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PPAPDC1B and WHSC1L1 Are Common Drivers of the 8p11-12 Amplicon, Not Only in Breast Tumors But Also in Pancreatic Adenocarcinomas and Lung Tumors

2013; Elsevier BV; Volume: 183; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2013.07.028

ISSN

1525-2191

Autores

Sardar Faisal Mahmood, Nadège Gruel, Rémy Nicolle, Elodie Chapeaublanc, Olivier Delattre, François Radvanyi, Isabelle Bernard‐Pierrot,

Tópico(s)

RNA modifications and cancer

Resumo

Amplification of the 8p11-12 chromosomal region is a common genetic event in many epithelial cancers. In breast cancer, several genes within this region have been shown to display oncogenic activity. Among these genes, the enzyme-encoding genes, PPAPDC1B and WHSC1L1, have been identified as potential therapeutic targets. We investigated whether PPAPDC1B and WHSC1L1 acted as general driver genes, thereby serving as therapeutic targets in other tumors with 8p11-12 amplification. By using publicly available genomic data from a panel of 883 cell lines derived from different cancers, we identified the cell lines presenting amplification of both WHSC1L1 and PPAPDC1B. In particular, we focused on cell lines derived from lung cancer and pancreatic adenocarcinoma and found a correlation between the amplification of PPAPDC1B and WHSC1L1 with their overexpression. Loss-of-function studies based on the use of siRNA and shRNA demonstrated that PPAPDC1B and WHSC1L1 played a major role in regulating the survival of pancreatic adenocarcinoma and small-cell lung cancer–derived cell lines, both in anchorage-dependent and anchorage-independent conditions, displaying amplification and overexpression of these genes. We also demonstrated that PPAPDC1B and WHSC1L1 regulated xenograft growth in these cell lines. Finally, quantitative RT-PCR experiments after PPAPDC1B and WHSC1L1 knockdown revealed exclusive PPAPDC1B and WHSC1L1 gene targets in small-cell lung cancer and pancreatic adenocarcinoma–derived cell lines compared with breast cancer. Amplification of the 8p11-12 chromosomal region is a common genetic event in many epithelial cancers. In breast cancer, several genes within this region have been shown to display oncogenic activity. Among these genes, the enzyme-encoding genes, PPAPDC1B and WHSC1L1, have been identified as potential therapeutic targets. We investigated whether PPAPDC1B and WHSC1L1 acted as general driver genes, thereby serving as therapeutic targets in other tumors with 8p11-12 amplification. By using publicly available genomic data from a panel of 883 cell lines derived from different cancers, we identified the cell lines presenting amplification of both WHSC1L1 and PPAPDC1B. In particular, we focused on cell lines derived from lung cancer and pancreatic adenocarcinoma and found a correlation between the amplification of PPAPDC1B and WHSC1L1 with their overexpression. Loss-of-function studies based on the use of siRNA and shRNA demonstrated that PPAPDC1B and WHSC1L1 played a major role in regulating the survival of pancreatic adenocarcinoma and small-cell lung cancer–derived cell lines, both in anchorage-dependent and anchorage-independent conditions, displaying amplification and overexpression of these genes. We also demonstrated that PPAPDC1B and WHSC1L1 regulated xenograft growth in these cell lines. Finally, quantitative RT-PCR experiments after PPAPDC1B and WHSC1L1 knockdown revealed exclusive PPAPDC1B and WHSC1L1 gene targets in small-cell lung cancer and pancreatic adenocarcinoma–derived cell lines compared with breast cancer. Oncogene activation, through gene amplification resulting in overexpression, contributes to malignant transformation in many solid tumors.1Albertson D.G. Gene amplification in cancer.Trends Genet. 