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

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Abstract

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/p21Cip−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/p21Cip−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.

Introduction

The Ras/Raf/MEK/ERK cascade couples signals from cell surface receptors to transcription factors, which regulate gene expression. A diagrammatic overview of the Ras/Raf/MEK/ERK pathway is presented in Fig. 1. This pathway is often activated in certain tumors by mutations or overexpression of upstream molecules such as BCR-ABL and epidermal growth factor receptor (EGFR). The Raf/MEK/ERK pathway also has profound effects on the regulation of apoptosis by the post-translational phosphorylation of apoptotic regulatory molecules including Bad, caspase 9 and Bcl-2. Depending upon the stimulus and cell type, this pathway can transmit signals which regulate apoptosis and cell cycle progression (Steelman et al., 2004a, Steelman et al., 2004b). A survey of the literature documents the daily increase in our understanding of this pathway, as there are multiple members of the kinase, transcription factor, apoptotic regulator and caspase executioner families, which can be activated or inactivated by protein phosphorylation. Raf, either through downstream MEK and ERK or independently of MEK and ERK, can induce the phosphorylation of proteins which control apoptosis. Additional signal transduction pathways interact with the Raf/MEK/ERK pathway to positively or negatively regulate its activity. Abnormal activation of this pathway occurs in human cancer due to mutations at Ras and B-Raf as well as genes in other pathways (e.g., PI3K, PTEN, Akt), which serve to regulate Raf activity. The Raf/MEK/ERK pathway also influences chemotherapeutic drug resistance as ectopic activation of Raf induces resistance to doxorubicin and paclitaxel in breast cancer cells. Mutations at B-Raf have been detected in various malignancies including melanoma, thyroid and breast cancer (Garnett and Marais, 2004). For all the above reasons, the Raf/MEK/ERK and PI3K/Akt pathways are important pathways to target for therapeutic intervention. Inhibitors of Ras, Raf, MEK, PI3K, PDK, Akt, mTOR and some downstream targets have been developed and many are currently in clinical trials. Naturally, some inhibitors are better than others and certain “specific” inhibitors actually inhibit multiple kinases.

Ras is a small GTP-binding protein, which is the common upstream molecule of several signaling pathways including Raf/MEK/ERK, PI3K/Akt and RalEGF/Ral (Peyssonnaux et al., 2000). Three Ras proteins have been identified, namely Ha-Ras, Ki-Ras and N-Ras. Ras proteins show varying abilities to activate the Raf/MEK/ERK and PI3K/Akt cascades. For example, Ki-Ras has been associated with the Raf/MEK/ERK pathway while Ha-Ras is associated with PI3K/Akt activation (Yan et al., 1998). Different mutation frequencies have been observed between Ras genes in human cancer (Ki-Ras>Ha-Ras). It is important to realize that Ki-Ras is the more frequently mutated Ras isoform in human cancer.

For Ras to be targeted to the cell membrane, it must be farnesylated by farnesyl transferase (Ha-, Ki- and N-Ras) or geranylgeranylated by geranylgeranyl transferase (N-and Ki-Ras). Farnesylation and geranylgeranylation both occur on the same cysteine residue. Ras preferentially undergoes farnesylation; however, in the presence of farnesylation inhibitors, N-Ras and Ki-Ras can undergo gernylgernylation. Farnesylation and geranylgeranylation are important for targeting Ras to the cell membrane. Ha-Ras and N-Ras can also undergo palmitoylation with Ha-Ras having two palmitoylation sites and N-Ras having one palmitoylation site. Ki-Ras appears to lack a palmitoylation site. It is believed that palmitoylation has a role in plasma membrane microlocalization. These post-translational modifications are important as they represent sites for therapeutic intervention which will be discussed later.

Following binding of cytokines, growth factors or mitogens to their appropriate receptors, activation of the coupling complex Shc/Grb2/SOS occurs. Upon stimulation by Shc/Grb2/SOS, the inactive Ras exchanges GDP for GTP and undergoes a conformational change and becomes active. The GTP-bound active Ras can then recruit Raf to cell membrane (see Fig. 2).

