Role of the PTEN protein in the multidrug resistance of prostate cancer cells

At present, there is no doubt that the signal transduction pathway P13K/Akt/PTEN/mTOR, controlled by phosphatidylinositol-3-kinase, is involved in tumor cell resistance to a number of drugs. Another well-known mechanism determining drug resistance in tumors is associated with the activity of drug transporters of the ABC superfamily (first of all, P-glycoprotein (Pgp), MRP1, BCRP, and LRP). Several mechanisms of cell defense can simultaneously operate in one cell. The interplay of different mechanisms involved in drug resistance is poorly understood. The PC3 and DU145 human prostate cell lines were used to show that the PTEN functional status determined the cell resistance to some drugs and that correlated with the levels of MRP1 and BCRP. Pgp was not involved in drug resistance of these cells. Introduction of PTEN into PTEN-deficient PC3 cells, as well as rapamycin treatment, inhibited Akt and mTOR and sensitized cells to doxorubicin and vinblastine. Exogenous PTEN altered the MRP1 and BCRP expression. The results indicate that at least two mechanisms of drug resistance operate in prostate cancer cells: the PI3K/Akt/PTEN/mTOR pathway and an elevated MRP1 expression. The mechanisms are interconnected: PTEN and mTOR signaling is involved in MRP1 and BCRP expression regulation.     

Participation of mTOR in the regulation of multidrug resistance of tumor cells

Molecular mechanisms of the influence of PI3K/Akt/PTEN/mTOR-signaling pathway on survival of tumor cells treated with cytotoxic drugs was studied using rapamycin (Rapa), mTOR specific inhibitor, and 9 human tumor cell lines of different origin and with different Akt kinase activity. Three of these cell lines were selected for drug resistance due to P-glycoprotein (Pgp or ABCB1) overexpression. Rapa inhibited phosphorylation of mTOR downstream effectors. Rapa sensitivity of the cells was Akt-dependent but did not correlate with ABCB1 overexpression. Suppression of mTOR function increased drug resistance in 8 out of 9 cell lines studied. The influence of Rapa on the ABC-transporter gene expression was examined. It was shown that in half of the cell lines studied Rapa exerted differential effects on the amount of ABC-transporter proteins: in some cases the protein amount decreased and in others, increased. The amount of mRNA remained unchanged. These data suggest that mTOR can regulate ABC transporters at the level of translation.     

Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance

The Ras/Raf/MEK/ERK and PI3K/PTEN/AKT signaling cascades play critical roles in the transmission of signals from growth factor receptors to regulate gene expression and prevent apoptosis. Components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf, PI3K, PTEN, Akt). Also, mutations occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. These pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of elevated activated Akt levels to phosphorylate and inactivate Raf-1. We have investigated the genetic structures and functional roles of these two signaling pathways in the malignant transformation and drug resistance of hematopoietic, breast and prostate cancer cells. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell-lineage-specific effects. Induced Raf expression can abrogate the cytokine dependence of certain hematopoietic cell lines (FDC-P1 and TF-1), a trait associated with tumorigenesis. In contrast, expression of activated PI3K or Akt does not abrogate the cytokine dependence of these hematopoietic cell lines, but does have positive effects on cell survival. However, activated PI3K and Akt can synergize with activated Raf to abrogate the cytokine dependence of another hematopoietic cell line (FL5.12) which is not transformed by activated Raf expression by itself. Activated Raf and Akt also confer a drug-resistant phenotype to these cells. Raf is more associated with proliferation and the prevention of apoptosis while Akt is more associated with the long-term clonogenicity. In breast cancer cells, activated Raf conferred resistance to the chemotherapeutic drugs doxorubicin and paclitaxel. Raf induced the expression of the drug pump Mdr-1 (a.k.a., Pgp) and the Bcl-2 anti-apoptotic protein. Raf did not appear to induce drug resistance by altering p53/p21^Cip−1 expression, whose expression is often linked to regulation of cell cycle progression and drug resistance. Deregulation of the PI3K/PTEN/Akt pathway was associated with resistance to doxorubicin and 4-hydroxyl tamoxifen, a chemotherapeutic drug and estrogen receptor antagonist used in breast cancer therapy. In contrast to the drug-resistant breast cancer cells obtained after overexpression of activated Raf, cells expressing activated Akt displayed altered (decreased) levels of p53/p21^Cip−1. Deregulated expression of the central phosphatase in the PI3K/PTEN/Akt pathway led to breast cancer drug resistance. Introduction of mutated forms of PTEN, which lacked lipid phosphatase activity, increased the resistance of the MCF-7 cells to doxorubicin, suggesting that these lipid phosphatase deficient PTEN mutants acted as dominant negative mutants to suppress wild-type PTEN activity. Finally, the PI3K/PTEN/Akt pathway appears to be more prominently involved in prostate cancer drug resistance than the Raf/MEK/ERK pathway. Some advanced prostate cancer cells express elevated levels of activated Akt which may suppress Raf activation. Introduction of activated forms of Akt increased the drug resistance of advanced prostate cancer cells. In contrast, introduction of activated forms of Raf did not increase the drug resistance of the prostate cancer cells. In contrast to the results observed in hematopoietic cells, Raf may normally promote differentiation in prostate cells which is suppressed in advanced prostate cancer due to increased expression of activated Akt arising from PTEN mutation. Thus in advanced prostate cancer it may be advantageous to induce Raf expression to promote differentiation, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK-induced proliferation. These signaling and anti-apoptotic pathways can have different effects on growth, prevention of apoptosis and induction of drug resistance in cells of various lineages which may be due to the expression of lineage-specific factors.     