2006; 22: 447-455Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 2Albertson D.G. Collins C. 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Transforming properties of 8p11-12 amplified genes in human breast cancer.Cancer Res. 2010; 70: 8487-8497Crossref PubMed Scopus (36) Google Scholar Among these, WHSC1L1 has been shown to have oncogenic properties in lung cancer cell lines displaying 8p11-12 amplification,13Tonon G. Wong K.K. Maulik G. Brennan C. Feng B. Zhang Y. Khatry D.B. Protopopov A. You M.J. Aguirre A.J. Martin E.S. Yang Z. Ji H. Chin L. Depinho R.A. High-resolution genomic profiles of human lung cancer.Proc Natl Acad Sci U S A. 2005; 102: 9625-9630Crossref PubMed Scopus (339) Google Scholar and FGFR1 has been shown to be overexpressed when amplified and to play an oncogenic role in bladder and colorectal cancers.14Williams S.V. Platt F.M. Hurst C.D. Aveyard J.S. Taylor C.F. Pole J.C. Garcia M.J. Knowles M.A. High-resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma.Genes Chromosomes Cancer. 2010; 49: 642-659PubMed Google Scholar, 23Nakao K. Mehta K.R. Fridlyand J. Moore D.H. Jain A.N. Lafuente A. Wiencke J.W. Terdiman J.P. Waldman F.M. High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization.Carcinogenesis. 2004; 25: 1345-1357Crossref PubMed Scopus (180) Google Scholar, 24Tomlinson D.C. Lamont F.R. Shnyder S.D. Knowles M.A. Fibroblast growth factor receptor 1 promotes proliferation and survival via activation of the mitogen-activated protein kinase pathway in bladder cancer.Cancer Res. 2009; 69: 4613-4620Crossref PubMed Scopus (103) Google Scholar, 25Veltman J.A. Fridlyand J. Pejavar S. Olshen A.B. Korkola J.E. DeVries S. Carroll P. Kuo W.L. Pinkel D. Albertson D. Cordon-Cardo C. Jain A.N. Waldman F.M. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.Cancer Res. 2003; 63: 2872-2880PubMed Google Scholar In addition to FGFR1, two genes from the 8p11-12 amplicon, WHSC1L1 and PPAPDC1B, are particularly interesting in terms of their potential as treatment targets. Indeed, these two genes encode enzymes, which could be targeted by small molecules blocking the catalytic site. Wolf-Hirschorn syndrome candidate 1-like 1 (WHSC1L1; alias NSD3) is a member of the nuclear receptor–binding SET domain (NSD) family of histone methyl transferases.14Williams S.V. Platt F.M. Hurst C.D. Aveyard J.S. Taylor C.F. Pole J.C. Garcia M.J. Knowles M.A. High-resolution analysis of genomic alteration on chromosome arm 8p in urothelial carcinoma.Genes Chromosomes Cancer. 2010; 49: 642-659PubMed Google Scholar, 23Nakao K. Mehta K.R. Fridlyand J. Moore D.H. Jain A.N. Lafuente A. Wiencke J.W. Terdiman J.P. Waldman F.M. High-resolution analysis of DNA copy number alterations in colorectal cancer by array-based comparative genomic hybridization.Carcinogenesis. 2004; 25: 1345-1357Crossref PubMed Scopus (180) Google Scholar, 24Tomlinson D.C. Lamont F.R. Shnyder S.D. Knowles M.A. Fibroblast growth factor receptor 1 promotes proliferation and survival via activation of the mitogen-activated protein kinase pathway in bladder cancer.Cancer Res. 2009; 69: 4613-4620Crossref PubMed Scopus (103) Google Scholar, 25Veltman J.A. Fridlyand J. Pejavar S. Olshen A.B. Korkola J.E. DeVries S. Carroll P. Kuo W.L. Pinkel D. Albertson D. Cordon-Cardo C. Jain A.N. Waldman F.M. Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.Cancer Res. 2003; 63: 2872-2880PubMed Google Scholar, 26Angrand P.O. Apiou F. Stewart A.F. Dutrillaux B. Losson R. Chambon P. NSD3, a new SET domain-containing gene, maps to 8p12 and is amplified in human breast cancer cell lines.Genomics. 2001; 74: 79-88Crossref PubMed Scopus (134) Google Scholar, 27Bergemann A.D. Cole F. Hirschhorn K. The etiology of Wolf-Hirschhorn syndrome.Trends Genet. 