Ha-ras Oncogene Research Articles

C/EBP\u03b2 deletion in oncogenic Ras skin tumors is a synthetic lethal event

Therapeutic targeting of specific genetic changes in cancer has proven to be an effective therapy and the concept of synthetic lethality has emerged. CCAAT/enhancer-binding protein-β (C/EBPβ), a basic leucine zipper transcription factor, has important roles in cellular processes including differentiation, inflammation, survival, and energy metabolism. Using a genetically engineered mouse model, we report that the deletion C/EBPβ in pre-existing oncogenic Ha-Ras mouse skin tumors in vivo resulted in rapid tumor regression. Regressing tumors exhibited elevated levels of apoptosis and p53 protein/activity, while adjacent C/EBPβ-deleted skin did not. These results indicate that the deletion of C/EBPβ de-represses p53 in oncogenic Ras tumors but not in normal wild-type Ras keratinocytes, and that C/EBPβ is essential for survival of oncogenic Ras tumors. Co-deletion of C/EBPβ and p53 in oncogenic Ras tumors showed p53 is required for tumor regression and elevated apoptosis. In tumors, loss of a pathway that confers adaptability to a stress phenotype of cancer/tumorigenesis, such as DNA damage, could result in selective tumor cell killing. Our results show that oncogenic Ras tumors display a significant DNA damage/replicative stress phenotype and these tumors have acquired a dependence on C/EBPβ for their survival. RNAseq data analysis of regressing tumors deleted of C/EBPβ indicates a novel interface between p53, type-1 interferon response, and death receptor pathways, which function in concert to produce activation of extrinsic apoptosis pathways. In summary, the deletion of C/EBPβ in oncogenic Ras skin tumors is a synthetic lethal event, making it a promising target for future potential anticancer therapies.

Restrained Terminal Differentiation and Sustained Stemness in Neonatal Skin by Ha-Ras and Bcl-2.

Nonmelanoma skin cancer is the most frequently diagnosed cancer in the United States. Deregulation of bcl-2 and ras family members is commonly observed in nonmelanoma skin cancer. It has been previously demonstrated that simultaneous bcl-2 and Ha-ras gene expression in keratinocytes results in resistance to cell death induced by ultraviolet radiation and enhanced multistep skin carcinogenesis. In this study, we aimed to elucidate the central roles of Ha-Ras and Bcl-2 in maintaining epidermal homeostasis. To assess the effect of deregulated Ha-Ras and Bcl-2 on skin differentiation, we have generated skin-specific transgenic mouse model constitutively expressing both oncogenic Ha-Ras and Bcl-2. Ectopic expression of Ha-Ras and Bcl-2 in newborn double transgenic epidermal keratinocytes induced abnormal epidermal differentiation accompanied by increased cell proliferation and suppressed apoptotic cell death, which resulted in thickened and wrinkled skin morphology in neonate skins. Expression of epidermal differentiation marker cytokeratin 1 was decreased. Expression of other differentiation markers loricrin and filaggrin was also decreased and delayed to be detected only in the upper stratum granulosum, whereas the proliferative markers cytokeratin 14 and cytokeratin 6, which are expressed in constitutively proliferative basal layer and stem cell niches such as hair follicles or neoplastic lesions, respectively, were highly expressed. The abnormal expression of epidermal cytokeratins suggests that Ha-Ras and Bcl-2 suppress the terminal differentiation and sustain the stem cell-like features in epidermal keratinocytes.

PATZ1 is a target of miR-29b that is induced by Ha-Ras oncogene in rat thyroid cells.

