human IGF1R with high affinity and neutralizes the function of IGF1R in cancer cells. MK-0646 inhibits the binding of IGF1 to IGF1R in cells, blocks receptor activation and
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downstream signaling pathways (PI3K/AKT and MAPK) and suppresses the biological effects mediated by IGF1R [31]. Preclinical studies showed that MK-0646 has selective potent and efficacious activity against IGF1R autophosphorylation and IGF1- and IGF2mediated tumor cell proliferation [32]. Furthermore, MK-0646 was shown to display significant antitumor activity in multiple cancers (breast cancer, rhabdomyosarcoma, non-small cell lung cancer, etc.), cell lines and mouse xenograft models [33]. MK0646 induces IGF1R down-regulation in vivo and in vitro; however, the specific molecular mechanisms responsible for this process are not fully known. The decrease can be attributed to membrane receptor internalization or degradation that perhaps involves the ubiquitin–proteasome degradation pathway [34,35]. It was suggested that this process might lead to augmented therapeutic effects [21,36,37]. In addition, co-administration of MK-0646 with other anticancer agents, such as taxanes, enhanced the in vitro and in vivo antitumor activity of MK-0646. Several clinical trials evaluating MK-0646, alone and in combination with additional anticancer agents are currently ongoing in patients with various types of solid tumors and in patients with multiple myeloma. A preliminary Phase I study in nine patients with advanced solid tumors reported that MK-0646 was well tolerated and inhibited IGF1R pathway signaling and cell proliferation [19]. In the current study, we demonstrated that treatment with MK-0646 caused a significant decrease in IGF1R expression in endometrial cancer cell lines corresponding to the Type 1 and 2 subcategories. Moreover, MK-0646 reduced the IGF1-induced phosphorylation of IGF1R. We also examined the effect of MK-0646 on the activation of the IGF signaling pathways and showed that MK-0646 reduced the IGF1-induced AKT and ERK phosphorylation. Similar results were reported in breast cancerderived MCF-7 cells [31,38]. Next, we examined the effect of MK-0646 on apoptosis. Our results indicate that treatment with IGF1 reduced the levels of cleaved PARP and caspase-3 and lowered apoptosis levels. Addition of MK-0646 increased cleaved protein levels, thus increasing apoptosis and preventing IGF1 from exerting its anti-apoptotic effect. Furthermore, we showed that addition of MK-0646 significantly lowered the percentage of dividing cells compared to cells treated with IGF1 alone, thus preventing IGF1 from exerting its proliferative effect. Co-treatment with IGF1 and MK-0646 consistently reduced the IGF1-stimulated proliferation of ECC-1 and USPC-1 cells in MTT assays. Finally, we assessed the ability of MK-0646 to enhance the cytotoxic effect achieved by chemotherapy. The combination of MK-0646 and cisplatin decreased the growth of Type 1 and Type 2 endometrial cancer cells, compared with cisplatin alone. Therefore, our results indicate that MK-0646 enhances chemotherapy inhibition in endometrial cancer. The importance of the IGF1R in cancer cell growth may be related to both its proliferative and anti-apoptotic functions. We demonstrated that by blocking the IGF1R, MK-0646 neutralized the IGF1R-mediated signal transduction and as a result, inhibited endometrial cancer cell growth either by suppressing cell proliferation or by promoting cell death. Moreover, we showed that MK-0646 potentiated the effect of chemotherapy. The relative contribution of these effects to the anti-cancer activity of MK-0646 in in vivo models remains to be determined. Although, in vivo studies showed significant response to IGFIR targeted therapy, there was relatively low response in clinical trials. In a recent review, Beserga suggested several possible reasons for the failure of IGF1R targeted therapies in clinical trials, including constitutive activation of the PI3K pathway, absence or very low levels of insulin receptor substrate-I, activation of the insulin receptor by IGF2 that bypasses the IGFIR inhibition, nuclear translocation of the IGFIR and, most importantly, the existence of sub-
