Transformation Of NIH 3T3 Cells Research Articles

Abstract C140: RIT1 mutations define a new genetic subset of lung adenocarcinoma.

Abstract Lung adenocarcinoma is comprised of distinct mutational subtypes characterized by mutually exclusive oncogenic mutations in RTK/RAS pathway members KRAS, EGFR, BRAF, and ERBB2, and translocations involving ALK, RET, and ROS1. Identification of these oncogenic events has transformed therapy of lung adenocarcinoma via application of therapies targeted towards specific genetic lesions in stratified patient populations. However, such mutations have been identified in only ∼55% of lung adenocarcinoma cases in the United States, suggesting other mechanisms of malignancy may be involved in the remaining cases. We analyzed exome sequencing data from 413 lung adenocarcinomas and identified somatic mutations in the small GTPase gene RIT1 in ∼2% of samples. Mutations in RIT1 cluster in a hotspot near the switch II domain of the protein that is homologous to the Q61 region in RAS genes. RIT1 switch II domain mutations are mutually exclusive with all other known lung adenocarcinoma driver mutations. Ectopic expression of mutated RIT1 induces activation of AKT and ERK signaling pathways and transforms NIH3T3 cells in vitro and in vivo. These data identify RIT1 as a driver oncogene in a specific subset of lung adenocarcinomas. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C140. Citation Format: Alice Berger, Marcin Imielinski, Fujiko Duke, Jeremiah Wala, Nathan Kaplan, Geng-Xian Shi, Douglas Andres, Matthew Meyerson. RIT1 mutations define a new genetic subset of lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C140.

Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling.

Erratum: Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells

Erratum: Combined effects of 60 Hz electromagnetic field exposure with various stress factors on cellular transformation in NIH3T3 cells

Mutation of the Salt Bridge-forming Residues in the ETV6-SAM Domain Interface Blocks ETV6-NTRK3-induced Cellular Transformation

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

Expression of DJ-1 in Endometrial Cancer

DJ-1 was originally cloned as a putative oncogene capable of transforming NIH3T3 cells in cooperation with H-Ras or c-Myc, which has been implicated in the pathogenesis of some solid tumors. The aim of this study was to investigate the expression and clinical significance of DJ-1 in endometrial cancer and study its effect on cell proliferation and apoptosis in endometrial cancer Ishikawa cells. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the DJ-1 expression in 100 surgical specimens of endometrial cancer tissues, paired tumor-adjacent tissues, and 30 surgical specimens of normal endometrium tissues. The proliferation variety of endometrial cancer Ishikawa cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay after transfecting the interference plasmid pGPU6/GFP/neo-DJ-1-shRNA into Ishikawa cells. Real-time polymerase chain reaction and Western blotting were used to evaluate the effect of interference plasmid on target gene expression. Apoptosis rate was determined by flow cytometry. DJ-1 expression in endometrial cancer tissues was higher than in tumor-adjacent tissues and normal endometrial tissues. At the same time, it was associated with signs of cancer progression, including differentiation, myometrial invasion depth, and presence of lymph node metastasis. Knocking down DJ-1 promoted the apoptosis of Ishikawa cells. High DJ-1 expression seems to be negatively correlated with apoptosis. Meanwhile, it may be part of the mechanisms for the development, invasion, and metastasis in endometrial cancer.

Drinking Water Disinfection Byproduct Iodoacetic Acid Induces Tumorigenic Transformation of NIH3T3 Cells

Iodoacetic acid (IAA) and iodoform (IF) are unregulated iodinated disinfection byproducts (DBPs) found in drinking water. Their presence in the drinking water of China has not been documented. Recently, the carcinogenic potential of IAA and IF has been a concern because of their mutagenicity in bacteria and genotoxicity in mammalian cells. Therefore, we measured their concentrations in Shanghai drinking water and assessed their cytotoxicity, genotoxicity, and ability to transform NIH3T3 cells to tumorigenic lines. The concentrations of IAA and IF in Shanghai drinking water varied between summer and winter with maximum winter levels of 2.18 μg/L IAA and 0.86 μg/L IF. IAA with a lethal concentration 50 (LC50) of 2.77 μM exhibited more potent cytotoxicity in NIH3T3 cells than IF (LC50 = 83.37 μM). IAA, but not IF, induced a concentration-dependent DNA damage measured by γ-H2AX staining and increased tail moment in single-cell gel electrophoresis. Neither IAA nor IF increased micronucleus frequency. Prolonged exposure of NIH3T3 cells to IAA increased the frequencies of transformed cells with anchorage-independent growth and agglutination with concanavalin A. IAA-transformed cells formed aggressive fibrosarcomas after inoculation into Balb/c nude mice. This study demonstrated that IAA has a biological activity that is consistent with a carcinogen and human exposure should be of concern.

