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Insulin-like growth factor (IGF)-I receptor (IGF-IR) signaling is required for carcinogenicity and progression of several cancers but the function of this pathway and its utility as a therapeutic target have not been studied comprehensively in biliary tract carcinomas (BTC). We investigated the immunohistochemical expression of elements of the IGF axis, matrilysin, overexpression of p53 and the methylation status of the IGFBP-3 promoter in 80 surgically resected BTC. We also assessed the effect of IGF-IR blockade on signal transduction, proliferation and survival in three BTC cell lines using a new tyrosine kinase inhibitor, BMS-536924, and dominant negative IGF-IR (IGF-IR/dn). The effects of IGF-IR blockade was also studied in nude mouse xenograft models. IGF-I was expressed in 60% and IGF-II in 50% of tumors. High expression was associated with tumor size. IGF-IR was expressed in 69% of the cases and was associated with advanced stage and matrilysin expression. Hypermethylation of the IGFBP-3 promoter was detected in 41% of BTC and was inversely correlated with p53 expression. BMS-536924 blocked autophosphorylation of IGF-IR and both Akt and ERK activation by both IGF-I and insulin. BMS-536924 suppressed proliferation and tumorigenicity in vitro in a dose-dependent fashion. This inhibitor upregulated chemotherapy-induced apoptosis in a dose-dependent fashion. Moreover, IGF-IR blockade was effective against tumors in mice. IGF-IR might identify a subset of BTC with a particularly aggressive phenotype and is a candidate therapeutic target in this disease. BMS-536924 might have significant therapeutic utility. (Cancer Sci 2012; 103: 252–261)
Biliary tract carcinomas (BTC) have one of the worst outcomes of all malignancies in Asia, Europe and the USA. Owing to its non-specific presenting symptoms, BTC is generally diagnosed late in the disease course.(1) Complete surgical resection is the only curative treatment for BTC, but surgery is often not possible for these advanced diseases.(2–4) Therefore, we must to seek new therapeutic options for this disease.
Recent advances in molecular cancer research have brought new therapeutic strategies targeting these signals into routine clinical usage. Growth factor receptors are one such group of targets and their activity can be blocked by tyrosine kinase inhibitors (TKI) or mAb. Insulin-like growth factor (IGF)-I receptor (IGF-IR) is one such candidate molecular target.(5,6)
Binding of the ligands IGF-I and IGF-II to IGF-IR causes receptor autophosphorylation and activates multiple signaling pathways, including ERK and the phosphatidylinositide 3-kinase (PI3-K)/Akt-1 axis.(7) Activation of IGF-IR is regulated by multiple factors, including IGF binding proteins (IGFBP) and IGF-2 receptors.(8–10)
Dysregulation of the IGF system has been implicated in the proliferation of numerous neoplasms.(6,11) Mutations or chromosomal amplifications of IGF-IR are rare; however, the regulation of IGF-IR expression is closely associated with the function of several oncogenes and tumor suppressor genes.(11) Although wild-type p53 inhibits IGF-IR expression, mutant p53 can induce IGF-IR expression.(12) Elevation of serum IGF-I increases the risk of developing several cancers.(9) IGF-IR signaling is also important in tumor dissemination through the control of migration, angiogenesis, invasion and metastasis.(13,14) The findings outlined above suggest a potential basis for tumor selectivity in therapeutic applications in gastrointestinal cancers.
There is, however, only limited information about the IGF/IGF-IR axis in BTC. In immunohistochemical studies, gallbladder carcinoma (GBC) expressed IGF-I in 45%, IGF-II in 25% and IGF-IR in 95% of cases with BTC(15) and all intrahepatic cholangiocarcinomas expressed both IGF-I and IGF-IR.(16) Several human BTC cells express IGF-IR.(16–18)
IGFBP-3, which is the most abundant IGFBP in the circulation, has both IGF-dependent and IGF-independent antiproliferative and proapoptotic effects on several cancers.(19) IGFBP-3 promoter methylation and gene silencing have been reported in cancers.(20,21) IGFBP-3 is induced by wild-type p53,(22) and promoter methylation at the p53 regulatory element causes gene silencing resistant to p53.(23) Thus, it is important to analyze the relationship between IGFBP-3 promoter methylation and expression of IGF-IR, its ligands and p53 in BTC.
