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 p110 alpha, p110 beta, p110 gamma and p110 delta. These isoforms combine with several regulatory subunits; for p110 alpha, beta and delta, the standard regulatory subunit is p85, for p110 gamma, it is p101. PI3Ks play important roles in human cancer. PIK3CA, the gene encoding p110 alpha, 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 p110 alpha 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 p110 alpha. One of these mechanisms operates largely independently of binding to p85, the other abolishes the requirement for an interaction with Ras. The non-alpha isoforms of p110 do not show cancer-specific mutations. However, they are often differentially expressed in cancer and, in contrast to p110 alpha, wild-type non-alpha 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 p110 alpha constitute an important goal and challenge for current drug development.

Constitutively active Rheb induces oncogenic transformation.

Rheb (Ras-homolog enriched in brain) is a component of the phosphatidylinositol 3-kinase (PI3K) target of rapamycin (TOR) signaling pathway, functioning as a positive regulator of TOR. Constitutively active mutants of Rheb induce oncogenic transformation in cell culture. The transformed cells are larger and contain more protein than their normal counterparts. They show constitutive phosphorylation of the ribosomal protein S6 kinase and the eukaryotic initiation factor 4E-binding protein 1, two downstream targets of TOR. The TOR-specific inhibitor rapamycin strongly interferes with transformation induced by constitutively active Rheb, suggesting that TOR activity is essential for the oncogenic effects of mutant Rheb. Rheb-induced transformation is also dependent on a C-terminal farnesylation signal that mediates localization to a cellular membrane. An engineered N-terminal myristylation signal can substitute for the farnesylation. Immunofluorescence localizes wild-type and mutant Rheb to vesicular structures in the cytoplasm, overlapping with the endoplasmic reticulum.

Oncogenic signaling of class I PI3K isoforms.

The catalytic subunits of class I PI3Ks comprise four isoforms: p110alpha, p110beta, p110delta and p110gamma. Cancer-specific gain-of-function mutations in p110alpha have been identified in various malignancies. Cancer-specific mutations in the non-alpha isoforms of class I PI3K have not yet been identified, however overexpression of either wild-type p110beta, p110gamma or p110delta is sufficient to induce cellular transformation in chicken embryo fibroblasts. The mechanism whereby these non-alpha isoforms of class I mediate oncogenic signals is unknown. Here we show that potently transforming class I isoforms signal via Akt/mTOR, inhibit GSK3beta and cause degradation of FoxO1. A functional Erk pathway is required for p110gamma and p110beta transformation but not for transformation by p110delta or the H1047R mutant of p110alpha. Transformation and signaling by p110gamma and p110beta are sensitive to loss of interaction with Ras, which acts as a membrane anchor. Mutations in the C2 domain of p110delta reduce transformation, most likely by interfering with membrane association. Several small molecule inhibitors potently and specifically inhibit the oncogenic signaling and transformation of each of the class I PI3K, and, when used in combination with MEK inhibitors, can additively reduce the transformation induced by p110beta and p110gamma.

Phosphorylation by Akt disables the anti-oncogenic activity of YB-1.

The Y box-binding protein 1 (YB-1) is a DNA/RNA-binding protein that regulates mRNA transcription and translation. It is a major component of free messenger ribonucleoprotein particles and, at higher concentrations, blocks protein synthesis. In chicken embryo fibroblasts, overexpression of YB-1 confers a specific resistance to oncogenic cellular transformation by phosphoinositide 3-kinase (PI3K) or Akt/PKB. Recent studies have identified YB-1 as a direct substrate of Akt. The functional significance of Akt-mediated phosphorylation remains largely unknown. We generated YB-1 mutants in the Akt phosphorylation consensus sequence to explore the effect of phosphorylated YB-1 in PI3K-induced transformation. In contrast to wild-type YB-1, the phosphomimetic S99E mutant no longer interferes with cellular transformation. This mutant has reduced affinity for the cap of mRNAs and fails to inhibit cap-dependent translation. The data suggest that phosphorylation by Akt disables the inhibitory activity of YB-1 and thereby enhances the translation of transcripts that are necessary for oncogenesis. Overexpression of wild-type YB-1 overrides inactivation by Akt and maintains inhibition of protein synthesis and resistance to transformation.

A short N-terminal sequence of PTEN controls cytoplasmic localization and is required for suppression of cell growth.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an important negative regulator of cell growth and a tumor suppressor. Its growth-attenuating activity is based on the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PIP3), an essential second messenger for the phosphoinositide 3-kinase/Akt signaling pathway. This activity may require localization of PTEN to cytoplasmic membranes. Yet PTEN can also localize to the cell nucleus where its functions remain unclear. Here we present data that define a short sequence in the N-terminal region of PTEN required for cytoplasmic localization. We will refer to this sequence as cytoplasmic localization signal (CLS). It could function as a non-canonical signal for nuclear export or as a cytoplasmic retention signal of PTEN. Mutations within the CLS induce nuclear localization and impair growth suppressive activities of PTEN while preserving lipid phosphatase activity. We propose that nuclear localization of PTEN is not compatible with plasma membrane-targeted growth suppressive functions of PTEN.

