Ras Proto-oncogene Research Articles

622. Oncolytic Adenoviruses Loaded With Active Drugs as a Novel Drug Delivery System for Cancer Therapy

L-carnosine (β-Ala-His) is a naturally occurring histidine dipeptide, normally found in brain, kidney and in large amounts in muscle. L-carnosine has biological functions, including antioxidant activity, ability to chelate metal ions, as well as anti-inflammatory and anti-senescence properties. Recent studies have demonstrated that 50-100 mM of L-carnosine decreases cell proliferation in a colon cancer cell line HCT116, bearing a mutation in codon 13 of the RAS proto-oncogene. In addition, pre-treatment with L-carnosine decreases the intracellular concentration of Adenosine Triphosphate (ATP) and Reactive Oxygen Species (ROS) and inhibits the cell cycle progression in the G1 phase. The proto-oncogene KRAS is mutated in a wide array of human cancers and is important both in tumour progression and resistance to anticancer drugs. To overcome treatment limitations due to the high intracellular concentration required we have hypothesized that L-carnosine can be conjugated on the capsid of oncolytic viruses. Oncolytic viruses are viruses that are able to replicate specifically in and destroy tumor cells and this property is either inherent or genetically-engineered. The association of viruses with specific drugs, would increase the efficacy of the treatment of human neoplasia due to the synergistic action of virus and drug. First we have developed a strategy to conjugate peptides on viral capsid, based on electrostatic interaction. Then, using different cancer cell lines we found that oncolytic virus coated with L-carnosine with a tail of positively charged polylysine was able to enhance a positive anticancer synergistic effect. Finally, in order to investigate the molecular mechanisms underlying the effect of tumor reduction by oncolytic virus coated with modified L-carnosine, we have used three different approaches. First, we have examined, in samples with virus alone, or in combination with L-carnosine, the oncolytic replication by evaluating the E1A expression, second the apoptotic mechanism by expression of specific genes and at end the autophagy regulation via the amount of LC3-II. In conclusion, we have developed a model to use oncolytic adenovirus as a scaffold to deliver active drugs. Once validated the proposed model could be used as a novel drug delivery system for cancer therapy.

4EBP1/eIF4E and p70S6K/RPS6 axes play critical and distinct roles in hepatocarcinogenesis driven by AKT and N-Ras protooncogenes in mice

Concomitant expression of activated forms of AKT and Ras in the mouse liver (AKT/Ras) leads to rapid tumor development via strong activation of the mTORC1 pathway. mTORC1 functions via regulating p70S6K/RPS6 and 4EBP1/eIF4E cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of RPS6 pathway via Rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras induced hepatocarcinogenesis. Combined treatment with Rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong anti-neoplastic effect was successfully recapitulated by ablating Raptor, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the protooncogene whose activity is specifically inhibited by 4EBP1, resulted in HCC development in cooperation with activated Ras. Mechanistically, we identified the ENTPD5/AK1/CMPK1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. Conclusions: Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras protooncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human hepatocellular carcinoma.

Polymorphism T81C in H-RAS Oncogene Is Associated With Disease Progression in Imatinib (TKI) Treated Chronic Myeloid Leukemia Patients.

BackgroundMammalian cells contain three functional RAS proto-oncogenes, known as H-RAS, K-RAS, and N-RAS, which encode small GTP-binding proteins in terms of p21rass. RAS genes have been elucidated as major participants in the development and progression of cancer. A single nucleotide polymorphism (SNP) at H-RAS cDNA position 81 T→C (rs12628) has been found to be associated with the risk of many human cancers like gastrointestinal, oral, colon, bladder and thyroid carcinomas. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to chronic myeloid leukemia as well, and we conducted this study to test the hypothesis in Indian population.MethodH-RAS polymorphism was studied in 100 chronic myeloid leukemia (CML) patients and 100 healthy controls by restriction fragmentation length polymorphism (RFLP-PCR). Associations between polymorphism and clinicopathological features of CML patients were investigated.ResultsIn CML patients, the TT, TC and CC genotype frequency was 38%, 61% and 1% respectively, compared to 92%, 8% and 0% in healthy controls respectively. Compared to TT genotype, CT was significantly associated with increased risk of CML (odds ratio (OR): 8.4, P < 0.00001). There was a statistically significant correlation of H-RAS polymorphism with phases (P < 0.0003), molecular response (P < 0.0001), hematological response (P < 0.04) and thrombocytopenia (P < 0.003). However, there was no correlation of this polymorphism found with other clinical parameters.ConclusionH-RAS T81C polymorphism was found to be associated with CML risk and prognosis of CML. These results suggest that C heterozygosis may be considered a potential risk factor for CML development in the North Indian population.