The insulin receptor (InsR) is also a key component of the IGF system. InsR activation leads cell proliferation in addition to glucose metabolism. In addition to insulin, InsR can bind IGF-II and initiate mitogenic signaling.(24) IGF-IR and InsR can form hybrid receptors that bind IGF at physiologic concentrations. InsR and IGF-IR/InsR hybrid receptors might also be involved in cancer biology as both insulin and IGF-I contribute to the development and progression of adenomatous polyps.(25)
We have reported that matrix metalloproteinase-7 (MMP-7, matrilysin) plays a key role in the progression of BTC.(26) Active MMP-7 is correlated with depth of invasion and advanced stage and downregulation of MMP-7 expression by siRNA results in a significant decrease in vitro invasiveness. Matrilysin is distinguished from other MMP by several unique characteristics: broad spectrum of proteolytic activity; ability to activate other MMP; and production by cancer cells but not stromal cells.(27,28) Moreover, we have reported a positive feedback loop between the IGF/IGF-IR axis and matrilysin in the progression and invasiveness of gastrointestinal cancers.(13)
Several possible approaches to blocking IGF-IR signaling have been reported. Humanized mAbs are available for IGF-IR,(29,30) and some are in clinical trials. TKI for IGF-IR have been developed, including NVP-AEW541.(31) The orally available compound BMS-536924, 1H-(benzimidazol-2-yl)-1H-pyridin-2-one, is a novel TKI for IGF-IR/InsR.(32,33) We have also constructed two dominant negative inhibitors for IGF-IR (IGF-IR/dn; IGF-IR/482st and IGF-IR/950st), which are active as plasmids and recombinant adenovirus vectors in gastrointestinal malignancies.(34–37) IGF-IR/482st encodes a truncated extracellular domain of IGF-IR and, therefore, should result in a secreted form that affects neighboring cells in addition to the transduced cells (a bystander effect).
In the present study, we analyzed the IGF axis in human BTC and assessed the impact of IGF-IR blockade on growth, apoptosis induction and in vivo therapeutic efficacy in subcutaneous xenografts.
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In the current study, IGF-positivity in carcinoma cells was associated with tumor size in all types of BTC. IGF-II-positivity tended to correlate with high T-stage and advanced overall stage. IGF-IR-positivity was seen immunohistochemically in over 60% of patients, and was associated with advanced tumor stage. Moreover, IGF-II and IGF-IR expression were correlated to one another in all types of BTC and IGF-I ligand and receptor expression were associated in GBC, suggesting possible aberrant activation of IGF-IR by paracrine/autocrine loops. We found an association of p53-positivity and IGF-IR in GBC, in agreement with published data that mutated p53 upregulates IGF-IR expression. Thus, the IGF-IR axis might contribute to the aggressive phenotype of tumor cells, resulting in the progression of BTC.
Hypermethylation of the IGFBP-3 promoter was detected in approximately 40% of the BTC studied. Thus, the activities of IGF might be upregulated, at least in part, by epigenetic silencing of IGFBP-3 in these cancers. IGFBP-3 methylation was detected more frequently in p53-negative tumors than in p53-positive tumors, suggesting that IGFBP-3 methylation might be more important in p53 wild-type tumors than in p53 mutated tumors, which might have downregulated IGFBP-3 through other mechanisms.
Matrilysin is revealed to play a key role in the development of BTC.(26) Interestingly, IGF-IR and matrilysin were related to each other in all types of BTC. IGFBP activity can be modulated by IGFBP proteases, and there are at least three classes of such proteases: cathepsins, kallikreins and MMP.(39) MMP-7 cleaves all IGFBP and thus activates IGF-IR signaling.(39,40) The results of the present study combined with previous research showing an IGF-IR/matrilysin positive feedback loop in gastrointestinal carcinoma,(13) indicate that both molecules might contribute to the progression of BTC.
