IGFBP3 Modulates Lung Tumorigenesis and Cell …...Finally, the treatment response to CDDP chemotherapy was evaluated under conditions of IGFBP3 overexpression.Igfbp3-null mice had

Oncogenes and Tumor Suppressors

IGFBP3 Modulates Lung Tumorigenesis and CellGrowth through IGF1 SignalingYong Antican Wang1, Yunguang Sun1,2, Joshua Palmer1, Charalambos Solomides3,Li-Ching Huang4, Yu Shyr4, Adam P. Dicker1, and Bo Lu1

Abstract

Insulin-like growth factor binding protein 3 (IGFBP3) mod-ulates cell growth through IGF-dependent and -independentmechanisms. Reports suggest that the serum levels of IGFBP3are associated with various cancers and that IGFBP3 expressionis significantly decreased in cisplatin (CDDP)-resistant lungcancer cells. Based on these findings, we investigated whetherIgfbp3 deficiency accelerates mouse lung tumorigenesis and ifexpression of IGFBP3 enhances CDDP response by focusing onthe IGF1 signaling cascade. To this end, an Igfbp3-null mousemodel was generated in combination with KrasG12D to comparethe tumor burden. Then, IGF-dependent signaling was assessedafter expressing wild-type or a mutant IGFBP3 without IGFbinding capacity in non–small cell lung cancer (NSCLC) cells.Finally, the treatment response to CDDP chemotherapy wasevaluated under conditions of IGFBP3 overexpression. Igfbp3-null mice had increased lung tumor burden (>2-fold) and only

half of human lung cancer cells survived after expression ofIGFBP3, which corresponded to increased cleaved caspase-3(10-fold), inactivation of IGF1 and MAPK signaling. In addi-tion, overexpression of IGFBP3 increased susceptibility toCDDP treatment in lung cancer cells. These results, for the firsttime, demonstrate that IGFBP3 mediates lung cancer progres-sion in a KrasG12D mouse model. Furthermore, overexpressionof IGFBP3 induced apoptosis and enhanced cisplatin responsein vitro and confirmed that the suppression is in part byblocking IGF1 signaling.

Implications: These findings reveal that IGFBP3 is effective inlung cancer cells with high IGF1 signaling activity and imply thatrelevant biomarkers are essential in selecting lung cancer patientsfor IGF1-targeted therapy. Mol Cancer Res; 15(7); 896–904. �2017AACR.

IntroductionLung cancer is the leading cause of cancer-related deaths world-

wide, with non–small cell lung cancer (NSCLC) representingapproximately 85% of all cases (1, 2). Many patients with NSCLCdevelop resistance to current chemotherapy, radiotherapy ortargeted therapy, which leads to decreased survival (3). To gaina better understanding of underlying mechanisms of treatmentresistance, researchers have studied insulin-like growth factor(IGF) signaling, which plays a central role in cellular growth,differentiation, and proliferation. Numerous cancers are associ-ated with aberrant IGF signaling, including lung cancer (4). Inparticular, IGF1, an abundant and ubiquitous polypeptidegrowth factor (5), is known to bind a transmembrane receptorcalled IGF1R and activate the PI3K–AKT and MAPK–ERK cascadethat activates proliferation and blocks apoptosis (6).

The bioavailability and bioactivity of IGF1 is controlled byIGFBP3, one of a family of six IGFBPs and themost abundant IGFbinding protein inhuman serum. IGF1maybecome "inactivated"after it binds with IGFBP3, which sequesters IGF1 in the extra-cellularmilieu, thereby inhibiting itsmitogenic and antiapoptoticactions (7). IGFBP3 also has IGF-independent bioactivities, prob-ably mediated by other receptors or signaling molecules (8, 9).

According to early epidemiological studies, lower IGFBP3levels were associated with a greater risk of various cancers,including prostate cancer (10), colorectal cancer (11), and lungcancer (12). It appears that high level of IGFBP3 is a protectivefactor and is associated with good prognosis in patients withadvanced NSCLC (13–15). No cases of IGFBP3 gene deletion inhumans have been reported, but hypermethylation of the IGFBP3promoter is involved in loss of IGFBP3 expression in NSCLC, andmay be related to the development of acquired treatment resis-tance (16–19). Although a prior report has shown that hetero-zygously deleted Igfbp3 transgenic mice withmutant IGF1 expres-sion have higher lung tumorigenesis, there was no increasedtumorigenesis in homozygously deleted Igfbp3 mice comparedwith wild-type Igfbp3 control, which was unexplained (20). Ourprevious data from lung cancer cell lines suggested that lungcancer cells express significantly lower levels of IGFBP3 whenthey develop resistance to cisplatin and radiation (21). Hence, wehypothesize that loss of IGFBP3will accelerate progressionof lungcancer and confer therapeutic resistance, while overexpressionwill sensitize treatment response.