The mammalian Raf gene family consists of A-Raf, B-Raf and Raf-1 (C-Raf). Raf is a serine/threonine (S/T) kinase and is normally activated by a complex series of events including: (i) recruitment to the plasma membrane mediated by an interaction with Ras (Yan et al., 1998); (ii) dimerization of Raf proteins (Luo et al., 1996); (iii) phosphorylation/dephosphorylation on different domains (Fabian et al., 1993); (iv) disassociation with the Raf kinase inhibitory protein (RKIP) (Yeung et al., 1999; Dhillon et al., 2002) and (v) association with scaffolding complexes (e.g., kinase suppressor of Ras, (KSR) (Blalock et al., 1999; Lee and McCubrey, 2002; Chang et al., 2003a) (see Fig. 2). Raf activity is further modulated by chaperonin proteins including Bag1, 14-3-3 (Fantl et al., 1994) and heat shock protein 90 (Hsp90) (Blagosklonny, 2002).

There are at least 13 regulatory phosphorylation sites on Raf-1 (Steelman et al., 2004a, Steelman et al., 2004b). Some of these sites (e.g., S43, S259 and S621) are phosphorylated when Raf-1 is inactive. This allows 14-3-3 to bind Raf-1 and confer a configuration which is inactive (see Fig. 2). Upon cell stimulation, S621 becomes transiently dephosphorylated by a phosphatase not yet identified. Phosphatases such as protein phosphatase 2A (PP2A) dephosphorylate S259 (Dhillon et al., 2002). 14-3-3 then disassociates from Raf-1. This allows Raf-1 to be phosphorylated at S338, Y340 and Y341, which renders Raf-1 active. A Src family kinase is likely responsible for phosphorylation at Y340 and Y341 (Marais et al., 1997). The phosphatases, which dephosphorylate S621 and other Raf phosphorylation sites, excluding S259, are unknown.

Y340 and Y341, the phosphorylation targets of Src family kinases, are conserved in A-Raf (Y299 and Y300), but are replaced with aspartic acid (D) at the corresponding positions in B-Raf (D492 and D493) (Fabian et al., 1993; Marais et al., 1997). The negatively charged aspartic acid residues mimic activated residues, which makes B-Raf highly active. Maximal activation of Raf-1 and A-Raf requires both Ras and Src activity while B-Raf activation is Src-independent (Marais et al., 1997). Interestingly, as will be discussed later, a greater number of mutations are detected at B-Raf than either Raf-1 or A-Raf, in human cancer. This may have resulted from a simpler mode of activation and selection of cells containing B-Raf mutations.

The S338 residue present in Raf-1 is conserved among the three Raf isoforms; however, in B-Raf (S445), this corresponding site is constitutively phosphorylated (Mason et al., 1999). S338 phosphorylation on Raf-1 is stimulated by Ras and is dependent on p21-activated protein kinase (PAK) (Diaz et al., 1997). Other phosphorylation sites in Raf-1 that may modulate its activity include: S43, S339, T491, S494, S497, S499, S619 and S621. Protein kinase C (PKC) has been shown to activate Raf and induce cross-talk between PKC and Raf/MEK/ERK signaling pathways (Kolch et al., 1993). S497 and S499 were identified as the target residues on Raf-1 for PKC phosphorylation. However, other studies suggest that these sites are not necessary for Raf-1 activation (Barnard et al., 1998).

Raf activity is negatively regulated by phosphorylation on the CR2 regulatory domain. Akt and protein kinase A (PKA) phosphorylate S259 on Raf-1 and inhibit its activity (Rommel et al., 1999). Furthermore, Akt or the related serum/glucocorticoid regulated kinase phosphorylate B-Raf on S364 and S428 and inactivate its kinase activity (Guan et al., 2000; Zhang et al., 2001). These S-phosphorylated Rafs associate with 14-3-3 and become inactive.

Recently a scaffolding protein, RKIP has been shown to inhibit Raf-1 activity (Yeung et al., 1999; Corbit et al., 2003). RKIP is a member of the phosphatidylethanolamine-binding protein family. This multi-gene family is evolutionally conserved and has related members in bacteria, plants and animals (Corbit et al., 2003). Interesting RKIP can bind Raf or MEK/ERK complex but not to Raf, MEK and ERK together. Various isoforms of PKC have been shown to phosphorylate RKIP on S153 which results in the disassociation of Raf and RKIP. The role of RKIP in metastasis will be discussed later.

