Expression Of Oncogenic H-Ras Research Articles

Expression of oncogenic HRAS in human Rh28 and RMS-YM rhabdomyosarcoma cells leads to oncogene-induced senescence

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level—eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a “quiet” genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.

Evolution of telomere maintenance and tumour suppressor mechanisms across mammals.

Mammalian species differ dramatically in telomere biology. Species larger than 5-10 kg repress somatic telomerase activity and have shorter telomeres, leading to replicative senescence. It has been proposed that evolution of replicative senescence in large-bodied species is an anti-tumour mechanism counteracting increased risk of cancer due to increased cell numbers. By contrast, small-bodied species express high telomerase activity and have longer telomeres. To counteract cancer risk due to longer lifespan, long-lived small-bodied species evolved additional telomere-independent tumour suppressor mechanisms. Here, we tested the connection between telomere biology and tumorigenesis by analysing the propensity of fibroblasts from 18 rodent species to form tumours. We found a negative correlation between species lifespan and anchorage-independent growth. Small-bodied species required inactivation of Rb and/or p53 and expression of oncogenic H-Ras to form tumours. Large-bodied species displayed a continuum of phenotypes requiring additional genetic 'hits' for malignant transformation. Based on these data we refine the model of the evolution of tumour suppressor mechanisms and telomeres. We propose that two different strategies evolved in small and large species because small-bodied species cannot tolerate small tumours that form prior to activation of the telomere barrier, and must instead use telomere-independent strategies that act earlier, at the hyperplasia stage.This article is part of the theme issue 'Understanding diversity in telomere dynamics'.

Does Harvey-Ras gene expression lead to oral squamous cell carcinoma? A clinicopathological aspect.

Background:Harvey-Ras (H-Ras) is an important guanosine triphosphatase protein for the regulation of cellular growth and survival. Altered Ras signaling has been observed in different types of cancer either by gene amplification and/or mutation. The H-Ras oncogene mutations are well reported, but expression of the H-Ras gene is still unknown.Objective:This study aimed to examine both protein and messenger-RNA (mRNA) expressions of H-Ras in oral squamous cell carcinoma (OSCC) and analyzed the association with risk habits and the clinicopathological profile of cases.Methodology:A total of 65 tissue specimens of OSCC (case group) and equal number of normal tissues (control group) were included in this study. H-Ras protein and mRNA expressions were analyzed using immunohistochemical and quantitative real time-polymerase chain reaction techniques, respectively.Results:The H-Ras protein was significantly overexpressed in the oral carcinoma group compared to the normal group (P = 0.03). Most of the OSCC cases showed positive staining with moderate expression, while negative and moderate staining was high in the control group. The majority of H-Ras positive cases were found in individuals with multiple risk habits including tobacco chewing. The risk of H-Ras positivity was 1.46 times higher in smokers than non-smokers. H-Ras positivity increased in cases affected with buccal mucosa site and higher grade of carcinoma. Relative mRNA level of H-Ras was significantly elevated in oral carcinoma as compared with the control group (P ≤ 0.001). Protein and mRNA levels of H-Ras in case group was poorly correlated.Conclusion:H-Ras oncogene expression was markedly higher in oral carcinoma, and it can be a prognostic marker and target for an effective molecular therapy.

Quantitative Proteomics Reveals Fundamental Regulatory Differences in Oncogenic HRAS and Isocitrate Dehydrogenase (IDH1) Driven Astrocytoma

