Am J Cancer Res 2018;8(5):792-809 www.ajcr.us /ISSN:2156-6976/ajcr0077452 Original Article ERBB3, IGF1R, and TGFBR2 expression correlate with PDGFR expression in glioblastoma and participate in PDGFR inhibitor resistance of glioblastoma cells Kang Song1,2*, Ye Yuan1,2*, Yong Lin1,2, Yan-Xia Wang1,2, Jie Zhou1,2, Qu-Jing Gai1,2, Lin Zhang1,2, Min Mao1,2, Xiao-Xue Yao1,2, Yan Qin1,2, Hui-Min Lu1,2, Xiang Zhang1,2, You-Hong Cui1,2, Xiu-Wu Bian1,2, Xia Zhang1,2, Yan Wang1,2 1Department of Pathology, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China; 2Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400038, China. *Equal contributors. Received April 6, 2018; Accepted April 9, 2018; Epub May 1, 2018; Published May 15, 2018 Abstract: Glioma, the most prevalent malignancy in brain, is classified into four grades (I, II, III, and IV), and grade IV glioma is also known as glioblastoma multiforme (GBM). Aberrant activation of receptor tyrosine kinases (RTKs), including platelet-derived growth factor receptor (PDGFR), are frequently observed in glioma. Accumulating evi- dence suggests that PDGFR plays critical roles during glioma development and progression and is a promising drug target for GBM therapy. However, PDGFR inhibitor (PDGFRi) has failed in clinical trials, at least partially, due to the activation of other RTKs, which compensates for PDGFR inhibition and renders tumor cells resistance to PDGFRi. Therefore, identifying the RTKs responsible for PDGFRi resistance might provide new therapeutic targets to syner- getically enhance the efficacy of PDGFRi. In this study, we analyzed the TCGA glioma database and found that the mRNA expressions of three RTKs, i.e. ERBB3, IGF1R, and TGFBR2, were positively correlated with that of PDGFR. Co-immunoprecipitation assay indicated novel interactions between the three RTKs and PDGFR in GBM cells. Moreover, concurrent expression of PDGFR with ERBB3, IGF1R, or TGFBR2 in GBM cells attenuated the toxicity of PDGFRi and maintained the activation of PDGFR downstream targets under the existence of PDGFRi. Thus, ERBB3, IGF1R, and TGFBR2 might participate in PDGFRi resistance of GBM cells. Consistent with this notion, combination of PDGFRi with inhibitor targeting either ERBB3 or IGF1R more potently suppressed the growth of GBM cells than each inhibitor alone. The positive correlations of PDGFR with ERBB3, IGF1R, and TGFBR2 were further confirmed in 66 GBM patient samples. Intriguingly, survival analysis showed that ERBB3 predicted poor prognosis in GBM pa- tients with high PDGFRA expression. Altogether, our work herein suggested that ERBB3, IGF1R, and TGFBR2 were responsible for PDGFRi resistance and revealed that ERBB3 acted as potential prognostic marker and therapeutic target for GBM with high PDGFRA expression. Keywords: Glioblastoma, resistance, PDGFR, ERBB3, IGF1R, TGFBR2 Introduction 3]. The Cancer Genome Atlas (TCGA) project has unveiled critical genetic alterations in glio- Glioma is a prevalent malignancy in brain and ma [4] and provides important rationales for pathologically classified into four grades, i.e. I, target therapies. In most of GBM cases, genetic II, III, and IV, according to the 2016 World profiling reveals aberrant activation of signaling Health Organization (WHO) classification of pathways mediated by receptor tyrosine kinas- central nervous system tumors [1]. Grade IV es (RTKs) [4-6], including the platelet-derived glioma is the most malignant form of glioma growth factor receptor (PDGFR) [5, 6]. PDGFR is and also known as glioblastoma multiforme a transmembrane receptor with 5 immunoglob- (GBM) [1]. Despite the progression of surgical ulin-like repeats in the extracellular domain and and pharmacological therapies, GBM is still an a tyrosine kinase domain in the intracellular intractable disease and the average survival domain. Two PDGFR members have been iden- time of GBM patients is only about one year [2, tified: PDGF receptor α (PDGFRA) and PDGF RTKs involved in resistance to PDGFR inhibitor Table 1. Clinicopathological information of tion of AXL (AXL Receptor Tyrosine Kinase) patients causes resistance to EGFR-targeted therapy in Clinical Feature Sample Amount lung cancer [23]. Therefore, the activation of alternative RTKs might be also responsible for Grade IV 66 the failure of PDGFRi and identifying these Gender Male 41 alternatively RTKs could provide new therapeu- Female 25 tic targets to synergetically enhance the inhibi- Age ≤ 50 31 tory effect of PDGFRi. > 50 35 Total 66 So far, few RTKs have been reported to partici- pate in the development of PDGFRi resistance in GBM cells, but TCGA database provides us a receptor β (PDGFRB) [7]. Upon the binding of powerful tool to systematically