In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.

Abstract 543: A loss of function Cbl-c mutation in a murine breast cancer model.

Abstract Receptor Tyrosine Kinase (RTK) signaling is essential for normal biological processes and inappropriate RTK activity can result in malignant transformation. Cbl proteins are a family of RING finger ubiquitin ligases that negatively regulate a variety of RTKs, including EGFR, MET, RET and components of the TCR pathway and disruption of this negative regulation can lead to tumor initiation and progression. Recent studies have identified a growing number of Cbl mutations associated with human myeloid neoplasias and epithelial tumors. Cbl-c is the most recently identified human Cbl protein and is expressed exclusively in epithelial cells. Recently, a novel Cbl-c cDNA has been isolated from a ductal carcinoma which developed in a C3(1)SV40Tag mouse, a murine model of breast cancer driven by Large T antigen. This mutant cDNA encodes a Cbl-c protein which lacks the catalytic domain of Cbl-c, thereby resembling known Cbl family mutations associated with myeoloid neoplasias. Genomic analysis of both parental and transgenic lines shows no evidence of germline mutation indicating that this mutation is likely somatic and may contribute to malignant transformation in this animal model. In overexpression studies, this mutant Cbl-c protein fails to mediate ubiquitination and degradation of activated EGFR and acts as a dominant negative protein, preventing ubiquitination of the EGFR by wild type Cbl proteins. The mutant Cbl-c protein also enhances transformation of NIH 3T3 cells by SV40 Large T antigen and through subsequent analysis of transforming capacity and signal pathway regulation, we are assessing interacting partners of Cbl-c and the potential role of this mutation in tumorigenesis. This mutant Cbl-c protein is the first functional evidence for the involvement of Cbl-c in transformation. Citation Format: Stephen C. Kales, Philip E. Ryan, Marion Nau, Jeffrey E. Green, Stanley Lipkowitz. A loss of function Cbl-c mutation in a murine breast cancer model. [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 543. doi:10.1158/1538-7445.AM2013-543

Abstract 5310: Activation of FGFR3 by genomic fusion in urothelial carcinoma of the bladder.

Abstract FGFR3 is frequently activated by mutation in urothelial carcinoma of the bladder. We demonstrate here a further method of activation, by gene fusion following chromosomal re-arrangement. Four fusions with transforming acid coiled-coil 3 (TACC3) resulting from 4p16.3 re-arrangement were identified. A fifth fusion following t(4;7) was with BAI1-associated protein 2-like 1 (BAIAP2L1), also known as IRTKS (Insulin Receptor Kinase Substrate) on chromosome 7. The FGFR3-TACC3 fusions involve four different genomic re-arrangements resulting in three different transcripts. The fusion proteins show constitutive phosphorylation, activation of the MAPK pathway and transform NIH-3T3 cells. All have lost the final exon of FGFR3, which includes the PLCγ1 binding site. Thus PLCγ1 pathway activation and known PLCγ1-induced effects in normal urothelial cells were not induced. The FGFR3-BAIAP2L1 fusion retains most of BAIAP2L1 including the IRSp53/MIM domain (IMD) that mediates actin binding and Rac interaction, while the FGFR3-TACC3 fusions all retain the TACC domain that mediates microtubule binding. As cells containing these fusion genes are highly sensitive to FGFR-selective agents, detection of fusion genes may be useful in selecting patients for targeted therapy. Citation Format: Sarah V. Williams, Carolyn D. Hurst, Margaret A. Knowles. Activation of FGFR3 by genomic fusion in urothelial carcinoma of the bladder. [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 5310. doi:10.1158/1538-7445.AM2013-5310

Abstract 2327: Oncogenic K-Ras and H-Ras differentially regulate cancer stem cell-like properties via repression of non-canonical Wnt signaling.