The regulatory transcriptional factor PATZ1 is constantly downregulated in human thyroid cancer where it acts as a tumour suppressor by targeting p53-dependent genes involved in Epithelial-Mesenchymal Transition and cell migration. The aim of the present work was to elucidate the upstream signalling mechanisms regulating PATZ1 expression in thyroid cancer cells. The bioinformatics search for microRNAs able to potentially target PATZ1 led to the identification of several miRNAs. Among them we focused on the miR-29b since it was found upregulated in rat thyroid differentiated cells transformed by the Ha-Ras oncogene towards a high proliferating and high migratory phenotype resembling that of anaplastic carcinomas. Functional assays confirmed PATZ1 as a target of miR-29b, and, consistently, an inverse correlation between miR-29b and PATZ1 protein levels was found upon induction of Ha-Ras oncogene expression in these cells. Interestingly, restoration of PATZ1 expression in rat thyroid cells stably expressing the Ha-Ras oncogene decreased cell proliferation and migration, indicating a key role of PATZ1 in Ras-driven thyroid transformation. Together, these results suggest a novel mechanism regulating PATZ1 expression based on the upregulation of miR-29b expression induced by Ras oncogene.

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity.

BackgroundMutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.MethodsSuppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-rasval12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7–4) harboring the inducible Ha-rasval12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.ResultsRetinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-rasval12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells “7-4” by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.ConclusionsWe confirmed that RbAp46 is a Ha-rasval12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1155-7) contains supplementary material, which is available to authorized users.

Increase in incidence of hepatic tumors caused by oncogenic somatic mutation in mice maternally exposed to inorganic arsenic and the multigenerational and transgenerational effects of inorganic arsenic.

Inorganic arsenic is a natural environmental contaminant and known to be a human carcinogen. Although rodent models are pivotal in elucidating the mode of action of arsenic, it has been difficult to verify the carcinogenicity of arsenic in rodents until recently. Waalkes et al. (Toxicol Appl Pharmacol 2003; 186:7-17) reported that maternal exposure to arsenite increases the incidence of hepatic tumors in the male pups of C3H mice in adulthood. This finding indicated that the gestational period is vulnerable to arsenic. Using the same experimental model, we found that maternal arsenite exposure increases the incidence of hepatic tumors caused by a somatic mutation of the C61A Ha-ras gene, which encodes an activated oncogenic Ha-ras protein. The G:C to T:A transversion is attributable to oxidative stress. Our further studies of gpt delta transgenic mice, which enable detection of in vivo mutation, and genome-wide analysis of DNA methylation levels using the methylated DNA immunoprecipitation-CpG island microarray method suggest that oxidative-stress-induced mutation and DNA methylation changes are involved in the tumor augmentation in the pups maternally exposed to arsenic. Our recent study has also suggested that maternal arsenic exposure increases the incidence of hepatic tumors even in the grandchildren (the F2 generation). Consideration should be given to multigenerational and transgenerational effects of maternal exposure in future studies.

Universal intracellular transducer ras and its role in the development of drosophila

Ras genes were first identified in the 1960s as transforming oncogenes that caused tumors in rats infected with Harvey and Kirsten sarcoma viruses (Ha-ras and Ki-ras oncogenes, accordingly). Subsequently, transforming ras genes were found in human cancer cells. Further investigations of neuroblastoma cells resulted in the finding of the third ras gene in the human, which was called N-ras. Ras gene products play an important role in the processes of cellular proliferation and differentiation and are controlled by receptor tyrosine kinases. Using drosophila as a model object allowed us to perform a successful genetic analysis while studying the functions of ras genes. Three polytene chromosome bands were detected in D. melanogaster with the help of the v-Ha ras sampling. According to Bridges' map, all three bands (Dras1, Dras2, Dras3) were mapped to regions 85D, 64B, and 62B of chromosome 3. Among them, only Drasl has a common origin with ras genes of mammals. Although there are numerous investigations of the role played by ras genes in the de- velopment of insects, this problem is still not fully understood. The importance of ras gene variations in the course of the evolutionary process has been insufficiently studied as well. Currently, Ras target proteins are actively identified, their signal pathways, as well as effects of influencing these pathways in the drosophila tissues, are studied in the cells of yeast and mammals. The main functions of Ras protein is in the signaling pathways controlling mutations during drosophila's morphogenesis and the connections of ras gene with phenotypic symptoms of tumors.

