Apolipoprotein E Promotes Invasion in Oral Squamous Cell Carcinoma
2017; Elsevier BV; Volume: 187; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2017.06.016
ISSN1525-2191
AutoresSangeeta Jayakar, Olivier Loudig, Margaret Brandwein‐Gensler, Ryung S. Kim, Thomas J. Ow, Berrin Ustun, Thomas M. Harris, Michael B. Prystowsky, Geoffrey Childs, Jeffrey E. Segall, Thomas J. Belbin,
Tópico(s)Cell Adhesion Molecules Research
ResumoOral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using in vitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho–extracellular signal–regulated kinase 1/2, phospho–c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7. Oral squamous cell carcinoma (OSCC) patients generally have a poor prognosis, because of the invasive nature of these tumors. In comparing transcription profiles between OSCC tumors with a more invasive (worst pattern of tumor invasion 5) versus a less invasive (worst pattern of tumor invasion 3) pattern of invasion, we identified a total of 97 genes that were overexpressed at least 1.5-fold in the more invasive tumor subtype. The most functionally relevant genes were assessed using in vitro invasion assays with an OSCC cell line (UM-SCC-1). Individual siRNA knockdown of 15 of these 45 genes resulted in significant reductions in tumor cell invasion compared to a nontargeting siRNA control. One gene whose knockdown had a strong effect on invasion corresponded to apolipoprotein E (APOE). Both matrix degradation and the number of mature invadopodia were significantly decreased with APOE knockdown. APOE knockdown also resulted in increased cellular cholesterol, consistent with APOE's role in regulating cholesterol efflux. APOE knockdown resulted in decreased levels of phospho–extracellular signal–regulated kinase 1/2, phospho–c-Jun N-terminal kinase, and phospho-cJun, as well as decreased activator protein 1 (AP-1) activity. Expression of matrix metalloproteinase 7 (MMP7), an AP-1 target, was also significantly decreased. Our findings suggest that APOE protein plays a significant role in OSCC tumor invasion because of its effects on cellular cholesterol and subsequent effects on cell signaling and AP-1 activity, leading to changes in the expression of invasion-related proteins, including MMP7. Head and neck cancers are the sixth most common malignancy worldwide, with >650,000 new cases each year.1Sasahira T. Kirita T. Kuniyasu H. Update of molecular pathobiology in oral cancer: a review.Int J Clin Oncol. 2014; 19: 431-436Crossref PubMed Scopus (75) Google Scholar Oral cavity squamous cell carcinomas (OSCCs) contribute to 264,000 of these cases, resulting in >128,000 deaths annually.1Sasahira T. Kirita T. Kuniyasu H. Update of molecular pathobiology in oral cancer: a review.Int J Clin Oncol. 2014; 19: 431-436Crossref PubMed Scopus (75) Google Scholar Despite advances in current treatment options, the 5-year survival for head and neck cancer patients has remained at 50%, a number that has not improved in the past 30 years. The poor prognosis associated with OSCC disease is due, in part, to the highly invasive and metastatic abilities of these tumor cells. OSCC patients often present with significant local invasion of tumor through the epithelial basement membrane into the underlying muscle, bone, or salivary tissue. The extent of tumor invasion directly affects patient prognosis and is an important factor in choosing treatment.2Jimenez L. Jayakar S.K. Ow T.J. Segall J.E. Mechanisms of invasion in head and neck cancer.Arch Pathol Lab Med. 2015; 139: 1334-1348Crossref PubMed Scopus (44) Google Scholar, 3Brandwein-Gensler M. Teixeira M.S. Lewis C.M. Lee B. Rolnitzky L. Hille J.J. Genden E. Urken M.L. Wang B.Y. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.Am J Surg Pathol. 