Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.     

Signaling pathways regulating TC21-induced tumorigenesis

TC21(R-Ras2), a Ras-related GTPase with transforming potential similar to H-, K- and N-Ras, is implicated in the pathogenesis of human cancers. Transforming growth factor beta (TGF-beta), a cytokine that plays a significant role in modulating tumorigenesis, normally prevents uncontrolled cell proliferation but paradoxically induces proliferation in H-Ras-transformed cancer cells. Although TC21 activates some pathways that mediate cellular transformation by the classical Ras proteins, the mechanisms through which TC21 induces tumor formation and how TGF-beta regulates TC21 transformed cells is not known. To better understand the role of TC21 in cancer progression, we overexpressed an activated G23V mutant of TC21 in a nontumorigenic murine mammary epithelial (EpH4) cell line. Mutant TC21-expressing cells were significantly more oncogenic than cells expressing activated G12V H-Ras both in vivo and in vitro. TC21-induced transformation and proliferation required activation of p38 MAPK, mTOR (the mammalian target of rapamycin), and phosphoinositide 3-kinase but not Akt/PKB. Transformation by TC21 rendered EpH4 cells insensitive to the growth inhibitory effects of TGF-beta, and the soft agar growth of these cells was increased upon TGF-beta stimulation. Despite losing responsiveness to TGF-beta-mediated growth inhibition, both Smad-dependent and independent pathways remained intact in TC21-transformed cells. Thus, overexpression of active TC21 in EpH4 cells induces tumorigenicity through the phosphoinositide 3-kinase, p38 MAPK, and mTOR pathways, and these cells lose their sensitivity to the normal growth inhibitory role of TGF-beta.     

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.     

The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.     

H-Ras Mediates the Inhibitory Effect of Epidermal Growth Factor on the Epithelial Na+ Channel

The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na⁺ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na⁺ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.     

Overexpression of RasN17 fails to neutralize endogenous Ras in MCF7 breast cancer cells

Receptor tyrosine kinases of the ErbB family have been implicated in the onset/progression of a number of neoplasias. In these diseases, ErbB receptor expression may be accompanied by constitutive activation caused by molecular alterations, overexpression, or ligand binding. An important signaling route activated by these receptors that has been linked to the stimulation of cell proliferation is the Ras route. Here we have investigated the action of a mutant Ras form, H-RasN17, on the proliferation of the breast cancer epithelial cell line MCF7 cells. In these cells expression of RasN17 failed to affect serum or ErbB receptor-stimulated proliferation. Analysis of the action of RasN17 indicated that overexpression of this mutant form of Ras did not affect neuregulin or protein kinase C-induced activation of Erk1/2. In addition, RasN17 failed to prevent activation of endogenous N-Ras and H-Ras even though the levels of the latter were much lower than those of the RasN17 form. The failure of RasN17 to prevent endogenous Ras activation did not appear to be due to deficient processing or sorting of the mutated form. These data indicated that the action of RasN17 as a bona fide inhibitor of Ras depends on the cell type and requires detailed analysis of the biochemical and biological properties of RasN17, particularly with respect to the activation of endogenous Ras.     