Abstract 320: New metastatic murine prostate cancer cell lines and preclinical model of human prostate cancer.

Abstract Defining oncogene-specific molecular pathways required for the induction and maintenance of prostate cancer is essential for the rational design of new therapies. Identifying molecular predictors of prostate cancer patient survival is important for patient therapeutic substratification. We generated murine prostate cancer cell lines via selective transduction with a single oncogene (c-Myc, Ha-Ras, NeuT, and v-Src). These cell lines demonstrate contact-independent growth characteristics in soft agar, and have the substantial growth advantage over primary prostate epithelial cells. Each of the prostate lines grew tumor in immune competent mice after subcutaneous injection, and develop lung metastasis in c-Myc, Ha-Ras, and v-Src cell line groups. RNA samples were prepared from the these cell lines, Microarray analysis identified a total of 2635 out of 22115 genes that were significantly altered in expression (at least two-fold change) in oncogene over-expressing cell lines when compared with prostate epithelial cell control samples. The distinct oncogene expression signature was compared to gene signatures obtained from other published databases to identify similarities to human prostate cancer phenotypes. Comparisons were performed against gene signatures representative of differential expression in advanced state vs. early stage prostate cancer, high grade vs low grade prostate cancer, recurrent vs. nonrecurrent, the results indicate a significant degree of similarity between the prostate oncogene expression signature and high-grade disease; between the oncogene expression signature and the advanced stage disease phenotype. Interrogation of two distinct cohorts of Prostate cancer patient samples using the oncogene signature demonstrated an ability to distinguish tumor from normal prostate with a predictive value for prostate cancer of 98 - 99%. The signature provided in blinded study independent substratification of reduced recurrence free survival by Kaplan-Meier analysis. The generation of new oncogene-specific prostate cancer cell lines that recapitulate human prostate cancer gene expression, that metastasize in immune-competent mice, are a valuable new resource for testing targeted therapy. The molecular signatures identified herein provide further value over current markers of prediction and outcome. Citation Format: Xiaoming Ju, Richard G. Pestell. New metastatic murine prostate cancer cell lines and preclinical model of human prostate cancer. [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 320. doi:10.1158/1538-7445.AM2013-320

Ras-induced Up-regulation of CTP:Phosphocholine Cytidylyltransferase α Contributes to Malignant Transformation of Intestinal Epithelial Cells

Cancer cells have enhanced lipogenic capacity characterized by increased synthesis of fatty acids and complex lipids, including phosphatidylcholine (PC). As the rate-limiting enzyme in the CDP-choline pathway for PC synthesis, CTP:phosphocholine cytidylyltransferase α (CCTα) is implicated in the provision of membranes and bioactive lipids necessary of cell proliferation. In this study, we assessed the role of CCTα in malignant intestinal epithelial cells transformed with activated H-ras (IEC-ras). Three IEC-ras clones had significant up-regulation CCTα expression, but PC synthesis and in vitro activity of CCTα were similar to control IEC. RNA interference of CCTα in adherent IEC-ras did not affect PC synthesis, confirming that the enzyme was relatively inactive. However, CCTα silencing in ras-transformed IEC reduced anchorage-independent growth, a criterion for malignant transformation, as well as tumorigenicity in mice. Relative to their adherent counterparts, detached IEC-ras had increased PC synthesis that was attenuated by inducible CCTα silencing. Detachment of IEC-ras was accompanied by increased CCTα phosphorylation and cytosolic enzyme activity. We conclude that the expanded pool of CCTα in IEC-ras is activated by detachment. This provides the increased PC biosynthetic capacity that contributes to malignant transformation of intestinal epithelial cells when detached from the extracellular matrix.

