The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p.

The JC12 monoclonal antibody recognizes a previously unknown nuclear protein that showed a restricted distribution in normal tonsil and was also overexpressed in a subset of diffuse large B-cell lymphomas. Using this reagent, we expression cloned cDNAs encoding its antigenic target and identified this protein as a novel putative transcription factor, FOXP1. The FOXP1 protein sequence contains predicted domains characteristic of transcription factors, including a winged helix DNA-binding motif, a second potential DNA-binding motif, a C(2)H(2) zinc finger, nuclear localization signals, coiled-coil regions, PEST sequences, and potential transactivation domains. The FOXP1 gene has been mapped to chromosome 3p14.1, a region that commonly shows loss of heterozygosity in a wide range of tumors and which is reported to contain a tumor suppressor gene(s). Using tissue arrays and immunohistochemistry, we demonstrate that both the FOXP1 mRNA and protein are widely expressed in normal tissues. The levels of FOXP1 mRNA were compared in paired normal and tumor tissues (from the same patient) using a tissue array containing cDNAs extracted from 68 samples taken from kidney, breast, prostate, uterus, ovary, cervix, colon, lung, stomach, rectum, small intestine, and from nine cancer cell lines. Differences in FOXP1 mRNA expression between normal and tumor samples were observed in 51% of cases. Most striking was the comparative loss of expression in 73% of colon tumors and comparative overexpression of FOXP1 mRNA in 75% of stomach tumors. Analysis of the FOXP1 mRNA expression in normal tissues (not taken from cancer patients) indicated that loss of FOXP1 expression may occur in some histologically normal tissues adjacent to tumors. Immunohistochemical analysis of FOXP1 protein expression was performed on 128 solid tumors, including 16 renal, 9 breast, 12 lung, 20 colon, 21 stomach, 10 head and neck, 35 prostate, and 5 pancreatic cases. Complete loss of expression, increased expression, and cytoplasmic mislocalization of the predominantly nuclear FOXP1 protein were frequently observed in neoplastic cells. Our study identifies FOXP1 as a new candidate tumor suppressor gene localized to the chromosome 3p14.1 region.

[1]  T. Hunter,et al.  Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  A. Banham,et al.  The plasma cell associated antigen detectable by antibody VS38 is the p63 rough endoplasmic reticulum protein. , 1997, Journal of clinical pathology.

[3]  B. Seed,et al.  Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Kanehisa,et al.  A knowledge base for predicting protein localization sites in eukaryotic cells , 1992, Genomics.

[5]  K Autio,et al.  DNA copy number losses in human neoplasms. , 1999, The American journal of pathology.

[6]  R. Bassel-Duby,et al.  Transient expression of a winged-helix protein, MNF-beta, during myogenesis , 1997, Molecular and cellular biology.

[7]  W. Herr,et al.  Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain , 1994, Molecular and cellular biology.

[8]  M. L. Le Beau,et al.  An FHIT tumor suppressor gene? , 1998, Genes, chromosomes & cancer.

[9]  W. Sellers,et al.  Identification of a cyclin-cdk2 recognition motif present in substrates and p21-like cyclin-dependent kinase inhibitors , 1996, Molecular and cellular biology.

[10]  H. Jäckle,et al.  Regulatory elements controlling expression of the Drosophila homeotic gene fork head. , 1990, The EMBO journal.

[11]  S. Lam,et al.  High resolution chromosome 3p allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple, discontinuous sites of 3p allele loss and three regions of frequent breakpoints. , 2000, Cancer research.

[12]  Y. Nishizuka,et al.  Regulation of nuclear translocation of forkhead transcription factor AFX by protein kinase B. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[13]  J. Heitman,et al.  Nuclear protein localization. , 1991, Biochimica et biophysica acta.

[14]  J. Califano,et al.  11:40 AM: A Genetic Progression Model for Head and Neck Cancer: Implications for Field Cancerization , 1996 .

[15]  B. Dynlacht,et al.  p107 uses a p21CIP1-related domain to bind cyclin/cdk2 and regulate interactions with E2F. , 1995, Genes & development.

[16]  D. Jans,et al.  Regulation of protein transport to the nucleus: central role of phosphorylation. , 1996, Physiological reviews.

