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Major Action of Endogenous Lysyl Oxidase in Clear Cell Renal Cell Carcinoma Progression and Collagen Stiffness Revealed by Primary Cell Cultures

2016; Elsevier BV; Volume: 186; Issue: 9 Linguagem: Inglês

10.1016/j.ajpath.2016.05.019

ISSN

1525-2191

Autores

Vitalba Di Stefano, B Torsello, Cristina Bianchi, Ingrid Cifola, Eleonora Mangano, Giorgio Bovo, Valeria Cassina, Marco Schmidt, Roberta Corti, C Meregalli, Silvia Bombelli, Paolo Viganò, Cristina Battaglia, G Strada, R Perego,

Tópico(s)

Renal cell carcinoma treatment

Resumo

Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC. Human clear cell renal cell carcinoma (ccRCC) is therapy resistant; therefore, it is worthwhile studying in depth the molecular aspects of its progression. In ccRCC the biallelic inactivation of the VHL gene leads to stabilization of hypoxia-inducible factors (HIFs). Among the targets of HIF-1α transcriptional activity is the LOX gene, which codes for the inactive proenzyme (Pro-Lox) from which, after extracellular secretion and proteolysis, derives the active enzyme (Lox) and the propeptide (Lox-PP). By increasing stiffness of extracellular matrix by collagen crosslinking, Lox promotes tumor progression and metastasis. Lox and Lox-PP can reenter the cells where Lox promotes cell proliferation and invasion, whereas Lox-PP acts as tumor suppressor because of its Ras recision and apoptotic activity. Few data are available concerning LOX in ccRCC. Using an in vitro model of ccRCC primary cell cultures, we performed, for the first time in ccRCC, a detailed study of endogenous LOX and also investigated their transcriptomic profile. We found that endogenous LOX is overexpressed in ccRCC, is involved in a positive-regulative loop with HIF-1α, and has a major action on ccRCC progression through cellular adhesion, migration, and collagen matrix stiffness increment; however, the oncosuppressive action of Lox-PP was not found to prevail. These findings may suggest translational approaches for new therapeutic strategies in ccRCC. Renal cell carcinoma (RCC) represents approximately 3% of all human adult malignant neoplamsms1Rini B.I. Campbell S.C. Escudier B. Roussy I.G. Renal cell carcinoma.Lancet. 2009; 373: 1119-1132Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar and is resistant to radiation and chemotherapy. Nephrectomy may be curative but only in the early disease stages; however, approximately 40% of patients experience tumor progression and metastasis during follow-up.2Alonso A.H. García M.C. Enguita C.G. Is there a role for systemic targeted therapy after surgical treatment for metastases of renal cell carcinoma?.World J Nephrol. 2015; 4: 254-262Crossref Google Scholar Clear cell RCC (ccRCC) represents approximately 75% of kidney carcinoma,1Rini B.I. Campbell S.C. Escudier B. Roussy I.G. Renal cell carcinoma.Lancet. 2009; 373: 1119-1132Abstract Full Text Full Text PDF PubMed Scopus (1158) Google Scholar and in approximately 90% of sporadic forms ccRCC is correlated with biallelic inactivation of the VHL tumor suppressor gene, on chromosome 3p, by somatic mutations or hypermethylation.3Young A.C. Craven R.A. Cohen D. Taylor C. Booth C. Harnden P. Cairns D.A. Astuti D. Gregory W. Maher E.R. Knowles M.A. Joyce A. Selby P.J. Banks R.E. Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma.Clin Cancer Res. 2010; 15: 7582-7592Crossref Scopus (142) Google Scholar, 4Cancer Genome Atlas Research NetworkComprehensive molecular characterization of clear cell renal cell carcinoma.Nature. 2013; 499: 43-49Crossref PubMed Scopus (2298) Google Scholar VHL inactivation leads to stabilization of hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, or only HIF-2α in approximately 40% of cases because of deletion of HIF-1α locus, on chromosome 14q.5Shen C. Beroukhim R. Schumacher S.E. Zhou J. Chang M. Signoretti S. Kaelin W.G. Genetic and functional studies implicate HIF1 as a 14q kidney cancer suppressor gene.Cancer Discov. 