Joan M. Carboni

Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

Drugs that target the insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (IR) are currently under investigation for a variety of malignancies including breast cancer. Although we have previously reported that IGF-IR expression in primary breast tumors is common, the activation status of this receptor has not been examined in relation to survival. Phosphorylated IGF-IR/IR (P-IGF-IR/IR) and its downstream signaling partner phospho-S6 (P-S6) were evaluated immunohistochemically in tumor tissue microarrays representing 438 cases of invasive breast cancer. P-IGF-IR/IR (n = 114; P = 0.046) and total levels of IR (n = 122; P = 0.009) were indicative of poor survival, whereas total IGF-IR (n = 112; P = 0.304) was not. P-IGF-IR/IR and P-S6 were coordinately expressed in primary breast tumors (likelihood ratio, 11.57; P = 6.70 x 10(-4)). Importantly, P-IGF-IR/IR was detected in all breast cancer subtypes (luminal, 48.1%; triple negative, 41.9%; and HER2, 64.3%). In vitro, the IGF-IR/IR inhibitor BMS-536924 decreased phospho-RSK and P-S6, and significantly suppressed the growth of breast cancer cell lines MCF-7, SUM149, and AU565 representing the luminal, triple negative, and HER2 subtypes, respectively, in monolayer and soft agar. BMS-536924 also inhibited growth in tamoxifen resistant MCF-7 Tam-R cells while having little effect on immortalized normal breast epithelial cells. Thus, we can determine which patients have the activated receptor and provide evidence that P-IGF-IR/IR is a prognostic factor for breast cancer. Beyond this, P-IGF-IR/IR could be a predictive marker for response to IGF-IR and/or IR-targeted therapies, as these inhibitors may be of benefit in all breast cancer subtypes including those with acquired resistance to tamoxifen.

Insulin-Mediated Acceleration of Breast Cancer Development and Progression in a Nonobese Model of Type 2 Diabetes

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

In vitro and in vivo antitumor effects of the dual insulin-like growth factor-I/insulin receptor inhibitor, BMS-554417.

The insulin-like growth factor receptor (IGF-IR) and insulin receptor are either overactivated and/or overexpressed in a wide range of tumor types and contribute to tumorigenicity, proliferation, metastasis, and drug resistance. Here, we show that BMS-554417, a novel small molecule developed as an inhibitor of IGF-IR, inhibits IGF-IR and insulin receptor kinase activity and proliferation in vitro, and reduces tumor xenograft size in vivo. In a series of carcinoma cell lines, the IC50 for proliferation ranged from 120 nmol/L (Colo205) to >8.5 micromol/L (OV202). The addition of stimulatory ligands was unnecessary for the antiproliferative effect in MCF-7 and OV202 cells. BMS-554417 treatment inhibited IGF-IR and insulin receptor signaling through extracellular signal-related kinase as well as the phosphoinositide 3-kinase/Akt pathway, as evidenced by decreased Akt phosphorylation at Ser473. At doses that inhibited proliferation, the compound also caused a G0-G1 arrest and prevented nuclear accumulation of cyclin D1 in response to LR3 IGF-I. In Jurkat T-cell leukemia cells, this agent triggered apoptotic cell death via the mitochondrial pathway. BMS-554417 was orally bioavailable and significantly inhibited the growth of IGF1R-Sal tumor xenografts in vivo. BMS-554417 is a member of a novel class of IGF-IR/insulin receptor inhibitors that have potential clinical applications because of their antiproliferative and proapoptotic activity in vitro and in vivo.

Tumor Development by Transgenic Expression of a Constitutively Active Insulin-Like Growth Factor I Receptor

The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.

Constitutively Active Type I Insulin-Like Growth Factor Receptor Causes Transformation and Xenograft Growth of Immortalized Mammary Epithelial Cells and Is Accompanied by an Epithelial-to-Mesenchymal Transition Mediated by NF-κB and Snail

ABSTRACT Type I insulin-like growth factor receptor (IGF-IR) can transform mouse fibroblasts; however, little is known about the transforming potential of IGF-IR in human fibroblasts or epithelial cells. We found that overexpression of a constitutively activated IGF-IR (CD8-IGF-IR) was sufficient to cause transformation of immortalized human mammary epithelial cells and growth in immunocompromised mice. Furthermore, CD8-IGF-IR caused cells to undergo an epithelial-to-mesenchymal transition (EMT) which was associated with dramatically increased migration and invasion. The EMT was mediated by the induction of the transcriptional repressor Snail and downregulation of E-cadherin. NF-κB was highly active in CD8-IGF-IR-MCF10A cells, and both increased levels of Snail and the EMT were partially reversed by blocking NF-κB or IGF-IR activity. This study places IGF-IR among a small group of oncogenes that, when overexpressed alone, can confer in vivo tumorigenic growth of MCF10A cells and indicates the hierarchy in the mechanism of IGF-IR-induced EMT.

