id1-inducedigf-ii andits autocrine/endocrine promotionof ... · id1-inducedigf-ii andits...

Cancer Therapy: Preclinical

Id1-Induced IGF-II and Its Autocrine/Endocrine Promotion ofEsophageal Cancer Progression and Chemoresistance—Implications for IGF-II and IGF-IR–Targeted Therapy

Bin Li1,2, Sai Wah Tsao1,2, Kwok Wah Chan2,3, Dale L. Ludwig5, Ruslan Novosyadlyy5, Yuk Yin Li1,Qing Yu He4, and Annie L.M. Cheung1,2

AbstractPurpose: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.

ExperimentalDesign:Antibody array-based screeningwas used to identify differentially secreted growth

factors from Id1-overexpressing esophageal cancer cells. In vitro and in vivo assayswere 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 overexpres-

sion 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 mono-

clonal 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 promotemetastasis of circulating cancer cells. Targeting IGF-II and IGF-IR had significant suppressive

effects on tumor growth andmetastasis inmice. 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.

IntroductionTumor-secreted growth factors can affect tumor micro-

environment (1, 2), as well as stimulate the cancer cellsthemselves to proliferate and develop a more malignantphenotype in an autocrine manner (3–6). A variety ofgrowth factors and cytokines exert their effects on cancercells by activating the membrane-associated phosphoinosi-tide 3-kinase (PI3K)/AKT signaling pathway, which regu-

lates a wide spectrum of cellular functions, including pro-liferation, survival, angiogenesis, invasion, and metastasis.We previously reported that the PI3K/AKT signaling path-way can be induced by ectopic expression of Id1 (inhibitorof differentiation or DNA binding), leading to increasedproliferation and survival of esophageal cancer cells (7). TheId1 protein is a helix-loop-helix protein (8) found to beoverexpressed in many types of human cancer, includingesophageal squamous cell carcinoma (ESCC; ref. 9). Ourtissuemicroarray (TMA) study established that dysregulatedId1 expression is associatedwith tumor invasion andmetas-tasis in ESCC (10).

The mechanism by which Id1 stimulates the PI3K/AKTpathway is not clear. In this study, using growth factorantibody array, we provided the first evidence that Id1increases the expression and secretionof insulin-like growthfactor-II (IGF-II) in a variety of human cancer types. Moreimportantly, cancer cell-secreted IGF-II was found to medi-ate the effect of Id1 on the PI3K/AKT pathway. A series of invitro and in vivo experiments were performed to study theautocrine and endocrine function of IGF-II in promotingESCC growth and progression.

Authors' Affiliations: 1Department of Anatomy, 2Centre for CancerResearch; 3Department of Pathology, Li Ka Shing Faculty of Medicine,The University of Hong Kong, Pokfulam, Hong Kong SAR; 4Institute of LifeandHealthEngineering, JinanUniversity,Guangzhou,China; and 5ImCloneSystemsCorporation, awholly ownedsubsidiary of Eli Lilly &Co,NewYork,New York

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Annie L.M. Cheung, Department of Anatomy, LiKa Shing Faculty of Medicine, The University of Hong Kong, 21 SassoonRoad, Pokfulam, Hong Kong SAR, China. Phone: 852-2819-9293; Fax:852-2817-0857; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-2735

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 2651

on June 18, 2020. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst March 5, 2014; DOI: 10.1158/1078-0432.CCR-13-2735

http://clincancerres.aacrjournals.org/

The IGF system is an attractive target for cancer therapy(11, 12). Elevated expressions of insulin-like growth factor-type I receptor (IGF-IR), and its ligands IGF-I and IGF-II, hadbeen reported in human cancers, including ESCC, and werefound to be correlated with advanced tumor stage andmetastasis (13, 14), but treatment efficacy of IGF-IR block-ade in ESCC remains to be evaluated. The feasibility oftargeting the IGF-II-IGF-IR axis as potential treatmentoptions for ESCC was investigated in this study in vitro, aswell as in vivo using tumorigenesis and cancer metastasismouse models. Because treatment failure and cancer recur-rence may be attributed to chemoresistance during conven-tional chemotherapy, we also examined whether IGF-IRblockade can enhance the chemosensitivity of ESCC cells inanimal models.

Materials and MethodsCell lines and transfection

Human ESCC cell lines HKESC-3 (15), KYSE150,KYSE270, KYSE410 (16), and T.Tn (17) were used in thisstudy. The Id1-overexpressing cell lines HKESC-3-Id1,KYSE150-Id1, and T.Tn-Id1 and their correspondingcontrol cell lines HKESC-3-CON, KYSE150-CON, and T.Tn-CON were generated previously (18, 19). Details onreagents and plasmids used in gene overexpression andknockdown, and in generating KYSE150-Luc cell line, aregiven in Supplementary Materials and Methods.

Growth factor antibody arrayConditioned culture media from HKESC-3-Id1 and

HKESC-3-CON cells were analyzed using a human growthfactor antibody array that detects 41 growth factors (CatalogNo. AAH-GF-1, RayBiotech) according to the manufac-

turer’s instructions. The immunosignals were detected withthe ECL Plus system (Amersham) and exposed to BioMaxLight Film (Kodak).

In vitroELISA, quantitative real-timePCR,Westernblotanalysis, MTT, TUNEL, cell migration, invasion, andsoft agar assays

The concentration of human IGF-II in the culture medi-umandmouse serumwasmeasured using aNon-ExtractionIGF-II ELISA Kit (Diagnostic Systems Laboratories) accord-ing to the manufacturer’s instruction. Anti-IGF-II (R&DSystems) was used in immunoneutralization experiments.Details of assays were described in our previous studies (19,20). Western blot bands were quantified using ImageJsoftware. See Supplementary Materials and Methods fordetails of antibodies and real-time PCR.

