Oncogenic Ras Blocks Anoikis by Activation of a Novel Effector Pathway Independent of Phosphatidylinositol 3-Kinase

Activated Ras, but not Raf, causes transformation of RIE-1 rat intestinal epithelial cells, demonstrating the importance of Raf-independent effector signaling in mediating Ras transformation. To further assess the contribution of Raf-dependent and Raf-independent function in oncogenic Ras transformation, we evaluated the mechanism by which oncogenic Ras blocks suspension-induced apoptosis, or anoikis, of RIE-1 cells. We determined that oncogenic versions of H-, K-, and N-Ras, as well as the Ras-related proteins TC21 and R-Ras, protected RIE-1 cells from anoikis. Surprisingly, our analyses of Ras effector domain mutants or constitutively activated effectors indicated that activation of Raf-1, phosphatidylinositol 3-kinase (PI3K), or RalGDS alone is not sufficient to promote Ras inhibition of anoikis. Treatment of Ras-transformed cells with the U0126 MEK inhibitor caused partial reversion to an anoikis-sensitive state, indicating that extracellular signal-regulated kinase activation contributes to inhibition of anoikis. Unexpectedly, oncogenic Ras failed to activate Akt, and treatment of Ras-transformed RIE-1 cells with the LY294002 PI3K inhibitor did not affect anoikis resistance or growth in soft agar. Thus, while important for Ras transformation of fibroblasts, PI3K may not be involved in Ras transformation of RIE-1 cells. Finally, inhibition of epidermal growth factor receptor kinase activity did not overcome Ras inhibition of anoikis, indicating that this autocrine loop essential for transformation is not involved in anoikis protection. We conclude that a PI3K- and RalGEF-independent Ras effector(s) likely cooperates with Raf to confer anoikis resistance upon RIE-1 cells, thus underscoring the complex nature by which Ras transforms cells.     

Protein kinase Ciota is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C iota (PKCiota) has been implicated in Ras signaling, however, a role for PKCiota in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCiota is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCiota in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCiota (kdPKCiota) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCiota in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCiota. Our data demonstrate that PKCiota is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCiota, and Rac1.     

Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the Raf-MEK-extracellular signal-regulated kinase pathway

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.     

Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.     

Acetylcholine muscarinic m1 receptor regulation of cyclic AMP synthesis controls growth factor stimulation of Raf activity.

Acetylcholine muscarinic m2 receptors (m2R) couple to heterotrimeric Gi proteins and activate the Ras/Raf/mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase in Rat 1a cells. In contrast to the m2R, stimulation of the acetylcholine muscarinic m1 receptor (m1R) does not activate the Ras/Raf/mitogen-activated protein kinase regulatory pathway in Rat 1a cells but rather causes a pronounced inhibition of epidermal growth factor and platelet-derived growth factor receptor activation of Raf. In Rat 1a cells, m1R stimulation of phospholipase C beta and the marked rise in intracellular calcium stimulated cyclic AMP (cAMP) synthesis, resulting in the activation of protein kinase A. Stimulation of protein kinase A inhibited Raf activation in response to growth factors. Platelet-derived growth factor receptor stimulation of phosphatidylinositol 3-kinase activity was not affected by either m1R stimulation or protein kinase A activation in response to forskolin-stimulated cAMP synthesis. GTP loading of Ras in response to growth factors was unaffected by protein kinase A activation but was partially inhibited by carbachol stimulation of the m1R. Therefore, protein kinase A action at the Ras/Raf activation interface selectively inhibited only one branch of the signal transduction network initiated by tyrosine kinases. Specific adenylyl cyclases responding to different signals, including calcium, with enhanced cAMP synthesis will regulate Raf activation in response to Ras.GTP. Taken together, the data indicate that G protein-coupled receptors can positively and negatively regulate the responsiveness of tyrosine kinase-stimulated mitogenic response pathways.     