Approximately 30% of lung cancers have RASmutations, 91%of NSCLC KRAS mutations involve codon 12, and the G12Dsubstitution represents 21% of mutations at this codon

1Department of Radiation Oncology, Thomas Jefferson University, Philadelphia,Pennsylvania. 2Department of Pathology, Medical College of Wisconsin, Mil-waukee, Wisconsin. 3Department of Pathology, Thomas Jefferson University,Philadelphia, Pennsylvania. 4Center for Quantitative Sciences, Vanderbilt Uni-versity Medical Center, Nashville, Tennessee.

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

Corresponding Author: Bo Lu, Thomas Jefferson University. G-301 BodineCancer Center, 111 S. 11th Street Philadelphia, PA 19107. Phone: 215-955-6705;Fax: 215-503-0013; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-16-0390

�2017 American Association for Cancer Research.

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(COSMIC; ref. 22). To test our hypothesis, we crossed KrasG12D

mutation driven lung cancer transgenicmicewith Igfbp3 knockoutmice to determine whether loss of IGFBP3 would change thetumor behavior. We also investigated the therapeutic response inhuman NSCLC cells with overexpression of IGFBP3. We foundthat knockout of IGFBP3 will promote lung tumorigenesis in vivo,while overexpression of IGFBP3 can suppress cell growth andenhance cisplatin sensitivity in NSCLC cell lines in part throughblocking IGF1 signaling.

Materials and MethodsMouse models and genotyping protocols

All experiments were performed according to protocolsapproved by the Institutional Animal Care and Use Committee(IACUC) of Thomas Jefferson University and complied with theGuide for the Care and Use of Laboratory Animals. KrasG12D

mutated mice were obtained from JAX (Strain Name: 129S/Sv-Krastm3Tyj/J Stock Number: 008185). They were maintained in aheterozygous state (KrasG12D/þ) because homozygosity for the KrasLA2 allele is embryonically lethal as described (23). The KrasG12D/þ

mice were crossed with Igfbp3 knockout mice (Igfbp3�/�, kindlyprovidedbyLexiconPharmaceuticals; ref. 24) togenerate Igfbp3þ/�:KrasG12D/þ F1mice, inbredof F1 to get F2 (Igfbp3þ/þ:KrasG12D/þ andIgfbp3�/�:KrasG12D/þ) mice, and then backcrossed to the Kras back-ground through at least six generations.

Screening of founder animals and their offspring was per-formed by PCR with genomic DNA isolated from tail clippingsusing the following primer sets: Igfbp3 wild-type allele, 50-TGCAGGCAGCCTAAGCACCTACCTC-30 and 50- CCCAGGGTC-CATTTTCCAACCTT -30; Igfbp3 deletion allele, 50-TAAGGTTCTC-CAGACCTCAAAGTG-30 and 50-CCCTAGGAATGCTCGTCAAGA-30; products length: wild-type ¼ 164 bp, mutant ¼ 288 bp. PCRcycling, 94�C for 3minutes, 35 cycles of 94�C for 30 seconds, 52�Cfor 30 seconds, 72�C for 30 seconds, and then 72�C for 10minutes; Kras wild-type allele, 50-TGCACAGCTTAGTGA-GACCC-30 and 50-GACTGCTCTCTTTCACCTCC-30; Kras-mutantallele, 50-TGCACAGCTTAGTGAGACCC -30 and 50-GGAG-CAAAGCTGCTATTGGC-30; products length: wild-type¼ 220 bp,mutant ¼ 390 bp, heterozygote ¼ 220 bp and 390 bp. PCRcycling, 94�C for 3 minutes, 35 cycles of 94�C for 30 seconds,64�C for 1 minutes, 72�C for 1 minutes, and then 72�C for 10minutes. Details of genotyping can be found from JAX standardprotocol for Kras (https://www.jax.org/strain/008185) and fromLexicon Pharmaceuticals for Igfbp3 (24).

Characterization of mouse lung cancerPaired littermates of F2 (Igfbp3þ/þ:KrasG12D/þ and Igfbp3�/�:

KrasG12D/þ) were sacrificed ranging from ages 4 to 7months. Afterpreliminary analysis of F2 mice, we sacrificed 5-month-oldIgfbp3þ/þ:KrasG12D/þ and Igfbp3�/�:KrasG12D/þmice that had beenbackcrossed to S129 background for representative analysis. Thelung tissue was immediately removed after the mice were sacri-ficed and visible pleural nodules were counted directly. Non-contrast whole-body CT imaging of 4- to 6-month-old micewithout respiratory or cardiac gating was performed using theMicroCAT II small-animal CT scanner (Siemens ImTek Inc.) at 80kVp and 500 mA (25). The DICOM data were imported to theMIM6.5 (MIM Software Inc.) for tumor contouring and 3Dreconstruction with different colors using the combination ofmanual segmentation and semiautomated contouring methods(26). These analyses were consistent between two independent

operators and performed in a blinded manner. Histology andquantification of lung tumor burden was conducted by corefacility and a pathologist.