NVP-BEZ235, a dual PI3K/mTOR inhibitor,induces cell death through alternate routes in prostate cancer cells depending on the PTEN genotype

Deregulation of the PI3K-AKT/mTOR pathway due to mutation of the tumor suppressor gene PTEN frequently occurs in human prostate cancer and is therefore considered to be an attractive therapeutic target. Here, we investigated how the PTEN genotype affected the antitumor effect of NVP-BEZ235 in human prostate cancer cells. In this setting, NVP-BEZ235 induced cell death in a PTEN-independent manner. NVP-BEZ235 selectively induced apoptotic cell death in the prostate cancer cell line DU145, which harbors wild-type PTEN; however, in the PC3 cell line, which is PTEN-null, treatment with NVP-BEZ235 resulted in autophagic cell death. Consistently, NVP-BEZ235 treatment did not result in the cleavage of caspase-3; instead, it resulted in the conversion of LC3-I to LC3-II, indicating autophagic cell death; these results suggest that an alternate mechanism of cell death is induced by NVP-BEZ235 in PTEN-null prostate cancer cells. Based on our findings, we conclude that the PTEN/PI3K/Akt pathway is critical for prostate cancer survival, and targeting PI3K signaling by NVP-BEZ235 may be beneficial in the treatment of prostate cancer, independent of the PTEN genotype.     

Involvement of Akt and mTOR in chemotherapeutic- and hormonal-based drug resistance and response to radiation in breast cancer cells

Elucidating the response of breast cancer cells to chemotherapeutic and hormonal based drugs and radiation is clearly important as these are common treatment approaches. Signaling cascades often involved in chemo-, hormonal- and radiation resistance are the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways. In the following studies we have examined the effects of activation of the Ras/PI3K/PTEN/Akt/mTOR cascade in the response of MCF-7 breast cancer cells to chemotherapeutic- and hormonal-based drugs and radiation. Activation of Akt by introduction of conditionally-activated Akt-1 gene could result in resistance to chemotherapeutic and hormonal based drugs as well as radiation. We have determined that chemotherapeutic drugs such as doxorubicin or the hormone based drug tamoxifen, both used to treat breast cancer, resulted in the activation of the Raf/MEK/ERK pathway which is often associated with a pro-proliferative, anti-apoptotic response. In drug sensitive MCF-7 cells which have wild-type p53; ERK, p53 and downstream p21^Cip-1 were induced upon exposure to doxorubicin. In contrast, in the drug resistant cells which expressed activated Akt-1, much lower levels of p53 and p21^Cip1 were induced upon exposure to doxorubicin. These results indicate the involvement of the Ras/PI3K/PTEN/Akt/mTOR, Ras/Raf/MEK/ERK and p53 pathways in the response to chemotherapeutic and hormonal based drugs. Understanding how breast cancers respond to chemo- and hormonal-based therapies and radiation may enhance the ability to treat breast cancer more effectively.     

