PIK3CA mutation, but not PTEN loss of function, determines the sensitivity of breast cancer cells to mTOR inhibitory drugs.

The phosphatidylinositol 3-kinase (PI3K) pathway is commonly activated in breast cancers due to frequent mutations in PIK3CA, loss of expression of PTEN or over-expression of receptor tyrosine kinases. PI3K pathway activation leads to stimulation of the key growth and proliferation regulatory kinase mammalian target of rapamycin (mTOR), which can be inhibited by rapamycin analogues and by kinase inhibitors; the effectiveness of these drugs in breast cancer treatment is currently being tested in clinical trials. To identify the molecular determinants of response to inhibitors that target mTOR via different mechanisms in breast cancer cells, we investigated the effects of pharmacological inhibition of mTOR using the allosteric mTORC1 inhibitor everolimus and the active-site mTORC1/mTORC2 kinase inhibitor PP242 on a panel of 31 breast cancer cell lines. We demonstrate here that breast cancer cells harbouring PIK3CA mutations are selectively sensitive to mTOR allosteric and kinase inhibitors. However, cells with PTEN loss of function are not sensitive to these drugs, suggesting that the functional consequences of these two mechanisms of activation of the mTOR pathway are quite distinct. In addition, a subset of HER2-amplified cell lines showed increased sensitivity to PP242, but not to everolimus, irrespective of the PIK3CA/PTEN status. These selective sensitivities were confirmed in more physiologically relevant three-dimensional cell culture models. Our findings provide a rationale to guide selection of breast cancer patients who may benefit from mTOR inhibitor therapy and highlight the importance of accurately assessing the expression of PTEN protein and not just its mutational status.

Rictor is a novel target of p70 S6 kinase-1.

The rapamycin-insensitive companion of mammalian target of rapamycin (mTOR) (Rictor) is a key member of mTOR complex-2 (mTORC2), which phosphorylates the AGC kinases Akt/PKB, PKC and SGK1 at a C-terminal hydrophobic motif. We identified several novel sites on Rictor that are phosphorylated, including Thr1135, which is conserved across all vertebrates. Phosphorylation of this site on Rictor is stimulated by amino acids and growth factors through a rapamycin-sensitive signaling cascade. We demonstrate here that Rictor is a direct target of the ribosomal protein S6 kinase-1 (S6K1). Rictor phosphorylation at Thr1135 does not lead to major changes in mTORC2-kinase activity. However, phosphorylation of this site turns over rapidly and mediates 14-3-3 binding to Rictor and mTORC2, providing possibility for altered interactions of the complex. These findings reveal an unexpected signaling input into mTORC2, which is regulated by amino acids, growth factors and rapamycin.

Murine Ksr interacts with MEK and inhibits Ras-induced transformation.

BACKGROUND: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway. RESULTS: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK. CONCLUSIONS: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.

The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells.

P110delta, a novel phosphoinositide 3-kinase in leukocytes.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been implicated in signal transduction through tyrosine kinase- and heterotrimeric G-protein-linked receptors. We report herein the cloning and characterization of p110delta, a novel class I PI3K. Like p110alpha and p110beta, other class I PI3Ks, p110delta displays a broad phosphoinositide lipid substrate specificity and interacts with SH2/SH3 domain-containing p85 adaptor proteins and with GTP-bound Ras. In contrast to the widely distributed p110alpha and beta, p110delta is exclusively found in leukocytes. In these cells, p110alpha and delta both associate with the p85alpha and beta adaptor subunits and are similarly recruited to activated signaling complexes after treatment with the cytokines interleukin 3 and 4 and stem cell factor. Thus, these class I PI3Ks appear not to be distinguishable at the level of p85 adaptor selection or recruitment to activated receptor complexes. However, distinct biochemical and structural features of p110delta suggest divergent functional/regulatory capacities for this PI3K. Unlike p110alpha, p110delta does not phosphorylate p85 but instead harbors an intrinsic autophosphorylation capacity. In addition, the p110delta catalytic domain contains unique potential protein-protein interaction modules such as a Pro-rich region and a basic-region leucine-zipper (bZIP)-like domain. Possible selective functions of p110delta in white blood cells are discussed.

R-Ras can activate the phosphoinositide 3-kinase but not the MAP kinase arm of the Ras effector pathways.