Here, we used a new IGF-IR-TKI, BMS-536924, for the accurate dissection of the responsible signaling pathways. Even in low concentrations, this agent suppressed colony formation efficiency and enhanced chemotherapy-induced apoptosis in vitro. In an in vitro study, another IGF-IR-TKI, NVP-AEW541, is also reported to suppress the growth of BTC cell lines;(17) however, its effects were slightly different from our results. Its activity was lower in GBC. NVP-AEW541 dephosphorylated both IGF-IR and Akt, but not ERK. Combined with gemcitabine, NVP-AEW541 exerted synergistic effects, while the combination with 5-FU was only additive.(17) Moreover, we revealed that not only TKI but also IGF-IR/dn effectively suppressed xenograft growth in mice. These results indicate that IGF-IR blockade is a promising treatment for BTC.
A major hurdle to the targeting of IGF-IR is the close homology to InsR.(41) Therefore, it is believed that any strategy designed to block IGF-IR signaling has to have specificity for IGF-IR without significant influence on InsR signaling. In contrast, an important role for InsR in regulating IGF action, as either a hybrid or holoreceptor, has been reported.(42) Moreover, increased insulin sensitivity in breast cancer has been observed by targeting IGF-IR.(43) Agents targeting all of the receptors responsible for IGF signaling might be necessary to disrupt the malignant phenotype regulated by this growth factor receptor family. BMS-536924, a TKI potent against both IGF-IR and InsR,(32) might not only be an advantage but a prerequisite in treating cancers. In this study, although BMS-536924 blocked both IGF-IR and InsR signals, and showed marked anti-tumor effects both in vitro and in vivo, it did not affect either body weight or blood glucose concentration.
Therefore, there are two opposing strategies for blockade IGF-IR signaling. One is to avoid adverse effects by shutting down only IGF-IR signaling without influencing InsR signaling. Such selective IGF-IR blockade can be achieved by IGF-IR-mAb or IGF-IR/dn, for prevention of recurrence or maintenance of remission. The other approach is to achieve maximum anti-tumor effects by blocking both receptors simultaneously, using BMS-536924. This latter approach might be significantly more effective, but potentially also more toxic than the former.
Meanwhile, several mechanisms for resistance to BMS-536924 have been reported. Although IGF and IGF-IR are highly expressed in BMS-536924-sensitive sarcoma cell lines, IGFBP-3 and IGFBP-6 are highly expressed in primary BMS-536924-resistant cell lines.(44) IGFBP are elevated 7–15-fold in cells with acquired resistance to BMS-536924 compared with parental sensitive cells. Overexpression of epidermal growth factor receptor (EGFR) and its ligands in the resistant cells might represent another mechanism for resistence.(44)
Many breast cancer tumors that achieve an initial response to trastuzumab ultimately acquire resistance to it. One mechanism of resistance is overexpression of IGF-IR(45) and another is the formation of IGF-IR/Her2 heterodimers.(46) These data suggested that IGF-IR blockade might be specifically effective for trastuzumab resistant tumors. As four cholangiocarcinoma cell lines are reported to express EGFR, HGFR and IGF-IR,(18) there are several potentially crosstalking signals between the IGF-IR and other receptors. Dual targeting TKI or combination strategies of IGF-IR inhibitors with other targeted therapies might achieve improved patient outcomes.(47)
In this study, we demonstrated that IGF-IR might be a marker of advanced disease and that the IGF/IGF-IR axis might contribute to a particularly aggressive phenotype in BTC (Fig. 5). IGF-IR blockade with BMS-536924 or IGF-IR/dn suppresses tumorigenicity and tumor survival both in vitro and in animal models. In addition, BMS-536924 has the advantage of being orally bioavailable. This study thus validates IGF-IR as a therapeutic target in human biliary tract malignancies and suggests that BMS-536924 might be a promising anticancer therapeutic for this disease.
Figure 5. Insulin-like growth factor (IGF)/IGF-I receptor (IGF-IR) axis in the progression of biliary tract carcinomas. Aberrant activation of IGF-IR is suggested by overexpressions of IGF ligands and receptor in tumor cells, simultaneously (IGF/IGF-IR autocrine or paracrine loops). Moreover, mutant p53 or IGF binding protein (IGFBP)-3 promoter hypermethylation reduced expressions of IGFBP-3, and IGF-IR signals upregulated matrilysin expression, which lysed IGFBP-3 (IGF-IR/matrilysin positive-feedback), which lead much free-form of IGF ligands. Strategies of targeting IGF-IR, BMS-536924 and dominant negative IGF-IR could inhibit tumor progression of biliary tract carcinomas.
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