Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells

Mantle cell lymphoma (MCL) is a hematologic neoplasm characterised by the t(11;14)(q13;q32) translocation leading to aberrant cyclin D1 expression. The cell functions of cyclin D1 depend on its partners and/or subcellular distribution, resulting in different oncogenic properties. We observed the accumulation of cyclin D1 in the cytoplasm of a subset of MCL cell lines and primary cells. In primary cells, this cytoplasmic distribution was correlated with a more frequent blastoid phenotype. We performed immunoprecipitation assays and mass spectrometry on enriched cytosolic fractions from two cell lines. The cyclin D1 interactome was found to include several factors involved in adhesion, migration and invasion. We found that the accumulation of cyclin D1 in the cytoplasm was associated with higher levels of migration and invasiveness. We also showed that MCL cells with high cytoplasmic levels of cyclin D1 engrafted more rapidly into the bone marrow, spleen, and brain in immunodeficient mice. Both migration and invasion processes, both in vivo and in vitro, were counteracted by the exportin 1 inhibitor KPT-330, which retains cyclin D1 in the nucleus. Our data reveal a role of cytoplasmic cyclin D1 in the control of MCL cell migration and invasion, and as a true operator of MCL pathogenesis.


Cell adhesion on fibronectin or HS-5 stromal cells was assessed with the Vybrant™ Cell
Adhesion Assay Kit (V-13181, Molecular Probes) as previously described 2 .

Construction of GFP/mCherry and luciferase-expressing MCL cell lines
Phoenix™ Ampho cells and 293T cells were maintained in DMEM supplemented with 10% FBS at 37°C, under an atmosphere containing 5% CO2 . Retroviral particles were generated by plating 3 × 10 6 Phoenix™ cells per 10 cm dish. On the day after plating, cells were transfected with 10 μg of pMSCV-Luc2-PKG-Neo-IRES-GFP plasmid 3

Immunoprecipitation of cyclin D1-interacting proteins and separation by electrophoresis
We obtained cell extracts enriched in cytosolic proteins by lysing cultured JeKo1 and U266 cells (5 x 10 7 cells) in an IP buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP40, 1 mM sodium orthovanadate and a cocktail of protease inhibitors (P-8340, Sigma-Aldrich). We then subjected these extracts (5 mg of protein) to immunoprecipitation with 10 μg of anti-cyclin D1 Ab (sc-718, Santa Cruz Biotechnology). This Ab is directed against the canonical form of cyclin D1 expressed in MCL and some MM cells. Samples were diluted in detergent-free Laemmli buffer and proteins were separated by SDS-PAGE in a 12% precast gel (Gebagel; Gene Bio-Applications, Paris, France). The gel was fixed by incubation in 30% ethanol, 10% acetic acid for 15 min, washed in MilliQ water for 15 min and stained with Coomassie blue, with the EZBlue gel-staining reagent (Sigma-Aldrich).

Trypsin digestion of proteins
Gel lanes were manually cut into 20 pieces, which were then processed by two successive rounds of washing in 50 mM NH4HCO3 in ACN (acetonitrile)/H2O (v/v), rehydration in 100 mM NH4HCO3, and dehydration in 100% ACN. Gel slices were subjected to reduction in 65 mM DTT (dithiothreitol) at 37°C, followed by alkylation in 135 mM iodoacetamide in the dark at room temperature. Gel slices were then washed/dehydrated/rehydrated as before and subjected to trypsin digestion (modified trypsin from Promega France, 4 ng/ml in 50 mM NH4HCO3) overnight at 37°C. The digested peptides were then extracted in two successive steps involving the addition of ACN/H2O/trifluoroacetic acid (TFA) (70/30/1). Supernatants were collected and concentrated in a SpeedVac.