BACKGROUND: The small GTPase R-Ras displays a less potent transforming activity than the closely related Ras oncogene products. Although R-Ras has been reported to interact with c-Raf1 and Ral-GDS in vitro, the pathways by which it exerts its effects on cellular proliferation are not known. RESULTS: Both Ras and R-Ras interact with phosphoinositide (PI) 3-kinase in vitro, and induce elevation of the levels of PI 3-kinase lipid products in intact cells. Unlike Ras, R-Ras does not activate Raf or mitogen-activated protein (MAP) kinase in cells. In co-transfection assays, the serine/threonine protein kinase PKB (or Akt) is effectively stimulated by R-Ras, Ras, mutants of Ras that activate PI 3-kinase but not other effectors, and activated forms of PI 3-kinase. Ras and R-Ras stimulate PKB/Akt through a non-autocrine mechanism that involves PI 3-kinase. The constitutive activation of PI 3-kinase alone is sufficient to activate PKB/Akt, but not the MAP kinase ERK or the stress-activated protein kinase, Jun N-terminal kinase. Transformation assays in fibroblasts suggest that PKB/Akt and Raf are part of distinct oncogenic signalling pathways. CONCLUSIONS: Both the Raf-MAP kinase and PI 3-kinase-PKB/Akt pathways are activated by Ras, but only the PI 3-kinase-PKB/Akt pathway is activated by R-Ras. PI 3-kinase, and downstream targets such as PKB/Akt, are likely to be essential mediators of transformation induced by R-Ras. PI 3-kinase, as well as Raf, is thus implicated also in Ras transformation.

Networks of interaction of p120cbl and p130cas with Crk and Grb2 adaptor proteins.

P120cbl, the product of the c-cbl proto-oncogene, has previously been shown to become tyrosine phosphorylated following EGF stimulation of cells, and to bind constitutively to the SH3 domain of the adaptor protein Grb2. Here we show that another adaptor protein, Crk, binds through its SH2 domain to tyrosine phosphorylated p120cbl. In addition, Crk becomes phosphorylated on tyrosine and serine following EGF treatment of PC12 and other cell lines. In unstimulated cells, while Grb2 is not bound to any tyrosine phosphoprotein, Crk is bound via its SH2 domain to tyrosine phosphorylated p130cas, the Crk-associated v-Src substrate. Following EGF treatment, Crk dissociates from p130cas, possibly due to a higher affinity of Crk SH2 for p120cbl compared with p130cas. Interaction between Grb2 and p120cbl increases threefold following EGF treatment of cells; in vitro, this induction of Grb2 association with unphosphorylated p120cbl can be mimicked by the addition of tyrosine phosphorylated Shc, suggesting a transfer of information between the SH2 and SH3 domains of Grb2. These data indicate that adaptor proteins can exchange binding partners in response to stimuli, and that different adaptor proteins can bind to the same partners by different mechanisms.

Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras-GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

Phosphatidylinositol 3' kinase: one of the effectors of Ras.

Ras proteins are proto-oncogene products that are critical components of signalling pathways leading from cell surface receptors to control of cellular proliferation, morphology and differentiation. the ability of Ras to activate the MAP kinase pathway through interaction with the serine/threonine kinase Raf is now well established. However, recent work has shown that Ras can also interact directly with the catalytic subunit of phosphatidylinositol 3' kinase and is involved in control of the lipid kinase in intact cells. A model is presented in which both tyrosine phosphoprotein interaction with the regulatory p85 subunit and Ras. GTP interaction with the catalytic p110 subunit is required to achieve optimal activation of phosphatidylinositol 3'kinase in response to extracellular stimuli. The ability of Ras to regulate phosphatidylinositol 3' kinase may be important both in Ras control of cellular morphology through the actin cytoskeleton and also in Ras control of DNA synthesis.

Downregulation of the Ras activation pathway by MAP kinase phosphorylation of Sos.

Formation of a complex of the nucleotide exchange factor Sos, the SH2 and SH3 containing adaptor protein Grb2/Sem-5 and tyrosine phosphorylated EGF receptor and Shc has been implicated in the activation of Ras by epidermal growth factor (EGF) in fibroblasts: related mechanisms for activation of Ras operate in other cell types. An increase in the apparent molecular weight of Sos has been reported to occur after several minutes of receptor stimulation due to phosphorylation by mitogen-activated protein (MAP) kinases. We report here that treatment of human peripheral blood T lymphoblasts with phorbol esters causes a similar shift in mobility of Sos. This modification of Sos does not alter its ability to bind Grb2, but correlates with strong inhibition of the binding of the Sos/Grb2 complex to tyrosine phosphorylated sequences, either a tyrosine phosphopeptide in cell lysates or p36 in intact cells. This effect, along with the mobility shift of Sos, can be mimicked in vitro by phosphorylation of Sos by the mitogen-activated protein kinase, ERK1. A novel negative feedback mechanism therefore exists whereby activation of MAP kinases through Ras results in the uncoupling of the Sos/Grb2 complex from tyrosine kinase substrates without blocking the interaction of Sos with Grb2.