In vivo tumorigenesis and metastasis experimentsAnimal experiments were conducted and subcutaneous

tumor volume and metastatic tumor nodules in the lungsof mice were quantified as previously described (19, 20).Details are provided in Supplementary Materials andMethods.

ImmunohistochemistryA TMA containing 35 cases of human ESCC in duplicated

cores (Catalog no. ES802, Biomax), sections of tumorxenografts and mouse lung tissues were processed forimmunohistochemistry and TUNEL (terminal deoxynu-cleotidyl transferase–mediated dUTP nick end labeling)assay. Staining intensity (for Id1, IGF-II, and phosphory-lated AKT; p-AKT), Ki-67 proliferative index, TUNEL-posi-tive apoptotic index, and CD31-positive microvessel den-sity (MVD) were assessed as previously described (19, 20).See Supplementary Materials and Methods for details.

Statistical analysisThe data were expressed as themean� SD and compared

using ANOVA. Correlations of IGF-II, Id1 and p-AKT in theTMA were assessed using Spearman rank correlation coef-ficient. P values < 0.05 were deemed significant. All in vitroexperiments were repeated at least three times.

ResultsCancer cell-secreted growth factors mediate theactivation of the PI3K/AKT signaling pathway and theinduction of cell proliferation by Id1

We investigated the involvement of autocrine mechan-isms in the inductionof PI3K/AKTby Id1.We found that theHKESC-3-Id1 conditioned medium (CM) induced proteinexpression of p-AKT and cell proliferation in the parentalHKESC-3 cells; the AKT activation was attenuated whenthe proteins in the conditioned medium were denatured(Fig. 1A and B). These results suggest that Id1-overexpres-sing ESCC cells had increased secretion of growth factor(s)that served as autocrine stimulator of the PI3K/AKT path-way and cancer progression.

Translational RelevanceEsophageal cancer ranks as the sixth most frequent

cause of cancer death in the world. There is an urgentneed for a better understanding of the underlyingmechanisms to develop more effective therapeuticstrategies for this highly lethal disease. This studyuncovers an autocrine/endocrine link between Id1 andthe PI3K/AKT pathway involving IGF-II and IGF-IR,which promotes esophageal cancer progression andmetastasis. Cixutumumab is currently investigated inclinical trials for the treatment of non–small cell lungcancer and hepatocellular carcinoma. Here, we showthat systemic treatment with cixutumumab can signif-icantly inhibit esophageal cancer growth and metas-tasis, as well as sensitize tumor xenografts to fluoro-uracil and cisplatin treatment in vivo. The present studytherefore provides important preclinical data support-ing the clinical application of cixutumumab systemictherapy both as a single agent and in combinationwith cytotoxic chemotherapeutic drugs.

Li et al.

Clin Cancer Res; 20(10) May 15, 2014 Clinical Cancer Research2652




Id1 induces IGF-II expression and secretionWeperformed antibody array-based screening to identify

growth factors that were differentially secreted by Id1-over-expressing ESCC cells. The results showed a higher concen-tration of IGF-II in the HKESC-3-Id1 CM than in theHKESC-3-CON CM (Fig. 1C and Supplementary Fig. S1).

Western blot analysis of the cell lysates and conditionedmedium showed increased IGF-II expression and secretionin three different Id1-overexpressing ESCC cell lines (Fig.1D), and this effect was successfully abrogated by shIGF-II(Fig. 1E and F). We also performed transient transfectionexperiments to confirm that Id1 induced the production

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Figure 1. Identification of the growth factors secreted from Id1-overexpressing cells. A, the HKESC-3 cells treated with the conditioned medium (CM)from HKESC-3-Id1 (with and without heat inactivation), and HKESC-3-CON cells for 1 hour were harvested for Western blot analysis. B, HKESC-3cells were incubated for up to 72 hours in conditioned medium from HKESC-3-CON and HKESC-3-Id1, and their growth rates determined using MTT assay.C, growth factor antibody array was incubated with the serum-free conditioned medium from HKESC-3-Id1 and HKESC-3-CON cells. The representativeIGF-II and internal control (IgG) spots showed higher concentration of IGF-II in the HKESC-3-Id1 conditioned medium. D, Western blot analysis ofIGF-II in the cell lysates and conditioned medium obtained from three Id1-overexpressing ESCC cell lines and the respective vector control cell lines whichhad been kept in serum-free medium for 24 hours. E and F, KYSE150-CON-shCON, KYSE150-Id1-shCON, and KYSE150-Id1-shIGF-II cells werecompared for the expression level of IGF-II in cell lysate (E) and the conditioned medium (F) by Western blot and ELISA, respectively. G, Western blotanalysis of IGF-II in cell lysates and conditionedmediumof KYSE150 cells transfectedwith different doses of pcDNA3-Id1 expression vector or siRNA againstId1. ��, P < 0.01; �� P < 0.001 compared with control.

Id1-IGF-II-IGF-IR-AKT Signaling and Targeted Cancer Therapy

www.aacrjournals.org Clin Cancer Res; 20(10) May 15, 2014 2653




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and secretion of IGF-II in a dose-dependent manner, andthat siRNA knockdown of Id1 reduced IGF-II expression(Fig. 1G). To exclude the off-target effect of siRNA, a secondRNA interference sequence was used to confirm the effectsof Id1 knockdown on IGF-II expression (SupplementaryFig. S2). The induction of IGF-II by Id1 was further con-firmed using a panel of human cancer cell lines, includinggastric, colon, liver, and prostate cancer (SupplementaryFig. S3). Furthermore, manipulation of Id1 expression inESCC cells via overexpression or short hairpin RNA knock-down resulted in corresponding changes in expression ofIGF-II mRNA (Supplementary Fig. S4), which indicate thatId1 regulates IGF-II at mRNA level.

