Endocytosis of very low-density lipoproteins: an unexpected mechanism for lipid acquisition by breast cancer cells

We previously described the expression of CD36 and LPL by breast cancer (BC) cells and tissues and the growth-promoting effect of VLDL observed only in the presence of LPL. We now report a model in which LPL is bound to a heparan sulfate proteoglycan motif on the BC cell surface and acts in concert with the VLDL receptor to internalize VLDLs via receptor-mediated endocytosis. We also demonstrate that gene-expression programs for lipid synthesis versus uptake respond robustly to triglyceride-rich lipoprotein availability. The literature emphasizes de novo FA synthesis and exogenous free FA uptake using CD36 as paramount mechanisms for lipid acquisition by cancer cells. We find that the uptake of intact lipoproteins is also an important mechanism for lipid acquisition and that the relative reliance on lipid synthesis versus uptake varies among BC cell lines and in response to VLDL availability. This metabolic plasticity has important implications for the development of therapies aimed at the lipid dependence of many types of cancer, in that the inhibition of FA synthesis may elicit compensatory upregulation of lipid uptake. Moreover, the mechanism that we have elucidated provides a direct connection between dietary fat and tumor biology.­. We previously described the expression of CD36 and LPL by breast cancer (BC) cells and tissues and the growth-promoting effect of VLDL observed only in the presence of LPL. We now report a model in which LPL is bound to a heparan sulfate proteoglycan motif on the BC cell surface and acts in concert with the VLDL receptor to internalize VLDLs via receptor-mediated endocytosis. We also demonstrate that gene-expression programs for lipid synthesis versus uptake respond robustly to triglyceride-rich lipoprotein availability. The literature emphasizes de novo FA synthesis and exogenous free FA uptake using CD36 as paramount mechanisms for lipid acquisition by cancer cells. We find that the uptake of intact lipoproteins is also an important mechanism for lipid acquisition and that the relative reliance on lipid synthesis versus uptake varies among BC cell lines and in response to VLDL availability. This metabolic plasticity has important implications for the development of therapies aimed at the lipid dependence of many types of cancer, in that the inhibition of FA synthesis may elicit compensatory upregulation of lipid uptake. Moreover, the mechanism that we have elucidated provides a direct connection between dietary fat and tumor biology.­. The dependence of several tumor cell types, including breast cancer (BC), on a supply of FAs to maintain proliferation is well established (1Menendez J.A. Lupu R. Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis.Nat. Rev. Cancer. 2007; 7: 763-777Crossref PubMed Scopus (2024) Google Scholar). The literature emphasizes de novo lipid synthesis, and more recently, FFA uptake via CD36, as the mechanisms used to satisfy this dependency. De novo lipogenesis produces the saturated long-chain FA palmitate (C16:0), most of which is used for plasma membrane phospholipid synthesis. The low rates of FA synthesis observed in most nonmalignant tissues has positioned fatty acid synthase (FASN) as a therapeutic target, prompting efforts to develop FASN inhibitors (2Kinlaw W.B. Baures P. Lupien L. Davis W. Kuemmerle N. Fatty acids and breast cancer: make them on site or have them delivered.J. Cell. Physiol. 2016; 231: 2128-2141Crossref PubMed Scopus (36) Google Scholar). In this context, we and others have observed that the provision of exogenous FFAs to cancer cells permits evasion of the cytotoxic effects of FASN inhibition, revealing the ability of cancer cells to take up, as well as synthesize, lipids (3Kuemmerle N.B. Rysman E. Lombardo P.S. Flanagan A.J. Lipe B.C. Wells W.A. Pettus J.R. Froehlich H.M. Memoli V.A. Morganelli P.M. et al.Lipoprotein lipase links dietary fat to solid tumor cell proliferation.Mol. Cancer Ther. 2011; 10: 427-436Crossref PubMed Scopus (184) Google Scholar, 4Zaidi N Lupien L Kuemmerle N Kinlaw W Swinnen J Smans