Cell culture, recombinant adenoviral vector, and siRNAAll human lung cancer cell lines were received with their

standard Cell Line Authentication and Characterization [H460was bought from ATCC, HCC2429 was kindly provided by Dr.Tao Dang (Vanderbilt University, Nashville, TN); ref. 27], andthe cells for all experiments were recovered from the cryopres-ervation batches of the 2 to 6 passages (in 1 month afterreceipt). All cells were subcultured in RMPI1640 (Invitrogen)supplemented with 10% fetal bovine serum (GE Healthcare),100 units/mL penicillin, and 100 mg/mL streptomycin (Gibico)not more than 6 months, and tested for Mycoplasma contam-ination every 2 months (28). Adenoviral recombinant humanIGFBP3 and IGFBP3GGG were gifts from Dr. Youngman Oh(Virginia Commonwealth University, Richmond, VA) (29).Recombinant human IGF1 (Cat. 8335-G1-01M) and IGFBP3(Cat. 8874-B3) were purchased from R&D. MTS (Promega)assay on 96-well plate was used for cell viability test.

Western blot analysisCultured cells were lysed with M-PER (Thermo Fisher, Cat.

78501) protein extraction reagent with protease and phosphataseinhibitor cocktail. Cell lysates were centrifuged at 9,000� g for 10minutes at 4�C. Supernatants were transferred to clean microcen-trifuge tubes, frozen on dry ice, and thawed on ice. Total proteinconcentrations in the lysates were determined using the PierceBCA Protein Assay Kit (Thermo Fisher, Cat. 23250). Equalamounts of total proteins (30 mg/lane) were loaded on a 10%SDS-PAGE. Membranes were subsequently incubated with vari-ous primary antibodies. To investigate IGF1 signaling, all cells had15 minutes treatment of recombinant human IGF1 (30 ng/mL,unless stated otherwise) post serum starvation prior to stop of cellculture. To better determine the difference of gene expressionlevels between groups, some bands were normalized by actin andsemiquantified by ImageJ (30).

Mouse plasma collection and ELISA analysis of IGFBP3Before treatment, mouse peripheral venous blood (250 mL)

was collected in the EDTA-treated Eppendorf tube and thencentrifuged at 1500 � g for 10 minutes. Supernatant plasma wascollected and stored at �20�C until use. Plasma IGFBP3 levelswere then analyzedusing amouseELISAkit (R&D,Cat:MGB300).To determine the tissue protein abundance of IGFBP3, equalvolumes of mouse stomach tissue total proteins (2 mL)were diluted to the same 300 mL for mouse IGFBP3 ELISA.The abundance of IGFBP3 was calculated by the equa-tion: AbundanceðppmÞ ¼ ðIGFBP3=Total proteinÞ � 150. Concen-trations of IGFBP3 in conditioned medium from cells infectedwith IGFBP3 adenovirus were quantitated by human IGFBP3ELISA Kit (R&D, Cat: DGB300).

Statistical analysisData are presented as mean � SD, unless stated otherwise.

Prism6 software was used for comparison of lung tumor burdenbetween same age mice groups. A two-tailed Mann–Whitney(Wilcoxon rank sum) test was used unless stated otherwise, andP value <0.05 was considered statistically significant. All analyseswere double-checked by statisticians using R software.

IGFBP3 Inhibits NSCLC through IGF1 Signaling

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https://www.jax.org/strain/008185


ResultsIgfbp3 knockout accelerates KrasG12D-induced lung cancergrowth

To determine the role of IGFBP3 in lung cancer, we generatedKrasG12D-driven lung tumors in Igfbp3 knockout mice, as shownin Fig. 1A. Genotyping identified KrasG12D/þ and Igfbp3�/� off-spring as shown in Fig. 1B. We further used mouse IGFBP3 ELISAkit to validate Igfbp3 knockout. As shown in Fig. 1C, the plasmalevels of IGFBP3 were similar between the original Igfbp3þ/þmice

and the backcrossed Igfbp3þ/þ:KrasG12D/þ mice (close to thereference concentration 364 ng/mL), while the knockout miceplasma contained almost no IGFBP3 signal. We also comparedthe abundance of local IGFBP3 in the whole-tissue lysate of micestomachs because gastric mucosa has a relatively high level ofIGFBP3 expression and less interference in contrast with lungtissue which has surgical bleeding (18). There was no significantdifference in IGFBP3 abundance between the Igfbp3þ/þ (14.8 �6.3 ppm) and Igfbp3þ/þ:KrasG12D/þ (16.6 � 3.2 ppm) mice, andIGFBP3 was not detectable in the knockout mice. All of theseresults confirmed that Igfbp3 was successfully knocked out in theKrasG12D/þ background.

