News and Publications

Division of Oncovirology

Peter K. Vogt, Ph.D., Division Head

Mutations in growth regulatory genes are the causative triggers of cancer. Most of these mutations are somatic; they result in aberrantly functioning positive and negative regulators of cell growth that together determine the neoplastic phenotype. The broad goal of the Division of Oncovirology is to understand the molecular mechanisms by which these growth regulators induce cancer.

The work of the division focuses on 2 groups of oncogenes. The first group codes for nuclear proteins that function as transcriptional activators or transcriptional repressors. The second group codes for cytoplasmic signaling components that are connected directly or indirectly to phosphatidylinositol-3-kinase.

Oncogenic Transcription Factors

P.K. Vogt, J. Li, M. Aoki, I. Bottoli, S.-L. Fu, K. Hollen, B.-H. Jiang, U. Kruse, Y. Ma, M. Nishizawa, B. Nowakowski

All nuclear proteins coded for oncogenes are transcription factors. BZip proteins and winged helix proteins form 2 large families of these transcriptional regulators.

BZIP PROTEINS

BZip proteins are defined by a leucine zipper dimerization domain located adjacent to a DNA-binding domain of mainly basic amino acids. Important families of the BZip proteins are the AP-1 transcription factors, which include Jun, Fos, and their close relatives, and the ATF proteins, which include CREB and its relatives. Many of the BZip proteins can form heterodimers, and these protein-protein associations create a broad range of combinatorial specificities that show preferential binding to variants of a basic DNA-target motif.

Oncogenic transformation induced by these transcription factors raises 2 fundamental questions. The first concerns the protein-protein interactions that mediate the activating signal from transcription factor to basic transcriptional machinery. The second deals with the differentially regulated genes that control the neoplastic phenotype of the cell. The question about signal transduction to the basic transcriptional machinery remains largely unanswered. Some important regulators of BZip proteins have been determined, but important links between basic transcriptional machinery and BZip proteins remain to be defined. More rapid progress is being made in determining and characterizing differentially regulated target genes.

Using directional tag polymerase chain reaction subtraction, we obtained a first generation of Jun targets. A hormone-regulatable Jun construct has enabled us to distinguish between direct targets whose promoters are regulated by Jun and indirect targets that are targets of targets. Hormone-induced activity of Jun leads to upregulation of direct targets in the absence of de novo protein synthesis. Differential expression of mRNAs for indirect targets depends on de novo protein synthesis. Several of the direct targets have functions related to cellular growth control, and the expression of these targets parallels the levels of transforming activity in diverse Jun mutants.

In collaboration with scientists at Digital Gene Technologies, La Jolla, California, we have used the total gene analysis method to isolate a second and more complete generation of Jun targets. The total gene analysis method has greater sensitivity for detection of messages present in small amounts and better quantification than do alternative techniques based on DNA microchips and microarrays. The long-range challenge of the target search, however, lies in functional studies that will divide differentially regulated genes into essential participants in oncogenesis and neutral bystanders. A small number of essential participants, differentially expressed in a normal target cell, should be able to induce neoplastic transformation.

WINGED HELIX PROTEINS

Members of the winged helix protein family share a conserved DNA-binding domain of about 110 amino acids. These proteins usually bind to DNA as monomers. Examples of oncoproteins belonging to this family are Qin, derived from the avian retrovirus avian sarcoma virus 31, and the human fusion protein PAX3-FKHR. The fundamental questions that need to be answered for this group of oncogenic transcription factors are the same as those for the BZip proteins; that is, we wish to define interactions between winged helix proteins and the basic transcriptional machinery and determine targets that are relevant to transformation.

Qin is an unusual oncoprotein. In the course of normal development, expression of Qin is confined to the telencephalon. Reporters carrying the consensus Qin-binding site are repressed by Qin. The repression is enhanced by mutations found in the viral version of Qin, which is more oncogenic than is its cellular counterpart. Paradoxically, these mutations lower the affinity of Qin for DNA. Oncogenicity is correlated with repression. Replacing the Qin repression domain with that of the Drosophila protein Engrailed retains oncogenic potential. In contrast, replacing the Qin repression domain with the activator domain of protein VP16 from herpes simplex virus results in a chimera that activates transcription from Qin-binding sites but is no longer oncogenic and induces a specific resistance to Qin-dependent transformation.