Characterization of a Novel Activated Ran GTPase Mutant and Its Ability to Induce Cellular Transformation

Ran (Ras-related nuclear) protein, a member of the Ras superfamily of GTPases, is best known for its roles in nucleocytoplasmic transport, mitotic spindle fiber assembly, and nuclear envelope formation. Recently, we have shown that the overexpression of Ran in fibroblasts induces cellular transformation and tumor formation in mice (Ly, T. K., Wang, J., Pereira, R., Rojas, K. S., Peng, X., Feng, Q., Cerione, R. A., and Wilson, K. F. (2010) J. Biol. Chem. 285, 5815-5826). Here, we describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Furthermore, Ran(K152A) expression in the human mammary SKBR3 adenocarcinoma cell line gives rise to an enhanced transformed phenotype and causes a robust stimulation of both ERK and the N-terminal c-Jun kinase (JNK). Microarray analysis reveals that the expression of the gene encoding SMOC-2 (secreted modular calcium-binding protein-2), which has been shown to synergize with different growth factors, is increased by at least 50-fold in cells stably expressing Ran(K152A) compared with cells expressing control vector. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth in NIH-3T3 cells and in SKBR3 cells and also inhibits cell invasion in fibroblasts. Collectively, our findings highlight a novel connection between the hyper-activation of the small GTPase Ran and the matricellular protein SMOC-2 that has important consequences for oncogenic transformation.

Abstract A21: DACH1 blocks prostate cancer cell growth and interleukin-6 signaling

Abstract In Drosophila, eye development is coordinately regulated by the retinal determination gene network (RDGN), mainly consisting of dachshund (dac), eyes absent (eya), and sin oculis (so). The mammalian homologues are Dach, Eya, and Six, respectively. RDGN, although best known for its role in eye specification, is essential for the development of many organs including retinal, kidney, brain, and limb in both flies and mammals. Recently, coordinate abnormal expression of the DACH1/Eya/Six complex has been observed in human tumors supporting a tumor promoting role of Six and Eya family members, and a tumor-suppressor function of DACH. Oncogenes sustain host-cell interactions, promoting invasiveness, recruiting inflammatory mediators and developing angiogenic networks. Ras and Myc promote tumor angiogenesis through up-regulation of VEGF and the inflammatory mediator, interleukin-8(IL-8) and interleukin-6 (IL-6). Our previous studies demonstrate DACH1 inhibits migration of MCF10A cells transformed with distinct oncogenes (Ha-Ras, c-Raf, c-Myc, and ErbB2) as well as the highly metastatic MDA-MB-231 human breast cancer cell line. An unbiased proteomic approach to identify candidate proteins regulated by DACH1 expression, using antibody array analysis, demonstrated DACH1 expression correlated with reduced secretion of IL-8 and the related chemokines IL-1, IL-6, GRO, and MIP1 (α, β, γ) from Ras or c-Myc transformed MCF-10A cells and repressed IL-8 promoter activity. Altered expressions of the IL-6 and IL-8 genes are seen in many cancers including prostate cancer. Hormone-resistant prostate carcinoma cell lines produce large amounts of IL-6 and IL-8 compared to hormone responsive prostate cancer cell lines. The expression of IL-8 and IL-6 are linked to poor prognosis and metastatic phenotype in prostate cancer. Recent finding demonstrated that IL-6 regulates the dynamic equilibrium between cancer stem cells and non-stem cancer cells in human breast and prostate cancer. IL-6 alone can transform non-tumorigenic benign prostate epithelial cells and further progress to invasive phenotype. To explore the role of DACH1 in androgen resistant prostate cancer, PC3 cells stably expressing DACH1 were established. DACH1 inhibited proliferation evaluated by MTT assay and growth curve. Microarray analyses demonstrated DACH1 inhibited mRNA expression of GRO, IL-6, IL-8 by 75%, 80% and 90%, respectively. However, there was no change of mRNA expression for IL-6 and IL-8 receptor. It has been reported that stable expression IL-6 in SV40T immortalized non-tumorigenic prostate epithelial cell line p69 induced EMT and acquired malignant phenotypes through autocrine loop IL6 and IGF-1R signaling. To further test the functional interaction of DACH1 with IL-6, a table cells expressing IL-6 alone or IL-6 with DACH1 were deployed. Ectopic expression of DACH1 attenuated activation of IGF-1 R by IL-6 as evaluated by phosphorylation of IGF-1R. Subcutaneous implantation study showed a 60% inhibition of in vivo tumor growth by DACH1. Together, our studies suggest that loss expression of DACH1 is an important cause of prostate tumorigenesis and restoration of DACH1 expression provides a potential therapeutic application. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A21.

