IGFBP3 Modulates Lung Tumorigenesis and Cell Growth

Published OnlineFirst March 22, 2017; DOI: 10.1158/1541-7786.MCR-16-0390

Oncogenes and Tumor Suppressors Molecular Cancer Research IGFBP3 Modulates Lung Tumorigenesis and Cell Growth through IGF1 Signaling Yong 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- increased lung tumor burden (>2-fold) and only half of human ulates cell growth through IGF-dependent and -independent lung cancer cells survived after expression of IGFBP3, which mechanisms. Reports suggest that the serum levels of IGFBP3 are corresponded to increased cleaved caspase-3 (10-fold), inactiva- associated with various cancers and that IGFBP3 expression is tion of IGF1 and MAPK signaling. In addition, overexpression of significantly decreased in cisplatin (CDDP)-resistant lung cancer IGFBP3 increased susceptibility to CDDP treatment in lung cancer cells. Based on these findings, we investigated whether Igfbp3 cells. These results, for the first time, demonstrate that IGFBP3 deficiency accelerates mouse lung tumorigenesis and if expression mediates lung cancer progression in a KrasG12D mouse model. of IGFBP3 enhances CDDP response by focusing on the IGF1 Furthermore, overexpression of IGFBP3 induced apoptosis and signaling cascade. To this end, an Igfbp3-null mouse model was enhanced cisplatin response in vitro and confirmed that the generated in combination with KrasG12D to compare the tumor suppression is in part by blocking IGF1 signaling. burden. Then, IGF-dependent signaling was assessed after expressing wild-type or a mutant IGFBP3 without IGF binding capacity Implications: These findings reveal that IGFBP3 is effective in in non–small cell lung cancer (NSCLC) cells. Finally, the treat- lung cancer cells with high IGF1 signaling activity and imply that ment response to CDDP chemotherapy was evaluated under relevant biomarkers are essential in selecting lung cancer patients conditions of IGFBP3 overexpression. Igfbp3-null mice had for IGF1-targeted therapy. Mol Cancer Res; 1–9. 2017 AACR.

Introduction The bioavailability and bioactivity of IGF1 is controlled by IGFBP3, one of a family of six IGFBPs and the most abundant IGF Lung cancer is the leading cause of cancer-related deaths world- binding protein in human serum. IGF1 may become "inactivated" wide, with non–small cell lung cancer (NSCLC) representing after it binds with IGFBP3, which sequesters IGF1 in the extra- approximately 85% of all cases (1, 2). Many patients with NSCLC cellular milieu, thereby inhibiting its mitogenic and antiapoptotic develop resistance to current chemotherapy, radiotherapy or actions (7). IGFBP3 also has IGF-independent bioactivities, prob- targeted therapy, which leads to decreased survival (3). To gain ably mediated by other receptors or signaling molecules (8, 9). a better understanding of underlying mechanisms of treatment According to early epidemiological studies, lower IGFBP3 resistance, researchers have studied insulin-like growth factor levels were associated with a greater risk of various cancers, (IGF) signaling, which plays a central role in cellular growth, including prostate cancer (10), colorectal cancer (11), and lung differentiation, and proliferation. Numerous cancers are associ- cancer (12). It appears that high level of IGFBP3 is a protective ated with aberrant IGF signaling, including lung cancer (4). In factor and is associated with good prognosis in patients with particular, IGF1, an abundant and ubiquitous polypeptide advanced NSCLC (13–15). No cases of IGFBP3 gene deletion in growth factor (5), is known to bind a transmembrane receptor humans have been reported, but hypermethylation of the IGFBP3 called IGF1R and activate the PI3K–AKT and MAPK–ERK cascade promoter is involved in loss of IGFBP3 expression in NSCLC, and that activates proliferation and blocks apoptosis (6). may be related to the development of acquired treatment resistance (16–19). Although a prior report has shown that hetero- zygously deleted Igfbp3 transgenic mice with mutant IGF1 expres- 1Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, sion have higher lung tumorigenesis, there was no increased Pennsylvania. 2Department of Pathology, Medical College of Wisconsin, Mil- tumorigenesis in homozygously deleted Igfbp3 mice compared waukee, Wisconsin. 3Department of Pathology, Thomas Jefferson University, 4 with wild-type Igfbp3 control, which was unexplained (20). Our Philadelphia, Pennsylvania. Center for Quantitative Sciences, Vanderbilt Uni- previous data from lung cancer cell lines suggested that lung versity Medical Center, Nashville, Tennessee. cancer cells express signiﬁcantly lower levels of IGFBP3 when Note: Supplementary data for this article are available at Molecular Cancer they develop resistance to cisplatin and radiation (21). Hence, we Research Online (http://mcr.aacrjournals.org/). hypothesize that loss of IGFBP3 will accelerate progression of lung Corresponding Author: Bo Lu, Thomas Jefferson University. G-301 Bodine cancer and confer therapeutic resistance, while overexpression Cancer Center, 111 S. 11th Street Philadelphia, PA 19107. Phone: 215-955-6705; will sensitize treatment response. Fax: 215-503-0013; E-mail: [email protected] Approximately 30% of lung cancers have RAS mutations, 91% doi: 10.1158/1541-7786.MCR-16-0390 of NSCLC KRAS mutations involve codon 12, and the G12D 2017 American Association for Cancer Research. substitution represents 21% of mutations at this codon