Coexisting KRAS and NRAS mutations in caecal adenocarcinoma and contiguous adenoma – a case report

Aim Activating mutations of KRAS and NRAS , isoforms of RAS proto-oncogenes which influence cell proliferation, differentiation and survival, arise in 35–40% of colorectal carcinoma (CRC), and are in recent years proven to convey resistance to anti-EGFR monoclonal antibody therapy in metastatic CRC. These mutations in CRCs are considered mutually exclusive through redundancy. Here we report and characterise a case of caecal CRC with coexisting KRAS and NRAS mutations. Methods An 81-year-old male, presenting with fever and right iliac fossa pain, underwent right hemicolectomy after CT diagnosis of 43 mm caecal mass. Tissue sections and immunohistochemical expression of CK7, CK20, CDX2, MSH2, MSH6, MLH1 and PMS2 were examined by two pathologists. Mutation analysis of KRAS , NRAS and BRAF was performed on lesional DNA using bidirectional Sanger sequencing of amplified PCR product. Results Low-grade CRC (T2N0MX) and contiguous sessile high-grade tubulovillous adenoma, both CDX2 + , CK20 + and CK7 – , were identified. Both lesions contained KRAS c.35G > T (G12 V) and NRAS c.34G > A (G12S) mutations, without immunohistochemical evidence of microsatellite instability. Conclusion We report, for the first time, coexisting KRAS and NRAS mutations in a single CRC. This may have therapeutic significance. Current understanding of RAS proto-oncogenes in pathogenesis and treatment response in CRCs is discussed.

4EBP1/eIF4E and p70S6K/RPS6 axes play critical and distinct roles in hepatocarcinogenesis driven by AKT and N-Ras proto-oncogenes in mice.

Concomitant expression of activated forms of v-akt murine thymoma viral oncogene homolog (AKT) and Ras in mouse liver (AKT/Ras) leads to rapid tumor development through strong activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway. mTORC1 functions by regulating p70S6K/ribosomal protein S6 (RPS6) and eukaryotic translation initiation factor 4E-binding protein 1/ eukaryotic translation initiation factor 4E (4EBP1/eIF4E) cascades. How these cascades contribute to hepatocarcinogenesis remains unknown. Here, we show that inhibition of the RPS6 pathway by rapamycin effectively suppressed, whereas blockade of the 4EBP1/eIF4E cascade by 4EBP1A4, an unphosphorylatable form of 4EBP1, significantly delayed, AKT/Ras-induced hepatocarcinogenesis. Combined treatment with rapamycin and 4EBP1A4 completely inhibited AKT/Ras hepatocarcinogenesis. This strong antineoplastic effect was successfully recapitulated by ablating regulatory associated protein of mTORC1, the major subunit of mTORC1, in AKT/Ras-overexpressing livers. Furthermore, we demonstrate that overexpression of eIF4E, the proto-oncogene whose activity is specifically inhibited by 4EBP1, resulted in hepatocellular carcinoma (HCC) development in cooperation with activated Ras. Mechanistically, we identified the ectonucleoside triphosphate diphosphohydrolase 5/ adenylate kinase 1/cytidine monophosphate kinase 1 axis and the mitochondrial biogenesis pathway as targets of the 4EBP1/eIF4E cascade in AKT/Ras and Ras/eIF4E livers as well as in human HCC cell lines and tissues. Complete inhibition of mTORC1 is required to suppress liver cancer development induced by AKT and Ras proto-oncogenes in mice. The mTORC1 effectors, RPS6 and eIF4E, play distinct roles and are both necessary for AKT/Ras hepatocarcinogenesis. These new findings might open the way for innovative therapies against human HCC.