populations in each cancer with different mutations and sensitivity to treatment [35]. Therefore, it has been suggested that predictive tumor biomarkers could assist in selecting patients who would benefit from the proposed treatment. Growing clinical evidence suggests a potential correlation between biomarkers related to the IGF1R pathway and clinical benefits from IGF1R-targeted therapies. High IGF1R expression and elevated circulating IGF1 levels were shown to be correlated with improved response to IGF1R-targeted therapies in clinical trials [39–41]. A recent study reported that increased nuclear localization of IGF1R is associated with better overall survival for patients treated with IGF1R antibody therapy [42]. In a recent review, Pollak suggested that IGF1R levels and the presence of IGF1R autocrine loops are possible predictive biomarkers for IGF1R-targeted therapy [43]. The author also suggested that the presence of activating mutations downstream of the IGF1R would induce resistance to IGF1R-targeted therapy. The present study validates the IGF-1R as a target for endometrial carcinoma therapy and complements our previous studies, clearly demonstrating that IGFIR inhibitors, including tyrosine kinase inhibitors and IGF1R antibodies, have potential therapeutic benefits in endometrial cancer treatment. Based on these results, it is expected that identification of predictive biomarkers will improve the clinical response to IGF-IR targeted therapy in patients with endometrial cancer and will provide the foundation for translational efforts directed at targeting the IGF axis in endometrial cancer. In summary, the current study indicates that treatment with MK-0646 may be a novel potential therapy for endometrial cancer. Future clinical and translational studies, along with identification of predictive biomarkers, will establish the best clinical settings and treatment combinations for the use of MK-0646 in cancer treatment.
Role of funding source This study was supported by a grant from Merck, Sharp and Dohme (MSD, Israel) to H.W., A.F., and I.B. None of the authors has received any payment or royalty from MSD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. I.B. and H.W. wish to thank the generous support of the Israel Cancer Research Fund (ICRF), Montreal, Canada.
Acknowledgements We would like to thank Drs. A.D. Santin and Y. Sharoni for providing cell lines. The authors wish to thank Merck, Sharp and Dohme (MSD, Israel) and the Israel Cancer Research Fund (ICRF, Montreal, Canada) for their generous support.
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Contents lists available at SciVerse ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
IGF1R-directed targeted therapy enhances the cytotoxic effect of chemotherapy in endometrial cancer Connie Bitelman a,1, Rive Sarfstein a,1, Menahem Sarig b,1, Zohar Attias-Geva a, Ami Fishman b, Haim Werner a, Ilan Bruchim b,⇑ a b
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba 44281, Israel
a r t i c l e
i n f o
Article history: Received 21 November 2012 Received in revised form 3 February 2013 Accepted 4 February 2013
Keywords: Endometrial cancer Uterine serous carcinoma IGF1 IGF1 receptor MK-0646
a b s t r a c t This study evaluated the potential ability of MK-0646 to inhibit IGF1-mediated biological actions and cell signaling events in Type 1 and Type 2 endometrial cancer. We found that MK-0646 treatment significantly decreased IGF1R expression. In addition, pretreatment with MK-0646 decreased the IGF1-induced phosphorylation of IGF1R, AKT and ERK. Apoptosis analyses showed that MK-0646 abolished the antiapoptotic effect of IGF1. Furthermore, MK-0646 treatment abolished the IGF1-stimulatory effect on proliferation and enhanced the cytotoxic effect of cisplatin. These findings indicate that specific inhibition of IGF1R could be a useful therapeutic approach for Type 1 and Type 2 endometrial cancer. Ó 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endometrial cancer is the most widespread gynecologic cancer in Western countries. Women have a 2–3% lifetime risk of developing the disease, and more than 40,000 new cases were diagnosed in 2009 in the United States alone [1]. The incidence of the disease has been increasing in recent years, presumably because of the growing obesity epidemic. Differences in molecular pathways, epidemiology