Scientific Report 2007

Molecular and Experimental Medicine

Division of Oncovirology

Molecular Genetics of Cancer

P.K. Vogt, A. Bader, D. Bai, A. Denley, A. Galkin, M. Gymnopoulos, J. Hart, F. Hosp, H. Jiang, P. Pavlickova, J. Shi, L. Zhao

The focus of our research is molecular mechanisms of carcinogenesis. We study viral and cellular oncoproteins and tumor suppressors, defining their functions in oncogenesis and identifying molecular targets for therapeutic intervention. In high-throughput screens, we look for small molecules that can interact with these targets and inhibit or reverse oncogenic cellular transformation.

Oncogenic Transformation

Oncogenic transformation of cells requires changes in gene activities, regulated at the level of transcription, translation, or posttranslational modification. These changes result in a gain of function for specific growth-promoting genes and a loss of function for growth-attenuating genes.

Phosphatidylinositol-3′-Kinase in Cancer

The discovery of cancer-specific mutations in PIK3CA, the gene that encodes the catalytic subunit p110α of phosphatidylinositol-3′-kinase (PI3K), was a breakthrough in cancer research. The finding that these mutants are highly restricted to 3 narrowly defined hot spots in the gene immediately suggested that the mutated p110α plays a causative role in cancer. We showed that the 3 hot-spot mutations, introduced individually into wild-type p110α, confer oncogenicity to the protein, making it capable of transforming cells in culture and inducing tumors in vivo. This gain of function is accompanied by enhanced enzymatic activity, constitutive activation of signaling by Akt/protein kinase B, and essential involvement of the target of rapamycin kinase in the oncogenic signaling pathway.

Rare Cancer-Specific Mutations in p110α

In addition to the hot-spot mutations, which account for about 80% of all cancer-mutated p110α, numerous rare mutations have been identified in diverse cancers. These rare mutations are distributed over the entire coding region of PIK3CA. We examined 15 of the rare mutations and found that 14 induce a gain of function that results in oncogenic transformation when the mutant protein is expressed in normal cells. The rare mutants also have increased catalytic activity and constitutively activate the Akt pathway. Rare mutants are, however, at least 10 times less oncogenic (as measured by the number of cell-transforming events per nanogram of DNA) than the hot-spot mutants. This reduced potency accounts for the rare occurrence of these mutants.

Multiple Molecular Mechanisms for Mutation-Induced Gain of Function in p110α

The protein p110α has several distinct structure-function domains: an N-terminal domain that binds the regulatory subunit, a Ras-binding domain, a C2 domain, a helical domain, and a C-terminal kinase domain. We have mapped hot-spot and rare mutations on a model structure of p110α. The locations of the mutations in the functional domains suggest at least 3 different molecular mechanisms for the gain of function. Mutations in the C2 domain increase the positive surface charge and thereby enhance recruitment to the plasma membrane. Mutations in the helical domain affect the interaction with a regulatory protein, probably p85. By interfering with the p85 interactions, these mutations relieve the inhibitory actions of p85 on p110α. Mutations in the kinase domain affect the position and the movement of the activation loop. They may lock the activation loop in the "on" position.

PI3K is an exceedingly attractive target for cancer therapy. Inhibitors specific to the cancer-derived mutations of PI3K would not affect normal PI3K signaling. The fact that PI3K is an enzyme and that the cancer-specific mutations result in gain of function greatly facilitates the design of effective inhibitors.

Small-Molecule Regulators of the Myc Network

Increased levels and enhanced function of the transcriptional regulator Myc are common in cancer. They result from gene amplification, elevated levels of transcription, and activated translation. In many cancers, a correlation exists between the gain of function in Myc, tumor grade, and poor prognosis, suggesting that Myc plays an important role in the causation and progression of cancer.

Myc is a transcription factor that functions only as a dimer with another protein, Max. The structure of the Myc-Max dimerization interface is known; single amino acid substitutions at critical sites can break or stabilize dimerization. In collaboration with D.L. Boger and K.D. Janda, Department of Chemistry, we have isolated several small molecules that interfere with the dimerization of Myc and Max. As a consequence, these molecules also prevent Myc DNA binding, Myc-dependent transcriptional activation, and Myc-induced oncogenic transformation.

The Myc-Max dimer belongs to a complex network that includes activators as well as repressors of transcription. All of the activators and repressors function as dimers with the Max protein, making Max the common denominator of the network. Max is also the only component of the network that can form homodimers, albeit weak and transcriptionally inactive homodimers. Small molecules that specifically stabilize the Max homodimer would trap this essential partner and make it unavailable for heterodimerization and for transcriptional regulatory activities. Such compounds would downregulate the entire network.

We have searched for small molecules that could bind specifically to Max and stabilize the Max homodimer while leaving Myc-Max dimerization unaffected. The search was performed in silico with the helix-loop-helix leucine zipper dimerization domain of Max and the National Cancer Institute diversity set of compounds. We used the software docking program AutoDock developed here at Scripps Research, and the computations were performed on the supercomputer at the University of California, San Diego. The candidates identified in silico were then screened by using fluorescence resonance energy transfer followed by cell-based assays for inhibition of Myc. The final compound that passed all these tests inhibits Myc-dependent cell growth, Myc-mediated transcriptional activation, and Myc-induced oncogenic transformation.

Publications

Bader, A.G., Kang, S., Vogt, P.K. Cancer-specific mutations in PI3KCA are oncogenic in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:1475, 2006.

Bader, A.G., Vogt, P.K. Leucine zipper transcription factors: bZIP proteins. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Ganten, G., Ruckpaul, K. (Eds.). Springer, New York, 2006, p. 964.

Denley, A., Gymnopoulos, M., Hart, J.R., Jiang, H., Zhao, L., Vogt, P.K. Biochemical and biological characterization of tumor-associated mutations of p110α. Methods Enzymol., in press.

Denning, G., Jean-Joseph, B., Prince, C., Durden, D.L., Vogt, P.K. A short N-terminal sequence of PTEN controls cytoplasmic localization and is required for suppression of cell growth. Oncogene 26:3930, 2007.

Fang, J., Meng, Q., Vogt, P.K., Zhang, R., Jiang, B.H. A downstream kinase of the mammalian target of rapamycin, p70S6K1, regulates human double minute 2 protein phosphorylation and stability. J. Cell. Physiol. 209:261, 2006.

Gymopoulos, M., Vogt, P.K. Rare, cancer-specific mutations in PIK3CA show gain of function. Proc. Natl. Acad. Sci. U. S. A. 104:5569, 2007.

Sawa, M., Hsu, T.-L., Itoh, T., Sugiyama, M., Hanson, S., Vogt, P.K., Wong, C.-H. Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo. Proc. Natl. Acad. Sci. U. S. A. 103:12371, 2006.

Vogt, P.K., Kang, S., Elsliger, M.-A., Gymnopoulos, M. Cancer-specific mutations in phosphatidylinositol 3-kinase. Trends Biochem. Sci. 32:342, 2007.