Effect ofUncariaandTabebuiaextracts on molecular epidemiological biomarkers in patients with colorectal cancer

The combined effect of surgical treatment and consumption of so-called “CoD™ tea” (containing Uncaria guianensis, U. tomentosa and Tabebuia avellanedae) on expression of c-myc, Ha-ras, Bcl-2, Ki-ras and p53 key onco/suppressor genes, the carbohydrate antigen (CA19-9) and carcino-embryonic antigen (CEA) tumour markers in blood samples of patients with colorectal cancer (CRC) were investigated. Expression of genes followed the effect of the surgical treatment combined with neoadjuvant chemotherapeutic treatment; this may predict the outcome of carcinoma. Moreover their expressions might show possible additional effect of supportive therapy, e.g. CoD™ consumption. The antioxidant capacity of blood was also examined. Blood samples were taken at the day of, and one week, 3, 6 and 12 months after the surgical treatment. During that period patients got 0.25 l standard portion of CoD™ tea three times a day. The surgical treatment and neoadjuvant therapy were able to suppress the expression of c-myc, Ha-ras, Bcl-2, Ki-ras, p53 genes up to the twelfth month. Moreover, CoD™ tea together with conventional treatment caused a strong decrease in the expression of c-myc and Ha-ras oncogenes in comparison to the non-consumer control.

Different metastasis promotive potency of small G-proteins RalA and RalB in in vivo hamster tumor model

BackgroundPreviously we have shown that oncogenic Ha-Ras stimulated in vivo metastasis through RalGEF-Ral signaling. RalA and RalB are highly homologous small G proteins belonging to Ras superfamily. They can be activated by Ras-RalGEF signaling pathway and influence cellular growth and survival, motility, vesicular transport and tumor progression in humans and in animal models. Here we first time compared the influence of RalA and RalB on tumorigenic, invasive and metastatic properties of RSV transformed hamster fibroblasts.MethodsRetroviral vectors encoding activated forms or effector mutants of RalA or RalB proteins were introduced into the low metastatic HET-SR cell line. Tumor growth and spontaneous metastatic activity (SMA) were evaluated on immunocompetent hamsters after subcutaneous injection of cells. The biological properties of cells, including proliferation, clonogenicity, migration and invasion were determined using MTT, wound healing, colony formation and Boyden chamber assays respectively. Protein expression and phosphorylation was detected by Westen blot analysis. Extracellular proteinases activity was assessed by substrate-specific zymography.ResultsWe have showed that although both Ral proteins stimulated SMA, RalB was more effective in metastasis stimulation in vivo as well as in potentiating of directed movement and invasion in vitro. Simultaneous expression of active RalA and RalB didn't give synergetic effect on metastasis formation. RalB activity decreased expression of Caveolin-1, while active RalA stimulated MMP-1 and uPA proteolytic activity, as well as CD24 expression. Both Ral proteins were capable of Cyclin D1 upregulation, JNK1 kinase activation, and stimulation of colony growth and motility. Among three main RalB effectors (RalBP1, exocyst complex and PLD1), PLD1 was essential for RalB-dependent metastasis stimulation.ConclusionsPresented results are the first data on direct comparison of RalA and RalB impact as well as of RalA/RalB simultaneous expression influence on in vivo cell metastatic activity. We showed that RalB activation significantly more than RalA stimulates SMA. This property correlates with the ability of RalB to stimulate in vitro invasion and serum directed cell movement. We also found that RalB-PLD1 interaction is necessary for the acquisition of RalB-dependent high metastatic cell phenotype. These findings contribute to the identification of molecular mechanisms of metastasis and tumor progression.

