Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma
2018; Elsevier BV; Volume: 59; Issue: 11 Linguagem: Inglês
10.1194/jlr.m083311
ISSN1539-7262
AutoresSrividya Velagapudi, Peter Schraml, Mustafa Yalcınkaya, Hella Anna Bolck, Lucia Rohrer, Holger Moch, Arnold von Eckardstein,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoClear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (125I-HDL and 125I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of 125I-HDL and 125I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells. Clear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (125I-HDL and 125I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of 125I-HDL and 125I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells. Classification of renal cell carcinoma (RCC) subtypes is based on histologically predominant cytoplasmic features [clear-cell RCC (ccRCC)], characteristic staining (chromophobe RCC), architectural features (papillary RCC), or specific molecular alterations (translocation RCC). ccRCC received its name from the microscopic appearance upon staining of formalin-fixed paraffin-embedded (FFPE) sections with H&E (1López J.I. Renal tumors with clear cells. A review.Pathol. Res. Pract. 2013; 209: 137-146Crossref PubMed Scopus (45) Google Scholar). The clear appearance of the cytoplasm is due to the accumulation of glycogen and lipids that are dissolved during routine processing with deparaffinization of FFPE sections using xylene and ethanol. The most prominent lipid stored in renal tumor cells is cholesterol, largely in the esterified form (2Gebhard R.L. Clayman R.V. Prigge W.F. Figenshau R. Staley N.A. Reesey C. Bear A. Abnormal cholesterol metabolism in renal clear cell carcinoma.J. Lipid Res. 1987; 28: 1177-1184Abstract Full Text PDF PubMed Google Scholar). The mechanisms for cholesterol accumulation in ccRCC cells are not well-understood. Three principle pathways have to be considered, two of which have been ruled out previously, namely, excessive cholesterol synthesis by the finding of decreased rather than increased activity of the rate-limiting enzyme, HMG-CoA reductase (3Wiley M.H. Howton M.M. Siperstein M.D. The quantitative role of the kidneys in the in vivo metabolism of mevalonate.J. Biol. Chem. 1977; 252: 548-554Abstract Full Text PDF PubMed Google Scholar), as well as abnormal cholesterol efflux (2Gebhard R.L. Clayman R.V. Prigge W.F. Figenshau R. Staley N.A. Reesey C. Bear A. Abnormal cholesterol metabolism in renal clear cell carcinoma.J. Lipid Res. 1987; 28: 1177-1184Abstract Full Text PDF PubMed Google Scholar). The third explanation is the most likely, excessive uptake of cholesterol from plasma lipoproteins beyond the capacity of utilization and processing. However, neither the lipoprotein classes nor the receptors and cellular pathways involved are well-characterized. ccRCC lacks the LDL receptor (LDLR), which is the main entry route for exogenous cholesterol into the majority of cells, including many tumor cells (4Clayman R.V. Bilhartz L.E. Spady D.K. Buja L.M. Dietschy J.M. Low density lipoprotein-receptor activity is lost in vivo in malignantly transformed renal tissue.FEBS Lett. 1986; 196: 87-90Crossref PubMed Scopus (21) Google Scholar). In contrast, the expression of both the VLDL receptor (VLDLR) and scavenger receptor BI (SR-BI) was found to be increased in ccRCC compared with the normal kidney tissue (5Sundelin J.P. Stahlman M. Lundqvist A. Levin M. Parini P. Johansson M.E. Boren J. Increased expression of the very low-density lipoprotein receptor mediates lipid accumulation in clear-cell renal cell carcinoma.PLoS One. 2012; 7: e48694Crossref PubMed Scopus (41) Google Scholar), and to mediate lipid uptake into ccRCC cells from VLDL and HDL, respectively (5Sundelin J.P. Stahlman M. Lundqvist A. Levin M. Parini P. Johansson M.E. Boren J. Increased expression of the very low-density lipoprotein receptor mediates lipid accumulation in clear-cell renal cell carcinoma.PLoS One. 2012; 7: e48694Crossref PubMed Scopus (41) Google Scholar, 6Xu G.H. Lou N. Shi H.C. Xu Y.C. Ruan H.L. Xiao W. Liu L. Li X. Xiao H.B. Qiu B. Up-regulation of SR-BI promotes progression and serves as a prognostic biomarker in clear cell renal cell carcinoma.BMC Cancer. 