Ras oncogene at 85D
Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is
additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin
regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). Expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies are observed in cotransfection assays with Rat-1 cells. Pak1(R299) does not inhibit transformation by the Raf oncogene, indicating that
inhibition is specific for Ras. Rat-1 cell lines expressing Pak1(R299) are highly resistant to Ras transformation, while cells expressing wild-type Pak1 are efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibits Ras transformation. Rac and Ras activation of JNK is inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK is inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibits Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling (Tang, 1997).
Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of either fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change, remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, it is proposed that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton (He, 1998).
The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes when expressed in the NIH 3T3 fibroblasts of athymic nude mice. The effector domain (amino acids 32-40 in H-Ras) has been shown to be essential for the interaction between Ras and a variety of effectors, which in mammals include Raf-1, PI3-K (phosphatidylinositol 3-kinase), RasGAPs, Ral guanine nucleotide exchange factors, MEK kinase 1, Rin1 and AF6/Rsb1. Point mutations within the effector domain of oncogenic ras generate mutants deficient in specific effector function and therefore activation of specific downstream signaling pathways. All mutants display comparable tumorigenic properties, but only V12S35 Ras, the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) pathway, induces tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK pathway in NIH 3T3 cells by mos or a constitutively active form of MEK is sufficient to induce metastasis, whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells express higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK occurs during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors do not show changes in the level of ERK1/2 activation, when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants are passaged one additional time in vivo, all mediate rapid tumor growth, but as before, only cells derived from V12S35 Ras-tumors form numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK activation, experimental metastasis appears to require constitutive activation of the ERK pathway (Webb, 1998).
Colon carcinomas commonly contain mutations in Ki-ras4B, but very rarely in Ha-ras, suggesting that
different Ras isoforms may have distinct functions in colon epithelial cell biology. Oncogenic Ki-ras4BVal-12, but not oncogenic Ha-rasVal-12, blocks the apicobasal polarization of colon epithelial cells by preventing normal glycosylation of the integrin beta1 chain of the collagen receptor. As a result, only the Ki-ras mutated cells exhibit altered cell-to-substratum attachment, whereas mutation of either Ras isoform activates mitogen-activated protein kinases. Intercellular adhesion proteins implicated in establishing basolateral polarity in colon epithelial cells are modulated by oncogenic Ki-Ras4BVal-12 proteins but not oncogenic Ha-RasVal-12 proteins. The embryonic adhesion protein carcinoembryonic antigen (CEA) is up-regulated at the mRNA and protein levels in each of three stable Ki-rasVal-12
transfectant lines but in none of three stable Ha-rasVal-12 transfectant lines. The elevated protein
levels of CEA in Ki-ras4BVal-12 transfectant cells are decreased by blocking expression of
Ki-ras4BVal-12 with antisense oligonucleotides. N-cadherin levels are decreased in only the Ki-ras
transfectants, whereas E-cadherin levels are unchanged. Ki-ras4BVal-12 transfectant cells do not polarize into cells with discrete apical and
basal regions and so can not restrict expression of CEA to the apical region. These unpolarized cells
display elevated levels of CEA all along their surface membrane where CEA mediates random,
multilayered associations of tumor cells. This aggregation is both calcium-independent and blocked by Fab' fragments of anti-CEA monoclonal antibody col-1. Trafficking of the lysosomal cysteine protease cathepsin B may also be altered when cell polarity cannot be established. Ki-ras4BVal-12
transfectant cells express 2-fold elevated protein levels of the lysosomal cysteine protease cathepsin B but do not up-regulate cathepsin B mRNA expression. One function of oncogenic c-Ki-Ras proteins in colon cancer progression may be to up-regulate CEA and thus to prevent the lateral adhesion of
adjacent colon epithelial cells that normally form a monolayer in vivo (Yan, 1997).