Abstract Cancer stem cells (CSCs) can cause cancer treatment failures and tumor recurrence due to their stem cell-like properties, such as the unlimited self-renewal, tumorigenicity, and chemo-resistance. Ras is the most common oncogene in human cancers, but its roles in stem cell-like properties have not been convincingly demonstrated. With a high degree of sequence homology as well as common sets of downstream effectors and upstream affecters, the three isoforms of Ras, N-, H- and K-Ras, have long been assumed to be functionally redundant. However, knockout of K-Ras, not N- or H-Ras, in mice leads to embryonic lethality, suggesting a non-redundant role of K-Ras in embryonic development. Herein, we report that oncogenic K-Ras, differentially from H-Ras, causes CSC-like properties in transformed mouse fibroblast and pancreatic cancer cells. When compared to the NIH3T3 cells with H-RasV12, the cells transformed with K-RasV12 demonstrated significantly enhanced sphere forming efficiency, elevated resistance toward cisplatin, and heightened sensitivity to the CSC inhibitor, salinomycin, while the RasGTPase, p-Erk and p-Akt activities are comparable. In the comparison with H-RasV12 transformed cells, K-RasV12 transformed NIH3T3 cells possessed significantly increased tumor initiating frequency in limited cell transplantation and in vivo serial transplantation assays. Through stem cell signaling-related genes focusing PCRarray, we further identified Frizzled 8 (Fzd8), a Wnt receptor and potential activator of non-canonical Wnt/Ca2+ signaling, was significantly down-regulated in K-RasV12 transformed NIH3T3 cells when compared to normal cells or H-RasV12- transformed cells. In clinical human pancreatic tissues, Fzd8 was dramatically reduced in malignant specimens, whereas normal tissue showed high expressions of Fzd8. Knockdown of K-Ras in pancreatic cancer cells led to significant increases in Fzd8 at RNA and protein levels, suggesting oncogenic K-Ras repress the expression of Fzd8. TOPFlash assay revealed that K-RasV12 transformed NIH3T3 had dramatically enhanced canonical beta-catenin activity when compared to normal or H-RasV12- transformed cells. Human pancreatic cancer cells with K-Ras knocked down showed significantly reduced canonical beta-catenin activity. In a syngenic mouse model, over-expression of Fzd8 in oncogenic K-Ras driven mouse pancreatic cancer cells delayed the orthotropic tumor formation, and further decreased peritoneal metastatic spreads. Our data collectively suggest that oncogenic K-Ras regulates the stem cell-like properties of cancer cells, differentially from oncogenic H-Ras, through repression of Fzd8-mediated non-canonical Wnt/Ca2+ signaling. Restoration of Fzd8 suppressed oncogenic K-Ras induced pancreatic tumorigenesis, providing an alternative way to target this “undruggable” oncogene. Citation Format: Man-Tzu Wang, Jacqueline Galeas, Cayde Ritchie, Frank McCormick. Oncogenic K-Ras and H-Ras differentially regulate cancer stem cell-like properties via repression of non-canonical Wnt signaling. [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 2327. doi:10.1158/1538-7445.AM2013-2327

Oncogenic FGFR3 gene fusions in bladder cancer

FGF receptor 3 (FGFR3) is activated by mutation or over-expression in many bladder cancers. Here, we identify an additional mechanism of activation via chromosomal re-arrangement to generate constitutively activated fusion genes. FGFR3–transforming acid coiled coil 3 (TACC3) fusions resulting from 4p16.3 re-arrangements and a t(4;7) that generates a FGFR3-BAI1-associated protein 2-like 1 (BAIAP2L1) fusion were identified in 4 of 43 bladder tumour cell lines and 2 of 32 selected tissue samples including the tumour from which one of the cell lines was derived. These are highly activated and transform NIH-3T3 cells. The FGFR3 component is identical in all cases and lacks the final exon that includes the phospholipase C gamma 1 (PLCγ1) binding site. Expression of the fusions in immortalized normal human urothelial cells (NHUC) induced activation of the mitogen-activated protein kinase pathway but not PLCγ1. A protein with loss of the terminal region alone was not as highly activated as the fusion proteins, indicating that the fusion partners are essential. The TACC3 fusions retain the TACC domain that mediates microtubule binding and the BAIAP2L1 fusion retains the IRSp53/MIM domain (IMD) that mediates actin binding and Rac interaction. As urothelial cell lines with FGFR3 fusions are extremely sensitive to FGFR-selective agents, the presence of a fusion gene may aid in selection of patients for FGFR-targeted therapy.

