Expression Profiling of Primary Tumors and Matched Lymphatic and Lung Metastases in a Xenogeneic Breast Cancer Model
2005; Elsevier BV; Volume: 166; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)62372-3
ISSN1525-2191
AutoresValérie Montel, To‐Yu Huang, Evangeline Mose, Kersi Pestonjamasp, David G. Jackson,
Tópico(s)HER2/EGFR in Cancer Research
ResumoUsing a purpose-designed experimental model, we have defined new, statistically significant, differences in gene expression between heavily and weakly metastatic human breast cancer cell populations, in vivo and in vitro. The differences increased under selection pressures designed to increase metastatic proficiency. Conversely, the expression signatures of primary tumors generated by more aggressive variants, and their matched metastases in the lungs and lymph nodes, all tended to converge. However, the few persisting differences among these selectively enriched malignant growths in the breast, lungs, and lymph nodes were highly statistically significant, implying potential mechanistic involvement of the corresponding genes. The evidence that has emerged from the current work indicates that selective enhancement of metastatic proficiency by serial transplantation co-purifies a subliminal gene expression pattern within the tumor cell population. This signature most likely includes genes participating in metastasis pathogenesis, and we document manageable numbers of candidates for this role. The findings also suggest that metastasis to at least two different organs occurs through closely similar genetic mechanisms. Using a purpose-designed experimental model, we have defined new, statistically significant, differences in gene expression between heavily and weakly metastatic human breast cancer cell populations, in vivo and in vitro. The differences increased under selection pressures designed to increase metastatic proficiency. Conversely, the expression signatures of primary tumors generated by more aggressive variants, and their matched metastases in the lungs and lymph nodes, all tended to converge. However, the few persisting differences among these selectively enriched malignant growths in the breast, lungs, and lymph nodes were highly statistically significant, implying potential mechanistic involvement of the corresponding genes. The evidence that has emerged from the current work indicates that selective enhancement of metastatic proficiency by serial transplantation co-purifies a subliminal gene expression pattern within the tumor cell population. This signature most likely includes genes participating in metastasis pathogenesis, and we document manageable numbers of candidates for this role. The findings also suggest that metastasis to at least two different organs occurs through closely similar genetic mechanisms. Metastasis, the spread of cancer cells from the primary tumor to distant organs and their treatment-resistant proliferation in multiple locations, remains a major clinical and biological challenge. It is known from previous work that tumor cells that make metastases can be propagated as cell lines that conserve their capabilities to produce secondary cancers in other organs.1Fidler IJ Kripke ML Metastasis results from preexisting variant cells within a malignant tumor.Science. 