Alfonso Bellacosa

Epithelial–mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/AKT pathways

Epithelial–mesenchymal transition (EMT) is an important process during development by which epithelial cells acquire mesenchymal, fibroblast-like properties and show reduced intercellular adhesion and increased motility. Accumulating evidence points to a critical role of EMT-like events during tumor progression and malignant transformation, endowing the incipient cancer cell with invasive and metastatic properties. Several oncogenic pathways (peptide growth factors, Src, Ras, Ets, integrin, Wnt/β-catenin and Notch) induce EMT and a critical molecular event is the downregulation of the cell adhesion molecule E-cadherin. Recently, activation of the phosphatidylinositol 3′ kinase (PI3K)/AKT axis is emerging as a central feature of EMT. In this review, we discuss the role of PI3K/AKT pathways in EMT during development and cancer with a focus on E-cadherin regulation. Interactions between PI3K/AKT and other EMT-inducing pathways are presented, along with a discussion of the therapeutic implications of modulating EMT in order to achieve cancer control.

AKT plays a central role in tumorigenesis

AKT is emerging as a central player in tumorigenesis. In this issue of PNAS, Mayo and Donner (1) report on yet another function of AKT, involving regulation of the Mdm2/p53 pathway. The first evidence pointing to a role of AKT in oncogenesis was provided by early studies of transforming viruses. A novel retrovirus, isolated from an AKR mouse T cell lymphoma (2), harbored transduced sequences of cellular origin (3). In 1991, our collaborative studies with Philip Tsichlis and Stephen Staal resulted in the cloning of the viral oncogene v- akt (4). The predicted oncoprotein contained viral Gag sequences fused to a kinase related to protein kinase C. The oncogenic potential of v-akt arises from the creation of a myristylation site at the amino terminus and consequent constitutive kinase activity (5). By different approaches, aimed at identifying novel protein kinases, two other groups independently cloned the identical cellular sequence at about the same time (6, 7). AKT is now known to define a family of closely related, highly conserved cellular homologues (reviewed in ref. 8). In human, these are designated AKT1 , AKT2 , and AKT3 , located at chromosomes 14q32, 19q13, and 1q44, respectively (reviewed in ref. 9). The encoded proteins are serine/threonine kinases belonging to the protein kinase B (PKB) family, and the AKT1, AKT2, and AKT3 proteins are also known as PKBα, PKBβ, and PKBγ, respectively. Each AKT family member contains an amino-terminal pleckstrin homology (PH) domain, a short α-helical linker, and a carboxyl-terminal kinase domain (8). PH domains exist in diverse signaling molecules and permit anchorage of proteins to the cell membrane via phospholipid interactions (10). The degree of functional redundancy between AKT1, AKT2, and AKT3 is currently unclear. Although each kinase responds similarly to various stimuli, their different tissue-specific expression patterns suggest distinct roles, …

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer.

The cyclin-dependent kinase inhibitor p27kip1 is a putative tumor suppressor for human cancer. The mechanism underlying p27kip1 deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27kip1-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27kip1, is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27kip1 in the cytoplasm, precluding p27kip1-induced G1 arrest. Conversely, the p27kip1-T157A mutant accumulates in cell nuclei and Akt does not affect p27kip1–T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27kip1 accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27kip1, secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27kip1 are functionally inactivated and the proliferation of breast cancer cells is sustained.

Transformation of hematopoietic cells by BCR/ABL requires activation of a PI-3k/Akt-dependent pathway

The BCR/ABL oncogenic tyrosine kinase activates phosphatidylinositol 3‐kinase (PI‐3k) by a mechanism that requires binding of BCR/ABL to p85, the regulatory subunit of PI‐3k, and an intact BCR/ABL SH2 domain. SH2 domain BCR/ABL mutants deficient in PI‐3k activation failed to stimulate Akt kinase, a recently identified PI‐3k downstream effector with oncogenic potential, but did activate p21 RAS and p70 S6 kinase. The PI‐3k/Akt pathway is essential for BCR/ABL leukemogenesis as indicated by experiments demonstrating that wortmannin, a PI‐3k specific inhibitor at low concentrations, suppressed BCR/ABL‐dependent colony formation of murine marrow cells, and that a kinase‐deficient Akt mutant with dominant‐negative activity inhibited BCR/ABL‐dependent transformation of murine bone marrow cells in vitro and suppressed leukemia development in SCID mice. In complementation assays using mouse marrow progenitor cells, the ability of transformation‐defective SH2 domain BCR/ABL mutants to induce growth factor‐independent colony formation and leukemia in SCID mice was markedly enhanced by expression of constitutively active Akt. In retrovirally infected mouse marrow cells, the BCR/ABL mutant lacking the SH2 domain was unable to upregulate the expression of c‐Myc and Bcl‐2; in contrast, expression of a constitutively active Akt mutant induced Bcl‐2 and c‐Myc expression, and stimulated the transcription activation function of c‐Myc. Together, these data demonstrate the requirement for the BCR/ABL SH2 domain in PI‐3k activation and document the essential role of the PI‐3k/Akt pathway in BCR/ABL leukemogenesis.

