Growth factor activation of MAP kinase requires cell adhesion.

The MAP kinase pathway is a major regulator of both normal and oncogenic growth. We report that activation of the MAP kinase ERK2 by serum or purified growth factors is strongly dependent on cell adhesion to extracellular matrix proteins. This effect is specific to soluble growth factors, since suspended cells still activate ERK2 in response to plating on fibronectin, and is reversible. Analysis of endogenous Ras and Raf show that these proteins are still activated by serum in suspended cells, whereas MEK activity is inhibited. Conversely, activation of ERK2 by activated mutants of Ras and Raf is still adhesion-dependent but activation by MEK is not. Consistent with these results, activated MEK enhances growth of ras-transformed cells in suspension but not when adherent. These results identify a novel synergism between cell adhesion- and growth factor-regulated pathways, and explain how oncogenic activation of MAP kinases induces both serum- and anchorage-independent growth.     

Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation,H-RasV12-Induced Senescence,and Transformation

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates the activation of the Raf/MEK/extracellular signal-regulated kinase (ERK) signal transduction pathway. KSR1 disruption in mouse embryo fibroblasts (MEFs) abrogates growth factor-induced ERK activation, H-Ras^V12-induced replicative senescence, and H-Ras^V12-induced transformation. Caveolin-1 has been primarily described as a major component of the coating structure of caveolae, which can serve as a lipid binding adaptor protein and coordinates the assembly of Ras, Raf, MEK, and ERK. In this study, we show that KSR1 interacts with caveolin-1 and is responsible for MEK and ERK redistribution to caveolin-1-rich fractions. The interaction between KSR1 and caveolin-1 is essential for optimal activation of ERK as a KSR1 mutant unable to interact with caveolin-1 does not efficiently mediate growth factor-induced ERK activation at the early stages of pathway activation. Furthermore, abolishing the KSR1–caveolin-1 interaction increases growth factor demands to promote H-Ras^V12-induced proliferation and has adverse effects on H-Ras^V12-induced cellular senescence and transformation. These data show that caveolin-1 is necessary for optimal KSR1-dependent ERK activation by growth factors and oncogenic Ras.     

Mechanisms of regulating the Raf kinase family

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.     

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway

Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.     

Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.     

Oncogenic K-Ras Regulates Bioactive Sphingolipids in a Sphingosine Kinase 1-dependent Manner

Sphingosine kinase 1 (SK1) is an important enzyme involved in the production of the bioactive lipid sphingosine 1-phosphate (S1P). SK1 is overexpressed in many forms of cancer, however, the contribution of SK1 to cancer progression is still unclear. One of the best characterized mutations found in several forms of human cancer is an activating point mutation in the Ras oncogene, which disrupts its GTPase activity and leads to stimulation of the MEK/ERK pathway. Because SK1 activity and subcellular localization have been shown to be regulated by ERK, we wished to investigate the effect of oncogenic Ras, a potent activator of the Raf/MEK/ERK pathway, on the activity of SK1 and sphingolipid metabolism. Using HEK293T cells transiently transfected with the K-RasG12V oncogene and both wild type and Sphk1(-/-) mouse embryonic fibroblasts stably infected with retroviral K-RasG12V, we found that K-RasG12V increases the production of S1P and decreases the production of ceramide in a SK1-dependent manner. In addition, we found that expression of the K-RasG12V oncogene leads to plasma membrane localization of SK1 and a reduction in cytosolic levels of SK1. This effect is likely mediated by the Raf/MEK/ERK pathway as constitutively active B-Raf or MEK1 are able to activate SK1, but constitutively active Akt1 is not. We believe this research has important implications for how sphingolipids may be contributing to oncogenic transformation and provide some of the first evidence for oncogenes inducing specific changes in sphingolipid metabolism through SK1 regulation.     

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.     