2005; 21: 188-195Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar The use of epigenetic targets, including histone methyltransferase, histone deacetylase, and DNA methyltransferase, is emerging as a potential method of cancer treatment, as reviewed by Morishita and di Luccio.28Morishita M. di Luccio E. Cancers and the NSD family of histone lysine methyltransferases.Biochim Biophys Acta. 2011; 1816: 158-163PubMed Google Scholar Phosphatidic acid phosphatase type 2–containing 1B (PPAPDC1B) is a lipid phosphatase.29Takeuchi M. Harigai M. Momohara S. Ball E. Abe J. Furuichi K. Kamatani N. Cloning and characterization of DPPL1 and DPPL2, representatives of a novel type of mammalian phosphatidate phosphatase.Gene. 2007; 399: 174-180Crossref PubMed Scopus (19) Google Scholar There is also growing interest in the use of phosphatases as therapeutic targets in various diseases, including diabetes, obesity, and cancer.30Saha S. Bardelli A. Buckhaults P. Velculescu V.E. Rago C. St Croix B. Romans K.E. Choti M.A. Lengauer C. Kinzler K.W. Vogelstein B. A phosphatase associated with metastasis of colorectal cancer.Science. 2001; 294: 1343-1346Crossref PubMed Scopus (583) Google Scholar, 31Tonks N.K. Muthuswamy S.K. A brake becomes an accelerator: pTP1B–a new therapeutic target for breast cancer.Cancer Cell. 2007; 11: 214-216Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 32Zhang Z.Y. Protein tyrosine phosphatases: prospects for therapeutics.Curr Opin Chem Biol. 2001; 5: 416-423Crossref PubMed Scopus (347) Google Scholar In addition, PPAPDC1B is a transmembrane protein and could, therefore, be targeted by antibodies. WHSC1L1 was first implicated in cancers with 8p11-12 amplification in the lung.13Tonon G. Wong K.K. Maulik G. Brennan C. Feng B. Zhang Y. Khatry D.B. Protopopov A. You M.J. Aguirre A.J. Martin E.S. Yang Z. Ji H. Chin L. Depinho R.A. High-resolution genomic profiles of human lung cancer.Proc Natl Acad Sci U S A. 2005; 102: 9625-9630Crossref PubMed Scopus (339) Google Scholar We subsequently showed that both PPAPDC1B and WHSC1L1 were involved in the survival and transformation of breast cancer cell lines with 8p11-12 amplification.17Bernard-Pierrot I. Gruel N. Stransky N. Vincent-Salomon A. Reyal F. Raynal V. Vallot C. Pierron G. Radvanyi F. Delattre O. Characterization of the recurrent 8p11-12 amplicon identifies PPAPDC1B, a phosphatase protein, as a new therapeutic target in breast cancer.Cancer Res. 2008; 68: 7165-7175Crossref PubMed Scopus (73) Google Scholar This involvement of WHSC1L1 in breast cancer was recently confirmed.22Yang Z.Q. Liu G. Bollig-Fischer A. Giroux C.N. Ethier S.P. Transforming properties of 8p11-12 amplified genes in human breast cancer.Cancer Res. 2010; 70: 8487-8497Crossref PubMed Scopus (36) Google Scholar In this study, we investigated whether these two genes, WHSC1L1 and PPAPDC1B, acted as general driver genes and could be considered as suitable treatment targets in tumors with 8p11-12 amplification other than breast cancers. We identified cell lines presenting amplifications of both WHSC1L1 and PPAPDC1B within the 8p11-12 region derived from cancers at various primary sites, from the cancer cell line panel of the Broad-Novartis Cancer Cell Line Encyclopedia (CCLE).33Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4856) Google Scholar We focused on the three cancers with the most derived cell lines displaying WHSC1L1 and PPAPDC1B amplification. We selected several of these cell lines to evaluate the role of these two genes in cell survival in vitro, in anchorage-dependent and in anchorage-independent conditions, and in tumor growth in vivo with xenograft models. PANC08.13 and DMS114 (pancreatic and lung