Published OnlineFirst June 19, 2014; DOI: 10.1158/1078-0432.CCR-13-1486 Clinical Cancer Biology of Human Tumors Research PBX1 Is a Favorable Prognostic Biomarker as It Modulates 13-cis Retinoic Acid–Mediated Differentiation in Neuroblastoma Nilay Shah1, Jianjun Wang2, Julia Selich-Anderson1, Garrett Graham3, Hasan Siddiqui1, Xin Li4, Javed Khan2, and Jeffrey Toretsky3 Abstract Purpose: Neuroblastoma is an embryonic childhood cancer with high mortality. 13-cis retinoic acid (13- cisRA) improves survival for some patients, but many recur, suggesting clinical resistance. The mechanism of resistance and the normal differentiation pathway are poorly understood. Three–amino-acid loop extension (TALE) family genes are master regulators of differentiation. Because retinoids promote differentiation in neuroblastoma, we evaluated TALE family gene expression in neuroblastoma. Experimental Design: We evaluated expression of TALE family genes in RA-sensitive and -resistant neuroblastoma cell lines, with and without 13-cisRA treatment, identifying genes whose expression correlates with retinoid sensitivity. We evaluated the roles of one gene, PBX1, in neuroblastoma cell lines, including proliferation and differentiation. We evaluated PBX1 expression in primary human neuroblas- toma samples by qRT-PCR, and three independent clinical cohort microarray datasets. Results: We confirmed that induction of PBX1 expression, and no other TALE family genes, was associated with 13-cisRA responsiveness in neuroblastoma cell lines. Exogenous PBX1 expression in neuroblastoma cell lines, mimicking induced PBX1 expression, significantly impaired proliferation and anchorage-independent growth, and promoted RA-dependent and -independent differentiation. Reduced PBX1 protein levels produced an aggressive growth phenotype and RA resistance. PBX1 expression correlated with histologic neuroblastoma subtypes, with highest expression in benign ganglioneuromas and lowest in high-risk neuroblastomas. High PBX1 expression is prognostic of survival, including in multivariate analysis, in the three clinical cohorts. Conclusions: PBX1 is an essential regulator of differentiation in neuroblastoma and potentiates retinoid- induced differentiation. Neuroblastoma cells and tumors with low PBX1 expression have an immature phenotype with poorer prognosis, independent of other risk factors. Clin Cancer Res; 20(16); 1–13. Ó2014 AACR. Introduction sents numerous therapeutic challenges. Infant patients Neuroblastoma is the most common extracranial pedi- commonly present with low-grade disease, often with atric solid tumor, representing 8% of childhood cancer spontaneous regression, and some patients are managed diagnoses but 15% of childhood cancer-related deaths expectantly with observation never requiring surgery (2). (1). The diverse clinical spectrum of neuroblastoma pre- However, there are no validated biomarkers to predict which patients can be safely observed in contrast to those who would benefit from surgery and chemotherapy. Authors' Affiliations: 1Center for Childhood Cancer and Blood Diseases, In contrast, >40% of patients present at diagnosis with The Research Institute of Nationwide Children's Hospital and The Ohio aggressive disease and distant metastases (1). Patients with State University College of Medicine, Columbus, Ohio; 2Oncogenomics Section, Advanced Technology Center, Pediatric Oncology Branch, Center high-risk disease receive multiagent chemotherapy, surgery, for Cancer Research, National Cancer Institute, NIH, Gaithersburg, Mary- high-dose chemotherapy with autologous hematopoietic land; 3Department of Oncology, Lombardi Comprehensive Cancer Center; and 4Department of Biostatistics, Bioinformatics and Biomathematics, stem cell rescue (HDC-aHSCR), radiotherapy, immuno- Georgetown University, Washington, D.C. therapy with chimeric anti-GD2 antibody ch14.18, IL2, cis Note: Supplementary data for this article are available at Clinical Cancer and GM-CSF, and the differentiation agent 13- retinoic Research Online (http://clincancerres.aacrjournals.org/). acid (13-cisRA). Despite intensive treatment and improved Corresponding Author: Nilay Shah, Nationwide Children's Hospital, 700 responses with the introduction of immunotherapy (3), up Children's Drive, RBII WA4023, Columbus, OH 43205. Phone: 614-722- to 40% of high-risk patient tumors progress during induc- 2876; Fax: 614-355-5895; E-mail: [email protected] tion chemotherapy (4) and 5-year disease-free survival doi: 10.1158/1078-0432.CCR-13-1486 remains <50% (5). Identification of MYCN amplification Ó2014 American Association for Cancer Research. as a prognostic biomarker of aggressive disease radically www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst June 19, 2014; DOI: 10.1158/1078-0432.CCR-13-1486 Shah et al. tissue. These varying but important roles make it critical to Translational Relevance evaluate these genes specifically in neuroblastoma to define Neuroblastoma arises from neural crest progenitor their roles in oncogenesis. cells, but the errors in differentiation that drive tumor- Here, we perform the first comprehensive analysis of igenesis remain cryptic. We identify PBX1 as a putative TALE family gene expression in neuroblastoma. We dem- critical regulator of differentiation in neuroblastoma. onstrate that, among TALE family genes, only PBX1 expres- Clinically, neuroblastoma presents diversely in children. sion is associated with responsiveness to 13-cisRA in neu- The majority of tumors in infants undergo spontaneous roblastoma. We confirm that PBX1 induces neuronal dif- involution or differentiation, but some patients require ferentiation and increases sensitivity to 13-cisRA. Further- chemotherapy to be cured. Most patients greater than 18 more, we show that PBX1 expression is directly associated months old, in contrast, present with high-risk meta- with decreased proliferation independently of 13-cisRA. static disease. Some patients benefit from multimodal Finally, we demonstrate that PBX1 expression in primary treatment including 13-cisRA, but there are no clinically human tumors is associated with low tumor grade and validated biomarkers that predict benefit for high-risk patient survival. PBX1 expression may thus serve as a bio- patients. Patient tumors with low PBX1 expression may marker in low-risk disease by identifying patients who may indicate more aggressive disease in both low-risk and be observed without intervention. In high-risk disease, high-risk neuroblastoma. Our work supports validation PBX1 may stratify those patients for whom current therapies of PBX1 expression as a risk factor for treatment strati- are ineffective, directing them to novel therapies. fication in patients with low-risk and high-risk neuro- blastoma, and also supports further studies into the Materials and Methods PBX1 regulation of and targeted therapeutics for treat- Cell lines ment-resistant disease. Cell lines SK-N-SH, LAN-5, IMR-32, SK-N-BE (2), and SK- N-RA were obtained from Javed Khan; SMS-KAN and SMS- KANR from Joanna Kitlinska [via Children’s Oncology Group (COG) MTA with Georgetown University, Washing- improved risk stratification and treatment of neuroblasto- ton, D.C.]; NBL-WS and LAI-5S from Susan Cohn (Univer- ma (6). Since the seminal discovery of MYCN, additional sity of Chicago, Chicago, IL); SHSY5Y from ATCC; CHLA- clinical and genetic biomarkers have been identified, but 15, CHLA-42, CHLA-90, CHLA-136, and LAN-6 from COG connection of these factors to neuroblastoma biology or Cell Culture Repository; and HEK293 T from OpenBioSys- tumor aggression remains cryptic, particularly in tumors tems. All were previously characterized (22–28). A table of without MYCN amplification (7, 8). the MYCN status of each cell line is in the Supplementary Neuroblastoma is a prototypic embryonic cancer with Methods. CHLA-15, CHLA-42, CHLA-136, and CHLA-90 demonstrated aberrations in normal developmental path- were grown in Iscove’s modified Dulbecco’s medium with ways (9, 10). The HOX genes are master regulators of 20% FBS (Hyclone) and 0.1% insulin, transferrin, and development in animals; select HOX gene expression, selenium (Corning). All other cell lines were grown in RPMI including HOXC6 and HOXC9, has been associated with with 10% FBS. All parental and modulated cell lines were neuroblastoma differentiation, response to 13-cisRA, and tested and authenticated by PowerPlex16 short tandem outcome (11, 12). HOX proteins critically interact with repeat (STR) analysis (Promega) by the Nucleic Acids Core cofactors, including members of the three–amino-acid loop laboratory at Nationwide Children’s Hospital (NCH), last extension (TALE) gene family. The TALE family genes in December 2013, and January 2014. Note that the SK-N- (PBX1-4, MEIS1-3, and PKOX1-2), like HOX genes, are RA cell line was initially labeled as SK-N-AS when received, critical to tissue differentiation (13), including retinoid- but has been characterized by STR analysis to be the SK-N- induced differentiation (14). Expression of PBX1-3 is reg- RA cell line, including aliquots of the original cell line ulated by retinoids in other cell types (15) and has been received from the Khan lab, all generated modulated cell shown to direct endogenous retinoid synthesis within the lines, and the aliquots of the cell lines at the end of the nervous system (16). A comprehensive study of the expres- studies. Dr. Khan’s lab has confirmed that the cell line