Oncogenic Ras Mutations Research Articles

A Molecular Signature for Oncogenic BRAF in Human Colon Cancer Cells is Revealed by Microarray Analysis

Sporadic colorectal cancer develops through a number of functional mutations. Key events are mutually exclusive mutations in BRAF or RAS oncogenes. Signatures for BRAF oncogene have been revealed in melanoma. In a previous study we have reported a molecular signature for HRAS and KRAS mutations in colorectal cell lines that also showed an EMT phenotype for HRAS. In this study we report a molecular profile for a BRAF oncogenic mutation BRAFV600E in colon using the Illumina 45,000 gene microarray. Key differentially expressed genes have been identified from the array analysis further verified by qPCR analysis. Ingenuity pathway analysis such as microsatellite instability, kinase signalling, apoptosis, WNT and Integrin signalling is presented. MutBRAF transforms cells through cross talk with developmental pathways Hedgehog and Wnt, as well as by deregulation of colorectal cancer related kinase pathways, like PI3K. Differential gene expression of BRAFV600E in colon as compared to those associated with RAS oncogenes is presented, as well as similarities and differences between oncogenic BRAF signatures in colon as compared to thyroid and melanoma are highlighted. Novel selected genes/pathways are validated in cell lines and clinical samples bearing BRAFV600E and may serve as markers/targets for personalised diagnosis/therapy/resistance of colorectal cancer.

The response to PAK1 inhibitor IPA3 distinguishes between cancer cells with mutations in BRAF and Ras oncogenes

While new drugs aimed at BRAF-mutated cancers are entering clinical practice, cells and tumors with activating Ras mutations are relatively resistant to those and quite a few other anti-cancer agents. This inspires the effort to reverse this resistance or to uncover new vulnerabilities in such resistant cancers. IPA3 has been originally identified as a small molecule inhibitor of p21-activated protein kinase 1 (PAK1), a candidate therapeutic target in human malignancies. We have tested a battery of melanoma and colon carcinoma cell lines that carry mutations in BRAF, NRAS and KRAS genes and have observed that those with NRAS and KRAS mutations are more sensitive to killing by IPA3. Genetic manipulations suggest that the differential response depends not just on these oncogenes, but also on additional events that were co-selected during tumor evolution. Furthermore, sublethal doses of IPA3 or ectopic expression of dominant-negative PAK1 sensitized Ras-mutated cells to GDC-0897 and AZD6244, which otherwise have reduced efficiency against cells with activated Ras. Dominant-negative PAK1 also reduced the growth of NRAS-mutated cells in confluent cultures, but, unlike IPA3, caused no significant toxicity. Although it remains to be proven that all the effects of IPA3 are exclusively due to inhibition of PAK1, our findings point to the existence of selective vulnerabilities, which are associated with Ras mutations and could be useful for better understanding and treatment of a large subset of tumors.

PI3 Kinase in Cancer: From Biology to Clinic

The discovery and clinical development of small-molecule inhibitors of the phosphatidylinositide 3-kinase (PI3 kinase) family of lipid kinases have marked a remarkable 20-year journey that follows the progressive developments in cancer biology over the last few decades: from hypothesis-driven, basic cancer research that began with viral oncogenesis and developed in the 1960s and 70s, through the discovery of individual mutated oncogenes and tumor suppressor genes in 1970 and 80s and the linkage of these cancer genes to signal transduction pathways in the 1990s, to all large-scale genome-wide sequencing, functional screening, and network biology efforts today. Thus, PI3 kinase research began with the discovery in 1985 of a new type of enzyme activity associated with viral oncogenesis. It benefited greatly from the discovery of wortmannin and LY294002 as PI3 kinase inhibitors and chemical tools in late 1980s to mid-90s. Alongside these tools, genetic validation of PI3 kinase as a target initially involved activation by upstream oncogenic receptor tyrosine kinases and RAS mutation, together with overexpression and amplification of the p110α catalytic isoform of PI3 kinase and frequent loss of the tumor suppressor and negative regulator of PI3 kinase activity, PTEN. As PI3 kinase drug development began, further stimulus came from the discovery through genome sequencing of mutations in PIK3CA, which encodes p110α and is the most frequently mutated kinase in the human genome. From these beginnings, there are now many PI3 kinase inhibitors in clinical trials and more in preclinical development. We review progress, current challenges, and future opportunities in this article.