cancer cell lines, respectively) cells were purchased from ATCC (LGC standard, Molsheim, France); DANG and HUPT4 (pancreatic cancer cell lines) cells were purchased from the Cell Line Service (Eppelheim, Germany) and the German Resource Centre for Biological Material (DSMZ, Braunschweig, Germany), respectively. PANC08.13 cells were grown in RPMI 1640 medium with 15% fetal bovine serum, supplemented with 2 mmol/L l-glutamine and 10 U/mL human insulin, adjusted to give final concentrations of 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mmol/L HEPES, and 1.0 mmol/L sodium pyruvate. DANG cells were grown in RPMI 1640 medium with 10% fetal bovine serum, supplemented with 2 mmol/L l-glutamine. HUPT4 cells were grown in 80% modified Eagle's medium (with Earle's salts), with 20% fetal bovine serum, 1% nonessential amino acids, and 1% sodium pyruvate. DMS114 cells were grown in Waymouth's MB 752/1 medium with 10% fetal bovine serum. All reagents were purchased from Life Technologies (Saint-Aubin, France). RNA was isolated from cell lines with RNeasy Mini kits (Qiagen, Courtaboeuf, France). Transient transfections were performed, using Lipofectamine RNAimax, according to the manufacturer's instructions (Invitrogen, Saint-Aubin, France), with 20 nmol/L siRNA. A negative control siRNA and siRNAs specific for PPAPDC1B and WHSC1L1 were purchased from Qiagen. For each gene, the siRNA was designed to knock down the expression of all known mRNA isoforms. The sequences of the sense-strand siRNA were as follows: PPAPDC1B-1, 5′-GAUGUUUGUUAUUGCAUUUTT-3′; PPAPDC1B-2, 5′-GCCGAUGUUUGUUAUUGCATT-3′; WHSC1L1-1, 5′-GGGUAUCCAUCAUCAAUCATT-3′; and WHSC1L1-2, 5′-CGAGAGUAUAAGGUCAUATT-3′. We knocked down the expression of human WHSC1L1 and PPAPDC1B genes in the DMS114 and DANG cell lines, using the Mission TRC human shRNA clone sets (pLKO.1-puro; Sigma, Saint-Quentin Fallavier, France), as previously described for breast cancer.17Bernard-Pierrot I. Gruel N. Stransky N. Vincent-Salomon A. Reyal F. Raynal V. Vallot C. Pierron G. Radvanyi F. Delattre O. Characterization of the recurrent 8p11-12 amplicon identifies PPAPDC1B, a phosphatase protein, as a new therapeutic target in breast cancer.Cancer Res. 2008; 68: 7165-7175Crossref PubMed Scopus (73) Google Scholar Cells expressing shRNA were selected on 2 μg/mL puromycin for 6 weeks for functional studies (cell proliferation and xenografts) and for 4 weeks after infection for RNA extraction. Reverse transcription was performed with 1 μg of total RNA, with the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Saint-Aubin, France), and PCR was performed in a Roche real-time thermal cycler with Roche Syber Green Master Mix (Roche, Meylan, France). The sequences of the primers used were as follows: PPADC1B, 5′-CCACTGTCTCTGATCTTCCTGG-3′ (sense) and 5′-GGTAAAGACGCCATTCAGAGCC-3′ (antisense); and WHSC1L1, 5′-CAGACGTTTCTGATGTGCAGTCC-3′ (sense) and 5′-CTCCAGGTGAAAGTGTTTGCAGC-3′ (antisense). PANC08.13 (2 × 105), HUPT4 (1.5 × 105), DANG (1.5 × 105), and DMS114 (3 × 105) cells were plated in a 6-well plate and transfected with 20 nmol/L siRNA. The cells were treated with trypsin for 72 hours (for DANG and DMS114) and 96 hours (for PANC08.13 and HUPT4) after transfection and stained with trypan blue, and viable cells were counted in triplicate with a Malassez hematocytometer (Roche, Meylan, France). We added 20,000 siRNA-transfected cells (PANC08.13, HUPT4, and DANG) in the corresponding growth medium and 0.35% agar to triplicate wells of a 12-well plate containing medium and 0.8% agar, 24 hours after transfection. The plates were incubated for 21 days, and colonies of at least 50 μm in diameter were scored as positive, on examination under a phase-contrast microscope equipped with a