2011; 1: 222-235Crossref PubMed Scopus (288) Google Scholar This stabilization determines a constitutive transcriptional activation of several target genes,6Semenza G.L. Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5295) Google Scholar, 7Keith B. Johnson R.S. Simon M.C. HIF1α and HIF2α: sibling rivalry in hypoxic tumour growth and progression.Nat Rev Cancer. 2011; 12: 9-22Crossref PubMed Scopus (306) Google Scholar many of which are involved in ccRCC progression4Cancer Genome Atlas Research NetworkComprehensive molecular characterization of clear cell renal cell carcinoma.Nature. 2013; 499: 43-49Crossref PubMed Scopus (2298) Google Scholar, 8Wozniak M.B. Le Calvez-kelm F. Abedi-ardekani B. Byrnes G. Durand G. Carreira C. Michelon J. Janout V. Holcatova I. Foretova L. Brisuda A. Lesueur F. McKay J. Brennan P. Scelo G. Integrative genome-wide gene expression profiling of clear cell renal cell carcinoma in Czech Republic and in the United States.PLoS One. 2013; 8: e57886Crossref PubMed Scopus (85) Google Scholar and also in its metabolic alterations.9Wettersten H.I. Hakimi A.A. Morin D. Bianchi C. Johnstone M.E. Donohoe D.R. Trott J.F. Aboud O.A. Stirdivant S. Neri B. Wolfert R. Stewart B. Perego R. Hsieh J.J. Weiss R.H. Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis.Cancer Res. 2015; 75: 2541-2552Crossref PubMed Scopus (187) Google Scholar Among HIF-1α target genes there is the lysyl oxidase (LOX) gene, on chromosome 5q23.2, that codes for a copper-dependent amine oxidase,10Barker H.E. Cox T.R. Erler J.T. The rationale for targeting the LOX family in cancer.Nat Rev Cancer. 2012; 12: 540-552Crossref PubMed Scopus (402) Google Scholar which is an important player in tumor progression and metastasis formation. LOX, after intracellular posttranslational modifications, results in an inactive 50-kDa proenzyme of 417 amino acids (Pro-Lox).11Trackman P.C. Bedell-Hogan D. Tang J. Kagan H.M. Post-translational glycosylation and proteolytic processing of a lysyl oxidase precursor.J Biol Chem. 1992; 267: 8666-8671Abstract Full Text PDF PubMed Google Scholar Once secreted in the extracellular matrix (ECM), Pro-Lox is proteolyzed by bone morphogenetic protein 1 and cleaved into the 32-kDa active enzyme (Lox) and the 18-kDa propeptide domain (Lox-PP).12Perryman L. Erler J.T. Lysyl oxidase in cancer research.Future Oncol. 2014; 10: 1709-1717Crossref PubMed Scopus (38) Google Scholar The extracellular active Lox enzyme catalyzes the covalent crosslinking of collagen and elastin in the ECM, increasing tensile strength and structural integrity of tissues, while producing hydrogen peroxide as a by-product.10Barker H.E. Cox T.R. Erler J.T. The rationale for targeting the LOX family in cancer.Nat Rev Cancer. 2012; 12: 540-552Crossref PubMed Scopus (402) Google Scholar The Lox action can increase stiffness of cancer ECM and promote cellular adhesion and migration, favoring progression, invasion, and metastasis as shown using tumor cell lines of breast, brain, and colon.13Payne S.L. Fogelgren B. Hess A.R. Seftor E.A. Wiley E.L. Fong S.F. Csiszar K. Hendrix M.J. Kirschmann D.A. Lysyl oxidase regulates breast cancer cell migration and adhesion through a hydrogen peroxide – mediated mechanism.Cancer Res. 2005; 65: 11429-11436Crossref PubMed Scopus (238) Google Scholar, 14Laczko R. Szauter K.M. Jansen M.K. Hollosi P. Muranyi M. Molnar J. Fong K.S. Hinek A. Csiszar K. Active lysyl oxidase (LOX) correlates with focal adhesion kinase (FAK)/paxillin activation and migration in invasive astrocytes.Neuropathol Appl Neurobiol. 2007; 33: 631-643Crossref PubMed Scopus (45) Google Scholar, 15Baker A. Bird D. Lang G. Cox T.R. Erler J.T. Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK.Oncogene. 2013; 32: 1863-1868Crossref PubMed Scopus (204) Google Scholar, 16Cox T.R. Rumney R.M. Schoof E.M. Perryman L. Hoye A.M. Agrawal A. Bird D. Latif N.A. Forrest H. Evans H.R. Huggins I.D. Lang G. Linding R. Gartland A. Erler J.T. The hypoxic cancer secretome induces pre-metastatic bone lesions through Lysyl Oxidase.Nature. 