Negative regulation of the alpha interferon-induced antiviral response by the Ras/Raf/MEK pathway

Interferon (IFN) is one of the molecules released by virus-infected cells, resulting in the establishment of an antiviral state within infected and neighboring cells. IFN-induced antiviral response may be subject to modulation by the cellular signaling environment of host cells which impact the effectiveness of viral replication. Here, we show that cells with an activated Ras/Raf/MEK signaling cascade allow propagation of viruses in the presence of IFN. Ras-transformed (RasV12) and vector control NIH 3T3 cells were infected with vesicular stomatitis virus (VSV) or an IFN-sensitive vaccinia virus (delE3L) in the presence of alpha interferon. While IFN protected vector control cells from infection by both viruses, RasV12 cells were susceptible to viral infection regardless of the presence of IFN. IFN sensitivity was restored in RasV12 cells upon RNA interference (RNAi) knockdown of Ras. We further investigated which elements downstream of Ras are responsible for counteracting IFN-induced antiviral responses. A Ras effector domain mutant that can only stimulate the Raf kinase family of effectors was able to suppress the IFN response and allow VSV replication. IFN-induced antiviral mechanisms were also restored in RasV12 cells by treatment with a MEK inhibitor (U0126 or PD98059). Moreover, by using RNAi to MEK1 and MEK2, we determined that MEK2, rather than MEK1, is responsible for suppression of the IFN response. In conclusion, our results suggest that activation of the Ras/Raf/MEK pathway downregulates IFN-induced antiviral response.     

Growth factor ligands of tyrosine kinase receptors activate the Rous sarcoma virus promoter by a Ras- and Raf-dependent mechanism

In PC12 cells, NGF and other ligands of tyrosine kinase receptors increased the activity of the Rous sarcoma virus (RSV) promoter. IGF-1 stimulated the RSV promoter in SH-SY5Y neuroblastoma cells. This promoter was also activated by oncogenic Ras and Raf in different cell types, and growth factor induction was inhibited by expression of dominant negative forms of ras and raf, showing that its effect is mediated by a Ras- and Raf-dependent mechanism. Therefore, the RSV promoter should not be used in the determination of transfection efficiency in the studies on regulation of gene expression by ligands of tyrosine kinase receptors, ras and raf.     

TC21 causes transformation by Raf-independent signaling pathways.

Although the Ras-related protein TC21/R-Ras2 has only 55% amino acid identity with Ras proteins, mutated forms of TC21 exhibit the same potent transforming activity as constitutively activated forms of Ras. Therefore, like Ras, TC21 may activate signaling pathways that control normal cell growth and differentiation. To address this possibility, we determined if regulators and effectors of Ras are also important for controlling TC21 activity. First, we determined that Ras guanine nucleotide exchange factors (SOS1 and RasGRF/CDC25) synergistically enhanced wild-type TC21 activity in vivo and that Ras GTPase-activating proteins (GAPs; p120-GAP and NF1-GAP) stimulated wild-type TC21 GTP hydrolysis in vitro. Thus, extracellular signals that activate Ras via SOS1 activation may cause coordinate activation of Ras and TC21. Second, we determined if Raf kinases were effectors for TC21 transformation. Unexpectedly, yeast two-hybrid binding analyses showed that although both Ras and TC21 could interact with the isolated Ras-binding domain of Raf-1, only Ras interacted with full-length Raf-1, A-Raf, or B-Raf. Consistent with this observation, we found that Ras- but not TC21-transformed NIH 3T3 cells possessed constitutively elevated Raf-1 and B-Raf kinase activity. Thus, Raf kinases are effectors for Ras, but not TC21, signaling and transformation. We conclude that common upstream signals cause activation of Ras and TC21, but activated TC21 controls cell growth via distinct Raf-independent downstream signaling pathways.     

Raf-independent deregulation of p38 and JNK mitogen-activated protein kinases are critical for Ras transformation

Activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, supporting the importance of Raf-independent pathways in mediating Ras transformation. The p38 and JNK mitogen-activated protein kinase cascades are activated by Ras via Raf-independent effector function. Therefore, we determined whether p38 and JNK activation are involved in Ras transformation of RIE-1 epithelial cells. Rather surprisingly, we found that pharmacologic inhibition of p38, together with Raf activation of ERK, was sufficient to mimic the morphologic and growth transformation caused by oncogenic Ras. p38 inhibition together with ERK activation also caused the same alterations in cyclin D1 and p21(CIP1) expression caused by Ras and induced an autocrine growth factor loop important for transformation. Finally, in contrast to p38, we found that JNK activation promoted Ras transformation, and that Ras deregulation of p38 and JNK was not mediated by activation of the Rac small GTPase. We conclude that a key action of Raf-independent effector pathways important for Ras transformation may involve inhibition of p38 and activation of JNK.     

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.     

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.