To determine the lung tumor burden of Igfbp3þ/þ:KrasG12D/þ vs.Igfbp3�/�:KrasG12D/þ, mice were sacrificed when they were 5months old. Tumor burden was measured through a number ofapproaches. First, we counted and graphed pleural lesions directlyand then nodules bigger than 1 mm in diameter from the wholetumor-bearing lungsweredissected,measuredwithdigital caliper,and graphed. As shown in representative pictures of one pairedmice (Fig. 2A), the number of pleural or dissected tumor noduleswas significantly increased in the Igfbp3�/�:KrasG12D/þ mice com-pared with the Igfbp3þ/þ:KrasG12D/þ mice (surface 27 vs. 10;dissected 57 vs. 13; Igfbp3þ/þ:KrasG12D/þ vs. Igfbp3�/�:KrasG12D/þ).

Because micro-CT is an effective tool to noninvasively measurethe growth of primary lung cancers and assess the tumor thera-peutic response in genetically engineered mice (25), we alsocontoured the tumor nodules and calculated tumor volumes onCT images. At first, we compared the volume from the CT versusthe volumemeasured with caliper after the dissection of the samenodules. As shown in Fig. 2B (top), tumor volumes of the samenodules measured by two methods are well-correlated linearly,and the pathological versus radiographical assessment of tumorburden reached a similar conclusion, as analyzed by Bland–Alt-man statistic method (31). The left and middle panels of Fig. 2Cshow color-marked nodules of representative axial and sagittalplanes of the CT images after validation of themicro-CT protocol,while the right panel of Fig. 2C shows color-rendered 3D recon-structions of tumors from the coronal plane with the same colorcode. Statistical analysis of tumor nodules and volumesmeasuredthrough micro-CT demonstrated that the median of total tumorvolume in the Igfbp3�/�:KrasG12D/þ mice increased significantly,by about 3-fold (Fig. 2D, right; medians, 20.01 vs. 59.13).

Increased tumor lesions in Igfbp3-deficient KrasG12D/þ miceCross-sections of tumor-bearing lungs following H&E staining

showed lung lesions of both groups (Igfbp3þ/þ:KrasG12D/þ vs.Igfbp3�/�:KrasG12D/þ) were high-grade adenomas or adenocarci-nomas (Fig. 3A left) and the tumor incidenceswere similar (100%at 4–6 months old). However, Igfbp3�/�:KrasG12D/þ mice havelarger tumors (Fig. 3C and statistic comparison of the percentageof tumor area to total lung area per tissue section in Fig. 3D,middle; Igfbp3þ/þ:KrasG12D/þ vs. Igfbp3�/�:KrasG12D/þ, P < 0.01),and/or more lesions than wild-type controls (Fig. 3C and statisticcomparisonof the tumornumber per tissue section in Fig. 3D, left;Igfbp3þ/þ:KrasG12D/þ vs. Igfbp3�/�:KrasG12D/þ P < 0.05). Immuno-histochemical (IHC) staining of Ki67 (a marker of cell prolifer-ation) showed that Igfbp3�/�:KrasG12D/þ mice have more positivestaining (Fig. 3A right, left are corresponding images of the samesectionH&E staining),withhigher Ki67 index indicating significanttumor proliferation than Igfbp3þ/þ:KrasG12D/þmice (Fig. 3B, tumorcells with Ki67 staining are depicted in low-magnification 200�

Figure 1.

Generation and identification of the Igfbp3�/�:KrasG12D/þ mouse lung cancermodel. A, Mouse breeding strategy. B, Mouse genotyping. C, Plasma levels ofIGFBP3 and the abundance of IGFBP3 in stomach total tissue lysates fromoriginal and backcrossed mice were analyzed by ELISA. For plasma assay,Welch two-sample t test, Igfbp3þ/þ vs. Igfbp3�/� P < 0.001; Igfbp3þ/þ:KrasG12D/þvs. Igfbp3�/�:KrasG12D/þ, P < 0.01. For tissue lysates assay,Welch two-sample t test, Igfbp3þ/þ vs. Igfbp3�/� P ¼ 0.056; Igfbp3þ/þ:KrasG12D/þvs. Igfbp3�/�:KrasG12D/þ, P < 0.05.

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Mol Cancer Res; 15(7) July 2017 Molecular Cancer Research898




and higher-magnification 400�; Fig. 3D, right; statistical compar-ison, Igfbp3þ/þ:KrasG12D/þ vs. Igfbp3�/�:KrasG12D/þ 400� P < 0.01,200� P < 0.0001). All these pathologic analysis implied that Igfbp3knockoutKrasG12Dmice havemore aggressive tumor growth thanwild-type.