K. Lipogenesis and lipolysis: the pathways exploited by the cancer cells to acquire fatty acids.Prog. Lipid Res. 2013; 52: 585-589Crossref PubMed Scopus (308) Google Scholar). We further demonstrated that BC cells and clinical BC tissues robustly express LPL, the principal enzyme for the hydrolysis of triglyceride (TG) carried in lipoproteins, as well as the cell-surface channel for FFA uptake, CD36. Others have begun to highlight the presence of LPL in and on the surface of cancer cells, the most prominent example being in chronic lymphocytic leukemia, where evidence supports LPL as a strong prognostic biomarker (5Kristensen L. Kristensen T. Abildgaard N. Royo C. Frederiksen M. Mourits-Andersen T. Campo E. Moller M.B. LPL gene expression is associated with poor prognosis in CLL and closely related to NOTCH1 mutations.Eur. J. Haematol. 2016; 97: 175-182Crossref PubMed Scopus (14) Google Scholar, 6Heintel D. Kienle D. Shehata M. Krober A. Kroemer E. Schwarzinger I. Mitteregger D. Le T. Gleiss A. Mannhalter C. et al.High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia.Leukemia. 2005; 19: 1216-1223Crossref PubMed Scopus (132) Google Scholar). The precise roles of LPL in cancer cells, however, are unresolved. LPL is best known as the enzyme responsible for the extracellular hydrolysis of TG carried in lipoproteins. LPL is produced by myocytes and adipocytes, secreted into the interstitial space, and transported to the capillary lumen (7Davies B.S. Beigneux A. Barnes R. Tu Y. Gin P. Weinstein M. Nobumori C. Nyren R. Olivecrona G. Bensadoun A. et al.GPIHP1 is responsible for the entry of lipoprotein lipase into capillaries.Cell Metab. 2010; 12: 42-52Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). For years, dogma held that secreted LPL was tethered to capillary endothelial cells by its heparin-binding domains and heparan sulfate proteoglycans (HSPGs) on the capillary surface (8Goldberg I.J. Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis.J. Lipid Res. 1996; 37: 693-707Abstract Full Text PDF PubMed Google Scholar). This belief was supported by the fact that LPL can be demarginated into plasma by heparin (9Korn E.D. Clearing factor, a heparin-activated lipoprotein lipase. I. Isolation and characterization of the enzyme from normal rat heart.J. Biol. Chem. 1955; 215: 1-14Abstract Full Text PDF PubMed Google Scholar) and by in vitro studies showing that LPL binds to HSPGs and that this interaction can be disrupted by the desulfation of HSPGs or digestion with heparinase or heparitinase (10Hoogewerf A.J. Cisar L.A. Evans D.C. Bensadoun A. Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation.J. Biol. Chem. 1991; 266: 16564-16571Abstract Full Text PDF PubMed Google Scholar, 11Parthasarathy N. Goldberg I. Sivaram P. Mulloy B. Flory D. Wagner W. Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.J. Biol. Chem. 1994; 269: 22391-22396Abstract Full Text PDF PubMed Google Scholar). An alternate model has come to light in recent years in which LPL is secreted into interstitial spaces, captured by glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) on the antiluminal surface of capillary endothelial cells, and shuttled to the luminal surface (12Beigneux A.P. Davies B. Gin P. Weinstein M. Farber E. Qiao X. Peale F. Bunting S. Walzem R. Wong J. et al.Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 plays a critical role in the lipolytic processing of chylomicrons.Cell Metab. 2007; 5: 279-291Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). Here, GPIHBP1 facilitates LPL binding to the luminal surface of the capillary wall, creating a “platform for lipolysis.” On this platform LPL mediates TG hydrolysis, releasing glycerol and FFAs that can be taken up through the cell-surface channel CD36 on adipocytes and myocytes. Apart from this lipolytic function, LPL may act as a noncatalytic bridge, promoting the uptake of lipoproteins via receptor-mediated endocytosis (13Blain J.F. Aumont N. Theroux L. Dea D. Poirier J. A polymorphism in lipoprotein lipase affects the severity of Alzheimer's disease pathophysiology.Eur. J. Neurosci. 