Retroviral oncogenes and TOR.

Retroviruses have recruited the catalytic subunit of PI 3-kinase and its downstream target, Akt, as oncogenes. These viruses cause tumors in animals and induce oncogenic transformation in cell culture. The oncogenicity of these viruses is specifically inhibited by rapamycin; retroviruses carrying other oncogenes are insensitive to this macrolide antibiotic. Rapamycin is an inhibitor of the TOR (target of rapamycin) kinase whose downstream targets include p70 S6 kinase and the negative regulator of translation initiation 4E-BP. Emerging evidence suggests that the TOR signals transmitted to the translational machinery are essential for oncogenic transformation by the PI 3-kinase pathway.

The growth-promoting activity of the Bad protein in chicken embryo fibroblasts requires binding to protein 14-3-3.

Phosphorylation of the Bad protein is a key regulatory event in the prevention of apoptosis by survival factors. Phosphorylated Bad binds to the cytosolic 14-3-3 protein and is sequestered from the apoptotic machinery of the mitochondrial membrane. To examine the role of Bad in cell growth and apoptosis in primary cultures, we produced stable Bad transfectants of chicken embryo fibroblasts (CEF). As expected, serum starvation of Bad transfectants promoted apoptosis. However, Bad-transfected CEF maintained in media with a high serum concentration were capable of anchorage-independent growth and grew to a higher saturation density than control CEF transfected with the empty vector. High dilutions of the infectious retroviral vector RCAS expressing Bad led to the formation of multilayered cell foci. The growth-promoting effects of Bad were dependent on the serine 136 phosphorylation site and correlated directly with binding of Bad to 14-3-3. These results suggest that phosphorylated Bad promotes cell growth and in oncogenic transformation may contribute to the neoplastic phenotype of the cell.

Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway has inherent oncogenic potential. It is up-regulated in diverse human cancers by either a gain of function in PI3K itself or in its downstream target Akt or by a loss of function in the negative regulator PTEN. However, the complete consequences of this up-regulation are not known. Here we show that insulin and epidermal growth factor or an inactivating mutation in the tumor suppressor PTEN specifically increase the protein levels of hypoxia-inducible factor (HIF) 1alpha but not of HIF-1beta in human cancer cell lines. This specific elevation of HIF-1alpha protein expression requires PI3K signaling. In the prostate carcinoma-derived cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced expression of HIF-1alpha was inhibited by the PI3K-specific inhibitors LY294002 and wortmannin in a dose-dependent manner. HIF-1beta expression was not affected by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3 cell line specifically inhibited the expression of HIF-1alpha but not that of HIF-1beta. In contrast to the HIF-1alpha protein, the level of HIF-1alpha mRNA was not significantly affected by PI3K signaling. Vascular endothelial growth factor reporter gene activity was induced by insulin in PC-3 cells and was inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1 dominant negative construct. Vascular endothelial growth factor reporter gene activity was also inhibited by expression of a dominant negative PI3K construct and by the tumor suppressor PTEN.

Oncogenic transformation induced by membrane-targeted Akt2 and Akt3.

The kinases Akt2, Akt3 and their myristylated variants, Myr-Akt2 and Myr-Akt3 were expressed by the RCAS vector in chicken embryo fibroblasts (CEF). Myr-Akt2 and Myr-Akt3 were strongly oncogenic, inducing multilayered foci of transformed cells. In contrast, wild-type Akt2 and Akt3 were only poorly transforming, their efficiencies of focus formation were more than 100-fold lower; foci appeared later and showed less multilayering. Addition of the myristylation signal not only enhanced oncogenic potential but also increased kinase activities. Myr-Akt2 and Myr-Akt3 also induced hemangiosarcomas in the animal, whereas wild type Akt2 and Akt3 were not oncogenic in vivo. Furthermore, Akt2, driven by the lck (lymphocyte specific kinase) promoter in transgenic mice, induced lymphomas. The oncogenic effects of Akt2 and Akt3 described here are indistinguishable from those of Akt1. The downstream targets relevant to oncogenic transformation are therefore probably shared by the three Akt kinases.

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.

Expression of a down-regulated target, SSeCKS, reverses v-Jun-induced transformation of 10T1/2 murine fibroblasts.

