Richard A. Franklin

Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy

The PI3K/Akt signal transduction cascade has been investigated extensively for its roles in oncogenic transformation. Initial studies implicated both PI3K and Akt in prevention of apoptosis. However, more recent evidence has also associated this pathway with regulation of cell cycle progression. Uncovering the signaling network spanning from extracellular environment to the nucleus should illuminate biochemical events contributing to malignant transformation. Here, we discuss PI3K/Akt-mediated signal transduction including its mechanisms of activation, signal transducing molecules, and effects on gene expression that contribute to tumorigenesis. Effects of PI3K/Akt signaling on important proteins controlling cellular proliferation are emphasized. These targets include cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors. Furthermore, strategies used to inhibit the PI3K/Akt pathway are presented. The potential for cancer treatment with agents inhibiting this pathway is also addressed.

Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine receptors to transcription factors: potential targeting for therapeutic intervention.

The Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. Depending upon the stimulus and cell type, this pathway can transmit signals, which result in the prevention or induction of apoptosis or cell cycle progression. Thus, it is an appropriate pathway to target for therapeutic intervention. This pathway becomes more complex daily, as there are multiple members of the kinase and transcription factor families, which can be activated or inactivated by protein phosphorylation. The diversity of signals transduced by this pathway is increased, as different family members heterodimerize to transmit different signals. Furthermore, additional signal transduction pathways interact with the Raf/MEK/ERK pathway to regulate positively or negatively its activity, or to alter the phosphorylation status of downstream targets. Abnormal activation of this pathway occurs in leukemia because of mutations at Ras as well as genes in other pathways (eg PI3K, PTEN, Akt), which serve to regulate its activity. Dysregulation of this pathway can result in autocrine transformation of hematopoietic cells since cytokine genes such as interleukin-3 and granulocyte/macrophage colony-stimulating factor contain the transacting binding sites for the transcription factors regulated by this pathway. Inhibitors of Ras, Raf, MEK and some downstream targets have been developed and many are currently in clinical trials. This review will summarize our current understanding of the Ras/Raf/MEK/ERK signal transduction pathway and the downstream transcription factors. The prospects of targeting this pathway for therapeutic intervention in leukemia and other cancers will be evaluated.

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal transduction pathways and the BCR-ABL oncoprotein in leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. These pathways have evolved regulatory proteins, which serve to limit their proliferative and antiapoptotic effects. Small molecular weight cell membrane-permeable drugs that target these pathways have been developed for leukemia therapy. One such example is imatinib mesylate, which targets the BCR-ABL kinase as well as a few structurally related kinases. This drug has proven to be effective in the treatment of CML patients. However, leukemic cells have evolved mechanisms to become resistant to this drug. A means to combat drug resistance is to target other prominent signaling components involved in the pathway or to inhibit BCR-ABL by other mechanisms. Treatment of imatinib-resistant leukemia cells with drugs that target Ras (farnysyl transferase inhibitors) or with the protein destabilizer geldanamycin has proven to be a means to inhibit the growth of resistant cells. This review will tie together three important signal transduction pathways involved in the regulation of hematopoietic cell growth and indicate how their expression is dysregulated by the BCR-ABL oncoprotein.

Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.

Kinases: positive and negative regulators of apoptosis.

Cells sense and respond to extracellular factors via receptors on the cell surface that trigger intracellular signaling pathways. The signals received by the receptors on hematopoietic cells often determine if the cell proliferates, survives or undergoes apoptosis. Apoptosis can be induced by almost any cytotoxic stimuli. These stimuli may be an absence of signals arising from cellular receptors, stimulation of specific ligand receptors on the cell surface, chemotherapeutic agents, and ionizing radiation or oxygen radicals, as well as a number of other factors. Cellular kinases and phosphatases participate in signaling cascades that influence this process. We review the ability of the calmodulin-dependent-kinases, I-κB kinases, PI3-kinases, Jak-kinases, PKC, PKA, and MAP kinase signaling pathways (Erk, Jnk, and p38), to influence the apoptotic process. In addition, we discuss the cross-talk that exists between signaling cascades that are pro-apoptotic and anti-apoptotic.

Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs.