Hsp90 may serve to stabilize activated Raf. Drugs such as geldanamycin inhibit Hsp90 and result in the rapid degradation of Raf (Blagosklonny, 2002). Raf is also a target of caspases. In this case, Raf is degraded and the cycle outlined in Fig. 2 is irreversibly broken.

The importance of Raf-1 in the Raf/MEK/ERK signal transduction pathway has come into question due to the discovery that B-Raf was a much more potent activator of MEK compared to Raf-1 and A-Raf. Many of the “functions” of Raf-1 persist in Raf-1 knock-out mice likely due to function of endogenous B-Raf (Mercer and Pritchard, 2003). Interestingly and controversially, it was recently proposed that B-Raf is not only the major activator of Mitogen-activated protein kinase/ERK kinase (MEK1), but B-Raf is also involved in Raf-1 activation. B-Raf may be temporally activated before Raf-1. However, there may be different subcellular localizations of B-Raf and Raf-1 within the cell that exert different roles in signaling and apoptotic pathways (Brummer et al., 2002). In some cases, B-Raf may transduce its signal through Raf-1. The reasons for this added step in the Raf/MEK/ERK kinase cascade are not obvious but may represent another layer of fine tuning.

MEK1 is a tyrosine (Y-) and S/T-dual specificity protein kinase (Alessi et al., 1994). Its activity is positively regulated by Raf phosphorylation on S residues in the catalytic domain. All three Raf family members are able to phosphorylate and activate MEK but different biochemical potencies have been observed (B-Raf>Raf-1⪢A-Raf) (Alessi et al., 1994). Activated MEK1 mutants have been constructed which will abrogate the cytokine dependence of hematopoietic cells and morphologically transform NIH-3T3 cells (Blalock et al., 2000). Another interesting aspect regarding MEK1 is that its predominate downstream target is ERK. In contrast, both upstream Raf and downstream ERK have multiple targets. Thus, therapeutic targeting of MEK1 is relatively specific.

Extracellular-signal-regulated kinases 1,2 (ERK) are S/T kinases and their activities are positively regulated by phosphorylation mediated by MEK1 and MEK2. ERKs can directly phosphorylate many transcription factors including Ets-1, c-Jun and c-Myc. ERK can also phosphorylate and activate the 90 kDa ribosomal S6 kinase (p90Rsk), which then leads to the activation of the transcription factor CREB (Chang et al., 2003a) (see Fig. 1). Moreover, through an indirect mechanism, ERK can lead to activation of the NF-κB transcription factor (nuclear factor immunoglobulin κ chain enhancer-B cell) by phosphorylating and activating inhibitor κB kinase. ERK1 and ERK2 are differentially regulated. ERK2 has been positively associated with proliferation while ERK1 may inhibit the effects of ERK2 in certain cells (Pouyssegur et al., 2002).

Recently, Raf-1 has been postulated to have non-enzymatic functions and serve as a docking protein (Hindley and Kolch, 2002) (see Fig. 3). Raf-1 has been proposed to have important functions at the mitochondrial membrane. Expression of membrane targeted Raf was shown to complement a BCR-ABL mutant in abrogating the cytokine dependence of hematopoietic cells (Salomoni et al., 1998). In these mutant BCR-ABL transfected cells, Bad was expressed in the hyperphosphorylated inactive form and released from the mitochondria into the cytosol. In contrast, in the cells containing the BCR-ABL mutant but lacking the membrane-targeted Raf-1, which were not cytokine-dependent, Bad was hypophosphorylated and present in the mitochondrial fraction. BCR-ABL may interact with mitochondrial targeted Raf-1 to alter the phosphorylation of Bad at the mitochondrial membrane and hence regulate (prevent) apoptosis in hematopoietic cells containing the BCR-ABL chromosomal translocation (Neshat et al., 2000). This survival mechanism was independent of MEK and ERK.

Ras and its downstream effector molecules affect the expression of many molecules which regulate cell cycle including p16Ink4a, p15Ink4b and p21Cip1, and can lead to premature cell cycle arrest at the G1 phase. This p15Ink4b/p16Ink4a or p21Cip1-mediated premature G1 arrest and subsequent senescence is dependent on the Raf/MEK/ERK pathway (Blalock et al., 1999; Malumbres et al., 2000).