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(COSMIC; ref. 22). To test our hypothesis, we crossed KrasG12D operators and performed in a blinded manner. Histology and mutation driven lung cancer transgenic mice with Igfbp3 knockout quantification of lung tumor burden was conducted by core mice to determine whether loss of IGFBP3 would change the facility and a pathologist. tumor behavior. We also investigated the therapeutic response in human NSCLC cells with overexpression of IGFBP3. We found Cell culture, recombinant adenoviral vector, and siRNA that knockout of IGFBP3 will promote lung tumorigenesis in vivo, All human lung cancer cell lines were received with their while overexpression of IGFBP3 can suppress cell growth and standard Cell Line Authentication and Characterization [H460 enhance cisplatin sensitivity in NSCLC cell lines in part through was bought from ATCC, HCC2429 was kindly provided by Dr. blocking IGF1 signaling. Tao Dang (Vanderbilt University, Nashville, TN); ref. 27], and the cells for all experiments were recovered from the cryopres- Materials and Methods ervation batches of the 2 to 6 passages (in 1 month after Mouse models and genotyping protocols receipt). All cells were subcultured in RMPI1640 (Invitrogen) All experiments were performed according to protocols supplemented with 10% fetal bovine serum (GE Healthcare), approved by the Institutional Animal Care and Use Committee 100 units/mL penicillin, and 100 mg/mL streptomycin (Gibico) (IACUC) of Thomas Jefferson University and complied with the not more than 6 months, and tested for Mycoplasma contam- Guide for the Care and Use of Laboratory Animals. KrasG12D ination every 2 months (28). Adenoviral recombinant human mutated mice were obtained from JAX (Strain Name: 129S/Sv- IGFBP3 and IGFBP3GGG were gifts from Dr. Youngman Oh Krastm3Tyj/J Stock Number: 008185). They were maintained in a (Virginia Commonwealth University, Richmond, VA) (29). þ heterozygous state (KrasG12D/ ) because homozygosity for the Kras Recombinant human IGF1 (Cat. 8335-G1-01M) and IGFBP3 þ LA2 allele is embryonically lethal as described (23). The KrasG12D/ (Cat. 8874-B3) were purchased from R&D. MTS (Promega) mice were crossed with Igfbp3 knockout mice (Igfbp3 / ,kindly assay on 96-well plate was used for cell viability test. þ provided by Lexicon Pharmaceuticals; ref. 24) to generate Igfbp3 / : þ þ þ þ KrasG12D/ F1 mice, inbred of F1 to get F2 (Igfbp3 / :KrasG12D/ and Western blot analysis þ Igfbp3 / :KrasG12D/ ) mice, and then backcrossed to the Kras back- Cultured cells were lysed with M-PER (Thermo Fisher, Cat. ground through at least six generations. 78501) protein extraction reagent with protease and phosphatase Screening of founder animals and their offspring was per- inhibitor cocktail. Cell lysates were centrifuged at 9,000 g for 10 formed by PCR with genomic DNA isolated from tail clippings minutes at 4 C. Supernatants were transferred to clean microcen- using the following primer sets: Igfbp3 wild-type allele, 50- trifuge tubes, frozen on dry ice, and thawed on ice. Total protein TGCAGGCAGCCTAAGCACCTACCTC-30 and 50- CCCAGGGTC- concentrations in the lysates were determined using the Pierce CATTTTCCAACCTT -30; Igfbp3 deletion allele, 50-TAAGGTTCTC- BCA Protein Assay Kit (Thermo Fisher, Cat. 23250). Equal CAGACCTCAAAGTG-30 and 