IGF-II expression is positively correlated with Id1 andp-AKT expressions in human esophageal tumorsImmunohistochemical results in TMA showed that

among the 21of 35 cancer specimens showingupregulation(i.e., moderate to strong immunostaining) of IGF-II, 15 and14 cases had upregulated Id1 and p-AKT expression, respec-tively (Supplementary Fig. S5 andSupplementary Table S1).Spearman rank correlation coefficient analysis indicated apositive correlation between IGF-II and Id1 expression (r¼0.461,P<0.005), andbetween IGF-II andp-AKT (r¼0.523,P < 0.005) in the ESCC specimens. These results suggest thatIGF-II expression is associated with upregulation of Id1 andp-AKT in human esophageal cancer.

Autocrine IGF-II mediates the tumor- and metastasis-promoting functions of Id1 through activation of thePI3K/AKT pathwayTo determine the significance and oncogenic role of Id1-

induced IGF-II in ESCC cells, neutralizing antibody againstIGF-II was added to the conditionedmedium fromHKESC-3-Id1 cells to block the functions of IGF-II. The resultsshowed that this treatment not only attenuated the sti-mulation of the PI3K/AKT pathway (Fig. 2A), but alsodecreased the proliferative (Fig. 2B), migratory (Fig. 2C),and survival (Fig. 2D) capabilities of HKESC-3-Id1 cells.Wealso studied the effects of cancer cell-derived IGF-II onanchorage-independent growth using soft agar assay. Asshown in Fig. 2E, the KYSE150 cancer cells were found tohave significantly enhanced colony formation ability whensuspended in a mixture of agar and KYSE150-Id1-shCONconditioned medium and plated on top of the base agarlayer.Moreover, in the cell invasion assay (Fig. 2F), parentalT.Tn and KYSE150 cells showed higher invasive abilitywhen suspended in the conditioned media of correspond-

ing Id1-overexpressing transfectants (i.e., T.Tn-Id1 andKYSE150-Id1-shCON). The effects on colony formationand cell invasion were abolished by knockdown of IGF-IIin Id1-overexpressing cells using shIGF-II or neutralizationof IGF-II in the conditioned medium (Fig. 2E and F).Furthermore, we found that treatmentwith the conditionedmedium from KYSE150 cells, which had higher endoge-nous Id1 expression (see Supplementary Fig. S6 showingendogenous Id1 and IGF-II expression levels in ESCC celllines), enhanced the ability of HKESC-3 cells to proliferate,migrate, and survive; the effects were abolished whenendogenous Id1 expression in KYSE150 cells was inhibitedby siRNA against Id1 (Supplementary Fig. S7).

The autocrine function of Id1-induced IGF-II in ESCCwas also investigated in vivo. As shown in Fig. 3A, KYSE150-Id1-shCON cells produced significantly larger tumors thanthe KYSE150-CON-shCON cells in nude mice (742.8 �142.9mm3vs. 516.5�101.8mm3onday 35;P<0.05), butthe stimulatory effect of Id1 on tumor growth was counter-acted in the IGF-II knockdown group. Comparison of thetumor xenografts indicated that the larger tumor size ofthe KYSE150-Id1-shCON group was due to increased cellproliferation and reduced apoptosis (Fig. 3B and Supple-mentary Fig. S8). Western blot analysis of the tumorxenografts showed that these changes were associated withthe higher expression levels of IGF-II and p-AKT in theKYSE150-Id1-shCON tumors (Fig. 3C). Moreover, themice bearing KYSE150-Id1-shCON xenografts had signif-icantly higher concentration of human IGF-II in the circu-lation compared with the other two groups (Fig. 3D),which suggests that IGF-II may function as an endocrinegrowth stimulator for esophageal cancer. Our results alsoshowed that shIGF-II successfully downregulated tumorand circulating IGF-II levels in vivo, thus abolishing theactivation of the PI3K/AKT pathway by Id1 (Fig. 3C andD).We also determined the contribution of IGF-II as a medi-ator of Id1-induced tumor metastasis using experimentalmetastasis assay. KYSE150-CON-shCON, KYSE150-Id1-shCON, or KYSE150-Id1-shIGF-II cells were intravenouslyinjected into nude mice through the tail vein. Six weekslater, numerous large metastatic nodules that showedpositive immunostaining for human-specific cytokeratin-8 were found in the lungs of the KYSE150-Id1-shCONgroup, but not in the KYSE150-CON-shCON andKYSE150-Id1-shIGF-II groups (Fig. 3E). These results there-fore support that IGF-II plays a significant role inmediatingthe Id1-promoted metastasis of human esophageal cancerin vivo.