Overexpression of activated Raf proteins is associated with such divergent responses as cell growth, cell cycle arrest or even apoptosis (Hoyle et al., 2000; Chang and McCubrey, 2001; Chang et al., 2002). The fate of the cells depends on the level and isoform of Raf kinase expressed. Ectopic overexpression of Raf proteins is associated with cell proliferation in cells including hematopoietic cells (Hoyle et al., 2000); erythroid progenitor cells (Sanders et al., 1998); and A10 smooth muscle cells (Cioffi et al., 1997). However, overexpression of activated Raf proteins is associated with cell cycle arrest in rat Schwann cells, mouse PC12 cells, human promyelocytic leukemia HL-60 cells, small cell lung cancer cell lines and some hematopoietic cells (Yen et al., 1994; Ravi et al., 1998; Lloyd et al., 1997). Depending on the Raf isoform, overexpression of Raf can lead to cell proliferation (A-Raf or Raf-1) or cell growth arrest (B-Raf) in NIH-3T3 fibroblast and FDC-P1 hematopoietic cells (Woods et al., 1997; Hoyle et al., 2000; Shelton et al., 2004). It is not clear why overexpression of the Raf gene can lead to such conflicting results, but it has been suggested that the opposite outcomes may be determined by the amount or activity of the particular Raf oncoprotein (Chang and McCubrey, 2001; Shelton et al., 2004).

NIH-3T3 cells have been transfected with the three different Raf genes. The introduced A-Raf molecule was able to upregulate the expression of cyclin D1, cyclin E, Cdk2 and Cdk4 and down-regulate the expression of Cdk inhibitor p27Kip1 (Woods et al., 1997). These changes induced the cells to pass through G1 phase and enter S phase. It should be remembered that A-Raf is the weakest Raf kinase and its role in cell proliferation is not clear. However, in B-Raf and Raf-1 transfected NIH-3T3 cells, there was also a significant induction of p21Cip1, which led to G1 arrest. Using cytokine-dependent FDC-P1 hematopoietic cells transfected with conditionally active mutant Raf-1, A-Raf and B-Raf genes as a model, we have demonstrated that moderate Raf activation, such as A-Raf and Raf-1, led to cell proliferation, which was associated with the induction of cyclin expression and Cdk activity. However, ectopic expression of the much more potent B-Raf led to apoptosis (Chang and McCubrey, 2001; Shelton et al., 2004).

An alternative explanation for the diverse proliferative results obtained with the three Raf genes is the different biological effects of A-Raf, B-Raf and Raf-1. The individual functions of these three different Raf proteins are not fully understood. Even though it has been shown that all three Raf proteins are activated by oncogenic Ras, target the same downstream molecules, i.e., MEK1 and MEK2, and use the same adapter protein for conformational stabilization; different biological and biochemical properties among them have been reported and their functions are not always compensatable (Wadewitz et al., 1993; Pritchard et al., 1996; Wojnowski et al., 1997; Kolch, 2001). It is safe to say that even as we learn more about the intricacies of these Raf molecules, we discover that there are more questions regarding their specificities and mechanism of activation.

Clearly Raf has many roles in kinase cascades and downstream transcription factors which regulate apoptosis. The Raf/MEK/ERK cascade and Raf by itself have diverse effects on key molecules involved in the prevention of apoptosis. For many years now, it has been known that the Raf/MEK/ERK pathway can phosphorylate Bad on S112 which contributes to its inactivation and subsequent sequesterization by 14-3-3 proteins (Zha et al., 1996). This allows Bcl-2 to form homodimers and an anti-apoptotic response is generated. Recently, it has been shown that the Raf/MEK/ERK cascade can phosphorylate caspase 9 on residue (T125) which contributes to the inactivation of this protein (Allan et al., 2003). Interesting, both Bad and caspase 9 are also phosphorylated, on different residues, by the Akt pathway indicating that the Raf/MEK/ERK and PI3K/Akt pathways can cross-talk and result in the prevention of apoptosis (Crdone et al., 1998). More controversially, Bcl-2 is also phosphorylated by the Raf/MEK/ERK cascade on certain residues, in the loop region, which has been associated with enhanced anti-apoptotic activity (Deng et al., 2001). As noted earlier, Raf has MEK- and ERK-independent functions at the mitochondrial membrane. For example, mitochondrial localized Raf can phosphorylate Bad, which results in its disassociation from the mitochondrial membrane.