P120RasGAP-Mediated Activation of c-Src Is Critical for Oncogenic Ras to Induce Tumor Invasion

Ras genes are the most common targets for somatic gain-of-function mutations in human cancers. In this study, we found a high incidence of correlation between Ras oncogenic mutations and c-Src activation in human cancer cells. We showed that oncogenic Ras induces c-Src activation mainly on the Golgi complex and endoplasmic reticulum. Moreover, we identified p120RasGAP as an effector for oncogenic Ras to activate c-Src. The recruitment of p120RasGAP to the Golgi complex by oncogenic Ras facilitated its interaction with c-Src, thereby leading to c-Src activation, and this p120RasGAP-mediated activation of c-Src was important for tumor invasion induced by oncogenic Ras. Collectively, our findings unveil a relationship between oncogenic Ras, p120RasGAP, and c-Src, suggesting a critical role for c-Src in cancers evoked by oncogenic mutations in Ras genes.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 5147: Glutamine addiction: Recognizing and harnessing the pathometabolic potential of Ras-mediated macropinocytosis

Abstract Oncogenic mutations in Ras-encoding genes are found in approximately 30% of all human tumors and are most prevalent in carcinomas of the pancreas, colon, lung and bladder. Oncogenic forms of Ras are trapped in a constitutively active state and, as a consequence, promote the persistent stimulation of effector pathways that control cell growth, survival and proliferation. A prominent phenotypic feature associated with oncogenic Ras expression is the stimulation of macropinocytosis, an endocytic process that mediates the uptake of extracellular fluid via large, heterogeneous vesicles known as macropinosomes. While in amoeboid organisms this process has been linked to nutrient internalization, the functional significance of oncogenic Ras-mediated macropinocytosis is unknown. We have found that cancer cells harboring oncogenic Ras utilize macropinocytosis to internalize extracellular albumin, a major constituent of extracellular fluids and a potentially rich source of amino acids. Furthermore, we have established that tumor cells harboring oncogenic Ras mutations display robust macropinocytosis in vivo. Utilizing cell biological approaches and amino acid quantification, we determined that extracellular albumin that is internalized via oncogenic Ras-stimulated macropinosomes is targeted for degradation leading to the intracellular production of albumin-derived amino acids. Cancer cells display a heightened dependence on glutamine due to their need to sustain rapid proliferation, bioenergetics and macromolecular biosynthesis. We have observed that in cells expressing oncogenic Ras, the adverse effects of glutamine deprivation on cell growth can be rescued by macropinocytosis-mediated uptake of extracellular albumin. Moreover, the capacity of macropinocytic inhibition to diminish this growth advantage is dependent on both glutamine and glutamine-derived metabolites. These results indicate that macropinocytosis may serve as a glutamine supply mechanism that is particularly critical under conditions where free glutamine is limiting. Together, these data implicate oncogenic Ras-mediated macropinocytosis in the regulation of cancer cell metabolism and point to the possibility of exploiting this process in the design of anti-cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5147. doi:1538-7445.AM2012-5147

Abstract 2225: GSK3β plays a significant role in Ras driven tumors potentially through an upstream effect on AKT

Abstract Pancreatic cancer is a highly aggressive cancer that is resistant to most drug therapies. Lung cancer is the number one cause of cancer deaths in the US and worldwide. Both of these tumors are known to have high levels of Ras oncogenic mutations (50 and 90% respectively). AKT is known to phosphorylate and inactive Glycogen Synthase Kinase 3 (GSK3). Additionally, GSK3 has been shown to play a role in oncogenesis in pancreatic cancer and lung cancer. Given the high level of AKT activity in transformed cells these two processes would appear to be at odds. However, much of the work on GSK3 has focused on the beta subunit. We have found that the alpha subunit plays a role in tumor proliferation and AKT activity. We see a significant effect of knockdown of GSK3β in Ras transformed pancreatic (Panc1, MiaPaca) and lung cancer (A549) cell lines in proliferation assays, with an approximately 70% decrease in growth compared to siControl cells. In contrast, the knockdown of the GSK3β subunit resulted in no decrease in cell proliferation in A549 cells compared to a 45% decrease in Panc 1 cells. Additionally, knockdown of GSK3β resulted in decreased levels of AKT activity by Western blot analysis as compared to siControl and GSK3β. Previous studies have focused on the role of the beta subunit of GSK3 in oncogenesis. Additionally, AKT has been studied as an upstream regulator rather than a downsteam target of GSK3. Our early studies suggest that the GSK3β subunit may play a significant role in the growth of Ras driven tumors cell lines upstream of AKT, and should be a potential target for therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2225. doi:1538-7445.AM2012-2225