2015; 522: 106-110Crossref PubMed Scopus (384) Google Scholar However, a tumor suppressor activity has also been ascribed to LOX, justifying its described up-regulation or down-regulation even in the same cancer types, such as prostate and head and neck tumors.17Payne S.L. Hendrix M.J. Kirschmann D.A. Paradoxical roles for lysyl oxidases in cancer — a prospect.J Cell Biochem. 2007; 101: 1338-1354Crossref PubMed Scopus (186) Google Scholar Although LOX was first described as an ECM protein, it also performs intracellular functions, and both active 32-kDa enzyme and 18-kDa propeptide can re-enter the cells by still unknown mechanisms. Active 32-kDa Lox, through reactive oxygen species by-products and integrin stimulation, promotes cell proliferation and invasion of colorectal carcinoma cell lines by activating phosphatidylinositol 3-kinase and the downstream FAK and Src signaling cascades.15Baker A. Bird D. Lang G. Cox T.R. Erler J.T. Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK.Oncogene. 2013; 32: 1863-1868Crossref PubMed Scopus (204) Google Scholar, 18Baker A. Cox T.R. Bird D. Lang G. Murray G.I. Sun X. Southall S.M. Wilson J.R. Erler J.T. The role of lysyl oxidase in SRC-dependent proliferation and metastasis of colorectal cancer.J Natl Cancer Inst. 2011; 103: 407-424Crossref PubMed Scopus (148) Google Scholar In fibroblasts, the nuclear localization of active Lox catalyzes the oxidation of H1 and H2 histone proteins, contributing to chromosome stability.19Li W. Nellaiappan K. Strassmaier T. Graham L. Thomas K.M. Kagan H.M. Localization and activity of lysyl oxidase within nuclei of fibrogenic cells.Proc Natl Acad Sci U S A. 1997; 94: 12817-12822Crossref PubMed Scopus (149) Google Scholar Vice versa, the 18-kDa Lox-PP, acts as a tumor suppressor, having Ras recision activity20Palamakumbura A.H. Jeay S. Guo Y. Pischon N. Sommer P. Sonenshein G.E. Trackman P.C. The propeptide domain of lysyl oxidase induces phenotypic reversion of ras-transformed cells.J Biol Chem. 2004; 279: 40593-40600Crossref PubMed Scopus (113) Google Scholar and apoptotic activity by blocking the mitogen-activated protein kinases/extracellular signal-related kinases pathways21Bais M.V. Nugent M.A. Stephens D.N. Sume S.S. Kirsch K.H. Gail E. Trackman P.C. Recombinant lysyl oxidase propeptide protein inhibits growth and promotes apoptosis of pre-existing murine breast cancer xenografts.PLoS One. 2012; 7: e31188Crossref PubMed Scopus (35) Google Scholar or impairing DNA repair pathways in the nucleus.22Bais M.V. Ozdener G.B. Sonenshein G.E. Trackman P.C. Effects of tumor-suppressor lysyl oxidase propeptide on prostate cancer xenograft growth and its direct interactions with DNA repair pathways.Oncogene. 2015; 34: 1928-1937Crossref PubMed Scopus (32) Google Scholar It has also been reported that in colon carcinoma cell lines LOX and HIF-1α potentiate each other to sustain tumor progression.23Pez F. Dayan F. Durivault J. Kaniewski B. Aimond G. Le Provost G.S. Deux B. Clézardine P. Sommer P. Pouysségur J. Reynaud C. The HIF-1 inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth.Cancer Res. 2011; 71: 1647-1657Crossref PubMed Scopus (113) Google Scholar All these findings suggest that LOX may have complex and different roles in tumorigenesis, depending on the transformation status of the cells,24Friesenhengst A. Pribitzer-Winner T. Schreiber M. Association of the G473A polymorphism and expression of lysyl oxidase with breast cancer risk and survival in European women: a hospital-based case-control study.PLoS One. 2014; 9: e105579Crossref PubMed Scopus (16) Google Scholar the molecular network, the cellular location, and the cell type in which it is active. Regarding ccRCC, currently few data have been reported concerning mainly LOX expression in RCC tissues25Young A.N. Amin M.B. Moreno C.S. Lim S.D. Cohen C. Petros J.A. Marshal F.F. Neish A.S. Expression profiling of renal epithelial neoplasms a method for tumor classification and discovery of diagnostic molecular markers.Am J Pathol. 