Oncogenic Ras Transformation Research Articles

G-quadruplex structural dynamics at MAPK12 promoter dictates transcriptional switch to determine stemness in breast cancer.

P38γ (MAPK12) is predominantly expressed in triple negative breast cancer cells (TNBC) and induces stem cell (CSC) expansion resulting in decreased survival of the patients due to metastasis. Abundance of G-rich sequences at MAPK12 promoter implied the functional probability to reverse tumorigenesis, though the formation of G-Quadruplex (G4) structures at MAPK12 promoter is elusive. Here, we identified two evolutionary consensus adjacent G4 motifs upstream of the MAPK12 promoter, forming parallel G4 structures. They exist in an equilibria between G4 and duplex, regulated by the binding turnover of Sp1 and Nucleolin that bind to these G4 motifs and regulate MAPK12 transcriptional homeostasis. To underscore the gene-regulatory functions of G4 motifs, we employed CRISPR-Cas9 system to eliminate G4s from TNBC cells and synthesized a naphthalene diimide (NDI) derivative (TGS24) which shows high-affinity binding to MAPK12-G4 and inhibits MAPK12 transcription. Deletion of G4 motifs and NDI compound interfere with the recruitment of the transcription factors, inhibiting MAPK12 expression in cancer cells. The molecular basis of NDI-induced G4 transcriptional regulation was analysed by RNA-seq analyses, which revealed that MAPK12-G4 inhibits oncogenic RAS transformation and trans-activation of NANOG. MAPK12-G4 also reduces CD44High/CD24Low population in TNBC cells and downregulates internal stem cell markers, arresting the stemness properties of cancer cells.

RAS induced senescence of skin keratinocytes is mediated through Rho-associated protein kinase (ROCK).

Cellular senescence is a well-documented response to oncogene activation in many tissues. Multiple pathways are invoked to achieve senescence indicating its importance to counteract the transforming activities of oncogenic stimulation. We now report that the Rho-associated protein kinase (ROCK) signaling pathway is a critical regulator of oncogene-induced senescence in skin carcinogenesis. Transformation of mouse keratinocytes with oncogenic RAS upregulates ROCK activity and initiates a senescence response characterized by cell enlargement, growth inhibition, upregulation of senescence associated β-galactosidase (SAβgal) expression, and release of multiple pro-inflammatory factors comprising the senescence-associated secretory phenotype (SASP). The addition of the ROCK inhibitor Y-27632 and others prevents these senescence responses and maintains proliferating confluent RAS transformed keratinocyte cultures indefinitely. Mechanistically, oncogenic RAS transformation is associated with upregulation of cell cycle inhibitors p15Ink4b , p16Ink4a , and p19Arf and downregulation of p-AKT, all of which are reversed by Y-27632. RNA-seq analysis of Y-27632 treated RAS-transformed keratinocytes indicated that the inhibitor reduced growth-inhibitory gene expression profiles and maintained expression of proliferative pathways. Y-27632 also reduced the expression of NF-κB effector genes and the expression of IκBζ downstream mediators. The senescence inhibition from Y-27632 was reversible, and upon its removal, senescence reoccurred in vitro with rapid upregulation of cell cycle inhibitors, SASP expression, and cell detachment. Y-27632 treated cultured RAS-keratinocytes formed tumors in the absence of the inhibitor when placed in skin orthografts suggesting that factors in the tumor microenvironment can overcome the drive to senescence imparted by overactive ROCK activity.

Ras-transformation reduce FAM20C expression and osteopontin phosphorylation.