2005; 29: 167-178Crossref PubMed Scopus (590) Google Scholar Depth of invasion of 2 to 4 mm in the oral tongue or floor of mouth is associated with 15% to 20% risk of metastasis to the cervical lymph nodes.4Spiro R.H. Huvos A.G. Wong G.Y. Spiro J.D. Gnecco C.A. Strong E.W. Predictive value of tumor thickness in squamous carcinoma confined to the tongue and floor of the mouth.Am J Surg. 1986; 152: 345-350Abstract Full Text PDF PubMed Scopus (466) Google Scholar As such, deep invasion into the tongue or mandible often requires extensive resection of oral cavity structures as part of therapy, contributing to significant patient morbidity and functional impact on speech and swallowing. Assessing the extent of tumor invasion using histological parameters in OSCC patients has shown some success in predicting patient outcome.3Brandwein-Gensler M. Teixeira M.S. Lewis C.M. Lee B. Rolnitzky L. Hille J.J. Genden E. Urken M.L. Wang B.Y. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.Am J Surg Pathol. 2005; 29: 167-178Crossref PubMed Scopus (590) Google Scholar, 5Brandwein-Gensler M. Smith R.V. Wang B. Penner C. Theilken A. Broughel D. Schiff B. Owen R.P. Smith J. Sarta C. Hebert T. Nason R. Ramer M. DeLacure M. Hirsch D. Myssiorek D. Heller K. Prystowsky M. Schlecht N.F. Negassa A. Validation of the histologic risk model in a new cohort of patients with head and neck squamous cell carcinoma.Am J Surg Pathol. 2010; 34: 676-688Crossref PubMed Scopus (152) Google Scholar, 6Li Y. Bai S. Carroll W. Dayan D. Dort J.C. Heller K. Jour G. Lau H. Penner C. Prystowsky M. Rosenthal E. Schlecht N.F. Smith R.V. Urken M. Vered M. Wang B. Wenig B. Negassa A. Brandwein-Gensler M. Validation of the risk model: high-risk classification and tumor pattern of invasion predict outcome for patients with low-stage oral cavity squamous cell carcinoma.Head Neck Pathol. 2013; 7: 211-223Crossref PubMed Scopus (106) Google Scholar, 7Almangush A. Bello I.O. Coletta R.D. Makitie A.A. Makinen L.K. Kauppila J.H. Pukkila M. Hagstrom J. Laranne J. Soini Y. Kosma V.M. Koivunen P. Kelner N. Kowalski L.P. Grenman R. Leivo I. Laara E. Salo T. For early-stage oral tongue cancer, depth of invasion and worst pattern of invasion are the strongest pathological predictors for locoregional recurrence and mortality.Virchows Arch. 2015; 467: 39-46Crossref PubMed Scopus (82) Google Scholar Brandwein-Gensler et al3Brandwein-Gensler M. Teixeira M.S. Lewis C.M. Lee B. Rolnitzky L. Hille J.J. Genden E. Urken M.L. Wang B.Y. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.Am J Surg Pathol. 2005; 29: 167-178Crossref PubMed Scopus (590) Google Scholar, 5Brandwein-Gensler M. Smith R.V. Wang B. Penner C. Theilken A. Broughel D. Schiff B. Owen R.P. Smith J. Sarta C. Hebert T. Nason R. Ramer M. DeLacure M. Hirsch D. Myssiorek D. Heller K. Prystowsky M. Schlecht N.F. Negassa A. Validation of the histologic risk model in a new cohort of patients with head and neck squamous cell carcinoma.Am J Surg Pathol. 2010; 34: 676-688Crossref PubMed Scopus (152) Google Scholar demonstrated that the histological pattern of tumor invasion (POI) contributed positively in predicting OSCC patient outcome as part of their risk assessment model. Specifically, they showed that the worst pattern of tumor invasion (WPOI), identified as the POI score taken from the most invasive portion of the tumor, was significantly associated with overall patient survival as well as local recurrence of disease.3Brandwein-Gensler M. Teixeira M.S. Lewis C.M. Lee B. Rolnitzky L. Hille J.J. Genden E. Urken M.L. Wang B.Y. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.Am J Surg Pathol. 2005; 29: 167-178Crossref PubMed Scopus (590) Google Scholar, 5Brandwein-Gensler M. Smith R.V. Wang B. Penner C. Theilken A. Broughel D. Schiff B. Owen R.P. Smith J. Sarta C. Hebert T. Nason R. Ramer M. DeLacure M. Hirsch D. Myssiorek D. Heller K. Prystowsky M. Schlecht N.F. Negassa A. Validation of the histologic risk model in a new cohort of patients with head and neck squamous cell carcinoma.Am J Surg Pathol. 