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PubMed2026

RAS-GTP Inhibition Overcomes Acquired Resistance to KRASG12C Inhibitors Mediated by Oncogenic and Wild-Type RAS Activation in Non-Small Cell Lung Cancer.

UNLABELLED: Small-molecule KRASG12C(OFF) inhibitors that bind to the inactive GDP-bound state of KRAS have demonstrated efficacy in patients with KRASG12C-mutant tumors, yet responses tend to be transient because of emergence of on-treatment resistance. Recently, RAS(ON) G12C-selective inhibitors, which bind to the active GTP-bound state of RAS, were described, and elironrasib is undergoing evaluation in multiple clinical trials. In this study, we generated resistant cell lines and patient-derived xenograft models to KRASG12C(OFF) and RAS(ON) G12C-selective inhibitors and interrogated resistance mechanisms using a multiomics strategy consisting of phosphoproteomics, whole-exome sequencing, and RNA sequencing combined with functional testing using small-molecule and CRISPR screens and RAS(ON) inhibitors being evaluated in clinical trials. Two models reactivated RAS signaling, either via KRASG12C gene amplification or NRASG13R mutation, and were vulnerable to dual inhibition by RAS(ON) G12C-selective and RAS(ON) multiselective inhibitors, RMC-4998 and RMC-7977. Two models, which lacked any discernable genomic alteration, acquired resistance associated with increased receptor tyrosine kinase activity and downstream persistent RAS activity and were sensitive to RAS-GTP inhibition by RMC-7977. Finally, one model displayed epithelial-mesenchymal transition, loss of RAS dependence, and acquired reliance on cell-cycle kinases and proteins associated with DNA damage response. This work highlights KRASG12C-selective inhibitor resistant states that parallel and complement clinical findings and demonstrate that a large subset could be overcome with a RAS(ON) multi-selective inhibitor as a stand-alone agent or in combination with other therapies. SIGNIFICANCE: Multi-omic characterization of resistance mechanisms to KRASG12C-selective inhibitors in non-small cell lung cancer provides insights that could inform precision medicine-based therapeutic approaches for improving the treatment of KRASG12C mutant tumors. See related article by Stern et al., p. 485.

Humans↗

PubMed2000

Divergent responses of ras-transfected and non-ras-transfected human keratinocytes to extracellular calcium.

Raising extracellular calcium (Ca(o)) induces terminal differentiation in cultured epidermal keratinocytes. The introduction of the ras oncogene into keratinocytes results in resistance to Ca(o)-mediated differentiation. To understand the signaling mechanism involved, we examined the Ca(o)-induced formation of inositol triphosphate (IP3) and changes in intracellular Ca2+ (Ca(i)) concentration in non-ras-transfected and ras-transfected HaCaT lines of human keratinocytes. When switched from 0.05- to 1.5-mM Ca(o) medium, the non-ras HaCaT line showed a rapid twofold increase in IP3 formation, whereas the IP3 level in the ras-transfected I-7 line was slightly affected. G-protein-coupled activation of phospholipase was intact in both lines, as evidenced by the generation of similar amounts of IP3 in response to addition of bradykinin or guanosine 5'-[gamma-thio]-triphosphate. Addition of 1.0 mM Ca(o) evoked similar Ca(i) responses in both non-ras- and ras-transfected cells: a transient elevation, followed by a sustained lower plateau. However, the two lines differed in their later responses: after being maintained in 1.0 mM Ca2+ for 24 h, the Ca(i) level was significantly lower in ras-transfected cells than in non-ras-transfected HaCaT cells. The Ca(o)-induced increase in Ca(i) in both lines was inhibited by the Ca2+ entry blocker SK&F 96365 or depolarization in high K+ bathing solution, demonstrating its dependence of calcium influx. The results suggest fundamental differences in the early signal that are generated in response to an increase in Ca(o) in ras-transfected keratinocytes, with the absence of a Ca(o)-induced rise in IP3--a signaling pathway defect that may play a role in the differentiation block the cells exhibit. In addition, the inability of ras-transfected cells to sustain a prolonged Ca(i) plateau may also contribute to their inability to differentiate in response to the Ca(o) signal.