and prognosis account for the two major groups of endometrial cancer, Type 1 and Type 2, with Type 1, the estrogen-related form, being the most frequent (more than 80% of cases) [2,3]. Type 1 tumors exhibit an endometrioid, well-differentiated morphology, and are usually associated with a relatively good prognosis, as opposed to Type 2 tumors which display a less differentiated phenotype, and have a worse prognosis. Uterine serous carcinoma (USC) constitutes the predominant histological class among Type 2 tumors [4,5]. USC represents 10% of all endometrial carcinomas, is considered to be a high grade tumor, and has the worst prognosis among endometrioid tumors [6]. A number of different genetic alterations have been detected in Type 1 endome-
⇑ Corresponding author. Address: Gynecologic Oncology Unit, Department of Obstetrics and Gynecology, Meir Medical Center, 59 Tschernichovsky Street, Kfar Saba, Israel. Tel.: +972 97472561; fax: +972 97471620. E-mail address: [email protected] (I. Bruchim). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.02.009
trial cancers, including microsatellite instability and mutations of the pTEN, KRAS2, and ß-catenin genes and near-diploid karyotype. In contrast, Type 2 tumors are associated with p53 and ERBB-2 (HER-2/neu) mutations and loss of heterozygosity on several chromosomes, and in most cases are not diploid [7–9]. Systemic hormonal and chemotherapeutic treatments represent the cornerstones of advanced and recurrent endometrial cancer management, although it is still characterized by a poor prognosis. Therefore, there is an urgent need to identify new therapeutic regimens that will prolong survival and transform response to treatment into cure. Recent research has focused on optimizing chemotherapy regimens, the development of alternative hormonal therapy strategies, and the introduction of targeted therapies. Moreover, ongoing trials are exploring multimodal therapies incorporating chemotherapy with novel molecular agents for advanced primary and recurrent disease. The insulin-like growth factors 1 and 2 (IGF1 and IGF2) are a family of mitogenic polypeptides with important roles in growth and differentiation. The biological actions of the IGFs are mediated by the IGF1 receptor (IGF1R), a transmembrane tyrosine kinase structurally related to the insulin receptor [10–12]. In addition to its central role in normal cell cycle progression, the IGF1R has a pivotal role in tumorigenesis [13,14]. Thus, most tumors and cancer-derived cell lines express high levels of IGF1R mRNA and protein, while suppression of IGF1R was shown to block IGF1stimulated cellular proliferation [15]. Several studies underline
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the crucial role of IGF1R action in endometrial cancer and the importance of altered IGF1R gene expression in the development of a malignant phenotype [16–18]. MK-0646 (Dalotuzumab, h7C10; Merck, Sharp and Dohme) is a recombinant, humanized IgG1 monoclonal antibody that binds with high affinity to IGF1R, leading to receptor internalization and subsequent degradation; hence, preventing its ligand-mediated activation. Recent in vivo and in vitro studies showed that MK-0646 exhibits potent antitumor activity in a number of malignancies. Preliminary data from recent Phase 1 clinical trials suggest that MK-0646 is safe and well-tolerated and showed clinical activity against advanced solid tumors [19]. The current study was designed to obtain preclinical data regarding the use of MK-0646 for treating endometrial cancer.
2.4. Cell cycle analysis Cells were serum-starved for 24 h and incubated in the presence or absence of MK-0646 and/or IGF1 for 72 or 120 h. After incubation, cells were washed with phosphate buffered saline, trypsinized, permeabilized with Triton X-100 (4%) and stained with propidium iodide (50 mg/ml). Stained cells were analyzed using a FACSort Flow Cytometer (Beckton-Dickinson, CA, USA).
3. Results The IGF1R gene has emerged in recent years as a promising therapeutic target. MK-0646 is a monoclonal anti-IGF1R antibody, currently in preclinical and clinical evaluations. In this study, we focused on three uterine carcinoma cell lines, which constitute validated models for Type 1 (ECC-1) and Type 2 (USPC-1 and USPC-2) endometrial cancers.
