M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 6 7 7 e6 9 2
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Receptor tyrosine kinase gene expression profiles of Ewing sarcomas reveal ROR1 as a potential therapeutic target in metastatic disease Jenny Potratza,*,1, Amelie Tillmannsa, Philipp Berninga, Eberhard Korschingb, Christiane Schaefera, Birgit Lechtapea,1, € ferc, Carolin Schleithoffa, Rebekka Unlanda, Karl-Ludwig Scha € rgensa, Uta Dirksena € ller-Tidowd, Heribert Ju Carsten Mu € nster, Albert-Schweitzer-Campus 1, 48149 Pediatric Hematology and Oncology, University Children’s Hospital Mu € nster, Germany Mu b €lische-Wilhelms Universita €t Mu € nster, Niels-Stensen-Strasse 12, 48149 Mu € nster, Institute of Bioinformatics, Westfa Germany c € sseldorf, Moorenstrasse 5, 40225 Du € sseldorf, Germany Institute of Pathology, University Medical Center Du a
d
Department of Inner Medicine IV, Hematology and Oncology, University Hospital Halle, Ernst-Grube-Strasse 40, 06120 Halle (Saale), Germany
A R T I C L E
I N F O
A B S T R A C T
Article history:
Receptor tyrosine kinases (RTKs) have provided molecular targets for the development of
Received 12 August 2015
novel, prognosis-improving agents in many cancers; however, resistances to these thera-
Received in revised form
pies occur. On the cellular level, one resistance mechanism is attributed to functional
11 December 2015
RTK redundancies and compensatory cross-signaling, leading to perception of RTKs as
Accepted 12 December 2015
signaling and target networks. To provide a basis for better exploitation of this network
Available online 20 December 2015
in Ewing sarcoma, we generated comprehensive qPCR gene expression profiles of RTKs in Ewing sarcoma cell lines and 21 untreated primary tumors. Key findings confirm
Keywords:
broad-spectrum RTK expressions with potential for signaling redundancy. Profile analyses
Ewing sarcoma
with regard to patient risk-group further revealed several individual RTKs of interest.
Metastasis
Among them, VEGFR3 and TIE1 showed high-level expressions and also were suggestive
Receptor tyrosine kinase
of poor prognosis in localized tumors; underscoring the relevance of angiogenic signaling
ROR1
pathways and tumor-stroma interactions in Ewing sarcoma. Of note, compared to localized
Therapeutic target
disease, tumors derived from metastatic disease were marked by global high-level RTK expressions. Nine individual RTKs were significantly over-expressed, suggesting contributions to molecular mechanisms of metastasis. Of these, ROR1 is being pursued as therapeutic target in leukemias and carcinomas, but un-characterized in sarcomas. We demonstrate expression of ROR1 and its putative ligand Wnt5a in Ewing sarcomas, and of an active ROR1 protein variant in cell lines. ROR1 silencing impaired cell migration in vitro. Therefore, ROR1 calls for further evaluation as a therapeutic target in metastatic
Abbreviations: MSC, mesenchymal stem cell; qPCR, real-time quantitative PCR; ROR1, receptor tyrosine kinase-like orphan receptor 1; RTK, receptor tyrosine kinase, for a listing of RTK abbreviations see Table A.1. * Corresponding author. Tel.: þ49 251 83 47732; fax: þ49 251 83 47735. E-mail address:
[email protected] (J. Potratz). 1 € nster, Albert-Schweitzer-Campus 1, 48149 Mu € nster. Present address. General Pediatrics, University Children’s Hospital Mu http://dx.doi.org/10.1016/j.molonc.2015.12.009 1574-7891/ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 6 7 7 e6 9 2
Ewing sarcoma; and described as a pseudo-kinase with several isoforms, underlines these additional complexities arising in our understanding of RTK signaling networks. ª 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1.