Overexpression of IGFBP3 inhibits survival in HCC2429 cellsthrough blocking IGF1 signaling

After we confirmed that Igfbp3 deficiency will promote lungtumorigenesis in vivo, we sought to determine how IGFBP3 exertsits antitumor effects on established lung cancer cells in vitro.Because IGFBP3 is known to bind to IGF1 and prevents IGF1

from activating IGF1R and its downstream signaling (32), twolung cancer cell lines HCC2429 and H460 were used for over-expressing of IGFBP3, as HCC2429 is an IGF-responsive cell linewhile H460 is not. In order to determine whether cell survivalinhibition depends on interaction between IGF1 and IGFBP3, weincluded the IGFBP3 GGG mutant (I56G, L80G, and L81G,mutant IGF binding sites) adenovirus, which expresses full-lengthIGFBP3 but has no binding affinity to IGF1 (Supplementary Fig.S1A). ELISA analyses, performed at 48 hours and 72 hours postvirus infection (control empty virus EV,wild-type IGFBP3,mutantIGFBP3GGG), demonstrated that IGFBP3 was highly expressedintracellularly and secreted extracellularly in both cell lines (Fig.

Figure 2.

Tumor burden of Igfbp3 deficient KrasG12D/þ mouse lung cancer model. A, Representative images of lung tumor burden in paired 5-month-old mice. Left, tumornodules at the pleural surface; right, tumor nodules dissected from tumor-bearing lungs. B, Comparison of two tumor volume measurement methods. Tumorvolumes of 146 nodules measured by micro-CT were compared with the same tumor dissections measured by caliper; their logarithmic volume data were used forline of equality (top) and Bland–Altman analysis (bottom). There is a good correlation between these two different measurements (r2 > 0.8) and a goodagreement asmost spots located betweenmean� 2� SD in Bland–Altman plot. C, Representative micro-CT images. Left, axial planes; middle, Sagittal planes; right,color-rendered 3D representations of tumor volumes from coronal planes, tumor area were marked with color circles. D, Statistical analysis of the number ofnodules and the total tumor volumesmeasured throughmicro-CT. Data are shown as scatter plot with a line representing themedian. Red dot represents themousehas increased total nodules and tumor volume from CT scan but shown fewer tumor nodules and smaller tumor area at the specific H&E tissue section inFig. 3D, Mann–Whitney test, � , P < 0.05; �� , P < 0.01.






4A). MTS assay also performed at 72 hours after adenovirusinfection and 15 minutes IGF1 (30 ng/mL) stimulation, weobserved a survival reduction of 40% in the wild-type IGFBBP3group and only 20% reduction in the IGFBP3 GGG group ofHCC2429. In contrast, neither virus affected the survival of H460(Fig. 4B and C left). Multiple signaling pathways were checked atthe same time showed that baseline signaling is different in thetwo cell lines (Supplementary Fig. S1B). Although both cellmodels showed high levels of phosphorylated ERK at basal level,compared with H460, HCC2429 showed active phosphorylatedIGF1R and AKT in empty virus infection groups. When IGFBP3 isoverexpressed, active IGF1R and ERK were significantly sup-pressed in HCC2429 while overexpression of the non IGF1-binding mutant IGFBP3 only partially inhibited ERK phosphor-ylation. Although theWestern blot images of AKTbands appear toshow no remarkable changes, quantitation by ImageJ revealed asignificant decrease in p-AKT/AKT in IGFBP3 group (normalized

p-AKT/AKT in Fig. S1B, 1:0.7:0.9). This indicated that IGFBP3infection can remarkably suppress AKT activation compared withempty virus control while the suppression was less in themutatedIGFBP3GGG group. No significant alteration of IGF1–MAPKpathway was seen in the H460. All these signaling changesresulted in increased apoptosis after ectopic IGFBP3 expressionin HCC2429, with apparent higher cleavage of caspase-3 (nor-malized cle.-CASP3/CASP3 in Supplementary Fig. S1B, 1:8.3:3.3).We also tested the potential suppressive effects of recombinanthuman IGFBP3 (rhIGFBP3) on these cell lines. As shown in Fig.4C (bottom), the cell survivalwas significantly decreased by about50% post 20 ng/mL treatment with exogenous rhIGFBP3 inHCC2429 compared with no effect on H460. All these datademonstrated that elevated levels of IGFBP3 may sequester IGF1and result in IGF1 signaling suppression, which can inhibitHCC2429 growth by blocking the PI3K and MAPK pathways.However, there must be other mechanisms contributing to cell

Figure 3.

Increased tumor lesions in Igfbp3-deficient KrasG12D/þ mice. A, Representative pathologic micrographs (400� magnification) of the lung tumor lesions by H&E(left) and the matched Ki67 IHC staining (right). B, Representative micrographs of Ki67 staining at unmatched sections with same magnification. Left, 200�magnification; Right, 400�magnification. C, Representative H&Ewhole-slide scan images shown tumor number and area difference.D, Statistical analysis of tumorlesions in sections from each mouse (identical section levels), number of total nodules (left), tumor area (middle), and Ki67-index (right) in similar size tumorsunder different magnification (200�, 19 images of Igfbp3þ/þ:KrasG12D/þ vs. 27 images of Igfbp3�/�:KrasG12D/þ; 400 � 4 images of Igfbp3þ/þ:KrasG12D/þ vs.12 images of Igfbp3�/�:KrasG12D/þ). Data are shown as scatter plot with lines representing the mean � SD. Red dot represents the mouse that has fewer tumornodules and smaller tumor area at that specific section but the total nodules and tumor volume from Fig. 2D shown increased tumor lesions. There is astatistical difference if we omit this mouse for tumor number comparison. Mann–Whitney test, � , P < 0.05; �� , P < 0.01; �� , P < 0.0001.