2001; 158: 1639-1651Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 26Takahashi M. Rhodes D.R. Furge K.A. Kanayama H. Kagawa S. Haab B.B. Teh B.T. Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification.Proc Natl Acad Sci U S A. 2001; 98: 9754-9759Crossref PubMed Scopus (348) Google Scholar, 27Cifola I. Spinelli R. Beltrame L. Peano C. Fasoli E. Ferrero S. Bosari S. Signorini S. Rocco F. Perego R. Proserpio V. Raimondo F. Mocarelli P. Battaglia C. Genome-wide screening of copy number alterations and LOH events in renal cell carcinomas and integration with gene expression profile.Mol Cancer. 2008; 7: 6Crossref PubMed Scopus (61) Google Scholar or cell lines.28Ross D.T. Scherf U. Eisen M.B. Perou C.M. Rees C. Spellman P. Iyer V. Jeffrey S.S. Van De Rijn M. Waltham M. Pergamenschikov A. Lee J.C. Lashkari D. Shalon D. Myers T.G. Weinstein J.N. Botstein D. Brown P.O. Systematic variation in gene expression patterns in human cancer cell lines.Nat Genet. 2000; 24: 227-235Crossref PubMed Scopus (1821) Google Scholar We performed, for the first time in human ccRCC, a comprehensive and detailed study of endogenous LOX expression and functions. To overcome the difficulties and limitations attributable to tissue heterogeneity, we took advantage of a consolidated in vitro model of primary cell cultures that we obtain from normal kidney and ccRCC tissues. We previously extensively characterized these primary cell cultures for their proteomic, cellular, and genomic features,29Perego R.A. Bianchi C. Corizzato M. Eroini B. Torsello B. Valsecchi C. Di Fonzo A. Cordani N. Favini P. Ferrero S. Pitto M. Sarto C. Magni F. Rocco F. Mocarelli P. Primary cell cultures arising from normal kidney and renal cell carcinoma retain the proteomic profile of corresponding tissues.J Proteome Res. 2005; 4: 1503-1510Crossref PubMed Scopus (34) Google Scholar, 30Bianchi C. Bombelli S. Raimondo F. Torsello B. Angeloni V. Ferrero S. Di Stefano V. Chinello C. Cifola I. Invernizzi L. Brambilla P. Magni F. Pitto M. Zanetti G. Mocarelli P. Perego R.A. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3.Am J Pathol. 2010; 176: 1660-1670Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 31Cifola I. Bianchi C. Mangano E. Bombelli S. Frascati F. Fasoli E. Ferrero S. Di Stefano V. Zipeto M.A. Magni F. Signorini S. Battaglia C. Perego R.A. Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.BMC Cancer. 2011; 11: 244Crossref PubMed Scopus (22) Google Scholar and we also investigated them for their transcriptomic profile. This in vitro model has been instrumental for the molecular and functional analysis of endogenous LOX related to ccRCC progression. Twenty-four patients with ccRCC (14 men and 10 women; median age, 71 years; range, 46 to 86 years) treated by surgery were enrolled in this study after written informed consent. Histologic types, grade, and tumor stage were defined according to World Health Organization classification.32Grignon D.J. Eble J.N. Bonsib S.M. Moch H. Clear cell renal cell carcinoma.in: Eble J.N. Sauter G. Epstein J.I. Sesterhenn I.A. WHO Classification of Tumours. Pathology and Genetics of Tumours of the Urinary System and Male Genital Organs. IARC Press, Lyon, France2004: 23-25Google Scholar Tumors were classified as 14pT1, 4pT2, 6pT3. Fuhrman grade were 2 G1, 16 G2, 5 G3, 1 not determined. The HIF-1α protein was overexpressed in 18 patients and not detectable in six. All procedures were approved by the local ethic committee. Primary cell cultures were obtained from specimens of tumor and matched renal cortex opposite to the tumor, collected after nephrectomy. The cellular composition was routinely evaluated by flow cytometry analysis as previously described.30Bianchi C. Bombelli S. Raimondo F. Torsello B. Angeloni V. Ferrero S. Di Stefano V. Chinello C. Cifola I. Invernizzi L. Brambilla P. Magni F. Pitto M. Zanetti G. Mocarelli P. Perego R.A. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3.Am J Pathol. 