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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Materials, cell lines, mice and tissue samples.  Anti-Akt1(c-20), anti-ERK1(K-23), anti-phospho-ERK1(E-4), ant-IGF-I(G-17) and anti-IGF-IRβ(C20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-phospho-Akt(Ser473) and anti-phospho-p44/42-MAPK(Thy202/Tyr204) were obtained from Cell-Signaling Technology (Beverly, MA, USA). Cisplatin and 5-fluorouracil (5-FU) were purchased from Sigma (St. Louis, MO, USA). Recombinant human IGF-I was purchased from R&D systems (Minneapolis, MN, USA). Human GBC cell lines TGBC-1TKB, TGBC-2TKB and TGBC-14TKB, and bile duct cancer cell lines TFK-1, HuH-28, MEC and TKKK were obtained from Riken Bioresource Center Cell Bank (Tsukuba, Japan). Cells were cultured in RPMI1640 or DMEM supplemented with 5–10% fetal bovine serum. Specific-pathogen-free female BALB/cAnNCrj-nu mice, 6-weeks-old, were purchased from Charles River (Yokohama, Japan). Mice were cared for and used according to our university’s guidelines.

Formalin-fixed, paraffin-embedded sections of 80 BTC (30 GBC, 30 extrahepatic bile duct carcinomas [BDC] and 20 carcinomas of the ampulla of Vater [CAV]) were used for immunohistochemical staining. All tumor specimens were obtained from patients who had undergone surgical treatment at Sapporo Medical University Hospital and affiliated hospitals. Sections containing the most invasive part of each tumor were used. Specimens for real-time PCR were immediately frozen in liquid nitrogen at the time of surgery and stored at −80°C. Histopathological features of the specimens were classified according to the seventh edition of the TNM classification system of the International Union Against Cancer. Informed consent was obtained from each subject. This study was approved by the Institutional Review Board of our university (Institutional Review Board approval number 22-135) and written informed consent was obtained from each subject.

BMS-536924 was kindly provided by Bristol-Myers Squibb (New York, NY, USA). Stock solution was prepared in DMSO and stored at −20°C. For oral administration to rodents, BMS-536924 was dissolved in a mixture of polyethylene glycol 400 (PEG400/water, 4:1 vol/vol) facilitated by stirring through the duration of dosing.

Immunohistochemical analysis.  Sections (5 μm) from formalin-fixed, paraffin-embedded tumor xenografts were prepared. After deparaffinization, endogenous peroxidase activity was blocked. Antibodies were applied after blocking with normal goat serum. Sections were incubated with the anti-rabbit secondary antibody (Santa Cruz Biotechnology) and a streptavidin-HRP followed by exposure to the diaminobenzidine tetrahydrochloride substrate (Dako, Glostrup, Denmark). The sections were counterstained in Mayer’s hematoxylin and mounted. Immunostaining signals were scored by two independent observers. Semiquantitative scores were given as the score of the percentage of positive cells plus the score of the staining intensity. The scoring criteria of the percentage of positive cells were as follows: score 0, 0–5% positive cancer cells; score 1, 6–25%; score 2, 26–50%; score 3, 51–75%; and score 4, 76–100% positive. The intensity score was given as follows: score 0, no staining; score 1, weak/equivocal; score 2, moderate; and score 3, strong staining. The final scores were from 0 to 7 and 4 or more was considered positive.

Quantitative DNA methylation analysis of insulin-like growth factor binding protein-3 by real-time PCR (MethyLight assay).  Sodium bisulfite treatment of genomic DNA and MethyLight assay were performed as described previously.(21,38) Primer sequences were 5′-GTTTCGGGCGTGAGTACGA-3′ and 5′-GAATCGACGCAAACACGACTAC-3′ for IGFBP-3 and 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′ and 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′ for β-actin. Probe sequences were 6FAM-5′-TCGGTTGTTTAGGGCGAAGTACGGG-3′-BHQ1 for IGFBP-3 and 6FAM-5′-CCAACACACAATAACAAACACA-3′-BHQ-1 for β-actin.

A percentage of methylated reference (i.e. degree of methylation) cutoff value of 4 was based on previously validated data.(38)

Western blotting.  Cells were cultured in serum-free medium for 24 h then stimulated with 20 ng/mL IGF-I or 10 nM insulin. Cell lysates were prepared as described previously.(34) Equal aliquots of lysates (100 μg) were separated by 4–20% SDS-PAGE and immunoblotted onto polyvinylidene Hybond-P membrane (Amersham, Arlington Heights, IL, USA). Analysis was performed using the indicated antibodies, and bands were visualized by ECL (Amersham).