Figure 2. Effects of Id1-induced IGF-II secretion on PI3K/AKT activation and on oncogenic properties of esophageal cancer cells. A–C, HKESC-3 cells weretreated with conditioned medium (CM) from HKESC-3-Id1, HKESC-3-CON, or HKESC-3-Id1 preincubated with IGF-II–neutralizing antibody for 1 hour.Western blot analysis (A), MTT assay (B), andwound-healing assay (C) showed that addition of anti-IGF-II (1 mg/mL) to theHKESC-3-Id1 conditionedmediumstrongly attenuated its stimulatory effects on the PI3K/AKT pathway, cell growth, and migration. D, HKESC-3 cells were exposed to various conditionedmedium for 12 hours, and then treatedwith 50 ng/mLof TNF-a for up to 36 hours. Note that the increase in resistance to TNF-a–induced apoptosis induced byHKESC-3-Id1 conditioned medium was attenuated by neutralization of IGF-II in the conditioned medium. E and F, ESCC cells were suspended in variousconditioned medium with or without the addition of 1 mg/mL IGF-II–neutralizing antibody. Soft agar (E) and chamber invasion assays (F) showed thatknockdown or neutralization of IGF-II attenuated the Id1-induced invasive and anchorage-independent growth abilities. �,P < 0.05; ��,P < 0.01; ��,P < 0.001compared with empty vector control.






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Figure 3. Effects of Id1-induced IGF-II on tumorigenicity and metastasis of esophageal cancer cells in nude mice. A, growth curves of subcutaneoustumors formed by KYSE150-CON-shCON, KYSE150-Id1-shCON or KYSE150-Id1-shIGF-II cells in nude mice (n ¼ 8 per group). B, Ki-67 proliferationand apoptosis indices of tumor xenografts. C, Western blot analysis confirmed elevated IGF-II protein levels in the KYSE150-Id1-shCON tumorxenografts, and successful knockdown of IGF-II in the KYSE-Id1-shIGF-II xenografts. D, serum IGF-II concentration was assessed by ELISA. E, KYSE150-CON-shCON, KYSE150-Id1-shCON, or KYSE150-Id1-shIGF-II cells were intravenously injected into nude mice through the tail vein (n ¼ 8 per group). Top,representative images of lungs harvested 6 weeks postinjection (metastatic nodules are indicated by arrows). Surface metastatic nodules in the lungswere counted, and the numbers presented separately for nodules below and above 1mm in diameter. A human-specific anti-cytokeratin-8 (CK-8)monoclonalantibody was used to distinguish human ESCC cells from the surroundingmouse pulmonary tissue (middle). Bottom, consecutive lung sections stained withhematoxylin and eosin (H&E). �, P < 0.05; ��, P < 0.01; ��, P < 0.001 compared with control group inoculated with KYSE150-CON-shCON cells.

Li et al.





Tumor-secreted IGF-II instigates growth andmetastasis of distant tumor cells in an endocrinemannerBecause the mice bearing Id1-expressing tumor xeno-

grafts showed elevated serum IGF-II (Fig. 3D), and exposureto exogenous IGF-II-containing culturemedia enhanced thein vitro tumorigenic and invasive potentials of esophagealcancer cells (Fig. 2E and F), we reasoned that, in addition toautocrine stimulation, tumor-derived IGF-II may also exertsystemic endocrine effects that facilitate tumor growth at adistant site. To test this hypothesis, we constructed an in vivoexperimental model (Fig. 4A) similar to that used by Wein-berg’s team in which more aggressive "instigator" cancercellswere injected into oneflankof the experimental animaland less aggressive "responder" cells into the contralateralflank (21). KYSE150-CON-shCON, KYSE150-Id1-shCON,and KYSE150-Id1-shIGF-II cells were subcutaneously inj-ected into the left flanks of different groups of nude mice tocompare their ability to instigate tumor formation of paren-tal KYSE150 cells inoculated subcutaneously into the rightflank 2weeks later (i.e., responder). As shown in Fig. 4B, theKYSE150-Id1-shCON group developed significantly largerresponding tumors than the KYSE150-CON-shCON group,but knockdown of IGF-II in the instigating tumor attenu-ated the growth of responding tumor to a level comparablewith that of the empty vector control group. We also foundincreased p-AKT expression (Fig. 4C), aswell as significantlyhigher Ki-67 proliferation and lower apoptotic indices inthe responding tumors of the KYSE150-Id1-shCON groupcompared with the KYSE150-Id1-shIGF-II and KYSE150-CON-shCON groups (Fig. 4D and Supplementary Fig. S9),suggesting that IGF-II secreted by primary tumors can exertendocrine influence to instigate the growthof less aggressivetumors at a distant site through activation of the PI3K/AKTsignaling pathway (Fig. 4E).To determine whether endocrine IGF-II can facilitate

the outgrowth of disseminated tumor cells, a modifiedexperimental metastasis assay was performed in whichparental KYSE150 cells were injected intravenouslyinto the tail vein of nude mice which were inoculated 2weeks earlier with KYSE150-CON-shCON, KYSE150-Id1-shCON, or KYSE150-Id1-shIGF-II subcutaneous xeno-grafts. Six weeks later, we found more metastatic nodulesin the lungs of the KYSE150-Id1-shCON group, than theother groups (Fig. 4F). These findings demonstrated thatId1-overexpressing tumors can facilitate metastatic colo-nization of disseminated cancer cells by secreting IGF-II.This aspect may have important therapeutic implicationsfor cancer treatment.

IGF-II neutralization suppresses esophageal cancergrowth in vivoTo investigate the antitumor effects of IGF-II-neutraliza-

tion on esophageal cancer, nudemice were inoculated withKYSE150 cells and given intratumoral injection of IGF-II-neutralizing antibody. As shown in Fig. 5A, treatment withIGF-II–neutralizing antibody for 15 days significantly sup-pressed tumor growth by approximately 49% without

detectable toxic effects on body weight, the lungs, liver, orkidneys (Supplementary Fig. S10A and S10B). Histologicevaluation showed that the suppressive effect of IGF-II–neutralizing antibody on the tumor growth was due todecreased cell proliferation rate and vascular supply, as wellas increased apoptosis (Supplementary Fig. S10C).