Bradykinin↗

PubMed1997

Targeted disruption of the epidermal growth factor receptor impairs growth of squamous papillomas expressing the v-ras(Ha) oncogene but does not block in vitro keratinocyte responses to oncogenic ras.

We have assessed the role of epidermal growth factor receptor (EGFR) signaling in biological responses to the v-ras(Ha) oncogene using primary keratinocytes from Egfr -/- mice and wild-type littermates. On the basis of several criteria, Egfr -/- keratinocytes were unresponsive to either acute or chronic exposure to several EGFR ligands but were stimulated to proliferate in response to several other mitogens. Although conditioned medium from primary keratinocytes transduced with v-ras(Ha) retrovirus (v-ras(Ha) keratinocytes) was a potent mitogen for wild-type but not Egfr -/- keratinocytes, v-ras(Ha) transduction of primary keratinocytes of either genotype resulted in a strong mitogenic response, arguing against an obligatory role for EGFR activation in v-ras(Ha)-mediated stimulation of keratinocyte proliferation. Infection with high-titer v-ras(Ha) retrovirus altered the keratin expression pattern in keratinocytes of both genotypes, suppressing differentiation-specific keratins K1 and K10 while activating aberrant expression of K8 and K18. In wild-type but not Egfr -/- cultures, K1 and K10 were also suppressed following infection at lower retroviral titers, presumably as a result of paracrine EGFR activation on uninfected cells present in these cultures. Squamous papillomas produced by grafting Egfr -/- v-ras(Ha) keratinocytes onto nude mice were only 21% of the size of wild-type v-ras(Ha) tumors, and a striking redistribution of S-phase cells was detected by immunostaining for bromodeoxyuridine. In Egfr -/- v-ras(Ha) papillomas, the fraction of total labeled nuclei detected in suprabasal layers was increased from 19 to 39%. In contrast, the basal layer labeling index of Egfr -/- papillomas was reduced to 34%, compared to 43% in wild-type tumors. Our results indicate that, although autocrine EGFR signaling is not required for keratinocyte responses to oncogenic ras in culture or benign tumor formation in nude mouse grafts, disruption of this pathway impairs growth of v-ras(Ha) papillomas by a mechanism that may involve alterations in keratinocyte cell cycle progression and/or migration in vivo.

Animals↗

PubMed1996

Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles.

Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras.

Alleles↗

PubMed2006

Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

Adult↗

PubMed1994

Differential Na+,K(+)-ATPase activity and cisplatin sensitivity between transformants induced by H-ras and those induced by K-ras.

We examined the differential effects of the H-ras oncogene and the K-ras oncogene on cisplatin sensitivity in murine NIH/3T3 cells transfected with these oncogenes. Although the NIH/3T3 cells transformed with H-ras oncogenes (EJ-NIH/3T3 and Ha8-21) showed an increased resistance to cisplatin compared to the parental NIH/3T3, the cell lines transformed with K-ras oncogenes (DT and 1,8DNP2-2-5) did not. Compared with NIH/3T3, the 2 H-ras transformants reduced both the accumulation of cisplatin and the Na+,K(+)-ATPase activity in the membrane fraction. On the other hand, we observed no significant difference in cellular accumulation of cisplatin or in Na+,K(+)-ATPase activity between parental NIH/3T3 and the K-ras transformants. Since these ras transformants did not affect the cellular metallothionein content, transcriptional level of DNA polymerase beta or activity of glutathione-S-transferase which is not associated with cisplatin sensitivity, these results suggest that cisplatin resistance is brought about by the H-ras oncogene, but not by K-ras, and that induction of cisplatin resistance by H-ras is mainly due to a reduction of cisplatin accumulation and an impairment of Na+,K(+)-ATPase activity in the membrane fraction.

3T3 Cells↗

PubMed1991

ras proteins and the ras-related signal transduction pathway.