Over the past decade, there has been an exponential increase in our knowledge of how cytokines regulate signal transduction, cell cycle progression, differentiation and apoptosis. Research has focused on different biochemical and genetic aspects of these processes. Initially, cytokines were identified by clonogenic assays and purified by biochemical techniques. This soon led to the molecular cloning of the genes encoding the cytokines and their cognate receptors. Determining the structure and regulation of these genes in normal and malignant hematopoietic cells has furthered our understanding of neoplastic transformation. Furthermore, this has allowed the design of modified cytokines which are able to stimulate multiple receptors and be more effective in stimulating the repopulation of hematopoietic cells after myelosuppressive chemotherapy. The mechanisms by which cytokines transduce their regulatory signals have been evaluated by identifying the involvement of specific protein kinase cascades and their downstream transcription factor targets. The effects of cytokines on cell cycle regulatory molecules, which either promote or arrest cell cycle progression, have been more recently examined. In addition, the mechanisms by which cytokines regulate apoptotic proteins, which mediate survival vs death, are being elucidated. Identification and characterization of these complex, interconnected pathways has expanded our knowledge of leukemogenesis substantially. This information has the potential to guide the development of therapeutic drugs designed to target key intermediates in these pathways and effectively treat patients with leukemias and lymphomas. This review focuses on the current understanding of how hematopoietic cytokines such as IL-3, as well as its cognate receptor, are expressed and the mechanisms by which they transmit their growth regulatory signals. The effects of aberrant regulation of these molecules on signal transduction, cell cycle regulatory and apoptotic pathways in transformed hematopoietic cells are discussed. Finally, anti-neoplastic drugs that target crucial constituents in these pathways are evaluated.

Roles of the RassRafsMEKsERK pathway in leukemia therapy

The Ras/Raf/mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway is often implicated in sensitivity and resistance to leukemia therapy. Dysregulated signaling through the Ras/Raf/MEK/ERK pathway is often the result of genetic alterations in critical components in this pathway as well as mutations at upstream growth factor receptors. Unrestricted leukemia proliferation and decreased sensitivity to apoptotic-inducing agents and chemoresistance are typically associated with activation of pro-survival pathways. Mutations in this pathway and upstream signaling molecules can alter sensitivity to small molecule inhibitors targeting components of this cascade as well as to inhibitors targeting other key pathways (for example, phosphatidylinositol 3 kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt/mammalian target of rapamycin (mTOR)) activated in leukemia. Similarly, PI3K mutations can result in resistance to inhibitors targeting the Ras/Raf/MEK/ERK pathway, indicating important interaction points between the pathways (cross-talk). Furthermore, the Ras/Raf/MEK/ERK pathway can be activated by chemotherapeutic drugs commonly used in leukemia therapy. This review discusses the mechanisms by which abnormal expression of the Ras/Raf/MEK/ERK pathway can contribute to drug resistance as well as resistance to targeted leukemia therapy. Controlling the expression of this pathway could improve leukemia therapy and ameliorate human health.

Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.

Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.

Suppression of PTEN function increases breast cancer chemotherapeutic drug resistance while conferring sensitivity to mTOR inhibitors.

Ectopic expression of mutant forms of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) lacking lipid (G129E) or lipid and protein (C124S) phosphatase activity decreased sensitivity of MCF-7 breast cancer cells, which have wild-type PTEN, to doxorubicin and increased sensitivity to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. Cells transfected with a mutant PTEN gene lacking both lipid and protein phosphatase activities were more resistant to doxorubicin than cells transfected with the PTEN mutant lacking lipid phosphatase activity indicating that the protein phosphatase activity of PTEN was also important in controlling the sensitivity to doxorubicin, while no difference was observed between the lipid (G129E) and lipid and protein (C124S) phosphatase PTEN mutants in terms of sensitivity to rapamycin. A synergistic inhibitory interaction was observed when doxorubicin was combined with rapamycin in the phosphatase-deficient PTEN-transfected cells. Interference with the lipid phosphatase activity of PTEN was sufficient to activate Akt/mTOR/p70S6K signaling. These studies indicate that disruption of the normal activity of the PTEN phosphatase can have dramatic effects on the therapeutic sensitivity of breast cancer cells. Mutations in the key residues which control PTEN lipid and protein phosphatase may act as dominant-negative mutants to suppress endogenous PTEN and alter the sensitivity of breast cancer patients to chemo- and targeted therapies.

Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (Δ) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as ΔA-Raf:ER was 20- to 200-fold more efficient than either ΔRaf-1:ER or ΔB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive ΔRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, ΔRaf:ER-infected cells. Abrogation of cytokine dependency by the activated ΔRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated ΔRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.