6 Corresponding author p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

To investigate the role of signaling by the small GTPase Ral, we have generated mice deficient for RalGDS, a guanine nucleotide exchange factor that activates Ral. We show that RalGDS is dispensable for mouse development but plays a substantial role in Ras-induced oncogenesis. Lack of RalGDS results in reduced tumor incidence, size, and progression to malignancy in multistage skin carcinogenesis, and reduced transformation by Ras in tissue culture. RalGDS does not appear to participate in the regulation of cell proliferation, but instead controls survival of transformed cells. Experiments performed in cells isolated from skin tumors suggest that RalGDS mediates cell survival through the activation of the JNK/SAPK pathway. These studies identify RalGDS as a key component in Ras-dependent carcinogenesis in vivo.

SummaryPoly-and oligodonal T cell stimuli like anti-CD3e monoclonal antibody or Staphylococcus aureus enterotoxin B (SEB), injected at doses that per se are not lethal, provoke acute death within less than 24 h, provided that endogenous glucocorticoids (GC) are depleted by adrenalectomy or by injection of saturating amounts of the GC receptor antagonist RU-38486 (mifepristone). Pharmacological doses of the GC agonist dexamethasone (DEX) alter the in vivo response of splenic V38 + T cells to SEB, thus impeding the expansion of such cells and causing their rapid (3 d) clonal deletion. In contrast, coadministration of RU-38486 counteracts a SEB-induced early (12 h) reduction of V38+CD4 + and V38+CD8 + spleen cells. In vivo T cell stimulation by injection of bacterial superantigen induces a rapid (peak at 90-120 rain) increase in corticosterone serum levels, suggesting that endogenous GC might control early T cell activation. Accordingly, kinetic studies revealed that RU-38486 has to be administered within 2 h after superantigen administration to exert its lethal effect. Similarly, exogenous GC must be injected during this critical phase (2 h) to rescue animals from acute death induced by coinjection of SEB and D-galactosamine (GAIN). Adrenalectomy, injection of RU-38486 and priming with GaIN per se provoke the programmed death of peripheral CD4 + and CD8 + T cells. Thus, three manipulations that sensitize mice for the lethal effect of T cell stimulation also exert a proapoptotic effect on peripheral T cells. In synthesis, endogenous and exogenous GC regulate T cell responses and determine the propensity of peripheral T cells to undergo apoptosis. SYnthetic glucocorticoid (GC) 1 agonists are used for therapy of a broad spectrum of organ-specific and generalized autoimmune diseases. In the same way, endogenous GC secreted by the adrenal glands may act as immunosuppressive and antiinflammatory agents that contend lifethreatening overreactions of the immune system, as well as autoaggressive responses (for reviews see references 1, 2). GC inhibit macrophage functions, including cytotoxic functions, processing, and presentation of antigen to T cells, inhibit the production of IL-2 and IFN-3, in T lymphocytes (3), shift T cell responses from the Thl to the Th2 type (4), decrease the activity of NK cells, induce programmed cell death in a variety of different immunologically relevant cells, including immature T and B cell precursors (for reviews see references 5, 6) and mature T cells (7), and inhibit the synthesis of a variety of proimflammatory cytokines (IL-1, IL-6, and TNF-o 0 (8). Immune activation frequently is associated with an in- crease in ACTH and GC secretion, and the susceptibility of certain animal strains to develop autoimmune diseases is linked to a deficient GC-mediated inhibition of immune function (2, 9-12).Recently, a synthetic steroid, KU-38486 (RU486, mifepristone), with potent antiprogestational and antiglucocorticoid activity has become available for clinical use as an abortifacient agent (13,14) an...