An 18-kDa protein (p18) was detected in lysates and conditioned medium from contact-arrested NIH
3T3 fibroblasts, but was not detected when the cells were transformed by the oncogene ras. Analysis
of transformation-defective cell clones generated after mutagenesis of the ras-retroviral vector used to
transduce the ras gene shows an inverse correlation between p18 expression and the degree of
transformation. p18 expression is high in non-transformed clones, intermediate in a partially
transformed clone, undetectable in fully transformed clones, and detectable only at the non-permissive
temperature in a clone that is cold-sensitive for ras transformation. In non-transformed cells, p18
expression varies with the degree of confluence. It is almost undetectable in medium from sparse,
proliferating cells, but increases as the cells approach confluence and peaks 2-4 days after
confluence. Microsequencing of partially purified p18 identifies it as the developmentally regulated
neurotrophic factor, pleiotrophin. Pleiotrophin is undetectable or almost
undetectable, in medium from fully transformed cells expressing the oncogenes v-src, truncated c-raf,
activated c-fms, or polyomavirus middle tumor antigen; it is low but easily detectable in medium from
SV40 large tumor antigen-expressing cells, which form soft agar colonies but not foci. Thus,
pleiotrophin expression in NIH 3T3 cells is associated with quiescence, and suppression of pleiotrophin
is related to oncogenic transformation (Corbley, 1997).
The brm and BRG-1 proteins (see Drosophila Brahma) are mutually exclusive subunits of the mammalian SWI-SNF complex. Within this complex, they provide the ATPase activity necessary for transcriptional regulation by nucleosome disruption. Both proteins have recently been found to interact with the p105Rb tumor suppressor gene product, suggesting a role for the mammalian SWI-SNF complex in the control of cell growth. The expression of brm, but not BRG-1, is negatively regulated by mitogenic stimulation, and growth arrest of mouse fibroblasts leads to increased accumulation of the brm protein. The expression of this protein is also down-regulated upon transformation by the ras oncogene. Re-introduction of brm into ras transformed cells leads to partial reversion of the transformed phenotype
by a mechanism that depends on the ATPase domain of the protein. These data suggest that increased levels of brm protein favour cell withdrawal from the cell cycle, whereas decreased expression of the brm gene may facilitate cellular transformation by various oncogenes. The persistence of BRG-1, but not brm, is essential for the viability of early embryonic cells. The persistence of BRG-1 in faster growing cells, and the accumulation of brm in cells that become slow growing or arrested, raises the possibility that a partial switch from BRG-1 to brm-containing SWI-SNF complexes occurs when cells slow their growth. brm may facilitate the expression of genes that are important for the maintenance of the quiescent or terminally differentiated state (Muchardt, 1998).
Several specific cell cycle activities are dependent on cell-substratum adhesion in nontransformed cells. The ability of the Ras oncoprotein to induce anchorage-independent growth is linked to its ability to abrogate this adhesion requirement. Ras signals via multiple downstream effector proteins; a synergistic combination of these proteins may be required for the highly altered phenotype of fully transformed cells. Ras may be viewed as a hub from which multiple pathways radiate. Studies are described on cell cycle regulation of anchorage-independent growth that utilize Ras effector loop mutants in NIH 3T3 and Rat 6 cells. Stable expression of activated H-Ras (12V) induces soft agar colony formation by both cell types, but each of three effector loop mutants (12V,35S, 12V,37G, and 12V,40C) is defective in producing this response. Of the three effector proteins for which there is evidence of a role in Ras-mediated transformation, the 12V,35S mutant binds to Raf but not PI(3)K or RalGDS; the 12V,37G mutant binds to RalGDS but not Raf or PI(3)K, and the 12V,40C mutant binds to PI(3)K but not Raf or RalGDS. Expression of all three possible pairwise combinations of these mutants synergizes to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V,35S-12V,37G and 12V,37G-12V,40C combinations are complementary in Rat 6 cells. Each individual effector loop mutant partially relieves adhesion dependence of pRB phosphorylation, cyclin E-dependent kinase activity, and expression of cyclin A in NIH 3T3 cells, but not for Rat 6 cells. The pairwise combinations of effector loop mutants that are synergistic in producing anchorage-independent growth in Rat 6 cells also lead to synergistic abrogation of the adhesion requirement for these cell cycle activities. The relationship between complementation in producing anchorage-independent growth and enhancement of cell cycle activities is not as clear in NIH 3T3 cells that express pairs of mutants, implying the existence of either thresholds for these activities or additional requirements in the induction of anchorage-independent growth. Ectopic expression of cyclin D1, E, or A synergizes with individual effector loop mutants to induce soft agar colony formation in NIH 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways in order to signal to the cell cycle machinery and that these pathways synergize to supplant the adhesion requirements of specific cell cycle events, leading to anchorage-independent growth (Yang, 1998).