2006; 24: 1245-1251Crossref PubMed Scopus (33) Google Scholar). In this role, LPL interacts with lipoproteins and a variety of different cell-surface proteins, including HSPGs and members of the LDL receptor family, including the VLDL receptor (VLDLR) (14Accioly M.T. Pacheco P. Maya-Monteiro C.M. Carrossini N. Robbs B.K. Oliveira S.S. Kaufmann C. Morgado-Diaz J.A. Bozza P.T. Viola J.P. Lipid bodies are reservoirs of cyclooxygenase-2 and sites of prostaglandin-E2 synthesis in colon cancer cells.Cancer Res. 2008; 68: 1732-1740Crossref PubMed Scopus (244) Google Scholar). The ability of LPL to serve as a bridge has been supported by both in vitro and in vivo experiments, including the work of Merkel et al. (15Merkel M. Kako Y. Radner H. Cho I.S. Ramasamy R. Brunzell J.D. Goldberg I.J. Breslow J.L. Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo.Proc. Natl. Acad. Sci. USA. 1998; 95: 13841-13846Crossref PubMed Scopus (139) Google Scholar), who showed that catalytically inactive LPL expressed in muscle could still bind to HSPGs and induce VLDL uptake. This function of LPL has not been previously reported in cancer cells. We previously described the expression of CD36 and LPL by BC cells and tissues and the growth-promoting effect of VLDL supplementation observed in BC cell lines only in the presence of LPL. We now describe the deployment of LPL in BC cells. Our data support a model in which LPL is bound to a heparin-like HSPG motif on the cell surface and acts in concert with the VLDLR to rapidly internalize intact lipoproteins via receptor-mediated endocytosis. We further observe substantial alterations in patterns of gene expression related to pathways for lipid acquisition (synthesis vs. uptake) in response to the availability of lipoproteins in tissue culture (TC) media and cellular LPL expression status. These findings highlight the importance of lipoprotein uptake as a method of lipid acquisition for cancer cells and demonstrate BC cell metabolic plasticity in response to nutrient availability. MCF-7, MDA-MB-231, BT-474, DU4475, SKBR3, and T47-D BC cells and HeLa cervical cancer cells were from the American Type Culture Collection and cultured in phenol red-containing HyClone RPMI-1640 media with 10% (v/v) heat-inactivated FBS (GE Healthcare Life Sciences) and 1% penicillin-streptomycin. MCF10A mammary epithelial cells were cultured in DMEM/F12 growth media (Invitrogen) supplemented with 5% horse serum (Invitrogen), 20 ng/ml epidermal growth factor (Peprotech), 0.5 mg/ml hydrocortisone, 100 ng/ml cholera toxin, 10 µg/ml insulin (Sigma-Aldrich), and 1% penicillin-streptomycin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Characteristics of the cell lines are in supplemental Table S1. Lipoprotein-depleted serum (LPDS) and matched-control FBS were from Kalen Biomedical. MDA-MB-231 shRNA cells were generated by lentiviral transduction using HuSH shRNA plasmid panels with pGFP-C-Lenti vectors and the Lenti-vpack packaging kit (TR30037) according to the manufacturer's guidelines (OriGene Technologies). Plasmids included four LPL shRNAs and a negative control (TL311692). Cells transduced with shLPL or scrambled shRNA were selected in puromycin. siRNAs targeting LPL or VLDLR (Sigma-Aldrich) were transfected into MDA-MB-231 BC cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) according to the manufacturer's guidelines. siRNAs used were LPL siRNA 1 (SASI_Hs01_00208454), LPL siRNA 2 (SASI_Hs01_00208455); VLDLR siRNA 1 (SASI_Hs02_00335553), VLDLR siRNA 2 (SASI_Hs01_00219062); and MISSION siRNA Universal Negative Control #1, SIC001 (Sigma-Aldrich). DiI-VLDL uptake, RNA expression by RT-PCR, and VLDLR protein expression by Western blot were assessed 96 h after transfection. Cells were lysed in RIPA buffer [20 mM Tris, 150 mM NaCl, 1% NP-40, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM NaPPi, 50 mM NaF, 10 mM Na-β-glycerophosphate (pH 7.4), Halt protease inhibitor cocktail (Pierce), and 1 mM Na3VO4 (New England Biolabs)]. Lysates were sonicated for 15 s and centrifuged at 17,000 g for 10 min at 4°C, and protein was quantified by BCA assay (Pierce). Protein was denatured in LDS sample buffer (GenScript) with 1.25% β-mercaptoethanol and heated to 95°C for 1 min. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were probed with antibodies against VLDLR (6A6; Santa Cruz Biotechnology) and vinculin (Cell Signaling Technology). Human LPL was expressed from a pCMV-6AC vector from OriGene (Cat. No. SC322258). An empty vector (pCMV Neo) served as the control. Plasmids were transfected using Lipofectamine 2000 DNA transfection reagent (Thermo Fisher Scientific). Pooled and clonal cell lines were selected by limiting dilution in neomycin (G418) and propagated in G418-containing media. Total RNA was subjected to on-column DNase digestion with the RNase-free DNase set (Qiagen, Cat. No. 79254), cDNA synthesis, and RT-PCR, as previously described. The following reagents were prepared as stock solutions in DMSO: GSK264220A, lipase inhibitor (50 mM; Cayman Chemical); Dynasore, inhibitor of dynamin 1 and 2 (80 mM; Sigma-Aldrich); and Hoechst 33342 nuclear stain (20 mM; Thermo Fisher Scientific). Other reagents were prepared and stored at 4°C until use: heparin sodium salt from porcine intestinal mucosa (50 mM stock solution in H2O; Sigma-Aldrich, Cat. No. H3149), human DiI-VLDLs (1 mg protein/ml; Alfa Aesar Chemicals), and LPL from bovine milk (Sigma-Aldrich, Cat. No. L2254). The protocol for preparing LPL from suspension (3.8 M ammonium sulfate, 0.02 M Tris HCl, pH 8.0) was adapted from Nishitsuji et al. (16Nishitsuji K. Hosono T. Uchimura K. Michikawa M. Lipoprotein lipase is a novel amyloid beta (Abeta)-binding protein that promotes glycosaminoglycan-dependent cellular uptake of Abeta in astrocytes.J. Biol. Chem. 2011; 286: 6393-6401Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Heparinase I from Flavobacterium (Sigma-Aldrich) was dissolved at 1 mg/ml in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 4 mM CaCl2, and 0.01% BSA and reconstituted in 20 mM Tris-HCl (pH 7.5), 4 mM CaCl2, and 0.01% BSA. Our mouse monoclonal anti-human LPL antibody (3Kuemmerle N.B. Rysman E. Lombardo P.S. Flanagan A.J. Lipe B.C. Wells W.A. Pettus J.R. Froehlich H.M. Memoli V.A. Morganelli P.M. et al.Lipoprotein lipase links dietary fat to solid tumor cell proliferation.Mol. Cancer Ther. 2011; 10: 427-436Crossref PubMed Scopus (184) Google Scholar) was conjugated to Alexa-Fluor 647 (AF-647) using the AF-647 antibody labeling kit (Thermo Fisher Scientific, Cat No. A20186). A monoclonal antibody directed against the HSNS4F5 HSPG motif, (GlcNS6S-IdoA2S)3, was supplied by Nicole Smits. The binding site from the single-chain NS4F5 antibody (NS4F5scFv) (17Smits N.C. Kurup S. Rops A.L. Ten Dam G.B. Massuger L.F. Hafmans T. Turnbull J.E. Spillmann D. Li J.P. Kennel S.J. et al.The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.J. Biol. Chem. 2010; 285: 41143-41151Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) was converted to a functional full-length human IgG2 antibody (NS4F5IgG). Briefly, the VH region of NS4F5scFv was linked to the human γ-2 constant region under the control of the human T lymphotropic virus elongation factor-1 promoter, whereas the VL region of NS4F5scFv was linked to the C-gene of the human lambda constant region under the control of a cytomegalovirus promoter. A nonfunctional isotype control (NS4F5null) was generated by modifying the VH sequence of NS4F5IgG (CARSGRKGRMR to CARSGSGGSGS). Heavy- and light-chain constructs of NS4F5 (NS4F5IgG, NS4F5null) were cotransfected into HEK-293 cells. Antibodies were purified by protein A-sepharose affinity chromatography and analyzed using SDS-PAGE and size-exclusion HPLC analysis using a TOSOH TSK gel SuperSW3000 column. Total RNA was isolated using RNeasy minicolumns from extracts prepared with QiaShredder (Qiagen RNeasy Kit). Concentration and purity of RNA were assessed by a NanoDrop DM-1000 spectrophotometer (NanoDrop Technologies). cDNA was produced using iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad) with 1 μg RNA template. Quantitative RT-PCR was performed using TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assays (Thermo Fisher Scientific). Primers are described in supplemental Table S2. qRT-PCR was performed on a Bio-Rad CFX 96 Real Time System C1000 Thermal Cycler with the following program: (1) 50°C × 2 min, (2) 90°C × 10 min, (3) 95°C × 15 s, (4) 60°C × 1 min + plate read, (5) Go to (3) 39 more cycles, (6) Melt curve 65°C to 95°C increment 0.5°C for 5 s + plate read. Relative gene expression is displayed in accordance with the 2−ΔΔCT method, normalized to cyclophilin. Where applicable, RT-PCR products were separated on 2% agarose gels prepared using 0.5× TBE with SYBR Safe DNA gel stain (Invitrogen) and visualized using a Bio-Rad Molecular Imager ChemiDoc XRS+ with Image Lab Software. Cells were detached with CellStripper reagent (Thermo Fisher Scientific). Viability was assessed using the Molecular Probes LIVE/DEAD stain kit with excitation at 405 nm (20 min, 1:3000 dilution). Cells were blocked with human IgG (1.25 mg/ml, 15 min; Sigma Aldrich). Surface staining of unfixed/unpermeabilized cells used the following antibodies: mouse monoclonal LPL-AF-647 (1 µg/ml, 30 min) and Dylight 650-NS4F5IgG or Dylight 650-NS4F5null (3.2 µg/ml, 30 min). All incubations were carried out at 4°C with PBS washes between each step. Analysis of intracellular antigens was achieved using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific). Following staining, cells were fixed with 0.5% formalin (20 min). Fluorescence was measured using a FACScan system on a MacsQuant-10 Flow Cytometer (Miltenyi Biotec), and data were analyzed using FlowJo 10 software (FlowJo, LLC). For LPL staining, the unstained control condition was verified as having equivalent fluorescent reads as a mouse IgG2b K Isotype Control APC antibody (Thermo Fisher Scientific). VLDLs labeled with DiI (1,1′-dioctadecyl-3,3,3′-tetramethyl-indocarbocyanine perchlorate) were from Alfa Aesar (Cat. No. J65568). VLDLs with protein- and lipid-specific labels were prepared using a technique adapted from Goulbourne et al. (18Goulbourne C.N. Gin P. Tatar A. Nobumori C. Hoenger A. Jiang H. Grovenor C. Adeyo O. Esko J. Goldberg I. et al.The GPIHBP1-LPL complex is responsible for the margination of triglyceride-rich lipoproteins in capillaries.Cell Metab. 2014; 19: 849-860Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Briefly, DiI-VLDLs were dialyzed into PBS containing 0.05 M sodium borate (pH 8.5) and incubated with DyLight 650 NHS-Ester (Thermo Fisher Scientific) at room temperature for 1.5 h. Nonreacted DyLight dye was removed by extensive dialysis against PBS. MDA-MB-231 cells were seeded subconfluently in 12-well plates. Growth media were replaced with RPMI-1640 containing 10% LPDS (Kalen) overnight, and cells were assayed for uptake of DiI-VLDL using methods adapted from Teupser (19Teupser D. Thiery J. Walli A. Seidel D. Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay.Biochim. Biophys. Acta. 1996; 1303: 193-198Crossref PubMed Scopus (68) Google Scholar). Briefly, cells were pretreated for 30 min in serum-free media prior to a 1 h incubation with 10 µg/ml DiI-VLDL. Surface-bound DiI-VLDL was removed with acid-wash buffer (0.5 M acetic acid with 150 mM NaCl, pH 2.5). Cells were washed with DPBS with calcium and magnesium, lysed in 1% SDS and 0.1 M NaOH, transferred to a black 96-well half-area plate (Greiner Bio-One), and assessed using a SpectraMax i3x microplate reader (Ex/Em: 520/580 nm; Molecular Devices). Fluorescence was corrected for protein. Cells were plated in complete media into 12-well plates, allowed to adhere overnight, and serum-starved for 1–8 h. Confluency was less than 70% at the time of treatment. All pretreatments (30 min) and VLDL uptake incubations (45 min) were carried out at 37°C in a humidified atmosphere containing 5% CO2, unless otherwise indicated. Cells were washed and then harvested by trypsinization. Samples were run on a ZE-5 flow cytometer (Bio-Rad). Data were analyzed using FlowJo 10 software (FlowJo, LLC). Cells were seeded subconfluently into an 8-chambered Lab-Tek coverglass (Nunc), allowed to adhere overnight, serum-starved for 1–8 h, and then assessed for DiI-VLDL uptake. Treatments with uptake-inhibiting reagents (30 min) were followed by a 45 min incubation with DiI-VLDL (5 µg/ml). Cells were rinsed with DPBS with calcium and magnesium and fixed with 3% paraformaldehyde in DPBS. Nuclei were stained with DAPI (Thermo Scientific) or Hoechst 33342 (Sigma-Aldrich). Images were acquired by confocal fluorescence microscopy with a Zeiss LSM 800 microscope equipped with a 63× oil-immersion objective with identical exposure settings for all experimental conditions. Images were processed uniformly across comparisons with Zen Lite 2.3 (Zeiss) and ImageJ 1.49 (Fiji). Cells were grown in chambered Lab-Tek coverglass wells (Nunc), washed with DPBS, stained with Hoechst 33342 (1 µg/ml), fixed in 3% paraformaldehyde in DPBS with calcium and magnesium, and incubated with LipidTOX Red Neutral Lipid Stain (1:800, 30 min at 37°C; Invitrogen). Lipase activity was measured using a fluorescent assay (20Basu D. Manjur J. Jin W. Determination of lipoprotein lipase activity using a novel fluorescent lipase assay.J. Lipid Res. 2011; 52: 826-832Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) using zwitterionic detergent [3-(N,N-dimethylmyristylammonio)propanesulfonate] and EnzChek lipase substrate, green fluorescent, 505/515 (250 µM stock; Sigma-Aldrich). Fluorescence was measured on a SpectraMax i3x plate reader and SoftMax Pro software from Molecular Devices (Ex/Em: 482/515 nm; 495 nm filter cutoff). Assays were carried out after a 10 min incubation at 37°C in black half-area 96-well plates (Greiner Bio-One) in 0.15 M NaCl, 20 mM Tris-HCl (pH 8.0), 0.0125% Zwittergent, and 1.5% FA-free BSA in a total volume of 100 µl. The average of no lipase controls was subtracted from raw values. Cells were seeded into 96-well plates, allowed to adhere overnight, treated with drugs, and cultured for 72 h at 37°C before viability assay using the ATP-based CellTiter-Glo (CTG) 2.0 Assay (Promega). Luminescence was read with the LMAX II luminometer (Molecular Devices). Background luminescence was calculated from CTG alone wells. Luminescence values (RLUs) were normalized to vehicle and compiled as a percentage of DMSO control. IC50 determination was performed using a nonlinear regression curve-fitting algorithm: log(inhibitor) versus response-variable (four-parameter) slope with Graph Pad Prism 6.0. Apoptosis was assessed using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and propidium iodide (Thermo Fisher Scientific). Flow cytometry data obtained with the eight-color MACSQuant-10 (Miltenyi Biotec) were analyzed using FlowJo software. Percentage apoptosis was the percentage Annexin V+ cells in the population. Statistical significance was evaluated using an unpaired two-tailed Student's t-test with Welch's correction or one-way ANOVA with correction for multiple comparisons (Dunnett's multiple-comparisons test) where applicable. P < 0.05 was deemed significant. Errors are presented as mean ± SD or SEM, as indicated. Breast tumor tissues stained with antibody directed against LPL displayed both cytoplasmic and cell-surface LPL staining (Fig. 1A). Using qRT-PCR, we established that LPL is variably expressed across a panel of human cancer cell lines (supplemental Fig. S1). We previously described LiSa-2 liposarcoma cells, HeLa cervical cancer cells, and DU4475 BC cells as high LPL-expressing and -secreting cell lines. Here, using flow cytometry with our AF-647-LPL antibody, we show that LPL is found to a variable degree on the surface of LPL-expressing cancer cells and at higher levels inside the cell, with the total LPL pool represented by the fixed/permeabilized condition (Fig. 1B). Cell-surface-bound LPL was displaceable by 500 µg/ml heparin, as quantified by flow cytometry (Fig. 1C) and immunocytochemistry using nonadherent DU4475 TNBC cells with a protocol designed to preserve cell-surface HSPGs (Fig. 1D). We predicted that LPL would be present in cells that express the gene but were surprised to find it in and on the surface of T47-D and MCF-7 LPL low to nonexpressing BC cell lines (Fig. 1E). We hypothesized that without LPL mRNA expression, the protein must be acquired from an exogenous source, such as the FBS of the TC media. The lipase assay confirmed that lipase activity was present in standard and matched-control FBS but not in lipoprotein-depleted FBS (Fig. 1F). We assessed the ability of T47-D cells to capture LPL from TC media by