Portal de Periódicos da CAPES

Hepatocyte-Derived Lipocalin 2 Is a Potential Serum Biomarker Reflecting Tumor Burden in Hepatoblastoma

2018; Elsevier BV; Volume: 188; Issue: 8 Linguagem: Inglês

10.1016/j.ajpath.2018.05.006

1525-2191

Laura Molina, Danielle Bell, Junyan Tao, Morgan Preziosi, Tirthadipa Pradhan‐Sundd, Sucha Singh, Minakshi Poddar, Jianhua Luo, Sarangarajan Ranganathan, Maria Chikina, Satdarshan P. Monga,

Cancer-related gene regulation

Hepatoblastoma (HB) is the most common pediatric liver malignant tumor. Previously, we reported co-activation of β-catenin and Yes-associated protein-1 (YAP1) in 80% of HB. Hepatic co-expression of active β-catenin and YAP1 via sleeping beauty transposon/transposase and hydrodynamic tail vein injection led to HB development in mice. Here, we identify lipocalin 2 (Lcn2) as a target of β-catenin and YAP1 in HB and show that serum Lcn2 values positively correlated with tumor burden. Lcn2 was strongly expressed in HB tumor cells in our mouse model. A tissue array of 62 HB cases showed highest LCN2 expression in embryonal and lowest in fetal, blastemal, and small cell undifferentiated forms of HB. Knockdown of LCN2 in HB cells had no effect on cell proliferation but reduced NF-κB reporter activity. Next, liver-specific Lcn2 knockout (KO) mice were generated. No difference in tumor burden was observed between Lcn2 KO mice and wild-type littermate controls after sleeping beauty transposon/transposase and hydrodynamic tail vein injection delivery of active YAP1 and β-catenin, although Lcn2 KO mice with HB lacked any serum Lcn2 elevation, demonstrating that transformed hepatocytes are the source of serum Lcn2. More blastemal areas and inflammation were observed within HB in Lcn2 KO compared with wild-type tumors. In conclusion, Lcn2 expressed in hepatocytes appears to be dispensable for the pathogenesis of HB. However, transformed hepatocytes secrete serum Lcn2, making Lcn2 a valuable biomarker for HB. Hepatoblastoma (HB) is the most common pediatric liver malignant tumor. Previously, we reported co-activation of β-catenin and Yes-associated protein-1 (YAP1) in 80% of HB. Hepatic co-expression of active β-catenin and YAP1 via sleeping beauty transposon/transposase and hydrodynamic tail vein injection led to HB development in mice. Here, we identify lipocalin 2 (Lcn2) as a target of β-catenin and YAP1 in HB and show that serum Lcn2 values positively correlated with tumor burden. Lcn2 was strongly expressed in HB tumor cells in our mouse model. A tissue array of 62 HB cases showed highest LCN2 expression in embryonal and lowest in fetal, blastemal, and small cell undifferentiated forms of HB. Knockdown of LCN2 in HB cells had no effect on cell proliferation but reduced NF-κB reporter activity. Next, liver-specific Lcn2 knockout (KO) mice were generated. No difference in tumor burden was observed between Lcn2 KO mice and wild-type littermate controls after sleeping beauty transposon/transposase and hydrodynamic tail vein injection delivery of active YAP1 and β-catenin, although Lcn2 KO mice with HB lacked any serum Lcn2 elevation, demonstrating that transformed hepatocytes are the source of serum Lcn2. More blastemal areas and inflammation were observed within HB in Lcn2 KO compared with wild-type tumors. In conclusion, Lcn2 expressed in hepatocytes appears to be dispensable for the pathogenesis of HB. However, transformed hepatocytes secrete serum Lcn2, making Lcn2 a valuable biomarker for HB. Hepatoblastoma (HB) is the most common pediatric liver malignant tumor, comprising about 1% of all pediatric cancers and accounting for 80% of pediatric liver cancer.1McLaughlin C.C. Baptiste M.S. Schymura M.J. Nasca P.C. Zdeb M.S. Maternal and infant birth characteristics and hepatoblastoma.Am J Epidemiol. 