Changes in expression of oncogenes and TP53 tumour suppressor gene as biomarkers in head and neck cancers

Despite modern diagnostic procedures and up-to-date therapy, the survival of head and neck tumour patients is unfavourable. This can be explained by several factors, one of which is the late recognition of the tumour. This study related to the changes in expression of the c-myc and Ha-ras oncogenes and the p53 tumour suppressor gene as biomarkers in head and neck cancer cases. The gene expressions were investigated on RNA gained from peripheral white blood cells of head and neck cancers patients before and after definitive treatment. The results were compared with those on a control group of patients with non-tumorous diseases. The gene expressions were significantly higher in the cancer group than that in the control group (volunteer medical staff and medical students). After definitive treatment, the expressions of all these genes were decreased in patients in whom there was no recurrence of the tumour, but enhanced in the event of recurrence. Such measurement may serve as reliable biomarkers to monitor tumour development and the efficiency of therapy. The method may also be useful for the early identification of populations exposed to noxe, which may lead to the development of head and neck cancers.

Abstract 129: Synthetic lethality induced by loss of PKC delta and mutated Ras

Abstract Table of ContentsSynthetic Lethality Induced by Loss of PKC d and Mutated RasAbstract Synthetic Lethality Induced by Loss of PKC δ and Mutated Ras Tongbo Zhu,* Lihua Chen,* Wei Du, Takanori Tsuji, and Changyan Chen Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston Abstract Synthetic lethal interaction between oncogenic Ha-ras and loss of PKC has been demonstrated. Recently, we reported that the concurrent knockdown of PKC α and β, via upregulating PKC δ, sensitizes cells with aberrant Ras signaling to apoptosis. As the continuation of the study, using the shRNA, we demonstrate that loss of PKC δ causes a lethal reaction in NIH3T3/Hras or prostate cancer DU145 cells that overexpress JNK. In this apoptotic process, PKC α and β are upregulated, and then associated with RACK1 (an adaptor for activated PKC) and JNK. Immunoblotting analysis shows that JNK is phosphorylated, accompanied with caspase 8 cleavage. The inhibition of JNK abrogates this apoptotic process triggered by PKC δ knockdown. Interestingly, without blocking PKC δ, the concurrent overexpression of wt- or CAT-PKCα and β is insufficient to induce apoptosis in the cells. Together with our previous findings, the data suggest that PKC α/β and δ function oppositely to maintain a balance that supports cells expressing v-ras to survive and prevents them from being eliminated through oncogenic stress-induced apoptosis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 129.

Synthetic Lethality Induced by Loss of PKC and Mutated Ras

Synthetic lethal interaction between oncogenic Ha-ras and loss of PKC has been demonstrated. Recently, the authors reported that the concurrent knockdown of PKC α and β, via upregulating PKC δ, sensitizes cells with aberrant Ras signaling to apoptosis. As a continuation of the study, using shRNA, the authors demonstrate that loss of PKC δ causes a lethal reaction in NIH3T3/Hras or prostate cancer DU145 cells that overexpress JNK. In this apoptotic process, PKC α and β are upregulated and then associated with RACK1 (an adaptor for activated PKC) and JNK. Immunoblotting analysis shows that JNK is phosphorylated, accompanied with caspase 8 cleavage. The inhibition of JNK abrogates this apoptotic process triggered by PKC δ knockdown. Interestingly, without blocking PKC δ, the concurrent overexpression of wt- or CAT-PKC α and β is insufficient to induce apoptosis in the cells. Together with the authors' previous findings, the data suggest that PKC α/β and δ function oppositely to maintain a balance that supports cells expressing v-ras to survive and prevents them from being eliminated through oncogenic stress-induced apoptosis.

