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The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration

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The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1752 Cancer Microenvironment and Immunology Research The Planar Cell Polarity Pathway Drives Pathogenesis of Chronic Lymphocytic Leukemia by the Regulation of B-Lymphocyte Migration Marketa Kaucka1, Karla Plevova2,4, Šarka Pavlova2,4, Pavlína Janovska1, Archana Mishra1, Jan Verner2,4, Jirina Prochazkov a1, Pavel Krejcí 1,5,6, Jana Kotaškova2,4, Petra Ovesna3, Boris Tichy2,4, Yvona Brychtova4, Michael Doubek2,4, Alois Kozubík1,5,Jirí Mayer2,4, Šarka Pospíšilova2,4, and Vítezslav Bryja1,5 Abstract The planar cell polarity (PCP) pathway is a conserved pathway that regulates cell migration and polarity in various contexts. Here we show that key PCP pathway components such as Vangl2, Celsr1, Prickle1, FZD3, FZD7, Dvl2, Dvl3, and casein kinase 1 (CK1)-e are upregulated in B lymphocytes of patients with chronic lymphocytic leukemia (CLL). Elevated levels of PCP proteins accumulate in advanced stages of the disease. Here, we show that PCP pathway is required for the migration and transendothelial invasion of CLL cells and that patients with high expression of PCP genes, FZD3, FZD7, and PRICKLE1, have a less favorable clinical prognosis. Our findings establish that the PCP pathway acts as an important regulator of CLL cell migration and invasion. PCP proteins represent an important class of molecules regulating pathogenic interaction of CLL cells with their microen- vironment. Cancer Res; 73(5); 1–11. Ó2012 AACR. Introduction proteins) are conserved in evolution and include Vang-like Wnt signaling pathways are crucial for cell-to-cell commu- protein 2 (Vangl2), cadherin EGF LAG 7-pass transmembrane nication, differentiation, and morphogenesis in embryonic G-type receptor (Celsr1), Frizzleds (FZD, in mammals mainly development. Dysfunction or deregulation of Wnt signaling FZD3, FZD6, and FZD7), which act as receptors, and cyto- accounts for a number of developmental defects, inherited plasmic components such as Disheveled (Dvl1-3 in mammals), diseases, and many types of cancer (1). The best-known Wnt Prickle-like proteins (mainly Prickle1), casein kinase 1e (CK1e), pathway—canonical or Wnt/b-catenin pathway—induces and the small GTPases Rho/Rac and downstream kinases b-catenin–dependent activation of T-cell factor/lymphoid ROCK/JNK. PCP proteins regulate cell polarity (3), convergent enhancer factor (TCF/LEF)-mediated transcription. However, extension movements during gastrulation and neurulation, Wnt ligands can activate other so-called noncanonical Wnt and polarity of the hair cells in the inner ear (3, 4). pathways, which are b-catenin–independent and biochemi- In the present study, we investigated the role of PCP proteins cally distinct from the canonical Wnt pathway (2). Among in chronic lymphocytic leukemia (CLL). CLL is characterized þ those, the pathway that regulates planar cell polarity (Wnt/ by clonal expansion and apoptosis dysregulation of CD5 B PCP pathway) is the best known (3). The core components of lymphocytes. Neoplastic CLL cells accumulate in blood, bone the Wnt/PCP pathway (referred here as PCP or polarity marrow, and lymphoid tissue. It is recognized that cell migra- tion and recirculation between peripheral blood and lymphoid niches in bone marrow and lymphoid tissue are important factors contributing to CLL biology (5–7) and that alterations 1 Authors' Affiliations: Institute of Experimental Biology, Faculty of Sci- in survival and proliferation of CLL are affected by their ence, 2CEITEC - Central European Institute of Technology, 3Institute of Biostatistics and Analyses, Masaryk University; 4Center of Molecular interaction with the microenvironment. Here, we show for the Biology and Gene Therapy, Department of Internal Medicine–Hematology first time that the core cassette of the PCP pathway composed and Oncology, University Hospital Brno and Medical Faculty MU; 5Depart- ment of Cytokinetics, Institute of Biophysics, Academy of Sciences of the of Celsr1, Prickle1, Vangl2, Dvl2, Dvl3, FZD3, and FZD7 is Czech Republic, Brno, Czech Republic; and 6Medical Genetics Institute, upregulated in B cells of patients with CLLs. We show that Cedars-Sinai Medical Center, Los Angeles, California PCP proteins, which are conserved regulators of the cell–cell Note: Supplementary data for this article are available at Cancer Research interaction and cell migration, are important mediators of CLL Online (http://cancerres.aacrjournals.org/). chemotactic responses and homing. K. Plevova and Š. Pavlova contributed equally to this work. Materials and Methods Corresponding Author: Vitezslav Bryja, Institute of Biophysics v.v.i., fi Academy of Sciences of the