Abstract 4420: Serum regulates reovirus-mediated cytopathy in K-Ras activated colorectal cancer and intestinal epithelial cell lines

Abstract Background: The oncolytic potency of replication competent REOvirus has been demonstrated in various cancers. The activity is most pronounced in Kras mutant cancer cells, which account for 30-40 % of all cancers. Herein we simultaneously utilized isogenic human derived colorectal cancer cell lines that differ only by the presence of mutant Kras and normal rat intestinal epithelial cells with inducible Kras to evaluate whether the presence of oncogenic Kras alters the sensitivity of colon cancer cells to reovirus. Methods: Reovirus was obtained from Oncolytics Biotech Inc. Rat IEC's harboring IPTG inducible mutant Ras oncogene (IEC-iKras) were treated with reovirus ± IPTG. The infectivity was maintained at a multiplicity of infection (MOI) of 2. MTT assay was performed at 72 hours to determine cell vability (CV). Similar experiments were also performed with HCT116 (Kras mut) and its isogenic derivative Hke3 [(Kras wild type (WT)]. Cells were cultured and treated in both serum rich (SR) and serum free (SF) media to determine the effect of growth factors on sensitivity to reovirus. Alterations in gene expression were determined by real time PCR and protein expression by western blot. Results: The activity of reovirus was observed in all cell lines studied. Reduction in CV was greater in Kras mutant HCT116 compared to WT Hke3 cells. Consistently, induction of Kras in IEC cells increased the potency of reovirus. Furthermore, this effect was more prominent in cells cultured in SF suggesting that growth factors in the serum may attenuate the effect of mutant Kras on reovirus sensitivity. Expression of the cell cycle regulator, p21 was preferentially increased upon reovirus infection in Kras mutant compared to WT cells, and in cells cultured in SF compared to SR conditions. Conclusion: Oncogenic Kras mutations increase the sensitivity of colon cancer cells to Reovirus. Clinical trials are underway testing reovirus in patients with Kras mutant metastatic colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4420. doi:1538-7445.AM2012-4420

Identification of Functionally Distinct TRAF Proinflammatory and Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (PI3K/MEK) Transforming Activities Emanating from RET/PTC Fusion Oncoprotein

Thyroid carcinomas that harbor RET/PTC oncogenes are well differentiated, relatively benign neoplasms compared with those expressing oncogenic RAS or BRAF mutations despite signaling through shared transforming pathways. A distinction, however, is that RET/PTCs induce immunostimulatory programs, suggesting that, in the case of this tumor type, the additional pro-inflammatory pathway reduces aggressiveness. Here, we demonstrate that pro-inflammatory programs are selectively activated by TRAF2 and TRAF6 association with RET/PTC oncoproteins. Eliminating this mechanism reduces pro-inflammatory cytokine production without decreasing transformation efficiency. Conversely, ablating MEK/ERK or PI3K/AKT signaling eliminates transformation but not pro-inflammatory cytokine secretion. Functional uncoupling of the two pathways demonstrates that intrinsic pro-inflammatory pathways are not required for cellular transformation and suggests a need for further investigation into the role inflammation plays in thyroid tumor progression.