2. Materials and methods
3.1. MK-0646 down-regulates IGF1R expression 2.1. Cell cultures and treatments Human endometrioid endometrial cancer (Type 1) and USC (Type 2) cell lines were used in this study. For endometrioid cancer, the ECC-1 cell line was used, and for USC, the USPC-1 and USPC-2 cell lines were used. ECC-1 cells were kindly provided by Dr. Y. Sharoni (Ben Gurion University, Beer Sheba, Israel) and USC cells by Dr. A. Santin (Yale University School of Medicine, New Haven, CT, USA). ECC-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l D-glucose (Gibco BRL, Paisley, Scotland) and USPC-1 and USPC2 cells were maintained in RPMI-1640 medium (Biological Industries Ltd., Beit Haemek, Israel). Cells were supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 50 mg/ml gentamicin sulfate, and 5.6 mg/l amphotericin B. All reagents were purchased from Biological Industries Ltd. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Human monoclonal IGF1R antibody MK-0646 (Dalotuzumab; Merck, Sharp and Dohme, Whitehouse Station, NJ, USA) was diluted in 20 mM histidine and 150 mM NaCl (20.95 g/l) and stored at 4 °C. In all experiments, cells were serum-starved for 24 h, after which they were treated with 10 lg/ml MK0646 in the presence or absence of IGF1 (50 ng/ml) (PeproTech Ltd., Rocky Hill, NJ, USA). In some experiments, cells were treated with 10 lg/ml MK-0646 for 5 h, or 24–144 h, separately and in combination with varying concentrations of cisplatin (1 lM, 5 lM or 10 lM) (Sigma–Aldrich Ltd., St. Louis, MO, USA). 2.2. Western immunoblots Cells were starved overnight and then incubated with MK-0646 in the presence or absence of IGF1 for the indicated periods. After incubation, cells were harvested and lysed in a buffer containing protease inhibitors (#9803, Cell Signaling Technology Beverly, MA, USA). Protein content was determined using the Bradford reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA) as a standard. Samples were electrophoresed through 10% or 5% SDS-PAGE gels, followed by blotting of the proteins onto nitrocellulose membranes. After blocking with either 5% skim milk and/or 3% BSA, the blots were incubated overnight with the antibodies listed below, washed and incubated with the appropriate horseradish peroxidase (HRP)conjugated secondary antibody. Antibodies against phospho-IGF1R (3024), IGF1R b-subunit (3027), phospho-AKT (9271), AKT (9272), phospho-ERK1/2 (9106), poly ADP ribose polymerase (PARP; 9542), and caspase-3 (9661), were obtained from Cell Signaling Technology. An antibody against ERK1 (K-23) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-tubulin (T5168) was purchased from Sigma–Aldrich Ltd. The secondary antibodies were HRP-conjugated goat anti-rabbit IgG (1:50,000) and donkey anti-mouse IgG (1:25,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Proteins were detected using the SuperSignal West PicoChemiluminescent Substrate (Pierce, Rockford, IL, USA). Tubulin expression was used as a loading control. 2.3. Proliferation measurements To determine the effect of MK-0646 on ECC-1 and USPC-1 cell proliferation, cells were seeded onto 24-well plates (2 104 ECC-1 cells/well and 5 104 USPC-2 cells/well). After 24 h, the cells were treated with either MK-0646 (10 lg/ml), IGF1 (80 ng/ml), or a combination of both for 24 and 48 h, in triplicate wells. Cell viability was assessed using a standard thiazolyl blue tetrazolium bromide (MTT) method [20]. The color developed was quantitated by measuring absorbance at a wavelength of 530 nm and reference wavelength of 630 nm on a Microplate Reader (Molecular Devices, SpectraMax 190, GMI, Inc., Minnesota, USA). Cell viability was expressed as a percentage of the optical density values obtained upon treatment relative to controls. In addition, we investigated a potential synergy between MK-0646 and cisplatin. To this end, cells were treated with MK-0646, cisplatin (1 lM, 5 lM or 10 lM), or a combination of MK-0646 with each dose of cisplatin for 120 h.
