The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epidermal growth factor receptor (EGFR) and BRAF. These findings potentially reveal an adaptive response during tumor evolution that is dependent on the mutational status of proximal genes in the RAS pathway and cellular context. Cell contextual differences in these adaptations may influence therapeutic responsiveness and alternative resistance mechanisms.

Endometrial polyps are common, yet the molecular mechanisms underlying their formation and progression remain unclear. We examined gene mutations possibly related to the pathogenesis of endometrial polyps, as well as to their clinical features. Four premenopausal patients with endometrial polyps, who were not under drug treatment, were recruited. Whole exomes of endometrial polyps and peripheral blood lymphocytes were analyzed by next‑generation sequencing, and somatic mutations were derived by subtraction. Then, 35 samples of endometrial polyps and 12 samples of atypical polypoid adenomyoma were newly recruited to validate the identified mutations by polymerase chain reaction‑reverse sequence specific oligonucleotide method. The mutations were also analyzed in separate stromal and glandular components of the polyps after laser‑capture microdissection. Whole exome sequencing revealed that KRAS mutations were the only type of mutation detectable in multiple cases (2/4). Targeted mutation analysis revealed that 16 of 35 samples (45.7%) of endometrial polyps harbored RAS mutations. Mutation‑positive cases exhibited a significantly higher number of endometrial polyps (3.25±2.70 vs. 1.74±0.87, P=0.045). Laser‑capture microdissection in NRAS‑mutated endometrial polyps revealed that both stromal and glandular components harbored RAS mutations. There was no RAS mutation in 12 samples of atypical polypoid adenomyoma. This is the first report demonstrating that pathogenic RAS mutations are frequent in non‑treated endometrial polyps. RAS mutations may have an important role in tumorigenesis and in the formation of multiple endometrial polyps.

It is now well established that in the human genome the canonical double helix coexists with folded G-quadruplex structures that are known to have important biological functions. In this review we summarize the current knowledge on quadruplex formation in the promoters of the ras genes that are mutated in about 30% of all human cancers. We describe the nuclear proteins that recognize these unusual DNA structures and discuss their function in transcription. We also examine the formation of G-quadruplexes in the 5'-untranslated region of the ras transcripts and conclude this review by reporting strategies that use either ras G-quadruplexes or proteins recognizing the ras G-quadruplexes as targets of anticancer small molecules.

Ras-GTPase-activating proteins (Ras-GAPs) have been implicated both as suppressors of Ras and as effectors in regulating cellular activities. To study whether Ras-GAPs have roles in tumor cell survival or not, mRNA levels of ras-related genes were measured in v-Ki-ras-transformed (DT) and the parental NIH/3T3 cells, using real-time PCR. mRNA levels of p120-Gap, Gap1(m), and PIK3CA were increased in DT cells compared with NIH/3T3 cells. p120-Gap and PIK3CA genes were induced by addition of serum or epidermal growth factor to serum-starved DT cells. Three anti-cancer drugs, an ERK kinase (MEK) inhibitor PD98059, a topoisomerase II poison doxorubicin (adriamycin), and a histone deacetylase inhibitor trichostatin A, selectively blocked the overexpression of p120-Gap and Gap1(m) genes in DT cells. These drugs also caused reversion of DT cells to the adherent shape associated with growth arrest. Our results suggest that p120-Gap and Gap1(m) genes provide important biomarkers for cancer therapies.

Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. Here we demonstrate that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers.

It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4(+) T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types.

The gene KRAS is commonly mutated in lung cancer to encode a constitutively active and oncogenic protein that is well established to initiate and maintain lung tumorigenesis. However, the remaining wild-type KRAS protein, or the other family members HRAS and NRAS, can still be activated in the presence of oncogenic KRAS. Moreover, loss of any one of these three genes has been shown to increase the sensitivity of mice to the carcinogen urethane, which induces Kras mutation-positive early lung lesions. To determine the contribution of progressively disrupting Hras and Nras genes on urethane lung tumorigenesis, mice with different combinations of wild-type and null alleles of Hras and Nras were exposed with urethane and tumor burden was assessed. As previously reported, loss of one allele of Hras increased the sensitivity of mice to this carcinogen, and this effect was further exacerbated by the loss of the second Hras allele. However, loss of one or both alleles of Nras failed to alter tumor burden, either in the absence or presence of Hras, after exposure to urethane. Additionally, no obvious difference between lung lesions in mice with wild-type versus null alleles was detected, suggesting that wild-type Ras proteins may exert a tumor suppressive effects at the time of initiation, although other interpretations are certainly possible. In summary, these data suggest that in some genetic backgrounds inactivation of different wild-type Ras genes can have different effects on urethane-induced lung tumorigenesis.

Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7) was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P = 0.04, Fisher's exact test). Thus, we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

RAS-MAPK signaling mediates processes critical to normal development including cell proliferation, survival, and differentiation. Germline mutation of RAS-MAPK genes lead to the Noonan-spectrum of syndromes. Here, we present a patient affected by a 6p-interstitial microdeletion with unknown underlying molecular etiology. Examination of 6p-interstitial microdeletion cases reveals shared clinical features consistent with Noonan-spectrum disorders including short stature, facial dysmorphia and cardiovascular abnormalities. We find the RAS-responsive element binding protein-1 (RREB1) is the common deleted gene in multiple 6p-interstitial microdeletion cases. Rreb1 hemizygous mice display orbital hypertelorism and cardiac hypertrophy phenocopying the human syndrome. Rreb1 haploinsufficiency leads to sensitization of MAPK signaling. Rreb1 recruits Sin3a and Kdm1a to control H3K4 methylation at MAPK pathway gene promoters. Haploinsufficiency of SIN3A and mutations in KDM1A cause syndromes similar to RREB1 haploinsufficiency suggesting genetic perturbation of the RREB1-SIN3A-KDM1A complex represents a new category of RASopathy-like syndromes arising through epigenetic reprogramming of MAPK pathway genes.

Oncolytic viruses exploit alterations in cancer cells to specifically infect cancer cells but not normal healthy cells. Previous work has shown that oncogenic Ras interferes with interferon (IFN) signaling to promote viral replication. Furthermore, inhibition of the Ras/Raf/MEK/ERK pathway at the level of Ras, MEK, or ERK was sufficient to restore IFN signaling. In order to identify genes that were commonly regulated by the inhibition of the Ras pathway and the IFN pathway, we treated NIH/3T3 cells that overexpress oncogenic Ras with the MEK inhibitor, U0126, or IFN-α for 6 h, and performed DNA microarray analysis (Gene Expression Omnibus accession number GSE49469). Here, we also provide additional information on the experimental and functional analysis of the genes responsive to U0126 and IFN.

RAS genes are among the most frequently mutated proto-oncogenes in cancer. However, how Ras stability is regulated remains largely unknown. Here, we report a regulatory loop involving the E3 ligase Nedd4-1, Ras, and PTEN. We found that Ras signaling stimulates the expression of Nedd4-1, which in turn acts as an E3 ubiquitin ligase that regulates Ras levels. Importantly, Ras activation, either by oncogenic mutations or by epidermal growth factor (EGF) signaling, prevents Nedd4-1-mediated Ras ubiquitination. This leads to Ras-induced Nedd4-1 overexpression, and subsequent degradation of the tumor suppressor PTEN in both human cancer samples and cancer cells. Our study thus unravels the molecular mechanisms underlying the interplay of Ras, Nedd4-1, and PTEN and suggests a basis for the high prevalence of Ras-activating mutations and EGF hypersignaling in cancer.

Mutation in RAS proteins is one of the most common genetic alterations observed in human and experimentally induced rodent cancers. In vivo, oncogenic mutations have been shown to occur at exons 12, 13, and 61, resulting in any 1 of 19 possible point mutations in a given tumor for a specific RAS isoform. While some studies have suggested a possible role of different mutant alleles in determining tumor severity and phenotype, no general consensus has emerged on the oncogenicity of different mutant alleles in tumor formation and progression. Part of this may be due to a lack of a single, signature pathway that shows significant alterations between different mutations. Rather, it is likely that subtle differences in the activation, or lack thereof, of downstream effectors by different RAS mutant alleles may determine the eventual outcome in terms of tumor phenotype. This paper reviews our current understanding of the potential role of different RAS mutations on tumorigenesis, highlights studies in model cell culture and in vivo systems, and discusses the potential of expression array and computational network modeling to dissect out differences in activated RAS genes in conferring a transforming phenotype.

Activating mutations of Ras genes are often observed in cancer. The protein products of the three Ras genes are almost identical. However, for reasons that remain unclear, KRAS is far more frequently mutated than the other Ras isoforms in cancer and RASopathies. We have quantified HRAS, NRAS, KRAS4A and KRAS4B protein abundance across a large panel of cell lines and healthy tissues. We observe consistent patterns of KRAS > NRAS»HRAS protein expression in cells that correlate with the rank order of Ras mutation frequencies in cancer. Our data provide support for the model of a sweet-spot of Ras dosage mediating isoform-specific contributions to cancer and development. We suggest that in most cases, being the most abundant Ras isoform correlates with occupying the sweet-spot and that HRAS and NRAS expression is usually insufficient to promote oncogenesis when mutated. However, our results challenge the notion that rare codons mechanistically underpin the predominance of KRAS mutant cancers. Finally, direct measurement of mutant versus wildtype KRAS protein abundance revealed a frequent imbalance that may suggest additional non-gene duplication mechanisms for optimizing oncogenic Ras dosage.

