Pathway crosstalk between Ras/Raf and PI3K in promotion of M-CSF-induced MEK/ERK-mediated osteoclast survival

While M-CSF-mediated MEK/ERK activation promotes osteoclast survival, the signaling pathway by which M-CSF activates MEK/ERK is unresolved. Functions for PI3K, Ras, and Raf have been implicated in support of osteoclast survival, although interaction between these signaling components has not been examined. Therefore, the interplay between PI3K, Ras and Raf in M-CSF-promoted MEK/ERK activation and osteoclast survival was investigated. M-CSF activates Ras to coordinate activation of PI3K and Raf/MEK/ERK, since Ras inhibition decreased PI3K activation and PI3K inhibition did not block M-CSF-mediated Ras activation. As further support for Ras-mediated signaling, constitutively active (ca) Ras promoted MEK/ERK activation and osteoclast survival, which was blocked by inhibition of PI3K or Raf. Moreover, PI3K-selective or Raf-selective caRas were only partially able to promote osteoclast survival when compared to parental caRas. We then examined whether PI3K and Raf function linearly or in parallel downstream of Ras. Expression of caPI3K increased MEK/ERK activation and promoted osteoclast survival downstream of M-CSF, supporting this hypothesis. Blocking Raf did not decrease osteoclast survival and MEK/ERK activation promoted by caPI3K. In addition, PI3K-selective Ras-mediated survival was not blocked by Raf inhibition. Taken together, our data support that Raf signaling is separate from Ras/PI3K signaling and PI3K signaling is separate from Ras/Raf signaling. These data therefore support a role for Ras in coordinate activation of PI3K and Raf acting in parallel to mediate MEK/ERK-promoted osteoclast survival induced by M-CSF.     

PI3K is required for insulin-stimulated but not EGF-stimulated ERK1/2 activation

The Ras/Raf/extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathway is known to cross-talk with other signaling pathways, including phosphatidylinositol 3-kinase (PI3K)/Akt pathway. However, the role of PI3K in ERK-1/2 activation induced by tyrosine kinase receptors was not fully understood. Here, we report that two structurally distinct PI3K inhibitors, wortmannin and LY294002, inhibited insulin-induced activation of ERK1/2 but had no effect on EGF-induced activation of ERK1/2 in hepatocellular carcinoma BEL-7402 and SMMC-7721 cells, breast cancer MCF-7 cells, and prostate cancer LNCaP cells. Although protein kinase C could act as a mediator between PI3K and ERK1/2, protein kinase C inhibitor chelerythrine chloride did not inhibit insulin-induced ERK1/2 activation. Both insulin- and EGF-induced ERK1/2 activation are strictly dependent on Ras activation, however, wortmannin only inhibited insulin-induced, but not EGF-induced Ras activation. These results indicate that PI3K plays different roles in the activation of Ras/ERK1/2 signaling by insulin and EGF, and that insulin-stimulated, but not EGF-stimulated, ERK1/2 and Akt signalings diverge at PI3K.     

Signal transduction of MEK/ERK and PI3K/Akt activation by hypoxia/reoxygenation in renal epithelial cells

The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.     

Hypoxia/Reoxygenation Stimulates Proliferation Through PKC-Dependent Activation of ERK and Akt in Mouse Neural Progenitor Cells

Cerebral ischemia increases neural progenitor cell proliferation and neurogenesis. However, the precise molecular mechanism is poorly understood. The present study was undertaken to determine roles of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt and their signaling pathways in neural progenitor cells exposed to hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion. Neural progenitor cells were isolated from postnatal mouse brain. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of MEK, but not by LY294002, a specific inhibitor of PI3K, whereas the Akt activation was blocked by LY294002, but not by U0126. Reoxygenation following 4-h hypoxia stimulated cell proliferation, which was dependent on ERK and Akt activation. Inhibitors of growth factor receptor (AG1478) and Src (PP2) and the antioxidant N-acetylcysteine did not affect activation of ERK and Akt, while the Ras and Raf inhibitors inhibited activation of ERK, but not Akt. PKC inhibitors inhibited both ERK and Akt activation. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt survival signaling pathways through a PKC-dependent mechanism. These pathways may be responsible for the repair process during ischemia/reperfusion.     