Family with sequence similarity 20, member C (FAM20C) is the main kinase of secreted phosphoproteins, including the multifunctional protein and cytokine, osteopontin (OPN). The phosphorylation of OPN varies greatly among cell types, tissues and species, and the different phospho-isoforms contribute to the multifunctionality of the protein. Expression of OPN is increased in human malignancies, and less phosphorylated isoforms of the protein have been associated with this phenotype. Here, we compared OPN from ras-transformed fibroblasts with that from their non-transformed parental cells, and found that OPN was less phosphorylated after ras-transformation. Furthermore, we demonstrated that expression of FAM20C mRNA was reduced five-fold in ras-transformed fibroblasts compared with non-transformed fibroblasts. Transfection with FAM20C of the ras-transformed fibroblasts restored the FAM20C mRNA expression but the phosphorylation of OPN was not increased proportionally. Likewise, the mRNA level of FAM20C was reduced in the malignant ras-transformed mammary cell line MCF10ACA1a compared with its non-transformed parental cell line MCF10A. These results suggest that expression of the FAM20C kinase is reduced after oncogenic ras-transformation, which potentially affects the phosphorylation of secreted phosphoproteins.

Cystine/glutamate antiporter xCT (SLC7A11) facilitates oncogenic RAS transformation by preserving intracellular redox balance

The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach, we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo by mitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

Annexin A2 Regulates AKT Upon H₂O₂-Dependent Signaling Activation in Cancer Cells.

Hydrogen peroxide (H2O2) is a main second messenger in oncogenic signaling networks including the Ras and the growth factor receptor pathways. This is achieved predominantly through the oxidation of redox-sensitive cysteine (Cys) residues in proteins resulting in changes to their structure and function. We previously identified annexin A2 (ANXA2) as a redox regulatory protein that plays an important cellular role during oxidative stress and also promoting tumorigenesis. Here we investigated the role of ANXA2 in the regulation of H2O2-dependent signaling that drives tumor progression. We show that depletion of ANXA2 leads to the enhanced activation of AKT following either EGF/EGFR stimulation or oncogenic Ras transformation. The phosphatase and tensin homologue (PTEN) protein negatively regulates the PI3K/AKT pathway. We demonstrate that ANXA2 via its reactive Cys-8 residue, binds to PTEN and that the co-expression of PTEN and ANXA2, but not ANXA2 Cys-8-Ala mutant, inhibits AKT phosphorylation on Ser 473. These results indicate that ANXA2 is important for PTEN regulation within the PI3K/AKT signaling cascade. Furthermore, we also reveal that ANXA2 inversely regulates the expression of the peroxidase, peroxiredoxin 2, in a reactive oxygen species dependent manner.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation.

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells.

Hypothemycin inhibits tumor necrosis factor-α production by tristetraprolin-dependent down-regulation of mRNA stability in lipopolysaccharide-stimulated macrophages

Hypothemycin, a resorcylic acid lactone polyketide, has been shown to inhibit oncogenic ras-transformation and T cell activation. In the present study, we investigated the effect of hypothemycin on tumor necrosis factor-α (TNF-α) production in macrophages and the molecular mechanisms involved in this effect. Hypothemycin potently suppressed the TNF-α production without affecting nitric oxide production in lipopolysaccharide (LPS)-stimulated macrophages. However, hypothemycin had no effect on the activity of TNF-α-converting enzyme, a key enzyme for converting membrane-bound pro-TNF-α into soluble TNF-α. Further study demonstrated that the stability of TNF-α mRNA was decreased by hypothemycin treatment. In addition, hypothemycin suppressed LPS-induced phosphorylation of p38 MAPK and ERK. Moreover, knockdown of tristetraprolin (TTP), which is an important trans-acting regulator of TNF-α mRNA stability and downstream target of p38 MAPK and ERK, reversed hypothemycin-mediated inhibition of TNF-α mRNA expression. Collectively, our results suggest that hypothemycin suppresses TNF-α production by TTP-dependent destabilization of TNF-α mRNA and this is mediated, at least in part, by blocking the activation of p38 MAPK and ERK.

Abstract 2708: The TWEAK-FN14 pathway promotes prostate cancer bone metastasis through the NFκB pathway.