2006; 163: 818-828Crossref PubMed Scopus (103) Google Scholar, 2Darbari A. Sabin K.M. Shapiro C.N. Schwarz K.B. Epidemiology of primary hepatic malignancies in U.S. children.Hepatology. 2003; 38: 560-566Crossref PubMed Scopus (228) Google Scholar Twenty percent of children will present with lung metastasis at diagnosis, and for this subset of patients, the prognosis is poor.3Czauderna P. Haeberle B. Hiyama E. Rangaswami A. Krailo M. Maibach R. Rinaldi E. Feng Y. Aronson D. Malogolowkin M. Yoshimura K. Leuschner I. Lopez-Terrada D. Hishiki T. Perilongo G. von Schweinitz D. Schmid I. Watanabe K. Derosa M. Meyers R. The Children's Hepatic tumors International Collaboration (CHIC)Novel global rare tumor database yields new prognostic factors in hepatoblastoma and becomes a research model.Eur J Cancer. 2016; 52: 92-101Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 4Perilongo G. Brown J. Shafford E. Brock P. De Camargo B. Keeling J.W. Vos A. Philips A. Pritchard J. Plaschkes J. Hepatoblastoma presenting with lung metastases: treatment results of the first cooperative, prospective study of the International Society of Paediatric Oncology on childhood liver tumors.Cancer. 2000; 89: 1845-1853Crossref PubMed Scopus (107) Google Scholar, 5Meyers R.L. Rowland J.R. Krailo M. Chen Z. Katzenstein H.M. Malogolowkin M.H. Predictive power of pretreatment prognostic factors in children with hepatoblastoma: a report from the Children's Oncology Group.Pediatr Blood Cancer. 2009; 53: 1016-1022Crossref PubMed Scopus (145) Google Scholar Treatment is dependent on staging, which is derived from a combination of radiographic and histologic findings. Although well-differentiated fetal HB can be cured with resection alone, less differentiated histologic subtypes, such as embryonal or crowded fetal, require more aggressive treatment with cytotoxic chemotherapy followed by surgical resection or liver transplantation.3Czauderna P. Haeberle B. Hiyama E. Rangaswami A. Krailo M. Maibach R. Rinaldi E. Feng Y. Aronson D. Malogolowkin M. Yoshimura K. Leuschner I. Lopez-Terrada D. Hishiki T. Perilongo G. von Schweinitz D. Schmid I. Watanabe K. Derosa M. Meyers R. The Children's Hepatic tumors International Collaboration (CHIC)Novel global rare tumor database yields new prognostic factors in hepatoblastoma and becomes a research model.Eur J Cancer. 2016; 52: 92-101Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar HB is unique in that multiple different histologic subtypes can exist within a single tumor.6Cairo S. Armengol C. De Reynies A. Wei Y. Thomas E. Renard C.A. et al.Hepatic stem-like phenotype and interplay of Wnt/beta-catenin and Myc signaling in aggressive childhood liver cancer.Cancer Cell. 2008; 14: 471-484Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar These histologic differences may be attributable to mutations that occur at various stages of hepatoblast development.7Dan Y.Y. Riehle K.J. Lazaro C. Teoh N. Haque J. Campbell J.S. Fausto N. Isolation of multipotent progenitor cells from human fetal liver capable of differentiating into liver and mesenchymal lineages.Proc Natl Acad Sci U S A. 2006; 103: 9912-9917Crossref PubMed Scopus (269) Google Scholar The most well-known mutations in HB are in CTNNB1 (β-catenin gene) and are seen in approximately 90% of human HB.8Koch A. Denkhaus D. Albrecht S. Leuschner I. von Schweinitz D. Pietsch T. Childhood hepatoblastomas frequently carry a mutated degradation targeting box of the beta-catenin gene.Cancer Res. 1999; 59: 269-273PubMed Google Scholar, 9Koch A. Weber N. Waha A. Hartmann W. Denkhaus D. Behrens J. Birchmeier W. von Schweinitz D. Pietsch T. Mutations and elevated transcriptional activity of conductin (AXIN2) in hepatoblastomas.J Pathol. 