A mechanism for synergy with combined mTOR and PI3 kinase inhibitors

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.     

Phosphatidylinositol 3-kinase/Akt stimulates androgen pathway through GSK3beta inhibition and nuclear beta-catenin accumulation

PI3K/Akt plays a critical role in prostate cancer cell growth and survival. Recent studies have shown that the effect of PI3K/Akt in prostate cells is mediated through androgen signaling. The PI3K inhibitor, LY294002, and a tumor suppressor, PTEN, negatively regulate the PI3K/Akt pathway and repress AR activity. However, the molecular mechanisms whereby PI3K/Akt and PTEN regulate the androgen pathway are currently unclear. Here, we demonstrate that blocking the PI3K/Akt pathway reduces the expression of an endogenous AR target gene. Moreover, we show that the repression of AR activity by LY294002 is mediated through phosphorylation and inactivation of GSK3beta, a downstream substrate of PI3K/Akt, which results in the nuclear accumulation of beta-catenin. Given the recent evidence that beta-catenin acts as a coactivator of AR, our findings suggest a novel mechanism by which PI3K/Akt modulates androgen signaling. In a PTEN-null prostate cancer cell line, we show that PTEN expression reduces beta-catenin-mediated augmentation of AR transactivation. Using the mutants of beta-catenin, we further demonstrate that the repressive effect of PTEN is mediated by a GSK3beta-regulated degradation of beta-catenin. Our results delineate a novel link among the PI3K, wnt, and androgen pathways and provide fresh insights into the mechanisms of prostate tumor development and progression.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.     

PI3 Kinase inhibition on TRAIL-induced apoptosis correlates with androgen-sensitivity and p21 expression in prostate cancer cells

TNF-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many types of cancer cells. TRAIL is considered a therapeutic target, therefore, it was of interest to examine molecular mechanisms that may modulate sensitivity to TRAIL signaling in prostate cancer cells. LNCaP cells were found to be relatively resistant to TRAIL induced cell death while PC3 cells were sensitive. PI3-kinase (PI3 K) inhibitors were able to render LNCaP cells sensitive to TRAIL but conferred resistance to PC3 cells. PI3 K inhibitors were associated with an increase in p21^{waf1, cip1} expression in PC3 cells where as p21 decreases in LNCaP cells suggesting that p21 may impart TRAIL resistance. Since androgen receptor (AR) signaling can be modulated by AKT, and p21 is an AR responsive gene, the impact of PI3 K inhibition on TRAIL sensitivity was evaluated in AR transfected PC3 cells (PC3AR). The expression of AR was significantly downregulated by PI3 K inhibition in LNCaP cells, which have an intact AR signaling axis. PC3AR cells expressed higher levels of p21 protein and were relatively resistant to TRAIL compared to control cells. Finally, using adenoviral p21 gene transfer we directly demonstrated that p21 can confer resistance to TRAIL-induced cell death. These results suggest that TRAIL resistance is not regulated simply by a PI3 K/AKT survival pathway associated with inactivating PTEN mutations but may also be modulated by downstream AR responsive targets such as p21. These findings may have significant clinical implications for the utility of TRAIL in the management of prostate cancer.     

Inhibition of mycobacterial infection by the tumor suppressor PTEN

The tumor suppressor PTEN is a lipid phosphatase that is frequently mutated in various human cancers. PTEN suppresses tumor cell proliferation, survival, and growth mainly by inhibiting the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. In addition to it role in tumor suppression, the PTEN-PI3K pathway controls many cellular functions, some of which may be important for cellular resistance to infection. Currently, the intersection between tumorigenic signaling pathways and cellular susceptibility to infection is not well defined. In this study we report that PTEN signaling regulates infection of both noncancerous and cancerous cells by multiple intracellular mycobacterial pathogens and that pharmacological modulation of PTEN signaling can affect mycobacterial infection. We found that PTEN deficiency renders multiple types of cells hyper-susceptible to infection by Mycoplasma and Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The lipid phosphatase activity of PTEN is required for attenuating infection. Furthermore, we found mycobacterial infection activates host cell Akt phosphorylation, and pharmacological inhibition of Akt or PI3K activity reduced levels of intracellular infection. Intriguingly, inhibition of mTOR, one of the downstream components of the Akt signaling and a promising cancer therapeutic target, also lowered intracellular Bacillus Calmette-Guérin levels in mammary epithelial cancer MCF-7 cells. These findings demonstrate a critical role of PTEN-regulated pathways in pathogen infection. The relationship of PTEN-PI3K-Akt mTOR status and susceptibility to mycobacterial infection suggests that the interaction of mycobacterial pathogens with cancer cells may be influenced by genetic alterations in the tumor cells.     