RAS oncogenic signal upregulates EZH2 in pancreatic cancer

The neoplastic transformation by mutant RAS is thought to require remodeling of expression of an entire set of genes. However, the underlying mechanism for initiation of gene expression remodeling in tumorigenesis remains elusive. This study was aimed to define the oncogenic role of EZH2, a histone modifier protein that is induced by oncogenic mutant RAS, using pancreatic cancers of transgenic rats exogenously expressing human mutant RAS. Immunohistochemical observation of preneoplastic or cancerous lesions in the animal model suggested that upregulation of Ezh2 protein is an initiating event in pancreatic carcinogenesis. MEK-inhibition or Elk-1-knockdown downregulated EZH2, and MEK-inhibition or EZH2-knockdown restored expression of a tumor suppressor, RUNX3 in human and rat pancreatic cancer cells activated by the oncogenic RAS. Furthermore, Elk-1- or EZH2-knockdown inhibited growth of the cancer cells. These results strongly suggested that the oncogenic RAS upregulates EZH2 through MEK-ERK signaling, resulted in downregulation of tumor suppressors including RUNX3 in pancreatic carcinogenesis.

MEK1/2 dual-specificity protein kinases: Structure and regulation

MEK1 and MEK2 are related protein kinases that participate in the RAS–RAF–MEK–ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, cell migration, differentiation, metabolism, and proliferation. Moreover, oncogenic mutations in RAS or B-RAF are responsible for a large proportion of human cancers. MEK1 is activated by phosphorylation of S218 and S222 in its activation segment as catalyzed by RAF kinases in an intricate process that involves a KSR scaffold. Besides functioning as a scaffold, the kinase activity of KSR is also required for MEK activation. MEK1 regulation is unusual in that S212 phosphorylation in its activation segment is inhibitory. Moreover, active ERK catalyzes a feedback inhibitory phosphorylation of MEK1 T292 that serves to downregulate the pathway.

Mechanistic modeling to investigate signaling by oncogenic Ras mutants

Mathematical models based on biochemical reaction mechanisms can be a powerful complement to experimental investigations of cell signaling networks. In principle, such models have the potential to find the behaviors that result from well-understood component interactions and their measurable properties, such as concentrations and rate constants. As cancer results from the acquisition of mutations that alter the expression level and/or the biochemistry of proteins encoded by mutated genes, mathematical models of cell signaling networks would also seem to have the potential to predict how these changes alter cell signaling to produce a cancer phenotype. Ras is commonly found in cancer and has been extensively characterized at the level of detail needed to develop such models. Here, we consider how biochemical mechanism-based models have been used to study mutant Ras signaling. These models demonstrate that it is clearly possible to use observable properties of individual reactions to predict how the entire system behaves to produce the high levels of signal that drive the cancer phenotype. These models also demonstrate differences in how models are developed and studied. Their evaluation suggests which approaches are most promising for future work.

Integrative transcriptional analysis between human and mouse cancer cells provides a common set of transformation associated genes

Mouse functional genomics is largely used to investigate relevant aspects of mammalian physiology and pathology. To which degree mouse models may offer accurate representations of molecular events underlining human diseases such as cancer is not yet fully established. Herein we compare gene expression signatures between a set of human cancer cell lines (NCI-60 cell collection) and a mouse cellular model of oncogenic K-ras dependent transformation in order to identify their closeness at the transcriptional level. The results of our integrative and comparative analysis show that in both species as compared to normal cells or tissues the transformation process involves the activation of a transcriptional response. Furthermore, the cellular mouse model of K-ras dependent transformation has a good degree of similarity with several human cancer cell lines and in particular with cell lines containing oncogenic Ras mutations. Moreover both species have similar genetic signatures that are associated to the same altered cellular pathways (e.g. Spliceosome and Proteasome) or to deregulation of the same genes (e.g. cyclin D1, AHSA1 and HNRNPD) detected in the comparison between cancer cells versus normal cells or tissues. In summary, we report one of the first in-depth analysis of global gene expression profiles of a K-ras dependent mouse cell model of transformation and a large collection of human cancer cells as compared to their normal counterparts. Taken together our findings show a strong correlation in the transcriptional and pathway alteration responses between the two species, therefore validating the use of the mouse model as an appropriate tool to investigate human cancer, and indicating that the comparative analysis, as described here, offers a useful approach to identify cancer-specific gene signatures.