2004; 204: 546-554Crossref PubMed Scopus (38) Google Scholar, 10Aretz S. Koch A. Uhlhaas S. Friedl W. Propping P. von Schweinitz D. Pietsch T. Should children at risk for familial adenomatous polyposis be screened for hepatoblastoma and children with apparently sporadic hepatoblastoma be screened for APC germline mutations?.Pediatr Blood Cancer. 2006; 47: 811-818Crossref PubMed Scopus (99) Google Scholar, 11Cairo S. Armengol C. Buendia M.A. Activation of Wnt and Myc signaling in hepatoblastoma.Front Biosci (Elite Ed). 2012; 4: 480-486Crossref PubMed Google Scholar Recently, overactivation of Yes-associated protein-1 (YAP1) was found in most HB. In fact, co-activation of β-catenin and YAP1 was reported in 80% of human HB.12Li H. Wolfe A. Septer S. Edwards G. Zhong X. Abdulkarim A.B. Ranganathan S. Apte U. Deregulation of Hippo kinase signalling in human hepatic malignancies.Liver Int. 2012; 32: 38-47Crossref PubMed Scopus (105) Google Scholar It is presumed that β-catenin and YAP1 activation results in nuclear translocation and binding to transcription factors T-cell factor 4 (TCF4) and TEA domain (TEAD), respectively, to initiate cell proliferation, growth, and differentiation.13Camargo F.D. Gokhale S. Johnnidis J.B. Fu D. Bell G.W. Jaenisch R. Brummelkamp T.R. YAP1 increases organ size and expands undifferentiated progenitor cells.Curr Biol. 2007; 17: 2054-2060Abstract Full Text Full Text PDF PubMed Scopus (935) Google Scholar, 14Tan X. Behari J. Cieply B. Michalopoulos G.K. Monga S.P. Conditional deletion of beta-catenin reveals its role in liver growth and regeneration.Gastroenterology. 2006; 131: 1561-1572Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 15Monga S.P. Pediaditakis P. Mule K. Stolz D.B. Michalopoulos G.K. Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration.Hepatology. 2001; 33: 1098-1109Crossref PubMed Scopus (215) Google Scholar Previously, we developed a murine model of HB using sleeping beauty transposon/transposase and hydrodynamic tail vein injection (SB-HTVI) to overexpress constitutively active forms of β-catenin (∆N90-β-catenin) and YAP1 (YAPS127A) in hepatocytes.16Tao J. Calvisi D.F. Ranganathan S. Cigliano A. Zhou L. Singh S. Jiang L. Fan B. Terracciano L. Armeanu-Ebinger S. Ribback S. Dombrowski F. Evert M. Chen X. Monga S.P. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.Gastroenterology. 2014; 147: 690-701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Co-expression of these two proto-oncogenes but not single expression led to HB development in YAP1–β-catenin mouse model with 100% penetrance.12Li H. Wolfe A. Septer S. Edwards G. Zhong X. Abdulkarim A.B. Ranganathan S. Apte U. Deregulation of Hippo kinase signalling in human hepatic malignancies.Liver Int. 2012; 32: 38-47Crossref PubMed Scopus (105) Google Scholar, 16Tao J. Calvisi D.F. Ranganathan S. Cigliano A. Zhou L. Singh S. Jiang L. Fan B. Terracciano L. Armeanu-Ebinger S. Ribback S. Dombrowski F. Evert M. Chen X. Monga S.P. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.Gastroenterology. 2014; 147: 690-701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar This finding suggests that an oncogenic promoter downstream of β-catenin and YAP1 may be involved in HB development. Here, we identify Lcn2 as one gene regulated by both β-catenin and YAP1 and up-regulated in HB in mice and patients. Lipocalin 2 (Lcn2), alias neutrophil gelatinase–associated lipocalin (NGAL), is a small protein that has been implicated in a variety of human cancers, including breast, pancreatic, gastric, colon, ovarian, and hepatocellular carcinomas.17Bauer M. Eickhoff J.C. Gould M.N. Mundhenke C. Maass N. Friedl A. Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer.Breast Cancer Res Treat. 2008; 108: 389-397Crossref PubMed Scopus (178) Google Scholar, 18Candido S. Maestro R. Polesel J. Catania A. Maira F. Signorelli S.S. McCubrey J.A. Libra M. Roles of neutrophil gelatinase-associated lipocalin (NGAL) in human cancer.Oncotarget. 2014; 5: 1576-1594Crossref PubMed Scopus (89) Google Scholar, 19Rodvold J.J. Mahadevan N.R. Zanetti M. Lipocalin 2 in cancer: when good immunity goes bad.Cancer Lett. 2012; 316: 132-138Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Roli L. Pecoraro V. Trenti T. Can NGAL be employed as prognostic and diagnostic biomarker in human cancers? a systematic review of current evidence.Int J Biol Markers. 2017; 32: e53-e61Crossref PubMed Scopus (28) Google Scholar It was first discovered less than a decade ago as a component of the innate immune system, present in neutrophils and other inflammatory cells as well as the epithelial lining of the respiratory and gastrointestinal tracts. Lcn2 participates in iron metabolism by binding siderophores and moving iron intracellularly and can deprive bacteria of iron, causing decreased bacterial growth.21Goetz D.H. Holmes M.A. Borregaard N. Bluhm M.E. Raymond K.N. Strong R.K. The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.Mol Cell. 2002; 10: 1033-1043Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar It is also a secretory protein present in both serum and urine of humans and animals and has been studied as a potential biomarker for acute kidney injury.22Wheeler D.S. Devarajan P. Ma Q. Harmon K. Monaco M. Cvijanovich N. Wong H.R. Serum neutrophil gelatinase-associated lipocalin (NGAL) as a marker of acute kidney injury in critically ill children with septic shock.Crit Care Med. 2008; 36: 1297-1303Crossref PubMed Scopus (284) Google Scholar, 23Mishra J. Ma Q. Kelly C. Mitsnefes M. Mori K. Barasch J. Devarajan P. Kidney NGAL is a novel early marker of acute injury following transplantation.Pediatr Nephrol. 2006; 21: 856-863Crossref PubMed Scopus (292) Google Scholar, 24Devarajan P. NGAL in acute kidney injury: from serendipity to utility.Am J Kidney Dis. 2008; 52: 395-399Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The role of Lcn2 in cancer has recently been under close scrutiny. Lcn2 is overexpressed in breast cancer and even more so in metastatic disease, directly correlating with worse patient outcomes.25Yang J. Bielenberg D.R. Rodig S.J. Doiron R. Clifton M.C. Kung A.L. Strong R.K. Zurakowski D. Moses M.A. Lipocalin 2 promotes breast cancer progression.Proc Natl Acad Sci U S A. 2009; 106: 3913-3918Crossref PubMed Scopus (267) Google Scholar, 26Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (238) Google Scholar, 27Wang Y. Zeng T. Neutrophil gelatinase-associated lipocalin protein as a biomarker in the diagnosis of breast cancer: a meta-analysis.Biomed Rep. 2013; 1: 479-483Crossref PubMed Google Scholar Conversely, overexpression of Lcn2 in pancreatic cancer is associated with antitumorigenic effect.28Tong Z. Kunnumakkara A.B. Wang H. Matsuo Y. Diagaradjane P. Harikumar K.B. Ramachandran V. Sung B. Chakraborty A. Bresalier R.S. Logsdon C. Aggarwal B.B. Krishnan S. Guha S. Neutrophil gelatinase-associated lipocalin: a novel suppressor of invasion and angiogenesis in pancreatic cancer.Cancer Res. 2008; 68: 6100-6108Crossref PubMed Scopus (136) Google Scholar, 29Xu B. Jin D.Y. Lou W.H. Wang