The Mechanisms of Differential Sensitivity to an Insulin-like Growth Factor-1 Receptor Inhibitor (BMS-536924) and Rationale for Combining with EGFR/HER2 Inhibitors

Overexpression and enhanced activity of insulin-like growth factor-I receptor (IGF-IR) in diverse tumor types make it an attractive target for cancer therapy. BMS-536924 is a potent small molecule inhibitor of IGF-IR, which shows antitumor activity in multiple tumor models, including sarcoma. To facilitate the development of IGF-IR inhibitors as cancer therapy, identification of biomarkers for selecting patients most likely to derive clinical benefit is needed. To do so, 28 sarcoma and neuroblastoma cell lines were screened for in vitro response to BMS-536924 to identify sensitive and resistant cell lines. Notably, Ewings sarcoma, rhabdomyosarcoma, and neuroblastoma are more responsive to BMS-536924, suggesting these specific subtypes may represent potential targeted patient subpopulations for the IGF-IR inhibitor. Gene expression and protein profiling were performed on these cell lines, and candidate biomarkers correlating with intrinsic and/or acquired resistance to BMS-536924 were identified. IGF-I, IGF-II, and IGF-IR were highly expressed in sensitive cell lines, whereas IGFBP-3 and IGFBP-6 were highly expressed in resistant lines. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in resistant cell lines may represent one possible resistance mechanism by the adaptation of IGF-IR-independent growth using alternative signaling pathways. Based on cross-talk between IGF-IR and EGFR pathways, combination studies to target both pathways were performed, and enhanced inhibitory activities were observed. These results provide a strategy for testing combinations of IGF-IR inhibitors with other targeted therapies in clinical studies to achieve improved patient outcomes. Further exploration of mechanisms for intrinsic and acquired drug resistance by these preclinical studies may lead to more rationally designed drugs that target multiple pathways for enhanced antitumor efficacy.

HER receptor signaling confers resistance to the insulin-like growth factor-I receptor inhibitor, BMS-536924

We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin, respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1 and HER-2 may overcome clinical resistance to IGF-IR inhibitors. [Mol Cancer Ther 2008;7(9):2589–98]

Insulin receptor isoform a and insulin-Like growth factor II as additional treatment targets in human osteosarcoma

Despite the frequent presence of an insulin-like growth factor I receptor (IGFIR)-mediated autocrine loop in osteosarcoma (OS), interfering with this target was only moderately effective in preclinical studies. Here, we considered other members of the IGF system that might be involved in the molecular pathology of OS. We found that, among 45 patients with OS, IGF-I and IGFBP-3 serum levels were significantly lower, and IGF-II serum levels significantly higher, than healthy controls. Increased IGF-II values were associated with a decreased disease-free survival. After tumor removal, both IGF-I and IGF-II levels returned to normal values. In 23 of 45 patients, we obtained tissue specimens and found that all expressed high mRNA level of IGF-II and >IGF-I. Also, isoform A of the insulin receptor (IR-A) was expressed at high level in addition to IGFIR and IR-A/IGFIR hybrids receptors (HR(A)). These receptors were also expressed in OS cell lines, and simultaneous impairment of IGFIR, IR, and Hybrid-Rs by monoclonal antibodies, siRNA, or the tyrosine kinase inhibitor BMS-536924, which blocks both IGFIR and IR, was more effective than selective anti-IGFIR strategies. Also, anti-IGF-II-siRNA treatment in low-serum conditions significantly inhibited MG-63 OS cells that have an autocrine circuit for IGF-II. In summary, IGF-II rather than IGF-I is the predominant growth factor produced by OS cells, and three different receptors (IR-A, HR(A), and IGFIR) act complementarily for an IGF-II-mediated constitutive autocrine loop, in addition to the previously shown IGFIR/IGF-I circuit. Cotargeting IGFIR and IR-A is more effective than targeting IGF-IR alone in inhibiting OS growth.

Differential Mechanisms of Acquired Resistance to Insulin-like Growth Factor-I Receptor Antibody Therapy or to a Small-Molecule Inhibitor, BMS-754807, in a Human Rhabdomyosarcoma Model

Agents targeting insulin-like growth factor-I receptor (IGF-IR), including antibodies and small-molecule inhibitors, are currently in clinical development for the treatment of cancers including sarcoma. However, development of resistance is a common phenomenon resulting in failures of anticancer therapies. In light of this problem, we developed two resistant models from the rhabdomyosarcoma cell line Rh41: Rh41-807R, with acquired resistance to BMS-754807, a small-molecule dual-kinase inhibitor targeting IGF-IR and insulin receptor (IR), and Rh41-MAB391R, with resistance to MAB391, an IGF-IR-blocking antibody. In addition, tumor xenograft models were established from Rh41 and Rh41-807R cell lines. Gene expression and DNA copy number analyses of these models revealed shared as well as unique acquired resistance mechanisms for the two types of IGF-IR inhibitors. Each resistant model used different signaling pathways as a mechanism for proliferation. Platelet-derived growth factor receptor α (PDGFRα) was amplified, overexpressed, and constitutively activated in Rh41-807R cells and tumors. Knockdown of PDGFRα by small interfering RNA in Rh41-807R resensitized the cells to BMS-754807. Synergistic activities were observed when BMS-754807 was combined with PDGFRα inhibitors in the Rh41-807R model in vitro. In contrast, AXL expression was highly elevated in Rh41-MAB391R but downregulated in Rh41-807R. Notably, BMS-754807 was active in Rh41-MAB391R cells and able to overcome resistance to MAB391, but MAB391 was not active in Rh41-807R cells, suggesting potentially broader clinical activity of BMS-754807. This is the first study to define and compare acquired resistance mechanisms for IGF-IR-targeted therapies. It provides insights into the differential acquired resistance mechanisms for IGF-IR/IR small-molecule inhibitor versus anti-IGF-IR antibody.

High IGF-IR Activity in Triple-Negative Breast Cancer Cell Lines and Tumorgrafts Correlates with Sensitivity to Anti–IGF-IR Therapy

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.