IGF-IR blockade inhibits esophageal cancer growthand metastasis in vivo

Because the biologic functions of IGF-II are mainlyexerted through IGF-IR, we next studied whether systemicblocking of IGF-IR may inhibit tumor growth in vivo.Cixutumumab, a fully human immunoglobulin G (IgG)-1 monoclonal antibody targeting human IGF-IR (22), wasinjected intraperitoneally into nude mice bearing ESCCtumor xenografts. We found a significant dose-dependentreduction of tumor volume 19 days after treatment, withdecreases of 66.4% and 76.6% for KYSE150 and KYSE270tumors, respectively, in the groups receiving 50 mg/kgcixutumumab treatment (Fig. 5B). Histologic comparisonof the tumor xenografts indicated that cixutumumabtreatment decreased the proliferation rate and enhancedapoptosis of cancer cells, as well as reduced tumor vascularsupply (Fig. 5C and Supplementary Figs. S11 and S12).Notably, Western blot and immunohistochemical analy-sis of tumor xenografts suggested that the inhibitoryeffects of cixutumumab on tumor growth were due toinactivation of the PI3K/AKT pathway (Fig. 5D and E). Theelevated expression of cleaved caspase-3 also confirmedthe increased apoptosis in the tumor cells of cixutumu-mab-treated mice (Fig. 5D). Furthermore, although cix-utumumab crossreacts with mouse IGF-IR (22), cixutu-mumab had no obvious adverse effects on the bodyweight and vital organs (Supplementary Fig. S13). Toevaluate the treatment efficacy of IGF-IR blockade onmetastasis, nude mice were intravenously injected withluciferase-expressing ESCC cells, then treated with cixutu-mumab. Bioluminescent imaging and histologic exami-nation showed marked lung metastasis in the controlgroups but significantly less metastatic activity in the lungsof the cixutumumab-treated groups (Fig. 6A–C). On thebasis of these in vivo data, we conclude that blockade ofIGF-IR with cixutumumab can significantly suppress thegrowth of tumor and metastasis.

Cixutumumab reverts acquired chemoresistance influorouracil-resistant ESCC sublines and enhances thechemosensitivity of ESCC cells in vivo

Development of drug resistance often occurs in patientstreated with conventional chemotherapeutic reagents. Tomimic the process by which patients acquire chemoresis-tance, fluorouracil (5-FU)-resistant ESCC sublines wereestablished by treating cells with increasing doses of 5-FU(up to 40 mmol/L) for over a year, and were highly resistantto 5-FU treatment (Fig. 6D). Our results showed that thefluorouracil-resistant cells had higher expression of thymi-dylate synthase than the parental cells (Supplementary Fig.S14A), and that cixutumumab treatment significantly






reduced the expressions of thymidylate synthase and p-AKTin the fluorouracil-resistant cells (Supplementary Fig.S14B). Cixutumumab alone could moderately inhibit theproliferation of the fluorouracil-resistant cells, and that theacquired chemoresistance in fluorouracil-resistant cells was

significantly abrogated by a combination of cixutumumaband 5-FU treatment (Fig. 6E). We then proceeded to inves-tigate the therapeutic potential of cixutumumab in combi-nation with conventional cytotoxic chemotherapeuticdrugs in vivo. Figure 6F shows that cixutumumab markedly

Responding tumor opposite to:

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Figure 4. Systemic endocrineinstigation of tumor growth andmetastasis by Id1-induced IGF-II.A, diagram depicting experimentalmodel and groups. B, growthcurves of KYSE150 tumorxenografts (responders) in theopposite flanks of mice thatharbored different types of"instigating" tumors (n ¼ 8). C,comparisonof p-AKTexpression inthe responder tumors by Westernblot analysis. D, comparison of Ki-67 and apoptotic indices inresponding tumors. E, model ofendocrine instigation of distanttumor growth by Id1-induced IGF-II. F, KYSE150 cells injectedintravenously into nude micebearing KYSE150-Id1-shCONtumor xenografts had highestpropensity to metastasize to thelungs (n¼ 8 per group). Top, grossexamination of lungs (metastaticnodules are indicated by arrows).Surface metastatic nodules in thelungs were counted, and thenumbers presented separatelyfor nodules below and above1 mm in diameter. Middle,immunohistochemical staining formetastatic human cancer cells inlung sections using anti-CK-8.Bottom, hematoxylin and eosin(H&E) staining of adjacent lungsections. �, P < 0.05; ��, P < 0.01;��, P < 0.001 compared withempty vector control group.

Li et al.





augmented the sensitivity of ESCC tumors to 5-FU andcisplatin.