2018; 18: 88Crossref PubMed Scopus (26) Google Scholar). The activity of vascular endothelial growth factor (VEGF) is increased in the majority of ccRCCs (7Masson N. Ratcliffe P.J. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways.Cancer Metab. 2014; 2: 3Crossref PubMed Google Scholar, 8Hakimi A.A. Reznik E. Lee C.H. Creighton C.J. Brannon A.R. Luna A. Aksoy B.A. Liu E.M. Shen R. Lee W. An integrated metabolic atlas of clear cell renal cell carcinoma.Cancer Cell. 2016; 29: 104-116Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) due to the constitutive activation of hypoxia-inducible factor (HIF)-1α by somatic mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL protein is a component of the E3-ubiquitin ligase complex that ubiquitylates HIF-1α and HIF-2α for proteasome-mediated degradation (7Masson N. Ratcliffe P.J. Hypoxia signaling pathways in cancer metabolism: the importance of co-selecting interconnected physiological pathways.Cancer Metab. 2014; 2: 3Crossref PubMed Google Scholar, 8Hakimi A.A. Reznik E. Lee C.H. Creighton C.J. Brannon A.R. Luna A. Aksoy B.A. Liu E.M. Shen R. Lee W. An integrated metabolic atlas of clear cell renal cell carcinoma.Cancer Cell. 2016; 29: 104-116Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 9Wiesener M.S. Munchenhagen P.M. Berger I. Morgan N.V. Roigas J. Schwiertz A. Jurgensen J.S. Gruber G. Maxwell P.H. Loning S.A. Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas.Cancer Res. 2001; 61: 5215-5222PubMed Google Scholar). Thus, the loss of VHL function leads to HIF-1α stabilization despite an adequately oxygenated tissue microenvironment, which in turn results in uncontrolled activation of HIF-target genes that regulate erythropoiesis (erythropoietin), angiogenesis (VEGF), glycolysis (glucose transporters and glycolytic pathway enzymes), and apoptosis (BNIP3) (8Hakimi A.A. Reznik E. Lee C.H. Creighton C.J. Brannon A.R. Luna A. Aksoy B.A. Liu E.M. Shen R. Lee W. An integrated metabolic atlas of clear cell renal cell carcinoma.Cancer Cell. 2016; 29: 104-116Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 9Wiesener M.S. Munchenhagen P.M. Berger I. Morgan N.V. Roigas J. Schwiertz A. Jurgensen J.S. Gruber G. Maxwell P.H. Loning S.A. Constitutive activation of hypoxia-inducible genes related to overexpression of hypoxia-inducible factor-1alpha in clear cell renal carcinomas.Cancer Res. 2001; 61: 5215-5222PubMed Google Scholar, 10Haase V.H. Regulation of erythropoiesis by hypoxia-inducible factors.Blood Rev. 2013; 27: 41-53Crossref PubMed Scopus (404) Google Scholar, 11Chan D.A. Sutphin P.D. Nguyen P. Turcotte S. Lai E.W. Banh A. Reynolds G.E. Chi J.T. Wu J. Solow-Cordero D.E. Targeting GLUT1 and the Warburg effect in renal cell carcinoma by chemical synthetic lethality.Sci. Transl. Med. 2011; 3: 94ra70Crossref PubMed Scopus (394) Google Scholar, 12Greijer A.E. van der Wall E. The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis.J. Clin. Pathol. 2004; 57: 1009-1014Crossref PubMed Scopus (597) Google Scholar). We have previously found that VEGF promotes the cell surface abundance of SR-BI in endothelial cells and thereby enhances the uptake of HDL into endothelial cells (13Velagapudi S. Yalcinkaya M. Piemontese A. Meier R. Norrelykke S.F. Perisa D. Rzepiela A. Stebler M. Stoma S. Zanoni P. VEGF-A regulates cellular localization of SR-BI as well as transendothelial transport of HDL but not LDL.Arterioscler. Thromb. Vasc. Biol. 2017; 37: 794-803Crossref PubMed Scopus (28) Google Scholar). Therefore, we hypothesized that increased activities of HIF-1α and hence VEGF promote the cell surface expression of SR-BI and thereby the uptake of HDL. To test this hypothesis, we combined immunohistochemical studies in human renal tumors with experiments in two ccRCC model cell lines and patient-derived ccRCC cell cultures. RCC patients were identified from the database of the Institute of Pathology and Molecular Pathology, University Hospital Zurich, Switzerland. All RCCs were histologically reevaluated by one pathologist (H.M.) and selected on the basis of H&E-stained tissue sections. The patient cohort and the construction of tissue microarrays (TMAs) of RCC were previously described (14Kononen J. Bubendorf L. Kallioniemi A. Barlund M. Schraml P. Leighton S. Torhorst J. 