Abstract Advanced prostate cancer is highly associated with castration resistance and the development of bone metastases. Understanding the mechanisms contributing to prostate cancer bone metastasis is needed in order to develop novel therapeutic agents. NFκB signaling has been implicated in prostate cancer progression. The Tweak-Fn14 axis, which activates both canonical and non-canonical NFκB pathways, has been shown to be up-regulated in many cancer types. However, the role of the Tweak-Fn14 signaling in prostate cancer progression has not been investigated. In this study, we show that oncogenic Ras transformation of the AR negative, DU145 cell line led to the acquisition of bone metastatic capability and associated increased expression of TWEAK and FN14. Knocking down FN14 using shRNAs, as well as blocking the NFκB pathway using an IKBα super repressor, in DU145/Rasb1 cells, significantly inhibited bone metastasis and improved survival. This inhibitory effect of FN14shRNA was fully rescued by activating the NFκB canonical pathway, but not the NFκB non-canonical pathway. FN14 expression is also high in the bone metastatic, AR negative prostate cancer PC3 cell line. Similarly, knocking-down FN14 in PC3 cells inhibited bone metastatic capacity following intracardiac inoculation in a xenograft model. TWEAK and FN14 are relatively low or undetectable in AR positive prostate cancer cell lines including Lncap and 22RV1. AR binding was detected in both TWEAK and FN14 promoters as determined by chromatin immunoprecipitation analysis in Lncap. Reporter assays demonstrated that AR binding inhibited FN14 and TWEAK transcription. The above experimental models were further supported by analyses of publically available expression data sets for clinical prostate cancer samples that showed FN14 expression was inversely correlated with AR gene expression signatures. In addition, individual FN14 RNA mean expression was higher in clinical metastasis of castrate (n=11) as compared to non-castrate (n=8) patients. We propose that down-regulation of AR activity under castrate conditions may lead to increased FN14 expression and NFκB activation, which provides a survival benefit for prostate cancer cells associated with disease progression. Therefore, targeting the TWEAK-FN14 pathway in prostate cancer patients provides potential, new preventive and therapeutic approaches. Citation Format: Juan Juan Yin, Yen-Nien Liu, Ben Barrett, Lei Fang, Ross Lake, Orla Casey, Heather Tillman, Yvona Ward, Kathleen Kelly. The TWEAK-FN14 pathway promotes prostate cancer bone metastasis through the NFκB pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2708. doi:10.1158/1538-7445.AM2013-2708

Tubulin Polymerization Promoting Protein 1 (Tppp1) Phosphorylation by Rho-associated Coiled-coil Kinase (Rock) and Cyclin-dependent Kinase 1 (Cdk1) Inhibits Microtubule Dynamics to Increase Cell Proliferation

Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

Combining ATR Suppression with Oncogenic Ras Synergistically Increases Genomic Instability, Causing Synthetic Lethality or Tumorigenesis in a Dosage-Dependent Manner

Previous studies indicate that oncogenic stress activates the ATR-Chk1 pathway. Here, we show that ATR-Chk1 pathway engagement is essential for limiting genomic instability following oncogenic Ras transformation. ATR pathway inhibition in combination with oncogenic Ras expression synergistically increased genomic instability, as quantified by chromatid breaks, sister chromatid exchanges, and H2AX phosphorylation. This level of instability was significantly greater than that observed following ATR suppression in untransformed control cells. In addition, consistent with a deficiency in long-term genome maintenance, hypomorphic ATR pathway reduction to 16% of normal levels was synthetic lethal with oncogenic Ras expression in cultured cells. Notably, elevated genomic instability and synthetic lethality following suppression of ATR were not due to accelerated cycling rates in Ras-transformed cells, indicating that these synergistic effects were generated on a per-cell-cycle basis. In contrast to the synthetic lethal effects of hypomorphic ATR suppression, subtle reduction of ATR expression (haploinsufficiency) in combination with endogenous levels of K-ras(G12D) expression elevated the incidence of lung adenocarcinoma, spindle cell sarcoma, and thymic lymphoma in p53 heterozygous mice. K-ras(G12D)-induced tumorigenesis in ATR(+/-)p53(+/-) mice was associated with intrachromosomal deletions and loss of wild-type p53. These findings indicate that synergistic increases in genomic instability following ATR reduction in oncogenic Ras-transformed cells can produce 2 distinct biological outcomes: synthetic lethality upon significant suppression of ATR expression and tumor promotion in the context of ATR haploinsufficiency. These results highlight the importance of the ATR pathway both as a barrier to malignant progression and as a potential target for cancer treatment.