Abstract PR01: H-RasG12V overexpression in the central nervous system leads to expansion of oligodendrocyte precursor cells and neural crest cells

Abstract Somatic mutations in Ras proto-oncogenes have been found in a large number of different cancers. In addition, germline mutations in the H-Ras gene have been shown to be the causative genetic alteration underlying Costello Syndrome (CS). Costello Syndrome belongs to the large group of Ras/MAPK pathway diseases commonly termed Rasopathies. CS patients exhibit craniofacial and cardiac abnormalities, mental retardation and are prone to develop cancers including neural crest derived neoplasias. Since Costello Syndrome is a rare disease, the effects of H-RasG12V expression on the development of the central nervous system remain largely uncharacterized. Towards this goal, we have established a zebrafish model suitable for live imaging and cost-effective drug screening. Using the Gal4/UAS system we have enforced expression of H-RasG12V in the central nervous system starting at early neurulation stages. H-RasG12V expressing embryos develop hyperplasia of the forebrain and the spinal cord and show an outgrowth in the dorsal tail region around 3 days post fertilization (dpf). In situ hybridisation revealed an increase in oligodendrocyte precursor cells, similar to what has been reported for zebrafish neurofibromin 1 mutants. In addition, cells expressing the neural crest markers Sox10 and FoxD3 accumulate lateral to the neural tube and notochord. Time-lapse imaging revealed that H-RasG12V expressing cells indeed migrate out of the spinal cord. This migration was ectopic, occuring several hours after normal neural crest migration. Furthermore, coinciding with the accumulation of neural crest like cells around the neural tube, the intersomitic vasculature hyperbranches to form new blood vessels towards domains of H-RasG12V expressing cells. Taking advantage of the early onset and obvious phenotypes in our H-RasG12V overexpression model we performed a pilot drug screen to identify potential therapeutic compounds for Costello Syndrome. We found the Mek1/Mek2 inhibitor PD98059 successfully inhibited hyperplasia of the forebrain and the spinal cord, with normal numbers of oligodendrocyte precursor cells and neural crest cells. Thus, this zebrafish model will be a valuable tool to further investigate the mechanisms underlying CNS- abnormalities of Costello Syndrome patients, including the formation of neural crest derived neoplasias and will be useful to identify therapies against this disease. This abstract is also presented as Poster A3. Citation Format: Martin Distel, David Traver. H-RasG12V overexpression in the central nervous system leads to expansion of oligodendrocyte precursor cells and neural crest cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR01.

Neurofibromatosis type 1 (NF1) and associated tumors.

Neurofibromatosis type 1 (NF1) is a frequent neurocutaneous syndrome that predisposes for various benign and malignant tumors. Most characteristic are neurofibromas which occur in almost all NF1 patients at some point in lifetime. Although neurofibromas are benign tumors they can be disfiguring and plexiform neurofibromas may progress to malignant peripheral nerve sheath tumors. Overall survival rates of patients with these malignant tumors are poor. Other neoplasias frequently observed in NF1 patients are pilocytic astrocytomas, gastrointestinal stromal tumors, pheochromocytomas and juvenile myelomonocytic leukemia. Several other tumors have been reported in NF1 patients but it is unclear if there is a true association between the particular tumor type and NF1. Some of these tumors might be caused by a rare recessively inherited childhood cancer syndrome termed constitutive mismatch repair deficiency syndrome which shows certain phenotypic overlap with NF1 but includes a broad spectrum of tumors which usually do not occur in NF1. The development of NF1-associated tumors is largely explained by the underlying defect of the NF1 gene which results in activation of the RAS proto-oncogene- a key mechanism of tumorigenesis. Several downstream effectors of activated RAS as well as cooperating molecular pathways have been identified. These insights provide the basis to develop novel targeted treatment strategies which are urgently needed to improve the outcome for patients with NF1-associated malignancies.