Wang et al.





survival inhibition of IGFBP3 because GGG mutant could notfully restore cell survival inhibition (Fig. 4B).

IGFBP3 can sensitize lung cancer cells response to cisplatintreatment

Because IGFBP3 inhibits lung cancer cell survival and itsexpression is reduced in cisplatin-resistant lung cancer cells

(21), we further explored its potential to enhance the efficacy ofcisplatin chemotherapy. After IGFBP3 was overexpressed inHCC2429, and H460 which is relatively cisplatin resistant, aremarkable enhanced cisplatin response was found in both celllines compared with single CDDP treatment, and higher efficacyof CDDPwas found inHCC2429 through theMTS assay (Fig. 4C,left). At 48 hours after CDDP treatment, caspase-3 cleavage was

Figure 4.

Elevated levels of IGFBP3 inhibits cell survival and enhances cisplatin treatment response in selected lung cancer cells.A, ELISA analysis of IGFBP3 levels in H460 andHCC2429 cells infected with adenovirus (wild-type IGFBP3, mutant IGFBP3GGG or control empty virus EV). At 48 hours and 72 hours after infection, thecell culture supernatants of conditionmediumwere collected and the total protein were extracted for ELISA analysis. Cell culture medium RPMI1640with or without10% FBS are used as negative controls. All samples with wild-type or mutant IGFBP3 adenovirus infection have significant elevated levels of IGFBP3 (Studentt test Bonferroni-corrected, P < 0.05), however, for the comparison of IGFPB3 and EV at 72 hours of cell culture supernatant, P ¼ 0.068 for H460; P ¼ 0.058 forHCC2429. B, MTS cell survival assay was performed at 72 hours after virus infection. 2-way ANOVA Tukey's multiple comparisons test, adjusted P value�� ,P<0.01; �� ,P <0.0001.C,Cisplatin treatment response enhancement of elevated IGFBP3. Top, H460andHCC2429 cellswere treatedwith 20mmol/L cisplatin at24 hours post adenovirus infection, MTS assay was performed 48 hours after CDDP treatment. Bottom, H460 and HCC2429 cells were treated with 20 ng/mLrhIGFBP3 and/or 20 mmol/L cisplatin simultaneously, MTS assay was performed 48 hours and 72 hours after CDDP treatment. Two-way ANOVA Tukeymultiple comparisons test, adjusted P value; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001. D, Schematic diagram of the extracellular and intracellularmechanisms of IGFBP3 effects on cellular apoptosis and proliferation. Extracellular IGFBP3 secreted by autocrine or paracrine can inhibit IGF1 signaling bysequestering IGF1; while intracellular IGFBP3 can bind with other molecules, both of them can trigger apoptosis or inhibit cell growth.






noted in H460 treated with CDDP and it became more pro-nounced after the combined treatment (Supplementary Fig. S1C,left). CDDP-treated HCC2429 cells showed a significant increasein caspase-3 cleavage, whereas IGFBP3 overexpression has a mildeffect. Combination treatment resulted in a significant increase ofcaspase-3 cleavage (Supplementary Fig. S1C, right). We also usedthe recombinant human IGFBP3 (rhIGFBP3) on these two celllines to validate the enhancement effects of IGFBP3. As shownin Fig. 4C (bottom), exogenous rhIGFBP3 significantly decreasedcell survival in the combination group compared with CDDP orrhIGFB3 single treatment groups forHCC2429, while only CDDPcan effectively suppress H460 cell growth at 48 hours and 72hours.

We further analyzed signaling pathways as shown in Supple-mentary Fig. S1D. ForH460, the survival reductionwas associatedwith a decrease in AKT phosphorylation (CDDP). For HCC2429,we observed a remarkable decrease in IGF1R phosphorylationfollowing IGFBP3 overexpression, and we observed a significantdecrease in AKT phosphorylation following CDDP and/orIGFBP3 overexpression. Changes of ERK phosphorylation inHCC2429 were: (i) an increase after CDDP treatment; and (ii)a stronger decrease after IGFBP3 overexpression, which canreverse the increase after CDDP treatment. In conclusion, com-bining IGFBP3 overexpression and CDDP treatment increasessusceptibility to cisplatin treatment in both cell lines by furtherblocking PI3K and/or MAPK pathways. In HCC2429, both intra-cellular and extracellular elevated IGFBP3 can enhance CDDPefficacy.