Thymine DNA Glycosylase Is Essential for Active DNA Demethylation by Linked Deamination-Base Excision Repair

DNA methylation is a major epigenetic mechanism for gene silencing. Whereas methyltransferases mediate cytosine methylation, it is less clear how unmethylated regions in mammalian genomes are protected from de novo methylation and whether an active demethylating activity is involved. Here, we show that either knockout or catalytic inactivation of the DNA repair enzyme thymine DNA glycosylase (TDG) leads to embryonic lethality in mice. TDG is necessary for recruiting p300 to retinoic acid (RA)-regulated promoters, protection of CpG islands from hypermethylation, and active demethylation of tissue-specific developmentally and hormonally regulated promoters and enhancers. TDG interacts with the deaminase AID and the damage response protein GADD45a. These findings highlight a dual role for TDG in promoting proper epigenetic states during development and suggest a two-step mechanism for DNA demethylation in mammals, whereby 5-methylcytosine and 5-hydroxymethylcytosine are first deaminated by AID to thymine and 5-hydroxymethyluracil, respectively, followed by TDG-mediated thymine and 5-hydroxymethyluracil excision repair.

Activation of AKT Kinases in Cancer: Implications for Therapeutic Targeting

The AKT1, AKT2, and AKT3 kinases have emerged as critical mediators of signal transduction pathways downstream of activated tyrosine kinases and phosphatidylinositol 3-kinase. An ever-increasing list of AKT substrates has precisely defined the multiple functions of this kinase family in normal physiology and disease states. Cellular processes regulated by AKT include cell proliferation and survival, cell size and response to nutrient availability, intermediary metabolism, angiogenesis, and tissue invasion. All these processes represent hallmarks of cancer, and a burgeoning literature has defined the importance of AKT alterations in human cancer and experimental models of tumorigenesis, continuing the legacy represented by the original identification of v-Akt as the transforming oncogene of a murine retrovirus. Many oncoproteins and tumor suppressors intersect in the AKT pathway, finely regulating cellular functions at the interface of signal transduction and classical metabolic regulation. This careful balance is altered in human cancer by a variety of activating and inactivating mechanisms that target both AKT and interrelated proteins. Reprogramming of this altered circuitry by pharmacologic modulation of the AKT pathway represents a powerful strategy for rational cancer therapy. In this review, we summarize a large body of data, from many types of cancer, indicating that AKT activation is one of the most common molecular alterations in human malignancy. We also review mechanisms of activation of AKT kinases, examples of therapeutic modulation of the AKT pathway in animal models, and the current status of efforts to target molecular components of the AKT pathway for cancer therapy and, possibly, cancer prevention.

Cyclin D expression is controlled post-transcriptionally via a phosphatidylinositol 3-kinase/Akt-dependent pathway.

Cyclin D expression is regulated by growth factors and is necessary for the induction of mitogenesis. Herbimycin A, a drug that binds to Hsp90, induces the destruction of tyrosine kinases and causes the down-regulation of cyclin D and an Rb-dependent growth arrest in the G1phase of the cell cycle. We find that the induction of D-cyclin expression by serum and its repression by herbimycin A are regulated at the level of mRNA translation. Induction of cyclin D by serum occurs prior to the induction of its mRNA and does not require transcription. Herbimycin A repression is characterized by a decrease in the synthetic rate of D-cyclins prior to changes in mRNA expression and in the absence of changes in the half-life of the protein. This effect on D-cyclin translation is mediated via a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. PI 3-kinase inhibitors such as wortmannin and LY294002, and rapamycin, an inhibitor of FRAP/TOR, cause a decline in the level of D-cyclins, whereas inhibitors of mitogen-activated protein kinase kinase and farnesyltransferase do not. Cells expressing the activated, myristoylated form of Akt kinase, a target of PI 3-kinase, are refractory to the effects of herbimycin A or serum starvation on D-cyclin expression. These data suggest that serum induction of cyclin D expression results from enhanced translation of its mRNA and that this results from activation of a pathway that is dependent upon PI 3-kinase and Akt kinase.

Akt activation by growth factors is a multiple-step process: the role of the PH domain

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of β-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat α-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dtmax) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dtmin, was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, β-adrenergic receptor (β-AR) density, and β-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-β is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-β/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting β-AR signaling capacity.

Akt Is a Direct Target of the Phosphatidylinositol 3-Kinase ACTIVATION BY GROWTH FACTORS, v-src and v-Ha-ras, IN Sf9 AND MAMMALIAN CELLS

The Akt protooncogene encodes a serine-threonine protein kinase which is activated by growth factor-generated signals that are transduced via the phosphatidylinositol 3′-kinase (PI3-K). Earlier studies suggested that the activation of Akt by PI3-K may be mediated by the binding of D3-phosphorylated phosphoinositides to the Akt pleckstrin homology (PH) domain. On the basis of these studies, it was hypothesized that Akt is a direct PI3-K target. To test this hypothesis, we reconstituted the pathway of Akt activation in baculovirus-infected Sf9 cells. The results showed that Akt, which is normally catalytically inactive in these cells, was activated when coexpressed with the activated PI3-K. Moreover, they showed that activated forms of c-Ha-ras (v-Ha-ras) and c-src (v-src or srcY527F), two molecules that transduce growth factor-generated signals, also activate Akt in a PI3-K-dependent manner in Sf9 as well as NIH 3T3 cells. The activation of Akt by both growth factors and v-ras and v-src (or srcY527F) depends on the integrity of the Akt PH domain and carboxyl-terminal tail. These results show that Akt activation via the PI3-K can be faithfully reproduced in baculovirus-infected Sf9 cells. The same results support the hypothesis that Akt is a direct target of the PI3-K and identify cytoplasmic signaling molecules that may contribute to the transduction of PI3-K/Akt activation signals.