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The clinical and pathologic parameters of the tumors on the TMA are summarized in supplemental Table S1. For some cases, there was no information available. This study was approved by the local commission of ethics (KEK-ZH no. 2011-0072/4). TMA sections (2.5 μm) were transferred to glass slides followed by immunohistochemical analysis according to the Ventana (Tucson, AZ) automated protocols, and the antibodies used are listed in supplemental Table S2. The staining intensities were classified as absent (0), weak (1), moderate (2), and strong (3). For detailed analysis, TMAs were scanned using the NanoZoomer digital slide scanner (Hamamatsu Photonics K.K.). Tissue samples of patients were made available by the Tissue Biobank of the Department of Pathology and Molecular Pathology, University Hospital of Zurich, Switzerland upon approval of the local ethics commission (KEK-ZH-Nr. 2011-0072 and KEK-ZH-Nr. 2014-0614) and upon patients' written consent. H&E-stained sections of FFPE and fresh-frozen renal tissue specimens were reviewed by a pathologist with specialization in uropathology (H.M.). Sanger sequencing was employed to assess the mutation status of the VHL gene (c.341-1G>C) for the ccRCC primary tumor and the corresponding cell culture. DNA was isolated from FFPE punches from tumor tissue (three cylinders with a diameter of 0.6 mm) or a minimum of 10,000 cultured cells using the Maxwell® 16 DNA purification kits (Promega, Madison, WI). PCR and sequencing of VHL were performed as previously described (17Rechsteiner M.P. von Teichman A. Nowicka A. Sulser T. Schraml P. Moch H. VHL gene mutations and their effects on hypoxia inducible factor HIFalpha: identification of potential driver and passenger mutations.Cancer Res. 2011; 71: 5500-5511Crossref PubMed Scopus (77) Google Scholar). Fresh tissue samples were placed into sterile 50 ml conical tubes containing transport medium (RPMI) (Gibco, Waltham, MA) with 10% FCS (Gibco) and Antibiotic-Antimycotic® (Gibco). FFPE cell pellets from cultured cells were prepared as previously described (18Struckmann K. Mertz K.D. Steu S. Storz M. Staller P. Krek W. Schraml P. Moch H. pVHL co-ordinately regulates CXCR4/CXCL12 and MMP2/MMP9 expression in human clear-cell renal cell carcinoma.J. Pathol. 2008; 214: 464-471Crossref PubMed Scopus (55) Google Scholar) and compared with FFPE specimens of the corresponding primary tumor by immunohistochemistry. Cultures were maintained in K1 medium (19Zhao Y. Zhao H. Zhang Y. Tsatralis T. Cao Q. Wang Y. Wang Y. Wang Y.M. Alexander S.I. Harris D.C. Isolation and epithelial co-culture of mouse renal peritubular endothelial cells.BMC Cell Biol. 2014; 15: 40Crossref PubMed Scopus (15) Google Scholar, 20Taub M. Ü B. Chuman L. Rindler M.J. Saier Jr., M.H. Sato G. 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Cell Biol. 2003; 5: 64-70Crossref PubMed Scopus (288) Google Scholar) and cultured using the same conditions as mentioned for 786-O. G418 (0.5 mg/ml) (Gibco; 10131) was used as selection antibiotic. Both cell lines were authenticated by the authentication service of Microsynth (Balgach, Switzerland) and were previously used by our group (22Ruf M. Mittmann C. Nowicka A.M. Hartmann A. Hermanns T. Poyet C. van den Broek M. Sulser T. Moch H. Schraml P. pVHL/HIF-regulated CD70 expression is associated with infiltration of CD27+ lymphocytes and increased serum levels of soluble CD27 in clear cell renal cell carcinoma.Clin. Cancer Res. 2015; 21: 889-898Crossref PubMed Scopus (40) Google Scholar, 23Casagrande S. Ruf M. Rechsteiner M. Morra L. Brun-Schmid S. von Teichman A. Krek W. Schraml P. Moch H. The protein tyrosine phosphatase receptor type J is regulated by the pVHL-HIF axis in clear cell renal cell carcinoma.J. Pathol. 