(CD147) Is Induced by C/Ebpβ and Is Differentially Expressed in ALK+
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Laboratory Investigation (2017) 97, 1095–1102 © 2017 USCAP, Inc All rights reserved 0023-6837/17 EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK − anaplastic large-cell lymphoma Janine Schmidt1, Irina Bonzheim1, Julia Steinhilber1, Ivonne A Montes-Mojarro1, Carlos Ortiz-Hidalgo2, Wolfram Klapper3, Falko Fend1 and Leticia Quintanilla-Martínez1 Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK − cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK − ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK − ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as T-cell activation and proliferation suggest its involvement in the pathogenesis of ALCL. Laboratory Investigation (2017) 97, 1095–1102; doi:10.1038/labinvest.2017.54; published online 5 June 2017 Anaplastic lymphoma kinase-positive (ALK+) anaplastic ALCL.3–6 C/EBPβ belongs to the CCAAT/enhancer binding large-cell lymphoma (ALCL) is characterized by the translo- protein family of transcription factors and members of this cation t(2;5)(p23;q35) that juxtaposes the nucleophosmin family have been related to cellular processes such as (NPM) gene to the ALK gene leading to the generation of the differentiation of adipocytes and cells of the myelomonocytic chimeric protein NPM-ALK.1 Several signaling pathways, lineage,7,8 control of metabolism,9 inflammation,10,11 and including the JAK/STAT (Janus kinase/signal transducer and cellular proliferation.12 In particular, C/EBPβ has been shown activator of transcription) pathway, are activated by the to play a role in the activation and differentiation of constitutive expression of the NPM-ALK fusion protein, macrophages.13,14 In this context, it is important to note that leading to proliferation, prolonged tumor cell survival, C/EBPβ is capable of transdifferentiating B-cell and T-cell cytoskeletal rearrangement, and cell migration.2 A central progenitors in vitro, leading to loss of B- or T-cell-specific target gene of the JAK/STAT signaling pathway is the markers and to a rapid and efficient reprogramming of these transcription factor C/EBPβ, which is overexpressed in ALK+ cells to macrophages.15,16 1Institute of Pathology and Neuropathology and Comprehensive Cancer Center Tübingen, University Hospital Tübingen, Eberhard-Karls-University, Tübingen, Germany; 2Departamento de Patología, Centro Médico ABC (The American British Cowdray Medical Center), Mexico City, Mexico and 3Hematopathology Section, University Hospital Schleswig-Holstein Campus Kiel/Christian-Albrechts University Kiel, Kiel, Germany Correspondence: Professor L Quintanilla-Martinez, MD, Institute of Pathology and Neuropathology and Comprehensive Cancer Center Tübingen, University Hospital Tübingen, Eberhard-Karls-University, Liebermeisterstr. 8, 72076 Tübingen, Germany. E-mail: [email protected] Received 9 December 2016; revised 12 April 2017; accepted 14 April 2017 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 97 September 2017 1095 EMMPRIN expression in ALCL J Schmidt et al ALCL cells are supposed to derive from activated T cells, as diaminobenzidine detection kit (Ventana). ALCL primary indicated by the presence of clonal TCR-gene rearrangements cases had been previously studied in paraffin sections with a and the expression of interleukin-2 receptor alpha (CD25) panel of antibodies to assess tumor cell phenotype. All cases and CD30, a member of the tumor necrosis factor receptor were stained for CD30 (Dako, Glostrup, Denmark, dilution superfamily.17,18 Despite the T-cell genotype, ALK+ ALCL 1:50), ALK (Dako, dilution 1:400), C/EBPβ (C-19; Santa Cruz frequently demonstrates an unusual phenotype with loss of Biotechnology, Santa Cruz, CA, USA, dilution 1:200), and most T-cell markers, of the T-cell receptor (TCR) and CD147 (MEM-M6/1; Abcam, Cambridge, UK, dilution