In vitro cell growth.  Four thousand cells were seeded into a 96-well plate and each was treated with several concentrations of BMS-536924. Cell growth was measured using WST-1 reagent (Roche, Basel, Switzerland), as described previously.(37)

Colony forming activity.  Cells (3 × 103/plate) were seeded onto 60 mm culture plates and incubated for 24 h. The cells were then treated with BMS-536924 and were incubated for 14 days. After air-drying, cells were fixed with methanol and stained with Giemsa solution. Colonies containing 50 cells or more were counted.

Assessment for apoptosis.  Caspase-3 colorimetric protease assay was performed following the manufacturer’s protocol (MBL, Nagoya, Japan). In brief, the caspase-3 activity of lysates (100 μg) was measured by colorimetric reaction at 400 nm. TUNEL assays were performed with an in situ apoptosis detection kit (Takara, Kyoto, Japan) following the manufacturer’s protocol.

In vivo therapeutic efficacy in established tumors.  One × 106 TGBC-1TKB were subcutaneously injected into nude mice. After tumors were palpable (24 days after inoculation), animals were treated orally once daily for 2 weeks, either with BMS-536924 (70 mg/kg) or control. Mice were killed when tumors reached 2 cm in size or they developed clinically evident symptoms. Tumor diameters were serially measured with calipers and tumor volume was calculated using the formula: tumor volume (mm3) = (width2 × length)/2.

After TGBC-1TKB tumors became palpable, adenovirus-IGF-IR/dn or adenovirus-lacZ were injected intratumorally for five successive days. Mice were killed on the 47th day.

Statistical analysis.  The association between immunohistochemical expression and clinicopathological characteristics were assessed using the Mann–Whitney’s U-test and Fisher’s exact test. The results are presented as means ± SE for each sample. The statistical significance of differences was determined by Student’s two tailed t-test in two groups and done by one-way anova in multiple groups, and by two-factor factorial anova. P-values of <0.05 were considered to indicate statistical significance.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

Overexpression of insulin-like growth factor-axis in biliary tract carcinomas tissues. Figure 1 shows representative results of immunohistochemistry for IGF-I, IGF-II, IGF-IR, matrilysin and p53 in BTC. IGF-I-positivity was 60% in total, 57% in GBC, 60% in BDC and 65% in CAV. IGF-I-positivity was significantly correlated with tumor size (Table 1). IGF-II-positivity was 50% in total, 47% in GBC, 53% in BDC and 50% in CAV. IGF-II-positivity was significantly correlated with tumor size, advanced T-stage in GBC, and advanced tumor stage in both BDC and CAV. IGF-IR-positivity was 69% in total, 63% in GBC, 77% in BDC and 65% in CAV. IGF-IR-positivity was significantly correlated with advanced tumor stage in all types and with advanced T-stage in GBC.

image

Figure 1.  Immunohistochemical expression of insulin-like growth factor (IGF)-I (A–D), IGF-II (E–H), IGF-I receptor (I–L), matrilysin (M–P) and p53 (Q–T) in gallbladder carcinoma (A, E, I, M, Q), in bile duct carcinomas (B, D, F, H, J, L, N, P, R, T) and in carcinomas of the ampulla of Vater (C, G, K, O, S). Representative pictures show that IGF-I was positively stained in most tumors (A–C) but not all (D). IGF-II was positively immunostained in (E–G) but was negative in (H). IGF-IR was positive in most cancers (I–K) but was negative in some (L). Matrilysin was expressed in most cancers (M–O) but not all (P). Overexpressed p53 was detected in most tumors (Q–S) but not in (T).

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Table 1.   Expression of insulin-like growth factor (IGF) axis in biliary tract cancer tissues
 NIGF-IPIGF-IIPIGF-I receptorP
+ + + 
1713 1416 1911 
(a) Gallbladder cancer tissues
Sex
 Male171070.921†890.749†1160.713†
 Female13766785
Size
 <25 mm187110.019†5130.014†990.069†
 >25 mm1210293102
pT
 pT1a3120.260‡120.038‡120.028‡
 pT1b1010101
 pT28532644
 pT3169797124
 pT42202020
pN
 pN015780.231†5100.136†780.064†
 pN11510596123
pStage
 pStage I3120.140‡120.080‡120.001‡
 pStage II5231414
 pStage IIIA7433434
 pStage IIIB138576121
 pStage IVA2202020
Matrilysin
 −15780.231†5100.136†690.010†
 +1510596132
p53
 −17980.461†6110.145†890.040†
 +138585112
IGF-I
 −13   3100.028†580.018†
 +17  116143
IGF-II
 −16      790.021†
 +14    122
 NIGF-IPIGF-IIPIGF-I receptorP
+ + + 
1812 1614 237 
(b) Extrahepatic bile duct cancer tissues
Sex
 Male231490.734†12110.581†1850.814†
 Female7434352
Size
 <25 mm11380.008†290.005†740.200†
 >25 mm19154145163
pT
 pT18350.224‡260.059‡530.100‡
 pT25413232
 pT3159696132
 pT42202020
pN
 pN016880.206†6100.067†1060.061†
 pN114104104131
pStage
 pStage I6240.115‡240.032‡240.006‡
 pStage II1610679133
 pStage III6425160
 pStage IV2202020
Matrilysin
 −6420.545†240.261†240.016†
 +2414101410213
p53
 −161060.940†790.225†1150.256†
 +148695122
IGF-I
 −12   390.014†750.068†
 +18   135162
IGF-II
 −14      860.025†
 +16    151
 NIGF-IPIGF-IIPIGF-I receptorP
+ + + 
137 1010 137 
  1. †Chi-square. ‡Mann–Whitney’s U-test. N, number.