Conditional apoptosis induced by oncogenic ras in thyroid cells

Mutations of ras are tumor-initiating events for many cell types, including thyrocytes. To explore early consequences after oncogenic Ras activation, we developed a doxycycline-inducible expression system in rat thyroid PCCL3 cells. Beginning 3-4 days after H-Ras(v12) expression, cells underwent apoptosis. The H-Ras(v12) effects on apoptosis were decreased by a mitogen-activated protein kinase kinase (MEK1) inhibitor and recapitulated by doxycycline-inducible expression of an activated MEK1 mutant (MEK1(S217E/S221E)). As reported elsewhere, acute expression of H-Ras(v12) also induces mitotic defects in PCCL3 cells through ERK (extracellular ligand-regulated kinase) activation, suggesting that apoptosis may be secondary to DNA damage. However, acute activation of SAPK/JNK (stress-activated protein kinase/Jun N-terminal kinase) through acute expression of Rac1(v12) also triggered apoptosis, without inducing large-scale genomic abnormalities. H-Ras(v12)-induced apoptosis was dependent on concomitant activation of cAMP by either TSH or forskolin, in a protein kinase A-independent manner. Thus, coactivation of cAMP-dependent pathways and ERK or JNK (either through H-Ras(v12), Rac1(v12), or MEK1(S217E/S221E)) is inconsistent with cell survival. The fate of thyrocytes within the first cell cycles after expression of oncogenic Ras is dependent on ambient TSH levels. If both cAMP and Ras signaling are simultaneously activated, most cells will die. Those that survive will eventually lose TSH responsiveness and/or inactivate the apoptotic cascade through secondary events, thus enabling clonal expansion.     

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.     

A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

Rasosomes originate from the Golgi to dispense Ras signals

Ras proteins undergo an incompletely understood trafficking process in the cell. Rasosomes are protein nanoparticles of 80–100 nm diameter that carry lipidated Ras isoforms (H-Ras and N-Ras) as well as their effectors through the cytoplasm and near the plasma membrane (PM). In this study, we identified the subcellular origin of rasosomes and how they spread Ras proteins through the cell. We found no dependency of rasosome formation on galectins, or on the GDP-/GTP-bound state of Ras. We found that significantly more rasosomes are associated with forms of Ras that are localized to the Golgi, namely N-Ras or the singly palmitoylated H-Ras mutant (C181S). To explore the possibility that rasosome originate from the Golgi, we used photoactivatable (PA)-GFP-H-Ras mutants and showed that rasosomes bud from the Golgi in a two-step mechanism. Newly released rasosomes first move in an energy-dependent directed fashion and then convert to randomly diffusing rasosomes. Dual fluorescence time-lapse imaging revealed the appearance of dually labeled rasosomes, indicating a dynamic exchange of cytoplasmic and PM-associated Ras with rasosome-associated Ras. Finally, higher levels of rasosomes correlate with higher levels of ERK phosphorylation, a key marker of Ras downstream signaling. We suggest that H-Ras and N-Ras proteins exchange with rasosomes that can function as carriers of palmitoylated Ras and its signals.     

ADP-ribosylation of oncogenic Ras proteins by Pseudomonas aeruginosa exoenzyme S in vivo

The exoenzyme S (ExoS)-producing Pseudomonas aeruginosa strain, 388, and corresponding ExoS knock-out strain, 388Δ exoS , were used in a bacterial and mammalian co-culture system as a model for the contact-dependent delivery of ExoS into host cells. Examination of DNA synthesis and Ras ADP-ribosylation in tumour cell lines expressing normal and mutant Ras revealed a decrease in DNA synthesis concomitant with ADP-ribosylation of Ras proteins after exposure to ExoS-producing bacteria, but not after exposure to non-ExoS-producing bacteria. Examination of normal H-Ras, K-Ras and N-Ras by two-dimensional electrophoresis after exposure to bacteria revealed differences in the degree of ADP-ribosylation by ExoS, with H-Ras being modified most extensively. ADP-ribosylation of oncogenic forms of Ras was examined in vivo using cancer lines expressing mutant forms of H-, N- or K-Ras. The mutant Ras proteins were modified in a manner qualitatively similar to their normal counterparts. Using Ras/Raf-1 co-immunoprecipitation after co-culture, it was found that exposure to ExoS-producing bacteria caused a decrease in the amount of Raf-1 associated with EGF-activated Ras and oncogenic Ras. The results from this study indicate that ExoS ADP-ribosylates both normal and mutant Ras proteins in vivo and inhibits signalling through Ras.     