We first explored the effect of MK-0646 on IGF1R expression levels. To this end, ECC-1 and USPC-1 cells were seeded in full medium and upon reaching 100% confluence, were treated with MK0646 (10 lg/ml) for 24 or 48 h. The cells were then lysed and analyzed by Western blotting using an IGF1R b subunit antibody. Results obtained showed that treatment with MK-0646 caused a marked decrease in IGF1R levels in both cell lines at both time points (Fig. 1A). Of note, the levels of both IGF1R precursor (250kDa) and mature (97-kDa) forms were reduced. Specifically, MK0646 decreased IGF1R levels by 63% (24 h) and 48% (48 h) in ECC-1 cells and by 26% (24 h) and 34% (48 h) in USPC-1 cells versus untreated cells (100%; Fig. 1B). No changes in tubulin levels were observed in MK-0646-treated cells. In addition, we recently showed that IGF1R mRNA levels were correlated with the protein levels in both cell lines [21]. 3.2. MK-0646 decreases phosphorylation of the IGF1R and associated downstream signaling mediators In order to determine the ability of MK-0646 to block activation of the various cellular pathways normally activated by the IGF1R, phosphorylation levels of the receptor were tested using an antiphospho-IGF1R antibody. Results obtained showed that MK-0646 attenuated the IGF1-induced IGF1R phosphorylation in both ECC-1 [68% versus IGF1-treated cells (100%)] and USPC-1 cells [53% versus IGF1-treated cells (100%) (Fig. 2A and B)]. Next, the effects of MK-0646 on downstream signaling pathways induced by IGF1R were examined. Phosphorylation levels of AKT and ERK1/2 were measured as representatives of the PI3K/AKT/PKB and MAPK pathways, respectively. As expected, treatment with IGF1 for 10 min stimulated AKT phosphorylation in both cell types, while pretreatment with MK-0646 for 5 h attenuated this effect. No significant effect was seen on the total AKT level after treatment with MK-0646 and IGF1 or with IGF1 alone, after these short exposures (Fig. 2A). A similar trend was seen in both phosphorylation and expression of ERK1/2 after treatment with MK-0646 and/or IGF1. 3.3. MK-0646 abrogates the anti-apoptotic effect of IGF1 in endometrial cancer cells To investigate the effect of MK-0646 on apoptosis, cleaved PARP and caspase-3 were measured using Western blots. Cleavage of the 116-kDa PARP molecule into an 85-kDa band is considered a typical hallmark of early apoptosis [22]. Caspase-3 has been shown to be a key component of the apoptotic machinery. It is activated in apoptotic cells and cleaves several cellular proteins, including PARP. Caspase-3 is a protease that promotes the splitting of PARP
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Fig. 1. IGF1R levels. ECC-1 and USPC-1 cells were treated with MK-0646 for 24 h and 48 h. At the end of the incubation period, cells were lysed and whole-cell lysates (100 lg) were resolved on SDS-PAGE and immunoblotted with antibodies against IGF1R b-subunit and tubulin as described in Materials and methods. (A) Scanning densitometry analysis represents the IGF1R levels in response to treatment with MK-0646. MK-0646 decreased IGF1R after 24 h and 48 h. Bars represent IGF1R values normalized to the corresponding tubulin levels, (B) the figure shows the result of a typical experiment, repeated three times with similar results.
Fig. 2. IGF1-mediated signaling pathways. ECC-1 and USPC-1 cell were treated for 5 h with MK-0646 (or left untreated) and/or IGF1 during the last 10 min of the incubation period. Whole-cell lysates (100 lg) were resolved on SDS-PAGE and immunoblotted with antibodies against pIGF1R, TIGF1R, pAKT, TAKT, pERK1/2 and TERK1/2. (A) Optical density was expressed as phospho-IGF-IR values normalized to the corresponding total IGF1R levels and (B) the figure shows the result of a typical experiment, repeated three times with similar results.
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Fig. 3. IGF1-stimulated protection from apoptosis. Western blot analysis of the effect of MK-0646 on PARP (A) and caspase-3 (B) on ECC-1 and USPC-1 cells. Serum-starved cells were treated with IGF1 for the indicated times in the presence or absence of MK-0646. After incubation, cell lysates were prepared and electrophoresed (100 lg). Results are representative of three independent experiments.
after it itself undergoes cleavage into 17- and 19-kDa fragments. Evaluation of cleaved PARP and caspase-3 constitute bona fide markers of apoptosis. Results obtained revealed that after 24 h and 48 h, IGF1 treatment led to a reduction in cleaved PARP levels in both cell types (Fig. 3A). Treatment with MK-0646 for 24 h and 48 h abolished the anti-apoptotic effect of IGF1 in both cell lines, as indicated by the increased levels of cleaved PARP compared to IGF1-treated cells (Fig. 3A). In addition, IGF1 significantly decreased the levels of cleaved caspase-3, while MK-0646 attenuated this decrease. In ECC-1 cells, however, the ability of MK-0646 to reverse the effect of IGF1 on caspase-3 cleavage was seen only at 24 h (Fig. 3B).