1977; 197: 893-895Crossref PubMed Scopus (1178) Google Scholar, 2Fidler IJ Tumor heterogeneity and the biology of cancer invasion and metastasis.Cancer Res. 1978; 38: 2651-2660PubMed Google Scholar The heritable nature of this escalating problem, confirmed by the work of many investigators (reviewed by Fidler3Fidler IJ The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.Nat Rev Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3696) Google Scholar) and by our own work on spontaneous murine and human neoplasms of various histogenetic origins,4Price JE Carr D Tarin D Spontaneous and induced metastasis of naturally occurring tumors in mice: analysis of cell shedding into the blood.J Natl Cancer Inst. 1984; 73: 1319-1326PubMed Google Scholar, 5Tarin D Price JE Kettlewell MG Souter RG Vass AC Crossley B Mechanisms of human tumor metastasis studied in patients with peritoneovenous shunts.Cancer Res. 1984; 44: 3584-3592PubMed Google Scholar demonstrates that it is caused by a genetic disorder governing the behavior of the cancer cells. Also, this inherited behavior pattern, although disruptive of tissues and organs, is a highly coordinated process requiring the completion of several complicated steps in the correct order in time and space. Successful achievement of the metastatic event therefore implies the sequential and orderly mobilization of relevant gene pathways. Knowledge of the genes involved would be of considerable diagnostic, prognostic, and therapeutic value both in patients who have not yet developed clinically detectable metastases and in more advanced cases in which the limitation of further spread would be beneficial. We therefore used oligonucleotide microarrays to perform high throughput screening of global gene expression in tumors and metastases produced by a unique matched pair of human clonal cell lines of opposite metastatic capabilities, which we have derived from the same breast cancer line, MDA-MB-435,6Bao L Pigott R Matsumura Y Baban D Tarin D Correlation of VLA-4 integrin expression with metastatic potential in various human tumour cell lines.Differentiation. 1993; 52: 239-246Crossref PubMed Scopus (40) Google Scholar and confirmed to be isogenic by several methods, including chromosomal analysis7Urquidi V Sloan D Kawai K Agarwal D Woodman AC Tarin D Goodison S Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis.Clin Cancer Res. 2002; 8: 61-74PubMed Google Scholar and genetic fingerprinting. There are already some reports of high-density microarray profiling of gene expression patterns in metastatic human primary cancers and metastases,8Lapointe J Li C Higgins JP van de Rijn M Bair E Montgomery K Ferrari M Egevad L Rayford W Bergerheim U Ekman P DeMarzo AM Tibshirani R Botstein D Brown PO Brooks JD Pollack JR Gene expression profiling identifies clinically relevant subtypes of prostate cancer.Proc Natl Acad Sci USA. 2004; 101: 811-816Crossref PubMed Scopus (1072) Google Scholar, 9Ramaswamy S Ross KN Lander ES Golub TR A molecular signature of metastasis in primary solid tumors.Nat Genet. 2003; 33: 49-54Crossref PubMed Scopus (2056) Google Scholar, 10van de Vijver MJ He YD van't Veer LJ Dai H Hart AA Voskuil DW Schreiber GJ Peterse JL Roberts C Marton MJ Parrish M Atsma D Witteveen A Glas A Delahaye L van der Velde T Bartelink H Rodenhuis S Rutgers ET Friend SH Bernards R A gene-expression signature as a predictor of survival in breast cancer.N Engl J Med. 2002; 347: 1999-2009Crossref PubMed Scopus (5383) Google Scholar, 11LaTulippe E Satagopan J Smith A Scher H Scardino P Reuter V Gerald WL Comprehensive gene expression analysis of prostate cancer reveals distinct transcriptional programs associated with metastatic disease.Cancer Res. 2002; 62: 4499-4506PubMed Google Scholar, 12Dhanasekaran SM Barrette TR Ghosh D Shah R Varambally S Kurachi K Pienta KJ Rubin MA Chinnaiyan AM Delineation of prognostic biomarkers in prostate cancer.Nature. 