[17]  J. Casanova,et al.  X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy , 2001, Nature Genetics.

[18]  B. Johansson,et al.  Deletion of chromosome arm 3p in hematologic malignancies , 1997, Leukemia.

[19]  G. Bullock,et al.  Techniques in Immunocytochemistry , 1983 .

[20]  D. Lawrence,et al.  The design of peptide-based substrates for the cdc2 protein kinase. , 1995, The Biochemical journal.

[21]  Stefan Imreh,et al.  Combined LOH/CGH analysis proves the existence of interstitial 3p deletions in renal cell carcinoma , 2000, Oncogene.

[22]  P. Speight,et al.  Frequent gene deletions in potentially malignant oral lesions. , 1996, British Journal of Cancer.

[23]  Marshall W. Anderson,et al.  Homozygous deletions of human chromosome 3p in lung tumors. , 1997, Cancer research.

[24]  P. Tucker,et al.  DNA-binding properties and secondary structural model of the hepatocyte nuclear factor 3/fork head domain. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Klemsz,et al.  Genesis, a Winged Helix Transcriptional Repressor with Expression Restricted to Embryonic Stem Cells* , 1996, The Journal of Biological Chemistry.

[26]  R. Costa,et al.  Analysis of hepatocyte nuclear factor-3 beta protein domains required for transcriptional activation and nuclear targeting. , 1995, Nucleic acids research.

[27]  C. Junien,et al.  Report of the committee on chromosome and gene loss in human neoplasia , 1991 .

[28]  J. Darnell,et al.  HNF-3A, a hepatocyte-enriched transcription factor of novel structure is regulated transcriptionally. , 1990, Genes & development.

[29]  Weiguo Shu,et al.  Characterization of a New Subfamily of Winged-helix/Forkhead (Fox) Genes That Are Expressed in the Lung and Act as Transcriptional Repressors* , 2001, The Journal of Biological Chemistry.

[30]  J. Darnell,et al.  Hepatocyte nuclear factor 3/fork head or "winged helix" proteins: a family of transcription factors of diverse biologic function. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[31]  S. Uzan,et al.  Loss of Heterozygosity on the Short Arm of Chromosome 3 in Cervical Intra-Epithelial Neoplasia without Concomitant Cervical Carcinoma , 1998, Pathobiology.

[32]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[33]  K. Kaestner,et al.  Unified nomenclature for the winged helix/forkhead transcription factors. , 2000, Genes & development.

[34]  E. Dmitrovsky,et al.  Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[35]  B. Futcher,et al.  Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis , 2000 .

[36]  D. Galas,et al.  Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse , 2001, Nature Genetics.

[37]  S. Burley,et al.  Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5 , 1993, Nature.

[38]  L. Lim,et al.  Hepatocyte Nuclear Factor 3 / fork head Homolog 11 Is Expressed in Proliferating Epithelial and Mesenchymal Cells of Embryonic and Adult Tissues , 1996 .

[39]  A. Maitra,et al.  Loss of heterozygosity at 3p in benign lesions preceding invasive breast cancer. , 1999, The Journal of surgical research.

[40]  A. Gerdes,et al.  Chromosome abnormalities in benign hyperproliferative disorders of epithelial and stromal breast tissue , 1995, International journal of cancer.

[41]  James E. Darnell,et al.  Variety in the level of gene control in eukaryotic cells , 1982, Nature.

[42]  P. Silver,et al.  Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival , 1999, Oncogene.

[43]  B. Schulman,et al.  Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[44]  S. Rogers,et al.  PEST sequences and regulation by proteolysis. , 1996, Trends in biochemical sciences.

[45]  P. Vogt,et al.  The retroviral oncogene qin belongs to the transcription factor family that includes the homeotic gene fork head. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[46]  R. Tjian,et al.  A glutamine-rich hydrophobic patch in transcription factor Sp1 contacts the dTAFII110 component of the Drosophila TFIID complex and mediates transcriptional activation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[47]  K. Yao,et al.  Molecular Analysis of a Novel Winged Helix Protein, WIN , 1997, The Journal of Biological Chemistry.