Table 1 Effect of MK-0646 on the cell cycle in endometrial cancer cells. ECC-1 and USPC-1 cells were seeded in quadruplicate dishes, serum-starved for 24 h, and treated with MK-0646 (or left untreated, controls) for 120 h (ECC-1) and 72 h (USPC-1). Cell cycle distribution was assessed by FACS analysis. The values in the table denote mean ± SEM. Cell type/stage ECC1 G0/G1 S+G2/M USPC1 G0/G1 S+G2/M * **
DMSO (%) 86 ± 0.05 14 ± 0.4 89.4 ± 0.8 10.6 ± 0.8
IGF1 (%) 78 ± 0.04* 22 ± 0.09* 79 ± 0.7* 21 ± 0.6*
IGF1 + MK-0646 (%) 84 ± 0.05** 16 ± 0.02** 90.8 ± 0.8** 9.2 ± 0.7**
p < 0.01 versus control cells (DMSO). p 6 0.01 versus IGF1-treated cells.
3.4. MK-0646 inhibits the IGF1-stimulated cell cycle progression The effect of MK-0646 on cell cycle progression was examined in ECC-1 and USPC-1 cells using FACS analysis. In ECC-1 cells, IGF1 increased the proportion of cells at the S+G2/M phase from 14 ± 0.4% to 22 ± 0.09% (Table 1). The addition of MK-0646 on top of IGF1 caused a decrease in the proportion of cells at the S+G2/ M phase from 22 ± 0.09% to 16 ± 0.02%, and increased the proportion of cells at the G0/G1 phase from 78 ± 0.04% to 84 ± 0.5%. Similarly, in USPC-1 cells IGF1 treatment led to an increase in the number of cells at S+G2/M from 10.6 ± 0.8% to 21 ± 0.6% and decreased the proportion of cells at the G0/G1 phase from
89.4 ± 0.8% to 79 ± 0.7% (Table 1). Addition of MK-0646 on top of IGF1 reduced the proportion of cells at S+G2/M from 21 ± 0.6% to 9.2 ± 0.7% and increased the proportion of cells at the G0/G1 phase from 79.4 ± 0.7% to 90.8 ± 0.8%.
3.5. Effect of IGF1R inhibition on proliferation The effect of MK-0646 on proliferation rate was determined using the MTT method. Proliferation rates were examined
Fig. 4. Endometrial cancer cell proliferation. Cells were placed in 24-well plates at a density of 2 104 cells/well for ECC-1 (A) and 5 104 cells/well for USPC-2 cells (B). Cells were treated with IGF1, or IGF1 in combination with MK-0646 (or left untreated) for 24 h and 48 h, after which the proliferation rate was measured by MTT assays. The number of cells at time 0 was assigned a value of 100% and they were used as control. The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate samples. p 6 0.01 versus untreated cells and p 6 0.01 versus IGF1-treated cells.
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Fig. 5. Endometrial cancer cell proliferation with MK-0646 and varying doses of cisplatin. Cells were plated in 24-well plates at a density of 2 104 cells/well for ECC-1 (A) and 5 104 cells/well for USPC-1 cells (B). The number of cells at time 0 was assigned a value of 100% and they were used as control. The bars represent the mean ± SEM of three independent experiments, each performed in triplicate samples. p 6 0.05 versus untreated cells and p 6 0.05 versus cisplatin-treated cells.
following treatment of ECC-1 and USPC-2 cells with IGF1, MK-0646, or both, compared to untreated cells. Treatment with IGF1 stimulated proliferation in both cell lines after 24 h and 48 h (Fig. 4). Co-treatment with IGF1 and MK-0646 inhibited proliferation in both cell lines after 24 h and 48 h, compared to cells treated with IGF1 alone. Specifically, in ECC-1 cells (Fig. 4A), MK-0646 inhibited proliferation by 59.2% and 73.7% at 24 h and 48 h, respectively and in USPC-2 cells by 58.5% and 72.7%, respectively (Fig. 4B). Similar results were obtained in preliminary assays using trypan blue staining followed by hemocytometer counting. 3.6. MK-0646 potentiates the effect of cisplatin Finally, the effects of MK-0646 on the resistance of endometrial cancer cells against chemotherapeutic agents, as well as possible synergistic effects of co-treatment with MK-0646 and cisplatin were studied. ECC-1 and USPC-1 were treated with cisplatin in the presence or absence of MK-0646. Proliferation rates were measured after 120 h using the MTT method. Treatment with cisplatin had a major cytotoxic effect on both cell types (Fig. 5A). Thus, after treatment with 10 lM cisplatin alone, there was a 49.8% decrease in proliferation rate in ECC-1 cells (Fig. 5A) and a 39.4% decrease in USPC-1 cells (Fig. 5B) compared to control. Next, co-incubation experiments using MK-0646 and increasing doses of cisplatin were performed in ECC-1 cells. At every dose of cisplatin, the addition of MK-0646 decreased the number of surviving cells in a dose-dependent fashion (Fig. 5A). Treatment of both ECC-1 and USPC-1 cells with MK-0646 and 10 lM of cisplatin caused decreases in proliferation of 20.4% and 10.3%, respectively, compared to treatment with 10 lM of cisplatin alone (Fig. 5A and B). In summary, addition of MK-0646 to cisplatin in endometrial cancer cells enhanced the cytotoxic effects of the chemotherapeutic treatment.