D.S. Lipocalin-2 is associated with a good prognosis and reversing epithelial-to-mesenchymal transition in pancreatic cancer.World J Surg. 2013; 37: 1892-1900Crossref PubMed Scopus (15) Google Scholar The mechanisms by which Lcn2 may be involved in oncogenesis have yet to be determined. Lcn2 can bind to matrix metallopeptidase-9 and stabilize it to aid in tumor invasion and metastasis.18Candido S. Maestro R. Polesel J. Catania A. Maira F. Signorelli S.S. McCubrey J.A. Libra M. Roles of neutrophil gelatinase-associated lipocalin (NGAL) in human cancer.Oncotarget. 2014; 5: 1576-1594Crossref PubMed Scopus (89) Google Scholar, 26Fernandez C.A. Yan L. Louis G. Yang J. Kutok J.L. Moses M.A. The matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin complex plays a role in breast tumor growth and is present in the urine of breast cancer patients.Clin Cancer Res. 2005; 11: 5390-5395Crossref PubMed Scopus (238) Google Scholar Lcn2 induction may trigger inflammation within the tumor environment via the NF-κB pathway.30Iannetti A. Pacifico F. Acquaviva R. Lavorgna A. Crescenzi E. Vascotto C. Tell G. Salzano A.M. Scaloni A. Vuttariello E. Chiappetta G. Formisano S. Leonardi A. The neutrophil gelatinase-associated lipocalin (NGAL), a NF-kappaB-regulated gene, is a survival factor for thyroid neoplastic cells.Proc Natl Acad Sci U S A. 2008; 105: 14058-14063Crossref PubMed Scopus (167) Google Scholar, 31Mahadevan N.R. Rodvold J. Almanza G. Perez A.F. Wheeler M.C. Zanetti M. ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-kappaB-dependent manner.BMC Cancer. 2011; 11: 229Crossref PubMed Scopus (48) Google Scholar, 32Kim H.J. Ohk B. Kang W.Y. Seong S.J. Suk K. Lim M.S. Kim S.Y. Yoon Y.R. Deficiency of lipocalin-2 promotes proliferation and differentiation of osteoclast precursors via regulation of c-Fms expression and nuclear factor-kappa B activation.J Bone Metab. 2016; 23: 8-15Crossref PubMed Google Scholar Lcn2 may also regulate iron-dependent pathways that are essential for cancer growth.33Tang H.C. Chang P.C. Chen Y.C. Iron depletion strategy for targeted cancer therapy: utilizing the dual roles of neutrophil gelatinase-associated lipocalin protein.J Mol Model. 2016; 22: 32Crossref PubMed Scopus (4) Google Scholar Increasingly, Lcn2 has been identified as a nonspecific biomarker of inflammation and organ injury that could be useful in disease monitoring or even predictive of prognosis.20Roli L. Pecoraro V. Trenti T. Can NGAL be employed as prognostic and diagnostic biomarker in human cancers? a systematic review of current evidence.Int J Biol Markers. 2017; 32: e53-e61Crossref PubMed Scopus (28) Google Scholar Although Lcn2 has been found to be overexpressed in hepatocellular carcinoma, it has not been studied in HB and not known to be associated with β-catenin or YAP1 signaling. On the basis of our analysis depicting marked up-regulation of Lcn2 in the mouse model of HB, we hypothesized that Lcn2 would be overexpressed in murine and human HB samples. We also examined whether Lcn2 was contributing to β-catenin–YAP1–induced HB. Lcn2 expression in human HB tumors correlated with tumor differentiation, with increased expression in less differentiated tumors. Lcn2 was secreted from hepatocytes in murine HB, and serum Lcn2 correlated strongly with tumor burden. Although knocking out Lcn2 from hepatocytes in mice did not affect HB initiation or progression, it affected overall tumor inflammation and led to areas of distinct histologic findings. Our work offers strong support for the applicability of serum Lcn2 as a biomarker for HB burden and histologic subtypes. Macroscopic tumor nodules <7 weeks after YAP1–β-catenin