DiscussionIn this study, we provide the first evidence that Id1

induces IGF-II in a variety of cancer cells, and that tumor-

secreted IGF-II not only promotes local tumor growththrough autocrine stimulation of the PI3K/AKT pathway,but also acts systemically to instigate the growth of distanttumors and facilitates the metastasis of circulating esoph-ageal cancer cells in an endocrine manner. Furthermore,because our results showed that neutralization of IGF-IIand IGF-IR blockade had significant antitumor effects,

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Figure 5. Effects of IGF-II–neutralizing antibody and cixutumumab on suppressing growth of human ESCC xenograft in nude mice. A, intratumoralinjection with IGF-II–neutralizing antibody significantly suppressed the growth of KYSE150 tumor xenografts (n ¼ 8 per group). B, KYSE150 (left) andKYSE270 (right) cells were injected subcutaneously into the flanks of nude mice (n¼ 6 per group). Treatment of the mice with 25 or 50 mg/kg cixutumumabtwice a week significantly delayed the growth of the tumor xenografts. C, Ki-67 proliferation index, MVD, and apoptotic index of tumor xenografts ofnudemice treatedwith different dosages of cixutumumabor isotype IgG. D,Western blot analysis of expression levels of p-AKT and its downstream targets inthe tumor xenografts. E, immunohistochemistry confirmed that cixutumumab treatment significantly inhibited the activity of the PI3K/AKT pathway intumor xenografts. Bars, SD; �, P < 0.05; ��, P < 0.01; ��, P < 0.001 compared with isotype IgG-treated mice.






including enhancing chemosensitivity, and inhibiting tu-mor growth, and metastasis in animal models, we proposetargeting IGF-II or IGF-IR as a potentially promising strategyin esophageal cancer therapy.

Here, we have uncovered a novel mechanism in whichId1 activates the PI3K/AKT pathway through inductionand secretion of autocrine IGF-II. Our finding that Id1increases the expression and secretion of IGF-II is signif-icant because to our knowledge, the only other growthfactor reported to be induced by Id1 expression in cancercells is VEGF (23, 24). However, VEGF is unlikely to

account for the diverse tumor-promoting effects of Id1.Data from our and neutralization experiments stronglysupport IGF-II as a key growth factor that mediates theoncogenic functions of Id1.

IGF-II is overexpressed in a spectrum of human cancers,including colorectal, breast, ovarian, testicular, and livercancer (25–29). An earlier immunohistochemical studyshowed that 50% of ESCC tumors express IGF-II which isassociated with disease progression and recurrence (13).Incidentally, our TMA analysis showed a positive correla-tion between Id1 and IGF-II expressions in esophageal

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Figure 6. Effects of cixutumumabon suppressing metastasis andchemoresistance of humanesophageal cancer cells. A,treatment of the mice with 25 or 50mg/kg cixutumumab twice a weeksignificantly reduced the extent oflung metastasis (n ¼ 6 per group).Representative photos ofbioluminescent imaging ofthe mice 8 weeks after cellinjection were shown here. B,bioluminescent signals detected inthe region of the lungs of the micefrom different treatment groupswere analyzed. C, representativehematoxylin and eosin (H&E)-stained lung sections from thethree groups ofmice. D,MTT assaycomparing the viability offluorouracil-resistant (FR) ESCCsublineswith that of parental cells 4days after treatment with 5-FU (20mmol/L for KYSE150 cell lines, 10mmol/L for KYSE410 cell lines, and5 mmol/L for KYSE270 cell lines). E,FR cells were treated with 5-FU,alone or in combination withcixutumumab (100 mg/mL), and cellviability compared using MTTassay. F, nude mice xenograftedwith human ESCC cells KYSE150(left) and KYSE270 (right) weretreated with cixutumumab(25 mg/kg), 5-FU (20 mg/kg),cisplatin (2 mg/kg), cixutumumabplus 5-FU, or cixutumumab pluscisplatin twice weekly (n ¼ 6 pergroup). Bars, SD; �, P < 0.05;��, P < 0.01; ��, P < 0.001compared with control.


Li et al.




tumors. It was proposed that growth and metastasis oftumors can be governed on a systemic level by endocrinefactors released by tumors at distant sites (21). By demon-strating that nudemice bearing Id1-expressing tumor xeno-grafts had increased serum IGF-II, which exerted a systemicinstigating effect on tumor growth at distant sites, we haveprovided new evidence to support this concept. Further-more, our results also indicated that tumor-secreted IGF-IIcould promote the colonization and proliferation of blood-borne ESCC cells at secondary sites. These findings haveimportant implications in controlling cancer progression.In previous studies, including our own, the role of Id1 inmetastasis was thought to be limited to enhancing cellmigration, cell invasion, epithelial to mesenchymal transi-tion, and production of matrix metalloproteinase, whichare the key initial steps of metastasis (19, 30, 31). Here, wefurther demonstrated that Id1plays an important role in thefinal steps of themetastatic cascade by increasing circulatingIGF-II.Esophageal cancer is a highly lethal malignancy, and

development of new therapeutic intervention is urgentlyneeded. Anticancer reagents using neutralizing antibodiesto target growth factor and their receptors have emerged as anew class of effective therapeutics for human cancer.Because previous studies showed that as high as 60% ofESCC tumors expresses IGF-IR (13), inhibition of the IGFsignaling pathway was proposed as a potential therapeuticoption for ESCC. However, very little has been reportedabout the efficacy of IGF-II targeting in preclinical studies orclinical trials. Our current study provides the first evidencethat neutralizing IGF-II alone can significantly inhibit thegrowthof humanESCC tumor xenografts. Anumber of IGF-IR inhibitors are currently in clinical trials for the treatmentof solid tumors (32).We found that cixutumumab not onlyhad significant inhibitory effects on esophageal cancergrowth and metastasis, but also markedly enhanced thesensitivity of ESCC tumor xenograft to 5-FU and cisplatin,which are the mainstay of chemotherapeutic regimens foresophageal cancer. Our preclinical data therefore highlightthe therapeutic potential of cixutumumab in combinationwith cytotoxic drugs. Cixutumumab is currently being eval-uated in combination with paclitaxel in phase II trial assecond-line therapy formetastatic esophageal cancer (http://www.clinicaltrials.gov/ct2/show/NCT01142388?term¼A12þesophageal&rank¼1). The mechanisms by which cix-

utumumab increases the sensitivity of cancer cells to che-motherapeutics are not fully understood.