2010; 34: 676-688Crossref PubMed Scopus (152) Google Scholar This model has since been validated by other groups as a pathological tool for predicting patient outcome, suggesting its potential utility in determining and properly timing treatment options for OSCC patients.6Li Y. Bai S. Carroll W. Dayan D. Dort J.C. Heller K. Jour G. Lau H. Penner C. Prystowsky M. Rosenthal E. Schlecht N.F. Smith R.V. Urken M. Vered M. Wang B. Wenig B. Negassa A. Brandwein-Gensler M. Validation of the risk model: high-risk classification and tumor pattern of invasion predict outcome for patients with low-stage oral cavity squamous cell carcinoma.Head Neck Pathol. 2013; 7: 211-223Crossref PubMed Scopus (106) Google Scholar, 7Almangush A. Bello I.O. Coletta R.D. Makitie A.A. Makinen L.K. Kauppila J.H. Pukkila M. Hagstrom J. Laranne J. Soini Y. Kosma V.M. Koivunen P. Kelner N. Kowalski L.P. Grenman R. Leivo I. Laara E. Salo T. For early-stage oral tongue cancer, depth of invasion and worst pattern of invasion are the strongest pathological predictors for locoregional recurrence and mortality.Virchows Arch. 2015; 467: 39-46Crossref PubMed Scopus (82) Google Scholar Despite the evidence that such clinical and histological parameters might be used to identify tumors that exhibit an invasive phenotype, the underlying molecular events responsible for these characteristics in OSCC are not fully understood. The search for molecular changes affecting invasion in these tumors has been an active area of research. Herein, we present data to show that tumors that are highly invasive (WPOI5) express higher levels of apolipoprotein E (APOE) protein compared to less invasive tumors (WPOI3), and that this expression of APOE is important to the invasion phenotype of OSCC cells. Mechanistic studies in our model system suggest that APOE decreases the cholesterol content of OSCC cells, leading to changes in intracellular signaling and downstream expression of proteases, and thus contributing to increased tumor cell invasion. Oral cavity tumor tissue specimens were obtained from Montefiore Medical Center (Bronx, NY). Histological assessment was performed as described,3Brandwein-Gensler M. Teixeira M.S. Lewis C.M. Lee B. Rolnitzky L. Hille J.J. Genden E. Urken M.L. Wang B.Y. Oral squamous cell carcinoma: histologic risk assessment, but not margin status, is strongly predictive of local disease-free and overall survival.Am J Surg Pathol. 2005; 29: 167-178Crossref PubMed Scopus (590) Google Scholar and samples with WPOI scores of WPOI5 and WPOI3 were identified. Whole-genome gene expression was evaluated in OSCC tumor tissues using two approaches. RNA was extracted from flash-frozen sections of tumors and measured using the HumanHT-12 v4 Beadchip arrays (Illumina, San Diego, CA), as described previously.8Schlecht N.F. Brandwein-Gensler M. Smith R.V. Kawachi N. Broughel D. Lin J. Keller C.E. Reynolds P.A. Gunn-Moore F.J. Harris T. Childs G. Belbin T.J. Prystowsky M.B. Cytoplasmic ezrin and moesin correlate with poor survival in head and neck squamous cell carcinoma.Head Neck Pathol. 2012; 6: 232-243Crossref PubMed Scopus (28) Google Scholar, 9Lleras R.A. Smith R.V. Adrien L.R. Schlecht N.F. Burk R.D. Harris T.M. Childs G. Prystowsky M.B. Belbin T.J. Unique DNA methylation loci distinguish anatomic site and HPV status in head and neck squamous cell carcinoma.Clin Cancer Res. 2013; 19: 5444-5455Crossref PubMed Scopus (69) Google Scholar As an additional evaluation of RNA from the same WPOI5 and WPOI3 tumors, biopsy cores from the edges of the tumors in formalin-fixed, paraffin-embedded tissue blocks were obtained and subjected to RNA extraction.10Loudig O. Milova E. Brandwein-Gensler M. Massimi A. Belbin T.J. Childs G. Singer R.H. Rohan T. Prystowsky M.B. Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT).Nucleic Acids Res. 2007; 35: e94Crossref PubMed Scopus (29) Google Scholar, 11Loudig O. Brandwein-Gensler M. Kim R.S. Lin J. Isayeva T. Liu C. Segall J.E. Kenny P.A. Prystowsky M.B. Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.Hum Pathol. 