News and Publications

Division of Oncovirology

Peter K. Vogt, Ph.D, Division Head

Mutations in growth regulatory genes are the genetic causes of cancer. They lead to a gain of function in positive growth regulators and a loss of function in negative growth regulators. Combinations of these activating and inactivating mutations determine the oncogenic cellular phenotype. Research in the Division of Oncovirology focuses on 2 broad areas: (1) the mechanisms by which mutated growth regulators cause cancer and (2) protein-protein interactions as targets of novel anticancer drugs.

Molecular Genetics of Cancer

P.K. Vogt, J. Li, M. Aoki, T. Berg, I. Bottoli, S. Cohen, S.-L. Fu, B.-H. Jiang, B. Kempf, Y. Ma, D. Maslyar, M. Nishizawa, C. Sonderegger, A. Waha

ABERRANT TRANSCRIPTION AND CANCER

All transcription factors have oncogenic potential. One of the now classic examples is Jun, a member of the AP-1 complex of transcriptional regulators. A mutated viral form of the Jun protein induces transformation in cells in culture and in sarcomas in chickens. The oncogenesis of Jun is based on aberrant transcription of specific target genes. A search for such genes that are differentially expressed in Jun-transformed cells has identified several novel Jun targets. Of particular interest is heparin-binding epithelial growth factor--like growth factor. Its expression is correlated with transformation and with the transforming potential of various Jun mutants. Overexpression of the growth factor alone induces a partial transformed phenotype, including foci of morphologically altered cells in monolayer cultures and anchorage-independent growth. In Jun-induced transformation, the growth factor must cooperate with other transformation-specific targets to produce full transformation.

One of our goals is to identify both transformation-related targets and several other oncogenic transcription factors. These factors include the forkhead--winged helix proteins Qin, mesenchyme-forkhead 1, and chicken winged helix 2 and the human chimeric protein PAX3-FKHR. Qin is of particular interest because it is a transcriptional repressor, and repression is correlated with oncogenic activity. The immediate targets of Qin presumably are negative growth regulators. PAX3-FKHR is generated by a chromosomal rearrangement that occurs in alveolar rhabdomyosarcomas in children. A deletion analysis of PAX3-FKHR showed that both DNA binding and transcriptional activation are prerequisites of oncogenic transformation.

For our initial searches for transformation-related targets, we used conventional techniques such as differential display, directional tag polymerase chain reaction, and representational difference analysis. With the installation of a central DNA microchip facility at TSRI, more comprehensive searches will be done. Genetic and functional screens will reduce the number of candidate targets and help single out those genes that affect the growth behavior of the cell.

PHOSPHATIDYLINOSITOL 3-KINASE IN ONCOGENESIS AND DIFFERENTIATION

An oncogenic form of phosphatidylinositol 3-kinase (PI 3-kinase; catalytic subunit p110a) was isolated from the avian retrovirus ASV16. This subunit induces foci of transformed cells in monolayer cultures and hemangiosarcomas in animals. The mutational changes that activate the oncogenicity of the cellular PI 3-kinase all induce enhanced localization of the kinase to cellular membranes. As a consequence, p110a becomes constitutively active and independent of the regulatory subunit p85. Kinase activity is a prerequisite for oncogenicity. Additional nonkinase domains are also necessary for oncogenic transformation by mutated p110a. Their function remains to be determined.

A downstream target of PI 3-kinase is the serine-threonine kinase Akt. Akt is also potentially oncogenic. As is the case with PI 3-kinase, enzymatic activity and increased membrane localization are prerequisites for oncogenicity. Akt also induces exclusively hemangiosarcomas in chickens, and transdominant negative mutants of Akt interfere with oncogenic transformation induced by PI 3-kinase. The oncogenic signal of PI 3-kinase, therefore, depends on Akt.

In muscle differentiation, constitutively active and oncogenic PI 3-kinase induces enhanced differentiation, in contrast to other oncogenes such as src, ras, or myc that interfere with differentiation. PI 3-kinase is not only a stimulator of myogenesis; it is also required for muscle differentiation. Similarly, Akt is an essential component of the myogenic pathway and can substitute for dysfunctional PI 3-kinase. The same signal originating in PI 3-kinase proceeds through Akt and then induces opposite responses in fibroblasts and myoblasts: oncogenic transformation and uncontrolled growth in the former and exit from the cell cycle and differentiation in the latter.

ONCOGENIC WNT SIGNALS

Wnt is a secreted protein. It initiates a signal cascade that is conserved from Drosophila to humans, and various forms of Wnt control diverse developmental decisions. Important cytoplasmic components of Wnt signaling are ß-catenin, the kinase GSK-3ß, the adenomatous polyposis coli protein APC, and transducin. These proteins form a multiprotein complex that regulates cytoplasmic concentrations of ß-catenin. Inactivating mutations in APC or stabilizing mutations in ß-catenin can lead to increased levels of ß-catenin and to translocation of ß-catenin into the nucleus, where the protein combines with the high-mobility group proteins lymphoid-enhancing factor (LEF) to form transcriptional activators. Mutants of APC and ß-catenin are important causative factors in human colon cancer and melanoma.

To analyze the oncogenic effects of elevated levels of LEF-dependent transcriptional activation, we constructed chimeras in which LEF was covalently linked to various transactivation domains, including ß-catenin, the hormone-binding domain of the human estrogen receptor, and the transactivation domain of herpes simplex virus protein VP16. All these constructs induce oncogenic transformation in cell cultures. The constructs are being used to identify downstream target genes that control LEF-dependent transformation and are important in human cancer.

COMBINATORIAL CHEMISTRY AND ONCOGENIC GROWTH SIGNALS

Cellular signals, including the signals of oncogenes, are mediated by protein-protein interactions. The proteins involved in these interactions offer targets for screens designed to isolate small molecules that will alter such interactions and that will affect signal propagation. In collaboration with D. Boger and C.H. Wong, Department of Chemistry, we developed assays that use fluorescence resonance energy transfer to test combinatorial libraries for effects on specific protein-protein interactions. The interaction between the proteins Myc and Max is the first such system used to isolate potential inhibitors of the myc oncogene.

PUBLICATIONS

Aoki, M., Batista, O., Bellacosa, A., Tsichlis, P., Vogt, P.K. The Akt kinase: Molecular determinants of oncogenicity. Proc. Natl. Acad. Sci. U. S. A. 95:14950, 1998.

Aoki, M., Hecht, A., Kruse, U., Kemler, R., Vogt, P.K. Nuclear endpoint of Wnt signaling: Neoplastic transformation induced by transactivating lymphoid-enhancing factor 1. Proc. Natl. Acad. Sci. U. S. A. 96:139, 1999.

Fu, S.-L., Bottoli, I., Goller, M., Vogt, P.K. Heparin-binding epidermal growth factor-like growth factor,a v-Jun target gene, induces oncogenic transformation. Proc. Natl. Acad. Sci. U. S. A. 96:5716, 1999.

Goller, M.E., Kruse, U., Iacovoni, J.S., Vogt, P.K. Glutaredoxin is a direct target of oncogenic Jun. Oncogene 16:2945, 1998.

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 248:295, 1998.

Jiang, B.H., Aoki, M., Zheng, J.Z., Li, J., Vogt, P.K. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B. Proc. Natl. Acad. Sci. U. S. A. 96:2077, 1999.

Jiang, B.H., Zheng, J.Z., Vogt, P.K. An essential role of phosphatidylinositol 3-kinase in myogenic differentiation. Proc. Natl. Acad. Sci. U. S. A. 95:14179, 1998.