2010; 176: 1660-1670Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar The renal cortex cultures were >90% epithelial cells of proximal and distal tubule derivation, whereas the ccRCC cultures were >90% epithelial neoplastic cells that originate from the transformation of proximal tubular cells.30Bianchi C. Bombelli S. Raimondo F. Torsello B. Angeloni V. Ferrero S. Di Stefano V. Chinello C. Cifola I. Invernizzi L. Brambilla P. Magni F. Pitto M. Zanetti G. Mocarelli P. Perego R.A. Primary cell cultures from human renal cortex and renal-cell carcinoma evidence a differential expression of two spliced isoforms of Annexin A3.Am J Pathol. 2010; 176: 1660-1670Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 33Shen S.S. Krishna B. Chirala R. Amato R.J. Truong L.D. Kidney-specific cadherin, a specific marker for the distal portion of the nephron and related renal neoplasms.Mod Pathol. 2005; 18: 933-940Crossref PubMed Scopus (103) Google Scholar All the experiments were performed on cells at first or second passage. Subconfluent primary cell cultures were siRNA transfected with ON-TARGETplus SMART pool LOX siRNA (l-009810-00) or ON-TARGETplus Control Pool siRNA (d-001810-10-05) (Thermo Scientific Dharmacon, Lafayette, CO), using Interferin siRNA transfection reagent (Polyplus transfection; Thermo Scientific, Waltham, MA) according to the manufacturer's instructions. Transfected cells were analyzed after 48 hours. Total RNA samples were extracted from primary cell cultures using the miRNeasy Mini kit (Qiagen, Hilden, Germany). RNA samples were quantified by ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), checked for integrity on the 2100 Bioanalyzer instrument (Agilent Technologies, Palo Alto, CA), and stored at −80°C until use. A total of 150 ng of RNA samples, from eight ccRCC and eight cortex primary cell cultures, were processed with the Ambion Whole Transcript Expression Kit (Applied Biosystems, Foster City, CA) and the GeneChip Whole Transcript Terminal Labeling kit (Affymetrix, Santa Clara, CA), according to the manufacturers' protocols. Biotinylated single-stranded DNA targets were fragmented and hybridized for 16 hours at 45°C onto GeneChip Human Exon 1.0 ST Arrays (Affymetrix). After washing and staining, fluorescent microarray images were acquired using the GeneChip Scanner 3000 7 G (Affymetrix) and analyzed with the GeneChip Operating software version 1.2 (Affymetrix). Raw intensity (.CEL) files are available at Array Express repository under accession number E-MTAB-4074 (Array Express, http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4074, last accessed April 27, 2016). Probe signal intensities were quantified, normalized, and converted into gene expression values by the robust multiarray average procedure, and differentially expressed genes in ccRCC versus normal cortex cultures (DEG-PCs) were calculated by analysis of variance method using Partek Genomics Suite software version 3.5 (Partek Inc., St. Louis, MO). DEGs were selected based on a fold-change ≥2 and a P < 0.05, which was considered to indicate a statistically significant difference. Functional enrichment analysis on Gene Ontology (GO) biological processes terms was performed on our DEG-PCs by ToppGene Suite (ToppGene Suite, https://toppgene.cchmc.org, last accessed April 27, 2016). A false discovery rate (Benjiamin-Hochberg correction) <0.05 was applied to define significantly enriched biological processes, limiting to GO terms with gene numbers between 200 and 1000 (200 ≤ n ≤ 1000). We compared our DEG-PC list to the list of differentially expressed genes related to ccRCC tissues (2493 DEG tissues, derived from RNA sequence analysis) reported as supplementary data by Wozniak et al.8Wozniak M.B. Le Calvez-kelm F. Abedi-ardekani B. Byrnes G. Durand G. Carreira C. Michelon J. Janout V. Holcatova I. Foretova L. Brisuda A. Lesueur F. McKay J. Brennan P. Scelo G. Integrative genome-wide gene expression profiling of clear cell renal cell carcinoma in Czech Republic and in the United States.PLoS