DiscussionBecause IGFBP3 has been associated with tumorigenesis, the

ability of IGFBP3 to influence tumor growth by controlling IGFbioavailability has received intense interest (33). To characterizethe roles of IGFBP3, transgenic and knockoutmousemodels havebeen developed (20, 24). Although there is no report of sponta-neous tumor formation in Igfbp3-deficient mice, knockout Igfbp3mice would develop carcinogen-induced breast tumors signifi-cantly earlier than the wild-type, or severe lung metastasis of 4T1cells injection (34). Researchers further investigated IGFBP30sfunction under the oncogenic mutation background. Mehta andcolleagues observed the metastasis suppression function aftercrossing Igfbp3�/� mice with mice expressing human oncogenicMYC in the prostate (35). For lung cancer, only one group hasinvestigated the impact of IGFBP3 using a transgenic mousemodel carrying lung-specific human IGF1 and found that whileIGF1 is overexpressed, leading to activation of IGF1R, IGFBP3either inhibits or potentiates IGF1 actions in lung carcinogenesis(20). Because there are only expression-level changes but nomutation records of IGF1 and/or IGFBP3 related to human lungcancer in the COSMIC database, we sought to gain functionalinsights by using a different lung cancer driver mutationmodel toinvestigate the paradoxical nature of IGFBP3 through germlinedeletion. Although the membrane-bound ErbB family of recep-torsmediatemitogenic signals extracellularly, the RAS/RAF/MEK/ERK signaling pathway is one of the two major intracellularsignaling cascades that regulate cell proliferation and alsocross-talk with the other pathways (PI3K-AKT-mTOR; refs. 36,37). Because approximately 15% to 25% of patients with lungadenocarcinoma have tumor-associated KRAS mutations andG12D mutation represents 21% of KRAS mutations in NSCLC

(22), researchers have developed the KrasG12D transgenic mouse,which develops lung adenomas with 100% penetrance thateventually progress to high-grade adenocarcinomas and proneto develop resistance to cisplatin chemotherapy (38, 39). There-fore, the KrasG12D mutation was chosen as the driving force formouse lung tumorigenesis in our current study. After successfullygenerating KrasG12D lung tumors in Igfbp3 knockout mice (Fig. 1),we assessed lung tumor progression by micro-CT scan or exam-inations of tumor tissues. Gross photography combined withmicro-CT and pathologic analysis demonstrated the total tumorburden was significantly increased in Igfbp3�/�:KrasG12D/þ mice(Figs. 2 and 3). Our data reveal that deletion of Igfbp3 leads toaggressive tumor growth in a transgenic model of lung cancer.

From our in vivo mice data, it is reasonable to expect that theintracellular signaling of MAPK and PI3K–AKT, which are twodownstream pathways of IGF1, will be more activated withoutIGFBP3 to restrict the bioavailability of IGF1. For the mechanisticinvestigation on an in vitro cellular model, the proapoptoticproperty of IGFBP3 was confirmed by ectopic overexpression ofIGFBP3 in human lung cancer cell lines, which has different IGF1response capacity. A high level of IGFBP3 inhibited survival ofHCC2429 but did not affect H460 (Fig. 4A–C, top), as expectedthat HCC2429 is an IGF-responsive cell line. Exogenous recom-binant human IGFBP3 showed similar selective suppressioneffects on HCC2429 (Fig. 4C,bottom). This differential may bedue to the different baseline signaling and other mechanisms aswell. Although both cell models have activated ERK, HCC2429has constitutively phosphorylated IGF1R and AKT in contrast toH460 (Fig. S1B). IGFBP3 expression significantly suppressed theactivation of IGF1R, AKT, and ERK in HCC2429, while only veryweakly cleavage of caspase-3 was found inH460.Our findings areconsistent with the results published by Lee and colleagues (6).

Although many studies have reported IGFBP3 as a proapopto-tic molecule in an IGF-dependent manner, several studies suggestthat IGFBP3 interacts with its cellular receptors (IGFBP-3R/LRP1/TbRV) to induce apoptosis through caspase-8 independent ofIGF1 signaling (40, 41). We used a well-established GGG-mutantIGFBP3without IGF1binding capacity to investigate the potentialcontribution of IGF1-independent mechanisms. We foundmutant IGFBP3 could not fully restore cell survival inhibition(Fig. 4B) as well as phosphorylation or activation of IGF1R, AKT,and ERK (Supplementary Fig. S1B), which implies that blockingof the IGF1R signaling pathway could be one major mechanism;other signaling pathwaysmight also contribute to the cell survivalinhibition after IGFBP3 overexpression. As the schematic diagramshows (Fig. 4D), extracellular IGFBP3 in serum or secreted bycellular autocrine/paracrine can inhibit IGF1 signaling by seques-tering IGF1, blocking its binding with IGF1R and hence suppres-sing AKT and ERK activation-associated cell proliferation. Othersignaling pathways may also contribute, including IGFBP30s owncell membrane receptor IGFBP3R or LRP1, which may mediatedeath signaling directly through caspase-8; intracellular IGFBP3can suppress ERK activation directly (40, 41).