Mammalian ras genes may naturally acquire oncogenic transformation potential through some point mutations which result in the impairment of the normal ras protein functions, and which are localised in codons 12, 13 or 61. Mutationally activated ras alleles were found in a wide variety of human and carcinogen (including radiation)-induced animal malignancies. In man, myeloid leukemias are often associated with the presence of a mutationally activated ras gene (for review, see Bos JL (1989), Cancer Res 49:4682-4689). However, we failed till now in our attempts to detect oncogenic ras mutations in radiation-induced mouse myeloid leukemias. We thus have the feeling that ras might perhaps participate to tumorigenesis through another mechanism provoking a deregulation of the ras protein functions. In order to help evaluate such a possibility, we give here a very concise overview of the properties of the ras proteins and of their regulation by a variety of still hypothetical molecular switches. This overview does not include bibliographic references. Indeed, we gathered much of the information described below at the Cold Spring Harbor Symposium on "Function and Evolution of ras Proteins", May 9-13, 1990. Communications presented at Cold Spring Harbor Symposia may contain preliminary data and should not be cited in bibliographies. Another voluntary omission in this overview is that, for the sake of simplicity, we do not mention whether the data were obtained from experiments performed on H-, K- or N-ras. Details can be found in the published book of abstracts.

Animals↗

PubMed1998

Inhibition of K-ras-transformed rodent and human cancer cell growth via induction of apoptosis by irreversible inhibitors of Ras endoprotease.

Proteolytic removal of the carboxyl terminal tripeptide of Ras oncoproteins is important in the Ras function. Two chloromethyl ketones, BFCCMK and UM96001, designed to be the Ras C-terminal sequence-specific endoprotease inhibitors, at low micromolar concentrations (5.0 microM), potently inhibit the growth of ras-transformed rodent and human cancer cells, whereas untransformed NIH/3T3 cells are not affected under the same conditions. Furthermore, BFCCMK and UM96001 block more than 98% of the anchorage-independent clonogenic growth of ras-transformed rat and human cancer cells at low micromolar concentrations. The blocking of cancer cell growth may be due to the selective induction of apoptosis of ras-transformed cells by these inhibitors. These results provide the first experimental evidence that the endoproteolysis of Ras oncoproteins is important for the growth and apoptosis of ras-transformed cancer cells. Therefore, the Ras C-terminal sequence-specific endoprotease may be a potential new target for the treatment of human cancers induced by ras mutations.

3T3 Cells↗

PubMed1997

Ras-GTPase activating protein (GAP): a putative effector for Ras.

One attractive candidate for a Ras effector protein, other than the Raf kinases, is Ras-GAP. Indeed, recent literature suggests that besides the Raf/MAP kinase cascade, additional pathways must be stimulated to elicit a full biological response to Ras. Ras binds the COOH terminal domain of Ras-GAP, while the NH2 terminal domain appears to be essential for triggering downstream signals. Since Ras-GAP itself has no obvious enzymatic function that might explain a role in processes associated with proliferation, differentiation or apoptosis, candidates for downstream Ras-GAP effectors that fulfill this role remain to be identified. The newly found GAP-SH3 domain Binding Protein (G3BP) may be one of these. This review will briefly overview the candidates Ras effectors and discuss the results that position Ras-GAP as a critical effector downstream of Ras.

Animals↗

PubMed2005

Expression of a constitutively active mutant of M-Ras in normal bone marrow is sufficient for induction of a malignant mastocytosis/mast cell leukemia, distinct from the histiocytosis/monocytic leukemia induced by expression of activated H-Ras.