Abstract B61: Ha-RAS mutations in fibroblast of the cervical stroma of squamous neoplasias of the uterine cervix

Abstract The stroma is maintained, renovated, and repaired by resident fibroblasts, supports and instructs the epithelium, and is essential for epithelial function. Now recognized that stromal changes are necessary for the establishment of cancer and previously considered as a spectator in front of the clonal expansion and acquisition of malignant characteristics of tumor cells. Most studies on the stroma and t its relationship with the generation, progression and metastasis of cancer in which involve multiple steps of tumorigenesis and the microenvironment, have focused especially on cancers such as breast and others as cancer of prostate, gastric, colorectal and cervical cancer. Reports of cervical cancer specifically address issues such as clonality and genomic features in cervical carcinogenesis. However, the presence of mutations Ha-RAS oncogen in stromal cells and their contribution to the generation and progression of cervical squamous neoplasia has not yet been reported. The aim of this study was detect and determine the frequency of mutations Ha-RAS in stromal fibroblasts from biopsies of the transformation zone of the uterine cervix. We selected eight samples of fresh frozen tissue of patients diagnosed with cervical intraepithelial neoplasia squamous (CIN I, CIN II, CIN III), cancer in situ and invasive squamous cell carcinoma, tissue in which it was represented only the stroma, without evidence of residual tumoral epithelial component, and each of whom had cervical tissue in paraffin-embedded control confirming the presence of cervical squamous neoplasia. The detection of Ha-ras mutations in codon 12 was performed using a nonradioactive PCR-SSCP and restriction enzyme analysis. The mutation found for a substitution of a glycine by a valine at codon 12 of Ha-RAS was detected in alone sample (12.5%), CIN III. The non-detection of the GTC mutation in the remaining seven samples could be due to somatic mutation that this represented only a small fraction (approximately 15%) of total DNA analyzed (33), as well as a topographical heterogeneity likely distribution of these mutations. The presence of a point mutation of Ha-RAS oncogene in the stroma of one case studied, represents an important change, this mutation prevents the conversion of active to inactive form, and suggest an alternative pathway involving stromal genetic instability in the generation of intraepithelial neoplasia and progression of the cervical cancer. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B61.

Mutaciones Ha-RAS en fibroblastos del estroma cervical de neoplasias escamosas de cuello uterino

ObjectiveTo detect Ha-Ras oncogene mutations in stromal fibroblasts in biopsies in the cervical transformation zone. MethodsEight samples were selected of fresh frozen tissue taken from patients diagnosed with intraepithelial neoplasia of the cervix, squamous type (NIC I, NIC II, NIC III), cancer in situ, and invasive squamous cell carcinoma, tissue in which only stroma was represented, with no evidence of residual tumor epithelial component; from each, the control cervical paraffin fixed tissue was counted which confirmed the presence of squamous cervical neoplastic process. The detection of codon 12 Ha-RAS mutations was carried out with non radioactive PCR-SSCP and restriction enzyme analysis. ResultsThe revealed mutation that altered a glycine into a valine in the codon 12 of the Ha-RAS oncogene was detected in only one sample (12.5%), NIC III. ConclusionsThe presence of molecular alteration, punctual mutation of the Ha-RAS oncogene in the stroma of one of the cases analyzed represents an important change and suggests an alternate route that could involve stromal genetic stability in the generation of intraepithelial neoplasia and in the progression of cervical cancer.

Biology of urothelial tumorigenesis: insights from genetically engineered mice.