Salvianolic acid A reverses paclitaxel resistance in human breast cancer MCF-7 cells via targeting the expression of transgelin 2 and attenuating PI3 K/Akt pathway

Chemotherapy resistance represents a major problem for the treatment of patients with breast cancer and greatly restricts the use of first-line chemotherapeutics paclitaxel. The purpose of this study was to investigate the role of transgelin 2 in human breast cancer paclitaxel resistance cell line (MCF-7/PTX) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) indicated that transgelin 2 may mediate paclitaxel resistance by activating the phosphatidylinositol 3-kinase (PI3 K)/Akt signaling pathway to suppress MCF-7/PTX cells apoptosis. The reversal ability of SAA was confirmed by MTT assay and flow cytometry, with a superior 9.1-fold reversal index and enhancement of the apoptotic cytotoxicity induced by paclitaxel. In addition, SAA effectively prevented transgelin 2 and adenosine-triphosphate binding cassette transporter (ABC transporter) including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and breast cancer resistance protein (BCRP) up-regulation and exhibited inhibitory effect on PI3 K/Akt signaling pathway in MCF-7/PTX cells. Taken together, SAA can reverse paclitaxel resistance through suppressing transgelin 2 expression by mechanisms involving attenuation of PI3 K/Akt pathway activation and ABC transporter up-regulation. These results not only provide insight into the potential application of SAA in reversing paclitaxel resistance, thus facilitating the sensitivity of breast cancer chemotherapy, but also highlight a potential role of transgelin 2 in the development of paclitaxel resistance in breast cancer.     

Crosstalk of the EphA2 receptor with a serine/threonine phosphatase suppresses the Akt-mTORC1 pathway in cancer cells

Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways.     

Akt1 activation can augment hypoxia-inducible factor-1alpha expression by increasing protein translation through a mammalian target of rapamycin-independent pathway

The phosphoinositide 3-kinase (PI3K)/Akt pathway is commonly activated in cancer; therefore, we investigated its role in hypoxia-inducible factor-1alpha (HIF-1alpha) regulation. Inhibition of PI3K in U87MG glioblastoma cells, which have activated PI3K/Akt activity secondary to phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation, with LY294002 blunted the induction of HIF-1alpha protein and its targets vascular endothelial growth factor and glut1 mRNA in response to hypoxia. Introduction of wild-type PTEN into these cells also blunted HIF-1alpha induction in response to hypoxia and decreased HIF-1alpha accumulation in the presence of the proteasomal inhibitor MG132. Akt small interfering RNA (siRNA) also decreased HIF-1alpha induction under hypoxia and its accumulation in normoxia in the presence of dimethyloxallyl glycine, a prolyl hydroxylase inhibitor that prevents HIF-1alpha degradation. Metabolic labeling studies showed that Akt siRNA decreased HIF-1alpha translation in normoxia in the presence of dimethyloxallyl glycine and in hypoxia. Inhibition of mammalian target of rapamycin (mTOR) with rapamycin (10-100 nmol/L) had no significant effect on HIF-1alpha induction in a variety of cell lines, a finding that was confirmed using mTOR siRNA. Furthermore, neither mTOR siRNA nor rapamycin decreased HIF-1alpha translation as determined by metabolic labeling studies. Therefore, our results indicate that Akt can augment HIF-1alpha expression by increasing its translation under both normoxic and hypoxic conditions; however, the pathway we are investigating seems to be rapamycin insensitive and mTOR independent. These observations, which were made on cells grown in standard tissue culture medium (10% serum), were confirmed in PC3 prostate carcinoma cells. We did find that rapamycin could decrease HIF-1alpha expression when cells were cultured in low serum, but this seems to represent a different pathway.     

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.     