Extracellular Signal-Regulated Kinase 2-Dependent Phosphorylation Induces Cytoplasmic Localization and Degradation of p21Cip1

p21(Cip1) is an inhibitor of cell cycle progression that promotes G(1)-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21(Cip1) downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21(Cip1), promoting p21(Cip1) nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21(Cip1) revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21(Cip1) translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G(1)/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21(Cip1).

A novel p21Cip1 phosphorylation/degradation pathway playing a role in the ERK‐mediated cell proliferation signal

p21Cip1 is an inhibitor of cell cycle progression that promotes G1 phase arrest by direct binding to cyclin‐dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21Cip1 downregulation at the post‐translational level. We show that ERK2 interacts with and phosphorylates p21Cip1, promoting p21Cip1 nucleocytoplasmic translocation and ubiquitin‐dependent degradation. Phosphopeptide analysis of in vitro ERK2‐phosphorylated p21Cip1 revealed two phosphorylation sites, Thr57 and Ser130. Both the phosphorylation‐deficient T57A/S130A p21Cip1 mutant and the NES mutant, that are resistant to ERK2‐mediated downregulation, retain the ability to potently inhibit the G1/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by phosphorylation, translocation, and degradation of the cell cycle inhibitory protein, p21Cip1, suggesting an alternative mechanism of Ras‐ERK signaling‐mediated cell proliferation. This work was supported by grants M10503010002, M10601000164, and M10642040003.

The Cytoplasmic Deacetylase HDAC6 Is Required for Efficient Oncogenic Tumorigenesis

Histone deacetylase inhibitors (HDACI) are promising antitumor agents. Although transcriptional deregulation is thought to be the main mechanism underlying their therapeutic effects, the exact mechanism and targets by which HDACIs achieve their antitumor effects remain poorly understood. It is not known whether any of the HDAC members support robust tumor growth. In this report, we show that HDAC6, a cytoplasmic-localized and cytoskeleton-associated deacetylase, is required for efficient oncogenic transformation and tumor formation. We found that HDAC6 expression is induced upon oncogenic Ras transformation. Fibroblasts deficient in HDAC6 are more resistant to both oncogenic Ras and ErbB2-dependent transformation, indicating a critical role for HDAC6 in oncogene-induced transformation. Supporting this hypothesis, inactivation of HDAC6 in several cancer cell lines reduces anchorage-independent growth and the ability to form tumors in mice. The loss of anchorage-independent growth is associated with increased anoikis and defects in AKT and extracellular signal-regulated kinase activation upon loss of adhesion. Lastly, HDAC6-null mice are more resistant to chemical carcinogen-induced skin tumors. Our results provide the first experimental evidence that a specific HDAC member is required for efficient oncogenic transformation and indicate that HDAC6 is an important component underlying the antitumor effects of HDACIs.