Role of Shc in the activation of Ras in response to epidermal growth factor and nerve growth factor.

Treatment of the rat pheochromocytoma cell line PC12 with nerve growth factor (NGF) or epidermal growth factor (EGF) is known to result in activation of Ras. In response to EGF treatment, complexes form between Sos, Grb2 and tyrosine phosphorylated Shc and/or EGF receptor. In response to NGF treatment, complexes form between Sos, Grb2 and tyrosine phosphorylated Shc. While Shc is also found bound to the activated NGF receptor, Trk, no complexes were detectable that contained both Trk and Grb2 or Sos. In streptolysin O permeabilised cells, a tyrosine phosphopeptide, EGFR-Y1068P, which binds to the SH2 domain of Grb2, totally blocks growth factor induced formation of complexes between Grb2 and Shc or EGF receptor, and also blocks activation of nucleotide exchange on Ras. At low concentrations, another tyrosine phosphopeptide, TRK-Y490P, which binds to the SH2 domain of Shc, blocks growth factor induced formation of complexes between Shc and the EGF receptor or Trk, but fails to block activation of nucleotide exchange on Ras. Higher concentrations of TRK-Y490P inhibit tyrosine phosphorylation of Shc and the formation of Shc complexes with Grb2: this results in strong inhibition of Ras activation by NGF and partial inhibition of Ras activation by EGF. These data demonstrate that the formation of a trimeric complex between tyrosine phosphorylated Shc, Grb2 and Sos is the key event in the activation of Ras in response to NGF. The binding of Sos to tyrosine phosphorylated receptor, via Grb2 may also contribute to Ras activation by EGF but not NGF, while stable complex formation between Shc and receptors is not necessary for Ras activation by either growth factor.

Phosphatidylinositol-3-OH kinase as a direct target of Ras.

Ras (p21ras) interacts directly with the catalytic subunit of phosphatidylinositol-3-OH kinase in a GTP-dependent manner through the Ras effector site. In vivo, dominant negative Ras mutant N17 inhibits growth factor induced production of 3' phosphorylated phosphoinositides in PC12 cells, and transfection of Ras, but not Raf, into COS cells results in a large elevation in the level of these lipids. Therefore Ras can probably regulate phosphatidylinositol-3-OH kinase, providing a point of divergence in signalling pathways downstream of Ras.

Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.

The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

The growth factor IL-2 activates p21ras proteins in normal human T lymphocytes.

The T cell growth factor IL-2 induces T cell progression through the cell cycle and ultimately controls T cell mitosis. Here we show that the guanine nucleotide-binding proteins p21ras may be involved in IL-2 signal transduction pathways. IL-2 causes a rapid and prolonged activation of p21ras in both murine and human T cells. The concentration-dependence of IL-2-mediated stimulation of p21ras correlated with IL-2 stimulation of T cell proliferation, which indicates that p21ras activity can be controlled by signals generated via the interaction between IL-2 and its high affinity cellular receptor. These results suggest that p21ras may play a role in the regulation of T cell growth by IL-2.

Stimulation of p21ras upon T-cell activation.

External signals that control the activity of proteins encoded by the ras proto-oncogenes have not previously been characterized. It is now shown that stimulation of the antigen receptor of T lymphocytes causes a rapid activation of p21ras. The mechanism seems to involve a decrease in the activity of GAP, the GTPase-activating protein, on stimulation of protein kinase C. In lymphocytes, p21ras may therefore be an important mediator of the action of protein kinase C.

Production of monoclonal antibodies to placental alkaline phosphatase: preliminary characterisation includes identification of one antibody reactive with routinely fixed histological preparations.