The fluorouracil-resistant cells used in this study showedhigh expression of thymidylate synthase, which is stronglyassociated with 5-FU chemoresistance (33). Activation ofthe AKT pathway in 5-FU chemoresistant ESCC cells andtumor xenografts has been reported (34, 35), and a recentstudy showed that AKT is upstream of thymidylate synthasein the 5-FU signaling events (36). Therefore, our datashowing significant suppression of p-AKT and thymidylatesynthase by cixutumumab treatment suggest that inhibitingthe PI3K/AKT pathway and thymidylate synthase expres-sionmay be the mechanisms by which cixutumumab helpscancer cells overcome 5-FU resistance.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. Li, S.W. Tsao, D.L. Ludwig, A.L.M. CheungDevelopment of methodology: B. Li, S.W. Tsao, K.W. ChanAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): B. Li, K.W. Chan, A.L.M. CheungAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): B. Li, K.W. Chan, Y.Y. Li, A.L.M. CheungWriting, review, and/or revision of the manuscript: B. Li, K.W. Chan,D.L. Ludwig, A.L.M. CheungAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): R. Novosyadlyy, Y.Y. Li, Q.Y. HeStudy supervision: A.L.M. Cheung

AcknowledgmentsThe authors thank Prof. Yutaka Shimada (University of Toyama, Toyama,

Japan) andDSMZ for theKYSE cell lines, Prof. G. Srivastava andDr. JudyYam(Department of Pathology,University ofHongKong,HongKong, China) forthe HKESC-3 cell line and luciferase expression vector, respectively, and Dr.Hitoshi Kawamata (Dokkyo University School of Medicine, Tochigi, Japan)for the T.Tn cell line. In vivo imaging data were acquired using equipmentmaintained by the University of Hong Kong Li Ka Shing Faculty of MedicineFaculty Core Facility.

Grant SupportThis work was supported by the Research Grants Council of the Hong

Kong SAR, China (GRF Project Nos. HKU 762610M and HKU 763111M)and The University of Hong Kong SRT Cancer research program andCRCG (Project Nos. 20067176143, 200807176012, 201007176305, and201211159003).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedOctober 4, 2013; revised January 23, 2014; accepted February 18,2014; published OnlineFirst March 5, 2014.

References1. Polyak K, Haviv I, Campbell IG. Co-evolution of tumor cells and their

microenvironment. Trends Genet 2009;25:30–8.2. Elkabets M, Gifford AM, Scheel C, Nilsson B, Reinhardt F, Bray MA,

et al. Human tumors instigate granulin-expressing hematopoietic cellsthat promote malignancy by activating stromal fibroblasts in mice.J Clin Invest 2011;121:784–99.

3. Rick FG, Schally AV, Szalontay L, Block NL, Szepeshazi K, Nadji M,et al. Antagonists of growthhormone-releasing hormone inhibit growthof androgen-independent prostate cancer through inactivation of ERKand Akt kinases. Proc Natl Acad Sci U S A 2012;109:1655–60.

4. Xie Q, Bradley R, Kang L, Koeman J, Ascierto ML, Worschech A, et al.Hepatocyte growth factor (HGF) autocrine activation predicts sensi-tivity to MET inhibition in glioblastoma. Proc Natl Acad Sci U S A2012;109:570–5.

5. Mo W, Chen J, Patel A, Zhang L, Chau V, Li Y, et al. CXCR4/CXCL12mediate autocrine cell- cycle progression in NF1-associated malig-nant peripheral nerve sheath tumors. Cell 2013;152:1077–90.

6. Chatterjee S, Heukamp LC, Siobal M, Schottle J, Wieczorek C, PeiferM, et al. Tumor VEGF:VEGFR2 autocrine feed-forward loop triggersangiogenesis in lung cancer. J Clin Invest 2013;123:1732–40.






7. Li B, Cheung PY, Wang X, Tsao SW, Ling MT, Wong YC, et al. Id-1activation of PI3K/Akt/NFkappaB signaling pathway and its signifi-cance in promoting survival of esophageal cancer cells. Carcinogen-esis 2007;28:2313–20.

8. Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins incancer. Nat Rev Cancer 2005;5:603–14.

9. Wong YC, Wang X, Ling MT. Id-1 expression and cell survival. Apo-ptosis 2004;9:279–89.

10. Yuen HF, Chan YP, Chan KK, Chu YY, Wong ML, Law SY, et al. Id-1and Id-2 are markers for metastasis and prognosis in oesophagealsquamous cell carcinoma. Br J Cancer 2007;97:1409–15.

11. Zha J, Lackner MR. Targeting the insulin-like growth factor receptor-1R pathway for cancer therapy. Clin Cancer Res 2010;16:2512–7.

12. Heidegger I, Pircher A, Klocker H, Massoner P. Targeting the insulin-like growth factor network in cancer therapy. Cancer Biol Ther2011;11:701–7.

13. ImsumranA, Adachi Y, YamamotoH, Li R,WangY,MinY, et al. Insulin-like growth factor-I receptor as a marker for prognosis and a thera-peutic target in human esophageal squamous cell carcinoma. Carci-nogenesis 2007;28:947–56.

14. Chava S, Mohan V, Shetty PJ, Manolla ML, Vaidya S, Khan IA, et al.Immunohistochemical evaluation of p53, FHIT, and IGF-II geneexpression in esophageal cancer. Dis Esophagus 2012;25:81–7.