The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause
cellular transformation were investigated using partial loss of function mutants of Ras and activated and
inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires
activation of other direct effectors in addition to the MAP kinase kinase kinase Raf; transformation is inhibited by
inactivation of the PI 3-kinase pathway. In fact, PI3-kinase interacts with Ras.GTP but not with Ras.GDP and is activated both in vitro and in vivo as a result of this interaction. Actin rearrangement correlates with the ability of Ras mutants
to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling,
while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of
activated Ras to stimulate PI 3-kinase, in addition to its stimulation of Raf, is therefore important in Ras transformation of
mammalian cells and essential in Ras-induced cytoskeletal reorganization. It thus apprears that at least two effector pathways need to be activated by Ras for transformation to occur. These pathways could include Raf plus PI 3-kinase, or others (Rodriguez-Viciana, 1997).
The familial melanoma gene (INK4a/MTS1/CDKN2) encodes potent tumor suppressor activity.
Although mice null for the ink4a homolog develop a cancer-prone condition, a pathogenetic link to
melanoma susceptibility has yet to be established. Mice with melanocyte-specific
expression of activated H-rasG12V on an ink4a-deficient background develop spontaneous cutaneous
melanomas after a short latency and with high penetrance. Consistent loss of the wild-type ink4a allele
is observed in tumors arising in ink4a heterozygous transgenic mice. No homozygous deletion of the
neighboring ink4b gene is detected. Moreover, as in human melanomas, the p53 gene remains in a
wild-type configuration with no observed mutation or allelic loss. These results show that loss of ink4a
and activation of Ras can cooperate to accelerate the development of melanoma; they provide the first
in vivo experimental evidence for a causal relationship between ink4a deficiency and the pathogenesis
of melanoma. This mouse model affords a system in which to identify and analyze pathways
involved in tumor progression against the backdrop of genetic alterations encountered in human
melanomas (Chin, 1997).
Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals
through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces
premature senescence involving the p53 and p16(INK4a) tumor suppressors. Constitutive activation of MEK (a component of the
MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts.
Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and
transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to
constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the
transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as
tumor suppressors (Lin, 1998).
Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of
the most efficient inducers of apoptosis is the death receptor Fas. Oncogenic Ras (H-Ras) downregulates Fas expression and
renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent.
Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known
anti-apoptotic effect of the downstream PKB/Akt kinase (Drosophila homolog Akt1) may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic
potential of H-ras may reside in its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis (Peli, 1999).
Advanced malignancy in tumors represents the phenotypic endpoint of successive genetic lesions that affect the function and regulation
of oncogenes and tumor-suppressor genes. The established tumor is maintained through complex and poorly understood host-tumor
interactions that guide processes such as angiogenesis and immune sequestration. The many different genetic alterations that accompany
tumor genesis raise questions as to whether experimental cancer-promoting mutations remain relevant during tumor maintenance. Melanoma genesis and maintenance are shown to be strictly dependent upon expression of H-RasV12G in a doxycycline-inducible
H-Ras12G mouse melanoma model null for the tumor suppressor INK4a. Withdrawal of doxycycline and H-RasV12G down-regulation
results in clinical and histological regression of primary and explanted tumors. The initial stages of regression involves marked
apoptosis in the tumor cells and host-derived endothelial cells. Although the regulation of vascular endothelial growth factor (VEGF) is
found to be Ras-dependent in vitro, the failure of persistent endogenous and enforced VEGF expression to sustain tumor viability
indicates that the tumor-maintaining actions of activated Ras extend beyond the regulation of VEGF expression in vivo. These results
provide genetic evidence that H-RasV12G is important in both the genesis and maintenance of solid tumors (Chin, 1999).
Transformation by oncogenic Ras requires the function of the Rho family GTPases. Ras-transformed cells have elevated
levels of RhoA-GTP, which functions to inhibit the expression of the cell cycle inhibitor p21/Waf1. These high levels of Rho-GTP are not
a direct consequence of Ras signaling but are selected for in response to sustained ERK-MAP kinase signaling. While the elevated
levels of Rho-GTP control the level of p21/Waf, they no longer regulate the formation of actin stress fibers in transformed cells. The sustained ERK-MAP kinase signaling resulting from transformation by oncogenic Ras down-regulates ROCK1 and Rho-kinase,
two Rho effectors required for actin stress fiber formation. The repression of Rho-dependent stress fiber formation by ERK-MAP kinase signaling contributes to the increased motility of Ras-transformed fibroblasts. Overexpression of the ROCK target LIM kinase restores actin stress fibers and
inhibits the motility of Ras-transformed fibroblasts. A model is proposed in which Ras and Rho signaling pathways cross-talk to promote signaling pathways
favoring transformation (Sahai, 2001).