evaluate the PDGF ligand, PDGFRA and PDGFRB form homo- relationships of PDGFR with other RTKs. In this or hetero-dimer and undergo autophosphoryla- study, we hypothesized that RTKs concurrently tion to activate downstream targets, inclu- expressed with PDGFR might contribute to ding PI3K (phosphatidylinositol-4,5-bisphos- PDGFRi resistance with high probability. The phate 3-kinase)/AKT (protein kinase B) and analyses on TCGA glioma database and a MAPK (mitogen-activated protein kinases)/ cohort containing 66 GBM patients revealed ERK1/2 (extracellular signal-regulated kinases tight relationships of PDGFR with three RTKs, i.e. ERBB3, IGF1R, and TGFBR2. Further cellu- 1/2), which results in cell proliferation, survival, lar experiments indicated that the three RTKs migration, and oncogenesis [8]. Remarkably, were indeed involved in PDGFRi resistance and PDGFRA overexpression has been widely de- the combination of PDGFRi with inhibitor tar- tected in all stages of glioma, and the activa- geting either ERBB3 or IGF1R might represent tion of PDGF/PDGFR signaling pathway is pivot- a novel therapeutic strategy to treat GBM ally involved in the initiation and progression of patients. glioma [9, 10]. Materials and methods The critical involvement of PDGFR in glioma makes PDGFR inhibitor (PDGFRi) promising Patient samples drug to treat glioma, especially PDGFR-de- pendent GBM. So far, several anti-tumor agents 66 GBM samples were obtained from patients targeting PDGFR have been developed, such as diagnosed and treated in Southwest Hospital Imatinib (Gleevec®), Sorafenib (Nexavar®), (Chongqing, China) (Table 1). All patients under- Nilotinib (Tasigna®), and Sunitinib (Sutent®). went surgical resection from January 2014 Although the data from in vitro and animal through December 2016. Specimens were experiments support the potent inhibitory fixed in 4% buffered formaldehyde solution effects of PDGFRi on GBM cells [11, 12], clini- after surgical removal, and then paraffin- cal trials of single PDGFRi have failed to show embedded. Pathohistological diagnoses were encouraging anti-tumor effects [13-16], which independently made by two neuropathologi- might result from the rapid emergence of resis- sts according to “The 2016 World Health tance to PDGFRi [17-19]. Multiple mechanisms Organization Classification of Tumors of the on resistance to RTK-targeted therapy have Central Nervous System” [1]. This study was been identified, including mutation of the active carried out according to the principles of the site, amplification of the targeted RTK, and acti- Helsinki Declaration and all protocols have been approved by the ethics committee of vation of alternative RTKs [18-20]. Indeed, co- Southwest Hospital, Third Military Medical activation of alternative RTKs renders tumor University (TMMU). cells resistance to inhibitor targeting original RTK [21]. Additionally, activation of c-MET (MET TCGA glioma database Proto-Oncogene, Receptor Tyrosine Kinase) and ERBB3 (Erb-B2 Receptor Tyrosine Kinase A group of 669 patient specimens from TCGA_ 3) leads to tolerance of lung cancer cells to GBMLGG database and 538 patient specimens Gefitinib, an inhibitor targeting EGFR (Epidermal from TCGA_GBM database (http://gliovis.bioin- Growth Factor Receptor) [22]. Similarly, activa- fo.cnio.es/) were utilized to evaluate the expres- 793 Am J Cancer Res 2018;8(5):792-809 RTKs involved in resistance to PDGFR inhibitor sion correlation, the clinical relevance, and the and recorded by fluoroanalyzer with OD 450 nm Gene Set Enrichment assay of RTKs. at day 3. Cell culture Cell migration and invasion LN229 and 293FT cell lines were purchased The upper chamber (Millicell 8.0 μm PET) was from ATCC (VA, USA), and primary GBM cells coated with 15 µl of Matrigel (CORNING, (GBM-1) were established from the tumor spec- 354234, Bedford, MA 01730, USA) for invasion imen of GBM patient treated in Southwest assay, and not coated for migration assay. Hospital (TMMU, Chongqing, China) [24]. All 3×104 cells in 200 µl of serum-free medium cells were grown in Dulbecco’s modified Eagle’s were seeded into the upper chamber and the medium (Gibco, NY14072, USA) supplemented lower chamber was added with 500 µl growth with 10% (v/v) fetal bovine serum (Gibco, medium supplemented with 10% FBS. After 10270-106, EU-Approved) and 1% (v/v) incubation at 37°C with 5% CO2 for 24 hr for Penicillin-Streptomycin (HyClone, SV30010, invasion assay and 12 hr for migration assay, Austria) at 37°C in a humidified incubator with the cells were fixed with 4% paraformaldehyde 5% CO2. (PFA) followed by crystal violet staining. Non- invading cells were removed with a cotton Plasmid constructs, lentivirus production, and swab, and the stained cells