2011; 42: 1911-1922Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Whole-genome gene expression was then measured using the whole-genome cDNA-Mediated Annealing, Selection, Extension and Ligation (DASL) array platform (Illumina), as described previously.11Loudig O. Brandwein-Gensler M. Kim R.S. Lin J. Isayeva T. Liu C. Segall J.E. Kenny P.A. Prystowsky M.B. Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.Hum Pathol. 2011; 42: 1911-1922Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Gene expression data were log2 transformed, and a t-test was performed comparing the same five WPOI3 and four WPOI5 tumor samples from the both data sets. Genes overexpressed in WPOI5 tumors, as determined by both approaches, were selected that had P < 0.05, and a minimum fold change of 1.5 in both Beadchip and DASL analyses. The overall false-discovery rate based on permutation of the group labels was 1%. All microarray gene expression data were deposited in the National Center for Biotechnology Information Gene Expression Omnibus public data repository (http://www.ncbi.nlm.nih.gov/geo; accession number GSE79795, last accessed October 18, 2016). The oral cavity squamous cell carcinoma cell line UM-SCC-1 was obtained from Dr. Thomas Carey (University of Michigan, Ann Arbor), and PCI-15B was provided by Dr. Jeffrey Myers (University of Texas MD Anderson Cancer Center, Houston, TX). Cells were grown in Dulbecco's modified Eagle's medium (DMEM)/high-glucose [catalog number (Cat.) SH30243; Hyclone/Thermo Fisher, Waltham, MA] supplemented with 10% fetal bovine serum (Cat. 16000; Thermo Fisher Scientific), 100 U/mL penicillin and 100 μg/mL streptomycin (Cat. 15140-122; Thermo Fisher Scientific), 100 μmol/L nonessential amino acids (Cat. 11140-050; Thermo Fisher Scientific), and 2 mmol/L l-glutamine (Cat. 25030-081; Thermo Fisher Scientific). Cells were grown in medium containing 50 μg/mL Plasmocure (Cat. Ant-pc; InvivoGen, San Diego, CA) for 2 weeks, and then maintained in medium without Plasmocure. All cells were grown at 37°C with 5% CO2. Cells were plated at a density of 5 × 104 cells in antibiotic-free complete medium for 24 hours before siRNA transfection in 6-well plates in a volume of 2 mL. At the time of transfection, culture medium was replaced with fresh antibiotic-free complete medium containing siGENOME oligos at a final concentration of 50 nmol/L with 1 μL of Dharmafect Reagent 4 (Cat. T-2004-02; GE Dharmacon, Lafayette, CO); cells were incubated for 72 hours without changing medium. Knockdowns were confirmed by Western blot analysis (see below). In the case of JUN knockdowns, cells were incubated at 48 hours before the invasion assay, and knockdowns were confirmed by real-time PCR, as described below. siRNA oligos used were as follows: siGENOME Nontargeting siRNA Pool No. 2, Cat. D-001206-14-05, sequences: 5′-UAAGGCUAUGAAGAGAUAC-3′, 5′-AUGUAUUGGCCUGUAUUAG-3′, 5′-AUGAACGUGAAUUGCUCAA-3′, and 5′-UGGUUUACAUGUCGACUAA-3′; Human JUN siGENOME SMARTpool, Cat. M-003268-03-0005, sequences: 5′-UGGAAACGACCUUCUAUGA-3′, 5′-UAACGCAGCAGUUGCAAAC-3′, 5′-GAGCGGACCUUAUGGCUAC-3′, and 5′-AAGUCAUGAACCACGUUAA-3′; Human matrix metalloproteinase 7 (MMP7) siGENOME SMARTpool, Cat. M-003782-01-0010, sequences: 5′-GGAACAGGCUCAGGACUAU-3′, 5′-GCUCAAGGACUAUCUCAAGA-3′, 5′-GAGAUGCUCACUUCGAUGA-3′, and 5′-CGGAGGAGAUGCUCACUUC-3′; Human APOE siGENOME SMARTpool, Cat. M-006470-00-0005; Human APOE siGENOME siRNA (individual oligos): siAPOE-01, Cat. D-006470-01-0005, sequence: 5′-AGACAGAGCCGGAGCCCGA-3′; siAPOE-02, Cat. D-006470-02-0005, sequence: 5′-GCGCGGACAUGGAGGACGU-3′; siAPOE-03, Cat. D-006470-03-0010, sequence: 5′-GCGCGCGGAUGGAGGAGAU-3′; siAPOE-04, and Cat. D-006470-04-0010, sequence: 5′-CUGCGUUGCUGGUCACAUU-3′. All siRNA oligos were from GE Dharmacon. Invasion assays were performed using BD BioCoat Matrigel Invasion Chambers (Cat. 08-774-122; BD Biosciences/Fisher, Franklin Lakes, NJ) after siRNA transfection. Invasion chambers were hydrated and equilibrated for 2 hours before