Functional profiling: from microarrays via cell-based assays to novel tumor relevant modulators of the cell cycle.

Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.

[1]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[2]  James M. Roberts,et al.  Regulation of the cytoskeleton: an oncogenic function for cdk inhibitors? , 2004, Nature Reviews Cancer.

[3]  A. Poustka,et al.  Systematic subcellular localization of novel proteins identified by large‐scale cDNA sequencing , 2000, EMBO reports.

[4]  T. Barrette,et al.  ONCOMINE: a cancer microarray database and integrated data-mining platform. , 2004, Neoplasia.

[5]  J. Hartley,et al.  DNA cloning using in vitro site-specific recombination. , 2000, Genome research.

[6]  John L Cleveland,et al.  Myc pathways provoking cell suicide and cancer , 2003, Oncogene.

[7]  F. Balkwill.  Cancer and the chemokine network , 2004, Nature Reviews Cancer.

[8]  Stefan Wiemann,et al.  LIFEdb: a database for functional genomics experiments integrating information from external sources, and serving as a sample tracking system , 2004, Nucleic Acids Res..

[9]  Bin Tian,et al.  A large-scale analysis of mRNA polyadenylation of human and mouse genes , 2005, Nucleic acids research.

[10]  Cathy H. Wu,et al.  InterPro, progress and status in 2005 , 2004, Nucleic Acids Res..

[11]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[12]  R. Dalla‐Favera,et al.  Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor. , 1998, Genes & development.

[13]  L. Pemberton,et al.  A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B , 2002, The EMBO journal.

[14]  Keiji Tanaka,et al.  A novel protein‐conjugating system for Ufm1, a ubiquitin‐fold modifier , 2004, The EMBO journal.

[15]  R. Fildes.  Journal of the Royal Statistical Society (B): Gary K. Grunwald, Adrian E. Raftery and Peter Guttorp, 1993, “Time series of continuous proportions”, 55, 103–116.☆ , 1993 .

[16]  J. McCubrey,et al.  Regulation of cell cycle progression and apoptosis by the Ras/Raf/MEK/ERK pathway (Review). , 2003, International journal of oncology.

[17]  Yudong D. He,et al.  Gene expression profiling predicts clinical outcome of breast cancer , 2002, Nature.

[18]  R. Harte,et al.  Identification of cell surface and secreted proteins essential for tumor cell survival using a genetic suppressor element screen , 2004, Oncogene.

[19]  M. Rubin,et al.  PRC17, a novel oncogene encoding a Rab GTPase-activating protein, is amplified in prostate cancer. , 2002, Cancer research.

[20]  M Vingron,et al.  Identification and Classification of Differentially Expressed Genes in Renal Cell Carcinoma by Expression Profiling on a Global Human 31 , 500-Element cDNA Array , 2001 .

[21]  David Botstein,et al.  SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data , 2003, Nucleic Acids Res..

[22]  T. Poggio,et al.  Multiclass cancer diagnosis using tumor gene expression signatures , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Stefan Wiemann,et al.  High-throughput protein analysis integrating bioinformatics and experimental assays. , 2004, Nucleic acids research.

[24]  D. Hanahan,et al.  The Hallmarks of Cancer , 2000, Cell.

[25]  M. Robinson,et al.  γ-Synergin: An Eh Domain–Containing Protein That Interacts with γ-Adaptin , 1999 .

[26]  M. You,et al.  Differentially expressed genes associated with mouse lung tumor progression , 2002, Oncogene.

[27]  O. Albagli-Curiel.  Ambivalent role of BCL6 in cell survival and transformation , 2003, Oncogene.

[28]  J. Zhang,et al.  Identification and characterization of DPZF, a novel human BTB/POZ zinc finger protein sharing homology to BCL-6. , 2001, Biochemical and biophysical research communications.

[29]  Guohua Pan,et al.  Local Regression and Likelihood , 1999, Technometrics.

[30]  Wolfgang Huber,et al.  From ORFeome to biology: a functional genomics pipeline. , 2004, Genome research.

[31]  Long Yu,et al.  Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues. , 2002, Gene expression.

[32]  Mina J. Bissell,et al.  Putting tumours in context , 2001, Nature Reviews Cancer.

[33]  F. Piano,et al.  Gene Clustering Based on RNAi Phenotypes of Ovary-Enriched Genes in C. elegans , 2002, Current Biology.

[34]  Ash A. Alizadeh,et al.  Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling , 2000, Nature.

[35]  Christian A. Rees,et al.  Molecular portraits of human breast tumours , 2000, Nature.

[36]  Martin Vingron,et al.  Gene expression in kidney cancer is associated with cytogenetic abnormalities, metastasis formation, and patient survival. , 2005, Clinical cancer research : an official journal of the American Association for Cancer Research.

[37]  G. Rubin,et al.  Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[38]  R. Razavi,et al.  Mitogen-activated Protein Kinase Phosphorylates and Targets Inducible cAMP Early Repressor to Ubiquitin-mediated Destruction* , 2001, The Journal of Biological Chemistry.

[39]  H. Himmelbauer,et al.  An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division , 2004, Nature.

[40]  L. Neckers,et al.  Functional proteomic screens reveal an essential extracellular role for hsp90α in cancer cell invasiveness , 2004, Nature Cell Biology.

[41]  Jian Yu,et al.  Large-scale cDNA transfection screening for genes related to cancer development and progression. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[42]  D. Leprince,et al.  The Tumor Suppressor Gene HIC1 (Hypermethylated in Cancer 1) Is a Sequence-specific Transcriptional Repressor , 2004, Journal of Biological Chemistry.

[43]  M. Wand.  Local Regression and Likelihood , 2001 .

[44]  C. Sherr,et al.  Principles of Tumor Suppression , 2004, Cell.

[45]  A. Poustka,et al.  A microscope‐based screening platform for large‐scale functional protein analysis in intact cells , 2003, FEBS letters.

[46]  Cheryl Jorcyk,et al.  Alterations in gene expression profiles during prostate cancer progression: functional correlations to tumorigenicity and down-regulation of selenoprotein-P in mouse and human tumors. , 2002, Cancer research.

[47]  H. Mewes,et al.  Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs. , 2001, Genome research.

[48]  N. Perrimon,et al.  Genome-Wide RNAi Analysis of Growth and Viability in Drosophila Cells , 2004, Science.

[49]  B. Garvik,et al.  Principles for the Buffering of Genetic Variation , 2001, Science.

[50]  N. Shikama,et al.  Functional Interaction between Nucleosome Assembly Proteins and p300/CREB-Binding Protein Family Coactivators , 2000, Molecular and Cellular Biology.

[51]  S. L. Wong,et al.  A Map of the Interactome Network of the Metazoan C. elegans , 2004, Science.

[52]  S. Mizuno,et al.  A microplate assay for quantitation of anchorage-independent growth of transformed cells. , 1995, Analytical biochemistry.

[53]  Stefan Wiemann,et al.  High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic. , 2004, Genome research.