RGS6 suppresses cellular transformation by oncogenic Ras through a Tip60‐Dnmt1‐apoptosis dependent mechanism (609.14)

Silencing of tumor suppressor genes by DNA methyltransferase 1 (Dnmt1), dysregulated in human cancers, is essential for oncogenic transformation by the Ras protooncogene. Here, we provide evidence that Regulator of G protein Signaling 6 (RGS6) suppresses Ras‐induced cellular transformation by facilitating Tip60‐mediated Dnmt1 degradation and promoting apoptosis. Employing mouse embryonic fibroblasts (MEFs) from wild type (WT) and RGS6‐/‐ mice, we found that Ras induced up‐regulation of RGS6, which in turn blocked Ras‐induced cellular transformation. RGS6 suppresses cellular transformation by down regulating Dnmt1 leading to inhibition of Dnmt1‐mediated anti‐apoptotic activity. Further experiments showed that RGS6 is a scaffolding protein for Dnmt1 and Tip60 required for Tip60‐mediated acetylation of Dnmt1 and Dnmt1 ubiquitylation and degradation. Indeed, RGS6 loss promotes Dnmt1 up‐regulation in a mouse model of breast cancer possibly contributing to accelerated tumorigenesis observed in RGS6‐/‐ mice. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogenesis providing new insights into our understanding of the mechanisms regulating Ras‐induced cellular transformation and Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target in cancer treatment.Grant Funding Source: Supported by NCI CA161882 (RAF), HHSN261200433000C (CB), CA090367 (DEQ)

New insights into the diagnosis of nodular goiter.

Preoperative diagnostic investigations of nodular goiter are based on two main examinations: ultrasonography of the thyroid gland and ultrasound-guided fine-needle aspiration biopsy. So far, FNAB has been the best method for the differentiation of nodules, but in some cases it fails to produce a conclusive diagnosis. Some of the biopsies do not provide enough material to establish the diagnosis, in some other biopsies cytological picture is inconclusive.Determining the eligibility of thyroid focal lesions for surgery has been more and more often done with molecular methods. The most common genetic changes leading to the development of thyroid cancer include mutations, translocations and amplifications of genes, disturbances in gene methylation and dysregulation of microRNA. The mutations of Ras proto-oncogenes and BRAF gene as well as disturbances of DNA methylation in promoter regions of genes regulating cell cycle (e.g. hypermethylation of RASSF1A gene and TIMP-3 gene) play an important role in the process of neoplastic transformation of thyreocyte. The advances in molecular biology made it possible to investigate these genetic disturbances in DNA and/or RNA from peripheral blood, postoperative thyroid tissue material and cytology specimens obtained through fine-needle aspiration biopsy of focal lesions in the thyroid gland.As it became possible to analyze the mutations and methylation of genes from cell material obtained through fine-needle aspiration biopsy, it would be beneficial to introduce the techniques of molecular biology in the pre-operative diagnosis of nodular goiter as a valuable method, complementary to ultrasonography and FNAB. The knowledge obtained from molecular studies might help to determine the frequency of follow-up investigations in patients with nodular goiter and to select patients potentially at risk of developing thyroid cancer, which would facilitate their qualification for earlier strumectomy.

RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated Dnmt1 degradation and promoting apoptosis

The RAS protooncogene has a central role in regulation of cell proliferation, and point mutations leading to oncogenic activation of Ras occur in a large number of human cancers. Silencing of tumor-suppressor genes by DNA methyltransferase 1 (Dnmt1) is essential for oncogenic cellular transformation by Ras, and Dnmt1 is overexpressed in numerous human cancers. Here we provide new evidence that the pleiotropic regulator of G protein signaling (RGS) family member RGS6 suppresses Ras-induced cellular transformation by facilitating Tip60-mediated degradation of Dmnt1 and promoting apoptosis. Employing mouse embryonic fibroblasts from wild-type and RGS6(-/-) mice, we found that oncogenic Ras induced upregulation of RGS6, which in turn blocked Ras-induced cellular transformation. RGS6 functions to suppress cellular transformation in response to oncogenic Ras by downregulating Dnmt1 protein expression leading to inhibition of Dnmt1-mediated anti-apoptotic activity. Further experiments showed that RGS6 functions as a scaffolding protein for both Dnmt1 and Tip60 and is required for Tip60-mediated acetylation of Dnmt1 and subsequent Dnmt1 ubiquitylation and degradation. The RGS domain of RGS6, known only for its GTPase-activating protein activity toward Gα subunits, was sufficient to mediate Tip60 association with RGS6. This work demonstrates a novel signaling action for RGS6 in negative regulation of oncogene-induced transformation and provides new insights into our understanding of the mechanisms underlying Ras-induced oncogenic transformation and regulation of Dnmt1 expression. Importantly, these findings identify RGS6 as an essential cellular defender against oncogenic stress and a potential therapeutic target for developing new cancer treatments.

The natural product honokiol inhibits calcineurin inhibitor-induced and Ras-mediated tumor promoting pathways

Although calcineurin inhibitors (CNIs) are very useful in preventing allograft rejection, they can mediate a rapid progression of post-transplantation malignancies. The CNI cyclosporine A (CsA) can promote renal tumor growth through activation of the proto-oncogene ras and over-expression of the angiogenic cytokine VEGF; the ras activation also induces over-expression of the cytoprotective enzyme HO-1, which promotes survival of renal cancer cells. Here, we show that the natural product honokiol significantly inhibited CsA-induced and Ras-mediated survival of renal cancer cells through the down-regulations of VEGF and HO-1. Thus, honokiol treatment may help to prevent tumor-promoting effects of CsA in transplant patients.

Abstract 608: Quinacrine and sorafenib as potential combination for anaplastic thyroid carcinoma.

Abstract Anaplastic thyroid cancer (ATC) comprises ∼2% of all thyroid cancers and its median survival rate remains poor. ATC is frequently resistant to conventional therapy and therefore it is essential to expand the number of treatment options for ATC. Proto-oncogenes RET, RAS and BRAF are some of the best targets described in thyroid cancer. Sorafenib is a small molecule multi-kinase inhibitor that inhibits RAF, MEK and ERK kinases among other targets and is therefore being evaluated in several phase II/III clinical trials in thyroid cancer. Quinacrine, a potent small molecule inhibitor of NFKB signaling, is currently being evaluated in phase II clinical trials. We have previously shown the effectiveness of quinacrine in combination with cytotoxic drugs in hepatocellular and colon carcinoma cells acts by inhibiting NFKB, Mcl-1, and angiogenesis in tumor cell lines that are deficient in p53. Here, we evaluate the efficacy of quinacrine in combination with sorafenib on a panel of human ATC cells. Quinacrine as a single agent effectively inhibits growth and promote apoptosis of ATC cells in vitro in a dose-dependent manner as assessed by CellTiter-Glo and sub-G1 analysis respectively. Combinatorial dose-response modulation of quinacrine with sorafenib suggests a synergistic drug-drug interaction with respect to growth stasis of ATC cells in vitro, as defined by Chou-Talalay. Western blot analysis suggest that the anti-apoptotic Bcl-2 family member Mcl-1, over-expressed in a number of solid tumors, is efficiently down-regulated in the ATC cell line 8505C by the combination of quinacrine and sorafenib. We also observe that the active form of transcription factor Stat3 is down-regulated by both quinacrine and sorafenib. In contrast to chloroquine that inhibits autophagy, our previous results have not shown that quinacrine's anti-tumor efficacy involves alterations in autophagy. Furthermore, sorafenib and quinacrine significantly improve survival in a mouse thyroid orthotopic in vivo xenograft model of ATC. We are currently exploring the detailed molecular mechanism of the quinacrine and sorafenib drug synergy and associated anti-tumor activity in vitro and in vivo. In addition, we are exploring the possibility of performing in vivo assays of the therapy combination with clinical samples. Our findings suggest that quinacrine in combination with sorafenib may be a novel and potentially cost effective therapeutic strategy for the treatment ATC. Citation Format: Junaid Abdulghani, Jean-Nicolas Gallant, Tiffany Whitcomb, David Dicker, David Goldenberg, Charles D. Smith, Niklas Finnberg, Wafik S. El-Deiry. Quinacrine and sorafenib as potential combination for anaplastic thyroid carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 608. doi:10.1158/1538-7445.AM2013-608