Ras-mutant Cancer Cells Display B-Raf Binding to Ras That Activates Extracellular Signal-regulated Kinase and Is Inhibited by Protein Kinase A Phosphorylation

The small G protein Ras regulates proliferation through activation of the mitogen-activated protein (MAP) kinase (ERK) cascade. The first step of Ras-dependent activation of ERK signaling is Ras binding to members of the Raf family of MAP kinase kinase kinases, C-Raf and B-Raf. Recently, it has been reported that in melanoma cells harboring oncogenic Ras mutations, B-Raf does not bind to Ras and does not contribute to basal ERK activation. For other types of Ras-mutant tumors, the relative contributions of C-Raf and B-Raf are not known. We examined non-melanoma cancer cell lines containing oncogenic Ras mutations and express both C-Raf and B-Raf isoforms, including the lung cancer cell line H1299 cells. Both B-Raf and C-Raf were constitutively bound to oncogenic Ras and contributed to Ras-dependent ERK activation. Ras binding to B-Raf and C-Raf were both subject to inhibition by the cAMP-dependent protein kinase PKA. cAMP inhibited the growth of H1299 cells and Ras-dependent ERK activation via PKA. PKA inhibited the binding of Ras to both C-Raf and B-Raf through phosphorylations of C-Raf at Ser-259 and B-Raf at Ser-365, respectively. These studies demonstrate that in non-melanocytic Ras-mutant cancer cells, Ras signaling to B-Raf is a significant contributor to ERK activation and that the B-Raf pathway, like that of C-Raf, is a target for inhibition by PKA. We suggest that cAMP and hormones coupled to cAMP may prove useful in dampening the effects of oncogenic Ras in non-melanocytic cancer cells through PKA-dependent actions on B-Raf as well as C-Raf.     

Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin

Ras activation induces a variety of cellular responses that depend on the specific activated effector, the intensity and amplitude of its activation, and the cellular type. Transient activation followed by a sustained but low signal of the Ras/Raf/MEK/ERK pathway is a common feature of cell proliferation in many systems. On the contrary, sustained, high activation is linked with either senescence or apoptosis in fibroblasts and to differentiation in neurones and PC12 cells. The temporal regulation of the pathway is relevant and not only depends on the specific receptor activated but also on the presence of diverse modulators of the pathway. We review here evidence showing that calcium (Ca(2+)) and calmodulin (CaM) are able to regulate the Ras/Raf/MEK/ERK pathway. CaM-binding proteins (CaMBPs) as Ras-GRF and CaM-dependent protein kinase IV (CaMKIV) positively modulate ERK1/2 activation induced by either NGF or membrane depolarisation in neurones. In fibroblasts, CaM binding to EGF receptor and K-Ras(B) may be involved in the downregulation of the pathway after its activation, allowing a proliferative signalling.     

MEK1 activation by PAK: a novel mechanism

Extracellular signal-Regulated Kinase (ERK) controls a variety of cellular processes, including cell proliferation and cell motility. While oncogenic mutations in Ras and B-Raf result in deregulated ERK activity and proliferation and migration in some tumor cells, other tumors exhibit elevated ERK signaling in the absence of these mutations. Here we provide evidence that PAK can directly activate MEK1 by a mechanism distinct from conventional Ras/Raf mediated activation. We find that PAK phosphorylation of MEK1 serine 298 stimulates MEK1 autophosphorylation on the activation loop, and activation of MEK1 activity towards ERK in in vitro reconstitution experiments. Serines 218 and/or 222 in the MEK1 activation loop are required for PAK-stimulated MEK1 activity towards ERK. MEK2, which is a poor target for PAK phosphorylation in cells, is not activated in this manner. Tissue culture experiments verify that this mechanism is used in suspended fibroblasts expressing mutationally activated PAK1. We speculate that aberrant signaling through PAK may directly induce anchorage-independent MEK1 activation in tumor cells lacking oncogenic Ras or Raf mutations, and that this mechanism may contribute to localized MEK signaling in focal contacts and adhesions during cell adhesion or migration.     