Abstract 3856: Loss fo N or K-Ras B does not mimic the effects of farnesy transferase inhibited osteosarcoma or 293T cells

Abstract Ras proteins are small GTPases that regulate growth, differentiation, survival, and cell death. Prenylation, the addition of either a farnesyl (15 carbon chain lipid) or geranylgeranyl (20 carbon chain lipid) group, allows Ras and other small GTPases to anchor into the plasma membrane, a necessary step for Ras to activate multiple signaling pathways, including the MAPK cascades. The specific CAAX motif at the C terminus of a protein determines whether it can be farnesylated, geranylgeranylated, or both. There are four highly homologous Ras proteins: K-Ras A, K-Ras B, N-Ras, and H-Ras, each containing a CAAX motif that allows farnesylation. K-Ras B and N-Ras can be alternately geranylgeranylated (GGd) if farnesylation is blocked by a specific small molecule (FTI). Oncogenic Ras mutations are found commonly in cancer, and Ras also can be a major mediator of signals initiated by activated receptor tyrosine kinases (RTKs). Since RTK signaling plays a major role in the biology of sarcomas, we were interested in the mechanisms by which FTIs might affect osteosarcoma cell growth and survival. Osteosarcoma cell lines have variable responses to FTI treatment, including reduced proliferation and programmed cell death at a range of concentrations from 1 nM to 100 nM (Hall et al. 2010). Further, 293T fibroblast cells also have decreased cell viability and increased subG1 and G2/M cell cycle arrest with FTI treatment. 293T cells respond similarly to OS187 our most sensitive osteosarcoma cell line. To assess Ras activity in response to FTI, we co-precipitated activated Ras with the Ras target protein Raf, then probed by western for Ras. Surprisingly, Ras activity was increased in response to FTI in both osteosarcoma cell lines and 293T cells. We also found that the activity of downstream effectors, ERK and p38 MAPK, also increased with FTI (Hall et al, 2010). It was unclear whether the changes in cell growth and survival were due to decrease in farnesylated Ras or the increase in geranylgeranylated Ras. To determine if loss of N-Ras or K-Ras B activity could account for reduced cell yield following FTI, we knocked down each of these proteins in our most responsive osteosarcoma cell line, OS187, and in 293T cells. There was no significant difference in cell yield or cell cycle when either of these proteins was knocked down. To determine if alternate prenylation of N or K-Ras B are responsible for decreased cell yield and/or cell cycle arrest, mutant forms of these proteins will need to be transduced into OS187 and 293T cells for further analysis. In conclusion, FTI caused growth inhibition and cell death for both osteosarcoma cells and 293T cells. FTIs increased Ras activation and the activity of downstream effectors. Since knocking out N or K-Ras B did not reproduce any of these effects, the effects of FTI are not due to the loss of farnesylated Ras. These observations suggest that the effects may be due to increased geranylgeranylated N Ras or K-Ras B. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3856. doi:10.1158/1538-7445.AM2011-3856

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

RAF Inhibitor-Induced KSR1/B-RAF Binding and Its Effects on ERK Cascade Signaling

RAF kinase inhibitors can induce ERK cascade signaling by promoting dimerization of RAF family members in the presence of oncogenic or normally activated RAS. This interaction is mediated by a dimer interface region in the RAF kinase domain that is conserved in members of the ERK cascade scaffold family, kinase suppressor of RAS (KSR). In this study, we find that most RAF inhibitors also induce the binding of KSR1 to wild-type and oncogenic B-RAF proteins, including V600E B-RAF, but promote little complex formation between KSR1 and C-RAF. The inhibitor-induced KSR1/B-RAF interaction requires direct binding of the drug to B-RAF and is dependent on conserved dimer interface residues in each protein, but, unexpectedly, is not dependent on binding of B-RAF to activated RAS. Inhibitor-induced KSR/B-RAF complex formation can occur in the cytosol and is observed in normal mouse fibroblasts, as well as a variety of human cancer cell lines. Strikingly, we find that KSR1 competes with C-RAF for inhibitor-induced binding to B-RAF and, as a result, alters the effect of the inhibitors on ERK cascade signaling.

Dual Mode of Oncogenesis

Oncogenic Ras mutants stimulate the first kinase in the MAPK pathway (Raf) and prevent SUMOylation-mediated inhibition of the second kinase, MEK.