4. Discussion The IGF pathway has been implicated in the etiology of several epithelial malignancies, including breast, colon, prostate and gynecologic cancers [23]. Various technologies are currently being employed to down-regulate IGF1R expression and signaling with the purpose of achieving improved response to cancer therapy. Among the different IGF1R targeting strategies, the most promising approaches to date are the antagonistic antibodies and the small molecule kinase inhibitors. A number of reports showed a correlation between components of the IGF system and endometrial cancer risk [24–26]. Moreover, several studies reported significant increases in IGF1R expression in endometrial cancer [16,27] and correlation to disease grade and stage [17,28]. We have recently demonstrated that a specific IGF1R tyrosine kinase inhibitor (NVP-AEW541, Novartis Pharma, Basel, Switzerland) and a fully human monoclonal antibody against the IGF1R (IMC-A12, ImClone Systems, New York, USA), effectively blocked IGF1R activity in endometrial cancer cell lines and abolished the IGF1-mediated signaling cascades [21,29]. IGF1R targeting for cancer therapy is currently being evaluated in over 100 clinical trials and by more than 30 drugs in clinical and laboratory studies [30]. In recent years, several IGF1R inhibitors have been developed, some of which were evaluated in Phase I-III clinical trials as monotherapy, as well as in combination with chemotherapy, radiotherapy, and/or antibodybased therapy. Results of these clinical trials in solid tumors have shown variable responses to IGF1R-directed therapies and recent Phase III trials in unselected patients were discontinued due to futility. MK-0646 is a recombinant, humanized IgG1 monoclonal antibody that binds to the human IGF1R with high affinity and neutralizes the function of IGF1R in cancer cells. MK-0646 inhibits the binding of IGF1 to IGF1R in cells, blocks receptor activation and
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downstream signaling pathways (PI3K/AKT and MAPK) and suppresses the biological effects mediated by IGF1R [31]. Preclinical studies showed that MK-0646 has selective potent and efficacious activity against IGF1R autophosphorylation and IGF1- and IGF2mediated tumor cell proliferation [32]. Furthermore, MK-0646 was shown to display significant antitumor activity in multiple cancers (breast cancer, rhabdomyosarcoma, non-small cell lung cancer, etc.), cell lines and mouse xenograft models [33]. MK0646 induces IGF1R down-regulation in vivo and in vitro; however, the specific molecular mechanisms responsible for this process are not fully known. The decrease can be attributed to membrane receptor internalization or degradation that perhaps involves the ubiquitin–proteasome degradation pathway [34,35]. It was suggested that this process might lead to augmented therapeutic effects [21,36,37]. In addition, co-administration of MK-0646 with other anticancer agents, such as taxanes, enhanced the in vitro and in vivo antitumor activity of MK-0646. Several clinical trials evaluating MK-0646, alone and in combination with additional anticancer agents are currently ongoing in patients with various types of solid tumors and in patients with multiple myeloma. A preliminary Phase I study in nine patients with advanced solid tumors reported that MK-0646 was well tolerated and inhibited IGF1R pathway signaling and cell proliferation [19]. In the current study, we demonstrated that treatment with MK-0646 caused a significant decrease in IGF1R expression in endometrial cancer cell lines corresponding to the Type 1 and 2 subcategories. Moreover, MK-0646 reduced the IGF1-induced phosphorylation of IGF1R. We also examined the effect of MK-0646 on the activation of the IGF signaling pathways and showed that MK-0646 reduced the IGF1-induced AKT and ERK phosphorylation. Similar results were reported in breast cancerderived MCF-7 cells [31,38]. Next, we examined the effect of MK-0646 on apoptosis. Our results indicate that treatment with IGF1 reduced the levels of cleaved PARP and caspase-3 and lowered apoptosis levels. Addition of MK-0646 increased cleaved protein levels, thus increasing apoptosis and preventing IGF1 from exerting its anti-apoptotic effect. Furthermore, we showed that addition of MK-0646 significantly