Glioblastoma multiformes (GBMs) are high-grade astrocytomas and the most common brain malignancies. Primary GBMs are often associated with disturbed RAS signaling, and expression of oncogenic HRAS results in a malignant phenotype in glioma cell lines. Secondary GBMs arise from lower-grade astrocytomas, have slower progression than primary tumors, and contain IDH1 mutations in over 70% of cases. Despite significant amount of accumulating genomic and transcriptomic data, the fundamental mechanistic differences of gliomagenesis in these two types of high-grade astrocytoma remain poorly understood. Only a few studies have attempted to investigate the proteome, phosphorylation signaling, and epigenetic regulation in astrocytoma. In the present study, we applied quantitative phosphoproteomics to identify the main signaling differences between oncogenic HRAS and mutant IDH1-driven glioma cells as models of primary and secondary GBM, respectively. Our analysis confirms the driving roles of the MAPK and PI3K/mTOR signaling pathways in HRAS driven cells and additionally uncovers dysregulation of other signaling pathways. Although a subset of the signaling changes mediated by HRAS could be reversed by a MEK inhibitor, dual inhibition of MEK and PI3K resulted in more complete reversal of the phosphorylation patterns produced by HRAS expression. In contrast, cells expressing mutant IDH1 did not show significant activation of MAPK or PI3K/mTOR pathways. Instead, global downregulation of protein expression was observed. Targeted proteomic analysis of histone modifications identified significant histone methylation, acetylation, and butyrylation changes in the mutant IDH1 expressing cells, consistent with a global transcriptional repressive state. Our findings offer novel mechanistic insight linking mutant IDH1 associated inhibition of histone demethylases with specific histone modification changes to produce global transcriptional repression in secondary glioblastoma. Our proteomic datasets are available for download and provide a comprehensive catalogue of alterations in protein abundance, phosphorylation, and histone modifications in oncogenic HRAS and IDH1 driven astrocytoma cells beyond the transcriptomic level.

High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis.

Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRAS(G12D) driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70 kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets.

DN-03 * MUTANT IDH1-DRIVEN CELLULAR TRANSFORMATION INCREASES RAD51-MEDIATED HOMOLOGOUS RECOMBINATION AND TEMOZOLOMIDE RESISTANCE

BACKGROUND: IDH1 mutation is noted in roughly 75% of all lower-grade glioma and is considered to be an early and driving event in the genesis of these tumors. In clinical studies IDH1 mutation correlates with increased overall survival, progression-free survival, and upfront response to TMZ implying that mutant IDH1 directly contributes to drug sensitivity. IDH1 mutant gliomas, however, have a different underlying biology than comparable IDH1 WT glioma, complicating any analysis of the contribution of IDH1 mutation to drug sensitivity. To more accurately determine if and how IDH1 mutation alters drug sensitivity, we employed a system widely for the study of cellular transformation and IDH biology. In this system normal human astrocytes immortalized by introduction of virally-encoded E6, E7 and hTERT can be transformed by additional expression of oncogenic H-Ras or by mutant IDH1, the latter of which induces epigenetic changes similar to those noted in lower-grade glioma. We therefore used these cells to determine if mutant IDH1 expression and/or the transformative events it sets in place were sufficient to alter TMZ sensitivity. METHODS: E6/E7/hTERT-immortalized, untransformed human astrocytes, and the same cells transformed by prior introduction of oncogenic V12H-Ras, were infected with lentiviral constructs encoding WT or mutant (R132H) IDH1, then monitored for transformation status and, following TMZ exposure (100 uM, 3hrs), for DNA damage and repair, cell cycle response, and TMZ sensitivity. RESULTS: The TMZ-sensitive immortalized, untransformed parental cells exhibited DNA damage (H2AX foci) and a prolonged G2 cell cycle arrest that began 2 days after TMZ exposure and persisted for 5 days. The same cells transformed by mutant IDH1 exhibited the same levels of initial DNA damage and cell cycle arrest as controls, although both events resolved significantly faster in the IDH1 mutant cells in association with increased clonagenic survival. The increases in DNA damage processing, cell cycle progression, and clonagenicity were unique to cells transformed by mutant IDH1, and were not noted following introduction of mutant IDH1 into Ras-transformed or established GBM cells. The changes were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic suppression of Rad51, the rate limiting protein in HR. CONCLUSIONS: Mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and increase repair of TMZ-induced DNA damage and TMZ resistance. Inhibitors of HR may therefore be a viable means to further enhance TMZ response in IDH1 mutant glioma. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