(c) Carcinoma tissues of the ampulla of Vater
Sex
 Male10730.500†640.328†730.500†
 Female10644664
Size
 <25 mm13670.022†490.029†760.177†
 >25 mm7706161
pT
 pT15230.167‡140.093‡140.167‡
 pT26423333
 pT38625362
 pT41101010
pN
 pN010640.500†460.328†550.175†
 pN110736482
pStage
 pStage IA3120.137‡120.049‡030.002‡
 pStage IB4221313
 pStage IIA4311322
 pStage IIB8626280
 pStage IV1101010
Matrilysin
 −6420.664†240.314†150.007†
 +149586122
p53
 −12840.608†570.325†750.392†
 +8535362
IGF−I
 −7   250.175†340.151†
 +13  85103
IGF−II
 −10      460.029†
 +10    91

Insulin-like growth factor-I-positivity was significantly correlated with IGF-II-positivity in GBC and BDC (P = 0.028 and 0.014, respectively). IGF-I-positivity was significantly correlated with IGF-IR-positivity in GBC and tended to be associated with the receptor in BDC (P = 0.018 and 0.068, respectively). IGF-II-positivity was significantly correlated with IGF-IR-positivity in GBC, BDC and CAV (P = 0.021, 0.025 and 0.029, respectively). IGF-IR-positivity was significantly correlated with matrilysin positivity in GBC, BDC and CAV (P = 0.010, 0.016 and 0.007, respectively). IGF-IR-positivity was significantly correlated with p53-positivity in GBC (P = 0.040).

The results indicated that the IGF axis might play an important role in tumor development of human BTC and that IGF-IR might interact with p53 in GBC and with matrilysin in BTC.

Hypermethylation of the insulin-like growth factor binding protein-3 promoter in biliary tract carcinomas tissues.  Hypermethylation of the IGFBP-3 promoter was observed in 41% of BTC, 43% in GBC, 37% in BDC and 45% in CAV (Table 2). IGFBP-3 methylation was not correlated with clinicopathological characteristics or expression of the IGF-axis. IGFBP-3 hypermethylation was detected more frequently in p53-negative tumors than in p53-positive tumors in GBC, BDC and CAV (P = 0.016, 0.021 and 0.025, respectively). Preliminary data indicates that IGFBP-3 hypermethylation is inversely associated with expression of IGFBP-3 mRNA (data not shown). Then, we wanted to assess the possibility of IGF-IR as a molecular target in human BTC.

Table 2.   Methylation of the IGFBP-3 promoter in biliary tract cancer tissues
 HypermethylatedHypomethylatedFisher’s exact test
Gallbladder cancer tissues13 (43%)17 (57%) 
 p53(+)211= 0.009
 p53(−)116
Extrahepatic bile duct cancer tissues11 (37%)19 (63%) 
 p53(+)212= 0.021
 p53(−)97
Carcinoma tissues of the ampulla of Vater9 (45%)11 (55%) 
 p53(+)17P = 0.025
 p53(−)84

Blockade of signal transduction.  All seven human BTC cell lines expressed IGF-IR detected by real-time PCR (data not shown). To investigate the effect of BMS-536924 on IGF/receptor signaling, we examined three BTC cell lines. First, we evaluated BMS-536924 activity in TGBC-1TKB by Western blotting. One μM BMS-536924 blocked IGF-I-induced phosphorylation of IGF-IR completely (Fig. 2A). This TKI blocked both basal phosphorylation of Akt-1 and ERK and that induced by IGF-I, in a dose-dependent manner. Similarly, in both 2TKB and 14TKB cells, BMS-536924 reduced ligand-induced IGF-IR autophosphorylation and phosphorylation of Akt-1 and ERK with dose dependency.

image

Figure 2.  BMS-536924 blocked insulin-like growth factor (IGF)-I/insulin signals of three biliary tract carcinomas cell lines by Western blotting. (A) BMS-536924 reduced IGF-I induced IGF-IR autophosphorylation of all cell lines. BMS-536924 blocked both Akt and ERK signals. (B) BMS-536924 also blocked insulin-induced InsR autophosporylation and the downstream signals. Ins, insulin; pIGF-IR, phosphorylated IGF-IR; tIGF-IR, total IGF-IR; pAkt, phosphorylated Akt-1; tAkt, total Akt-1; pERK, phosphorylated ERK-1/-2; tERK, total ERK-1/-2; pInsR, phosphorylated InsR; tInsR, total InsR.