PAK1 is a novel MEK-independent raf target controlling expression of the IAP survivin in M-CSF-mediated osteoclast survival

As activation of the Ras/Raf/MEK/ERK pathway is a critical component of M-CSF-promoted osteoclast survival, determining specific mechanism by which M-CSF activates this signal transduction pathway is paramount towards advancing treatment of pathological conditions resulting in increased bone turnover. The p21 activated kinase PAK1 modulates activation of the Raf/MEK/ERK pathway by either directly activating Raf or priming MEK for activation by Raf. Therefore a role for PAK1 in M-CSF-mediated activation of the MEK/ERK pathway controlling osteoclast survival was assessed. Here we show that PAK1 is activated by M-CSF in a Ras-dependent mechanism that promotes osteoclast survival. Surprisingly, PAK1 did not modulate Raf activation or Raf-mediated MEK activation. M-CSF mediated activation of Raf was required for PAK1 activation and osteoclast survival promoted by PAK1. This survival response was MEK-independent as expression of constitutively active MEK did not rescue osteoclasts from apoptosis induced by blocking PAK1 function. Functionally, PAK1 promoted osteoclast survival by modulating expression of the IAP family member Survivin. M-CSF therefore functions to promote PAK1 activation as a novel MEK-independent Raf target to control Survivin-mediated osteoclast survival.     

Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.     

Raf-induced cell cycle progression in human TF-1 hematopoietic cells

Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to beta-estradiol-regulated DeltaRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21(Cip1), which are associated with G(1) progression. Activated DeltaRaf-1:ER and DeltaA-Raf:ER but not DeltaB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16(Ink4a), a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16(Ink4a) suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.     

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.     

DA-Raf,a dominant-negative antagonist of the Ras–ERK pathway,is a putative tumor suppressor

Activating mutations of RAS genes, particularly KRAS, are detected with high frequency in human tumors. Mutated Ras proteins constitutively activate the ERK pathway (Raf–MEK–ERK phosphorylation cascade), leading to cellular transformation and tumorigenesis. DA-Raf1 (DA-Raf) is a splicing variant of A-Raf and contains the Ras-binding domain (RBD) but lacks the kinase domain. Accordingly, DA-Raf antagonizes the Ras–ERK pathway in a dominant-negative fashion and suppresses constitutively activated K-Ras-induced cellular transformation. Thus, we have addressed whether DA-Raf serves as a tumor suppressor of Ras-induced tumorigenesis. DA-Raf(R52Q), which is generated from a single nucleotide polymorphism (SNP) in the RBD, and DA-Raf(R52W), a mutant detected in a lung cancer, neither bound to active K-Ras nor interfered with the activation of the ERK pathway. They were incapable of suppressing activated K-Ras-induced cellular transformation and tumorigenesis in mice, in which K-Ras-transformed cells were transplanted. Furthermore, although DA-Raf was highly expressed in lung alveolar epithelial type 2 (AE2) cells, its expression was silenced in AE2-derived lung adenocarcinoma cell lines with oncogenic KRAS mutations. These results suggest that DA-Raf represents a tumor suppressor protein against Ras-induced tumorigenesis.     

Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.     

Tumor Suppressor PTEN Inhibits Integrin- and Growth Factor–mediated Mitogen-activated Protein (MAP) Kinase Signaling Pathways

The tumor suppressor PTEN dephosphorylates focal adhesion kinase (FAK) and inhibits integrin-mediated cell spreading and cell migration. We demonstrate here that expression of PTEN selectively inhibits activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. PTEN expression in glioblastoma cells lacking the protein resulted in inhibition of integrin-mediated MAP kinase activation. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)- induced MAPK activation were also blocked. To determine the specific point of inhibition in the Ras/Raf/ MEK/ERK pathway, we examined these components after stimulation by fibronectin or growth factors. Shc phosphorylation and Ras activity were inhibited by expression of PTEN, whereas EGF receptor autophosphorylation was unaffected. The ability of cells to spread at normal rates was partially rescued by coexpression of constitutively activated MEK1, a downstream component of the pathway. In addition, focal contact formation was enhanced as indicated by paxillin staining. The phosphatase domain of PTEN was essential for all of these functions, because PTEN with an inactive phosphatase domain did not suppress MAP kinase or Ras activity. In contrast to its effects on ERK, PTEN expression did not affect c-Jun NH₂-terminal kinase (JNK) or PDGF-stimulated Akt. Our data suggest that a general function of PTEN is to down-regulate FAK and Shc phosphorylation, Ras activity, downstream MAP kinase activation, and associated focal contact formation and cell spreading.     

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.