The RAS/RAF and PI3K/PTEN signaling pathways play central roles in hepatocarcinogenesis. KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN are key cancer-related genes in the RAS/RAF and PI3K/PTEN signaling pathways. Genetic alterations in these genes often lead to the dysregulation of the two cascades. Little is known regarding the frequency of hotspot mutations in these critical components among Chinese patients with hepatocellular carcinoma (HCC). In the current study, 57 somatic hotspot mutations in 36 HCCs samples collected from Chinese patients using direct DNA sequencing method were examined. Two cases of KRAS somatic mutations (KRAS codon 61; Gln to His) were identified among 36 HCCs (5.6%). However, no mutations were found in the NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes. These findings indicated that point mutations in the KRAS gene, but not mutations in NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes, at a somatic level contribute to the abnormal activation of the RAS/RAF and PI3K/PTEN pathways in HCC.

The interconnected Ras/ERK and PI3K/AKT pathways play a central role in colorectal tumorigenesis, and they are targets for elucidating mechanisms involved in attempts to induce colon cancer cell death. Both ginger (Zingiber officinale) and honey have been shown to exhibit anti-tumor and anti-inflammation properties against many types of cancer, including colorectal cancer. However, there are currently no reports showing the combined effect of these two dietary compounds in cancer growth inhibition. The aim of this study was to evaluate the synergistic effect of crude ginger extract and Gelam honey in combination as potential cancer chemopreventive agents against the colorectal cancer cell line HT29.

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.

Adenoid cystic carcinoma (AdCC), one of the most common salivary gland carcinomas, usually has a fatal outcome. Epidermal growth factor receptor (EGFR) pathway gene mutations are important in predicting a patient's prognosis and estimating the efficacy of molecular therapy targeting the EGFR pathway. In this study of salivary gland AdCC (SAdCC), we looked for gene mutations in EGFR, RAS family (KRAS, HRAS, and NRAS), PIK3CA, BRAF, and AKT1, using a highly sensitive single-base extension multiplex assay, SNaPshot. Out of 70 cases, EGFR pathway missense mutations were found in 13 (18.6%): RAS mutations in 10 (14.3%), EGFR in one (1.4%), and PIK3CA in 5 (7.1%). None of the cases showed an EGFR deletion by direct sequencing. Concurrent gene mutations were found in three cases (4.3%). EGFR pathway mutations were significantly associated with a shorter disease-free (p = 0.011) and overall survival (p = 0.049) and RAS mutations were as well; (p = 0.010) and (p = 0.024), respectively. The gene fusion status as determined by a FISH assay had no significant association with mutations of the genes involved in the EGFR pathway. In conclusion, EGFR pathway mutations, especially RAS mutations, may be frequent in SAdCC, and associated with a poor prognosis for the patient.

Twenty-nine cDNAs encoding Ras-like family small GTPases (RSGs) were cloned and sequenced from Nilaparvata lugens. Twenty-eight proteins are described here: 3 from Rho, 2 from Ras, 9 from Arf and 14 from Rabs. These RSGs from N.lugens have five conserved G-loop motifs and displayed a higher degree of sequence conservation with orthologues from insects. RT-qPCR analysis revealed NlRSGs expressed at all life stages and the highest expression was observed in hemolymph, gut or wing for most of NlRSGs. RNAi demonstrated that eighteen NlRSGs play a crucial role in nymphal development. Nymphs with silenced NlRSGs failed to molt, eclosion or development arrest. The qRT-PCR analysis verified the correlation between mortality and the down-regulation of the target genes. The expression level of nuclear receptors, Kr-h1, Hr3, FTZ-F1 and E93 involved in 20E and JH signal pathway was impacted in nymphs with silenced twelve NlRSGs individually. The expression of two halloween genes, Cyp314a1 and Cyp315a1 involved in ecdysone synthesis, decreased in nymphs with silenced NlSar1 or NlArf1. Cyp307a1 increased in nymphs with silenced NlArf6. In N.lugens with silenced NlSRβ, NlSar1 and NlRab2 at 9th day individually, 0.0% eclosion rate and almost 100.0% mortality was demonstrated. Further analysis showed NlSRβ could be served as a candidate target for dsRNA-based pesticides for N.lugens control.

Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras(G13D), that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.