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In both 1TKB and 2TKB (Fig. 2B), 1 μM BMS-536924 also reduced the insulin-induced phosphorylation of InsR and its downstream signal activity with dose dependency. Thus, BMS-536924 effectively interrupted both IGF-I and insulin induced signals in BTC.

Reduction of cell growth in vitro.  BMS-536924 reduced the growth on plastic of all BTC cells in a dose-dependent manner, as analyzed by the WST-1 assay (Fig. 3A). Moreover, BMS-536924 dramatically reduced the in vitro tumorigenicity of all cells dose dependently as assessed by colony formation assay (Fig. 3B). These results indicate that BMS-536924 effectively blocks the carcinogenicity and proliferation of BTC.

image

Figure 3.  BMS-536924 reduces in vitro growth and colony formation and enhances the effect of chemotherapy of BTC. (A) BMS-536924 reduced growth of three cell lines on plastic with dose dependency, detected by WST-1 assay. Without circle, control; open circle, 1 μM BMS-536924; closed circle, 10 μM BMS-536924. (B) Colony formation assay shows that BMS-536924 reduces in vitro tumorigenicity, in a dose-dependent manner. (C, D) In TGBC-1TKB (C) and TGBC-2TKB (D), caspase-3 assay showed that BMS-536924 enhanced chemotherapy-induced apoptosis (analyzed by two-factor factorial anova). bsl00084, control; bsl00066, 1 μM BMS-536924; ○, 5 μM BMS-536924; •, 10 μM BMS-536924.

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Effect on survival.  The effects of BMS-536924 in combination with chemotherapy were assessed. All of the tested drugs, 5-FU, gemcitabine and cisplatin, induced caspase-3 activity in TGBC-1TKB (Fig. 3C). BMS-536924 enhanced 5-FU-induced apoptosis synergistically and both gemcitabine-induced and cisplatin-induced apoptosis additively. In TGBC-2TKB, BMS-536924 upregulated drug-induced apoptosis synergistically for all three chemotherapies (Fig. 3D). These results suggest that BMS-536924 enhances the effects of chemotherapy.

BMS-536924 suppressed tumors in mice.  To assess the effect of this drug on cancer in vivo, TGBC-1TKB cells were inoculated into nude mice and allowed to form evident tumors. Oral administration of 70 mg/kg BMS-536924 significantly inhibited tumor growth in mice (Fig. 4A).

image

Figure 4.  The effect of BMS-536924 on TGBC-1TKB established subcutaneous tumors in nude mice. (A) 1 × 106 1TKB cells were injected subcutaneously on day −24. After tumors were palpable (day 0), animals were treated orally once daily for 2 weeks, either with BMS-536924 (closed circle) or with control (open circle) (n = 15 in each group). (B) TUNEL assay showed that BMS-536924 upregulated apoptosis. (C) There is no difference in either murine weight or serum glucose concentration at death. NS, no significant difference.

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To evaluate the effect of BMS-536924 on apoptosis induction in tumors, TUNEL assays were performed (Fig. 4B). BMS-536924 upregulated apoptosis in xenografts tumors. The treatment did not have adverse effects on the body weight of mice or the glucose levels at the time of death (Fig. 4C), suggesting tolerable toxicity.

Effect of dominant negative insulin-like growth factor-I receptor on biliary tract carcinomas in mice.  To assess the effect of IGF-IR/482st on BTC in vivo, TGBC-1TKB cells were inoculated in nude mice and allowed to form evident tumors. Intra-tumoral injection of adenovirus-IGF-IR/482st for five successive days markedly suppressed tumor growth without influencing body weight or glucose concentration at death (Table 3). These results suggest that IGF-IR might be a candidate molecular target for human BTC.

Table 3.   Effect of dominant negative insulin-like growth factor-I reactor on TGBC1TKB xenografts on nude mice
 Control (mean ± SE)IGF-IR/dn (mean ± SE) 
  1. NS, no significant difference.

Tumor volume (mm3)2158.9 ± 369.9765.8 ± 435.2P = 0.0349
Murine body weight (g)23.3 ± 0.621.7 ± 0.7NS
Glucose (mg/dL)164.7 ± 9.9150.0 ± 23.8NS

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

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.

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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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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References

This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology and from the Ministry of Health, Labour and Welfare, Japan.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References