associated molecules,19 as well as the unusual expression of 1:1500). Eighty-one ALK+ ALCL cases were stained from mono- and histiocytic surface antigens. The monocytic tissue microarrays, whereas 34 cases were stained from whole markers CD13 and CD33 as well as CD68, and rarely the sections. In addition, as control for the normal stain of myeloid antigen CD15 have been detected in ALCL cells20,21 CD147, three tonsils and two lymph nodes were used. In and their expression is more prevalent in ALK+ ALCL.22 addition, 22 cases were stained for PD-L1 (28-8, Abcam, On the basis of the central role of C/EBPβ in ALK+ ALCL dilution 1:50). The use of human tissue samples was approved pathogenesis and on the fact that C/EBPβ has the potential to by the local ethics committee of the University of Tübingen transdifferentiate thymocytes to macrophages, we wanted to (approval 620/2011BO2). investigate whether the overexpression of C/EBPβ is respon- sible for the unique phenotype of ALCL. Therefore, extensive Virus Production and Lentiviral Infection surface antigen expression profiles of two ALK+ ALCL cell For production of the replication-defective lentiviral virions, lines with and without specific knockdown of C/EBPβ were the packaging cell line HEK293T was co-transfected with the generated and selected antigens further examined in primary packaging plasmid pCMVΔR8.9, the envelope plasmid ALCL cases. containing the G-Protein of vesicular stomatitis virus and the transfer vector (pFUGW).24 The transfer vector includes MATERIALS AND METHODS the reporter gene EGFP driven by an internal ubiquitine-C Cell Culture and Patient Samples promoter and the specific shRNA under the control of the The ALK+ ALCL (t(2;5)(p23;q35)-positive) cell lines human H1 promoter. The pFUGW derivate vectors with the SUDHL-1, SUP-M2, and SR786; the ALK − ALCL cell line specific C/EBPβ-shRNA inserted were named ‘pF-C/EBPβ,’ Mac-1; the mantle cell lymphoma cell lines Rec-1, Jeko-1, and vectors without the shRNA insert were called ‘pF-S.’ The Granta; the promyelocytic leukemia cell line HL-60; the lentiviral infection of KiJK and Karpas 299 cells (1 × 106 cells diffuse large B-cell lymphoma cell line SUDHL-4; the per ml) was performed in 12-well plates in the presence of multiple myeloma cell line KMS-12; the T-cell acute Polybren (8 μg/ml; Sigma, St Luis, MO, USA). Cells were lymphoblastic lymphoma cell line Jurkat; and the Burkitt centrifuged at 800 g and 32 °C for 90 min. Subsequently the lymphoma cell line Raji were cultured as previously cells were incubated for 24 h at 37 °C in a CO2 incubator. described.3 HeLa and HEK293T cells were cultured in DMEM (Invitrogen GmbH, Darmstadt, Germany) supple- Cytofluorimetric Analysis of Infected Cells mented with 10% fetal bovine serum (FCS; PAA Laboratories Three days after infection, transduction efficiency and cell GmbH, Pasching, Austria) and 100 μg/ml penicillin/strepto- viability of infected cells and control cells were analyzed by mycin (Invitrogen). One hundred fifteen formalin-fixed, flow cytometry. Cells were washed in phosphate-buffered paraffin-embedded ALCL primary samples (97 ALK+ and saline (PBS; Applied Biosystems Foster City, CA, USA) and 18 ALK − ) were collected from the files of the Institute of resuspended in PBS with 5% FCS and 1 μg/ml Propidium Pathology and Neuropathology, University Hospital Tübin- iodide (PI; Sigma, ST. Luis, MO, USA). Infected cells were gen, Germany, Institute of Pathology, University of Kiel, detected by means of GFP positivity and the percentage of Germany, and Department of Pathology, the American dead cells was determined using PI. Data were evaluated with British Cowdray Medical Center, Mexico City, Mexico. All WinMDI 2.9 software. cases were comprehensively immunophenotyped, as part of the diagnostic work-up, and were classified following the RNA Isolation and Real-Time Quantitative PCR recommendations of the World Health