Abstract 4292: RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines.

Abstract Elevated levels of Ras activity are a prominent feature in human lung cancer. In approximately 20% of lung cancer cases, Ras activation is achieved by mutation of the Ras proto-oncogene. Here, we explore the possible contribution of RasGRP3 to Ras signaling in lung cancer. RasGRP3 is a Ras guanyl nucleotide exchange factor which has been shown to be expressed in prostate tumor and melanoma cell lines, contributing to the level of Ras activation, cell proliferation, and tumor xenograft growth. Here, we report that RasGRP3 is expressed in multiple human lung cancer cell lines but not in normal human bronchial epithelial cells. Likewise, initial analysis reveals its expression in human lung tumors but not in normal lung tissue. Suppression of endogenous RasGRP3 expression in lung cancer cell lines showing reduced H-Ras-GTP and K-Ras-GTP formation as well as Akt phosphorylation downstream from epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulation. Additionally, suppression of RasGRP3 inhibited translocation of the NF-kB p50 and p65 subunits from the cytoplasm to the nuclear fraction and modulated several other transcriptional factors as well. Conversely, expression of exogenous RasGRP3 elevated H-Ras-GTP and K-Ras-GTP and activated AKT phosphorylation in the lung cancer H496 cell line, which itself only expresses RasGRP3 at low levels. These changes in signaling were mirrored at the cellular level. Downregulation of endogenous RasGRP3 inhibited cell proliferation, impeded cell migration and reduced colony formations in soft agar. Our results suggest that RasGRP3 may provide an addition targetable signaling element in lung cancer. Citation Format: Jing Yang, Timothy Esch, Dazhi Yang, Peter Blumberg. RasGRP3 contributes to Ras signaling, growth, and migration of human lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4292. doi:10.1158/1538-7445.AM2013-4292

Aneurysmal Rupture of the Internal Carotid Artery in a Presumed Neurofibromatosis Type I Patient

Carotid artery aneurysms are uncommon but have the potential of catastrophic clinical events. The aneurysms, as we know, lead to severe neurologic symptoms like embolization and even sudden death from rupture. Aneurysms of the carotid artery result from injury or may be a long term sequel of spontaneous dissection. It could be also caused by congenital arterial anomalies such as Neurofibromatosis type I (NF-I). NF-I, so-called von Recklinghausen disease, is an autosomal dominant disorder affecting one per 3,5004000 live births. NF-I is developed by mutations in, or deletions of, the NF-I gene that is located on 17q11.2. This gene controls neurofibromin, a GTPaseactivating protein that is thought to have a possible tumor-suppressor function by maintaining the ras proto-oncogene in an inactive form. Over 250 mutations in the NF-I gene have been identified. The inheritance is autosomal dominant type with 50% of the cases representing new mutations. Clinical features of NF-I include multiple hyperpigmented macules (so-called cafe′au lait spots), various benign and malignant tumors including neurofibroma, iris hamartomas (so-called Lisch nodules), and freckling in the axillary or inguinal regions (so-called Crowe’s sign), and abnormalities of blood vessels. Vascular abnormalities, mostly in the form of aneurysms or stenoses, affect mediumand large-sized vessels and are presented in NF-I. The renal artery is the most frequently involved one, but abdominal aortic coarctation, internal carotid artery aneurysms, and