Recently, Raf-1 was shown to interact with mammalian sterile 20-like kinase (MST-2) and prevent its dimerization and activation (O’Neill et al., 2004). MST-2 is a kinase, which is activated by pro-apoptotic agents such as staurosporine and Fas ligand. Raf-1 but not B-Raf binds MST-2. Depletion of MST-2 from Raf-1–/– cells abrogated sensitivity to apoptosis. Overexpression of MST-2 increased sensitivity to apoptosis. It was proposed that Raf-1 might control MST-2 by sequestering it into an inactive complex. This complex of Raf-1:MST-2 is independent of MEK and downstream ERK. Raf-1 can also interact with the apoptosis signal related kinase (ASK1) to inhibit apoptosis (Du et al., 2004). ASK1 is a general mediator of apoptosis and it is induced in response to a variety of cytotoxic stresses including TNF, Fas and reactive oxygen species. ASK1 appears to be involved in the activation of the JNK and p38 MAP kinases. This is another interaction of Raf-1 which is independent of MEK and ERK.

A common feature of cells transformed by Raf is the expression of growth factors, which often have an autocrine effect. NIH-3T3 cells transformed by activated Raf secrete heparin-binding Epidermal Growth Factor (hbEGF) (McCarthy et al., 1995). Hematopoietic cells transformed by activated Raf genes often express GM-CSF, which has autocrine growth factor effects (McCarthy et al., 1997; McCubrey et al., 1998, McCubrey et al., 2001; Hoyle et al., 2000; Shelton et al., 2003a, Shelton et al., 2003b). Kaposi's sarcoma transformed B cells, which express elevated levels of B-Raf, express high levels of vascular endothelial growth factor (VEGF) (Akula et al., 2005). Recently, it has been shown that B-Raf increases the infectivity of Kaposi's Sarcoma Virus (Akula et al., 2004). One mechanism responsible for this enhancement of viral infection by B-Raf is its ability to induce VEGF expression (Ford et al., 2004; Hamden et al., 2004, Hamden et al., 2005). Many growth factor genes contain in their promoter regions binding sites for transcription factors phosphorylated by the Raf/MEK/ERK pathway (Chang et al., 2003a). Thus aberrant Raf expression may establish an autocrine loop, which results in the continuous stimulation of cell growth. Alternatively, the VEGF expression induced by Raf can promote angiogenesis. Raf-induced growth factor expression will contribute to both the prevention of apoptosis as well as chemotherapeutic drug resistance as growth factor expression has been associated with both the prevention of apoptosis and drug resistance (Alexia et al., 2004).

After ligand-induced activation of specific receptors, PI3K can be activated by two mechanisms. First, a phosphorylated Y residue on the receptor serves as a docking site for the p85 regulatory subunit of PI3K (Chang et al., 2003b). This recruits the catalytic subunit of PI3K, p110, to this complex. Alternatively, upon activation of the cytokine receptor by the appropriate ligand, the Shc protein binds the receptor to enable the Grb-2 and Sos proteins to form a complex which results in the activation of Ras. Ras is then able to induce the membrane translocation and activation of p110 subunit of PI3K.

Activated PI3K converts phosphatidylinositol 4,5 phosphate (PIP2) into phosphatidylinositol 3,4,5 phosphate (PIP3) which results in the membrane localization of phosphoinositol-dependent kinase-1 (PDK1) via its pleckstrin homology (PH) domain. Akt is also recruited to the lipid-rich plasma membrane by its PH domain and is phosphorylated at residues by T308 and S473 by PDK1 and an unidentified kinase, respectively. An overview of the PI3K/Akt pathway is presented in Fig. 4.

Akt is the primary mediator of PI3K-initiated signaling; it has a number of downstream substrates that can each contribute to the onset of cancer. Among these targets are Bad, procaspase-9, Iκ-K, the forkhead family of transcription factors (FKHR/AFX), GSK-3, p21CIP1 and others (Nicholson and Anderson, 2002; Chang et al., 2003b). It is worth noting that Akt can cause the activation of specific substrates (e.g., Iκ-K and CREB) or may mediate the inactivation of other proteins (e.g., Raf, Bad, procaspase-9, FOXO3 and GSK-3).