lowered the percentage of dividing cells compared to cells treated with IGF1 alone, thus preventing IGF1 from exerting its proliferative effect. Co-treatment with IGF1 and MK-0646 consistently reduced the IGF1-stimulated proliferation of ECC-1 and USPC-1 cells in MTT assays. Finally, we assessed the ability of MK-0646 to enhance the cytotoxic effect achieved by chemotherapy. The combination of MK-0646 and cisplatin decreased the growth of Type 1 and Type 2 endometrial cancer cells, compared with cisplatin alone. Therefore, our results indicate that MK-0646 enhances chemotherapy inhibition in endometrial cancer. The importance of the IGF1R in cancer cell growth may be related to both its proliferative and anti-apoptotic functions. We demonstrated that by blocking the IGF1R, MK-0646 neutralized the IGF1R-mediated signal transduction and as a result, inhibited endometrial cancer cell growth either by suppressing cell proliferation or by promoting cell death. Moreover, we showed that MK-0646 potentiated the effect of chemotherapy. The relative contribution of these effects to the anti-cancer activity of MK-0646 in in vivo models remains to be determined. Although, in vivo studies showed significant response to IGFIR targeted therapy, there was relatively low response in clinical trials. In a recent review, Beserga suggested several possible reasons for the failure of IGF1R targeted therapies in clinical trials, including constitutive activation of the PI3K pathway, absence or very low levels of insulin receptor substrate-I, activation of the insulin receptor by IGF2 that bypasses the IGFIR inhibition, nuclear translocation of the IGFIR and, most importantly, the existence of sub-
populations in each cancer with different mutations and sensitivity to treatment [35]. Therefore, it has been suggested that predictive tumor biomarkers could assist in selecting patients who would benefit from the proposed treatment. Growing clinical evidence suggests a potential correlation between biomarkers related to the IGF1R pathway and clinical benefits from IGF1R-targeted therapies. High IGF1R expression and elevated circulating IGF1 levels were shown to be correlated with improved response to IGF1R-targeted therapies in clinical trials [39–41]. A recent study reported that increased nuclear localization of IGF1R is associated with better overall survival for patients treated with IGF1R antibody therapy [42]. In a recent review, Pollak suggested that IGF1R levels and the presence of IGF1R autocrine loops are possible predictive biomarkers for IGF1R-targeted therapy [43]. The author also suggested that the presence of activating mutations downstream of the IGF1R would induce resistance to IGF1R-targeted therapy. The present study validates the IGF-1R as a target for endometrial carcinoma therapy and complements our previous studies, clearly demonstrating that IGFIR inhibitors, including tyrosine kinase inhibitors and IGF1R antibodies, have potential therapeutic benefits in endometrial cancer treatment. Based on these results, it is expected that identification of predictive biomarkers will improve the clinical response to IGF-IR targeted therapy in patients with endometrial cancer and will provide the foundation for translational efforts directed at targeting the IGF axis in endometrial cancer. In summary, the current study indicates that treatment with MK-0646 may be a novel potential therapy for endometrial cancer. Future clinical and translational studies, along with identification of predictive biomarkers, will establish the best clinical settings and treatment combinations for the use of MK-0646 in cancer treatment.
Role of funding source This study was supported by a grant from Merck, Sharp and Dohme (MSD, Israel) to H.W., A.F., and I.B. None of the authors has received any payment or royalty from MSD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. I.B. and H.W. wish to thank the generous support of the Israel Cancer Research Fund (ICRF), Montreal, Canada.
Acknowledgements We would like to thank Drs. A.D. Santin and Y. Sharoni for providing cell lines. The authors wish to thank Merck, Sharp and Dohme (MSD, Israel) and the Israel Cancer Research Fund (ICRF, Montreal, Canada) for their generous support.
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