Oncogenic Ras-transformed human fibroblasts exhibit differential changes in contraction and migration in 3D collagen matrices

Tractional force exerted by tissue cells in 3D collagen matrices can be utilized for matrix remodeling or cell migration. The interrelationship between these motile processes is not well understood. The current studies were carried out to test the consequences of oncogenic Ras (H-RasV12) transformation on human fibroblast contraction and migration in 3D collagen matrices. Beginning with hTERT-immortalized cells, we prepared fibroblasts stably transformed with E6/E7 and with the combination HPV16 E6/E7 and H-RasV12. Oncogenic Ras-transformed cells lost contact inhibition of cell growth, formed colonies in soft agar and were unable to make adherens junctions. We observed no changes in the extent or growth factor dependence of collagen matrix contraction (floating or stress-relaxation) by oncogenic Ras-transformed cells. On the other hand, transformed cells in nested collagen matrices lost not only growth factor selectivity, but also cell–matrix density-dependent inhibition of migration. These findings demonstrate differential regulation of collagen matrix contraction and cell migration in 3D collagen matrices.

NADPH oxidase 1 plays a critical mediating role in oncogenic Ras-induced vascular endothelial growth factor expression

Reactive oxygen species (ROS)-generating enzyme Nox1 is important in the induction of oncogenic Ras transformation phenotypes, but it is not defined whether Nox1 is involved in Ras-induced upregulation of vascular endothelial growth factor (VEGF), a potent stimulator of tumor angiogenesis. Here we describe that ablation of the Nox1 activity by Nox1 small-interference RNAs (siRNAs) or diphenylene iodonium (DPI) inhibited synthesis of both VEGF proteins and VEGF mRNAs in K-Ras transformed normal rat kidney (KNRK) cells. Nox1siRNAs and DPI suppressed extracellular signal-regulated kinase (ERK)-dependent phosphorylation of a transcription factor Sp1 and Sp1 binding to a VEGF promoter. Furthermore, tumors derived from Nox1siRNA-transfected KNRK cells markedly decreased neovascularization. The Nox1 activity was required for VEGF production in human colon cancer CaCO-2 cells, as in the case of KNRK cells. However, since overexpression of Nox1 in normal rat kidney cells failed to induce VEGF, the Nox1 activity alone was not sufficient to upregulate VEGF expression, which suggests that unlike the previously proposed model, Nox1 may act in concert with other effectors integrated into the Ras network. We propose that Nox1 mediates oncogenic Ras-induced upregulation of VEGF and angiogenesis by activating Sp1 through Ras-ERK-dependent phosphorylation of Sp1.

Analysis of Ras‐induced oncogenic transformation of NIH‐3T3 cells using differential‐display 2‐DE proteomics

Ras proteins control at least three crucial signalling networks responsible for several cellular processes including anchorage independence, survival, and proliferation. Point mutations in one of the three ras genes are frequent in human tumours. In these tumours, Ras oncoproteins contribute significantly to the malignant phenotype, including deregulation of tumour-cell growth, apoptosis and invasiveness, and the ability to induce angiogenesis. Although significant strides have been made in understanding Ras biology, the collaborative actions of Ras effectors are still poorly understood. Here, we describe a proteomics approach to study global changes in protein expression in Ras-transformed NIH3T3 cells. We exploited 2-D difference gel electrophoresis (DIGE) for pre-separation fluorescent protein labelling with three separate dyes to reduce gel-to-gel variability, to increase sensitivity and dynamic range of protein detection, and to enhance quantification of dysregulated proteins. Proteins dysregulated (> 1.5-fold) by oncogenic Ras transformation reported to be implicated in Ras-regulated pathways include S-methyl-5-thioadenosine phosphorylase, stress-induced-phosphoprotein 1, galectin-1, annexin A7 (synexin), 60S acidic ribosomal protein P0, serine/threonine protein phosphatase type 1 (PP1alpha) and prohibitin. Significantly, we report for the first time the expression of the newly discovered cytokine IL-25 (or IL-17E) in mouse embryonic fibroblast cells and its down-regulation (2.1-fold) upon Ras-induced oncogenic transformation.

Proteomics analyses of ovarian caner using genetically defined human ovarian cancer models

Using a genetically defined human ovarian cancer model, we have analyzed the protein expression profile of human ovarian cancer cells established by oncogenic RAS transformation. Cells were immortalized by retroviral transfection of SV40 t/T antigens and the catalytic subunit of telomerase (hTERT). Careful analyses of protein targets associated with oncogenic transformation have enabled us to identify several novel signaling pathways that play important roles in oncogenic transformation of human ovarian epithelial cells.