Czech Republic, Kralovopolska 135, Brno Cell isolation, puri cation, and cell treatments CZ-612 65, Czech Republic. Phone: 420-549493291; Fax: 420- Primary B cells from previously untreated patients with 541211214; E-mail: [email protected] CLLs or healthy volunteers were separated using gradient doi: 10.1158/0008-5472.CAN-12-1752 centrifugation followed by non–B-cell depletion (RosetteSep Ó2012 American Association for Cancer Research. B Cell Enrichment Kit and Human CD3þ Depletion Kit; www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst January 21, 2013; DOI: 10.1158/0008-5472.CAN-12-1752 Kaucka et al. StemCell Technologies; or MACS B cell Isolation Kit II; Miltenyi subsets of patients with CLLs. To confirm that the levels of Biotec) according to the manufacturer's instructions. Flow PCP components are indeed altered in CLLs, we collected cytometry was conducted for evaluation of CD5 and CD19 RNA samples from purified peripheral blood B cells (CD19þ expression on purified cells (tricolor panel: CD45-TRI-COLOR, cell population purity 95%) of control individuals (n ¼ 6) MHCD45065, Invitrogen, CD5-FITC, A08932, CD19-PE, A07769, and patients with CLL (n ¼ 93). qRT-PCR analysis showed Beckman Coulter). For further analyses, samples with the statistically increased mRNA levels of 3 PCP genes, CELSR1, purity higher than 95% B cells were used. The cell line MEC1 PRICKLE1, and FZD7, in CLL samples and identified that 2 was obtained from DSMZ, cell line was tested by DSMZ using other genes VANGL2 and FZD3 have high levels in a subset molecular genetic methods; MEC1 cells were cultured in RPMI- of patients with CLLs [Fig. 1A—normalized to the expression 1640 supplemented with 10% FBS and antibiotics at 37oC and of b2-microglobulin; Supplementary Fig. S1A—normalized to 5% CO2. b-actin]. Subsequent analysis of the protein level by Western blotting Transwell assay confirmed increased protein expression of the PCP proteins The chemotaxis assay was conducted in HTS Transwell-96 Vangl2, Dvl2, Dvl3, Prickle1, and CK1e (Fig. 1B, quantified well plates (Corning Incorporated) with 5.0-mm pore size in Fig. 1C) in patients with CLLs. The level of b-catenin did polycarbonate membranes following the manufacturer's not differ between CLLs and healthy cells (Fig. 1B), and we instructions. A total of 0.5 Â 106 cells (primary CLL or MEC1 failed to detect activated (dephosphorylated) b-catenin (not cells) were seeded in the Transwell upper insert, which was shown), which suggest that the Wnt/b-catenin pathway is either nontreated or coated with a human umbilical vein not active in CLL cells. All CLL samples tested had high ex- endothelial cells monolayer (HUVEC). Cells in the insert were pression of LEF1 and Ror1 (Fig. 1B), which were previously treated with antibodies or inhibitors as described in Supple- identified as abundantly and consistently expressed in CLL mentary Material. Cells were incubated overnight in full medi- cells (9, 10). Flow cytometry detection (Fig. 1D) further con- o um (including 10% fetal calf serum) at 37 C and 5% CO2, and firmed increased cell surface levels of Ror1, FZD3, Celsr1, and after 18 hours the migration toward the chemokine was Vangl2 in comparison to healthy B cells. analyzed by a Coulter Counter (model FN, Coulter Next, we asked whether the levels of PCP proteins are Electronics; Figs. 2A–C, 3A–D, and 4A–C; Supplementary Figs. stable or they change during the disease course. When we S2B and S4E–S4H) or by C6 flow cytometer (Accuri; Figs. 2E– compared samples of 6 patients with CLLs collected at 2 G, 3E–G, and 4D; Supplementary Fig. S2E). The migration index different time points (t1 and t2;Fig.1E),wewereableto was calculated as the number of cells (treated or untreated) detect changes in the levels of PCP proteins in CLL cells from migrating in response to the chemokine divided by the number individual time points. Specifically, we observed a clear of cells migrating toward the control medium only. In case of increase in Dvl2, Dvl3, and Prickle1 levels in patients fol- very variable migration indexes (in primary CLL cells), cell lowed in time, which is quantified in Supplementary Fig. numbers were normalized to the chemokine-only condition S1B. The most prominent changes were observed mainly in and expressed as "relative migration." patients, who progressed from Rai stages (11) 0/I/II to the stage III or IV (see patients C, H, P). The levels of LEF1 and In vivo experiments b-catenin do not change (Fig. 1E). Nonirradiated mice (8- to 16-week-old) nonobese diabetic/ In summary, using several methods, we provide evidence severe combined immunodeficient (NOD/SCID) IL2Rg-null that a host of PCP proteins are overexpressed in B cells of (NSG) mice were used for transplantation experiments. Freshly patients with
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