Ras-mediated Cell Transformation Research Articles

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation.

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3' untranslated region (3' UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3'UTR APA sites, leading to mRNA isoforms that differ substantially in 3'UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRAS G12V . Perturbations of isoform expression with long 3'UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3'UTR isoform contributes to the secreted Timp2 protein much more than the short 3'UTR isoform. The short and long 3'UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRAS G12V . Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3'UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

Aurora-B and Survivin Expression in Head and Neck Cancer: Correlation with Clinicopathologic Features and Patient Survival

Aurora-B and survivin are the members of chromosomal passenger complex, which regulates cell division. The kinase activity of aurora-B is stimulated by survivin. Their activities are responsible for oncogenic Ras-mediated cell transformation and accelerated tumor cell growth. It has been shown that high expressions of aurora-B and survivin are correlated with malignant behaviors of head and neck cancer. However, according to our knowledge, there is no study to evaluate the prognostic value of both markers on patient survival.

Aurora-B/AIM-1 kinase activity is involved in Ras-mediated cell transformation

Aurora-B, previously known as AIM-1, is a conserved eukaryotic mitotic protein kinase. In mammals, this kinase plays an essential role in chromosomal segregation processes, including chromosome condensation, alignment, control of spindle checkpoints, chromosome segregation, and cytokinesis. Aurora-B is overexpressed in various cancer cells, suggesting that the kinase activity perturbs chromosomal segregation processes. Its forced overexpression induces chromosomal number instability and progressive tumorigenicity in rodent cells in vitro and in vivo. Nevertheless, based on focus formation in BALB/c 3T3 A31-1-1 cells, Aurora-B is not oncogenic. Here, we show that Aurora-B kinase activity augments Ras-mediated cell transformation. RNA interference with short hairpin RNA inhibits transformation by Ras and its upstream oncogene Src, but not by the downstream oncogene Raf. In addition, the inner centromere protein, which is a passenger protein associated with Aurora-B, has a similar ability to potentiate the activity of oncogenic Ras. These data indicate that elevated Aurora-B activity promotes transformation by oncogenic Ras by enhancing oncogenic signaling and by converting chromosome number-stable cells to aneuploid cells.

A Ras-like Tumor Suppressor

Another Ras-like tumor suppressor has been identified by Ellis et al. , adding to a growing list of heretical members of the Ras superfamily of small GTP-binding proteins. Rig (Ras-related inhibitor of cell growth) was identified through a bioinformatic search of the human expressed sequence tag database for GTP-binding proteins. It is most similar to Noey2, another Ras-like protein with tumor suppressing properties. As predicted by the Rig amino acid sequence, the protein is modified by farnesyl and geranylgeranyl isoprenoid lipids. It also inhibited Ras-mediated cell transformation when expressed in cultured cells. Rig expression is limited to human brain and heart tissue, and its loss of expression was observed in various neural tumor-derived cell lines and primary human astrocytomas. The mechanism by which loss of Rig expression causes tumorigenesis remains to be determined, although it may involve regulation of Ras effector molecules such as the kinase Raf. C. A. Ellis, M. D. Vos, H. Howell, T. Vallecorsa, D. W. Fults, G. J. Clark, Rig is a novel Ras-related protein and potential neural tumor suppressor. Proc. Natl. Acad. Sci. U.S.A. 99 , 9876-9881 (2002). [Abstract] [Full Text]

Transforming Growth Factor-β1 Promotes Invasiveness after Cellular Transformation with Activated Ras in Intestinal Epithelial Cells

Invasion is a defining event in carcinoma progression. In general, invasive carcinoma is characterized by an epithelial–fibroblastoid conversion associated with loss of cell–cell adhesion receptors such as E-cadherin and β-catenin. We report here that TGF-β1 promotes the invasiveness by modulating the alterations of cellular plasticity including a loss of cell–cell contact in Ras-transformed epithelial cells. In order to examine the role of TGF-β1 in the Ras-induced responses, intestinal epithelial cells expressing a conditionally activated Ha-RasVal12 (RIE-iRas cells) were used in this study. Induced expression of activated Ha-RasVal12 caused morphologic transformation of the RIE-iRas cells with an increase in vimentin expression and a decrease of E-cadherin levels. There was also redistribution of β-catenin from the cytoplasm to the nucleus after the induction of Ras. TGF-β1 treatment enhanced both the decrease in E-cadherin levels and the redistribution of β-catenin. Interestingly, the activation of Ras markedly decreased the level of TGF-β receptor type II (TβRII) in RIE-iRas cells. However, the expression of plasminogen activator inhibitor-1, which is known to be transcriptionally induced by TGF-β1, was strongly induced by TGF-β1 despite the marked downregulation of TβRII. The induction of Ha-RasVal12 markedly increased the invasiveness in RIE-iRas cells, as evaluated by a collagen type I-coated Boyden-chamber assay, and the Ras-mediated invasiveness was significantly enhanced by TGF-β1 treatment. Expression of a dominant-negative form of TβRII in the RIE-iRas cells abrogated both growth-inhibitory and invasion responses to TGF-β1. Collectively, these results suggest that TGF-β1 and oncogenic Ras collaborate in promoting cellular invasiveness in intestinal epithelial cells. The enhancement of invasiveness was correlated with decreased E-cadherin levels and subcellular distribution of β-catenin. The enhancement of oncogenic Ras-mediated cell transformation by TGF-β1 occurs via TβRII.