2013; 229: 525-534Crossref PubMed Scopus (9) Google Scholar). Human aortic endothelial cells (HAECs) from Cell Applications Inc. (304-05a) were cultured in endothelial cell basal medium (LONZA Clonetics CC-3156) with 5% fetal bovine serum (Gibco), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Sigma-Aldrich), supplemented with singleQuots (LONZA Clonetics CC-4176 or ATCC PCS-100-041). Hepatocellular carcinoma cells (Huh7) from JCRB (0403) and human renal proximal tubular epithelial cell line, HK-2 (provided by R. Wüthrich Clinic for Nephrology, Department of Internal Medicine, University Hospital Zurich, Switzerland), were cultured in DMEM with 10% fetal bovine serum (Gibco), 100 U/ml of penicillin, and 100 μg/ml of streptomycin (Sigma-Aldrich). LDL (1.019 < d < 1.063 g/ml) and HDL (1.063 < d < 1.21 g/ml) were isolated from fresh normolipidemic plasma of blood donors by sequential ultracentrifugation as described previously (24Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6479) Google Scholar, 25Rohrer L. Cavelier C. Fuchs S. Schluter M.A. Volker W. von Eckardstein A. Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells.Biochim. Biophys. Acta. 2006; 1761: 186-194Crossref PubMed Scopus (55) Google Scholar). LDL and HDL were radioiodinated with Na125I by the McFarlane monochloride procedure modified for lipoproteins (25Rohrer L. Cavelier C. Fuchs S. Schluter M.A. Volker W. von Eckardstein A. Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells.Biochim. Biophys. Acta. 2006; 1761: 186-194Crossref PubMed Scopus (55) Google Scholar, 26Freeman M. Ekkel Y. Rohrer L. Penman M. Freedman N.J. Chisolm G.M. Krieger M. Expression of type I and type II bovine scavenger receptors in Chinese hamster ovary cells: lipid droplet accumulation and nonreciprocal cross competition by acetylated and oxidized low density lipoprotein.Proc. Natl. Acad. Sci. USA. 1991; 88: 4931-4935Crossref PubMed Scopus (176) Google Scholar). Specific activities between 300–900 cpm/ng of protein were obtained. All assays were performed in RPMI-1640 (Sigma) containing 25 mmol/l HEPES and 0.2% BSA instead of serum (referred to as assay medium). Where indicated, cells were pretreated with sorafenib tosylate (Selleckchem; 90 nM) or sunitinib malate (Selleckchem; 80 nM) for 30 min or with VEGF-A (Sigma; 25 ng/ml) or anti-SR-BI neutralizing antibody (1:500; Novus NB400-113) or anti-IgG control (1:500, Santa Cruz-2027) for 1 h at 37°C. Following treatments, the cells were incubated with 10 μg/ml of radio-iodinated HDL (125I-HDL) or radio-iodinated LDL (125I-LDL) in the absence or presence of a 40 times excess of nonlabeled HDL and LDL, respectively, for 1 h at 37°C for association experiments. At the end of the cell association step, the cells were washed twice with Tris-BSA buffer, followed by two washes with PBS containing CaCl2 and MgCl2 and then lysed in 0.1 N NaOH buffer. Specific cellular association was calculated by subtracting the values obtained in the presence of excess unlabeled HDL or LDL (unspecific) from those obtained in the absence of unlabeled HDL and LDL (total), respectively. For the pulse-chase experiments, 50,000 cells were seeded in 24-well plates and cultured for 48 h. Then the cells were pulsed for 1 h with 10 μg/ml of 125I-HDL or 125I-LDL at 37°C in the presence or absence of the respective unlabeled lipoprotein for competition and determination of specific interactions. After 1 h of pulse incubation, the cells were either directly processed for the measurement of association or were washed three times with assay medium, chased for 1, 2, or 4 h at 37°C with the assay medium containing 10 μg/ml of unlabeled HDL or LDL. At the end of each chase period, the cells were handled as described above for the cellular association experiments. In addition, the media were collected and subjected to precipitation with trichloroacetic acid (TCA). Radioactivity was counted by Perkin Elmer γ-counter. Precipitated radioactivity was postulated to reflect nondegraded lipoproteins, whereas radioactivity in the supernatant was considered to reflect degraded lipoproteins (27Goldstein J.L. Brunschede G.Y. Brown M.S. Inhibition of proteolytic degradation of low density lipoprotein in human fibroblasts by chloroquine, concanavalin A, and Triton WR 1339.J. Biol. Chem. 1975; 250: 7854-7862Abstract Full Text PDF PubMed Google Scholar). The amount of radioactivity in each fraction of the well (cell associated, TCA supernatant, and TCA precipitated) was calculated by normalizing to the specific