injection (n = 3 per mouse; three mice) and normal livers from noninjected mice (n = 3) were used for mRNA isolation and subjected to Affymetrix gene array using chromatin immunoprecipitation (ChIP) R430 version 2.0 (Affymetrix, Santa Clara, CA). The full data set is available at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo; accession number GSE112485). Microarray data were normalized using the expresso function from the affy Bioconductor package.34Gautier L. Cope L. Bolstad B.M. Irizarry R.A. affy–analysis of Affymetrix GeneChip data at the probe level.Bioinformatics. 2004; 20: 307-315Crossref PubMed Scopus (3862) Google Scholar Probes were mapped to genes using the custom brain array CDF.35Sandberg R. Larsson O. Improved precision and accuracy for microarrays using updated probe set definitions.BMC Bioinformatics. 2007; 8: 48Crossref PubMed Scopus (125) Google Scholar Differential expression was determined using the limma Bioconductor package, with robust linear models.36Ritchie M.E. Phipson B. Wu D. Hu Y. Law C.W. Shi W. Smyth G.K. limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Crossref PubMed Scopus (15316) Google Scholar A list of genes that were significantly (q value < 0.1) up-regulated (at least 1.5-fold) in tumor samples relative to control livers was compiled. Genome-wide scan for TCF4-TEAD target genes was performed to identify genes containing both TEAD and TCF4 binding sites in their promoters. Potential TCF4 targets were taken from a combination of genome-wide TCF4 ChIP experiments as compiled by the ChIP Enrichment Analysis (CHEA) database.37Lachmann A. Xu H. Krishnan J. Berger S.I. Mazloom A.R. Ma'ayan A. ChEA: transcription factor regulation inferred from integrating genome-wide ChIP-X experiments.Bioinformatics. 2010; 26: 2438-2444Crossref PubMed Scopus (536) Google Scholar Because the CHEA database does not contain any data for TEAD, potential TEAD targets were extracted from a genome-wide scan of TEAD consensus sites as provided by the Homer Known Motifs genome track.38Heinz S. Benner C. Spann N. Bertolino E. Lin Y.C. Laslo P. Cheng J.X. Murre C. Singh H. Glass C.K. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities.Mol Cell. 2010; 38: 576-589Abstract Full Text Full Text PDF PubMed Scopus (6335) Google Scholar Genes that had a TEAD consensus site within 2000 upstream and 500 downstream of the transcription start site were considered. Using the above two approaches, a significant overlap of genes was found (hypergeometric P = 0.01), resulting in five top candidates, of which Lcn2 was one and was chosen for additional, in-depth analysis. All mouse studies were performed in accordance with NIH's Guide for the Care and Use of Laboratory Animals39Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research Council: Guide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar and under an approved animal protocol by the Institutional Animal Use and Care Committee at the University of Pittsburgh. Lcn2-floxed mice were used with permission from Dr. Jack Cowland and generously supplied by Dr. Grace Gao (Rutgers University, New Brunswick, NJ). Lcn2-floxed mice were crossed with albumin-Cre mice to generate liver-specific Lcn2 knockout (KO) or Lcn2 KO mice. Lcn2 KO mice or wildtype (WT) littermate controls were injected with plasmids for both ∆N90–β-catenin and YAP S127A at 7 to 8 weeks of age using the concentrations and HTVI technique described in our original publication.16Tao J. Calvisi D.F. Ranganathan S. Cigliano A. Zhou L. Singh S. Jiang L. Fan B. Terracciano L. Armeanu-Ebinger S. Ribback S. Dombrowski F. Evert M. Chen X. Monga S.P. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.Gastroenterology. 2014; 147: 690-701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar At 6 to 7 weeks post injection, the mice were sacrificed. Serum and livers were harvested from all animals and stored at −80°C. Liver sections were placed in 10% formalin for 48 hours and then transferred to 70% ethanol. Sections were processed and embedded into paraffin blocks. After transfection, the cell media was aspirated, and radioimmunoprecipitation assay buffer that contained Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA) was added to each well. HepG2 cells were lyzed in radioimmunoprecipitation assay buffer, and three wells of each sample were pooled for optimal protein concentration, which was determined by bicinchoninic acid assay (Thermo Fisher Scientific). Aliquots of 40 μg were denatured by boiling for 9 minutes in 2× Laemmli Sample Buffer (Bio-Rad, Hercules, CA) and loaded in precast 4% to 15% gradient gels (Bio-Rad) for electrophoresis. Protein was transferred onto a nitrocellulose membrane via a semidry transfer method using the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked in 5% nonfat dry milk in blotto (Tris-buffered saline that contained 0.1% Tween 20) overnight at 4°C. The primary antibodies used were as follows: rabbit monoclonal anti-LCN2 (ab125075, 1:1000, Abcam, Cambridge, MA), rabbit monoclonal anti-Yap (ab52771, 1:1000, Abcam), mouse monoclonal anti–β-catenin (1:1000, BD Biosciences, San Jose, CA), rabbit polyclonal anti-p65 (1:200, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal phosphor–Ser536-p65 (1:1000, Cell Signaling, Danvers, MA), and mouse monoclonal anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000, Sigma-Aldrich, St. Louis, MO). Each primary antibody was incubated overnight at 4°C, except anti-GAPDH, which was added for 2 hours at room temperature. Membranes were washed with blotto and incubated with rabbit (1:15,000) or mouse (1:50,000) horseradish peroxidase–secondary antibody for 2 hours. Bands were visualized by Super Signal West Femto kit (Thermo Fisher Scientific). Frozen livers from a previously published YAP–β-catenin model were used.16Tao J. Calvisi D.F. Ranganathan S. Cigliano A. Zhou L. Singh S. Jiang L. Fan B. Terracciano L. Armeanu-Ebinger S. Ribback S. Dombrowski F. Evert M. Chen X. Monga S.P. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.Gastroenterology. 2014; 147: 690-701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar RNA was extracted with Trizol (Ambion, Carlsbad, CA). A DNase kit (Ambion) was used to remove contaminating genomic DNA and cDNA synthesis kit (Invitrogen, Carlsbad, CA) was used for reverse transcription. Real-time PCR was performed on cDNA using SybrGreen. Primers were purchased from Integrated DNA Technologies (Coralville, IA). Mouse-specific Lcn2 primers used were as follows: 5′-TTTCACCCGCTTTGCCAACT-3′ (forward), 5′-GTCTCTGCGCATCCCAFTCA-3′ (reverse). Additional primers used include the following: inducible nitric oxide synthase: 5′-TCACGCTTGGGTCTTGTTCA-3′ (forward) and 5′-GGGGAGCCATTTTGGTGACT-3′ (reverse); MYC: 5′-CCTAGTGCTGCATGAGGAGA-3′ (forward) and 5′-TCCACAGACACCACATCAATTT-3′ (reverse); IL-6: 5′-ATCAGGAAATTTGCCTATTGAAA-3′ (forward) and 5′-CCAGGTAGCTATGGTACTCCAGA-3′ (reverse); interferon-γ: 5′-ATCTGGAGGAACTGGCAAAA-3′ (forward) and 5′-TTCAAGACTTCAAAGAGTCTGAGGTA-3′ (reverse); and IL-1β: 5′-AGTTGACGGACCCCAAAAG-3′ (forward) and 5′-AGCTGGATGCTCTCATCAGG-3′ (reverse). Mouse-specific GAPDH primers used were as follows: 5′-AACTTTGGCATTGTGGAAGG-3′ (forward) and 5′-ACACATTGGGGGTAGGAACA-3′ (reverse). Blood from all mice was collected at time of sacrifice. Serum was then used