measuring grid. Six-week-old female Swiss nu/nu mice were obtained from Charles River Laboratories (Saint Germain sur l'Arbresle, France) and reared in the animal facility of the Curie Institute in specified pathogen-free conditions. Animals were housed and cared for in accordance with the institutional guidelines of the French National Ethics Committee (Ministère de l'Agriculture et de la Pêche, Direction Départementale des Services Vétérinaires, Paris, France) under the supervision of investigators with the required accreditation. We injected 9 × 105 DMS114 and 2 × 106 DANG cells s.c. into the flank (dorsal region) of each mouse. Tumor formation was monitored for up to 60 days, and tumor size was determined weekly with Vernier calipers: two perpendicular diameters were used to estimate tumor volume according to the formula, ab2/2, where a is the largest and b is the smallest diameter. For PPAPDC1B and WHSC1L1, mRNA level (Affymetrix or RNA sequencing signal) and DNA copy number [single-nucleotide polymorphism (SNP) array, normalized signal] were compared in cell lines and in tumors, by Pearson's correlation test (Affymetrix, Paris, France). The levels of mRNA for PPAPDC1B and WHSC1L1 in nonamplified cell lines were compared with those in cell lines with an unchanged 8p-DNA copy number, in Student's t-tests. We evaluated the results of the functional experiments with the Student's t-test and the Wilcoxon rank-sum test for in vitro and in vivo experiments, respectively. Both gene-centered mRNA data (custom-made Agilent chips) and copy number data (Agilent comparative genomic hybridization arrays) were obtained for 112 squamous cell carcinomas from The Cancer Genome Atlas consortium34Cancer Genome Atlas Research NetworkComprehensive genomic characterization of squamous cell lung cancers.Nature. 2012; 489: 519-525Crossref PubMed Scopus (2922) Google Scholar (https://tcga-data.nci.nih.gov/tcga/dataAccessMatrix.htm?mode=ApplyFilter&diseaseType=LUSC, last accessed March 5, 2013). Copy number data for 65 small-cell lung carcinomas (SCLCs; Affymetrix SNP 6.0 arrays, Paris, France) and normalized gene expression for 15 of these tumors (RNA sequencing) were obtained from Bashyam et al.35Bashyam M.D. Bair R. Kim Y.H. Wang P. Hernandez-Boussard T. Karikari C.A. Tibshirani R. Maitra A. Pollack J.R. Array-based comparative genomic hybridization identifies localized DNA amplifications and homozygous deletions in pancreatic cancer.Neoplasia. 2005; 7: 556-562Abstract Full Text PDF PubMed Scopus (173) Google Scholar Gene expression (Affymetrix u133 plus 2.0) and copy number data (Affymetrix SNP 6.0 arrays) for 883 cell lines were obtained from the CCLE.33Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4856) Google Scholar For the analysis of copy number data, we defined gene amplification as a normalized SNP array log2 copy number ratio of more than one with respect to normal DNA (more than four copies), and gain as a normalized SNP array log2 copy number ratio between 0.6 and 1 (between three and four copies). We investigated the role of PPAPDC1B and WHSC1L1 as driver genes and potential therapeutic targets for tumors with 8p11-12 amplification from sites other than the breast. We began by identifying cancer-derived cell lines presenting PPAPDC1B and WHSC1L1 amplification and overexpression. We made use of publicly available data concerning gene copy number obtained with Affymetrix SNP6 arrays for 883 cancer-derived cell lines, and data concerning gene expression were obtained with the Affymetrix U133plus2.0 DNA array for most of these cell lines (807/883) from the CCLE.33Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4856) Google Scholar Amplification of both PPAPDC1B and WHSC1L1 in the 8p11-12 