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Purpose: To investigate the autocrine/endocrine role of Id1-induced insulin-like growth factor-II (IGF-II) in esophageal cancer, and evaluate the potential of IGF-II- and IGF-type I receptor (IGF-IR)-targeted therapies.Experimental Design: Antibody array-based screening was used to identify differentially secreted growth factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assays were performed to confirm the induction of IGF-II by Id1, and to study the autocrine and endocrine effects of IGF-II in promoting esophageal cancer progression. Human esophageal cancer tissue microarray was analyzed for overexpression of IGF-II and its correlation with that of Id1 and phosphorylated AKT (p-AKT). The efficacy of intratumorally injected IGF-II antibody and intraperitoneally injected cixutumumab (fully human monoclonal IGF-IR antibody) was evaluated using in vivo tumor xenograft and experimental metastasis models.Results: Id1 overexpression induced IGF-II secretion, which promoted cancer cell proliferation, survival, and invasion by activating AKT in an autocrine manner. Overexpression of IGF-II was found in 21 of 35 (60%) esophageal cancer tissues and was associated with upregulation of Id1 and p-AKT. IGF-II secreted by Id1-overexpressing esophageal cancer xenograft could instigate the growth of distant esophageal tumors, as well as promote metastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive effects on tumor growth and metastasis in mice. Cixutumumab treatment enhanced the chemosensitivity of tumor xenografts to fluorouracil and cisplatin.Conclusions: The Id1–IGF-II–IGF-IR–AKT signaling cascade plays an important role in esophageal cancer progression. Blockade of IGF-II/IGF-IR signaling has therapeutic potential in the management of esophageal cancer. Clin Cancer Res; 20(10); 2651–62. ©2014 AACR.

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PDF file - 1419KB Supplementary Fig S1. Human growth factor antibody array analysis of the conditioned media from HKESC-3-CON and HKESC-3-Id1 cells. Supplementary Fig S2. Effect of knockdown Id1 expression on IGF2 level using a second RNAi sequence. Supplementary Fig S3. Effect of ectopic Id1 expression on IGF2 level in multiple cancer types. Supplementary Fig S4. Effect of Id1 manipulation on IGF2 mRNA expression in ESCC cells. Supplementary Fig S5. IGF2, Id1, and p-AKT expressions in human esophageal cancer. Supplementary Fig S6. Western blot analysis of Id1 and IGF2 expressions in ESCC cell lines. Supplementary Fig S7. Effects of Id1 knockdown on autocrine stimulation of esophageal cancer cell proliferation, migration, and survival. Supplementary Fig S8. Ki-67 and TUNEL staining of tumor xenografts from three groups of mice inoculated with KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 cells, respectively. Supplementary Fig S9. Ki-67 and TUNEL staining in "responder" tumors of different groups of mice bearing KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1- shIGF2 "instigating" tumors, respectively. Supplementary Fig S10. Anti-tumor effects of IGF2-neutralizing antibody in vivo. Supplementary Fig S11. Immunohistochemical analysis of tumor xenografts treated with cixutumumab or the isotype IgG. Supplementary Fig S12. TUNEL staining of tumor xenografts from two groups of mice inoculated with KYSE150 (A) and KYSE270 (B) respectively. Supplementary Fig S13. Evaluation of toxic effects of cixutumumab in nude mice. A, comparison of body weight of cixutumumab-treated mice and isotype IgG-treated control mice. B, histological examination of lung, liver and kidney specimens (H&E stained). Supplementary Fig S14. Increased thymidylate synthase expression in FR cells was inhibited by cixutumumab.

PDF file - 8KB, Immunohistochemical analysis of expression levels of Id1, IGF2, and p-AKT in human ESCC specimens.

Supplementary Figure 3 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Supplementary Figure 4C-D from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Supplementary Figure 1 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Supplementary Figure 2 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

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. Cancer Res; 70(2); 741–51

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

Supplementary Figure Legends 1-5 from Insulin-Mediated Acceleration of Breast Cancer Development and Progression in a Nonobese Model of Type 2 Diabetes

Among the mechanisms implicated in the tumor-promoting effects of obesity, signaling by insulin-like growth factor-I (IGF-I) and insulin has received considerable attention. However, the emerging realization that obesity is associated with chronic inflammation has prompted other consideration of how the IGF-I axis may participate in cancer progression. In the present study, we used two mouse models of chronic (LID) and inducible (iLID) igf-1 gene deficiency in the liver to investigate the role of IGF-I in regulating the host microenvironment and colorectal carcinoma growth and metastasis in obese mice. Obese mice had a heightened inflammatory response in the liver, which was abolished in mice with chronic IGF-I deficiency (LID). In control animals changes to the hepatic microenvironment associated with obesity sustained the presence of tumor cells in the liver and increased the incidence of hepatic metastases after intrasplenic/portal inoculation of colon carcinoma cells. These changes did not occur in LID mice with chlonic IGF-1 deficiency. In contrast, these changes occured in iLID mice with acute IGF-1 deficiency, in the same manner as the control animals, revealing a fundamental difference in the nature of the requirement for IGF-1 on tumor growth and metastasis. In the setting of obesity, our findings imply that IGF-1 is critical to activate and sustain an inflammatory response in the liver that is needed for hepatic metastasis, not only through direct, paracrine effect on tumor cell growth, but also through indirect effects involving the tumor microenvironment. Cancer Res; 70(1); 57–67

Supplementary Figure 4A-B from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.