cellular association of the no chase of parental 786-O cells or primary ccRCC cells (represents initial radioactivity for the chase points). Total RNA was isolated using TRI reagent (Sigma T9424) according to the manufacturer's instructions. Genomic DNA was removed by digestion using DNase (Roche) and RNase inhibitor (Ribolock; Thermo Scientific). Reverse transcription was performed using M-MLVRT (Invitrogen; 200 U/μl) following the standard protocol as described by the manufacturer. Quantitative PCR was done with Lightcycler FastStart DNA Master SYBR Green I (Roche) using gene-specific primers, as mentioned in the supplemental information. The 786-O and 786-O-VHL cells were reverse transfected with siRNA (Ambion Silencer Select; Life Technologies) targeted to LDLR (s224006, s224007, s4), VLDLR [siGENOME SMARTpool siRNA D-003721-02; ON-TARGET plus human VLDLR (7436), Dharmacon], neuropilin-1 (NRP1) (s16844, s16843), or nonsilencing control (4390843, silencer select or siGENOME control siRNA D-001220-01-20, Dharmacon or ON-TARGET control siRNA D-01810-10-20, Dharmacon) at a final concentration of 5 nmol/l using Lipofectamine RNA iMAX transfection reagent (Invitrogen; 13778150) in an antibiotic-free medium. All experiments were performed 72 h posttransfection and efficiency of transfection was confirmed with at least two siRNAs against each gene using quantitative RT-PCR. Cells were lysed in RIPA buffer [10 mmol/l Tris (pH 7.4), 150 mmol/l NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and complete EDTA (Roche)] with protease inhibitors (Roche). Equal amounts of protein were separated on SDS-PAGE and trans-blotted onto PVDF membrane (GE Healthcare). Membranes were blocked in appropriate blocking buffer recommended for the antibody (TBS-T supplemented with 5% milk) and incubated overnight on a shaker at 4°C with primary antibodies in the same blocking buffer. Membranes were incubated for 1 h with a HRP-conjugated secondary antibody (Dako) in the blocking buffer. Membranes were further incubated with chemiluminescence substrate for 1 min (Pierce ECL Plus; Thermo Scientific) and imaged using Fusion Fx (Vilber). The expression of LDLR (1:1,000, ab30532; Abcam), VLDLR (1:1,000, NBP1-78162; Novus), SR-BI (1:1,000, NB400-131; Novus), and NRP1 (1:1,000, ab81321; Abcam) were evaluated and compared with the expression of TATA binding protein (TBP) (1:1,000, ab51841; Abcam), which was used as a loading control. Biotinylation of intact cells was performed using 20 mg/ml EZ-Link Sulfo-NHS-S-S-Biotin (Thermo Scientific) in the cold for 1 h with mild shaking and quenched with ice-cold 50 mM Tris (pH 7.4). Cells were lysed in RIPA buffer (total cell lysate), and 200–500 μg of lysates were incubated with 20 μl of BSA-blocked streptavidin beads suspension (GE Healthcare) for 16 h at 4°C and pelleted by centrifugation; the pellet represents surface proteins. Proteins were dissociated from the pellet by boiling with SDS loading buffer, analyzed by SDS-PAGE, and immunoblotted with SR-BI antibody (NB400-131; Novus), TBP (ab51841; Abcam) used as intracellular control, and Na+/K+-ATPase (1:200, Santa Cruz-21712) used as cell surface control. Cells were cultured at a density of 5,000 cells per well in a 96-well plate for 72 h. After transfecting with either siRNA against NRP1 or LDLR or nonsilencing controls for 60 h or blocking with SR-BI neutralizing antibody for 1 h, the cells were treated overnight with 50 μg/ml of HDL or LDL. Following the overnight treatment, the supernatant was removed and cells were washed twice with PBS. The cells were then incubated with 30 μl of MTT solution (5 mg/ml in PBS, M5655; Sigma) diluted in 270 μl of DMEM for 30 min. The resultant formazan salts were extracted with DMSO and absorbance intensity was read at 550 nm and reference wavelength at 650 nm (DMSO). The rate of cell proliferation was calculated relative to the 786-O parental cell line. Contingency table analysis and Pearson's chi-square tests were used to analyze the associations between protein expression patterns and clinical parameters. Overall survival rates were determined according to the Kaplan-Meier method and analyzed for statistical differences using a log-rank test. The data