region, as defined by a normalized SNP array log2 copy number ratio of more than one with respect to normal DNA (more than four copies), was detected in 26 cancer cell lines (Figure 1 and Supplemental Table S1). These cell lines included six derived from breast cancers (11.5% of the 52 breast cancer cell lines studied: MDAMB134VI, CAL120, JIMT1, ZR75.1, CAMA1, and YMB1), three pancreatic cell lines (7.5% of the 40 studied: DANG, SNU213, and PANC08.13), nine lung cancer cell lines (5.6% of the 158 studied: DMS114, NCIH2444, NCIH1703, CORL88, NCIH1581, HCC95, DMS454, NCIH520, and NCIH1648), two large intestine cancer cell lines (3.9% of the 51 studied: SW837 and SNUC1), three hematopoietic cancer cell lines (1.8% of the 164 studied: BV173, KG1, and OCILY19), one liver cancer cell line (3.7% of the 27 studied: SNU761), one stomach cancer cell line (2.7% of the 37 studied: LSMU), and one bone cancer cell line (4% of the 25 studied: G292CLONEA141B1). We focused on cell lines derived from pancreatic and lung cancers, which were (after breast cancer, for which PPAPDC1B and WHSC1L1 have already been shown to act as driver genes) the two cancers with the most derived cell lines displaying PPAPDC1B and WHSC1L1 amplification, at 7.5% and 5.6%, respectively. Moreover, two other lung cell lines (Calu3 and NCIH1733) presented a gain, as shown by their normalized SNP array log2 copy number ratios, which were between 0.6 and 1 (more than three copies but less than four copies) (Supplemental Table S1). We found that, as in breast cell lines, there was a direct relationship between DNA copy number and expression level for both PPAPDC1B and WHSC1L1 in lung and pancreatic cell lines (Pearson's r > 0.6, P < 0.0001), indicating that DNA copy number had a significant effect on gene expression (Figure 2A). Consistent with this finding, as in breast cancer–derived cell lines, PPAPDC1B and WHSC1L1 were significantly more strongly expressed in cell lines derived from lung and pancreatic cancers that displayed amplification than in cell lines from these sources without amplification (Student's t-test, P < 0.05) (Figure 2B). We then investigated whether these correlations between gene amplification and gene overexpression were also relevant in the tumors from which the cell lines were derived. Pancreatic cell lines with PPAPDC1B and WHSC1L1 amplification were derived from pancreatic ductal adenocarcinomas, in which PPAPDC1B and WHSC1L1 have been shown to be gained in two different studies: in 10% of the tumors (4 of 39)15Birnbaum D.J. Adélaïde J. Mamessier E. Finetti P. Lagarde A. Monges G. Viret F. Goncalvès A. Turrini O. Delpero J.R. Iovanna J. Giovannini M. Birnbaum D. Chaffanet M. Genome profiling of pancreatic adenocarcinoma.Genes Chromosomes Cancer. 2011; 50: 456-465Crossref PubMed Scopus (98) Google Scholar and in 7% of the tumors (1 of 13).36Aguirre A.J. Brennan C. Bailey G. Sinha R. Feng B. Leo C. Zhang Y. Zhang J. Gans J.D. Bardeesy N. Cauwels C. Cordon-Cardo C. Redston M.S. DePinho R.A. Chin L. High-resolution characterization of the pancreatic adenocarcinoma genome.Proc Natl Acad Sci U S A. 2004; 101: 9067-9072Crossref PubMed Scopus (233) Google Scholar WHSC1L1 mRNA levels have been shown to be correlated with DNA copy number in pancreatic adenocarcinoma (P = 0.039), but PPAPDC1B expression was not evaluated in this study.36Aguirre A.J. Brennan C. Bailey G. Sinha R. Feng B. Leo C. Zhang Y. Zhang J. Gans J.D. Bardeesy N. Cauwels C. Cordon-Cardo C. Redston M.S. DePinho R.A. Chin L. High-resolution characterization of the pancreatic adenocarcinoma genome.Proc Natl Acad Sci U S A. 2004; 101: 9067-9072Crossref PubMed Scopus (233) Google Scholar To our knowle

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