50-CCCTAGGAATGCTCGTCAAGA- amounts of total proteins (30 mg/lane) were loaded on a 10% 30; products length: wild-type ¼ 164 bp, mutant ¼ 288 bp. PCR SDS-PAGE. Membranes were subsequently incubated with vari- cycling, 94C for 3 minutes, 35 cycles of 94C for 30 seconds, 52C ous primary antibodies. To investigate IGF1 signaling, all cells had for 30 seconds, 72C for 30 seconds, and then 72C for 10 15 minutes treatment of recombinant human IGF1 (30 ng/mL, minutes; Kras wild-type allele, 50-TGCACAGCTTAGTGA- unless stated otherwise) post serum starvation prior to stop of cell GACCC-30 and 50-GACTGCTCTCTTTCACCTCC-30; Kras-mutant culture. To better determine the difference of gene expression allele, 50-TGCACAGCTTAGTGAGACCC -30 and 50-GGAG- levels between groups, some bands were normalized by actin and CAAAGCTGCTATTGGC-30; products length: wild-type ¼ 220 bp, semiquantified by ImageJ (30). mutant ¼ 390 bp, heterozygote ¼ 220 bp and 390 bp. PCR cycling, 94C for 3 minutes, 35 cycles of 94C for 30 seconds, Mouse plasma collection and ELISA analysis of IGFBP3 m 64C for 1 minutes, 72C for 1 minutes, and then 72C for 10 Before treatment, mouse peripheral venous blood (250 L) minutes. Details of genotyping can be found from JAX standard was collected in the EDTA-treated Eppendorf tube and then protocol for Kras (https://www.jax.org/strain/008185) and from centrifuged at 1500 g for 10 minutes. Supernatant plasma was Lexicon Pharmaceuticals for Igfbp3 (24). collected and stored at 20 C until use. Plasma IGFBP3 levels were then analyzed using a mouse ELISA kit (R&D, Cat: MGB300). Characterization of mouse lung cancer To determine the tissue protein abundance of IGFBP3, equal þ þ þ Paired littermates of F2 (Igfbp3 / :KrasG12D/ and Igfbp3 / : volumes of mouse stomach tissue total proteins (2 mL) þ KrasG12D/ ) were sacrificed ranging from ages 4 to 7 months. After were diluted to the same 300 mL for mouse IGFBP3 ELISA. preliminary analysis of F2 mice, we sacrificed 5-month-old The abundance of IGFBP3 was calculated by the equa- þ þ þ þ Igfbp3 / :KrasG12D/ and Igfbp3 / :KrasG12D/ mice that had been tion: AbundanceðppmÞ¼ðIGFBP3=Total proteinÞ150. Concen- backcrossed to S129 background for representative analysis. The trations of IGFBP3 in conditioned medium from cells infected lung tissue was immediately removed after the mice were sacri- with IGFBP3 adenovirus were quantitated by human IGFBP3 ficed and visible pleural nodules were counted directly. Non- ELISA Kit (R&D, Cat: DGB300). contrast whole-body CT imaging of 4- to 6-month-old mice without respiratory or cardiac gating was performed using the Statistical analysis MicroCAT II small-animal CT scanner (Siemens ImTek Inc.) at 80 Data are presented as mean SD, unless stated otherwise. kVp and 500 mA (25). The DICOM data were imported to the Prism6 software was used for comparison of lung tumor burden MIM6.5 (MIM Software Inc.) for tumor contouring and 3D between same age mice groups. A two-tailed Mann–Whitney reconstruction with different colors using the combination of (Wilcoxon rank sum) test was used unless stated otherwise, and manual segmentation and semiautomated contouring methods P value <0.05 was considered statistically significant. All analyses (26). These analyses were consistent between two independent were double-checked by statisticians using R software.