sets for all in vitro experiments were performed with the GraphPad Prism 7.02 software. Data sets from independent experiments were pooled and the statistical tests were chosen based on the number of groups being compared (two or more than two). All the in vitro tests in this work are based either on the Mann-Whitney t-test or Kruskal-Wallis followed by Dunn's posttest. Values are expressed as mean ± SEM. P < 0.05 was regarded as significant and P > 0.05 was regarded as not significant. Immunostaining was performed on RCC TMAs for the major apolipoproteins of HDL and LDL, apoA-I and apoB, respectively (Fig. 1A, B), as well as SR-BI (Fig. 1C). Based on the staining intensities, expression levels in tumors were graded from 0 to 3 for apoA-I and SR-BI expression, and from 0 to 2 for apoB expression, as staining intensities were generally lower. Increased immunoreactivity with anti-apoA-I or anti-SR-BI antibodies, but not immunoreactivity with anti-apoB antibodies, significantly differentiated 175 ccRCC tissues from papillary RCC (Table 1).TABLE 1Quantification of apolipoproteins and SR-BI expression in RCC TMASubtypeProtein of InterestStaining IntensityccRCC [% (n)]Chromophobe [% (n)]Papillary [% (n)]apoA-I05.1 (9)012 (3)118.9 (33)50 (3)44 (11)224.6 (43)16.7 (1)32 (8)351.4 (90)33.3 (2)12 (3)apoB021.7 (38)16.7 (1)24 (6)162.3 (109)50 (3)68 (17)216 (28)33.3 (2)8 (2)SR-BI018.7 (31)80 (4)78.9 (15)125.3 (42)20 (1)10.5 (2)227.7 (46)05.3 (1)328.3 (47)05.3 (1)Percent and absolute (n) frequencies of anti-apoA-I, anti-apoB, and anti-SR-BI staining intensities among RCC subtypes. Open table in a new tab Percent and absolute (n) frequencies of anti-apoA-I, anti-apoB, and anti-SR-BI staining intensities among RCC subtypes. Strong cytoplasmic apoA-I and SR-BI expression (staining intensity of 2 and 3) was seen in approximately 75% and 56% of ccRCCs. Moderate and weak cytoplasmic apoB expression was observed in 78% of ccRCCs. These data indicate that the presence of apolipoproteins is characteristic in the majority of ccRCCs (Table 1). We next evaluated the associations of the lipoprotein immunoreactivities in ccRCCs with tumor stage (pT) and ISUP grade. Only anti-apoB immunoreactivity was significantly associated with late tumor stage (supplemental Table S3, P = 0.0371). apoA-I (P = 0.025) and apoB (P = 0.0006), but not SR-BI, expression was significantly correlated with higher ISUP grade (supplemental Table S4). We used the Kaplan-Meier method and log-rank test to evaluate any associations of apoA-I, apoB, and SR-BI immunoreactivity with overall survival (supplemental Fig. S1a–c). Only anti-apoA-I expression was significantly correlated with worse patient outcome (P = 0.0407, supplemental Fig. S1a). This association lost statistical significance upon multivariate analysis taking into account tumor stage and grade. Loss of function of the VHL protein leads to stabilization of HIF-α in ccRCC. Therefore, we statistically evaluated the associations of lipoprotein immunoreactivity with markers of the VHL/HIF axis, namely HIF-1α (supplemental Table S5) and HIF-1α targets CA9 and GLUT1 (supplemental Tables S6, S7), as well as microvessel density recorded by CD34 abundance (supplemental Table S8). Interestingly, the immunoreactivity for apoA-I showed significant positive associations with each of the four markers (HIF-1α, P = 0.0078; CA9, P = 0.0272; GLUT1, P = 0.0175; and CD34, P < 0.001). apoB immunoreactivity was significantly and positively associated with microvessel density (P = 0.0197) and nuclear HIF-1α staining (P = 0.0049). However, SR-BI immunoreactivity showed no significant association with any marker. To unravel the origin of lipoprotein accumulation in ccRCC, we performed in vitro assays in two ccRCC cell lines that lack functional VHL (786-O and RCC4) and their stably transfected VHL wild-type counterparts (786-O-VHL, RCC4-O-VHL). To further corroborate our findings, we utilized patient-derived ccRCC and normal epithelial kidney cell cultures that were established from surgical tissue specimens. Histological and genotypic comparison verified the resemblance of the patient-derived ccRCC and normal epithelial cell cu
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