for enzyme-linked immunosorbent assay (ELISA) for Lcn2 using the Mouse Lipocal-2/NGAL Quantikine ELISA kit (catalog number MLCN20; R&D Systems, Minneapolis, MN). Assays were performed as per manufacturer's instructions. All patient sample analysis was performed under an exempt protocol by the Institutional Review Board at the Children's Hospital of Pittsburgh. A tissue array of 69 deidentified HB cases collected from the Children's Hospital of Pittsburgh was assessed for LCN2 expression using immunohistochemistry. Slides were deparaffinized by passing through xylene, graded alcohol, and deionized water.16Tao J. Calvisi D.F. Ranganathan S. Cigliano A. Zhou L. Singh S. Jiang L. Fan B. Terracciano L. Armeanu-Ebinger S. Ribback S. Dombrowski F. Evert M. Chen X. Monga S.P. Activation of beta-catenin and Yap1 in human hepatoblastoma and induction of hepatocarcinogenesis in mice.Gastroenterology. 2014; 147: 690-701Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar Slides were rinsed with phosphate-buffered saline, immersed in citrate buffer at a pH of 6.0, and microwaved at 60% power. Three percent hydrogen peroxide was applied for 10 minutes, and slides were rinsed with phosphate-buffered saline. Samples were blocked for 30 minutes using Superblock (ScyTek Laboratories Inc., Logan, UT) and incubated with LCN2 antibody (HPA002695, 1:100, Sigma-Aldrich) at room temperature for 60 minutes. Slides were rinsed and incubated with biotin-conjugated secondary rabbit antibody (Chemicon, Temecula, CA) for 30 minutes. Signal was detected using Vectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA) and developed with diaminobenzidine (Vector Laboratories). All slides were then counterstained with Shandon hematoxylin solution (Thermo Fisher Scientific). Slides were dehydrated using graded alcohol (95%, 100%) and xylene before being coverslipped. The tissue array was reviewed by a pediatric pathologist (S.R.) and signal positivity determined using a 0- to 3-point grading scale, with zero indicating no expression and 3 being highest expression. Samples of normal liver included on the same tissue array were used as baseline controls. The histologic subgroup of each sample was determined by cell morphologic features and arrangement. LCN2 cytoplasmic and nuclear immunoreactivity was recorded for each histologic component of each sample. The same immunohistochemistry protocol was used to analyze the expression of several markers in paraffin-embedded mouse liver samples. The following antibodies were used: Myc-tag (CS2276S, 1:1000, Cell Signaling, Danvers, MA), proliferating cell nuclear antigen (sc-56, 1:4000, Santa Cruz Biotechnology, Santa Cruz, CA), cyclin-D1 (ab134175, 1:200, Abcam), glutamine synthetase (GS; G2781, 1:4000, Sigma-Aldrich), CD45 (sc-53665, 1:100, Santa Cruz Biotechnology), F4/80 (MCA497GA, 1:100, Serotech, Hercules, CA), and neutrophil elastase (ab68672, 1:1500, Abcam). HepG2 cells, a human HB cell line with a known 116–amino acid deletion in β-catenin, were grown in Eagle's minimum essential media, supplemented with 10% fetal bovine serum. Fifty percent confluent HepG2 cells plated in 6-well plates (200,000 cells per well) were transfected with siRNA against LCN2 (catalog number AM51331; identification number 121013; Ambion), β-catenin (catalog number 6225s; Cell Signaling Technology), Yap1 (catalog number 4392420; identification number s20368; Ambion), or scrambled RNA (catalog number AM4611; Ambion) using INTERFERin siRNA Transfection Reagent (catalog number 409-10; Polyplus Transfection, Illkirch, France) as recommended by the manufacturer and incubated for 72 hours. All knock-downs were per