addition of cells in DMEM in a 24-well plate, and by adding DMEM inside the chambers with incubation in a 37°C incubator. Cells were detached with Accutase (Cat. S-1100-1; BioExpress/Fisher, Kaysville, UT) and counted. OSCC cells were centrifuged, resuspended in serum-free medium (0.7% bovine serum albumin/DMEM), and plated into the upper well of the invasion chamber at a density of 100,000 cells in a volume of 0.5 mL. The lower chamber of the transwell assay contained 1 mL of 0.1 nmol/L mouse epidermal growth factor (Cat. 53003018; Invitrogen, Carlsbad, CA) diluted in 0.7% bovine serum albumin/DMEM. Invasion chambers were incubated at 37°C for 24 hours. Cells were then fixed with formalin for 15 minutes, and stained with 0.2% crystal violet for 10 minutes. Cells that did not invade through to the underside of the membrane were removed by scraping. The filters were excised, applied to a glass coverslip, and imaged using a flatbed scanner (Epson America, Long Beach, CA); the percentage area of filter covered by invading cells was quantified using ImageJ software version 1.49 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). For each siRNA experiment, tumor cell invasion was normalized to the percentage invasion of cells treated with a nontargeting siRNA. All individual experiments were performed in triplicate. To collect total RNA for expression analysis, cultured cells were washed in ice-cold phosphate-buffered saline, scraped, and lysed in RLT buffer using the RNeasy Mini Kit (Cat. 74104; Qiagen, Valencia, CA). Total RNA was purified, according to the manufacturer's protocol. RNA quality was assessed using an Agilent 2100 BioAnalyzer and an RNA 6000 Nano Kit (Agilent Technologies, Wilmington, DE), according to the manufacturer's instructions. Real-time quantitative RT-PCR was performed using the TaqMan RNA-to-CT 1-Step Kit (Cat. 4392938; Applied Biosystems/Thermo Fisher Scientific). All gene expression measurements were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase as an internal control. Cells were washed in cold phosphate-buffered saline, and lysed in a 2X SDS Laemmli buffer containing protease inhibitor (Cat. 11836170-001; Roche, Basel, Switzerland), phosphatase inhibitor cocktail I (Cat. 524624; EMD Chemicals, Billerica, MA), and phosphatase inhibitor cocktail II (Cat. P5726; Sigma, St. Louis, MO). Lysates were passed through a 28-gauge needle to shear DNA, boiled for 5 minutes, and loaded into an SDS-PAGE gel to run for approximately 90 minutes at 120 V. Extracellular APOE protein was measured using 30 μL of cell culture supernatant, adding 10X SDS loading buffer, boiling for 5 minutes, and running on gel, as above. Proteins were transferred onto a nitrocellulose membrane (Cat. 1620112; Bio-Rad, Hercules, CA) and then blocked for 1 hour at room temperature in an Odyssey blocking buffer (Cat. 927-40000; LI-COR Biosciences, Lincoln, NE). Primary antibodies were diluted in blocking buffer, according to the manufacturer's instructions, and incubated overnight at 4°C on a rocking shaker. Blots were then washed three times in tris-buffered saline with Tween 20, incubated with secondary antibodies IRDye 680RD donkey anti-mouse (1:20,000) (Cat. 926-68072; LI-COR Biosciences) and IRDye 800RD donkey anti-rabbit (1:5000) (Cat. 926-32213; LI-COR Biosciences) in blocking buffer for 1 hour at room temperature, washed in tris-buffered saline with Tween 20, and imaged using an Odyssey scanner (LI-COR Biosciences). Images were analyzed using ImageJ software version 1.49. Primary antibodies and their respective dilutions used for Western blot analysis were as follows: apolipoprotein E antibody (1:500) (Cat. Ab52607) from Abcam (Cambridge, UK); P44/42 mitogen-activated protein kinase [MAPK; extracellular signal–regulated kinase (ERK) 1/2] antibody (Cat. 9102S), phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) antibody (Cat. 9101S), c-Jun antibody (Cat. 9165S), phospho-c-Jun (Ser63) antibody (Cat. 