Although chemotherapy and targeted therapy demonstrates aremarkable therapeutic benefit in NSCLCs, the development ofresistance is inevitable for many patients. One possible reasonof chemoresistance in NSCLC might be the epigenetic inacti-vation of IGFBP3 because CDDP treatment could induce DNAhypermethylation (42, 43).Previously, we found a significantreduction of IGFBP3 by promoter methylation in cisplatin-resistant cells (21). Similarly, other observations revealed that

Wang et al.





hypermethylation-derived IGFBP3 deficiency mediates resis-tances to cisplatin or other DNA damaging agents in NSCLC(44, 45). Therefore, we compared the CDDP response with orwithout increased level of IGFBP3 and found that combinationtreatment resulted in a significant increase of CDDP suscepti-bility in both lung cancer cells (Fig. 4C) or at least in HCC2429when combined with rhIGFBP3 treatment (Fig. 4C, bottom).This discovery adds to our previous findings suggesting thepossibility of a therapeutic approach for sensitizing lung cancerto chemo- or radiotherapy by targeting the IGF1R, especiallyIGFBP3 (21). Our data are consistent with other reports of theassociation of IGFBP3 with therapy resistance through variousmechanisms. For example, IGFBP3 could sensitize antiestro-gen-resistant breast cancer cells by preventing the antiapoptoticfunction of GRP78 (29); activation of the IGF1R pathwayassociated with loss of IGFBP3 also involved in acquiredresistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) inNSCLC (46, 47), and even to the newly developed mutant-selective third-generation EGFR-TKIs (48). Given that plati-num-based chemotherapy still used as the first-line drugs forNSCLCs and targeted therapy will be more widely used inclinical practice, to maximize the efficacy or reverse acquiredresistance, a combination therapy with targeting IGF1 signalingsuch as IGFBP3 might be an attractive treatment option in thenear future.

There are several limitations of our research. AlthoughMAPK isone of the major intracellular signaling pathways, PI3K–AKT andother important downstream pathways could not be representedin this Kras mice model. Hence, more lung cancer models withdifferent driver mutations should be used to test IGFBP3 func-tions in these transgenic mice. And the lung tissue-specificKrasG12D mutation and Igfbp3 knockout model will be a bettermodel to investigate the precise effects of IGFBP3 on lung cancer.In addition, only two lung cancer cell lines (HCC2429 andH460)maynot fully reflect that heterogeneity of theNSCLC though thesecell lines were chosen for their IGF1 signaling according to ourexperimental design, which confirmed that the efficacy of IGFBP3depends on the activity of IGF1 signaling in part. More studies areneeded to decipher the mechanism by which death pathways areconnected to IGFBP3.

Using a unique mouse model we demonstrated the role ofIGFBP3 in lung carcinogenesis and to translate our findings fromtransgenic mice into clinical cancer therapy, we validated ourresults on IGF1-sensitive (HCC2429) versus -resistant (H460)

human lung cancer cell lines anddemonstrated tumor response toelevated levels of IGFBP3. Our results extend the findings ofIGFBP3 with different cell lines, and our findings are concordantwith our prior research and others in the literature. In summary,our findings indicate a tumor-suppressive effect of IGFBP3 onlung tumor growth and its synergistic effect with cisplatin, whichsuggests that the IGF1 and IGFBP3 pathways harbor multipletherapeutic targets to overcome therapy resistance in NSCLC, andIGFBP3 may be the most promising. We also demonstrate thatIGFBP3 can only be effective in lung cancer cells that have veryactive IGF1 signaling, which implies that relevant biomarkers areessential for selecting lung cancer patient for IGF1-target cancertherapy. Continuing work will focus on IGF1-dependent and-independent pathways in the Igfbp3:KrasG12D mice model andon in vivo therapy-resistant mechanisms.

Disclosure of Potential Conflicts of InterestJ.D. Palmer is an assistant professor at Ohio State University. Y. Shyr is a

consultant/advisory board member for Aduro Biotech, Janssen, Novartis, andRoche. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Y. Sun, B. LuDevelopment of methodology: Y.A. Wang, Y. SunAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y.A. Wang, Y. Sun, J. Palmer, A.P. DickerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y.A.Wang, Y. Sun, C. Solomides, L.-C. Huang, Y. ShyrWriting, review, and/or revision of the manuscript: Y.A. Wang, Y. Sun,J. Palmer, A.P. Dicker, B. LuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.-C. Huang, A.P. DickerStudy supervision: Y. Sun, B. Lu

AcknowledgmentsThe authors are grateful to Ms. Jennifer Wilson (Writing Center of Thomas

Jefferson University) for her suggestions, editing, and writing assistance.

Grant SupportThis work was supported by the Jefferson Dean's Transformational Science

Award.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 3, 2016; revised November 3, 2016; accepted March 16,2017; published OnlineFirst March 22, 2017.

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IGFBP3 Modulates Lung Tumorigenesis and Cell …...Finally, the treatment response to CDDP chemotherapy was evaluated under conditions of IGFBP3 overexpression.Igfbp3-null mice had