Expression of constitutively activated M-Ras in normal murine bone-marrow cells was sufficient to induce the factor-independent, in vitro growth and differentiation of colonies of macrophages and neutrophils, and the generation of immortal lines of factor-independent mast cells, and, upon in vivo injection of the transduced cells, a fatal mastocytosis/mast-cell leukemia. In contrast, expression of constitutively activated H-Ras in bone-marrow cells resulted in the in vitro growth, in the absence of exogenous factors, of colonies that contained only macrophages and of lines of cells resembling dendritic cells, and, upon in vivo injection of the transduced cells, a fatal histiocytosis/monocytic leukemia. Macrophages generated by bone-marrow cells expressing activated M-Ras or activated H-Ras differed morphologically, the latter appearing more activated, a difference abrogated by an inhibitor of Erk activation. Inhibition of either Erk or PI3 kinase blocked the capacity of both activated M-Ras and activated H-Ras to support proliferation and viability. However, inhibition of p38 MAPK activity suppressed proliferation of bone-marrow cells expressing activated H-Ras, but enhanced that of bone-marrow cells expressing activated M-Ras. Thus, expression of either activated M-Ras or H-Ras in normal hematopoietic cells was sufficient for transformation but each resulted in the generation of distinct lineages of cells.

Animals↗

PubMed1995

Ras farnesyltransferase inhibitors suppress the phenotype resulting from an activated ras mutation in Caenorhabditis elegans.

Attachment of Ras protein to the membrane, which requires farnesylation at its C terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. We use Caenorhabditis elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, as a model system to test previously identified farnesyltransferase inhibitors. We show here that two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene, which act downstream and upstream of let-60 ras, respectively, in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors.

Alkyl and Aryl Transferases↗

PubMed1996

p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues.

Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids. Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S-transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions. However, Arg786, Lys831, and Arg925 were observed to be essential for binding to Ras-GTP but not for protein structure. RasGAP residues 890-902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras. This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.

Adenylyl Cyclases↗

PubMed1999

Estrogen inducibility of c-Ha-ras transcription in breast cancer cells. Identification of functional estrogen-responsive transcriptional regulatory elements in exon 1/intron 1 of the c-Ha-ras gene.

Although mutation of ras gene is rare in human breast cancer, overexpression of normal c-Ha-ras gene is frequently observed. Using a mouse mammary metastasis model consisting of genetically related mammary tumor sublines with variant metastatic potential, we have previously (i) demonstrated a direct correlation between c-Ha-ras mRNA and protein levels and metastatic potential and (ii) identified a novel hormone-responsive transcriptional regulatory element in intron 1 of the mouse c-Ha-ras gene that contains the consensus half-site of a glucocorticoid response element and flanking consensus half-sites for estrogen response element. Here, we have examined the functionality of intron 1 sequence in context of upstream sequences by using transient transfection assays with plasmids expressing chloramphenicol acetyltransferase. Intron 1 sequence and sequences similar to intron 1 element located in exon 1 function as transcriptional regulatory elements that confer hormonal inducibility to chloramphenicol acetyltransferase gene expression both independently and in context of 5'-flanking sequences. Measurement of c-Ha-ras transcription rates and protein expression by nuclear run-on and metabolic labeling assays showed a 5-12-fold enhancement, respectively, following treatment with 17beta-estradiol that was blunted by ICI 182,780 in the nonmetastatic variant. In contrast, constitutive overexpression of c-Ha-ras transcripts and protein in the metastatic subline was unaffected by estrogen and ICI 182,780. Gel shift assays demonstrated specific interaction of c-Ha-ras exon 1 sequence with nuclear proteins of human breast cancer MCF-7 cells with formation of two complexes, one of which contains estrogen receptor. Our data demonstrate a direct (i) interaction of c-Ha-ras sequence with estrogen receptor and (ii) stimulatory effect of estrogen on c-Ha-ras gene transcription and suggest that alteration in transcriptional regulation of c-Ha-ras gene by estrogen may play an important role in progression of breast cancer.

Animals↗

PubMed2001

A direct interaction between oncogenic Ha-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependent transformation of epithelial cells.