Activation of H-Ras and Rac1 correlates with epidermal growth factor-induced invasion in Hs578T and MDA-MB-231 breast carcinoma cells

There is considerable experimental evidence that hyperactive Ras proteins promote breast cancer growth and development including invasiveness, despite the low frequency of mutated forms of Ras in breast cancer. We have previously shown that H-Ras, but not N-Ras, induces an invasive phenotype mediated by small GTPase Rac1 in MCF10A human breast epithelial cells. Epidermal growth factor (EGF) plays an important role in aberrant growth and metastasis formation of many tumor types including breast cancer. The present study aims to investigate the correlation between EGF-induced invasiveness and Ras activation in four widely used breast cancer cell lines. Upon EGF stimulation, invasive abilities and H-Ras activation were significantly increased in Hs578T and MDA-MB-231 cell lines, but not in MDA-MB-453 and T47D cell lines. Using small interfering RNA (siRNA) to target H-Ras, we showed a crucial role of H-Ras in the invasive phenotype induced by EGF in Hs578T and MDA-MB-231 cells. Moreover, siRNA-knockdown of Rac1 significantly inhibited the EGF-induced invasiveness in these cells. Taken together, this study characterized human breast cancer cell lines with regard to the relationship between H-Ras activation and the invasive phenotype induced by EGF. Our data demonstrate that the activation of H-Ras and the downstream molecule Rac1 correlates with EGF-induced breast cancer cell invasion, providing important information on the regulation of malignant progression in mammary carcinoma cells.     

Synergistic effect of curcumin on epigallocatechin gallate-induced anticancer action in PC3 prostate cancer cells

Epigallocatechin gallate (EGCG) and curcumin are well known to naturally-occurring anticancer agents. The aim of this study was to verify the combined beneficial anticancer effects of curcumin and EGCG on PC3 prostate cancer cells, which are resistant to chemotherapy drugs and apoptosis inducers. EGCG showed weaker inhibitory effect on PC3 cell proliferation than two other prostate cancer cell lines, LNCaP and DU145. Co-treatment of curcumin improved antiproliferative effect of EGCG on PC3 cells. The protein expressions of p21 were significantly increased by the co-treatment of EGCG and curcumin, whereas it was not changed by the treatment with each individual compound. Moreover, treatments of EGCG and curcumin arrested both S and G2/M phases of PC3 cells. These results suggest that the enhanced inhibitory effect of EGCG on PC3 cell proliferation by curcumin was mediated by the synergic up-regulation of p21-induced growth arrest and followed cell growth arrest. [BMB Reports 2015; 48(8): 461-466]     

Regulation of endothelial cell differentiation and transformation by H-Ras

Angiogenesis is regulated by growth factors which activate tyrosine kinase receptors leading to the activation of a number of intracellular signaling pathways. The specific function of H-Ras during FGF-2 stimulated endothelial cell differentiation, defined as invasive growth and formation of branching networks in fibrin gels, was investigated by using conditionally immortalized endothelial cell lines induced to express H-Ras mutants. Expression of inhibitory N17Ras did not impair differentiation in response to FGF-2 and TNF-alpha. The farnesyltransferase inhibitor FTI-277 inhibited farnesylation of Ras but did not inhibit differentiation of human microvascular endothelial cells or mouse brain endothelial cells. In contrast, activated V12Ras inhibited endothelial cell differentiation and cells displayed a transformed phenotype with an increased rate of proliferation and loss of contact inhibited growth. Furthermore, V12Ras expressing endothelial cells grew as solid tumors when injected subcutaneously into mice. Our data suggest that, in endothelial cells, H-Ras activity is not required for differentiation. However, this activity must be tightly regulated as aberrant activity can disturb the ability of endothelial cells to undergo differentiation.     