Line 10T1/2 mouse fibroblast overexpressing the v-Jun oncoprotein were morphologically altered, grew into multilayered foci in culture and formed colonies when suspended in agar. The growth rate of the v-Jun-transformed 10T1/2 cells was not changed significantly from that of the untransformed parental cells, but the saturation density of the transformed cultures exceeded that of normal controls by a factor of 2. mRNA extracted from v-Jun-transformed 10T1/2 cells was analysed for differential gene expression with DNA micro-array technology. One of the targets downregulated by v-Jun was identified as SSeCKS (Src-suppressed C kinase substrate). Re-expression of SSeCKS in v-Jun-transformed fibroblasts reversed the transformed phenotype of the cells. Their ability to form foci was reduced to background levels, the number and size of agar colonies was lowered by a factor of 10 and the saturation density was significantly diminished. However, expression of SSeCKS had little effect on the morphology of v-Jun-transformed 10T1/2 cells. These data suggest that the SSeCKS protein has growth-attenuating properties. Down-regulation of SSeCKS may be essential for Jun-induced transformation.

A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt.

The oncoproteins P3k (homolog of the catalytic subunit of class IA phosphoinositide 3-kinase) and Akt (protein kinase B) induce oncogenic transformation of chicken embryo fibroblasts. The transformed cells show constitutive phosphorylation of the positive regulator of translation p70S6 kinase (S6K) and of the eukaryotic initiation factor 4E-BP1 binding protein (4E-BP1), a negative regulator of translation. Phosphorylation activates S6K and inactivates 4E-BP1. A mutant of Akt that retains kinase activity but does not induce phosphorylation of S6K or of 4E-BP1 fails to transform chicken embryo fibroblasts, suggesting a correlation between the oncogenicity of Akt and phosphorylation of S6K and 4E-BP1. The macrolide antibiotic rapamycin effectively blocks oncogenic transformation induced by either P3k or Akt but does not reduce the transforming activity of 11 other oncoproteins. Rapamycin inhibits the kinase mTOR, an important regulator of translation, and this inhibition requires binding of the antibiotic to the immunophilin FKBP12. Displacement of rapamycin from FKBP12 relieves the inhibition of mTOR and also restores P3k-induced transformation. These data are in accord with the hypothesis that transformation by P3k or Akt involves intervention in translational controls.

The oncogenic potential of the high mobility group box protein Sox3.

Sox proteins belong to the superfamily of high mobility group (HMG) proteins. Sox3 is expressed predominantly in the immature neuroepithelium. Ectopic expression of Sox3 causes oncogenic transformation of chicken embryo fibroblasts (CEFs). The oncogenicity of Sox3 is correlated with nuclear localization and transcriptional regulatory activity; mutants containing deletions in the HMG box or the transactivation domain fail to induce foci of transformation. These observations suggest that Sox proteins can induce aberrant cell growth and strengthen the link of HMG proteins to oncogenesis.

Oncogenic transformation by the FOX protein Qin requires DNA binding.

Some functions of the Qin oncoprotein are not dependent on DNA binding. In order to test the requirement for DNA binding in Qin-induced oncogenic transformation, site directed mutations were introduced in the winged helix (WH) DNA binding domain of the Qin protein. In cellular Qin (c-Qin), the glycine at position 233 was either deleted or substituted with the amino acids aspartic acid, alanine, glutamic acid, asparagine, proline or lysine. The same position carries aspartic acid in the viral Qin protein (v-Qin). The adjacent residues, threonine 232 and lysine 234, were separately mutated to proline. Several additional amino acid substitutions believed to be involved in DNA contacts were introduced at the following c-Qin positions: asparagine 189, histidine 193, serine 196 or arginine 236. Most of the substitutions reduced DNA binding of Qin, one mutation, H193A, completely abolished DNA binding, and another mutation, T232P, increased DNA binding affinity. Mutant H193A failed to transform chicken embryo fibroblasts (CEF), all other mutants, even those showing minimal DNA binding, retained oncogenicity for CEF. The efficiencies of focus formation induced by these mutant proteins in cell culture were not significantly different from that of wild type. However, the rate of focus development and the size of foci induced by the Qin mutants were greater with strong DNA binders than with weak DNA binders. Transdominant negative constructs consisting of the winged helix domain of cQin or v-Qin interfered with focus formation induced by full length Qin proteins. These results suggest that DNA binding is a prerequisite for transformation by Qin, and strong DNA binding is related to accelerated transformation in CEF.

The new serine-threonine kinase, Qik, is a target of the Qin oncogene.

The winged helix transcription factor Qin is the avian homolog of the mammalian brain factor 1 (BF-1) and has the potential to act as an oncogenic protein. We used representational difference analysis to identify genes that are differentially expressed in chicken embryo fibroblasts (CEF) transformed by Qin. One of the up-regulated Qin targets identified in this analysis is a serine-threonine kinase termed Qik (Qin-induced kinase). Qik belongs to the AMPK/SNF1 kinase family. It is a ubiquitously expressed protein and is upregulated rapidly after a hormone-regulated form of Qin is activated. In vitro kinase tests demonstrate that Qik is capable of autophosphorylation. Elevated levels of Qik transcripts are also observed in Src-transformed cells, suggesting that Src and Qin share some targets.