15. Hu YC, Lam KY, Law SY, Wan TS, Ma ES, Kwong YL, et al. Estab-lishment, characterization, karyotyping, and comparative genomichybridization analysis of HKESC-2 and HKESC-3: two newly estab-lished human esophageal squamous cell carcinoma cell lines. CancerGenet Cytogenet 2002;135:120–7.

16. Shimada Y, Imamura M, Wagata T, Yamaguchi N, Tobe T. Character-ization of 21 newly established esophageal cancer cell lines. Cancer1992;69:277–84.

17. KawamataH, Furihata T,Omotehara F, Sakai T,HoriuchiH, ShinagawaY, et al. Identification of genes differentially expressed in a newlyisolated human metastasizing esophageal cancer cell line, T.Tn-AT1,by cDNA microarray. Cancer Sci 2003;94:699–706.

18. Hui CM, Cheung PY, Ling MT, Tsao SW, Wang X, Wong YC, et al. Id-1promotes proliferation of p53-deficient esophageal cancer cells. Int JCancer 2006;119:508–14.

19. Li B, Tsao SW, Li YY, Wang X, Ling MT, Wong YC, et al. Id-1 promotestumorigenicity and metastasis of human esophageal cancer cellsthrough activation of PI3K/AKT signaling pathway. Int J Cancer2009;125:2576–85.

20. Li B, Li YY, Tsao SW, Cheung AL. Targeting NF-kappaB signalingpathway suppresses tumor growth, angiogenesis, and metastasis ofhuman esophageal cancer. Mol Cancer Ther 2009;8:2635–44.

21. McAllister SS, Gifford AM, Greiner AL, Kelleher SP, Saelzler MP, InceTA, et al. Systemic endocrine instigation of indolent tumor growthrequires osteopontin. Cell 2008;133:994–1005.

22. Rowinsky EK, Youssoufian H, Tonra JR, Solomon P, Burtrum D,Ludwig DL. IMC-A12, a human IgG1 monoclonal antibody to theinsulin-like growth factor I receptor. Clin Cancer Res 2007;13:5549s-55s.

23. Ling MT, Lau TC, Zhou C, Chua CW, Kwok WK, Wang Q, et al.Overexpressionof Id-1 inprostate cancer cells promotes angiogenesisthrough the activation of vascular endothelial growth factor (VEGF).Carcinogenesis 2005;26:1668–76.

24. Dong Z, Wei F, Zhou C, Sumida T, Hamakawa H, Hu Y, et al. SilencingId-1 inhibits lymphangiogenesis through down-regulation of VEGF-Cin oral squamous cell carcinoma. Oral Oncol 2011;47:27–32.

25. Cullen KJ, Smith HS, Hill S, Rosen N, Lippman ME. Growth factormessenger RNA expression by human breast fibroblasts from benignand malignant lesions. Cancer Res 1991;51:4978–85.

26. Kim HT, Choi BH, Niikawa N, Lee TS, Chang SI. Frequent loss ofimprinting of the H19 and IGF-II genes in ovarian tumors. Am J MedGenet 1998;80:391–5.

27. Nonomura N, Miki T, Nishimura K, Kanno N, Kojima Y, Okuyama A.Altered imprinting of the H19 and insulin-like growth factor II genes intesticular tumors. J Urol 1997;157:1977–9.

28. Schirmacher P, Held WA, Yang D, Chisari FV, Rustum Y, Rogler CE.Reactivation of insulin-like growth factor II during hepatocarcinogen-esis in transgenic mice suggests a role in malignant growth. CancerRes 1992;52:2549–56.

29. Zhang L, Zhou W, Velculescu VE, Kern SE, Hruban RH, Hamilton SR,et al. Gene expression profiles in normal and cancer cells. Science1997;276:1268–72.

30. Fong S, Itahana Y, Sumida T, Singh J, Coppe JP, Liu Y, et al. Id-1 as amolecular target in therapy for breast cancer cell invasion and metas-tasis. Proc Natl Acad Sci U S A 2003;100:13543–8.

31. MinnAJ,GuptaGP,Siegel PM,BosPD, ShuW,Giri DD, et al. Genes thatmediate breast cancer metastasis to lung. Nature 2005;436:518–24.

32. Scagliotti GV, Novello S. The role of the insulin-like growth factorsignalingpathway in non-small cell lung cancer andother solid tumors.Cancer Treat Rev 2012;38:292–302.

33. Rustum YM. Thymidylate synthase: a critical target in cancer therapy?Front Biosci 2004;9:2467–73.

34. You F, Aoki K, Ito Y, Nakashima S. AKT plays a pivotal role in theacquisition of resistance to 5-fluorouracil in human squamous carci-noma cells. Mol Med Rep 2009;2:609–13.

35. YoshidaT,Miyoshi T, Seike J, YamaiH, Takechi H, YuasaY, et al. Geneexpression changes in a chemoresistant model with human esoph-ageal cancer xenografts using cDNA microarray. Anticancer Res2009;29:1163–8.

36. Vinod BS, Antony J, Nair HH, Puliyappadamba VT, Saikia M, Naraya-nan SS, et al. Mechanistic evaluation of the signaling events regulatingcurcumin-mediated chemosensitization of breast cancer cells to 5-fluorouracil. Cell Death Dis 2013;4:e505.


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2014;20:2651-2662. Published OnlineFirst March 5, 2014.Clin Cancer Res Bin Li, Sai Wah Tsao, Kwok Wah Chan, et al.

Targeted Therapy−Implications for IGF-II and IGF-IR−−Esophageal Cancer Progression and Chemoresistance

Id1-Induced IGF-II and Its Autocrine/Endocrine Promotion of

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