The interplay between epigenetic silencing, oncogenic KRas and HIF-1 regulatory pathways in control of BNIP3 expression in human colorectal cancer cells

Bcl-2/adenovirus E1B-19kDa-interacting protein 3 (BNIP3) is an important mediator of cell survival and a member of the Bcl-2 family of proteins that regulate programmed cell death and autophagy. We have previously established a link between the expression of oncogenic HRas and up-regulation of BNIP3 and the control of autophagy in cancer cells. However, in view of varied expression of BNIP3 in different tumor types and emerging uncertainties as to the role of epigenetic silencing, oncogenic regulation and the role of BNIP3 in cancer are still poorly understood.In the present study we describe profound effect of KRas on the expression of methylated BNIP3 in colorectal cancer cells and explore the interplay between HIF-1, hypoxia pathway and oncogenic KRas in this context. We observed that BNIP3 mRNA remains undetectable in aggressive DLD-1 cells harboring G13D mutant KRAS and HT-29 colorectal cancer cells unless the cells are exposed to demethylating agents such as 5-aza-2′-deoxycytidine. Following this treatment BNIP3 expression remains uniquely dependent on the Ras activity. We found that hypoxia or pharmacological activation of HIF-1 alone contributes to, but is not sufficient for efficient induction of BNIP3 mRNA transcription in cells lacking mutant KRas activity. The up-regulation of BNIP3 by KRas in this setting is mediated by the MAPK pathway, and is attenuated by the respective inhibitors (PD98059, U0126). Thus, we demonstrate the novel mechanism where activity of Ras is essential for 5-aza-2′-deoxycytidine-mediated BNIP3 expression. Moreover, we found that 5-aza-2′-deoxycytidine-mediated or enforced up-regulation of BNIP3 in DLD-1 cells results in KRas-dependent resistance to 5-Fluorouracil.

Optimization of oncogene expression through intra‐population competition

Although functional roles have been assigned to many genes, e.g. those involved in cell-cycle regulation, growth signaling, or cancer, considerably less is known about the quantitative relationship between gene expression levels and outcome. We devised an intra-population competition to study oncogene dosage. Cell populations were engineered to express a range of H-Ras oncogene levels. Cells with different levels of H-Ras then "competed" for an increased share of the total cell population. Using flow cytometry to track the population composition over time, we determined the relationship between the different H-Ras oncogene expression levels and the net proliferation rate. Under culture conditions in which wild-type Ras activation was suppressed, we found that increased and maximal net proliferation occurred when the H-Ras G12V oncogene was expressed at a level 1.2-fold that of wild-type Ras. As the H-Ras G12V expression levels increased above this optimal level, proliferation rates decreased. Our findings suggest that the tumor evolution process may optimize gene expression levels for maximal cell proliferation. In principle, engineered intra-population competitions can be used to determine proliferation rates associated with the level of any ectopically expressed gene. The approach also may be used to determine proliferation rates associated with different cell species in a heterogeneous population or to improve the proliferation rate of a cell line. We also envision that the tracking of intra-population competitions could be utilized to investigate the evolution of tumors in the body.

Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x=1) to fluid-like (x=2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

C4ST-1/CHST11-controlled chondroitin sulfation interferes with oncogenic HRAS signaling in Costello syndrome