2001; 412: 822-826Crossref PubMed Scopus (1441) Google Scholar but no consensus has yet emerged on any groups of genes that are consistently involved. This may be due to the masking effects resulting from comparisons between samples from individuals of different genetic backgrounds. The work presented below makes progress from previous approaches by using a tightly controlled, well characterized, xenograft model of breast cancer metastasis.7Urquidi V Sloan D Kawai K Agarwal D Woodman AC Tarin D Goodison S Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis.Clin Cancer Res. 2002; 8: 61-74PubMed Google Scholar, 13Goodison S Kawai K Hihara J Jiang P Yang M Urquidi V Hoffman RM Tarin D Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein.Clin Cancer Res. 2003; 9: 3808-3814PubMed Google Scholar This investigative system facilitated direct examination of differences between primary tumors and matched metastases in the lungs and lymph nodes from the same animal and thus eliminated the noise from biological variations between different individuals. To our knowledge, this is the first study to systematically investigate gene expression patterns in matched metastases from both of these organs in the same host. In addition, this investigation provides new data on dynamic gene expression patterns in vivo and in vitro of metastasis-competent and incompetent human cell populations within the same parent tumor, opening a window on the effects of tumor-host interactions on behavior. Such comparison is not possible in samples excised from clinical tumor specimens. Technical advances are also incorporated in this work: tumor cell lineages were labeled with green fluorescent protein (GFP) to enhance accuracy of selection of primary and secondary tumor tissue for analysis. Also, to evaluate the initial screening results, we conducted extensive laboratory studies and computational (training and test) procedures and validated the expression levels of a number of genes of interest. Our findings indicated that the genes expressed in primary tumors generated by metastasis-competent cell populations differed clearly from those in their metastasis-incompetent counterparts. In contrast, the patterns in metastases were similar to the primary tumors from which they originated, and metastases in the lungs displayed remarkably similar gene expression patterns to those in the lymph nodes of the same animal. Additionally, the patterns observed in tumors and metastases differed from those seen in the parental cell lines in vitro, indicating that the host microenvironment is an active participant in tumor progression and metastasis. These findings have significant implications for defining mechanisms of metastasis and for designing novel effective therapy. They also contribute a manageable list of candidate genes from which to choose targets for interventional studies on mechanisms involved in the process. The NM-2C5 and M-4A4 lines were isolated in our laboratory from the MDA-MD-435 breast cancer cell line as described by Bao et al6Bao L Pigott R Matsumura Y Baban D Tarin D Correlation of VLA-4 integrin expression with metastatic potential in various human tumour cell lines.Differentiation. 1993; 52: 239-246Crossref PubMed Scopus (40) Google Scholar and subsequently transduced with an enhanced green fluorescent protein-expressing vector.13Goodison S Kawai K Hihara J Jiang P Yang M Urquidi V Hoffman RM Tarin D Prolonged dormancy and site-specific growth potential of cancer cells spontaneously disseminated from nonmetastatic breast tumors as revealed by labeling with green fluorescent protein.Clin Cancer Res. 2003; 9: 3808-3814PubMed Google Scholar These monoclonal cell lines were routinely cultured in RPMI 1640 medium supplemented with 10% newborn calf serum (Invitrogen, Carlsbad, CA), penicillin, and streptomycin at 37°C in a humidified atmosphere