One. 2013; 8: e57886Crossref PubMed Scopus (85) Google Scholar Comparison between GO biological processes enriched in ccRCC primary cultures and ccRCC tissues was performed using the ToppCluster tool, applying a false discovery rate threshold of 0.05 (ToppCluster, https://toppcluster.cchmc.org, last accessed April 27, 2016).34Kaimal V. Bardes E.E. Tabar S.C. Jegga A.G. Aronow B.J. ToppCluster: a multiple gene list feature analyzer for comparative enrichment clustering and network-based dissection of biological systems.Nucleic Acids Res. 2010; 38: W96-W102Crossref PubMed Scopus (249) Google Scholar Meta-analysis for LOX gene expression was performed by using Oncomine Research Edition web tool (Oncomine, www.oncomine.org, last accessed October 13, 2015) comparing seven different ccRCC data sets (Oncomine identifications: Beroukhim, Gumz, Higginz, Jones, Lenburg, Vasselli, Yusenko). A total of 1 μg of RNA was reverse transcribed with the high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA). TaqMan gene expression real-time PCR assays were performed in duplicate for each sample, following manufacturer's instructions using the indicated commercial kits (Lox: Hs00942480_m1; TGF-ß3: Hs01086000_m1; Snail: Hs00195591_m1; Zeb-2: Hs00207691_m1; E-Cadherin: Hs01023894_m1; GAPDH: Hs99998805_m1; Applied Biosystem) and an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems). The data were expressed as 2−ΔCT or 2−ΔΔCT when referred to a calibrator considered equal to 1. Immunohistochemistry (IHC) was performed on matched ccRCC and cortex deparaffinated tissue sections of eight different patients' specimens. After retrieval and blocking, the sections were incubated overnight at 4°C with two different primary polyclonal rabbit antibodies against human LOX. The former, defined antibody A, is able to recognize the active Lox 32-kDa peptide and the Pro-Lox 50 kDa peptide (dilution, 1:200; 31238, Abcam, Cambridge, UK); the latter, here called antibody B, recognizes the Lox-PP 18-kDa peptide and the Pro-Lox peptide (dilution, 1:200; NBPI-30012, Novus Biologicals, Littleton, CO). Renal sections were then incubated with secondary anti-rabbit antibody (dilution, 1:100; Dako, Glostrup, Denmark) coupled with horseradish peroxidase and then color was developed with 3-3′-diaminobenzidine. A semiquantitative evaluation of ccRCC tissue staining was performed, by two independent pathologists, in tissue slides at ×200 magnification using ImageJ software version 1.50i (NIH, Bethesda, MD; http://imagej.nih.gov/ij) Cell Counter function and taking into account intensity and subcellular distribution of staining. About 1200 cells were analyzed in five different fields for each of the eight different tissue specimens. Immunofluorescence was performed on primary cell cultures as we previously described35Bianchi C. Torsello B. Di Stefano V. Zipeto M. Facchetti R. Bombelli S. Perego R.A. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton.Exp Cell Res. 2013; 319: 2091-2102Crossref PubMed Scopus (8) Google Scholar using antibody A (dilution, 1:200), antibody B (dilution, 1:200), antibody against Paxillin (dilution, 1:50; clone 349, Becton Dickinson, San Jose, CA), and the Alexa Fluor 488 or 594 conjugated anti-rabbit or anti-mouse antibodies (dilution, 1:100; Invitrogen, Carlsberg, CA). Nuclei were counterstained with mounting DAPI (Invitrogen). Immunofluorescence images were obtained with confocal microscope Zeiss LSM710, using 63× objective, equipped with Zen software version 2009 (ZEISS Italia, Arese, Italy). Primary cell cultures were lyzed in buffer containing 50 mmol/L Tris pH 7.4, Nonidet P40, 0.25% deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, protease inhibitor cocktail, and phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich, Milan, Italy), and nuclear and nuclear-free fraction preparations were performed as previously described.35Bianchi C. Torsello B. Di Stefano V. Zipeto M. Facchetti R. Bombelli S. Perego R.A. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton.Exp Cell Res. 2013; 319: 2091-2102Crossref PubMed Scopus (8) Google Scholar Thirty micrograms of protein of cellular lysates was separated on NuPage 4% to 12% Bis-Tris precast gels (Invitrogen) and transferred to nitrocellulose membranes. To evaluate the secreted LOX proteins, primary cell cultures were grown until semiconfluence in defined media (Dulbecco's modified Eagle's medium F12, 5 μg/mL of insulin, 5 μg/mL of transferrin, 5 ng/mL of sodium selenite, 36 ng/mL of hydrocortisone, 40 pg/mL of triiodothyronine, 20 ng/mL of epidermal growth factor; all from Sigma-Aldrich) that were collected, centrifuged at 530 × g, and left overnight at −20°C with four volumes of ethanol 100%. These media were then centrifuged twice at 13,000 × g for 30 minutes at 4°C, and the whole protein pellets were suspended in loading buffer for gel separation and membrane transfer as for cell culture lysates. The membranes, after blocking, were probed overnight at 4°C with antibodies against LOX (antibody A, dilution, 1:500; antibody B, dilution, 1:500), β-actin (dilution, 1:1000; A2066, Sigma), HIF-1α (dilution, 1:500; NB100-132, Novus Biological), N-cadherin (dilution, 1:1000; 32/N, Becton Dickinson), E-cadherin (dilution, 1:1000; Becton Dickinson), paxillin (dilution, 1:1000; 2542, Cell Signaling, Boston, MA), phosho-Y118 paxillin (dilution, 1:1000; 2541, Cell Signaling), vimentin (dilution, 1:1000; V9, Dako), and α-smooth muscle actin (dilution 1:1000; 14A, Dako). Secondary antibodies coupled with horseradish peroxidase and SuperSignal West Dura Detection System (Pierce, Rockford, IL) were used to detect protein bands. Densitometric values of specific bands were normalized with corresponding β-actin band intensities, unless otherwise specified. The protein bands of culture media were normalized by the number of cells collected from each plate. ccRCC cells were stained with anti-cytokeratin fluorescein isothiocyanate antibody (dilution, 1:10; CK-6H5, Miltenyi Biotec, Bergisch Gladbach, Germany) 48 hours after LOX siRNA or control siRNA transfection. Fluorescence-activated cell sorting analysis was performed with MOFLOS ASTRIOS and data analyzed by Kaluza software version 1.2 (Beckman Coulter, Miami, FL). The acquisition process was stopped when 20,000 events were collected in the population gate. Apoptosis was assessed using Caspase-Glo 3/7 Assay (Promega, Madison, WI), following manufacturer's instruction. A total of 2 × 104 cells of LOX silenced cultures have been seeded on 96-well plate in duplicate for each sample. Luminescence was recorded on spectrometer Victor Wolla C1420 (Perkin Elmer, Woltham, MA) at the wave length of 485 ex/527em nm and expressed as relative caspase 3/7 activity with respect to the control considered equal to 1. Cell proliferation of LOX silenced and control ccRCC primary cultures was determined in triplicate for each sample (2 × 105 cells) by MTT assay (Sigma-Aldrich). After medium withdrawal, 0.5 mg/mL of MTT solution was added to each well, and cells were incubated at 37°C for an hour and a half, then dimethyl sulfoxide was added and the final products quantified at 570 nm. The optical density value of (LOX silenced sample/nonspecific siRNA sample) ×100 indicated the percentage of proliferating cells with respect to the control. Migration activity was evaluated by Boyden chamber assay and wound healing assay as previously described.35Bianchi C. Torsello B. Di Stefano V. Zipeto M. Facchetti R. Bombelli S. Perego R.A. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton.Exp Cell Res. 2013; 319: 2091-2102Crossref PubMed Scopus (8) Google Scholar In the Boyden chamber assay, 2 × 104 LOX silenced and control ccRCC primary cells were seeded in duplicate in the upper chamber of a Transwell device, equipped with 8-μm microporous membranes (Corning Inc., New York, NY) and Dulbecco's modified Eagle's medium low glucose with 5% fetal bovine serum. The lower compartments of Transwells we

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