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

We explored the crosstalk between cell survival (phosphatidylinositol 3-kinase (PI3K)/Akt) and mitogenic (Ras/Raf/MEK/extracellular signal-regulated kinase (ERK)) signaling pathways activated by an epidermal growth factor (EGF) and analyzed their sensitivity to small molecule inhibitors in the PI3K-mutant estrogen receptor (ER)-positive MCF7 and T47D breast cancer cells. In contrast to MCF7 cells, ERK phosphorylation in T47D cells displayed resistance to MEK inhibition by several structurally different compounds, such as U0126, PD 098059 and PD 198306, MEK suppression by small interfering RNA (siRNA) and was also less sensitive to PI3K inhibition by wortmannin. Similar effect was observed in PI3K-wild type ER-positive BT-474 cells, albeit to a much lesser extent.MEK-independent ERK activation was induced only by ErbB receptor ligands and was resistant to inhibition of several kinases and phosphatases that are known to participate in the regulation of Ras/mitogen-activated protein kinase (MAPK) cascade. Although single agents against PDK1 or Akt did not affect EGF-induced ERK phosphorylation, a combination of PI3K/Akt and MEK inhibitors synergistically suppressed ERK activation and cellular growth. siRNA-mediated silencing of class I PI3K or Akt1/2 genes also significantly decreased U0126-resistant ERK phosphorylation.Our data suggest that in T47D cells ErbB family ligands induce a dynamic, PI3K/Akt-sensitive and MEK-independent compensatory ERK activation circuit that is absent in MCF7 cells. We discuss candidate proteins that can be involved in this activation circuitry and suggest that PDZ-Binding Kinase/T-LAK Cell-Originated Protein Kinase (PBK/TOPK) may play a role in mediating MEK-independent ERK activation.     

Roles of ERK, PI3 kinase, and PLC-γ pathways induced by overexpression of translationally controlled tumor protein in HeLa cells

We reported previously that translationally controlled tumor protein (TCTP) is a cytoplasmic repressor of Na,K-ATPase in HeLa cells. In the current study, we showed that TCTP overexpression using adenovirus as vehicle, induced partial inhibition of Na,K-ATPase; phosphorylation of EGFR tyrosine residues 845, 992, 1068, and 1148; activation of Ras/Raf/ERK pathway; activation of PI3K/Akt pathway; and phosphorylation of PLC-γ in HeLa cells. Specific inhibition of PI3K/Akt pathway in contrast to the inhibition of ERK, significantly decreased TCTP overexpression-induced survival signal. Inhibition of PLC-γ pathway significantly decreased TCTP overexpression-induced cell migration but inhibition of ERK had less effect. These results suggest that TCTP plays a key physiological role in cell survival through Akt pathway and migration through PLC-γ pathway.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.     

A3 adenosine receptor activation inhibits cell proliferation via phosphatidylinositol 3-kinase/Akt-dependent inhibition of the extracellular signal-regulated kinase 1/2 phosphorylation in A375 human melanoma cells

Adenosine exerts its effects through four subtypes of G-protein-coupled receptors: A(1), A(2A), A(2B), and A(3). Stimulation of the human A(3) receptor has been suggested to influence cell death and proliferation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Due to their importance, the cross-talk between these two pathways has been investigated. Here, we show that the A(3) adenosine receptor agonist Cl-IB-MECA stimulates PI3K-dependent phosphorylation of Akt leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation. The response to Cl-IB-MECA was not blocked by A(1), A(2A), or A(2B) receptor antagonists, although it was abolished by A(3) receptor antagonists. Furthermore, the response to Cl-IB-MECA was generated at the cell surface, since the inhibition of A(3) receptor expression, by using small interfering RNA, abolished agonist effects. Using A375 cells, we show that A(3) adenosine receptor stimulation results in PI3K-dependent phosphorylation of Akt, leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation.     

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells

There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer.In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells.Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.     

Analysis of Ras-dependent signals that prevent caspase-3 activation and apoptosis induced by cytokine deprivation in hematopoietic cells

In hematopoietic cells, Ras has been implicated in signaling pathways that prevent apoptosis triggered by deprivation of cytokines, such as interleukin-3 (IL-3). However, the mechanism whereby Ras suppresses cell death remains incompletely understood. We have investigated the role of Ras in IL-3 signal transduction by using the cytokine-dependent BaF3 cell line. Herein, we show that the activation of the pro-apoptotic protease caspase-3 upon IL-3 removal is suppressed by expression of activated Ras, which eventually prevents cell death. For caspase-3 suppression, the Raf/extracellular signal-regulated kinase (ERK)- or phosphatidylinositol 3-kinase (PI3-K)/Akt-mediated signaling pathway downstream of Ras was required. However, inhibition of both pathways did not block activated Ras-dependent suppression of cell death-associated phenotypes, such as nuclear DNA fragmentation. Thus, a pathway that is independent of both Raf/ERK and PI3-K/Akt pathways may function downstream of Ras, preventing activated caspase-3-initiated apoptotic processes. Conditional activation of c-Raf-1 also suppressed caspase-3 activation and subsequent cell death without affecting Akt activity, providing further evidence for a PI3-K/Akt-independent mechanism.     