In addition, this pathway also includes phosphatases that serve to negatively regulate the growth-promoting effects of PI3K activity. The phosphatases PTEN and SHIP-1/2 can remove the phosphates from PIP3 (Steelman et al., 2004b). Mutations in these phosphatases, which eliminate their activity, can lead to tumor progression. Consequently, the genes encoding these phosphatases are referred to as anti-oncogenes or tumor suppressor genes.

Amplification of ras proto-oncogenes and activating mutations that lead to the expression of constitutively active Ras proteins are observed in approximately 30% of human cancers (Flotho et al., 1999; Stirewalt et al., 2001). Recent studies have indicated that B-Raf is mutated in approximately 7% of all cancers (Garnett and Marais, 2004). However, this frequency may change as more and diverse tumors are examined for B-Raf mutation.

For many years, the Raf oncogenes were not thought to be frequently mutated in human cancer and most attention to abnormal activation of this pathway was dedicated to Ras mutations which can regulate both the Raf/MEK/ERK and PI3K/Akt pathways. However, recently it was shown that B-Raf is frequently mutated in certain types of cancer, especially melanoma (27–70%), papillary thyroid cancer (36–53%), colorectal cancer (5–22%) and ovarian cancer (30%) (Davis et al., 2002; Garnett and Marais, 2004; Libra et al., 2005). The reasons for mutation at B-Raf and not Raf-1 or A-Raf are not entirely clear. Based on the mechanism of activation of B-Raf, it may be easier to select for B-Raf than either Raf-1 or A-Raf mutations. As stated previously, activation of B-Raf would require one genetic mutation whereas activation of either Raf-1 or A-Raf would require two genetic events. It has been proposed recently that the structure of B-Raf, Raf-1 and A-Raf may dictate the ability of mutations to occur at these molecules, which can permit the selection of activated oncogenic forms (Fransen et al., 2004; Garnett and Marais, 2004; Wan et al., 2004). These predictions have arisen from determining the crystal structure of B-Raf (Wan et al., 2004). Like many enzymes, B-Raf is proposed to have small and large lobes, which are separated, by a catalytic cleft. The structural and catalytic domains of B-Raf and the importance of the size and positioning of the small lobe may be critical in its ability to be stabilized by certain activating mutations. In contrast, the precise substitutions in A-Raf and Raf-1 are not predicted to result in small lobe stabilization thus preventing the selection of mutations at A-Raf and Raf-1, which would result in activated oncogenes (Wan et al., 2004). Raf-1 has been known for years to interact with Hsp90. Hsp90 may stabilize activated Raf-1, B-Raf and A-Raf. The role that Hsp90 plays in the mutation and selection of activated Raf mutation is highly speculative and very intriguing (see Fig. 5).

The most common B-Raf mutation is a change at nucleotide 600 which converts a valine to a glutamic acid (V600E) (Garnett and Marais, 2004). This B-Raf mutation accounts for over 90% of the B-Raf mutations found in melanoma and thyroid cancer. It has been proposed that B-Raf mutations may occur in certain cells, which express high levels of B-Raf due to hormonal stimulation. Certain hormonal signaling will elevate intracellular cAMP levels, which result in B-Raf activation, which leads to proliferation. Melanocytes and thyrocytes are two such cell types, which have elevated B-Raf expression as they are often stimulated by the appropriate hormones (Busca et al., 2000). Moreover, it now thought that B-Raf is the more important kinase in the Raf/MEK/ERK cascade (Garnett and Marais, 2004), thus mutation at B-Raf activates downstream MEK and ERK. In some models, wild-type (WT) and mutant B-Raf activates Raf-1, which in turn activates MEK and ERK (Garnett and Marais, 2004).

In some cells, B-Raf mutations are believed to be initiating events and not sufficient for full-blown neoplastic transformation (Rajagopalan et al., 2002; Yuen et al., 2002). Moreover, there appears to be cases where certain B-Raf mutations (V600E) and Ras mutations are not permitted in the transformation process as they might result in hyperactivation of Raf/MEK/ERK signaling and expression, which leads to cell cycle arrest (Davis et al., 2002). In contrast, there are other situations, which depend on the particular B-Raf mutation and require both B-Raf and Ras mutations for transformation. The B-Raf mutations in these cases result in weaker levels of B-Raf activity (Davis et al., 2002; Yuen et al., 2002).