Costello syndrome is a pediatric genetic disorder linked to oncogenic germline mutations in the HRAS gene. The disease is characterized by multiple developmental abnormalities, as well as predisposition to malignancies. Our recent observation that heart tissue from patients with Costello syndrome showed a loss of the glycosaminoglycan chondroitin-4-sulfate (C4S) inspired our present study aimed to explore a functional involvement of the chondroitin sulfate (CS) biosynthesis gene Carbohydrate sulfotransferase 11/Chondroitin-4-sulfotransferase-1 (CHST11/C4ST-1), as well as an impaired chondroitin sulfation balance, as a downstream mediator of oncogenic HRAS in Costello syndrome. Here we demonstrate a loss of C4S, as well as a reduction in C4ST-1 mRNA and protein expression, in primary fibroblasts from Costello syndrome patients. We go on to show that expression of oncogenic HRAS in normal fibroblasts can repress C4ST-1 expression, whereas interference with oncogenic HRAS signaling in Costello syndrome fibroblasts elevated C4ST-1 expression, thus identifying C4ST-1 as a negatively regulated target gene of HRAS signaling. Importantly, we show that forced expression of C4ST-1 in Costello fibroblasts could rescue the proliferation and elastogenesis defects associated with oncogenic HRAS signaling in these cells. Our results indicate reduced C4ST-1 expression and chondroitin sulfation imbalance mediating the effects of oncogenic HRAS signaling in the pathogenesis of Costello syndrome. Thus, our work identifies C4ST-1-dependent chondroitin sulfation as a downstream vulnerability in oncogenic RAS signaling, which might be pharmacologically exploited in future treatments of not only Costello syndrome and other RASopathies, but also human cancers associated with activating RAS mutations.

Abstract 1681: Synergistic induction of Mek1/2 and Nox-1 by histone deacetylase inhibitors leads to differential induction of reactive oxygen species for mediating selective apoptosis of oncogenic H-Ras-expressing cells

Abstract The goal of this study was to investigate roles of the ERK pathway and Nox-1 in the pro-apoptotic ability of oncogenic H-Ras to enhance histone deacetylase inhibitor (HDACI)-induced cell death. Oncogenic induction of the ras genes is widely involved in human cancers. Our previous studies revealed that expression of oncogenic H-Ras increases susceptibility of human and mouse cells to HDACIs, such as FK228 and trichostatin A, for inducing selective apoptosis. In this study, we used the mouse embryo fibroblast 10T1/2-TR-H-ras cell system, in which ectopic expression of oncogenic H-Ras(V12) is controlled by addition of tetracycline into cultures, and paired oncogenic H-Ras-expressing and parental human epithelial cells. We pursued the roles of Mek1/2 activation, Nox-1 elevation, and reactive oxygen species (ROS) production in FK228-induced selective cell death of oncogenic H-Ras-expressing cells versus counterpart cells. We verified that Nox-1 elevation and ROS played essential roles in oncogenic H-Ras-induced cell proliferation and morphological transformation. We also detected that FK228 treatment and expression of oncogenic H-Ras synergistically induced Mek1/2 activation, leading to differentially elevated Nox-1 and ROS and contributing to induction of selective apoptosis of oncogenic H-Ras-expressing cells versus counterpart cells. FK228 treatment also resulted in mitochondrial ROS production, and mitochondrial ROS contributed to Mek1/2 activation, bypassing Raf, to induce Erk1/2 activation for elevating Nox-1 and ROS, leading to apoptosis. Accordingly, Mek1/2 and Nox-1 played important pro-apoptotic roles in ROS-mediated cell death. Thus, Mek1/2 and Nox-1 should be considered as potential targets in designing therapeutic protocols using FK228 in ROS-mediated cell killing to treat cancer cells acquiring Ras activation. 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 1681. doi:10.1158/1538-7445.AM2011-1681

Kita Driven Expression of Oncogenic HRAS Leads to Early Onset and Highly Penetrant Melanoma in Zebrafish

BackgroundMelanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Methodology and Principal FindingsUsing the combinatorial Gal4 –UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2–4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1–3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.Conclusions and SignificanceThis indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

Malignant Transformation of Non-Neoplastic Barrett's Epithelial Cells through Well-Defined Genetic Manipulations