Loss of activating EGFR mutant gene contributes to acquired resistance to EGFR tyrosine kinase inhibitors in lung cancer cells

Non-small-cell lung cancer harboring epidermal growth factor receptor (EGFR) mutations attains a meaningful response to EGFR-tyrosine kinase inhibitors (TKIs). However, acquired resistance to EGFR-TKIs could affect long-term outcome in almost all patients. To identify the potential mechanisms of resistance, we established cell lines resistant to EGFR-TKIs from the human lung cancer cell lines PC9 and11-18, which harbored activating EGFR mutations. One erlotinib-resistant cell line from PC9 and two erlotinib-resistant cell lines and two gefitinib-resistant cell lines from 11-18 were independently established. Almost complete loss of mutant delE746-A750 EGFR gene was observed in the erlotinib-resistant cells isolated from PC9, and partial loss of the mutant L858R EGFR gene copy was specifically observed in the erlotinib- and gefitinib-resistant cells from 11-18. However, constitutive activation of EGFR downstream signaling, PI3K/Akt, was observed even after loss of the mutated EGFR gene in all resistant cell lines even in the presence of the drug. In the erlotinib-resistant cells from PC9, constitutive PI3K/Akt activation was effectively inhibited by lapatinib (a dual TKI of EGFR and HER2) or BIBW2992 (pan-TKI of EGFR family proteins). Furthermore, erlotinib with either HER2 or HER3 knockdown by their cognate siRNAs also inhibited PI3K/Akt activation. Transfection of activating mutant EGFR complementary DNA restored drug sensitivity in the erlotinib-resistant cell line. Our study indicates that loss of addiction to mutant EGFR resulted in gain of addiction to both HER2/HER3 and PI3K/Akt signaling to acquire EGFR-TKI resistance.     

Regulation of mTOR and p70 S6 kinase by the muscarinic M4 receptor in PC12 cells

The G_i-coupled M₄ muscarinic acetylcholine receptor (mAChR) has recently been shown to stimulate the survival of PC12 cells through the PI3K/Akt/tuberin pathway. Since mTOR and p70S6K are critical components in activating translation which lie downstream of tuberin, we examined the ability of M₄ mAChR to regulate these targets in PC12 cells. Carbachol (CCh) dose-dependently stimulated both mTOR and p70S6K phosphorylations and these responses were abolished by pertussis toxin pretreatment, indicating the involvement of the G_i-coupled M₄ mAChR. Phosphorylations of both mTOR and p70S6K were effectively blocked upon inhibition of PI3K by wortmannin. As compared to similar responses elicited by the nerve growth factor (NGF), the M₄ mAChR-induced activation of Akt/tuberin/mTOR/p70S6K occurred in a relatively transient manner. Although inhibition of protein phosphatase 2A by okadaic acid augmented the transient effects of CCh on Akt/tuberin phosphorylations, it failed to significantly prolong these responses. The total protein level of PTEN (tumor suppressor gene phosphatase and tensin homologue deleted on chromosome ten) was attenuated upon NGF, but not CCh treatment. This indicates that downregulation of PTEN may help to sustain the phosphorylation of Akt/tuberin by NGF. Collectively, these findings suggest that PP2A and PTEN may be involved in fine tuning the regulation of Akt/tuberin/mTOR/p70S6K in PC12 cells by M₄ mAChR and TrkA, respectively.     

The Raf/MEK/ERK pathway can govern drug resistance,apoptosis and sensitivity to targeted therapy

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on proliferation, drug resistance, prevention of apoptosis and sensitivity to signal transduction inhibitors were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf and Akt activation. Drug resistant cells were isolated from FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells in the presence of doxorubicin. Activation of Raf-1, in the drug resistant FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells, increased the IC₅₀ for doxorubicin 80-fold, whereas activation of Akt-1, by itself, had no effect on the doxorubicin IC₅₀. However, Akt-1 activation enhanced cell proliferation and clonogenicity in the presence of chemotherapeutic drugs. Thus the Raf/MEK/ERK pathway had profound effects on the sensitivity to chemotherapeutic drugs, and Akt-1 activation was required for the long-term growth of these cells as well as resistance to chemotherapeutic drugs. The effects of doxorubicin on the induction of apoptosis in the drug resistant cells were enhanced by addition of either mTOR and MEK inhibitors. These results indicate that targeting the Raf/MEK/ERK and PI3K/Akt/mTOR pathways may be an effective approach for therapeutic intervention in drug resistant cancers that have mutations activating these cascades.