Expression of p21/Waf1 following mitogenic stimulation is dependent on the ERK-MAP kinase pathway. In the
control of cell cycle progression, p21/Waf1 has a dual role: it serves as an assembly factor for active complexes of D-type cyclins and their cyclin-dependent kinases
(CDKs) and is an inhibitor of CDK2. In studies employing transient assays of Ras-driven proliferation, the absence of signaling
through RhoA results in the Ras-driven ERK-MAP kinase pathway, inducing levels of p21/Waf1 that are inhibitory to cell cycle progression.
signaling through RhoA is required to suppress growth inhibitory levels of p21/Waf1 in Ras-transformed Swiss-3T3 cells and in two human
colorectal cancer cell lines. In these human cell lines, inhibition of the Raf/MEK/ERK and PI-3-kinase Ras effector pathways does not affect Rho-GTP levels, thus
supporting the model that Rho activity is determined by selection; however, the levels of Rho-GTP in these cells compared with untransformed cells could not be
determined due to the lack of genotypically matched controls. It is proposed that cells with high levels of Rho activity are selected
for because they counteract the high levels of p21/Waf1 induced by oncogenic Ras and proliferate, while cells with low Rho activity remain growth arrested by high
p21/Waf1 levels. Interestingly, the Ras-transformed Swiss-3T3 cells have higher levels of p21/Waf1 than parental non-transformed cells. However, these cells
proliferate presumably because the levels of p21/Waf1 are such that they enable the assembly of active cyclin D-CDK complexes rather than inhibit CDK2. It is proposed that the elevated levels of Rho-GTP in the transformed cells set a threshold level of p21/Waf1 that is compatible with proliferation (Sahai, 2001).
The spectrum of tumors associated with oncogenic Ras in humans often differs from those in mice either treated with carcinogens or
engineered to sporadically express oncogenic Ras, suggesting that the mechanism of Ras transformation may be different in humans. Ras primarily
stimulates three main classes of effector proteins, Rafs, PI3-kinase, and RalGEFs, with Raf generally being the most potent at
transforming murine cells. Using oncogenic Ras mutants that activate single effectors as well as constitutively active effectors, it has been found that
the RalGEF, and not the Raf or PI3-kinase pathway, is sufficient for Ras transformation in human cells. Thus, oncogenic Ras may
transform murine and human cells by distinct mechanisms, and the RalGEF pathway -- previously deemed to play a secondary role in Ras transformation
-- could represent a new target for anti-cancer therapy (Hamad, 2002).
tob (Drosophila homolog: Tob) is a member of an emerging family of genes with antiproliferative function. Tob is rapidly phosphorylated at Ser 152, Ser 154, and
Ser 164 by Erk1 and Erk2 upon growth-factor stimulation. Oncogenic Ras-induced transformation and growth-factor-induced cell
proliferation are efficiently suppressed by mutant Tob which carries alanines but not glutamates, thereby mimicking phosphoserines at these sites.
Wild-type Tob inhibits cell growth when the three serine residues are not phosphorylated but is less inhibitory when the serines are
phosphorylated. Because growth of Rb-deficient cells is not affected by Tob, Tob appears to function upstream of Rb. Intriguingly,
cyclin D1 expression is elevated in serum-starved tob-/- cells. Reintroduction of wild-type Tob and mutant Tob with serine-to-alanine but not to
glutamate mutations on the Erk phosphorylation sites in these cells restores the suppression of cyclin D1 expression. Finally, the S-phase population is significantly
increased in serum-starved tob-/- cells as compared with that in wild-type cells. Thus, Tob inhibits cell growth by suppressing cyclin D1 expression,
which is canceled by Erk1- and Erk2-mediated Tob phosphorylation. It is proposed that Tob is critically involved in the control of early G1 progression (Suzuki, 2002).
Pten heterozygous (Pten+/-) mice develop increased papilloma numbers and show decreased carcinoma latency time in comparison with controls after skin treatment with dimethyl benzanthracene (DMBA) and tetradecanoyl-phorbol acetate (TPA). H-ras mutation is normally a hallmark of DMBA-TPA-induced skin tumors, but 70% of carcinomas from Pten+/- mice do not exhibit this mutation, and in all cases have lost the wild-type Pten allele. Tumors that retain the Pten wild-type allele also have H-ras mutations, indicating that activation of H-ras and complete loss of Pten are mutually exclusive events in skin carcinomas. Mitogen-activated protein kinase (MAPK) is consistently activated in the tumors with H-ras mutations, but is strongly down-regulated in Pten-/- tumors, suggesting that this pathway is dispensable for skin carcinoma formation. These data have important implications in designing individual therapeutic strategies for the treatment of cancer (Mao, 2004).