A rare point mutation in the Ras oncogene in hepatocellular carcinoma

The Ras gene is one of the oncogenes most frequently detected in human cancers, and codes for three proteins (K-, N-, and H-Ras). The aim of this study was to examine the mutations in codons 12, 13 and 61 of the three Ras genes in cases of human hepatocellular carcinoma (HCC). Paired samples of HCC and corresponding non-malignant liver tissue were collected from 61 patients who underwent hepatectomy. A dot-blot analysis was used to analyze the products of the polymerase chain reaction (PCR) amplification of codons 12, 13, and 61 of K-, N- and H-Ras for mutations. Only one mutation (K-Ras codon 13; Gly to Asp) was detected among the 61 patients. Interestingly, this patient had a medical history of surgery for both gastric cancer and right lung cancer. No mutations were found in codons 12 and 61 of K-Ras or codons 12, 13 and 61 of the N-Ras and H-Ras genes in any of the HCCs or corresponding non-malignant tissues. These findings indicated that the activation of Ras proto-oncogenes by mutations in codons 12, 13, and 61 does not play a major role in hepatocellular carcinogenesis.

Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma

The discovery of potent inhibitors of the BRAF proto-oncogene has revolutionized therapy for melanoma harboring mutations in BRAF, yet NRAS-mutant melanoma remains without an effective therapy. Because direct pharmacological inhibition of the RAS proto-oncogene has thus far been unsuccessful, we explored systems biology approaches to identify synergistic drug combination(s) that can mimic RAS inhibition. Here, leveraging an inducible mouse model of NRAS-mutant melanoma, we show that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) activates apoptosis but not cell-cycle arrest, which is in contrast to complete genetic neuroblastoma RAS homolog (NRAS) extinction, which triggers both of these effects. Network modeling pinpointed cyclin-dependent kinase 4 (CDK4) as a key driver of this differential phenotype. Accordingly, combined pharmacological inhibition of MEK and CDK4 in vivo led to substantial synergy in therapeutic efficacy. We suggest a gradient model of oncogenic NRAS signaling in which the output is gated, resulting in the decoupling of discrete downstream biological phenotypes as a result of incomplete inhibition. Such a gated signaling model offers a new framework to identify nonobvious coextinction target(s) for combined pharmacological inhibition in NRAS-mutant melanomas.

Combination therapy targeting Raf-1 and MEK causes apoptosis of HCT116 colon cancer cells

Members of the Ras protooncogene family are mutated in approximately 75% of colon cancers. The Raf kinases (Raf-1, b-Raf and a-Raf) directly interact with Ras and serve as mediators of mitogenic signals. Expression of the constitutively active alleles of Raf or Ras gene families results in oncogenesis in a number of model systems. Previous studies emphasized the importance of Raf-1 and b-Raf in preventing apoptosis in addition to their roles in cell growth. In the present study, we examined whether inhibition of the Raf-1 or b-Raf kinase decreases cell growth and increases apoptosis in colon cancer cells. c-Raf and b-Raf were depleted in colon cancer cell lines, such as HCT116, HT29 and Colo205, containing Ras or b-Raf mutations by RNA interference (RNAi). The results showed that colon cancer cells with activating Ras mutations undergo apoptosis following Raf-1 inhibition, as determined by cell cycle analysis and the release of cytochromec. Moreover, in b-Raf mutant colon cancers, the inhibition of b-Raf as compared to Raf-1 is crucial for cancer cell death. There is increasing evidence for both MEK-independent Raf signaling and Raf-independent MEK signaling. Thus, we investigated whether targeting multiple points of the mitogen-activated protein kinase (MAPK) pathway with a MEK inhibitor and Raf RNAi increases cancer cell death. The results showed that combination therapy, inhibiting Raf and MEK kinases simultaneously, increased apoptosis in colon cancer cells. Taken together, our data demonstrate that combination therapy targeting the MAPK pathway at two distinct points, Raf kinase and MEK, has greater efficacy in increasing cancer cell death and is likely to improve therapeutic outcomes for patients.