Introduction
Ewing sarcoma is a rare cancer, but the second most common sarcoma of bone in childhood and adolescence (Bernstein et al., 2006; Gurney et al., 1999). Metastasis at diagnosis is the most significant adverse prognostic factor (Cotterill et al., 2000; Ladenstein et al., 2010; Paulussen et al., 1998). Risk-adapted and metastasis-targeted therapeutic strategies therefore are key aims of current translational research. Given the complex series of events adding to the metastatic process, it is plausible that molecular players and consequently targets should be distinct from those active in localized tumors (Krishnan et al., 2005; Valastyan and Weinberg, 2011). At the same time it remains undefined, whether primary genetic determinants predestine to metastasis, or whether secondary events that generate a metastatic tumor sub-clone prevail. Several microarray gene expression studies aimed to identify prognostic and/or metastatic gene expression signatures in Ewing sarcoma (Ohali et al., 2004; Schaefer et al., 2008; Volchenboum et al., 2015; Zambelli et al., 2010). So far, while focusing on individual signature genes, validations from a comprehensive gene pathway or gene family perspective are lacking. Receptor tyrosine kinases (RTKs) and their dysregulation represent key oncogenic and tumor-maintaining events in many cancers (Gschwind et al., 2004; Lemmon and Schlessinger, 2010). Furthermore, their ligand-mediated activation and intrinsic kinase activities are well amenable to therapeutic inhibition, making RTKs highly investigated drug targets. Multiple ligand-blocking antibodies and small molecule kinase inhibitors are in development and clinical € nne et al., 2009). Because application (Gschwind et al., 2004; Ja oncogenic RTKs are not principally unique to cancer entities € nne et al., 2009; Lemmon and Schlessinger, 2010), rare dis(Ja eases such as Ewing sarcoma can profit from therapeutic advances in more prevalent cancers. This was demonstrated in an advent of pre-clinical and clinical IGF1R-targeted investigations in pediatric sarcomas, stimulated by remarkable responses of Ewing sarcoma patients in early phase trials (Olmos et al., 2010; Tolcher et al., 2009). More recently, distinct RTK functions in the metastatic cellular capacities and consequently targeting potential are reaching focus. RTKs interact with integrin and cadherin cell adhesion molecules to induce and exert pro-migratory and pro-invasive signals through intracellular FAK and SRC kinases (Steeg, 2006). In Ewing sarcoma, examples are PDGFR and EGFR signaling identified as metastasis-associated pathways, and AXL and PTK7 found over-expressed in a poor prognosis gene signature (Ohali et al., 2004; Schaefer et al., 2008). ERBB4 was shown to interact with E-cadherin to initiate FAK signaling, and therapeutic SRC inhibition impaired cell
migration and invasion (Mendoza-Naranjo et al., 2013; Shor et al., 2007). RTKs may therefore provide molecular targets also for metastasis-directed therapeutic strategies. Despite broad and confirmed molecular bases, resistances to RTK-targeted therapies occur. Including Ewing sarcoma, where subsequent clinical trials of IGF1R-targeted antibodies demonstrated no more than modest activity, and concluded that additional markers predictive of benefit were required (Juergens et al., 2011; Pappo et al., 2011). As one resistance mechanism to RTK-targeted strategies, signaling redundancies, cross-talk, and mutual compensation of signaling € nne et al., 2009; Lemmon and input have been identified (Ja Schlessinger, 2010; Stommel et al., 2007). In glioblastoma and lung cancer, resistance to EGFR-targeted agents was conferred by redundant IGF1R downstream signaling, as well as by compensatory MET amplification, maintaining ERBB3 signaling input (Chakravarti et al., 2002; Engelman et al., 2007; Jun et al., 2014). Vice versa, resistance of pediatric rhabdomyosarcoma (RMS) cell lines to IGF1R inhibitors was attributed to over-expression of EGFR and MET receptors. In keeping, in both RMS and Ewing sarcoma cell lines, IGF1R inhibitor sensitivity was restored by simultaneous silencing of RON, a MET family RTK (F. Huang et al., 2010; Potratz et al., 2010). RTK signaling hence emerges as an interdependent cellular network of therapeutic relevance. As this introduction points out, the family of RTKs makes for prime candidates of therapeutic targets, including metastasis-directed targets; with the draw-back of our to date insufficient understanding of confounding RTK network interactions. Objective of this work therefore was to provide a basis for systematic exploration of the RTK network in Ewing sarcoma. Comprehensive RTK gene expression profiles of Ewing sarcoma cell lines and untreated primary tumors were generated. To incorporate metastasis-specific aspects, profiles were characterized with respect to tumor origin from localized or metastatic disease.
2.
Methods
2.1.
Tumor samples and clinical data
21 samples of untreated Ewing sarcoma primary tumors were available. Diagnosis was based on standard histopathologic criteria and on EWS-FLI1 or EWS-ERG fusion transcripts. All patients were enrolled in clinical trials of the German Cooperative Ewing Sarcoma Study Group of the German Society of Pediatric Hematology and Oncology (GPOH), 19 patients into EURO-E.W.I.N.G.99 and one patient each into CESS86 and EICESS92, respectively. Trials were in compliance with the
M O L E C U L A R O N C O L O G Y 1 0 ( 2 0 1 6 ) 6 7 7 e6 9 2
Declaration of Helsinki, with approval of concerned ethics committees and patient’s written informed consent obtained before registration of patients. In this cohort, all patients with metastatic disease presented with metastasis not restricted to the lung, but to bone/bone marrow or other sites, defining them as high-risk R3 patients. In four cases diagnostic staging showed more than five bone metastases. In these cases tissue samples were from the diagnostic biopsy site. Clinical survival data were available for 20 of 21 patients and survival by age and by (high-risk) metastatic status as key prognostic factors reflected published cohorts (Figure A.1)(Ladenstein et al., 2010; Le Deley et al., 2014; Paulussen et al., 1998, 2008). For risk-group assignment and further characteristics see Table 1.
2.2.