We describe the production and preliminary characterisation of a set of monoclonal antibodies (MAbs) raised against placental alkaline phosphatase (PLAP). Different forms of antigen presentation, PLAP or PLAP-like expressing whole cells, placental membranes or purified PLAP were used to immunise BALB/c mice. Initial screening was carried out against the immunising material by ELISA, against fresh frozen placental sections by immunostaining and against purified PLAP using an enzyme capture assay. The most successful fusions were those following whole cell immunisation, producing 27 antibodies that all reacted with both the placental and testicular form of enzyme. These all showed a broadly similar pattern of reactivity when tested against a range of human malignant cell lines. Further characterisation identified one antibody, 8B6, as strongly reactive with formalin-fixed paraffin-embedded placental sections. This antibody also performed well when tested against a range of normal and malignant routinely fixed tissue sections. Of 14 antibodies analysed for immunoglobulin isotype, 10 were of the IgGI subclass. In competitive binding studies with 7 antibodies to discriminate epitopes, at least 4 distinct binding sites were identified. By Scatchard analysis on 4 of these antibodies, binding constants of 3 were within the range 3.5-5.3 x 10(-9)M. Unusually the 4th antibody appeared to recognise 2 separate antigen sites with binding constants of 2.1 and 7.5 x 10(-9)M. In a preliminary study to compare patterns of reactivity of a selection of the new antibodies with a limited number of sera from smokers and seminoma patients, results indicate their potential for further typing within the placental group of enzymes.

Placental-type alkaline phosphatase in cervical neoplasia.

Monoclonal antibodies reactive with placental-type alkaline phosphatase have formed the basis of methods for detection of this oncodevelopmental antigen in patients with pre-invasive and invasive cervical neoplasia, with or without evidence of papilloma virus infection. Disease-related elevations of placental-type alkaline phosphatase were not observed in patients' sera. Solubilised cervical smears or biopsy material, and cervical mucus swabs, often contained substantial amounts of this isoenzyme; however, there was no significant difference between any of the patient and control groups. Thus, serological and smear test assays for placental-type alkaline phosphatase were not useful in differential diagnosis of cervical lesions. However, its presence in most biopsy specimens, often at high levels, indicated possible application for in vivo radioimmunoimaging studies of invasive or metastatic cervical cancer.

Adoptive T cell immunotherapy of MSV-induced tumours in nude mice. Part I. Biology of tumour regression and recurrence.

The induction of immunity to progressively growing murine sarcoma virus (MSV) tumours in nude (nu/nu) mice by reconstitution with immune T cells from syngeneic (+/+) donors has been studied. Whole spleen cell preparations served as the source of immune T cells. Transfer of immune, but not of normal, spleen cells resulted in partial or apparently complete regression of primary tumours and a related moderate to considerable extension of survival time. The dose, the time in days between immunization and transfer, as well as timing of the spleen cells in relation to tumour cell challenge, were all factors which influenced the effectiveness of the protective inocula. An unexpected consequence of even the very effective primary immunotherapy regimens, was secondary tumour development after varying tumour-free intervals. This was most frequently manifest as tumour recurrences at the original injection site either on their own or in combination with distant metastases. Such a relatively high frequency of tumour reappearance and metastatic spread contrasts markedly with the rare instances of secondary regrowth in normal immunocompetent mice. The present reconstitution system may therefore provide a new model for studying the inhibitory or stimulatory properties of T cells with respect to tumour regression and dissemination.

Adoptive T cell immunotherapy of MSV-induced tumours in nude mice. Part II. Sequential analysis of serum immune complexes and blocking activity in reconstituted mice in relation to tumour biology.

In seven separate experiments, nude (nu/nu) mice carrying established murine sarcoma virus (MSV) tumours were reconstituted with syngeneic (+/+) immune splenic T cells. These immune protected mice were randomly divided to provide smaller groups for serial exsanguination. At various time points mice were individually bled and CIC concentration and blocking activity of each individual serum was determined. Control sera were obtained from nu/nu and adult +/+ mice inoculated with tumour cells only, and from nu/nu mice protected with normal +/+ spleen cells. In all the mice studied, CIC and blocking appeared to be mutually independent parameters throughout the MSV tumour course. On the other hand, in immune protected mice considered alone or together with the control groups, CIC and time after tumour cell inoculation, but not tumour size, were significantly correlated. A significant relationship between blocking and tumour size was also established, although this only applied to immune protected mice. However, analysis of the combined data from sequentially bled immune protected mice in relation to different phases of tumour behaviour, did not support the notion that blocking, and more particularly the persistence of CIC, contribute to tumour regrowth and dissemination.