2361S), stress-activated protein kinase/JNK antibody (Cat. 9258S), and phospho–stress-activated protein kinase/JNK (Thr183/Tyr185) antibody (Cat. 4668S), all diluted 1:1000 and from Cell Signaling Technology (Danvers, MA); and β-tubulin antibody (1:5000) (Cat. T4026) and β-actin antibody (1:20,000) (Cat. A5441) from Sigma. Matrix degradation and invadopodia assays were performed in glass-bottom MatTek dishes (catalog number P35G-1.5-14-C; MatTek, Ashland, MA) that were coated with Alexa Fluor 405–labeled gelatin (Cat. A-30000; Thermo Fisher Scientific), as described.12Oser M. Yamaguchi H. Mader C.C. Bravo-Cordero J.J. Arias M. Chen X. Desmarais V. van Rheenen J. Koleske A.J. Condeelis J. Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation.J Cell Biol. 2009; 186: 571-587Crossref PubMed Scopus (287) Google Scholar, 13Jimenez L. Sharma V.P. Condeelis J. Harris T. Ow T.J. Prystowsky M.B. Childs G. Segall J.E. MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma.Arch Pathol Lab Med. 2015; 139: 1349-1361Crossref PubMed Scopus (20) Google Scholar Cells were plated onto the fluorescently labeled gelatin and allowed to attach for 4 hours. Immunofluorescence was performed, as described previously.13Jimenez L. Sharma V.P. Condeelis J. Harris T. Ow T.J. Prystowsky M.B. Childs G. Segall J.E. MicroRNA-375 suppresses extracellular matrix degradation and invadopodial activity in head and neck squamous cell carcinoma.Arch Pathol Lab Med. 2015; 139: 1349-1361Crossref PubMed Scopus (20) Google Scholar For invadopodia measurements, cells were fixed in 4% paraformaldehyde for 20 minutes, permeabilized with 0.05% Triton X-100, blocked in 1% bovine serum albumin/1% fetal bovine serum, and then incubated with antibodies for cortactin (Cat. 05-180; Millipore) and Tks5 (Cat. Sc-30122; Santa Cruz, Dallas, TX) for 1 hour at room temperature. Cells were incubated with secondary antibodies (Cy5 anti-rabbit and Cy3 anti-mouse) for 1 hour at room temperature. Both cells and matrix were imaged on an inverted Olympus IX71 fluorescent microscope (Olympus America, Center Valley, PA) at 60× objective in the DAPI, tetrarhodamine isothiocyanate, and Cy5 channels to view the Alexa Fluor 405–labeled matrix, cortactin, and Tks5, respectively. Total cellular cholesterol was measured using the Amplex Red cholesterol assay kit (Cat. A12216; Thermo Fisher Scientific). Cells were lysed in a buffer containing 150 mmol/L NaCl, 1 mmol/L EGTA, 0.1 mmol/L MgCl2, 10 mmol/L HEPES (pH 7.4), and 0.5% Triton X-100. Protease inhibitor (Roche) was added to the lysis buffer immediately before lysing the cells. Cells were scraped, sonicated for 15 seconds, and then incubated on ice for 30 minutes. Lysates were centrifuged at 4°C for 15 minutes at full speed, and supernatant was collected. Cholesterol was quantified in the supernatant, according to the instructions of the manufacturer (Thermo Fisher Scientific). Total protein was quantified in the samples using a Pierce BCA Protein assay (catalog number 23225; Thermo Fisher Scientific) for normalization. Changes in the phosphorylation of signaling proteins were screened using a phospho-kinase antibody array (Cat. ARY003B; R&D Systems, Minneapolis, MN). OSCC cells were transfected with siRNA for APOE, or with a nontargeting siRNA, as described above. After 72 hours, cells were detached with Accutase and counted. Cells were lysed in a volume of 1 mL lysis buffer per 1 × 107 cells, according to the manufacturer's instructions. Cell lysates were incubated on ice for 30 minutes and then centrifuged at top speed, and protein was quantified using the Pierce BCA Protein assay. Total protein (600 μg) from each sample was diluted and incubated with the phospho-kinase array membrane. Membranes were imaged on an Odyssey scanner and analyzed using ImageStudio Lite software version 5.0 (LI-COR Biosciences). AP-1 transcriptional activity was measured using a luciferase reporter construct provided by Dr. Ahmad Waseem (Queen Mary University of