Cell lines
Ewing sarcoma cell lines and Rh30 alveolar rhabdomyosarcoma cells were from the institutional cell line bank. A673 cell line derivates with stable shRNA silencing of EWS-FLI1 (EWS-FLI1-off ) or ERG control (EWS-FLI1-on) were kindly provided by Prof. S. Lessnick (Huntsman Cancer Centre, University of Utah) and previously described (Smith et al., 2006). HeLa cervix carcinoma, CAPAN-1 pancreatic adenocarcinoma, HL60 myeloid leukemia and 697 acute lymphoblastic leukemia (ALL) cell lines were from ATCC (Manassas, VA). All lines were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Darmstadt, Germany) and regularly tested to be free of mycoplasma contamination. MSC cultures were derived from tumor-negative bone marrow aspirates of five Ewing sarcoma, two rhabdomyosarcoma, and two leukemia patients. Mononuclear cells were isolated from bone marrow by Ficoll density gradient centrifugation and cultured in uncoated tissue culture flasks in DMEM medium (high glucose and pyruvate) supplemented with 10% FBS, 2 mM L-glutamin (Life Technologies, Darmstadt, Germany) and 3 ng/ml recombinant human basic fibroblast growth factor (PeproTech, Rocky Hill, NJ). Adherent MSC were selected by repetitive medium changes. Osteogenic and adipogenic differentiation potential was confirmed as previously described (Ern et al., 2010). Healthy-donor peripheral blood mononuclear cells (PBMC) were isolated accordingly and directly processed for studies. Fibroblasts from skin biopsies were kindly provided by Prof. H. Omran (University € nster). They were cultured in DMEM Children’s Hospital Mu medium supplemented with FBS and 2 mM L-glutamine (Life Technologies, Darmstadt, Germany). All primary cells were collected and utilized following informed consent and approval by ethical committees.
2.3.
RNA isolation and cDNA preparation
Tumor RNA was extracted as described (Schaefer et al., 2008). Cell line RNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). MSC RNA was harvested after 1e3 passages (14e30 days). A total of 2 mg of RNA were reversetranscribed using random hexamers and M-MLV reverse transcriptase according to the manufacturer’s protocol (Promega, Madison, WI).
2.4.
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Primers and probes
For listings of RTK families and abbreviations see Table A.1. RTK primers and probes were previously described and validated (Eurogentec, Seraing, Belgium) (Table A.2) € ller-Tidow et al., 2004). For Wnt5a analyses the A&B Taq(Mu Man Gene Expression Assay was used (Applied Biosystems, Foster City, CA).
2.5.
Real-time quantitative reverse transcription PCR
High-throughput qPCR analysis was done in 384-well plate format using the Tecan Genesis RP150 automated pipetting € nnedorf, Switzerland) and an ABIPrism 7900 HT system (Ma Sequence Detection System (PerkineElmer/Applied Biosystems, Foster City, CA). PCR reactions were performed in duplicate and contained 250 nM of each primer and probe in a final volume of 12 ml. Real-time PCR conditions were 50 C/ 2 min, 95 C/10 min, followed by 40 cycles of 95 C/15 s and 60 C/1 min cDNA concentrations of cell line samples were adjusted to Ct values of GAPDH housekeeping control gene to ensure equal PCR amplification efficiencies. Relative gene expression levels were calculated by DDCt-method compared to GAPDH and a calibrator sample containing cDNA of HeLa, HL60, and TC32 cells. cDNA concentrations of tumor samples were highly unequal. Therefore, concentrations remained unadjusted and relative gene expressions were calculated based on standard curves generated by serial dilutions of the calibrator cDNA using SDS 2.1 software (Applied Biosystems, Foster City, CA) and normalized to GAPDH.
2.6.
Western blotting
Procedures and buffers were previously described (Potratz et al., 2010). Antibodies were: ROR1 goat polyclonal (1:250; R&D Systems, Minneapolis, MN); PARP rabbit polyclonal; phospho-Ser473-AKT rabbit polyclonal (both 1:100; Cell Signaling; Beverly, MA); DVL3 mouse monoclonal (1:750); actin goat polyclonal antibody (1:1000; both Santa Cruz Biotechnology, Santa Cruz, CA). Secondary HRP-antibodies were from Santa Cruz Biotechnology (anti-goat, 1:3000), BD Pharmingen (anti-rabbit, 1:5000) (Franklin Lakes, NJ), and Cell Signaling (anti-mouse, 1:5000). Densitometric analyses were performed using ImageJ software (version 1.49v) (Schneider et al., 2012).
2.7.
Flow cytometry
Cells were stained with 0.4 mg of ROR1 polyclonal antibody (R&D Systems, Minneapolis, MN) or IgG isotype control (Jackson Immunoresearch, Baltimore, PA) for 30 min at 4 C. After washing, 0.75 mg of FITC-conjugated secondary antibody (rabbit anti-goat IgG; Jackson Immunoreseach, Baltimore, PA) were added and incubated for 10 min at room temperature. Following additional washes, stained cells were fixed in PBS containing 1% paraformaldehyde (PFA) and analyzed on a FACSCantoII flow cytometer (BD Bioscience, Franklin Lakes, NJ) using FACS Diva software.
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Table 1 e Patient characteristics. No.
Sex
Age [years]
Disease extension
1 2 3 4
EE99 CESS86 EICESS92 EE99
f m m f
13 17 12 9
Localized Localized Localized Localized
>200 NA