BackgroundHuman Barrett's cancer cell lines have numerous, poorly-characterized genetic abnormalities and, consequently, those lines have limited utility as models for studying the early molecular events in carcinogenesis. Cell lines with well-defined genetic lesions that recapitulate various stages of neoplastic progression in Barrett's esophagus would be most useful for such studies.Methodology/Principal FindingsTo develop such model cell lines, we started with telomerase-immortalized, non-neoplastic Barrett's epithelial (BAR-T) cells, which are spontaneously deficient in p16, and proceeded to knock down p53 using RNAi, to activate Ras by introducing oncogenic H-RasG12V, or both. BAR-T cells infected with either p53 RNAi or oncogenic H-RasG12V alone maintained cell-to-cell contact inhibition and did not exhibit anchorage-independent growth in soft agar. In contrast, the combination of p53 RNAi knockdown with expression of oncogenic H-RasG12V transformed the p16-deficient BAR-T cells, as evidenced by their loss of contact inhibition, by their formation of colonies in soft agar, and by their generation of tumors in immunodeficient mice.Conclusions/SignificanceThrough these experiments, we have generated a number of transformed and non-transformed cell lines with well-characterized genetic abnormalities recapitulating various stages of carcinogenesis in Barrett's esophagus. These lines should be useful models for the study of carcinogenesis in Barrett's esophagus, and for testing the efficacy of chemopreventive and chemotherapeutic agents.

Differential induction of reactive oxygen species through Erk1/2 and Nox-1 by FK228 for selective apoptosis of oncogenic H-Ras-expressing human urinary bladder cancer J82 cells

This study sought to reveal mechanisms for differential regulation of reactive oxygen species (ROS) in histone deacetylase inhibitor FK228-induced selective apoptosis of oncogenic H-Ras-expressing human cancer cells. Human urinary bladder cancer J82 and oncogenic H-Ras-expressing J82 cells were used to reveal FK228-induced differential Erk1/2 activation, Nox-1 elevation, ROS production, glutathione (GSH) depletion, caspase activation, and apoptosis. Specific inhibitors were used to suppress Nox-1 activity and ROS production. Mek1/2 inhibitor was used to suppress Erk1/2 activation. Validated-specific siRNAs were used to knock down Nox-1. ROS levels, GSH levels, and caspase-3/7 activities were measured by GSH assay, flow cytometry and luminescence assays, respectively. Western blot analysis determined levels of Erk1/2 and Nox-1. Erk1/2, Nox-1, ROS, caspase-3/7, and cell death were differentially induced, whereas GSH was differentially depleted by FK228 in oncogenic H-Ras-expressing J82 versus parental cells. Blockage of the ERK pathway resulted in suppressing oncogenic H-Ras- and FK228-induced Nox-1 elevation, ROS production, caspase activation, and cell death. Knockdown of Nox-1 by specific siRNAs reduced FK228-induced ROS production, caspase activation, and cell death. Oncogenic H-Ras expression and FK228 treatment synergistically induced the ERK pathway, resulting in differentially increased Nox-1 elevation, ROS production, and GSH depletion, leading to differential caspase activation and cell death in oncogenic H-Ras-expressing J82 versus parental cells.

Abstract 1580: Differential induction of reactive oxygen species by histone deacetylase inhibitors for mediating selective apoptosis of oncogenic H-Ras-expressing cells