Mass spectrometry analysis
Mass spectrometry (MS) was performed with a nanoflow high-performance liquid chromatography (HPLC) system (LC Packings Ultimate 3000, Dionex) connected to a hybrid LTQ-OrbiTrap XL (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (New Objective, Woburn). Mobile phases A (99.9 % MilliQ water and 0.1% formic acid (v:v)) and B (99.9% acetonitrile and 0.1% formic acid (v:v)) for HPLC were delivered by the Ultimate 3000 nanoflow LC system (LC Packings, Dionex). We loaded 10 µl of prepared peptide mixture onto a trapping precolumn (5 mm × 300 μm i.d., 300 Å pore size, Pepmap C18, 5 μm), through which 2% buffer B was passed for three minutes, at a flow rate of 25 µl/min. This step was followed by reverse-phase separations at a flow rate of 0.250 µl/minute on an analytical column (15 cm × 300 μm i.d., 300 Å pore size, Pepmap C18, 5 μm, Dionex). A gradient of 2% to 90% buffer B was passed through the column for 105 min. The column was then washed with 90% buffer B for 16 min, and with 2% buffer B for 19 min before the loading of the next sample. Peptides were detected by direct elution from the HPLC column into the electrospray ion source of the mass spectrometer. An ESI voltage of 1.5 kV was applied to the HPLC buffer via the liquid junction provided by the nanoelectrospray ion source, and the ion transfer tube temperature was set at 200°C.
The MS instrument was operated in its data-dependent mode, with automatic switching between full-scan survey MS and consecutive MS/MS acquisition. Full-scan survey MS spectra (mass range 400 -2000) were acquired in the OrbiTrap section of the instrument, with a resolution of R = 60,000 at m/z 400; ion injection times were calculated for each spectrum so as to allow the accumulation of 10 6 ions in the OrbiTrap. The seven peptide ions yielding the most intense signals in each survey scan, with an intensity above 2,000 counts (to avoid triggering fragmentation too early during the peptide elution profile) and a charge state ≥ 2, were sequentially isolated at a target value of 10,000 and fragmented in the linear ion trap by collision-induced dissociation (CID). Normalised collision energy was set to 35%, with an activation time of 30 ms. Peaks selected for fragmentation were automatically placed on a dynamic exclusion list for 120 s, with a mass tolerance of ± 10 ppm to avoid prevent selection of the same ion for fragmentation more than once. The following parameters were used: the repeat count was set to 1, the exclusion list size limit was 500, singly charged precursors were rejected and the maximum injection time was set at 500 ms and 300 ms for full MS and MS/MS scan events, respectively. For an optimal duty cycle, the fragment ion spectra were recorded on the LTQ mass spectrometer in parallel with OrbiTrap full-scan detection. For OrbiTrap measurements, external calibration was performed before each injection series, to ensure an overall mass accuracy error of less than 5 ppm for the detected peptides. MS data were saved in a RAW file format, with XCalibur 2.0.7 and tune 2.4 (Thermo Fisher Scientific).

Data processing and identification of peptides and proteins
Data were analysed with Proteome Discoverer 1.2 software supported by Mascot  (Table S1 and S2).

Datamining
For the validation of mass spectrometry results, the two protein lists generated were compared with each other and with the list of proteins interacting with cyclin D1 in the Granta MCL cell line 6 . The proteins present in the three cell lines were then selected and analysed for gene ontology (GO) clustering, with DAVID Bioinformatics Resources 7 . For analysis of the cyclin D1 interactome in JeKo1 cells, we selected the 200 proteins with the highest peptide coverage percentages (Table S4) for further analysis with DAVID tools and then with the PANTHER database 8 , to determine their cellular function, associated biological process, cell component, and protein class. Possible protein-protein interactions were sought by using the selected 51 cytoskeleton-associated proteins to query the STRING database 9 . The data are summarized as a network showing current interactions, with network nodes representing proteins, and edges representing protein/protein interactions. Supplementary Fig. S1 A, cultured MCL cells were harvested. Nuclear (n) and cytosolic (c) protein extracts were prepared with the BioVision kit. The purified proteins were separated by SDS-PAGE and analysed by IB with the Abs indicated. The purity of each fraction was checked with Abs against PARP and BiP with these proteins also used as controls for gel loading. B, tumour cells from the indicated patients were purified. Nuclear and cytoplasmic fractions were obtained, separated by SDS-PAGE and the proteins were transferred onto nitrocellulose membranes. The membranes were incubated with the indicated Abs, which were also used to determine extract purity. C, proteins purified from JeKo1 cells were immunoprecipitated with an anticyclin D1 Ab. The bound (b) and unbound (u) fractions were resolved by SDS-PAGE and analysed by IB with anti-cyclin D1, anti-α-tubulin, and anti-β-actin Abs. An aliquot of the purified protein preparation (1/10) was analysed directly (input). D, RAMOS cells were transduced with 250 ng/ml TAT-cyclin D1 protein for 6 h (or treated with 0.9% NaCl as a control) and analysed by transmission electronic microscopy, as previously described 1 . The corresponding scale bars are indicated on each image. Supplementary Fig. S2 A, we coated 96-well plates with fibronectin (FBN) or used them as culture plates for HS-5 stromal cells. MCL cells were stained with calcein-AM and allowed to adhere to substrates for 4 h. Fluorescence was recorded before and after thorough washing, and the percentage of adherent cells was calculated and plotted on the graph (means ± s.d. for three independent experiments carried out in triplicate). B, cultured MCL cells were cytospun, fixed and permeabilized. They were then stained with an anti-XPO1 (sc-5595, Santa Cruz Biotechnologies) primary Ab and an AlexaFluor 488conjugated goat anti-rabbit IgG (Life Technologies) secondary Ab. Nuclei were counterstained with DAPI. Cells were analysed by confocal microscopy (Fluoview FV 1000 confocal microscope and Fluoview Viewer software, Olympus) at x 180 magnification. Whole-cell extracts were obtained and the proteins they contained were separated by electrophoresis and analysed by IB with the same anti-XPO1 Ab and an anti-β-actin Ab as a control. C, JeKo1 and Z138 cells were treated with 2 μM palbociclib (+) or vehicle (-) for 24 h and assayed for chemotaxis. Triplicate samples from two independent experiments were analysed. The percentages of migrating cells in palbociclib-treated samples and vehicle-treated samples were calculated. The means ± s.d. are plotted on the graph. ***, p < 0.001. Tables   Table S1. List of the proteins identified as interacting with cyclin D1 in JeKo1 cells (Table   S1_R1.xls) Table S2. List of the proteins identified as interacting with cyclin D1 in U266 cells (Table   S2_R1.xls)