Reactive oxygen species (ROS) are implicated in the pathophysiology of various diseases, including cancer. In this study, JunD, a member of the AP-1 family of transcription factors, is shown to reduce tumor angiogenesis by limiting Ras-mediated production of ROS. Using junD-deficient cells, JunD is demonstrated to regulate genes involved in antioxidant defense, H2O2 production, and angiogenesis. The accumulation of H2O2 in junD-/- cells decreases the availability of FeII and reduces the activity of HIF prolyl hydroxylases (PHDs) that target hypoxia-inducible factors-alpha (HIFalpha) for degradation. Subsequently, HIF-alpha proteins accumulate and enhance the transcription of VEGF-A, a potent proangiogenic factor. This study uncovers the mechanism by which JunD protects cells from oxidative stress and exerts an antiangiogenic effect. Furthermore, new insights are provided into the regulation of PHD activity, allowing immediate reactive adaptation to changes in O2 or iron levels in the cell (Gerald, 2004).
Production of ROS and hypoxic response are key players in the occurrence and progression of cancers. The junD-/- adult mice do not develop tumors spontaneously, suggesting that the protective effect of JunD may only be uncovered under stress conditions. A protective effect of JunD has been demonstrated in cells transformed by the Ras oncogene, one of the most frequently mutated oncogenes in human cancers. Ras-mediated transformation enhances ROS production, and treatment with antioxidant molecules decreases the proliferation rate of Ras-transformed cell lines. JunD has been shown to antagonize Ras-mediated transformation by modulating cell proliferation. The present study shows that overexpression of JunD decreases ROS production in Ras-transformed cells. Thus, it is proposed that the inhibitory effect of JunD on the proliferation of Ras-transformed cell lines is mediated in part through the decreased level of ROS. Moreover, Ras oncogene contributes to the growth of solid tumors by a direct effect on cell proliferation and by facilitating tumor angiogenesis. Indeed, transformation by Ras stabilizes HIF-1α and upregulates VEGF-A expression as well as other HIF target genes. Furthermore, Ras-induced stabilization of HIF-1α is mediated through inhibition of HIF hydroxylation. The data argue that ROS accumulation in Ras-transformed cells triggers PHD inhibition. JunD has a major effect on this process. Indeed, JunD decreases ROS production, restores PHD activity, and subsequently reduces significantly Ras-dependent tumor angiogenesis in vivo. Thus, JunD displays a protective role against Ras-mediated transformation by buffering cells to maintain the redox balance (Gerald, 2004).
Pancreatic ductal adenocarcinoma (PDA) constitutes a lethal disease that affects >30,000 people annually in the United States. Deregulation of Hedgehog signaling has been implicated in the pathogenesis of PDA. To gain insights into the role of the pathway during the distinct stages of pancreatic carcinogenesis, a mouse model was established in which Hedgehog signaling is activated specifically in the pancreatic epithelium. Transgenic mice survived to adulthood and developed undifferentiated carcinoma, indicating that epithelium-specific Hedgehog signaling is sufficient to drive pancreatic neoplasia but does not recapitulate human pancreatic carcinogenesis. In contrast, simultaneous activation of Ras and Hedgehog signaling caused extensive formation of pancreatic intraepithelial neoplasias, the earliest stages of human PDA tumorigenesis, and accelerated lethality. These results indicate the cooperation of Hedgehog and Ras signaling during the earliest stages of PDA formation. They also mark Hedgehog pathway components as relevant therapeutic targets for both early and advanced stages of pancreatic ductal neoplasia (Pasca di Magliano, 2007).