The development of protein farnesyltransferase and other ras-directed therapeutics for malignant diseases

Ras is a guanine nucleotide-binding protein that plays a critical role in cell growth and malignant transformation. Following stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/MAP kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated Ras proteins that remain locked in an active state, thereby relaying aberrant proliferative signals. After synthesis, Ras undergoes a series of post-translational modifications, the most important of which is the addition of a lipophilic farnesyl isoprenoid moiety in a reaction catalysed by the enzyme protein farnesyltransferase (FTase). The farnesylation of Ras allows it to interact with other proteins in close proximity to the plasma membrane. Inhibiting FTase would prevent Ras from maturing into its biologically active form and, therefore, FTase is of considerable interest as a potential therapeutic target. Various classes of FTase inhibitors have been identified that interfere with the farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines and inhibit the growth of human tumour xenografts. In transgenic mice with established tumours, with various ras mutations. FTase inhibitors cause regression in some tumours, which appears to be mediated through both apoptosis and cell cycle regulation. There is also ample, intriguing evidence indicating that FTase inhibitors are active in malignancies without ras mutations. In addition, there is experimental evidence indicating that FTase inhibitors enhance the cytotoxic effects of other therapeutic modalities including radiation and several types of cytotoxic chemotherapy. To date, FTase inhibitors have been well-tolerated in both animal studies and early clinical trials and do not seem to produce the generalised cytotoxic effects in normal tissues, that are a major limitation of most conventional anticancer agents. Clinical evaluations of FTase inhibitors to determine the feasibility of administering them as single agents and in combination with other therapeutic modalities on dose schedules that portend optimal therapeutic indices in preclinical studies are ongoing. However, because of the unique biological aspects of FTase, designing disease-directed Phase II and III evaluations of their effectiveness presents formidable challenges.

NF-kappa B is required for H-ras oncogene induced abnormal cell proliferation and tumorigenesis.

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription factors. In turn, these factors control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription factor NF-kappa B contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-kappa B activity by forced expression of a super-repressor form of I kappa B alpha, the major inhibitor of NF-kappa B, markedly decreased the growth rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo fibroblasts. Such clonally isolated cells overexpressing I kappa B alpha super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-kappa B in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-alpha treatment. In contrast, inhibition of NF-kappa B activity in non-transformed fibroblasts had minimal effect on growth, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-kappa B activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-kappa B in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity. Oncogene (2000) 19, 841 - 849.

Ras protein farnesyltransferase: A strategic target for anticancer therapeutic development.

Ras proteins are guanine nucleotide-binding proteins that play pivotal roles in the control of normal and transformed cell growth and are among the most intensively studied proteins of the past decade. After stimulation by various growth factors and cytokines, Ras activates several downstream effectors, including the Raf-1/mitogen-activated protein kinase pathway and the Rac/Rho pathway. In approximately 30% of human cancers, including a substantial proportion of pancreatic and colon adenocarcinomas, mutated ras genes produce mutated proteins that remain locked in an active state, thereby relaying uncontrolled proliferative signals. Ras undergoes several posttranslational modifications that facilitate its attachment to the inner surface of the plasma membrane. The first-and most critical-modification is the addition of a farnesyl isoprenoid moiety in a reaction catalyzed by the enzyme protein farnesyltransferase (FTase). It follows that inhibiting FTase would prevent Ras from maturing into its biologically active form, and FTase is of considerable interest as a potential therapeutic target. Different classes of FTase inhibitors have been identified that block farnesylation of Ras, reverse Ras-mediated cell transformation in human cell lines, and inhibit the growth of human tumor cells in nude mice. In transgenic mice with established tumors, FTase inhibitors cause regression in some tumors, which appears to be mediated through both apoptosis and cell cycle regulation. FTase inhibitors have been well tolerated in animal studies and do not produce the generalized cytotoxic effects in normal tissues that are a major limitation of most conventional anticancer agents. There are ongoing clinical evaluations of FTase inhibitors to determine the feasibility of administering them on dose schedules like those that portend optimal therapeutic indices in preclinical studies. Because of the unique biologic aspects of FTase, designing disease-directed phase II and III evaluations of their effectiveness presents formidable challenges.

Relevance of ER to the Development of Endometrial Hyperplasia and Adenocarcinoma.