Urothelium, one of the slowest cycling epithelia in the body, embodies a unique biological context for cellular transformation. Introduction of oncogenes into or removing tumor suppressor genes from the urothelial cells or a combination of both using the transgenic and/or knockout mouse approaches has provided useful insights into the molecular mechanisms of urothelial transformation and tumorigenesis. It is becoming increasingly clear that over-activation of the receptor tyrosine kinase (RTK) pathway, as exemplified by the constitutively activated Ha-ras oncogene, is both necessary and sufficient to initiate the low-grade, non-invasive urothelial carcinomas. Dosage of the mutated Ha-ras, but not concurrent inactivation of pro-senescence molecules p16Ink4a and p19Arf, dictates whether and when the low-grade urothelial carcinomas arise. Inactivation of both p53 and pRb, a prevailing paradigm previously proposed for muscle-invasive urothelial tumorigenesis, is found to be necessary but insufficient to initiate this urothelial carcinoma variant. Instead, downregulation in p53/pRb co-deficient urothelial cells of p107, a pRb family member, is associated with the genesis of the muscle-invasive bladder cancers. p53 deficiency also seems to be capable of cooperating with that of PTEN in eliciting invasive urothelial carcinomas. The genetically engineered mice have improved the molecular definition of the divergent pathways of urothelial tumorigenesis and progression, helped delineate the intricate crosstalk among different genetic alterations within a urothelium-specific context, identified new prognostic markers and novel therapeutic targets potentially applicable for clinical intervention, and provided in vivo platforms for testing preventive strategies of bladder cancer.

The Multifaceted Role of MTDH/AEG-1 in Cancer Progression

Cancer is the result of the progressive acquisition of multiple malignant traits through the accumulation of genetic or epigenetic alterations. Recent studies have established a functional role of MTDH (Metadherin)/AEG-1 (Astrocyte Elevated Gene 1) in several crucial aspects of tumor progression, including transformation, evasion of apoptosis, invasion, metastasis, and chemoresistance. Overexpression of MTDH/AEG-1 is frequently observed in melanoma, glioma, neuroblastoma, and carcinomas of breast, prostate, liver, and esophagus and is correlated with poor clinical outcomes. MTDH/AEG-1 functions as a downstream mediator of the transforming activity of oncogenic Ha-Ras and c-Myc. Furthermore, MTDH/AEG-1 overexpression activates the PI3K/Akt, nuclear factor kappaB (NFkappaB), and Wnt/beta-catenin signaling pathways to stimulate proliferation, invasion, cell survival, and chemoresistance. The lung-homing domain of MTDH/AEG-1 also mediates the adhesion of tumor cells to the vasculature of distant organs and promotes metastasis. These findings suggest that therapeutic targeting of MTDH/AEG-1 may simultaneously suppress tumor growth, block metastasis, and enhance the efficacy of chemotherapeutic treatments.

Nuclear LYRIC/AEG-1 interacts with PLZF and relieves PLZF-mediated repression

LYRIC/AEG-1 and its altered expression have been linked to carcinogenesis in prostate, brain and melanoma as well as promoting chemoresistance and metastasis in breast cancer. LYRIC/AEG-1 function remains unclear, although LYRIC/AEG-1 is activated by oncogenic HA-RAS, through binding of c-myc to its promoter, which in turn regulates the key components of the PI3-kinase and nuclear factor-kappaB pathways. We have identified the transcriptional repressor PLZF as an interacting protein of LYRIC/AEG through a yeast two-hybrid screen. PLZF regulates the expression of genes involved in cell growth and apoptosis including c-myc. Coexpression of LYRIC/AEG-1 with PLZF leads to a reduction in PLZF-mediated repression by reducing PLZF binding to promoters. We have confirmed that nuclear LYRIC/AEG-1 and PLZF interact in mammalian cells via the N- and C termini of LYRIC/AEG-1 and a region C terminal to the RD2 domain of PLZF. Both proteins colocalize to nuclear bodies containing histone deacetylases, which are known to promote PLZF-mediated repression. Our data suggest one mechanism for cells with altered LYRIC/AEG-1 expression to evade apoptosis and increase cell growth during tumourigenesis through the regulation of PLZF repression.