Peter K. Vogt

Oncogenic PI3K deregulates transcription and translation

There have long been indications of a role for PI3K (phosphatidylinositol 3-kinase) in cancer pathogenesis. Experimental data document a requirement for deregulation of both transcription and translation in PI3K-mediated oncogenic transformation. The recent discoveries of cancer-specific mutations in PIK3CA, the gene that encodes the catalytic subunit p110α of PI3K, have heightened the interest in the oncogenic potential of this lipid kinase and have made p110α an ideal drug target.

Class I PI3K in oncogenic cellular transformation

Class I phosphoinositide 3-kinase (PI3K) is a dimeric enzyme, consisting of a catalytic and a regulatory subunit. The catalytic subunit occurs in four isoforms designated as p110α, p110β, p110γ and p110δ. These isoforms combine with several regulatory subunits; for p110α, β and δ, the standard regulatory subunit is p85, for p110γ, it is p101. PI3Ks play important roles in human cancer. PIK3CA, the gene encoding p110α, is mutated frequently in common cancers, including carcinoma of the breast, prostate, colon and endometrium. Eighty percent of these mutations are represented by one of the three amino-acid substitutions in the helical or kinase domains of the enzyme. The mutant p110α shows a gain of function in enzymatic and signaling activity and is oncogenic in cell culture and in animal model systems. Structural and genetic data suggest that the mutations affect regulatory inter- and intramolecular interactions and support the conclusion that there are at least two molecular mechanisms for the gain of function in p110α. One of these mechanisms operates largely independently of binding to p85, the other abolishes the requirement for an interaction with Ras. The non-α isoforms of p110 do not show cancer-specific mutations. However, they are often differentially expressed in cancer and, in contrast to p110α, wild-type non-α isoforms of p110 are oncogenic when overexpressed in cell culture. The isoforms of p110 have become promising drug targets. Isoform-selective inhibitors have been identified. Inhibitors that target exclusively the cancer-specific mutants of p110α constitute an important goal and challenge for current drug development.

Continuous tissue culture cell lines derived from chemically induced tumors of Japanese quail

Several continuous tissue culture cell lines were established from methylcholanthrene-induced fibrosarcomas of Japanese quail. The lines consist either of fibroblastic elements, round refractile cells or polygonal cells. They show transformed characteristics in agar colony formation and hexose uptake, and most are tumorigenic. Their cloning efficiency in plastic dishes is not increased over that of normal quail embryo fibroblasts. The quail tumor cell lines do not produce endogenous avian oncoviruses and fail to complement the Bryan high titer strain of Rous sarcoma virus; those tested lack the p27 protein of avian oncoviruses. Most of the cell lines are susceptible to subgroup A avian sarcoma viruses, but are relatively resistant to viruses of subgroups C, E and F as compared to normal quail embryo fibroblasts.

Cancer-specific mutations in PIK3CA are oncogenic in vivo

The PIK3CA gene, coding for the catalytic subunit p110α of class IA phosphatidylinositol 3-kinases (PI3Ks), is frequently mutated in human cancer. Mutated p110α proteins show a gain of enzymatic function in vitro and are oncogenic in cell culture. Here, we show that three prevalent mutants of p110α, E542K, E545K, and H1047R, are oncogenic in vivo. They induce tumors in the chorioallantoic membrane of the chicken embryo and cause hemangiosarcomas in the animal. These tumors are marked by increased angiogenesis and an activation of the Akt pathway. The target of rapamycin inhibitor RAD001 blocks tumor growth induced by the H1047R p110α mutant. The in vivo oncogenicity of PIK3CA mutants in an avian species strongly suggests a critical role for these mutated proteins in human malignancies.

Helical domain and kinase domain mutations in p110α of phosphatidylinositol 3-kinase induce gain of function by different mechanisms

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is up-regulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in ≈30% of tumors of the prostate, breast, cervix, and endometrium. The most prominent of these mutants, represented by single amino acid substitutions in the helical or kinase domain, show a gain of enzymatic function, activate AKT signaling, and induce oncogenic transformation. We have carried out a genetic and biochemical analysis of these hot-spot mutations in PIK3CA. The results of this study suggest that the helical and kinase domain mutations trigger gain of function through different mechanisms. They show different requirements for interaction with the PI3K regulatory subunit p85 and with RAS-GTP. The gain of function induced by helical domain mutations is independent of binding to p85 but requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding but is highly dependent on the interaction with p85. We speculate that the contrasting roles of p85 and RAS-GTP in helical and kinase domain mutations reflect two distinct states of mutated p110α. These two states differ in mutation-induced surface charges and also may differ in conformational properties that are controlled by interactions with p85 and RAS-GTP. The two states do not appear mutually exclusive because the helical and kinase domain mutations act synergistically when present in the same p110α molecule. This synergism also supports the conclusion that the helical and kinase domain mutations operate by two different and independent mechanisms.

Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions

Abstract Polycations enhance the infectivity of avian sarcoma viruses for chick embryo fibroblast cultures up to 80-fold. The enhancement is restricted to members of avian tumor virus subgroups B, C, and D and to RSV(O). Subgroup A viruses are either unaffected or inhibited by polycations. The polycation-mediated enhancement is at least in part due to an increased adsorption rate of virus to cell, but may also involve penetration. Viral growth rates are not influenced by cationic polymers. Polyanions reduce the infectivity of avian sarcoma viruses and are also able to neutralize the virus-enhancing activity of polycations. An exception is dextran sulfate which causes virus inhibition only at low concentrations (less than 4 μg/ml), but enhances viral infectivity at higher concentrations. Extracts from normal or leukosis virus-infected chick embryo fibroblasts also enhance focus formation by certain avian sarcoma viruses. The characteristics of this enhancement are similar to that mediated by polycations.

jun:Oncogene and Transcription Factor

Publisher Summary This chapter discusses the structure of the cellular and retroviral jun genes and their relationship to other transcriptional regulators. The interaction between the jun and fos proteins, and the control and oncogenic potential of jun are also presented. In the cell the fos protein is associated with a cellular protein, p 39. Together with p 39, the fos binds sequence specifically to DNA and can trans-activate genes. By itself, however, the fos protein does not show specific DNA binding. DNA affinity precipitation and gel retardation assays in conjunction with immunoblots of the bound proteins have shown that the target site for the fos – p 39 complex is the AP-1 consensus sequence. The p 39 precipitated from the cell with anti- jun sera and p 39 precipitated with anti- fos serum, as part of the fos – p 39 complex have identical tryptic peptide maps. Therefore, p 39 and jun are considered to be identical.

Jun, the oncoprotein

Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.

Small-molecule antagonists of Myc/Max dimerization inhibit Myc-induced transformation of chicken embryo fibroblasts

Myc is a transcriptional regulator of the basic helix–loop–helix leucine zipper protein family. It has strong oncogenic potential, mutated or virally transduced forms of Myc induce lymphoid tumors in animals, and deregulated expression of Myc is associated with numerous types of human cancers. For its oncogenic activity, Myc must dimerize with the ubiquitously expressed basic helix–loop–helix leucine zipper protein Max. This requirement for dimerization may allow control of Myc activity with small molecules that interfere with Myc/Max dimerization. We have measured Myc/Max dimerization with fluorescence resonance energy transfer and have screened combinatorial chemical libraries for inhibitors of dimerization. Candidate inhibitors were isolated from a peptidomimetics library. Inhibition of Myc/Max interaction was validated by ELISA and electrophoretic mobility-shift assay. Two of the candidate inhibitors also interfere with Myc-induced oncogenic transformation in chicken embryo fibroblast cultures. Our work provides proof of principle for the identification of small molecule inhibitors of protein–protein interactions by using high-throughput screens of combinatorial chemical libraries.

Triple layer control : Phosphorylation, acetylation and ubiquitination of FOXO proteins

FOXO proteins are transcriptional regulators that control cell cycle progression, DNA repair, apoptosis, and defense against oxidative damage. These divergent functions of FOXO proteins are regulated by signal-induced, post-translational modifications. Phosphorylation of cytoplasmic FOXO at specific sites by JNK initiates translocation into the nucleus. Acetylation and deacetylation of nuclear FOXO affects the selection of transcriptional programs that are controlled by FOXO proteins. Activation of Akt by growth factors results in phosphorylation of nuclear FOXO at specific sites followed by additional phosphorylations mediated by other kinases. Akt-dependent phosphorylation reduces the DNA-binding activity of FOXO, interferes with binding to the co-activators p300/CBP, and inactivates the FOXO nuclear translocation signal. The Akt-phosphorylated FOXO is exported from the nucleus in a CRM1- and 14-3-3-dependent process. Cytoplasmic, Akt-phosphorylated FOXO interacts with the ubiquitin ligase Skp2 and is targeted for proteasomal degradation. The nuclear-cytoplasmic “FOXO shuttle” is driven by stress signals that result in nuclear import and FOXO transcriptional activity and growth signals that initiate nuclear export and proteasomal degradation of FOXO.