[48]  S. Pongor,et al.  Substrate specificity of CDC2 kinase from human HeLa cells as determined with synthetic peptides and molecular modeling. , 1994, Archives of biochemistry and biophysics.

[49]  A. Read,et al.  Allelic imbalance on chromosome 3p in oral dysplastic lesions: an early event in oral carcinogenesis. , 1996, Cancer research.

[50]  C. Ingles,et al.  Direct interaction between the transcriptional activation domain of human p53 and the TATA box-binding protein. , 1993, The Journal of biological chemistry.

[51]  D. Mason,et al.  IMMUNOCYTOCHEMICAL DETECTION OF T AND B CELL POPULATIONS IN ROUTINE BLOOD SMEARS , 1984, The Lancet.

[52]  M. Lerman,et al.  Identification of intragenic mutations in the von Hippel-Lindau disease tumour suppressor gene and correlation with disease phenotype. , 1994, Human molecular genetics.

[53]  Yalin H. Wei,et al.  Cloning and characterization of the t(X;II) breakpoint from a leukemic cell line identify a new member of the forkhead gene family , 1994, Genes, chromosomes & cancer.

[54]  M. Kozak.  An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. , 1987, Nucleic acids research.

[55]  H. Jäckle,et al.  The homeotic gene fork head encodes a nuclear protein and is expressed in the terminal regions of the Drosophila embryo , 1989, Cell.

[56]  B. Emanuel,et al.  Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma , 1993, Nature Genetics.

[57]  W. Sellers,et al.  Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes , 1999, Molecular and Cellular Biology.

[58]  J. G. Patton,et al.  Alternative splicing in the control of gene expression. , 1989, Annual review of genetics.

[59]  S. Naylor,et al.  Deletions of the short arm of chromosome 3 in solid tumors and the search for suppressor genes. , 1997, Advances in cancer research.

[60]  S. Tucker,et al.  The genetic locus NRC-1 within chromosome 3p12 mediates tumor suppression in renal cell carcinoma independently of histological type, tumor microenvironment, and VHL mutation. , 1999, Cancer research.

[61]  R. Medema,et al.  AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1 , 2000, Nature.

[62]  R. Berger,et al.  AF6q21, a novel partner of the MLL gene in t(6;11)(q21;q23), defines a forkhead transcriptional factor subfamily. , 1997, Blood.

[63]  W. Knöchel,et al.  Five years on the wings of fork head , 1996, Mechanisms of Development.

[64]  G. Tse,et al.  High frequency of chromosome 3p deletion in histologically normal nasopharyngeal epithelia from southern Chinese. , 2000, Cancer research.

[65]  B. Chadwick,et al.  FKHL15, a new human member of the forkhead gene family located on chromosome 9q22. , 1997, Genomics.

[66]  M. Gstaiger,et al.  Association of human CUL‐1 and ubiquitin‐conjugating enzyme CDC34 with the F‐box protein p45SKP2: evidence for evolutionary conservation in the subunit composition of the CDC34–SCF pathway , 1998, The EMBO journal.

[67]  D Weigel,et al.  The fork head domain: A novel DNA binding motif of eukaryotic transcription factors? , 1990, Cell.

[68]  A. Baxevanis,et al.  Interactions of coiled coils in transcription factors: where is the specificity? , 1993, Current opinion in genetics & development.

[69]  S. Frutiger,et al.  Binding sites for hepatocyte nuclear factor 3 beta or 3 gamma and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic alpha-amylase , 1995, Molecular and cellular biology.

[70]  Arndt Borkhardt,et al.  Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23) , 1997, Oncogene.

[71]  H. Ochs,et al.  The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3 , 2001, Nature Genetics.

[72]  G. Bratthauer,et al.  Genetic abnormalities in mammary ductal intraepithelial neoplasia‐flat type (“clinging ductal carcinoma in situ”) , 2000, Cancer.

[73]  J. Biegel,et al.  Fusion of PAX7 to FKHR by the variant t(1;13)(p36;q14) translocation in alveolar rhabdomyosarcoma. , 1994, Cancer research.

[74]  D. Reinberg,et al.  Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53 , 1994, Molecular and cellular biology.