Abstract This study is to understand the mechanisms involved in the reactive oxygen species (ROS)-mediated pro-apoptotic ability of H-Ras to enhance apoptosis induced by histone deacetylase inhibitors (HDACIs). Oncogenic induction of the ras genes is widely involved in human cancers. Expression of oncogenic H-Ras increases susceptibility of human and mouse cells to HDACIs, such as FK228 and trichostatin A, for inducing caspase activation and selective apoptosis. To understand the mechanisms for HDACI-induced differential activation of caspases and apoptosis in oncogenic H-Ras-expressing cells, we investigated the role and regulation of ROS in HDACI-induced cell death. We detected that expression of oncogenic H-Ras resulted in potentiating intracellular ROS machinery, increasing the susceptibility to HDACIs for enhancing the production of ROS, and depleting glutathione, leading to differential activation of caspases and cell death. Furthering our understanding of how oncogenic H-Ras augmented the generation of ROS, we detected that expression of oncogenic H-Ras was able to increase the level of Nox1, a homologue of the catalytic subunit of the superoxide-generating NADPH oxidase. Suppression of Nox1 by the specific inhibitor diphenylene iodonium or specific siRNAs significantly reduced ROS production induced by oncogenic H-Ras and HDACIs as well as HDACI-induced cell death of oncogenic H-Ras-expressing cells. Our results led us to a suggestion that increased ROS played a contributing role in the proapoptotic ability of oncogenic H-Ras to enhance HDACI-induced activation of caspases for inducing selective apoptosis in a dose-dependent manner; Nox1 played an important role in the ability of oncogenic H-Ras to potentiate ROS-producing machinery for enhancing cell susceptibility to HDACIs and, in turn, increasing ROS contents leading to caspase activation and selective apoptosis. Differential increases of intracellular ROS by oncogenic H-Ras to potentiate cell death should be seriously considered as a potential target in developing therapeutic strategies to control oncogenic H-Ras-involved human cancers by HDACIs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1580.

Epidermal expression of oncogenic ras subverts cytotoxic inflammation by skewing infiltrating myeloid cells towards an immunosuppressive phenotype. (100.16)

Abstract Cancer development is frequently accompanied by inflammation and immune activation. The nature of this inflammatory response, however, is non-uniform and can consist of cells with opposing functions. To characterize the innate immune response to neoplastic epithelial tissue, we used an epidermally-restricted transgenic mouse model of oncogenic H-Ras expression in which epidermal hyperplasia and premalignant progression induces aberrant myelopoesis and cutaneous hyperinflammation associated with the formation of myeloid resident epithelial microabscesses. Antibody mediated depletion of neutrophils concurrent with transgene activation ablated neutrophil infiltration and microabscesses resulting in increased epidermal thickening relative to isotype controls. Neutrophils isolated from mice expressing ras for 1 week killed keratinocytes in vitro, suggesting that the in vivo myeloid response to oncogenic ras expression is initially a cytotoxic one. Simultaneously, there is cutaneous upregulation of genes implicated in the activation, function and recruitment of myeloid derived suppressor cells (MDSC) such as GMCSF, GCSF, MMP9, VEGF, S100A8/9, CCL2, and IL1β. This coincides with upregulation of IL4Rα on myelocytes, increased percentage of Tregs, and decreased effector T cell responses from 1 to 2 weeks post H-Ras expression. These data suggest that the acute cytotoxic neutrophil response is replaced by a suppressor myeloid phenotype that could enhance neoplastic progression.

Expression of H-RASV12 in a zebrafish model of Costello syndrome causes cellular senescence in adult proliferating cells

Constitutively active, 'oncogenic' H-RAS can drive proliferation and transformation in human cancer, or be a potent inducer of cellular senescence. Moreover, aberrant activation of the Ras pathway owing to germline mutations can cause severe developmental disorders. In this study we have generated transgenic zebrafish that constitutively express low levels, or can be induced to express high levels, of oncogenic H-RAS. We observed that fish carrying the integrated transgene in their germline display several hallmarks of Costello syndrome, a rare genetic disease caused by activating mutations in the gene H-RAS, and can be used as a model for the disease. In Costello-like fish, low levels of oncogenic H-RAS expression are associated with both reduced proliferation and an increase in senescence markers in adult progenitor cell compartments in the brain and heart, together with activated DNA damage responses. Overexpression of H-RAS through a heat-shock-inducible promoter in larvae led to hyperproliferation, activation of the DNA damage response and tp53-dependent cell cycle arrest. Thus, oncogene-induced senescence of adult proliferating cells contributes to the development of Costello syndrome and provides an alternative pathway to transformation in the presence of widespread constitutively active H-RAS expression.