London, London, England). OSCC cells were initially transfected with siRNA for APOE-04 or nontargeting siRNA control. Forty-eight hours after initial siRNA transfection, cells were then transfected with 1.98 μg of a firefly luciferase reporter construct containing six repeats of a consensus AP-1 binding motif in the pGL4.26 vector (Promega, Madison, WI), as described in Brown et al,14Brown L. Waseem A. Cruz I.N. Szary J. Gunic E. Mannan T. Unadkat M. Yang M. Valderrama F. O'Toole E.A. Wan H. Desmoglein 3 promotes cancer cell migration and invasion by regulating activator protein 1 and protein kinase C-dependent-Ezrin activation.Oncogene. 2014; 33: 2363-2374Crossref PubMed Scopus (48) Google Scholar along with 0.02 μg of the Renilla luciferase construct pRL-CMV (Promega). FuGENE HD (Cat. PRE2311; Promega) was used as a transfection reagent. After 24 hours, the cells were lysed and luciferase signal was measured using a dual-luciferase reporter assay, according to the manufacturer's instructions (Cat. PR-E1910; Promega). To collect total RNA for expression analysis, cultured cells were washed in ice-cold phosphate-buffered saline, scraped, and lysed in RLT lysis buffer using the RNeasy Mini Kit (Cat. 74104; Qiagen). Total RNA was purified according to the manufacturer's protocol. RNA quality was assessed using an Agilent 2100 BioAnalyzer and RNA 6000 Nano Kit (Agilent Technologies, Wilmington, DE), according to the manufacturer's instructions. Real-time quantitative RT-PCR was performed using the TaqMan RNA-to-CT 1-Step Kit. All gene expression measurements were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase as an internal control. After validating reduced expression of APOE mRNA and assessing RNA quality for each sample, 20 to 50 μg of total RNA was provided to the New York Genome Center for RNA-sequencing (RNA-Seq) analysis. Two independent RNA preparations were used for the UMSCC-1 cell line, each containing a sample treated with siNT and siAPOE, for a total of four samples. RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Briefly, 500 ng of total RNA was purified by oligo-dT beads selecting for polyadenylated RNA species and fragmented by divalent cations under elevated temperature. The fragmented RNA underwent first-strand synthesis using reverse transcriptase with random priming. Second-strand synthesis was performed using DNA polymerase I and RNaseH. All cDNA fragments were subjected to end repair, adenylation of the 3′ ends, and ligation of adapters. The resulting cDNA libraries were enriched using eight cycles of PCR. Quality control of the library construction process consisted of assaying the final library size using the Agilent Bioanalyzer and quantifying the final library content by real-time PCR and PicoGreen (fluorescence) methods. A single observed peak between 250 and 350 bp indicated a properly constructed and amplified library ready for sequencing. RNA sequencing was performed on the HiSeq 2500 instrument using v4 SBS chemistry, according to the Illumina protocol, as described.15Bentley D.R. Balasubramanian S. Swerdlow H.P. Smith G.P. Milton J. Brown C.G. et al.Accurate whole human genome sequencing using reversible terminator chemistry.Nature. 2008; 456: 53-59Crossref PubMed Scopus (2519) Google Scholar Sequencing libraries were loaded onto the HiSeq 2500 flow cell for clustering on the cBot using the instrument-specific clustering protocol. Sequencing reads were aligned with STAR16Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. Batut P. Chaisson M. Gingeras T.R. STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (19131) Google Scholar version 2.4.0c, and genes annotated in Gencode v18 were quantified with featureCounts17Liao Y. Smyth G.K. Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics. 2014; 30: 923-930Crossref PubMed Scopus (8888) Google Scholar v1.4.3-p1. The DESeq2 package in Bioconductor was used for normali
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