The PAX3-FKHR protein of alveolar rhabdomyosarcoma unites the DNA-binding domains of PAX3 with the transactivation domains of the winged helix protein FKHR. Deletion analyses show that both DNA binding and transcriptional regulation are required for transformation in vitro. A reduction in transactivation potential of the fusion protein changes the morphology of the transformed cell.

Several winged helix genes and their corresponding proteins have been isolated from chicken cells. One of these, chicken winged helix protein 2, has been mutated to become rapidly transforming for chicken embryo fibroblasts. A comparison of such activating mutations in related transcription factors is instructive and helps detect functional changes required for overt oncogenicity.

THE ONCOGENE P3K

The oncogene p3k was isolated from avian sarcoma virus 16. It codes for a homolog of the p110 catalytic subunit of phosphatidylinositol-3-kinase. Cellular p110 is controlled by the p85 regulatory subunit; a protein characterized by multiple modular domains that mediate specific protein-protein interactions. The properties of p110 important in oncogenicity are enhanced binding to the plasma membrane and efficient translation leading to elevated levels of protein expression. Interaction with p85 is not required for oncogenicity, and mutational inactivation of the kinase activity results in loss of oncogenic potential, suggesting that the P3k protein must be enzymatically active for neoplastic transformation.

A downstream target of phosphatidylinositol-3-kinase is the serine/threonine kinase Akt. Like P3k, Akt can be mutated to become highly oncogenic. Localization at the plasma membrane and an active kinase domain are again prerequisites for transformation. A transdominant negative Akt specifically interferes with P3k-induced transformation, indicating that Akt is an essential mediator of the oncogenic signal issued by P3k. Both oncogenes, akt and p3k, share the same unexpectedly narrow tumor spectrum. They induce only hemangiosarcomas. The basis for this remarkable tissue specificity of transformation is being actively investigated.

PUBLICATIONS

Chang, H.W., Aoki, M., Fruman, D., Auger, K.R., Bellacosa, A., Tsichlis, P.N., Cantley, L.C., Roberts, T.M., Vogt, P.K. Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 276:1848, 1997.

Freyaldenhoven, B.S., Freyaldenhoven, M.P., Iacovoni, J.S., Vogt, P.K. Avian winged helix proteins CWH-1, CWH-2, and CWH-3 repress transcription from Qin binding sites. Oncogene 15:483, 1997.

Goller, M.E., Kruse, U., Iacovoni, J.S., Vogt, P.K. Glutaredoxin is a direct target of oncogenic Jun. Oncogene, in press.

Himly, M., Foster, D.N., Bottoli, I., Iacovoni, J.S., Vogt P.K. The DF-1 chicken fibroblast cell line: Transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses. Virology, in press.

Kruse, U., Iacovoni, J.S., Goller, M.E., Vogt, P.K. Hormone-regulatable neoplastic transformation induced by a Jun-estrogen receptor chimera. Proc. Natl. Acad. Sci. U.S.A. 94:12396, 1997.

Li, J., Thurm, H., Chang, H.W., Iacovoni, J.S., Vogt, P.K. Oncogenic transformation induced by the Qin protein is correlated with transcriptional repression. Proc. Natl. Acad. Sci. U.S.A. 94:10885, 1997.

Vogt, P.K., Chang, H.-W., Goller, M.E., Iacovoni, J.S., Kruse, U., Li, J., Scheidler, S. Onkogene: Fortschritte in Zytoplasma und Nukleus. Nova Acta Leopoldina 303:1, 1997.

Vogt, P.K., Li, J., Freyaldenhoven, B.S. Revelations of a captive: Retroviral Qin and the oncogenicity of winged helix proteins. Virology 238:1, 1997.