Differential Regulation of RasGAPs in Cancer

Ever since their discovery as cellular counterparts of viral oncogenes more than 25 years ago, much progress has been made in understanding the complex networks of signal transduction pathways activated by oncogenic Ras mutations in human cancers. The activity of Ras is regulated by nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), and much emphasis has been put into the biochemical and structural analysis of the Ras/GAP complex. The mechanisms by which GAPs catalyze Ras-GTP hydrolysis have been clarified and revealed that oncogenic Ras mutations confer resistance to GAPs and remain constitutively active. However, it is yet unclear how cells coordinate the large and divergent GAP protein family to promote Ras inactivation and ensure a certain biological response. Different domain arrangements in GAPs to create differential protein-protein and protein-lipid interactions are probably key factors determining the inactivation of the 3 Ras isoforms H-, K-, and N-Ras and their effector pathways. In recent years, in vitro as well as cell- and animal-based studies examining GAP activity, localization, interaction partners, and expression profiles have provided further insights into Ras inactivation and revealed characteristics of several GAPs to exert specific and distinct functions. This review aims to summarize knowledge on the cell biology of RasGAP proteins that potentially contributes to differential regulation of spatiotemporal Ras signaling.

Ras in Cancer and Developmental Diseases

Somatic, gain-of-function mutations in ras genes were the first specific genetic alterations identified in human cancer about 3 decades ago. Studies during the last quarter century have characterized the Ras proteins as essential components of signaling networks controlling cellular proliferation, differentiation, or survival. The oncogenic mutations of the H-ras, N-ras, or K-ras genes frequently found in human tumors are known to throw off balance the normal outcome of those signaling pathways, thus leading to tumor development. Oncogenic mutations in a number of other upstream or downstream components of Ras signaling pathways (including membrane RTKs or cytosolic kinases) have been detected more recently in association with a variety of cancers. Interestingly, the oncogenic Ras mutations and the mutations in other components of Ras/MAPK signaling pathways appear to be mutually exclusive events in most tumors, indicating that deregulation of Ras-dependent signaling is the essential requirement for tumorigenesis. In contrast to sporadic tumors, separate studies have identified germline mutations in Ras and various other components of Ras signaling pathways that occur in specific association with a number of different familial, developmental syndromes frequently sharing common phenotypic cardiofaciocutaneous features. Finally, even without being a causative force, defective Ras signaling has been cited as a contributing factor to many other human illnesses, including diabetes and immunological and inflammatory disorders. We aim this review at summarizing and updating current knowledge on the contribution of Ras mutations and altered Ras signaling to development of various tumoral and nontumoral pathologies.

Lonafarnib (SCH66336) improves the activity of temozolomide and radiation for orthotopic malignant gliomas

Malignant gliomas are highly lethal tumors resistant to current therapies. The standard treatment modality for these tumors, surgical resection followed by radiation therapy and concurrent temozolomide, has demonstrated activity, but development of resistance and disease progression is common. Although oncogenic Ras mutations are uncommon in gliomas, Ras has been found to be constitutively activated through the action of upstream signaling pathways, suggesting that farnesyltransferase inhibitors may show activity against these tumors. We now report the in vitro and orthotopic in vivo results of combination therapy using radiation, temozolomide and lonafarnib (SCH66336), an oral farnesyl transferase inhibitor, in a murine model of glioblastoma. We examined the viability, proliferation, farnesylation of H-Ras, and activation of downstream signaling of combination-treated U87 cells in vitro. Lonafarnib alone or in combination with radiation and temozolomide had limited tumor cell cytotoxicity in vitro although it did demonstrate significant inhibition in tumor cell proliferation. In vivo, lonafarnib alone had a modest ability to inhibit orthotopic U87 tumors, radiation and temozolomide demonstrated better inhibition, while significant anti-tumor activity was found with concurrent lonafarnib, radiation, and temozolomide, with the majority of animals demonstrating a decrease in tumor volume. The use of tumor neurospheres derived from freshly resected adult human glioblastoma tissue was relatively resistant to both temozolomide and radiation therapy. Lonafarnib had a significant inhibitory activity against these neurospheres and could potentate the activity of temozolomide and radiation. These data support the continued research of high grade glioma treatment combinations of farnesyl transferase inhibitors, temozolomide, and radiation therapy.