Identification and characterization of genes upregulated in cells transformed by v-Jun.

The transcription factor Jun (c-Jun) functions as a recipient of extracellular growth signals and converts them into patterns of gene expression. An oncogenic variant of c-Jun was isolated from the acutely transforming retrovirus ASV17. Overexpression of this viral Jun (v-Jun) induces transformation of chicken embryo fibroblasts (CEF) in culture and fibrosarcomas in chickens. v-Jun is a constitutively active form of c-Jun and transforms cells presumably by deregulating the expression of specific target genes. In this report, we describe six genes whose transcripts are upregulated in v-Jun-transformed CEF. Three of these genes show homology to known mammalian genes, to MAP kinase phosphatase 2 (MKP-2), to reversion-induced LIM protein (RIL) and to cytokine-inducible SH2-containing protein (CIS). Northern blot analysis, using CEF infected with various Jun mutants or an estrogen-regulatable Jun chimera, revealed distinct induction patterns of individual targets by v-Jun. The chicken RIL homolog showed an expression pattern tightly correlated with the activity of v-Jun. Its expression is also transformation-dependent, suggesting a role for this gene in v-Jun transformation. The newly identified v-Jun targets can serve as molecular markers in the v-Jun transformation process. Oncogene (2000) 19, 3537 - 3545

Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells.

Phosphatidylinositol 3-kinase (PI 3-kinase) is a signaling molecule that controls numerous cellular properties and activities. The oncogene v-p3k is a homolog of the gene coding for the catalytic subunit of PI 3-kinase, p110alpha. P3k induces transformation of cells in culture, formation of hemangiosarcomas in young chickens, and myogenic differentiation in myoblasts. Here, we describe a role of PI 3-kinase in angiogenesis. Overexpression of the v-P3k protein or of cellular PI 3-kinase equipped with a myristylation signal, Myr-P3k, can induce angiogenesis in the chorioallantoic membrane (CAM) of the chicken embryo. This process is characterized by extensive sprouting of new blood vessels and enlargement of preexisting vessels. Overexpression of the myristylated form of the PI 3-kinase target Akt, Myr-Akt, also induces angiogenesis. Overexpression of the tumor suppressor PTEN or of dominant-negative constructs of PI 3-kinase inhibits angiogenesis in the yolk sac of chicken embryos, suggesting that PI 3-kinase and Akt signaling is required for normal embryonal angiogenesis. The levels of mRNA for vascular endothelial growth factor (VEGF) are elevated in cells expressing activated PI 3-kinase or Myr-Akt. VEGF mRNA levels are also increased by insulin treatment through the PI 3-kinase-dependent pathway. VEGF mRNA levels are decreased in cells treated with the PI 3-kinase inhibitor LY294002 and restored by overexpression of v-P3k or Myr-Akt. Overexpression of VEGF by the RCAS vector induces angiogenesis in chicken embryos. These results suggest that PI 3-kinase plays an important role in angiogenesis and regulates VEGF expression.

v-Jun overrides the mitogen dependence of S-phase entry by deregulating retinoblastoma protein phosphorylation and E2F-pocket protein interactions as a consequence of enhanced cyclin E-cdk2 catalytic activity.

v-Jun accelerates G(1) progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G(1)/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic "clock" is reset. D-cyclin-cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16(INK4A) inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin-cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun-estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin-cdk, is likely to be the critical Rb kinase target of v-Jun.

The catalytic subunit of phosphoinositide 3-kinase: requirements for oncogenicity.

The retroviral oncogene p3k (v-p3k) of avian sarcoma virus 16 (ASV16) codes for the catalytic subunit of phosphoinositide (PI) 3-kinase, p110alpha. The v-P3k protein is oncogenic in vivo and in vitro; its cellular counterpart, c-P3k, lacks oncogenicity. Fusion of viral Gag sequences to the amino terminus of c-P3k activates the transforming potential. Activation can also be achieved by the addition of a myristylation signal to the amino terminus or of a farnesylation signal to the carboxyl terminus of c-P3k. A mutated myristylation signal was equally effective; it also caused a strong increase in the kinase activity of P3k. Mutations that inactivate lipid kinase activity abolish oncogenicity. The transforming activity of P3k is correlated with the ability to induce activating phosphorylation in Akt. Point mutations and amino-terminal deletions recorded in v-P3k were shown to be irrelevant to the activation of oncogenic potential. Interactions of P3k with the regulatory subunit of PI 3-kinase, p85, or with Ras are not required for transformation. These results support the conclusion that the oncogenicity of P3k depends on constitutive lipid kinase activity. Akt is an important and probably essential downstream component of the oncogenic signal from P3k.