Cells expressing oncogenic Ras proteins transmit a complex set of signals that ultimately result in constitutive activation of signaling molecules, culminating in unregulated cellular function. Although the role of oncogenic Ras in a variety of cellular responses including transformation, cell survival, differentiation, and migration is well documented, the direct Ras/effector interactions that contribute to the different Ras biological end points have not been as clearly defined. Observations by other groups in which Ras-dependent transformation can be blocked by expression of either dominant negative forms of Phosphatidylinositol (PI) 3-kinase or PTEN, a 3-phosphoinositide-specific phosphatase, support an essential role for PI 3-kinase and its lipid products in the transformation process. These observations coupled with the in vitro observations that the catalytic subunits of PI 3-kinase, the p110 isoforms, bind directly to Ras-GTP foster the implication that a direct interaction between an oncogenic Ras protein and PI 3-kinase are causal in the oncogenicity of mutant Ras proteins. Using an activated Ha-Ras protein (Y64G/Y71G/F156L) that fails to interact with PI 3-kinase, we demonstrate that oncogenic Ha-Ras does not require a direct interaction with PI 3-kinase to support anchorage-independent growth of IEC-6 epithelial cells. We do find, however, that IEC-6 cells expressing an oncogenic Ha-Ras protein that no longer binds PI 3-kinase are greatly impaired in their ability to migrate toward fibronectin.

Cell Movement↗

PubMed1992

The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP.

Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).

Amino Acid Sequence↗

PubMed1996

[Heterogeneity of GTPase-activating proteins for Ras in the regulation of Ras signal transduction pathway].

The proto-oncogene ras is an essential gene for the growth and the differentiation for various types of cells. Ras, ras gene product, is a GTP binding protein which controls the signal transduction by GTP hydrolysis. The ras gene is frequently activated by point mutations in various types of human cancers, which results in a decrease in the GTPase activity of its product. A GTPase-activating protein p120 (p120GAP) was identified as a factor which stimulates the GTPase of normal ras gene product p21 but not of the mutated. An NF1 gene was identified as a gene whose loss of function causes an onset of human disorder, neurofibromatosis type I. The NF1 gene encodes a protein which contains a region with a similarity to the catalytic domain of p120GAP. We recently purified a novel Ras GAP whose molecular weight and immunogenecity are different from those of p120GAP and NF1. We named the novel mammalian Ras GAP as Gap1m. Isolation and sequencing of Gap1m cDNA revealed that Gap1m is indeed a novel Ras GAP. We also succeeded in isolation of another novel Ras GAP gene, GapIII/Gap1IP4BP, which is closely related to Gap1m. Recently, it is shown that GapIII/Gap1IP4BP binds inositol-tetrakis phosphate compounds. The overview of these Ras GAP molecules is described.

Amino Acid Sequence↗

PubMed2001

Inhibition of Kirsten-ras expression in human colorectal cancer using rationally selected Kirsten-ras antisense oligonucleotides.

Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms.

Adenocarcinoma↗

PubMed1992

Alternate mechanisms of ras activation are complementary and favor and formation of ras-GTP.

The mechanisms of ras activation by mutations in residue 61 and in the NKXD guanine nucleotide-binding consensus sequence (ras residues 116-119) have been evaluated. Weakly transforming mutations that either reduce intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (61P) or enhance guanine nucleotide exchange rates (116H, 119E) were combined into the same H-ras proteins. The resulting double-mutant proteins exhibited significantly stronger transforming forming activities than are observed with each individual mutation, suggesting that the consequences of these two different mechanisms of activation favor maintenance of ras in the active form, which is GTP bound. In vivo nucleotide association analysis demonstrated a direct relationship between ras-GTP formation and transforming activity. Although both 61P and 61L mutations result in reduced intrinsic GTPase activity and loss of GAP stimulation in vitro, only H-ras(61L) exhibits strong transforming activity. While H-ras(61L) is found predominantly in the GTP-bound form, H-ras(61P) is predominantly complexed with GDP in vivo. Thus, in vitro GAP stimulation of GTPase activity does not directly correlate with transforming potential, suggesting that other ras-specific regulatory components may also be important in regulating the cycling of ras between CDP- and GTP-bound states.

3T3 Cells↗