The tumor suppressor DiRas3 forms a complex with H-Ras and C-RAF proteins and regulates localization, dimerization, and kinase activity of C-RAF

The maternally imprinted Ras-related tumor suppressor gene DiRas3 is lost or down-regulated in more than 60% of ovarian and breast cancers. The anti-tumorigenic effect of DiRas3 is achieved through several mechanisms, including inhibition of cell proliferation, motility, and invasion, as well as induction of apoptosis and autophagy. Re-expression of DiRas3 in cancer cells interferes with the signaling through Ras/MAPK and PI3K. Despite intensive research, the mode of interference of DiRas3 with the Ras/RAF/MEK/ERK signal transduction is still a matter of speculation. In this study, we show that DiRas3 associates with the H-Ras oncogene and that activation of H-Ras enforces this interaction. Furthermore, while associated with DiRas3, H-Ras is able to bind to its effector protein C-RAF. The resulting multimeric complex consisting of DiRas3, C-RAF, and active H-Ras is more stable than the two protein complexes H-Ras·C-RAF or H-Ras·DiRas3, respectively. The consequence of this complex formation is a DiRas3-mediated recruitment and anchorage of C-RAF to components of the membrane skeleton, suppression of C-RAF/B-RAF heterodimerization, and inhibition of C-RAF kinase activity.     

In-silico identification of small molecules targeting H-Ras and in-vitro cytotoxicity with caspase-mediated apoptosis in carcinoma cells

H-Ras oncogene plays a critical role in the transformation of normal cells to a malignant phenotype through constitutive activation of the GTP bound protein leading to uncontrolled cell proliferation in several human cancers. Thus, H-Ras oncoprotein serves as an excellent target for anticancer drug discovery. To identify novel H-Ras inhibitors, we performed structure-based virtual screening of the Maybridge HitFinder™ library using Schrodinger suite. Thirty ligands from the chemical library were identified as they showed preferential in silico binding initially to H-Ras proteins with Gly12Val, Gly13Asp, and Gly12Val-Gly13Asp mutations. Absorption, distribution, metabolism, excretion, and toxicity profile confirmed drug-like properties of the compounds. Three representative molecules were tested for antiproliferative effect on T24 urinary bladder carcinoma cell line, MCF-7 breast cancer cell line and HDF-7 normal dermal fibroblast cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Two compounds (Cmpds) showed antiproliferative activity exclusively in the cancer cell lines with minimal effect on the control HDF-7 cells. The effect of compound treatment on cell cycle progression, assessed by propidium iodide (PI) staining, depicted increased arrest of T24 cell line in the sub G1 phase. Further, Annexin-V PI dual staining and pan caspase inhibitor Z-VAD-fmk indicated caspase-dependent apoptotic activity of Cmpds 1 and 3. Our findings demonstrate caspase-dependent apoptotic activity of Cmpds 1 and 3 selectively against Gly12Val mutated T24 cancer cell line implicating a potential for treatment of bladder cancer. We envisage that these molecules may be promising candidates with potential therapeutic value in H-Ras mutation-associated cancers.     

Optimization of oncogene expression through intra-population competition

Although functional roles have been assigned to many genes, e.g. those involved in cell-cycle regulation, growth signaling, or cancer, considerably less is known about the quantitative relationship between gene expression levels and outcome. We devised an intra-population competition to study oncogene dosage. Cell populations were engineered to express a range of H-Ras oncogene levels. Cells with different levels of H-Ras then “competed” for an increased share of the total cell population. Using flow cytometry to track the population composition over time, we determined the relationship between the different H-Ras oncogene expression levels and the net proliferation rate. Under culture conditions in which wild-type Ras activation was suppressed, we found that increased and maximal net proliferation occurred when the H-Ras G12V oncogene was expressed at a level 1.2-fold that of wild-type Ras. As the H-Ras G12V expression levels increased above this optimal level, proliferation rates decreased. Our findings suggest that the tumor evolution process may optimize gene expression levels for maximal cell proliferation. In principle, engineered intra-population competitions can be used to determine proliferation rates associated with the level of any ectopically expressed gene. The approach also may be used to determine proliferation rates associated with different cell species in a heterogeneous population or to improve the proliferation rate of a cell line. We also envision that the tracking of intra-population competitions could be utilized to investigate the evolution of tumors in the body.