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IGFBP3 Inhibits NSCLC through IGF1 Signaling

þ þ þ Results and the backcrossed Igfbp3 / :KrasG12D/ mice (close to the reference concentration 364 ng/mL), while the knockout mice Igfbp3 knockout accelerates KrasG12D-induced lung cancer plasma contained almost no IGFBP3 signal. We also compared growth the abundance of local IGFBP3 in the whole-tissue lysate of mice To determine the role of IGFBP3 in lung cancer, we generated stomachs because gastric mucosa has a relatively high level of KrasG12D-driven lung tumors in Igfbp3 knockout mice, as shown þ IGFBP3 expression and less interference in contrast with lung in Fig. 1A. Genotyping identified KrasG12D/ and Igfbp3 / off- tissue which has surgical bleeding (18). There was no significant spring as shown in Fig. 1B. We further used mouse IGFBP3 ELISA þ þ difference in IGFBP3 abundance between the Igfbp3 / (14.8 kit to validate Igfbp3 knockout. As shown in Fig. 1C, the plasma þ/þ G12D/þ þ þ 6.3 ppm) and Igfbp3 :Kras (16.6 3.2 ppm) mice, and levels of IGFBP3 were similar between the original Igfbp3 / mice IGFBP3 was not detectable in the knockout mice. All of these results confirmed that Igfbp3 was successfully knocked out in the þ KrasG12D/ background. þ þ þ To determine the lung tumor burden of Igfbp3 / :KrasG12D/ vs. þ Igfbp3 / :KrasG12D/ , mice were sacrificed when they were 5 months old. Tumor burden was measured through a number of approaches. First, we counted and graphed pleural lesions directly and then nodules bigger than 1 mm in diameter from the whole tumor-bearing lungs were dissected, measured with digital caliper, and graphed. As shown in representative pictures of one paired mice (Fig. 2A), the number of pleural or dissected tumor nodules þ was significantly increased in the Igfbp3 / :KrasG12D/ mice com- þ þ þ pared with the Igfbp3 / :KrasG12D/ mice (surface 27 vs. 10; þ þ þ þ dissected 57 vs. 13; Igfbp3 / :KrasG12D/ vs. Igfbp3 / :KrasG12D/ ). Because micro-CT is an effective tool to noninvasively measure the growth of primary lung cancers and assess the tumor therapeutic response in genetically engineered mice (25), we also contoured the tumor nodules and calculated tumor volumes on CT images. At first, we compared the volume from the CT versus the volume measured with caliper after the dissection of the same nodules. As shown in Fig. 2B (top), tumor volumes of the same nodules measured by two methods are well-correlated linearly, and the pathological versus radiographical assessment of tumor burden reached a similar conclusion, as analyzed by Bland–Alt- man statistic method (31). The left and middle panels of Fig. 2C show color-marked nodules of representative axial and sagittal planes of the CT images after validation of the micro-CT protocol, while the right panel of Fig. 2C shows color-rendered 3D recon- structions of tumors from the coronal plane with the same color code. Statistical analysis of tumor nodules and volumes measured through micro-CT demonstrated that the median of total tumor þ volume in the Igfbp3 / :KrasG12D/ mice increased significantly, by about 3-fold (Fig. 2D, right; medians, 20.01 vs. 59.13).

þ Increased tumor lesions in Igfbp3-deficient KrasG12D/ mice Cross-sections of tumor-bearing lungs following H&E staining þ þ þ showed lung lesions of both groups (Igfbp3 / :KrasG12D/ vs. þ Igfbp3 / :KrasG12D/ ) were high-grade adenomas or adenocarcinomas (Fig. 3A left) and the tumor incidences were similar (100% þ at 4–6 months old). However, Igfbp3 / :KrasG12D/ mice have larger tumors (Fig. 3C and statistic comparison of the percentage of tumor area to total lung area per tissue section in Fig. 3D, þ þ þ þ middle; Igfbp3 / :KrasG12D/ vs. Igfbp3 / :KrasG12D/ ,P< 0.01), and/or more lesions than wild-type controls (Fig. 3C and statistic Figure 1. comparison of the tumor number per tissue section in Fig. 3D, left; fi / G12D/þ þ þ þ þ Generation and identi cation of the Igfbp3 :Kras mouse lung cancer Igfbp3 / :KrasG12D/ vs. Igfbp3 / :KrasG12D/ P < 0.05). Immuno- model. A, Mouse breeding strategy. B, Mouse genotyping. C, Plasma levels of histochemical (IHC) staining of Ki67 (a marker of cell prolifer- IGFBP3 and the abundance of IGFBP3 in stomach total tissue lysates from / G12D/þ original and backcrossed mice were analyzed by ELISA. For plasma assay, ation) showed that Igfbp3 :Kras mice have more positive Welch two-sample t test, Igfbp3þ/þ vs. Igfbp3/ P < 0.001; Igfbp3þ/þ: staining (Fig. 3A right, left are corresponding images of the same KrasG12D/þvs. Igfbp3/:KrasG12D/þ,P< 0.01. For tissue lysates assay, section H&E staining), with higher Ki67 index indicating significant þ/þ / þ/þ þ þ þ Welch two-sample t test, Igfbp3 vs. Igfbp3 P ¼ 0.056; Igfbp3 : tumor proliferation than Igfbp3 / :KrasG12D/ mice (Fig. 3B, tumor G12D/þ / G12D/þ < Kras vs. Igfbp3 :Kras ,P 0.05. cells with Ki67 staining are depicted in low-magnification 200

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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, tumor nodules at the pleural surface; right, tumor nodules dissected from tumor-bearing lungs. B, Comparison of two tumor volume measurement methods. Tumor volumes of 146 nodules measured by micro-CT were compared with the same tumor dissections measured by caliper; their logarithmic volume data were usedfor line of equality (top) and Bland–Altman analysis (bottom). There is a good correlation between these two different measurements (r2 > 0.8) and a good agreement as most spots located between mean 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 of nodules and the total tumor volumes measured through micro-CT. Data are shown as scatter plot with a line representing the median. Red dot represents the mouse has 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 in Fig. 3D, Mann–Whitney test, , P < 0.05; , P < 0.01.