Combinatorial gene targeting hTERT and BI-1 in CNE-2 nasopharyngeal carcinoma cell line

Nasopharyngeal carcinoma (NPC) is a common malignant tumor. In recent studies, we demonstrated that overexpression of the Bax inhibitor-1 (BI-1) induces cell transformation in NIH3T3 cells and that knockdown of BI-1 and human telomerase reverse transcriptase (hTERT) gene expression suppresses NPC cell proliferation and induces apoptosis. To evaluate the combination anti-tumor effects of siRNAs against hTERT and BI-1 in the CNE-2 NPC cell line, combined and separate short-hairpin (sh)RNA plasmids targeting hTERT and BI-1, respectively, were constructed. hTERT and BI-1 mRNA and protein levels were examined by real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation, colony formation and migration ability were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), soft agar and wound healing assay. Cell apoptosis was observed by flow cytometry, Hoechst 33258 staining and caspase-3 activity. hTERT, BI-1 and combined shRNA plasmids were injected into xenograft NPC tumor tissues, and expression of hTERT and BI-1 was detected by real-time PCR and immunohistochemistry. Tumor growth was measured by tumor volume and apoptosis in vivo was confirmed by TdT-mediated dUTP nick end-labeling (TUNEL). Our results showed that combined shRNA specific for hTERT and BI-1 markedly suppressed hTERT and BI-1 gene expression in vitro and in vivo. In addition, CNE-2 cell proliferation was inhibited in vitro as well as in vivo. Following the knockdown of the two gene expressions, CNE-2 exhibited a decrease in colony formation and migration ability and an increase in the apoptotic rate compared to the control groups. Our in vitro and in vivo study showed that the combinative silencing of the two genes enhanced the therapeutic effect compared to the silencing of each individual shRNA. These data suggested that combinatorial gene therapy targeting hTERT and BI-1 may be beneficial as a tumor therapy strategy against human NPCs.

Abstract B24: A p19Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and tumor suppression

Abstract Cellular senescence is an intrinsic tumor suppression mechanism that protects cells from neoplastic transformation by activated oncogenes. As such, strategies to enhance senescence could be a promising avenue for cancer prevention. The transcription factor (TF) CCAAT/enhancer binding protein β (C/EBPβ) is induced and post-translationally activated by oncogenic Ras signaling in primary mouse embryonic fibroblasts (MEFs) and cooperates with RB:E2F to implement RasV12-induced senescence. In contrast, immortalized cells lacking the tumor suppressor p19Arf, such as NIH 3T3 murine fibroblasts, bypass senescence and are transformed by RasV12. Our lab previously showed that C/EBPβ expression is down-regulated in association with RasV12 transformation of NIH 3T3 cells. Furthermore, p19Arf acts in a p53-independent manner to maintain C/EBPβ levels in RasV12-expressing fibroblasts. Thus, C/EBPβ functions as part of an Arf-dependent tumor suppressor network activated by oncogenic stress. The opposing effects of oncogenic Ras on Cebpb expression in primary MEFs and immortalized NIH 3T3 cells prompted us to investigate the mechanisms that regulate Cebpb gene transcription. The early growth response (Egr) family of zinc-finger TFs is composed of 4 members, Egr-1-4. Here we show that Egrs are also down-regulated in RasV12-transformed NIH 3T3 cells (3T3Ras) and their ectopic expression restores C/EBPβ levels. Over-expression of any of the Egr proteins causes growth arrest and senescence in 3T3Ras cells in a manner that is partially dependent on reactivation of C/EBPβ. Egr proteins stimulate transcription from a promoter-reporter construct containing ∼3 kb of the Cebpb 5' flanking region. The proximal Cebpb promoter contains several putative Egr binding motifs, and mutating three of these sites disrupts Egr-mediated transactivation of the Cebpb reporter. Chromatin immunoprecipitation assays showed that ectopically expressed and endogenous Egrs bind to the Cebpb proximal promoter. In addition, serum stimulation of normal NIH 3T3 cells induces C/EBPβ mRNA and protein levels, which peak between 2-5 hours and coincide with transient induction of Egr proteins. Moreover, Egrs are only weakly induced by serum in 3T3Ras cells compared to their robust induction in the parental cells, with the exception of Egr-4 which was not detected in either cell line. Thus, decreased Egr levels can account for RasV12-induced down-regulation of C/EBPβ in transformed fibroblasts. Furthermore, microarray data from Oncomine show a strong correlation between CEBPB and EGR expression in human tumor samples. Collectively, our findings identify the Egr-C/EBPβ axis as a critical pathway in determining the cellular response (transformation or senescence) to oncogenic Ras-p19Arf signaling. Citation Format: Jacqueline Salotti, Krisada Sakchaisri, Peter Johnson. A p19Arf-Egr-C/EBPβ axis underlying oncogene-induced senescence and tumor suppression. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B24.