Estrogen has an important role in both the etiology and treatment of hormone-dependent endometrial cancers, although the mechanism remains elusive. To definethe role of estrogen-mediated signaling we investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation via the [ &sup12; Val ] K-Ras mutant. This mutant enhanced the steady state level and transcriptional activity of ER. In addition, overexpression of both wild type K-Ras and ER transformed NIH3T3 cells. Co-expression of the progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of ER activation. The antisense oligomers complementary to ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation. To address whether ER activation is also important in the development of human endometrial cancers, we investigated ER and PR expression levels in premalignant and malignant endometrial lesions. The results suggested the implication of ER abundance in endometrial hyperplasias, though modulation of PR expression by ER was retained. G1 adenocarcinoma also expressed higher levels of ER while PR modulation by ER was abrogated. These data implied the importance of ER activitiesin endometrial hyperplasia and G1 adenocarcinoma development.

EPS8 and E3B1 transduce signals from Ras to Rac.

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.

Requirement of Estrogen Receptor Expression and Function for [12Val] K-Ras-Mediated NIH3T3 Cell Transformation

We investigated the biological significance of estrogen receptors (ERs) in NIH3T3 cell transformation by the [¹²Val] K-Ras mutant. This mutant enhanced the steady-level and transcriptional activity of ER. Coexpression of the progesterone receptor with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

Contribution of enhanced transcriptional activation by ER to [12Val] K-Ras mediated NIH3T3 cell transformation

We investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation by the [12Val] K-Ras mutant. This mutant enhanced the steady state level of ER. Cells expressing mutant K-Ras (K12V cell) were tumorigenic. To determine the role of ER accumulation in Ras-transformed cells, we developed cells (KwtER cells) that overexpressed both wild-type (wt) K-Ras and ER, and found these cells were also tumorigenic. E2 stimulated the transcriptional activity by ER dominantly in K12V cells. However, only partial activation of ER by E2 was seen in KwtER cells. In the presence of 10% serum in media, the activation of ER appeared only in transformed KtwER and K12V cells, suggesting that two independently transmitted signals, the E2-ER binding and the ER-AF1 activation, are necessary for ER activation and that the dominant activation of ER might be involved in Ras-mediated cell transformation. Co-expression of progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of the activation of ER. The antisense oligomers complementary to the ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.

Recombinant Expression of Caveolin-1 in Oncogenically Transformed Cells Abrogates Anchorage-independent Growth

Caveolae are plasma membrane-attached vesicular organelles. Caveolin-1, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes in vivo. Both caveolae and caveolin are most abundantly expressed in terminally differentiated cells: adipocytes, endothelial cells, and muscle cells. Conversely, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes such as v-abl and H-ras (G12V); caveolae are absent from these cell lines. However, its remains unknown whether down-regulation of caveolin-1 protein and caveolae organelles contributes to their transformed phenotype. Here, we have expressed caveolin-1 in oncogenically transformed cells under the control of an inducible-expression system. Regulated induction of caveolin-1 expression was monitored by Western blot analysis and immunofluorescence microscopy. Our results indicate that caveolin-1 protein is expressed well using this system and correctly localizes to the plasma membrane. Induction of caveolin-1 expression in v-Abl-transformed and H-Ras (G12V)-transformed NIH 3T3 cells abrogated the anchorage-independent growth of these cells in soft agar and resulted in the de novo formation of caveolae as seen by transmission electron microscopy. Consistent with its antagonism of Ras-mediated cell transformation, caveolin-1 expression dramatically inhibited both Ras/MAPK-mediated and basal transcriptional activation of a mitogen-sensitive promoter. Using an established system to detect apoptotic cell death, it appears that the effects of caveolin-1 may, in part, be attributed to its ability to initiate apoptosis in rapidly dividing cells. In addition, we find that caveolin-1 expression levels are reversibly down-regulated by two distinct oncogenic stimuli. Taken together, our results indicate that down-regulation of caveolin-1 expression and caveolae organelles may be critical to maintaining the transformed phenotype in certain cell populations.

Review Oncologic, Endocrine & Metabolic: Inhibitors of protein farnesylation: A new approach to cancer chemotherapy

The Ras protein functions at a critical point in the signalling pathway between extracellular growth regulatory messengers and the nuclear factors that are responsible for the initiation of cellular replication. The action of mutant, oncogenic Ras proteins is implicated in a significant fraction of human tumours. All Ras proteins appear to require post-translational lipidation for their function. The key lipidation event is the attachment of a farnesyl unit to a cysteine near the carboxyl terminus of the protein. Recently, the possibility of interfering with Ras-mediated cell transformation through inhibition of protein farnesylation has attracted significant attention. This review surveys the types of compounds that are reported to inhibit the enzyme protein farnesyltransferase and the biological effects of these compounds in vitro and in vivo.