of 5% CO2-95% air. Cell line LM3 was derived from a lung metastasis produced by M-4A4, and cell line CL16 was obtained from a lung metastasis made by descendants of LM3 after two more similar selection cycles in nude mice. Both are progressively more metastatic variants of the parent line (see below) grown under the same conditions in vitro. One million cells in 50 μl of a mixture of RPMI 1640 medium and ECM gel (Sigma Chemical Co., St. Louis, MO) were inoculated into the mammary fat pad of anesthetized mice. Animals were euthanized and autopsied at 3 to 4 months postinoculation when the primary tumors reached ∼20 mm in diameter. Metastasis formation was assessed by macroscopic observation of all major organs for secondary tumors and confirmed by histological examination. Metastasis was also confirmed by looking for fluorescence of incorporated GFP under blue light (λ = 490 nm), which is sufficiently sensitive to detect single cells. Only cell clusters (>1 mm) are regarded as true metastases. Tissues from primary tumors and metastases were snap-frozen and stored at −80°C until used for RNA or protein extraction. Protein detection and quantification were performed either on primary tumors or sera from tumor-bearing mice depending on the protein localization. Tumor homogenates were prepared in 20 mmol/L Tris-HCl, pH 8.0, 5 mmol/L CaCl2, 1 mmol/L phenylmethylsulfonyl fluoride, 15 μmol/L pepstatin A, and 0.05% (w/v) Brij 35. The Complete EDTA-free protease inhibitor cocktail (Boehringer Ingelheim Chemicals, Petersburg, VA) was added to the extraction buffer. The tissues were homogenized on ice, and the homogenates were centrifuged at 14,000 × g for 40 minutes (4°C). Protein quantitation of the supernatants was performed using the Coomassie Plus Protein Assay kit (Pierce, Rockford, IL). Twenty micrograms of denatured protein samples was separated on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis glycine gel, and proteins were transferred onto polyvinylidene difluoride membranes using a semi-dry apparatus (Bio-Rad, Life Science, Hercules, CA) according to the manufacturer's instructions. Immunodetection was performed as described previously7Urquidi V Sloan D Kawai K Agarwal D Woodman AC Tarin D Goodison S Contrasting expression of thrombospondin-1 and osteopontin correlates with absence or presence of metastatic phenotype in an isogenic model of spontaneous human breast cancer metastasis.Clin Cancer Res. 2002; 8: 61-74PubMed Google Scholar using the following specific antibodies: monoclonal anti-silver homolog (Pmel-17) (Neomarkers, Inc., Fremont, CA); monoclonal anti-MITF (Neomarkers, Inc.); polyclonal anti-α1-antichymotrypsin (DAKO, Carpinteria, CA); polyclonal anti-TRP1 (Santa Cruz Biotechnology, Santa Cruz, CA); monoclonal anti-osteopontin (Chemicon International, Inc., Temecula, CA); monoclonal anti-thrombospondin (BD Transduction Laboratories, San Jose, CA); and polyclonal anti-MMP-8 (Chemicon International, Inc). MMP-8 and OPN protein expressions were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits from Amersham Biosciences (San Francisco, CA) and Assay Designs (Ann Arbor, MI), respectively. Frozen sections of xenograft primary tumors were fixed in 4% buffered formaldehyde. Antigen retrieval was performed using the target retrieval solution at 1:10 dilution (DAKO). Endogenous peroxidase activity was blocked by incubation in 3% H2O2. Nonspecific binding of the antibodies to irrelevant proteins was blocked by incubation in 10% goat serum. Proteins of interest were targeted with the same antibodies used in the Western-blot experiments (see above). When monoclonal mouse antibodies were applied, a prior incubation of the section