and higher-magnification 400; Fig. 3D, right; statistical compar- from activating IGF1R and its downstream signaling (32), two þ þ þ þ ison, Igfbp3 / :KrasG12D/ vs. Igfbp3 / :KrasG12D/ 400 P < 0.01, lung cancer cell lines HCC2429 and H460 were used for over- 200 P < 0.0001). All these pathologic analysis implied that Igfbp3 expressing of IGFBP3, as HCC2429 is an IGF-responsive cell line knockout KrasG12D mice have more aggressive tumor growth than while H460 is not. In order to determine whether cell survival wild-type. inhibition depends on interaction between IGF1 and IGFBP3, we included the IGFBP3 GGG mutant (I56G, L80G, and L81G, Overexpression of IGFBP3 inhibits survival in HCC2429 cells mutant IGF binding sites) adenovirus, which expresses full-length through blocking IGF1 signaling IGFBP3 but has no binding affinity to IGF1 (Supplementary Fig. After we confirmed that Igfbp3 deficiency will promote lung S1A). ELISA analyses, performed at 48 hours and 72 hours post tumorigenesis in vivo, we sought to determine how IGFBP3 exerts virus infection (control empty virus EV, wild-type IGFBP3, mutant its antitumor effects on established lung cancer cells in vitro. IGFBP3GGG), demonstrated that IGFBP3 was highly expressed Because IGFBP3 is known to bind to IGF1 and prevents IGF1 intracellularly and secreted extracellularly in both cell lines (Fig.

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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&E whole-slide scan images shown tumor number and area difference. D, Statistical analysis of tumor lesions in sections from each mouse (identical section levels), number of total nodules (left), tumor area (middle), and Ki67-index (right) in similar size tumors under 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 tumor nodules and smaller tumor area at that specific section but the total nodules and tumor volume from Fig. 2D shown increased tumor lesions. There is a statistical difference if we omit this mouse for tumor number comparison. Mann–Whitney test, , P < 0.05; , P < 0.01; , P < 0.0001.

4A). MTS assay also performed at 72 hours after adenovirus p-AKT/AKT in Fig. S1B, 1:0.7:0.9). This indicated that IGFBP3 infection and 15 minutes IGF1 (30 ng/mL) stimulation, we infection can remarkably suppress AKT activation compared with observed a survival reduction of 40% in the wild-type IGFBBP3 empty virus control while the suppression was less in the mutated group and only 20% reduction in the IGFBP3 GGG group of IGFBP3GGG group. No significant alteration of IGF1–MAPK HCC2429. In contrast, neither virus affected the survival of H460 pathway was seen in the H460. All these signaling changes (Fig. 4B and C left). Multiple signaling pathways were checked at resulted in increased apoptosis after ectopic IGFBP3 expression the same time showed that baseline signaling is different in the in HCC2429, with apparent higher cleavage of caspase-3 (nor- two cell lines (Supplementary Fig. S1B). Although both cell malized cle.-CASP3/CASP3 in Supplementary Fig. S1B, 1:8.3:3.3). models showed high levels of phosphorylated ERK at basal level, We also tested the potential suppressive effects of recombinant compared with H460, HCC2429 showed active phosphorylated human IGFBP3 (rhIGFBP3) on these cell lines. As shown in Fig. IGF1R and AKT in empty virus infection groups. When IGFBP3 is 4C (bottom), the cell survival was significantly decreased by about overexpressed, active IGF1R and ERK were significantly sup- 50% post 20 ng/mL treatment with exogenous rhIGFBP3 in pressed in HCC2429 while overexpression of the non IGF1- HCC2429 compared with no effect on H460. All these data binding mutant IGFBP3 only partially inhibited ERK phosphor- demonstrated that elevated levels of IGFBP3 may sequester IGF1 ylation. Although the Western blot images of AKT bands appear to and result in IGF1 signaling suppression, which can inhibit show no remarkable changes, quantitation by ImageJ revealed a HCC2429 growth by blocking the PI3K and MAPK pathways. significant decrease in p-AKT/AKT in IGFBP3 group (normalized However, there must be other mechanisms contributing to cell

