Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL.
Emruli et al BMC Cancer (2016) 16:493 DOI 10.1186/s12885-016-2550-4 RESEARCH ARTICLE Open Access Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma Venera Kuci Emruli1, Roger Olsson2, Fredrik Ek2 and Sara Ek1* Abstract Background: Mantle cell lymphoma (MCL) is an aggressive disease with short median survival Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation Methods: An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway A functionally interesting target, vacuolar-type H+-ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry Results: We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11dependent activity Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced antiproliferative response in SOX11 low cells We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines Conclusions: We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo Keywords: V-ATPase, SOX11, Mantle cell lymphoma Background Mantle cell lymphoma (MCL) is an aggressive subtype of B cell lymphoma, with only year median survival [1] The disease is defined by the t(11;14) translocation, resulting in overexpression of the CCND1 gene and subsequent promotion of G1 to S cell cycle transition Additional specific disease marker include the neural * Correspondence: sara.ek@immun.lth.se Department of Immunotechnology, Lund University, Medicon Village, Scheelevägen 8, 223 87 Lund, Sweden Full list of author information is available at the end of the article transcription factor, SOX11, overexpressed in >95 % of MCL cases [2, 3] Despite recent major therapeutic progress, relapses are common and long-term survival remains poor, and novel targets with curative potential are sought among pathways important for MCL cell survival Functionally, MCL is characterized by a number of different genetic aberrations [4], and efforts have focused on targeting the constitutive NFĸB signaling [5], BTK [6] but also Wnt signaling Wnt signaling is of vital importance both for promotion of lymphomagenesis in © 2016 The Author(s) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Emruli et al BMC Cancer (2016) 16:493 MCL [7], but also for survival and evolution, as assessed by gene expression analysis [8, 9] In a previous siRNA screen, we identified the Wnt receptor FZD2 to be functionally active and affecting proliferation in MCL [10] Wnt is of importance in a wide variety of tumors and may be specifically interesting for development of therapies that target cancer stem cells [11], with limited off-target effects [12] This potential has recently been demonstrated in MCL where Wnt-targeting substances were particularly effective in eradication of lymphoma-initiating cells [13] We and others have shown that the neural transcriptional factor SOX11 is a highly specific diagnostic [2], functional [14–16], and prognostic antigen [17] SOX11 has been shown to act through a number of signaling pathways, including TGF-β signaling [14], plasmacytic differentiation [18], angiogenesis [19], but also Wnt [20] Homologous transcription factors to SOX11, the SOX C family, have also been shown to interact with Wnt [21] With the aim to identify novel targets for therapy in MCL through search at the intersection of SOX11/Wnt signaling, we performed a compound evaluation of an annotated library with 75 compounds interacting either as inhibitors or activators of the Wnt-signaling pathway, and investigated (i) the stand-alone effect and (ii) the SOX11-dependent effect on proliferation in MCL cells Results showed that among the evaluated 75 substances affecting Wnt-signaling, 15 compounds resulted in reduced proliferation in all the three different MCL cell lines evaluated Further, upon filtering for differential response in relation to SOX11 level, three substances directed to two different targets (V-ATPase and Dkk1) were identified Further validation studies were focused on substances targeting V-ATPase, and confirmed that both the V-ATPase specific inhibitors bafilomycin A1 and the analogue concanamycin A result in SOX11dependent growth reduction V-ATPase is a known regulator of intra- and extracellular pH, thus normal expression of this proton pump is of critical point for maintenance of ideal cellular pH [22] In this study, we show for the first time that V-ATPase inhibitors effectively reduce proliferation in MCL cells, are sensitive to SOX11 status and that V-ATPase is expressed on the surface of both primary MCL cells and cell lines, and thus an interesting therapeutic target Methods Cultivation of cell lines Three MCL cell lines, Z138, GRANTA-519 and JEKO-1, transfected with an inducible shRNA-vector were used to knock-down SOX11 through addition of μg/ml doxycycline (Sigma-Aldrich, Saint Louis, MO, USA) Briefly, cell lines were maintained as previously described [23] in tet-free R10 medium (RPMI-1640 (Life Technologies, Page of Grand Island, NY, USA) supplemented with 10 % tetapproved fetal bovine serum (Life Technologies) and 20 μM L-glutamine (Life Technologies)), and cultured under standard conditions (humidified atmosphere, % CO2, 37 °C) SOX11 protein expression was monitored over time by flow cytometry analysis, performed as previously described [24] Doxycycline was used to induce down-regulation of SOX11 Thus, SOX11 high cells are referred to as non-induced (SOX11IND-) and SOX11 low cells as induced (SOX11IND+) SOX11IND- cells express similar SOX11 level compared to non-transfected, wildtype cells All cell cultures were kept in log phase, at a density of 0.8–2 × 106 cells/ml Molecular substances and reagents Wnt pathway small molecule library was purchased from Enzo Life Sciences (BML-2838), dissolved in DMSO (10 mM) and stored at −80 °C Upon treatment of cells, the small molecules were resuspended and diluted in tet-free R10 medium and used immediately, or stored at +8 °C and consumed within a week Individual compounds bafilomycin A1 (ALX-380-063-M001) and concanamycin A (ALX-380-034-C025) were purchased from Enzo Life Sciences (Farmingdale, NY, USA) as dry powders and dissolved in DMSO Assessment of proliferation using thymidine incorporation Fifty thousand cells per well were plated in 96 well Cytostar-T plates (PerkinElmer, Waltham, MA, USA) and cultured for 24 h prior to treatment with a library of compounds interacting with the Wnt pathway (n = 75), at concentrations of 0.5, 2, and 10 μM Cell proliferation was evaluated 0, 24, 48 and 72 h after addition of small molecules, by measuring incorporation of [14C]thymidine (PerkinElmer) using a Wallac 1450 MicroBeta liquid scintillation counter (PerkinElmer) The relative proliferation is presented as relative to the non-treated control, at the specific time-point Each data-point is represented by a minimum of three replicates (Figs and 2) Cell viability measurements using Cell Titer Glow Fifty thousand cells per well were plated out in 96 well assay plates and cultured for 24 h before treatment with Wnt-targeting small molecule library (n = 75) for up to 48 h On the day of measurement, cells were equilibrated for 30 at room temperature, treated with CellTiterGlo reagent (Promega, Madison, WI, USA), mixed and allowed to equilibrate for additional 10 min, before the luminescent signal was recorded using a FLOUstar Omega (BMG Labtech, Ortenberg, Germany) instrument Transient knock-down of V-ATPase To obtain co-knock of V-ATPase and SOX11, Z138 SOX11IND+/SOX11IND- cells were transfected using an Emruli et al BMC Cancer (2016) 16:493 Fig V-ATPase is sensitive to SOX11 status Assessment of cell proliferation in stably transfected Z138 cells treated with different concentrations of (a) bafilomycin A1, and (b) concanamycin A, for 24 h Mean values (n = biological replicates per group) are normalized against corresponding non-treated control samples, and error bars indicate SEM The significance was determined by Student’s t-test (*P