with goat anti-mouse Ig Fab fragments (Jackson Immunoresearch Laboratories, West Grove, PA), which neutralized the Fc domain reactivity of the endogenous host Ig, was performed. The horseradish peroxidase-conjugated secondary antibody was visualized by diaminobenzidine substrate-chromogen 3,3′-diaminobenzidine (DAKO). Sections were counterstained with Hematoxylin-Gills No. 2 solution, dehydrated in alcohol, cleared in xylene, and mounted in Permount (Fisher Chemicals, Lake Forest, CA). Total RNA was extracted from cultured cells and frozen tissue samples with TRIzol reagent (Invitrogen) and cleaned with the DNA-free kit (Ambion, Austin, TX). RNA quality was assessed by running the samples on a native 1% agarose gel and on a Biogem analyzer (Agilent, Palo Alto, CA). cRNA was prepared in the University of California-San Diego Cancer Center Microarray facility as described by the standard Affymetrix microarray protocols. The cRNA was then hybridized to human HG-U133A GeneChip oligonucleotide arrays (Affymetrix, Santa Clara, CA), which interrogate approximately 22,000 transcripts. The arrays were scanned at 560 nm using an argon-ion confocal laser as the excitation source. The DAT files containing the scanned images of each microarray were individually inspected for quality control and digitized by Microarray Analysis Suite 5.0 (Affymetrix). The resultant CEL files containing the raw numerical data for signal intensity at probe level were collectively read and analyzed in dChip software.14Li C Wong WH Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection.Proc Natl Acad Sci USA. 2001; 98: 31-36Crossref PubMed Scopus (2721) Google Scholar, 15Li C, Wong WH: Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application. Genome Biol 2001, 2:RESEARCH0032. EpubGoogle Scholar Briefly, each microarray was normalized against a common baseline array using the “invariant probe set” method. After normalization, the model-based expression index of each gene was then calculated according to the PM-MM model. To identify candidate genes that were differentially expressed between any two group of arrays, a screening filter consisting of the following criteria was applied: 1) a fold change (fc) larger than 1.5 or 3; 2) two-tailed P values (paired t-test if applicable) smaller than 0.05; and 3) a minimal difference of 100 between the group mean of model-based expression index. The resultant lists of candidate genes were then sorted according to their corresponding P values with a cut-off of 0.005 to ensure high stringency of the analysis (Table 1, Table 2, Table 3, Table 4, Table 5, Table 6). For subsequent high-level analysis, candidate gene lists from six group-wise comparisons were combined and subjected to hierarchical clustering (centroid-linkage) or classification by linear discriminant analysis (LDA) within the R environment (http://www.R-project.org).Table 1Lists of Differentially Expressed Genes in the Horizontal and Vertical Comparisons: Nonmetastatic Tumor versus Metastatic Tumor (P < 0.005, fc > 1.5)P valuefcGene descriptionAccession no.Probe setTop 20 genes up-regulated in the nonmetastatic tumor (versus the metastatic tumor) 0.000023.48Cancer/testis antigen 2AJ012833.1215733_x_at 0.0000291.98Cancer/testis antigen 1AF038567.1211674_x_at 0.000032.17Cancer/testis antigen 1AJ275978.1217339_x_at 0.0000392.86Nucleotide binding protein 2 (MinD homolog, E. coli)NM_012225.1218227_at 0.0000482.72Sulfide quinone reductase-like (yeast)NM_021199.1217995_at 0.0000632.32Membrane-spanning 4-domains, subfamily A, member 3L35848.1210254_at 0.0000821.88HN1-likeAK023154.1212115_at 0.0000872.29Thrombospondin 1NM_003246.1201110_s_at 0.0000982.31Serologically defined