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survival inhibition of IGFBP3 because GGG mutant could not (21), we further explored its potential to enhance the efﬁcacy of fully restore cell survival inhibition (Fig. 4B). cisplatin chemotherapy. After IGFBP3 was overexpressed in HCC2429, and H460 which is relatively cisplatin resistant, a IGFBP3 can sensitize lung cancer cells response to cisplatin remarkable enhanced cisplatin response was found in both cell treatment lines compared with single CDDP treatment, and higher efﬁcacy Because IGFBP3 inhibits lung cancer cell survival and its of CDDP was found in HCC2429 through the MTS assay (Fig. 4C, expression is reduced in cisplatin-resistant lung cancer cells 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 and HCC2429 cells infected with adenovirus (wild-type IGFBP3, mutant IGFBP3GGG or control empty virus EV). At 48 hours and 72 hours after infection, the cell culture supernatants of condition medium were collected and the total protein were extracted for ELISA analysis. Cell culture medium RPMI1640 with or without 10% FBS are used as negative controls. All samples with wild-type or mutant IGFBP3 adenovirus infection have signiﬁcant elevated levels of IGFBP3 (Student t 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 for HCC2429. 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, H460 and HCC2429 cells were treated with 20 mmol/L cisplatin at 24 hours post adenovirus infection, MTS assay was performed 48 hours after CDDP treatment. Bottom, H460 and HCC2429 cells were treated with 20 ng/mL rhIGFBP3 and/or 20 mmol/L cisplatin simultaneously, MTS assay was performed 48 hours and 72 hours after CDDP treatment. Two-way ANOVA Tukey multiple comparisons test, adjusted P value; , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001. D, Schematic diagram of the extracellular and intracellular mechanisms of IGFBP3 effects on cellular apoptosis and proliferation. Extracellular IGFBP3 secreted by autocrine or paracrine can inhibit IGF1 signaling by sequestering IGF1; while intracellular IGFBP3 can bind with other molecules, both of them can trigger apoptosis or inhibit cell growth.

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IGFBP3 Inhibits NSCLC through IGF1 Signaling