Different B-Raf mutations have been mapped to various regions of the B-Raf protein. However, the mutations at the aforementioned 600 residue in B-Raf appear to be the most common. This mutation (V600E) results in activation of B-Raf and downstream MEK and ERK. Some of the other B-Raf mutations are believed to result in B-Raf molecules with impaired B-Raf activity, which must signal through Raf-1 (Garnett and Marais, 2004). Others mutations, such as D593V, may activate alternative signal transduction pathways (36). A diagram of the B-Raf mutations and effects of stabilization is presented in Fig. 5.

The relationship between dysregulated PI3K activity and the onset of cancer is well-documented. For example, a mutated version of the p85 subunit of PI3K has recently been isolated from a Hodgkin's Lymphoma-derived cell line (CO) (Jucker et al., 2002). Further evidence has shown that PI3K is the predominant growth-factor-activated pathway in LNCaP human prostate carcinoma cells (Lin et al., 1999). Other reports directly implicate PI3K activity in a variety of human tumors including breast cancer (Fry, 2001), lung cancer (Lin et al., 2001), melanomas (Krasilnikov et al., 1999) and leukemia (Martinez-Lorenzo et al., 2000) among others. Further evidence supports the notion that Akt (a.k.a., protein kinase B, PKB), a downstream kinase of PI3K, is also heavily involved in the malignant transformation of cells (Nicholson and Anderson, 2002). Taken together, these collective data endorse the substantial role that PI3K-signaling plays in oncogenesis. Moreover, targeted inhibition of the central components of this pathway appears to be an excellent choice for future drug formulations.

Recently, a role for RKIP in cancer was hypothesized. Certain advanced prostate cancers express lower amounts of RKIP than less malignant prostate cancer specimens (Fu et al., 2003; Keller et al., 2004). Inhibition of RKIP expression makes certain prostate cells more metastatic (Fu et al., 2003). The mechanism responsible for this increase in metastasis is believed to be due to the enhanced activity of the Raf/MEK/ERK signaling pathways. RKIP is not thought to alter the tumorigenic properties of prostate cancer cells; rather it is thought to be a suppressor of metastasis and may function by decreasing vascular invasion (Fu et al., 2004).

The role of the Raf/MEK/ERK pathway in prostate cancer remains controversial. The studies with RKIP suggest that increasing Raf activity, by inhibition of RKIP after phosphorylation by PKC, is somehow linked with metastasis in prostate cancer (Keller et al., 2004). However, the Raf/MEK/ERK pathway may be shut off in advanced prostate cancer due to the deletion of the PTEN gene, which normally regulates the activity of Akt by counterbalancing PI3K activity (Steelman et al., 2004a, Steelman et al., 2004b). In some cells, Akt may inhibit Raf-1 activity by phosphorylation of Raf-1 on S259.

Section snippets

DNA sequencing of the B-Raf gene in tumor samples

The sequence of the B-Raf gene in patient samples was performed as described in Libra et al. (2005). A detailed description of the recruitment of the patient samples is presented in Libra et al. (2005).

Cell lines and growth factors

Cells were maintained in a humidified 5% CO2 incubator with RPMI-1640 [(RPMI) Invitrogen, Carlsbad, CA, USA] complemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, GA, USA). The interleukin (IL-3)-dependent FL5.12 and FDC-P1 murine cell lines were cultured in this medium

Structure of the B-Raf gene in European-based cancer patients

We have begun to investigate the structure of the B-Raf gene in various human malignancies in a European-Based Cancer Group (Catania and Aviano, Italy) (Libra et al., 2005). It is important to compare the frequency of mutation of genes involved in cancer in different ethnic groups. Recently it has been observed that Japanese non-small cell lung cancer patients have a higher frequency of mutation at the EGFR gene which confers sensitivity to treatment with EGFR kinase inhibitors (Lynch et al.,

Summary

Over the past 25 years, there has been much progress in elucidating the involvement of the Ras/Raf/MEK/ERK and PI3K/PTEN/PDK/Akt cascades in etiology of human neoplasia and the induction of chemotherapeutic drug resistance. From initial seminal studies which elucidated the oncogenes present in avian and murine oncogenes, we learned that ErbB, Ras, Src, Abl, Raf, PI3K, Akt, Jun, Fos, Ets and NF-κB were originally cellular genes which were captured by retroviruses. Biochemical studies defined and

Acknowledgments

JAM and RAF have been supported in part by a grant from the NIH (R01098195).

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