Ras-mediated Loss of the Pro-apoptotic Response Protein Par-4 Is Mediated by DNA Hypermethylation through Raf-independent and Raf-dependent Signaling Cascades in Epithelial Cells

The apoptosis-promoting protein Par-4 has been shown to be down-regulated in Ras-transformed NIH 3T3 fibroblasts through the Raf/MEK/ERK MAPK pathway. Because mutations of the ras gene are most often found in tumors of epithelial origin, we explored the signaling pathways utilized by oncogenic Ras to down-regulate Par-4 in RIE-1 and rat ovarian surface epithelial (ROSE) cells. We determined that constitutive activation of the Raf, phosphatidylinositol 3-kinase, or Ral guanine nucleotide exchange factor effector pathway alone was not sufficient to down-regulate Par-4 in RIE-1 or ROSE cells. However, treatment of Ras-transformed RIE-1 or ROSE cells with the MEK inhibitors U0126 and PD98059 increased Par-4 protein expression. Thus, although oncogenic Ras utilizes the Raf/MEK/ERK pathway to down-regulate Par-4 in both fibroblasts and epithelial cells, Ras activation of an additional signaling pathway(s) is required to achieve the same outcome in epithelial cells. Methylation-specific PCR showed that the par-4 promoter is methylated in Ras-transformed cells through a MEK-dependent pathway and that treatment with the DNA methyltransferase inhibitor azadeoxycytidine restored Par-4 mRNA transcript and protein levels, suggesting that the mechanism for Ras-mediated down-regulation of Par-4 is by promoter methylation. Support for this possibility is provided by our observation that Ras transformation was associated with up-regulation of Dnmt1 and Dnmt3 DNA methyltransferase expression. Finally, ectopic Par-4 expression significantly reduced Ras-mediated growth in soft agar, but not morphological transformation, highlighting the importance of Par-4 down-regulation in specific aspects of Ras-mediated transformation of epithelial cells.

Maintenance of mouse trophoblast stem cell proliferation by TGF-β/activin

Mouse trophoblast stem (TS) cells can be grown indefinitely in vitro with FGF4 and embryonic fibroblast conditioned media (EFCM). Here, we report that the active protein components of EFCM include TGF-β and the related factor activin, and that long-term continuous TS cell proliferation is possible in media supplemented with only serum, FGF4, and TGF-β. As trophoblasts are an epithelial cell type, the promotion of TS cell proliferation represents an unusual function for TGF-β and activin since TGF-β in particular is well known as an inhibitor of nontransformed epithelial cell proliferation. Our data suggest that constitutive FGF signaling in TS cells selectively inhibits the ability of TGF-β to repress c- myc expression, a central component of the TGF-β cytostatic transcriptional response previously observed to be lost in other epithelial cell types upon oncogenic Ras transformation.

The superoxide-generating oxidase Nox1 is functionally required for Ras oncogene transformation.

The activated Ras oncogene can transform various mammalian cells and has been implicated in development of a high population of malignant human tumors. Recent studies suggest that generation of reactive oxygen species such as superoxide and H(2)O(2) is involved in cell transformation by the activated Ras. However, the nature of an oxidase participating in Ras-transformation is presently unknown. Here, we report that Ras oncogene up-regulates the expression of Nox1, a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase, via the mitogen-activated protein kinase kinase-mitogen-activated protein kinase pathway, and that small interfering RNAs designed to target Nox1 mRNA effectively blocks the Ras transformed phenotypes including anchorage-independent growth, morphological changes, and production of tumors in athymic mice. Therefore, we propose that increased reactive oxygen species generation by Ras-induced Nox1 is required for oncogenic Ras transformation.