PIK3R2 encodes a ubiquitous regulatory subunit (p85β) of PI3K, an enzyme that generates 3-polyphosphoinositides at the plasma membrane. PI3K activation triggers cell survival and migration. We found that p85β expression is elevated in breast and colon carcinomas and that its increased expression correlates with PI3K pathway activation and tumor progression. p85β expression induced moderate PIP 3 generation at the cell membrane and enhanced cell invasion. In accordance, genetic alteration of pik3r2 expression levels modulated tumor progression in vivo. Increased p85β expression thus represents a cellular strategy in cancer progression.A ctivation of class I PI3K is involved in the pathogenesis of cancer. PI3Ks are lipid kinases that phosphorylate membrane phosphoinositides [i.e., phosphatidylinositol (PtdIns)] to generate PtdIns(3,4)P 2 (PIP 2 ) and PtdIns(3,4,5)P 3 (PIP 3 ). PI3K is composed of a regulatory and a p110 catalytic subunit. Four genes encode the highly conserved p110 catalytic subunit (PIK3CA, CB, CD, and CG). p110α, β, and δ associate with p85 regulatory subunits and are activated mainly by growth factor receptors; p110γ associates with distinct regulatory subunits and is activated preferentially by G protein-coupled receptors (1-3). Three genes encode p85-type regulatory subunits: PIK3R1 (p85α, p55α, p50α), PIK3R2 (p85β), and PIK3R3 (p55γ). R1 and R2 are ubiquitously expressed and R3 expression is tissue-restricted (4).p85β is expressed at lower levels than p85α in most tissues (5-7). Whereas mice deficient in Pik3r2 develop normally and exhibit only moderate metabolic and immunological defects (7) Pik3r1 −/− mice die perinatally (8). p85α controls p110 stability and blocks p110 activity during quiescence (9). The inhibitory role of p85α on p110 activity explains why WT PIK3R1 expression is normally reduced in tumors, and that p85α mutations that relieve p110 from p85 inhibition have been found in cancer (10). Despite extensive analysis of p85α mutations in tumors (10-12), p85β involvement in cancer is less well studied. Here we analyzed the potential contribution of p85β in cancer.Results p85β Expression Is Increased in Breast and Colon Carcinomas. By using microarray technology, we performed a preliminary survey of the expression of the genes that form part of the PI3K pathway in a collection of clinical breast (n = 14) and colon carcinomas (n = 12). Comparison of PI3K subunit expression showed that mRNA levels of PIK3R2 (which encodes p85β) were increased in nearly half the carcinoma samples examined, whereas PIK3R1 (which encodes p85α) was decreased (Fig. S1). To study this finding in more detail, we compared 20 colon adenocarcinomas (CCs) and 35 breast carcinomas (BCs) with normal surrounding tissue. Tumor and normal samples had comparable numbers of epithelial cells, and normal tissue had a low percentage of malignant cells (0-10%).To evaluate p85β expression levels, we prepared extracts from normal and tumor samples and analyzed p85 levels by Western blot (WB). We generated anti-p85β Abs an...

Dysregulation of the PI3K/PTEN pathway is a frequent event in cancer, and PIK3CA and PTEN are the most commonly mutated genes after TP53. PIK3R1 is the predominant regulatory isoform of PI3K. PIK3R2 is an ubiquitous isoform that has been so far overlooked, but data from The Cancer Genome Atlas shows that increased expression of PIK3R2 is also frequent in cancer. In contrast to PIK3R1, which is a tumor-suppressor gene, PIK3R2 is an oncogene. We review here the opposing roles of PIK3R1 and PIK3R2 in cancer, the regulatory mechanisms that control PIK3R2 expression, and emerging therapeutic approaches targeting PIK3R2.Antagonistic Roles of PIK3R1/p85a and PIK3R2/p85b in Cancer PI3K enzymes are a conserved family of lipid kinases that phosphorylate the inositol 3 0 -OH groups of membrane phosphoinositides (PI). Class I PI3K enzymes convert PI(4,5)bisphosphate (PIP 2 ) into PI(3,4,5)trisphosphate (PIP 3 ), a second messenger. Class IA PI3K is composed of a heterodimer between a p110 catalytic subunit and a p85 regulatory subunit. Of the four PI3K catalytic subunit isoforms (PI3Ka, PI3Kb, PI3Kg, and PI3Kd), only PI3Ka and PI3Kb are expressed ubiquitously and are frequently altered in cancer [1,2]. Three different genes encode p85-type subunits: PIK3R1, PIK3R2, and PIK3R3, and these code for p85a (and alternative splice forms), p85b and p55g, respectively. PIK3R1 and PIK3R2 are broadly expressed, whereas PIK3R3 is selectively expressed in adult testis and the brain [1][2][3][4].PIK3R1/p85a is the most abundant isoform in normal tissues [5][6][7] but its expression is reduced in cancer. Conversely, PIK3R2/p85b expression levels are elevated in advanced cancer stages ([7,8]; data from The Cancer Genome Atlas, TCGA i ). Physiological activation of PI3K is induced by binding of p85 to activated receptor tyrosine kinases (RTKs) and is further enhanced by GTPases of the Ras family. Rho GTPases and G protein-coupled receptors also activate PI3Kb [1,2,9,10]. Classically, p85a and p85b have been considered to be similar proteins that associate with RTKs and with catalytic subunits that induce PI3K activation. This may represent an oversimplification because a growing number of studies support different and opposite functions of p85a and p85b in cancer [7,8]. In this review article we summarize available data on the function p85a and p85b, and attempt to make sense of the observation that PIK3R1 and PIK3R2 have opposing functions in tumor progression. This is important because distinct consequences of interfering with PIK3R1/p85a or PIK3R2/p85b should be considered in the design and development on new therapies. Domains, Localization, and Activities of PIK3R1 and PIK3R2p85 subunits control PI3K activation by modulating the stability, conformation, and localization of the catalytic subunit [4,11,12]. The primary structure of p85 includes an N-terminal region composed of an Src homology 3 (SH3) domain followed by a RhoGap HighlightsThe Cancer Genome Atlas Project revealed that specific changes in gene expression are hallmarks of som...

Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G 0 /G 1 transition) and again in late G 1 . The two ubiquitous PI3K isoforms (p110␣ and p110␤) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110␣ was activated first in G 0 /G 1 , followed by a minor p110␤ activity peak. In late G 1 , p110␣ activation preceded that of p110␤, which showed the maximum activity at this time. p110␤ activation required Ras activity, whereas p110␣ was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110␣ and -␤ activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110␣ in blocking early G 1 events. We show that inhibition of either p110␣ or p110␤ reduced cell cycle entry. These results reveal that PI3K␣ and -␤ present distinct activation requirements and kinetics in G 1 phase, with a selective action of PI3K␣ at the G 0 /G 1 phase transition. Nevertheless, PI3K␣ and -␤ both regulate S-phase entry.

Intravenous injections of 50 micrograms Staphylococcus aureus enterotoxin B (SEB) or bacterial lipopolysaccharide (LPS) are lethal, provided that mice are simultaneously sensitized with either N-galactosamine (GalN) or the anti-glucocorticoid RU-38486. Similar to the synthetic glucocorticoid (GC) receptor agonist dexamethasone, pharmacological doses of the immunomodulator linomide (quinoline-3-carboxamide) prevent death in all four models of lethal septic shock (LPS + GalN, LPS + RU-38486, SEB + GalN, and SEB + RU-38486) and inhibit the secretion of tumor necrosis factor, one of the major intermediate effector molecules of SEB and LPS toxicity. In this system, cyclosporine A (CsA), although effective in suppressing SEB toxicity, fails to counteract the lethal effect of LPS. This observation, together with the fact that linomide acts in the presence of excess amounts of GC receptor antagonist, indicates that linomide functions in a different way to that of known immunosuppressive agents like CsA and GC.

Peripheral blood T lymphocytes require two signals to enter and progress along the cell cycle from their natural quiescent state. The first activation signal is provided by the stimulation through the T cell receptor, which induces the synthesis of cyclins and the expression of the high affinity interleukin-2 receptor. The second signal, required to enter the S phase, is generated upon binding of interleukin-2 to the high affinity ␣␤␥ interleukin-2 receptor. However, resting T cells already express intermediate affinity ␤␥ interleukin-2 receptors. As shown here, T cell stimulation through intermediate affinity receptors is capable of inducing cell rescue from the apoptosis suffered in the absence of stimulation. Characterization of the signaling pathways utilized by ␤␥ interleukin-2 receptors in resting T cells, indicated that pp56 lck , but not Jak1 or Jak3, is activated upon receptor triggering. Compelling evidence is presented indicating that phosphatidylinositol 3-kinase associates with the intermediate affinity interleukin-2 receptor and is activated upon interleukin-2 addition. Bcl-x L gene was also found to be induced upon ␤␥ interleukin-2 receptor stimulation. Finally, pharmacological inhibition of phosphatidylinositol 3-kinase blocked both interleukin-2-mediated bcl-x L induction and cell survival. We conclude that ␤␥ interleukin-2 receptor mediates T-cell survival via a phosphatidylinositol 3-kinasedependent pathway, possibly involving pp56 lck and bcl-x L as upstream and downstream effectors, respectively.