Somatic activation of Ras occurs frequently in human cancers, including one-third of lung cancers. Activating Ras mutations also occur in the germline, leading to complex developmental syndromes. The precise mechanism by which Ras activation results in human disease is uncertain. This study describes the phenotype of a mouse engineered to harbor a germline oncogenic K-rasG12D mutation. This mouse exhibits early embryonic lethality due to a placental trophoblast defect. Reconstitution with a wild-type placenta rescues the early lethality, but mutant embryos still succumb to cardiovascular and hematopoietic defects. In addition, mutant embryos demonstrate a profound defect in lung branching morphogenesis associated with striking up-regulation of the Ras/mitogen-activated protein kinase (MAPK) antagonist Sprouty-2 and abnormal localization of MAPK activity within the lung epithelium. This defect can be significantly suppressed by lentiviral short hairpin RNA (shRNA)-mediated knockdown of Sprouty-2 in vivo. Furthermore, in the context of K-rasG12D-mediated lung tumorigenesis, Sprouty-2 is also up-regulated and functions as a tumor suppressor to limit tumor number and overall tumor burden. These findings indicate that in the lung, Sprouty-2 plays a critical role in the regulation of oncogenic K-ras, and implicate counter-regulatory mechanisms in the pathogenesis of Ras-based disease (Shaw, 2007).
Ras is mutated to remain in the active oncogenic state in many cancers. Since Ras has proven difficult to target therapeutically, a search was performed for secreted, druggable proteins induced by Ras that are required for tumorigenesis. Ras was found to induce the secretion of cytokine IL6 in different cell types, and knockdown of IL6, genetic ablation of the IL6 gene, or treatment with a neutralizing IL6 antibody retards Ras-driven tumorigenesis. IL6 appears to act in a paracrine fashion to promote angiogenesis and tumor growth. Inhibiting IL6 may therefore have therapeutic utility for treatment of cancers characterized by oncogenic Ras mutations (Ancrile, 2007).
Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16Ink4a gene. Melanomas also frequently exhibit constitutive activation of the Wnt/β-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. Contrary to expectations, stabilized β-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16Ink4a promoter. Significantly, in a novel mouse model for melanoma, stabilized β-catenin bypasses the requirement for p16Ink4a mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease (Delmas, 2007).
Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, it was shown that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS (K-ras). These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human familial adenomatous polyposis adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas (Phelps, 2009).
The p53 tumor suppressor limits proliferation in response to cellular stress through several mechanisms. This study tests whether the recently described ability of p53 to limit stem cell self-renewal suppresses tumorigenesis in acute myeloid leukemia (AML), an aggressive cancer in which p53 mutations are associated with drug resistance and adverse outcome. The approach combined mosaic mouse models, Cre-lox technology, and in vivo RNAi to disable p53 and simultaneously activate endogenous Kras(G12D)-a common AML lesion that promotes proliferation but not self-renewal. It was shown that p53 inactivation strongly cooperates with oncogenic Kras(G12D) to induce aggressive AML, while both lesions on their own induce T-cell malignancies with long latency. This synergy is based on a pivotal role of p53 in limiting aberrant self-renewal of myeloid progenitor cells, such that loss of p53 counters the deleterious effects of oncogenic Kras on these cells and enables them to self-renew indefinitely. Consequently, myeloid progenitor cells expressing oncogenic Kras and lacking p53 become leukemia-initiating cells, resembling cancer stem cells capable of maintaining AML in vivo. These results establish an efficient new strategy for interrogating oncogene cooperation, and provide strong evidence that the ability of p53 to limit aberrant self-renewal contributes to its tumor suppressor activity (Zhao, 2010).
Autophagy is a catabolic pathway used by cells to support metabolism in response to starvation and to clear damaged proteins and organelles in response to stress. Expression of a H-ras(V12) or K-ras(V12) oncogene up-regulates basal autophagy, which is required for tumor cell survival in starvation and in tumorigenesis. In Ras-expressing cells, defective autophagosome formation or cargo delivery causes accumulation of abnormal mitochondria and reduced oxygen consumption. Autophagy defects also lead to tricarboxylic acid (TCA) cycle metabolite and energy depletion in starvation. As mitochondria sustain viability of Ras-expressing cells in starvation, autophagy is required to maintain the pool of functional mitochondria necessary to support growth of Ras-driven tumors. Human cancer cell lines bearing activating mutations in Ras commonly have high levels of basal autophagy, and, in a subset of these, down-regulating the expression of essential autophagy proteins impaired cell growth. As cancers with Ras mutations have a poor prognosis, this 'autophagy addiction' suggests that targeting autophagy and mitochondrial metabolism are valuable new approaches to treat these aggressive cancers (Guo, 2011).
Previously work has identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). In this study RNAi-based epistasis experiments were performed and RAS-directed silencing was found to occur through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers (Wajapeyee, 2013).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Table of contents
Ras85D:
Biological Overview
| Regulation
| Protein Interactions
| Effects of Mutation
| Ras as Oncogene
| References
Society for Developmental Biology's Web server.