incubation with different amounts of FBS overnight (Fig. 1G). A FBS concentration-dependent increase was observed in cell-surface LPL in T47-D cells. LPL was also detected on cells incubated with media conditioned for 3 days by LPL-secreting LiSa-2 liposarcoma cells. LPL has been reported to bind to HSPGs, the (GlcNS6S-IdoA2S)3 or HSNS4F5 motif, in particular (11Parthasarathy N. Goldberg I. Sivaram P. Mulloy B. Flory D. Wagner W. Oligosaccharide sequences of endothelial cell surface heparan sulfate proteoglycan with affinity for lipoprotein lipase.J. Biol. Chem. 1994; 269: 22391-22396Abstract Full Text PDF PubMed Google Scholar, 17Smits N.C. Kurup S. Rops A.L. Ten Dam G.B. Massuger L.F. Hafmans T. Turnbull J.E. Spillmann D. Li J.P. Kennel S.J. et al.The heparan sulfate motif (GlcNS6S-IdoA2S)3, common in heparin, has a strict topography and is involved in cell behavior and disease.J. Biol. Chem. 2010; 285: 41143-41151Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Using qRT-PCR, we first determined that the cancer cell lines used in our studies did not express detectable levels of GPIHBP1, the one exception being DU4475 TNBC cells (not shown). Using flow cytometry and a Dylight 650-labeled antibody for HSNS4F5 (NS4F5IgG) we established that this binding site for LPL is present on the surface of cancer cells (supplemental Fig. S2A). This was confirmed visually using fluorescence confocal microscopy, shown by a representative image of HSNS4F5 staining on MDA-MB-231 BC cells (supplemental Fig. S2B). The abundance of HSNS4F5 on the cell surface was sensitive to confluence and nutrient supply (not shown). Knockdown of heparan sulfate 6-O-sulfotransferase 1 (HS6ST1), an enzyme responsible for 6-O-sulfation of heparan sulfate, reduced HSNS4F5 on the surface of MDA-MB-231 BC cells, supporting the specificity of the NS4F5IgG (supplemental Fig. S2C). Initial studies revealed that VLDL bind to and are rapidly internalized by cancer cells. This raised the question of whether the observed uptake represented the internalization of intact lipoproteins or that of FFA released by LPL-mediated hydrolysis of TG. To investigate this, the lipid and protein components of VLDLs were labeled with DiI and DyLight 650, respectively, and visualized using confocal microscopy (supplemental Fig. S3). As shown in the merged channel, the fluorescently labeled lipid and protein components of internalized VLDL particles coincide, indicating that intact lipoproteins are internalized. We characterized DiI-VLDL uptake using flow cytometry, confocal microscopy, and plate-based fluorescent assays. Using MDA-MB-231 BC cells, we show that DiI-VLDL uptake is time- and dose-dependent and temperature-sensitive (Fig. 2). MDA-MB-231 cells incubated at 4°C with DiI-labeled VLDL particles (5 µg/ml) bind VLDLs at the cell surface, while incubations at 37°C result in both binding and internalization (Fig. 2C, D). Binding of DiI-VLDL to the surface of MDA-MB-231 cells at 4°C was abrogated by excess unlabeled VLDL, indicating specificity consistent with a ligand-receptor interaction (supplemental Fig. S4). Rapid internalization of intact VLDL particles through a temperature-dependent process led us to investigate whether uptake was by receptor-mediated endocytosis. Treatment of MDA-MB-231 cells with Dynasore, an inhibitor of clathrin-coated pit-mediated endocytosis, resulted in a concentration-dependent decrease in DiI-VLDL uptake (Fig. 2E). We hypothesized that VLDL uptake was mediated by cell-surface HSPG-bound LPL acting as a noncatalytic bridge to facilitate the uptake of intact lipoprotein particles through receptor-mediated endocytosis. We assessed the dependence on HSPGs (directly) and LPL (indirectly) by first testing the effect of heparin on DiI-VLDL uptake. Heparin caused a concentration-dependent decrease in DiI-VLDL uptake by MDA-MB-231 cells (Fig. 3A). Heparinase I and/or III likewise reduced DiI-VLDL uptake (Fig. 3B). Confocal microscopy using DiI-VLDLs and DyLight 650-NS4F5IgG shows that both VLDLs and HSNS4F5 localize to the cell surface (in cells incubated at 4°C) (Fig. 3C). Treating cells with unlabeled NS4F5 antibody reduced DiI-VLDL uptake, as