colon cancer antigen 16BC001149.1221514_at 0.0000992.43Influenza virus NS1A binding proteinAF205218.1201362_at 0.0001012.13Guanine nucleotide binding protein (G protein), γ 11NM_004126.1204115_at 0.0001022.45Splicing factor, arginine/serine-rich 7, 35 kdNM_006276.2201129_at 0.0001062.33DnaJ (Hsp40) homolog, subfamily A, member 3NM_005147.1205963_s_at 0.0001121.65Kinesin family member 4ANM_012310.2218355_at 0.0001212.34Influenza virus NS1A binding proteinAB020657.1201363_s_at 0.0001243281.94Chondroitin sulfate proteoglycan 4 (melanoma-associated)NM_001897.1204736_s_at 0.0001322.36Regulator of G-protein signalling 10NM_002925.2204316_at 0.0001381.65Uracil-DNA glycosylaseNM_003362.1202330_s_at 0.00014421.5Melanoma antigen, family A, 1 (directs expression of antigen MZ2-E)NM_004988.1207325_x_at 0.0001451.86Heat shock protein 75NM_016292.1201391_atTop 20 genes up-regulated in the metastatic tumor (versus the nonmetastatic tumor) 0.0000013.44Protein kinase C-like 1NM_002741.1202161_at 0.00000210.81Serine (or cysteine) proteinase inhibitor, clade A, member 3NM_001085.2202376_at 0.00000310.34Collagen, type IX, α 1NM_001851.1222008_at 0.0000074.52Serine (or cysteine) proteinase inhibitor, clade F, member 1NM_002615.1202283_at 0.000016.29Aldehyde dehydrogenase 1 family, member A1NM_000689.1212224_at 0.000013.92Preferentially expressed antigen in melanomaNM_006115.1204086_at 0.000012.61Retinol dehydrogenase 11 (all-trans and 9-cis)NM_016026.1217775_s_at 0.0000132.32SH3-domain binding protein 4AF015043.1222258_s_at 0.0000153.57Dynein, cytoplasmic, intermediate polypeptide 1NM_004411.1205348_s_at 0.0000236.64Ribonuclease, RNase A family, 1 (pancreatic)NM_002933.1201785_at 0.0000282.14Proteasome (prosome, macropain) 28S subunit, non-ATPase, 8NM_002812.1200820_at 0.0000323.1Dudulin 2NM_018234.1218424_s_at 0.000043.23Likely ortholog of mouse semaF cytoplasmic domain-associated protein 3AL569804212915_at 0.0000484.11LIM domain proteinBE043700214175_x_at 0.0000511.63ADP-ribosylation factor interacting protein 2 (arfaptin 2)NM_012402.1202109_at 0.00005247.46G antigen 4NM_021123.1208235_x_at 0.0000522.2Protein tyrosine phosphatase type IVA, member 1BF576710200732_s_at 0.00006179.78G antigen 4NM_001476.1208155_x_at 0.0000612.41Eukaryotic translation initiation factor 2, subunit 1 α, 35 kdBC002513.1201143_s_at 0.0000623.55Retinoblastoma-associated factor 600AB007931.1211950_at Open table in a new tab Table 2Lists of Differentially Expressed Genes in the Horizontal and Vertical Comparisons: Nonmetastatic Tumor versus Lung Metastases (P < 0.005, fc > 1.5)P valuefcGene descriptionAccession no.Probe setTop 20 genes up-regulated in the nonmetastatic tumor (versus the lung metastases) 0.0000273.49Guanine nucleotide binding protein (G protein), gamma 11NM_004126.1204115_at 0.0000271.92Non-POU domain containing, octamer-bindingNM_007363.2200057_s_at 0.0000312.03Chromosome 11 hypothetical protein ORF3NM_020154.1217898_at 0.0000372.4Mahogunin, ring finger 1AB011116.1212576_at 0.0000418.62Similar to X-linked ribosomal protein 4 (RPS4X)AL137162217019_at 0.0000522.22Cytoplasmic FMR1-interacting protein 1BC005097.1208923_at 0.0000561.63Dual specificity phosphatase 4BC002671.1204015_s_at 0.0000582.43RNA binding protein S1, serine-rich domainNM_006711.1207939_x_at 0.000071.55Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult))NM_000454.1200642_at 0.0000722.6Synaptosomal-associated protein, 23 kdBC003686.1209130_at 0.0000811.91Succinate-CoA ligase, GDP-forming, β subunitAL050226.1215772_x_at 0.0000881.68PVVP2 periodic tryptophan protein homolog (yeast)U56085.1209336_at 0.0000892.01Similar to RPS3A (ribosomal protein S3A)AL356115216823_at 0.0000931.7Sialyltransferase 4C (β-galactoside α-2,3-sialyltransferase)NM_006278.1203759_at 0.000096477.5Cancer/testis antigen 1AF038567.1211674_x_at 0.0000962.74Regulator of G-protein signaling 10NM_002925.2204316_at 0.0000972.58TYRO3 protein tyrosine kinaseU05682.1211432_s_at 0.0000971.91ALEX3 proteinNM_016607.1217858_s_at 0.0001112.09Hypothetical gene supported by AK000185AK000185.1216644_at 0.0001152.12MAX interacting protein 1NM_005962.1202364_atTop 20 genes up-regulated in the lung metastases (versus the nonmetastatic tumor) 0.0000022.12DEAD (Asp-Glu-Ala-Asp) box polypeptide 39NM_005804.1201584_s_at 0.0000025.17*Refuted by Q-PCR.Neuroblastoma, suppression of tumorigenicity 1NM_005380.1201621_at 0.0000035.28Serine (or cysteine) proteinase inhibitor, clade F, member 1NM_002615.1202283_at 0.0000037.75Ribonuclease, RNase A family, 1 (pancreatic)NM_002933.1201785_at 0.0000043.72Neuroblastoma, suppression of tumorigenicity 1D2812437005_at 0.0000046.18OsteopontinM83248.1209875_s_at 0.0000052.03Adaptor-related protein complex 2, σ 1 subunitBC006337.1211047_x_at 0.0000068.71LIM domain proteinBC003096.1211564_s_at 0.00000913.92Baculoviral IAP repeat-containing 7 (livin)NM_022161.1220451_s_at 0.000013.06KIAA0930 proteinAK025608.1217118_s_at 0.0000111.65Cytochrome c oxidase subunit VlbNM_001863.2201441_at 0.00001313.79Ocular albinism 1 (Nettleship-Falls)NM_000273.1206696_at 0.0000172.69Six transmembrane epithelial antigen of the prostateNM_012449.1205542_at 0.0000173.4Retinoblastoma-associated factor 600AB007931.1211950_at 0.0000282.72G antigen 4NM_001476.1208155_x_at 0.0000246.76SlalyltransferaseNM_006456.1204542_at 0.0000272.36N-Acylsphingosine amidohydrolase (acid ceramidase) 1AI934569213702_x_at 0.0000283.62Arginase, type IIU75667.1203946_s_at 0.0000371.68Heme binding protein 1NM_015987.1218450_at 0.0000477.32Proteoglycan 1, secretory granuleJ03223.1201858_s_at* Refuted by Q-PCR. Open table in a new tab Table 3Lists of Differentially Expressed Genes in the Horizontal and Vertical Comparisons: Nonmetastatic Tumor (versus LN Metastases (P < 0.005, fc > 1.5)P valuefcGene descriptionAccession no.Probe setTop 20 genes up-regulated in the nonmetastatic tumor (versus the LN metastases) 0.0000241.56Dual specificity phosphatase 4BC002671.1204015_s_at 0.0000283.08Transforming growth factor, β-induced, 68 kdNM_000358.1201506_at 0.000031.99Ras-GTPase-activating protein SH3-domain-binding proteinBG500067201503_at 0.0000351.85Non-POU domain containing, octamer-bindingNM_007363.2200057_s_at 0.000041.56Superoxide dismutase 1, soluble (amyotrophic lateral sclerosis 1 (adult)NM_000454.1200642_at 0.0000482.54KIAA0843 proteinNM_014945.1205730_s_at 0.00005280.77Cancer/testis antigen 1AF038567.1211674_x_at 0.0000561.85Nuclear pore complex interacting proteinNM_006985.1204538_x_at 0.0000681.67Integrin, α 6NM_000210.1201656_at 0.0000743.11Plectin 1, intermediate filament binding protein 500 kdZ54367216971_s_at 0.000082.15DEAD (Asp-Glu-Ala-Asp) box polypeptide 41NM_016222.1217840_at 0.0000851.6ATPase, H+ transporting, lysosomal 16 kd, V0 subunit cNM_001694.1200954_at 0.0000881.96Cytoplasmic FMR1 interacting protein 1BC005097.1208923_at 0.0000891.91ParvulinBE674061214224_s_at 0.0000942.19Sialyltransferase 4C (β-galactoside α-2,3-sialyltransferase)NM_006278.1203759_at 0.0000952.36STIP1 homology and U-Box containing protein 1NM_005861.1217934_x_at 0.0000962.49Mahogunin, ring finger 1AB011116.1212576_at 0.0000992.23Nonmetastatic cells 4, protein expressed inAL523860212739_s_at 0.0001041.97MAX interacting protein 1NM_005962.1202364_at 0.0001212.54Regulator of G-protein signaling 10NM_002925.2204316_atTop 20 genes up-regulated in the LN metastases (versus the nonmetastatic tumor) 06.63OsteopontinM83248.1209875_s_at 