noted in H460 treated with CDDP and it became more pro- (22), researchers have developed the KrasG12D transgenic mouse, nounced after the combined treatment (Supplementary Fig. S1C, which develops lung adenomas with 100% penetrance that left). CDDP-treated HCC2429 cells showed a significant increase eventually progress to high-grade adenocarcinomas and prone in caspase-3 cleavage, whereas IGFBP3 overexpression has a mild to develop resistance to cisplatin chemotherapy (38, 39). There- effect. Combination treatment resulted in a significant increase of fore, the KrasG12D mutation was chosen as the driving force for caspase-3 cleavage (Supplementary Fig. S1C, right). We also used mouse lung tumorigenesis in our current study. After successfully the recombinant human IGFBP3 (rhIGFBP3) on these two cell generating KrasG12D lung tumors in Igfbp3 knockout mice (Fig. 1), lines to validate the enhancement effects of IGFBP3. As shown we assessed lung tumor progression by micro-CT scan or exam- in Fig. 4C (bottom), exogenous rhIGFBP3 significantly decreased inations of tumor tissues. Gross photography combined with cell survival in the combination group compared with CDDP or micro-CT and pathologic analysis demonstrated the total tumor þ rhIGFB3 single treatment groups for HCC2429, while only CDDP burden was significantly increased in Igfbp3 / :KrasG12D/ mice can effectively suppress H460 cell growth at 48 hours and 72 (Figs. 2 and 3). Our data reveal that deletion of Igfbp3 leads to hours. aggressive tumor growth in a transgenic model of lung cancer. We further analyzed signaling pathways as shown in Supple- From our in vivo mice data, it is reasonable to expect that the mentary Fig. S1D. For H460, the survival reduction was associated intracellular signaling of MAPK and PI3K–AKT, which are two with a decrease in AKT phosphorylation (CDDP). For HCC2429, downstream pathways of IGF1, will be more activated without we observed a remarkable decrease in IGF1R phosphorylation IGFBP3 to restrict the bioavailability of IGF1. For the mechanistic following IGFBP3 overexpression, and we observed a significant investigation on an in vitro cellular model, the proapoptotic decrease in AKT phosphorylation following CDDP and/or property of IGFBP3 was confirmed by ectopic overexpression of IGFBP3 overexpression. Changes of ERK phosphorylation in IGFBP3 in human lung cancer cell lines, which has different IGF1 HCC2429 were: (i) an increase after CDDP treatment; and (ii) response capacity. A high level of IGFBP3 inhibited survival of a stronger decrease after IGFBP3 overexpression, which can HCC2429 but did not affect H460 (Fig. 4A–C, top), as expected reverse the increase after CDDP treatment. In conclusion, com- that HCC2429 is an IGF-responsive cell line. Exogenous recom- bining IGFBP3 overexpression and CDDP treatment increases binant human IGFBP3 showed similar selective suppression susceptibility to cisplatin treatment in both cell lines by further effects on HCC2429 (Fig. 4C,bottom). This differential may be blocking PI3K and/or MAPK pathways. In HCC2429, both intra- due to the different baseline signaling and other mechanisms as cellular and extracellular elevated IGFBP3 can enhance CDDP well. Although both cell models have activated ERK, HCC2429 efficacy. has constitutively phosphorylated IGF1R and AKT in contrast to H460 (Fig. S1B). IGFBP3 expression significantly suppressed the activation of IGF1R, AKT, and ERK in HCC2429, while only very Discussion weakly cleavage of caspase-3 was found in H460. Our findings are Because IGFBP3 has been associated with tumorigenesis, the consistent with the results published by Lee and colleagues (6). ability of IGFBP3 to influence tumor growth by controlling IGF Although many studies have reported IGFBP3 as a proapopto- bioavailability has received intense interest (33). To characterize tic molecule in an IGF-dependent manner, several studies suggest the roles of IGFBP3, transgenic and knockout mouse models have that IGFBP3 interacts with its cellular receptors (IGFBP-3R/LRP1/ been developed (20, 24). Although there is no report of sponta- TbRV) to induce apoptosis through caspase-8 independent of neous tumor formation in Igfbp3-deficient mice, knockout Igfbp3 IGF1 signaling (40, 41). We used a well-established GGG-mutant mice would develop carcinogen-induced breast tumors signifi- IGFBP3 without IGF1 binding capacity to investigate the potential cantly earlier than the wild-type, or severe lung metastasis of 4T1 contribution of IGF1-independent mechanisms. We found 0 cells injection (34). Researchers further investigated IGFBP3 s mutant IGFBP3 could not fully restore cell survival inhibition function under the oncogenic mutation background. Mehta and (Fig. 4B) as well as phosphorylation or activation of IGF1R, AKT, colleagues observed the metastasis suppression function after and ERK (Supplementary Fig. S1B), which implies that blocking crossing Igfbp3 / mice with mice expressing human oncogenic of the IGF1R signaling pathway could be one major mechanism; MYC in the prostate (35). For lung cancer, only one group has other signaling pathways might also contribute to the cell survival investigated the impact of IGFBP3 using a transgenic mouse inhibition after IGFBP3 overexpression. As the schematic diagram model carrying lung-specific human IGF1 and found that while shows (Fig. 4D), extracellular IGFBP3 in serum or secreted by IGF1 is overexpressed, leading to activation of IGF1R, IGFBP3 cellular autocrine/paracrine can inhibit IGF1 signaling by seques- either inhibits or potentiates IGF1 actions in lung carcinogenesis tering IGF1, blocking its binding with IGF1R and hence suppres- (20). Because there are only expression-level changes but no sing AKT and ERK activation-associated cell proliferation. Other mutation records of IGF1 and/or IGFBP3 related to human lung signaling pathways may also contribute, including IGFBP30s own cancer in the COSMIC database, we sought to gain functional cell membrane receptor IGFBP3R or LRP1, which may mediate insights by using a different lung cancer driver mutation model to death signaling directly through caspase-8; intracellular IGFBP3 investigate the paradoxical nature of IGFBP3 through germline can suppress ERK activation directly (40, 41). deletion. Although the membrane-bound ErbB family of recep- Although chemotherapy and targeted therapy demonstrates a tors mediate mitogenic signals extracellularly, the RAS/RAF/MEK/ remarkable therapeutic benefit in NSCLCs, the development of ERK signaling pathway is one of the two major intracellular resistance is inevitable for many patients. One possible reason signaling cascades that regulate cell proliferation and also of chemoresistance in NSCLC might be the epigenetic inacti- cross-talk with the other pathways (PI3K-AKT-mTOR; refs. 36, vation of IGFBP3 because CDDP treatment could induce DNA 37). Because approximately 15% to 25% of patients with lung hypermethylation (42, 43).Previously, we found a significant adenocarcinoma have tumor-associated KRAS mutations and reduction of IGFBP3 by promoter methylation in cisplatin- G12D mutation represents 21% of KRAS mutations in NSCLC resistant cells (21). Similarly, other observations revealed that

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Wang et al.