IL2 - Cancer Research and Clinical Oncology for the Next Decade

ABSTRACT The next decade of cancer research will continue to focus on identification and effective targeting of somatic driver mutations. The first successfully targeted driver mutation in lung cancer is the mutated epidermal growth factor receptor (EGFR) discovered 8 years ago. Multiple prospective trials for patients with EGFR mutations comparing treatment with gefitinib and erlotinib versus combination chemotherapy have been reported. The prolongation in PFS and decreased toxicity with EGR-TKIs has led to their regulatory approval as initial systemic therapy in patients with non-small-cell lung cancer (NSCLC) and EGFR sensitizing mutations in many countries throughout the world. The second driver mutation successfully targeted is the ALK rearrangement in NSCLC. Five years have past since Dr. Mano et al. discovered a rearrangement between ALK and EML4 in adenocarcinomas of the lung that could transform NIH 3T3 cells. Further research has shown that additional ALK rearrangements can give rise to constitutively activated ALK tyrosine kinase in ∼5% of NSCLC patients. The ALK inhibitor, crizotinib, has been studied in NSCLC patients with prospectively identified ALK rearrangements in a series of phase I, II, and III trials. The prospective clinical studies administering crizotinib to NSCLC with ALK rearrangements have response rates of ∼60%, PFS of ∼10 months, and more than half of the patients are alive 2 years from starting crizotinib. The high response rates, prolongation in PFS and decreased toxicity with crizotinib compared with chemotherapy has led to the regulatory approval of crizotinib as systemic therapy in patients with NSCLC and ALK rearrangements in the United States in August 2011. The additional driver mutations targeted in NSCLC include KRAS, BRAF, HER2, and PIK3CA mutations, ROS1 and RET rearrangements as well as MET amplification. BRAF has been successfully targeted in melanoma and HER2 amplification in breast cancer. Multiplex genomic characterization efforts are identifying these driver mutations and multi-institutional trials with therapeutic agents directed against these genomic alterations are underway.

IS5-2 - Interaction of ELF-4 and NPM1 in AML

ABSTRACT ELF-4 is a transcription factor belonging to the ETS family, which is expressed in a wide variety of tissues including hematopoietic cells. It is reported that ELF-4 is essential for the development of NK/NKT cells, regulation of the cell cycle of hematopoietic stem cells, and was shown to be a transcriptional activator whose targets include cytokine genes, such as IL-3, GM-CSF, and IL-8. In this study, we aimed to test the role of ELF-4 in acute myeloid leukemia (AML), especially AML with normal karyotype. Tandem affinity purification assay revealed that nucleophosmin (NPM1) is one of the associated proteins with ELF-4, and that was confirmed using artificially expressed ELF-4 and NPM1 proteins. Co-expression of NPM1 reduced the activity of ELF-4, whereas mutation of NPM1 that is present in about 50% of AML with normal karyotype, increased the transcriptional activity of ELF-4. Along with the transcriptional activity, co-expression of wild type NPM1 decreased, and mutated NPM1 accelerated the transformation of NIH3T3 cells by the over expression of ELF-4. Although mutated NPM1 changes intracellular localization, from nucleus to cytoplasm, its mutation did not affect the localization of ELF-4. Results of the EMSA assay suggested that wild type NPM1 hampers the DNA binding of ELF-4. Since one of the targets of elf-4 is mdm2, a negative regulator of p53, we tested whether ELF-4 regulates the expression of HDM2, human version of mdm2. ChIP assay and luciferase assay using the promoter of HDM2 showed that ELF-4 binds to the one of the putative binding sites, and it transactivates the HDM2 promoter. Co-expression of wild type NPM1 reduced, and mutated NPM1 enhanced the binding of ELF-4 to its promoter. These results suggested that ELF-4 has an important role in the biology of AML, with a strong relationship with the mutational situation of the NPM1 gene.