0.0000073.4Ornithine decarboxylase 1NM_002539.1200790_at 0.0000072.2RAB27A, member RAS oncogene familyBE502030209514_s_at 0.0000113Baculoviral IAP repeat-containing 7 (livin)NM_022161.1220451_s_at 0.000016.23Sulfotransferase family, cytosolic, 1C, member 1AF026303.1205342_s_at 0.000013.94Preferentially expressed antigen in melanomaNM_006115.1204086_at 0.000013.67KIAA0930 proteinAK025608.1217118_s_at 0.000012.4Mitochondrial ribosomal protein L35NM_016622.1218890_x_at 0.00001339.88Chromosome 1 open reading frame 34BC004399.1210652_s_at 0.0000213.73GREB1 proteinNM_014668.1205862_at 0.0000232.45Malate dehydrogenase 1, NAD (soluble)NM_005917.1200978_at 0.0000232.24Adaptor-related protein complex 2, σ 1 subunitNM_021575.1208074_s_at 0.0000242.42N-Acylsphingosine amidohydrolase (acid ceramidase) 1U47674.1210980_s_at 0.0000252.07Adaptor-related protein complex 2, σ 1 subunitBC006337.1211047_x_at 0.0000271.9Sorting nexin 10NM_013322.1218404_at 0.00002811.05Aldehyde dehydrogenase 1 family, member A1NM_000689.1212224_at 0.000036.06Serine (or cysteine) proteinase inhibitor, clade F, member 1NM_002615.1202283_at 0.000032.92Neutral sphingomyelinase (N-SMase) activation associated factorNM_003580.1203269_at 0.0000324519.89G antigen 4NM_001473.1207663_x_at 0.0000322.36N-acylsphingosine amidohydrolase (acid ceramidase) 1AI934569213702_x_at Open table in a new tab Table 4Lists of Differentially Expressed Genes in the Horizontal and Vertical Comparisons: Metastatic Tumor versus Lung Metastases (P < 0.005, fc > 1.5)P valuefcGene descriptionAccession no.Probe setGenes up-regulated in the metastatic tumor (versus the lung metastases) 0.0007281.72*Refuted by Q-PCR.GalNAc-T1NM_020474.2201724_s_at 0.0018161.61Oxysterol binding protein-like 10NM_017784.1219073_s_at 0.0018681.87Solute carrier family 23 (nucleobase transporters), member 2AL389886209236_at 0.0025542.09Transmembrane, prostate androgen-induced RNANM_020182.1217875_s_at 0.002971.66Plasminogen activator, tissueNM_000930.1201860_s_at 0.003421.63Likely ortholog of rat GRP78-binding proteinNM_017870.1218834_s_at 0.0036641.66SRY (sex determining region Y)-box 4NM_003107.1201417_at 0.0046191.57Scavenger receptor class B, member 1NM_005505.1201819_at 0.0047061.6Hypothetical protein LOC283687AF249277.1210242_x_atGenes up-regulated in the lung metastases (versus the metastatic tumor) 0.000526.83Surfactant, pulmonary-associated protein CJ0355338691_s_at 0.0005762†Validated by Q-PCR.Neuroblastoma, suppression of tumorigenicity 1NM_005380.1201621_at 0.0010281.75Tight junction protein 1 (zona occludens 1)NM_003257.1202011_at 0.0011412.77Human HL14 gene encoding β-galactoside-binding lectin, 3 end, clone 2M14087.1216405_at 0.0015471.53Phosphoenolpyruvate carboxykinase 2 (mitochondrial)NM_004563.1202847_at 0.0016481.8†Validated by Q-PCR.Neuroblastoma, suppression of tumorigenicity 1D2812437005_at 0.001781751.48Surfactant, pulmonary-associated protein CBC005913.1211735_x_at 0.0021321.68VAMP (vesicle-associated membrane protein)-associated protein A, 33 kdAF154847.1208780_x_at 0.0025962.57New member of the thymosininterferon-inducible multigene familyAL133228216438_s_at 0.0032351.93Pilin-like transcription factorNM_012228.1218773_s_at 0.0034081.53Ets variant gene 5 (ets-related molecule)X76184.1216375_s_at 0.0035772.02Ubiquitin specific protease 1AW499935202412_s_at 0.0037741.68Apolipoprotein C-INM_001645.2204416_x_at 0.0039321.82Serine/arginine repetitive matrix 2AI655799208610_s_at 0.0041591.94Serotonin-7 receptor pseudogeneU86813.1216098_s_at 0.0045081.68Endothelin receptor type BM74921.1204271_s_at 0.0048892.37Transmembrane 4 superfamily member 2NM_004615.1202242_at*
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