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     

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.     

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.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

Targeting signal transduction pathways to eliminate chemotherapeutic drug resistance and cancer stem cells

Breast cancer is one of the most common cancers and affects nearly 1 in 7 women. We have demonstrated that targeting the CaM-K, Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be a novel approach to treat drug resistant breast cancer and eliminate cancer stem cells. Common chemotherapeutic drugs, such as doxorubicin, induce the CaM-K pathway which in turn, leads to activation of anti-apoptotic pathways such as Raf/MEK/ERK and PI3K/Akt. Some drug resistant breast cancers exhibited increased expression of CaM-KIV. CaM-K inhibitors synergized with doxorubicin to induce the death of all drug resistant breast cancers examined. Since CaM-Ks are known to result in activation of the Raf/MEK/ERK and PI3K/Akt pathways, we investigated the roles that these pathways exert in breast cancer drug resistance. CaM-K inhibitors suppressed ERK activation in response to doxorubicin in both drug sensitive and resistant cells. CaM-K inhibitors also suppressed ERK activation in response to FBS in the drug resistant cells suggesting dependence on the CaM-K pathway for proliferation. Both the Raf/MEK/ERK and PI3K/Akt pathways are involved in breast cancer drug resistance as they were detected at elevated, activated levels in the drug resistant cells and introduction of constitutively activated forms of Raf-1 and Akt-1 resulted in drug resistance. Drug resistant CICs were often hypersensitive to MEK and mTOR inhibitors, implicating important roles of these pathways in drug resistance. In summary, targeting these pathways may enhance therapy of drug resistant breast cancer and eliminate CICs.Breast cancer therapy is often limited by the occurrence of drug resistance which may be due to the re-emergence of CICs. The studies outlined in this proposal may identify a potentially novel role for CaM-Ks in drug resistance and metastasis and may lead to improved approaches to treat breast tumors by eliminating CICs. Our proposed studies are highly innovative as we will determine the involvement of the CaM-K pathway in breast cancer drug resistance, metastasis and CIC formation. Similar approaches have not been previously performed. Our studies may result in the discovery of novel methods to treat breast cancer by targeting the CaM-K pathway in combination with currently used and approved chemotherapeutic regimens to eliminate CICs which may be responsible for both drug resistance and metastasis.     

Dominant roles of the Raf/MEK/ERK pathway in cell cycle progression,prevention of apoptosis and sensitivity to chemotherapeutic drugs

The effects of the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways on cell cycle progression, gene expression, prevention of apoptosis and sensitivity to chemotherapeutic drugs were examined in FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells which are conditionally-transformed to grow in response to Raf-1 and Akt-1 activation by treatment with testosterone or tamoxifen respectively. In these cells we can compare the effects of normal cytokine vs. oncogene mediated signaling in the same cells by changing the culture conditions. Raf-1 was more effective than Akt-1 in inducing cell cycle progression and preventing apoptosis in the presence and absence of chemotherapeutic drugs. The normal cytokine for these cells, interleukin-3 induced/activated most downstream genes transiently, with the exception of p70S6K that was induced for prolonged periods of time. In contrast, most of the downstream genes induced by either the activate Raf-1 or Akt-1 oncogenes were induced for prolonged periods of time, documenting the differences between cytokine and oncogene mediated gene induction which has important therapeutic consequences. The FL/ΔAkt-1:ER*(Myr⁺) + ΔRaf-1:AR cells were sensitive to MEK and PI3K/mTOR inhibitors. Combining MEK and PI3K/mTOR inhibitors increased the induction of apoptosis. The effects of doxorubicin on the induction of apoptosis could be enhanced with MEK, PI3K and mTOR inhibitors. Targeting the Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways may be an effective approach for therapeutic intervention in those cancers which have upstream mutations which result in activation of these pathways.     

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.     

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.     

Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy

The effects of inhibition of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways and chemotherapeutic drugs on cell cycle progression and drug sensitivity were examined in cytokine-dependent FL5.12 hematopoietic cells. We examined their effects, as these cells resemble normal hematopoietic precursor cells as they do not exhibit “oncogene-addicted” growth, while they do display “cytokine-addicted” proliferation as cytokine removal resulted in apoptosis in greater than 80% of the cells within 48 h. When cytokine-dependent FL5.12 cells were cultured in the presence of IL-3, which stimulated multiple proliferation and anti-apoptotic cascades, MEK, PI3K and mTOR inhibitors transiently suppressed but did not totally inhibit cell cycle progression or induce apoptosis while chemotherapeutic drugs such as doxorubicin and paclitaxel were more effective in inducing cell cycle arrest and apoptosis. Doxorubicin induced a G₁ block, while paclitaxel triggered a G₂/M block. Doxorubicin was more effective in inducing cell death than paclitaxel. Furthermore the effects of doxorubicin could be enhanced by addition of MEK, PI3K or mTOR inhibitors. Cytokine-dependent cells which proliferate in vitro and are not “oncogene-addicted” may represent a pre-malignant stage, more refractory to treatment with targeted therapy. However, these cells are sensitive to chemotherapeutic drugs. It is important to develop methods to inhibit the growth of such cytokine-dependent cells as they may resemble the leukemia stem cell and other cancer initiating cells. These results demonstrate the enhanced effectiveness of targeting early hematopoietic progenitor cells with combinations of chemotherapeutic drugs and signal transduction inhibitors.     

Inhibition of GSK-3β activity can result in drug and hormonal resistance and alter sensitivity to targeted therapy in MCF-7 breast cancer cells

The PI3K/Akt/mTORC1 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance, and metastasis. One molecule regulated by this pathway is GSK-3β. GSK-3β is phosphorylated by Akt on S9, which leads to its inactivation; however, GSK-3β also can regulate the activity of the PI3K/Akt/mTORC1 pathway by phosphorylating molecules such as PTEN, TSC2, p70S6K, and 4E-BP1. To further elucidate the roles of GSK-3β in chemotherapeutic drug and hormonal resistance of MCF-7 breast cancer cells, we transfected MCF-7 breast cancer cells with wild-type (WT), kinase-dead (KD), and constitutively activated (A9) forms of GSK-3β. MCF-7/GSK-3β(KD) cells were more resistant to doxorubicin and tamoxifen compared with either MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In the presence and absence of doxorubicin, the MCF-7/GSK-3β(KD) cells formed more colonies in soft agar compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells. In contrast, MCF-7/GSK-3β(KD) cells displayed an elevated sensitivity to the mTORC1 blocker rapamycin compared with MCF-7/GSK-3β(WT) or MCF-7/GSK-3β(A9) cells, while no differences between the 3 cell types were observed upon treatment with a MEK inhibitor by itself. However, resistance to doxorubicin and tamoxifen were alleviated in MCF-7/GSK-3β(KD) cells upon co-treatment with an MEK inhibitor, indicating regulation of this resistance by the Raf/MEK/ERK pathway. Treatment of MCF-7 and MCF-7/GSK-3β(WT) cells with doxorubicin eliminated the detection of S9-phosphorylated GSK-3β, while total GSK-3β was still detected. In contrast, S9-phosphorylated GSK-3β was still detected in MCF-7/GSK-3β(KD) and MCF-7/GSK-3β(A9) cells, indicating that one of the effects of doxorubicin on MCF-7 cells was suppression of S9-phosphorylated GSK-3β, which could result in increased GSK-3β activity. Taken together, these results demonstrate that introduction of GSK-3β(KD) into MCF-7 breast cancer cells promotes resistance to doxorubicin and tamoxifen, but sensitizes the cells to mTORC1 blockade by rapamycin. Therefore GSK-3β is a key regulatory molecule in sensitivity of breast cancer cells to chemo-, hormonal, and targeted therapy.     

Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy

The effects of inhibition of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways and chemotherapeutic drugs on cell cycle progression and drug sensitivity were examined in cytokine-dependent FL5.12 hematopoietic cells. We examined their effects, as these cells resemble normal hematopoietic precursor cells as they do not exhibit “oncogene-addicted” growth, while they do display “cytokine-addicted” proliferation as cytokine removal resulted in apoptosis in greater than 80% of the cells within 48 h. When cytokine-dependent FL5.12 cells were cultured in the presence of IL-3, which stimulated multiple proliferation and anti-apoptotic cascades, MEK, PI3K and mTOR inhibitors transiently suppressed but did not totally inhibit cell cycle progression or induce apoptosis while chemotherapeutic drugs such as doxorubicin and paclitaxel were more effective in inducing cell cycle arrest and apoptosis. Doxorubicin induced a G₁ block, while paclitaxel triggered a G₂/M block. Doxorubicin was more effective in inducing cell death than paclitaxel. Furthermore the effects of doxorubicin could be enhanced by addition of MEK, PI3K or mTOR inhibitors. Cytokine-dependent cells which proliferate in vitro and are not “oncogene-addicted” may represent a pre-malignant stage, more refractory to treatment with targeted therapy. However, these cells are sensitive to chemotherapeutic drugs. It is important to develop methods to inhibit the growth of such cytokine-dependent cells as they may resemble the leukemia stem cell and other cancer initiating cells. These results demonstrate the enhanced effectiveness of targeting early hematopoietic progenitor cells with combinations of chemotherapeutic drugs and signal transduction inhibitors.     

Therapeutic resistance resulting from mutations in Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR signaling pathways

Chemotherapy remains a commonly used therapeutic approach for many cancers. Indeed chemotherapy is relatively effective for treatment of certain cancers and it may be the only therapy (besides radiotherapy) that is appropriate for certain cancers. However, a common problem with chemotherapy is the development of drug resistance. Many studies on the mechanisms of drug resistance concentrated on the expression of membrane transporters and how they could be aberrantly regulated in drug resistant cells. Attempts were made to isolate specific inhibitors which could be used to treat drug resistant patients. Unfortunately most of these drug transporter inhibitors have not proven effective for therapy. Recently the possibilities of more specific, targeted therapies have sparked the interest of clinical and basic researchers as approaches to kill cancer cells. However, there are also problems associated with these targeted therapies. Two key signaling pathways involved in the regulation of cell growth are the Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR pathways. Dysregulated signaling through these pathways is often the result of genetic alterations in critical components in these pathways as well as mutations in upstream growth factor receptors. Furthermore, these pathways may be activated by chemotherapeutic drugs and ionizing radiation. This review documents how their abnormal expression can contribute to drug resistance as well as resistance to targeted therapy. This review will discuss in detail PTEN regulation as this is a critical tumor suppressor gene frequently dysregulated in human cancer which contributes to therapy resistance. Controlling the expression of these pathways could improve cancer therapy and ameliorate human health.     

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.     

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.     

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.     

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.     

The role of the PTEN/mTOR axis in clinical response of rectal cancer patients

Background

Preoperative chemoradiotherapy has long been accepted as a method to improve survival and lifetime quality of rectal cancer patients. However, physiologic effects of these therapies largely depend on the resistance of cells to the radiation, type of chemotherapeutic agents and individual responses. As one of the signaling cascades involved in chemo- or radiation- resistance, the present study focused on several proteins involved in pTEN/Akt/mTOR pathway to explore their prognostic significance.

Materials and methods

Samples from advanced stage rectal cancer patients were analyzed to detect expression levels of pTEN/Akt/mTOR pathway related proteins pTEN, mLST8, REDD1, BNIP3, SAG and NOXA, together with p53, by RT-qPCR. Kaplan–Meier analysis was used to assess expression-survival relation and correlations among all proteins and clinicopathological features were statistically analyzed.

Results.

Except p53, none of the proteins showed prognostic significance. High p53 expression presented clear impact on overall survival and disease free survival. It was also significantly related to pathologic complete response. p53 showed high correlation to local recurrence as well. On the other hand, strong correlation was observed with PTEN expression and tumor response, but not with survival. High associations were also observed between mLST8/REDD1, PTEN and NOXA, confirming their role in the same cascade.

Conclusion

The contentious role of p53 as a prognostic biomarker in colorectal cancer was further affirmed, while PTEN and REDD1 could be suggested as potential candidates. Additionally, NOXA emerges as a conjunctive element for different signaling pathways.