hypermethylation-derived IGFBP3 deficiency mediates resis- human lung cancer cell lines and demonstrated tumor response to tances to cisplatin or other DNA damaging agents in NSCLC elevated levels of IGFBP3. Our results extend the findings of (44, 45). Therefore, we compared the CDDP response with or IGFBP3 with different cell lines, and our findings are concordant withoutincreasedlevelofIGFBP3 and found that combination with our prior research and others in the literature. In summary, treatment resulted in a significant increase of CDDP suscepti- our findings indicate a tumor-suppressive effect of IGFBP3 on bility in both lung cancer cells (Fig. 4C) or at least in HCC2429 lung tumor growth and its synergistic effect with cisplatin, which when combined with rhIGFBP3 treatment (Fig. 4C, bottom). suggests that the IGF1 and IGFBP3 pathways harbor multiple This discovery adds to our previous findings suggesting the therapeutic targets to overcome therapy resistance in NSCLC, and possibility of a therapeutic approach for sensitizing lung cancer IGFBP3 may be the most promising. We also demonstrate that to chemo- or radiotherapy by targeting the IGF1R, especially IGFBP3 can only be effective in lung cancer cells that have very IGFBP3 (21). Our data are consistent with other reports of the active IGF1 signaling, which implies that relevant biomarkers are association of IGFBP3 with therapy resistance through various essential for selecting lung cancer patient for IGF1-target cancer mechanisms. For example, IGFBP3 could sensitize antiestro- therapy. Continuing work will focus on IGF1-dependent and gen-resistant breast cancer cells by preventing the antiapoptotic -independent pathways in the Igfbp3:KrasG12D mice model and function of GRP78 (29); activation of the IGF1R pathway on in vivo therapy-resistant mechanisms. associated with loss of IGFBP3 also involved in acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in Disclosure of Potential Conflicts of Interest NSCLC (46, 47), and even to the newly developed mutant- J.D. Palmer is an Assistant Professor at Ohio State University. Y. Shyr is a selective third-generation EGFR-TKIs (48). Given that plati- consultant/advisory board member for Aduro Biotech, Janssen, Novartis, and fl num-based chemotherapy still used as the first-line drugs for Roche. No potential con icts of interest were disclosed by the other authors. NSCLCs and targeted therapy will be more widely used in Authors' Contributions clinical practice, to maximize the efficacy or reverse acquired Conception and design: Y. Sun, B. Lu resistance, a combination therapy with targeting IGF1 signaling Development of methodology: Y.A. Wang, Y. Sun such as IGFBP3 might be an attractive treatment option in the Acquisition of data (provided animals, acquired and managed patients, near future. provided facilities, etc.): Y.A. Wang, Y. Sun, J. Palmer, A.P. Dicker There are several limitations of our research. Although MAPK is Analysis and interpretation of data (e.g., statistical analysis, biostatistics, one of the major intracellular signaling pathways, PI3K–AKT and computational analysis): Y.A. Wang, Y. Sun, C. Solomides, L.-C. Huang, Y. Shyr other important downstream pathways could not be represented Writing, review, and/or revision of the manuscript: Y.A. Wang, Y. Sun, J. Palmer, A.P. Dicker, B. Lu in this Kras mice model. Hence, more lung cancer models with Administrative, technical, or material support (i.e., reporting or organizing different driver mutations should be used to test IGFBP3 func- data, constructing databases): L.-C. Huang, A.P. Dicker tions in these transgenic mice. And the lung tissue-specific Study supervision: Y. Sun, B. Lu KrasG12D mutation and Igfbp3 knockout model will be a better model to investigate the precise effects of IGFBP3 on lung cancer. Acknowledgments In addition, only two lung cancer cell lines (HCC2429 and H460) The authors are grateful to Ms. Jennifer Wilson (Writing Center of Thomas may not fully reflect that heterogeneity of the NSCLC though these Jefferson University) for her suggestions, editing, and writing assistance. cell lines were chosen for their IGF1 signaling according to our experimental design, which confirmed that the efficacy of IGFBP3 Grant Support depends on the activity of IGF1 signaling in part. More studies are This work was supported by the Jefferson Dean's Transformational Science Award. needed to decipher the mechanism by which death pathways are The costs of publication of this article were defrayed in part by the payment of connected to IGFBP3. page charges. This article must therefore be hereby marked advertisement in Using a unique mouse model we demonstrated the role of accordance with 18 U.S.C. Section 1734 solely to indicate this fact. IGFBP3 in lung carcinogenesis and to translate our findings from transgenic mice into clinical cancer therapy, we validated our Received November 3, 2016; revised November 3, 2016; accepted March 16, results on IGF1-sensitive (HCC2429) versus -resistant (H460) 2017; published OnlineFirst March 22, 2017.

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www.aacrjournals.org Mol Cancer Res; 2017 OF9

IGFBP3 Modulates Lung Tumorigenesis and Cell Growth through IGF1 Signaling

Yong Antican Wang, Yunguang Sun, Joshua Palmer, et al.

Mol Cancer Res Published OnlineFirst March 22, 2017.

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