Ras Promotes Growth by Alternative Splicing-Mediated Inactivation of the KLF6 Tumor Suppressor in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the fifth most prevalent cancer worldwide and the third most lethal. Dysregulation of alternative splicing underlies a number of human diseases, yet its contribution to liver cancer has not been explored fully. The Krüppel-like factor 6 (KLF6) gene is a zinc finger transcription factor that inhibits cellular growth in part by transcriptional activation of p21. KLF6 function is abrogated in human cancers owing to increased alternative splicing that yields a dominant-negative isoform, KLF6 splice variant 1 (SV1), which antagonizes full-length KLF6-mediated growth suppression. The molecular basis for stimulation of KLF6 splicing is unknown. In human HCC samples and cell lines, we functionally link oncogenic Ras signaling to increased alternative splicing of KLF6 through signaling by phosphatidylinositol-3 kinase and Akt, mediated by the splice regulatory protein ASF/SF2. In 67 human HCCs, there is a significant correlation between activated Ras signaling and increased KLF6 alternative splicing. In cultured cells, Ras signaling increases the expression of KLF6 SV1, relative to full-length KLF6, thereby enhancing proliferation. Abrogation of oncogenic Ras signaling by small interfering RNA (siRNA) or a farnesyl-transferase inhibitor decreases KLF6 SV1 and suppresses growth. Growth inhibition by farnesyl-transferase inhibitor in transformed cell lines is overcome by ectopic expression of KLF6 SV1. Down-regulation of the splice factor ASF/SF2 by siRNA increases KLF6 SV1 messenger RNA levels. KLF6 alternative splicing is not coupled to its transcriptional regulation. Our findings expand the role of Ras in human HCC by identifying a novel mechanism of tumor-suppressor inactivation through increased alternative splicing mediated by an oncogenic signaling cascade.

QS285. GLUT 1 and Hexokinase 2 Induction by Oncogenic Ras Requires Both the Phosphoinositide 3-Kinase and Map Kinase Pathways

Increased glucose metabolism is one of the most characteristic phenotypes of malignancy. Rat Intestinal Epithelial (RIE-1) cells that contain an isopropyl-1-thio-β-D-galactopyranoside (IPTG) inducible expression system for H-RasVal-12 was utilized to evaluate phenotypic changes associated with oncogenic Ras expression and cellular transformation. We have demonstrated that oncogenic H-Ras expression increases the committed steps of glucose metabolism, namely glucose uptake and phosphorylation in these cells.

Proapoptotic ability of oncogenic H-Ras to facilitate apoptosis induced by histone deacetylase inhibitors in human cancer cells

More than 35% of human urinary bladder cancers involve oncogenic H-Ras activation. In addition to tumorigenic ability, oncogenic H-Ras possesses a novel proapoptotic ability to facilitate the induction of apoptosis by histone deacetylase inhibitors (HDACI). HDACIs are a new class of anticancer agents and are highly cytotoxic to transformed cells. To understand the connection between the selectivity of HDACIs on transformed cells and the proapoptotic ability of oncogenic H-Ras to facilitate HDACI-induced apoptosis, we introduced oncogenic H-Ras into urinary bladder J82 cancer cells to mimic an acquisition of the H-ras gene activation in tumor development. Expression of oncogenic H-Ras promoted J82 cells to acquire tumorigenic ability. Meanwhile, oncogenic H-Ras increased susceptibility of J82 cells to HDACIs, including FR901228 and trichostatin A, for inducing apoptosis. The caspase pathways, the B-Raf and extracellular signal-regulated kinase pathway, p21(Cip1) and p27(Kip1), and core histone contents are regulated differently by FR901228 in oncogenic H-Ras-expressed J82 cells than their counterparts in parental J82 cells, contributing to the increased susceptibility to the induction of selective apoptosis. Our results lead us to a suggestion that HDACIs activate the proapoptotic ability of oncogenic H-Ras, indicating a potential therapeutic value of this new class of anticancer agents in the control of human urinary bladder cancer that has progressed to acquire oncogenic H-Ras.