Ras Protein Activation Is a Key Event in Activity-dependent Survival of Cerebellar Granule Neurons

Neuronal activity promotes the survival of cerebellar granule neurons (CGNs) during the postnatal development of cerebellum. CGNs that fail to receive excitatory inputs will die by apoptosis. This process could be mimicked in culture by exposing CGNs to either a physiological concentration of KCl (5 mm or K5) plus N-methyl-d-aspartate (NMDA) or to 25 mm KCl (K25). We have previously described that a 24-h exposure to NMDA (100 μm) or K25 at 2 days in vitro induced long term survival of CGNs in K5 conditions. Here we have studied the molecular mechanisms activated at 2 days in vitro in these conditions. First we showed that NMDA or K25 addition promoted a rapid stimulation of PI3K and a biphasic phosphorylation on Ser-473 of Akt, a PI3K substrate. Interestingly, we demonstrated that only the first wave of Akt phosphorylation is necessary for the NMDA- and K25-mediated survival. Additionally, we detected that both NMDA and K25 increased ERK activity with a similar time-course. Moreover, our results showed that NMDA-mediated activation of the small G-protein Ras is necessary for PI3K/Akt pathway activation, whereas Rap1 was involved in NMDA phosphorylation of ERK. On the other hand, Ras, but not Rap1, mediates K25 activation of PI3K/Akt and MEK/ERK pathways. Because neuroprotection by NMDA or K25 is mediated by Ras (and not by Rap1) activation, we propose that Ras stimulation is a crucial event in NMDA- and K25-mediated survival of CGNs through the activation of PI3K/Akt and MEK/ERK pathways.     

Role of EGF receptor transactivation in phosphoinositide 3-kinase-dependent activation of MAP kinase by GPCRs

Many G protein coupled receptors (GPCRs) cause phosphorylation of MAP kinases through transactivation of the epidermal growth factor receptor (EGF-R), leading to increased cell survival and growth, motility, and migration. Phosphoinositide 3-kinase (PI3K) is one of the important cell survival signaling molecules activated by EGF-R stimulation. However, the extent to which EGF-R transactivation is essential for GPCR agonist-stimulated PI3K activation is not known. Here we examined the mechanism of PI3K activation that elicits GPCR-mediated ERK1/2 activation by pathways dependent and/or independent of EGF-R transactivation in specific cell types. Immortalized hypothalamic neurons (GT1-7 cells) express endogenous gonadotropin-releasing hormone receptors (GnRH-R) and their stimulation causes marked phosphorylation of ERK1/2 and Akt (Ser 473) through transactivation of the EGF-R and recruitment of PI3K. In C9 hepatocytes, agonist activation of AT1 angiotensin II (AT1-R), lysophosphatidic acid (LPA), and EGF receptors caused phosphorylation of Akt through activation of the EGF-R in a PI3K-dependent manner. However, ERK1/2 activation by these agonists in these cells was independent of PI3K activation. In contrast, agonist stimulation of HEK 293 cells stably expressing AT1-R caused ERK1/2 phosphorylation that was independent of EGF-R transactivation but required PI3K activation. LPA signaling in these cells showed partial and complete dependence on EGF-R and PI3K, respectively. These data indicate that GPCR-induced ERK1/2 phosphorylation is dependent or independent of PI3K in specific cell types, and that the involvement of PI3K during ERK1/2 activation is not dependent solely on agonist-induced transactivation of the EGF-R.     

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.     

Allosteric modulation of Ras-GTP is linked to signal transduction through RAF kinase

Ras is a key signal transduction protein in the cell. Mutants of Gly(12) and Gln(61) impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln(61) in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.     

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.     

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/PTE N/Akt/mTO R, Ras/Raf/MEK/ERK and p53 pathways. In the following studies we have examined the effects of activation of the Ras/PI3K/PTE N/Akt/mTO R 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 proproliferative, 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/PTE N/Akt/mTO R, 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.Key words: Akt, ERK, mTOR, chemotherapeutic drugs, radiation     

Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells.

The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser(259), resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells.     

M-Ras induces Ral and JNK activation to regulate MEK/ERK-independent gene expression in MCF-7 breast cancer cells

Constitutive activation of M-Ras has previously been reported to cause morphologic and growth transformation of murine cells, suggesting that M-Ras plays a role in tumorigenesis. Cell transformation by M-Ras correlated with weak activation of the Raf/MEK/ERK pathway, although contributions from other downstream effectors were suggested. Recent studies indicate that signaling events distinct from the Raf/MEK/ERK cascade are critical for human tumorigenesis. However, it is unknown what signaling events M-Ras triggers in human cells. Using constitutively active M-Ras (Q71L) containing additional mutations within its effector-binding loop, we found that M-Ras induces MEK/ERK-dependent and -independent Elk1 activation as well as phosphatidylinositol 3 kinase (PI3K)/Akt and JNK/cJun activation in human MCF-7 breast cancer cells. Among several human cell lines examined, M-Ras-induced MEK/ERK-independent Elk1 activation was only detected in MCF-7 cells, and correlated with Rlf/M-Ras interaction and Ral/JNK activation. Supporting a role for M-Ras signaling in breast cancer, EGF activated M-Ras and promoted its interaction with endogenous Rlf. In addition, constitutive activation of M-Ras induced estrogen-independent growth of MCF-7 cells that was dependent on PI3K/Akt, MEK/ERK, and JNK activation. Thus, our studies demonstrate that M-Ras signaling activity differs between human cells, highlighting the importance of defining Ras protein signaling within each cell type, especially when designing treatments for Ras-induced cancer. These findings also demonstrate that M-Ras activity may be important for progression of EGFR-dependent tumors.