Molecular analysis of RAS-RAF tyrosine-kinase signaling pathway alterations in patients with plasma cell myeloma

In patients with plasma cell myeloma (PCM), interphase fluorescence in situ hybridization (FISH) detects prognostically relevant genetic alterations, for example, the prognostically adverse t(4;14)(p16;q32) or 17p deletions. Furthermore, mutations of the RAS protooncogene were suggested to be associated to myeloma pathogenesis.1 The RAS pathway inhibitor lonafarnib combined with the proteasome inhibitor bortezomib demonstrated synergistic cell death in human myeloma cells in association with downregulation of p-AKT in an in vitro setting.2 By whole-genome and whole-exome sequencing, Chapman et al.3 found frequent involvement of genes associated to the nuclear factor-kappaB pathway. Rare PCM cases were positive for BRAF mutations,3 which had previously been detected in solid tumors (for example, melanoma) and hematological neoplasms, for example, hairy cell leukemia.4

Essential Role of Cdc42 in Ras-Induced Transformation Revealed by Gene Targeting

The ras proto-oncogene is one of the most frequently mutated genes in human cancer. However, given the prevalence of activating mutations in Ras and its association with aggressive forms of cancer, attempts to therapeutically target aberrant Ras signaling have been largely disappointing. This lack of progress highlights the deficiency in our understanding of cellular pathways required for Ras-mediated tumorigenesis and suggests the importance of identifying new molecular pathways associated with Ras-driven malignancies. Cdc42 is a Ras-related small GTPase that is known to play roles in oncogenic processes such as cell growth, survival, invasion, and migration. A pan-dominant negative mutant overexpression approach to suppress Cdc42 and related pathways has previously shown a requirement for Cdc42 in Ras-induced anchorage-independent cell growth, however the lack of specificity of such approaches make it difficult to determine if effects are directly related to changes in Cdc42 activity or other Rho family members. Therefore, in order to directly and unambiguously address the role of Cdc42 in Ras-mediated transformation, tumor formation and maintenance, we have developed a model of conditional cdc42 gene in Ras-transformed cells. Loss of Cdc42 drastically alters the cell morphology and inhibits proliferation, cell cycle progression and tumorigenicity of Ras-transformed cells, while non-transformed cells or c-Myc transformed cells are largely unaffected. The loss of Cdc42 in Ras-transformed cells results in reduced Akt signaling, restoration of which could partially rescues the proliferation defects associated with Cdc42 loss. Moreover, disruption of Cdc42 function in established tumors inhibited continued tumor growth. These studies implicate Cdc42 in Ras-driven tumor growth and suggest that targeting Cdc42 is beneficial in Ras-mediated malignancies.

RAS signalling in the colorectum in health and disease

RAS proteins act as molecular switches between several homeostatic inputs and signal transduction pathways that regulate important cellular processes including cell growth, differentiation and survival. Activating mutations change the function of normal proto-oncogenic RAS proteins to oncogenic RAS proteins that trigger a wide range of downstream effectors altering expression of transcription factors that together stimulate cell proliferation and modulate apoptosis and differentiation. RAS genes are amongst the most frequently mutated genes in human cancers, in particular KRAS is mutated in 40-50% of colorectal cancers. Mutation of this gene has a significant impact on treatment management and patients' survival, particularly in relation to anti-EGFR therapy, which is only effective in KRAS wild-type cases. Here, we discuss the regulation of KRAS signalling in the colorectum, some of the post-transcriptional and post-translational modifications that control its activity, the mutations and other DNA alterations that are found in this tumour type and the implications that they have for disease progression and current drug treatments.