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.

Multidimensional investigation of HER2 in advanced urothelial carcinoma (UC).

4580 Background: Incidence of Her2 positivity and association with overall survival (OS) are controversial in advanced UC. Activating Her2 mutations have been identified in other cancers, but they have not been previously reported in UC. We determined Her2 status by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and copy number gain (CNG) via array CGH of primary UC tumors from patients (pts) with metastatic disease. Targeted Her2 sequencing was performed at known mutation hotspots, and mutation effect was investigated in vitro. Methods: Tissue microarrays of formalin fixed paraffin-embedded tumor from 98 UC pts treated with platinum-based combination chemotherapy for metastatic disease were evaluated for Her2 protein and for Her2 gene amplification by using standard clinical protocols. Positive staining was defined as an IHC score of 3+ or a FISH ratio of ≥2 using scoring criteria established for evaluation of breast cancer. Her2 CNG was evaluated by aCGH with cutoff log base 2 ratio > 0.9. Mutation status was validated by hME sequencing. OS was measured from start of treatment for metastatic disease. Association of OS and Her2 status was assessed by a Cox regression model. NIH-3T3 cells with Her2 V777L were assessed for growth, invasion, and Her2 kinase activation. Results: 22% of pts had 3+ Her2 staining by IHC. 21% of pts had FISH amplification. These were concordant in 78% of pts. CNG was identified in 16% and was concordant with FISH and IHC 85% and 88% of the time, respectively. Her2 status by any modality showed no significant association with OS in either univariate [HR=0.94, 95% CI: (0.52, 1.70), p=0.83] or multivariate [HR=1.12, 95% CI: (0.61, 2.06), p=0.72] analysis. Her2 mutations (V777L and L755S) were identified in 2 pts (2%). In vitro analysis of V777L results in transformation of NIH-3T3 cells, leading to increased growth, invasion on soft agar, and Her2 kinase constitutive activation. Conclusions: Her2 overexpression or amplification in the primary tumor does not predict OS in pts with metastatic UC. Other research has suggested that V777L sensitizes cells to lapatinib, while L755S leads to lapatinib resistance. These rare oncogenic Her2 mutations occur and may be therapeutic targets in selected pts.

Abstract A31: Roles of Nogo-B receptor (NgBR) in regulating Ras translocation and modulating breast cancer cell sensitivity to chemotherapeutic drug and irradiation

Abstract Ras, a critical oncogene, is one of small GTPase family members. It is active when bound to GTP and inactive when bound to GDP. The activation process needs membrane translocation of Ras because its activators are recruited by membrane receptors, such as EGF receptors. In addition, appropriate localization of activated Ras also is critical for interacting with the downstream effectors. Therefore, regulation of Ras membrane translocation is very important for Ras function. Nogo-B receptor (NgBR) is a type I receptor with a single transmembrane domain. Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro and blood vessel formation in Zebrafish. Here, we demonstrate that NgBR can bind and recruit farnesylated Ras to plasma membrane, which results in the activation of PI3K-Akt and Raf-MEK-ERK signaling pathways in human breast cancer cells. Direct interaction between NgBR and farnesylated Ras has been demonstrated by co-immunoprecipitation of MBP-NgBR from incubation of purified recombinant farnesylated Ras and MBP-NgBR protein. Knockdown of NgBR in MDA-MB-231 human breast cancer cells remarkably reduces the amount of membrane-associated Ras. These results suggest that NgBR is a potential modulator for Ras translocation. Knockdown of NgBR expression in MDA-MB-231 breast cancer cells decreases cancer cell colony formation, and abolishes EGF-stimulated Ras activation and phosphorylation of Akt in MDA-MB-231 cells. In addition, overexpression of NgBR caused the transformation of NIH-3T3 cells via increasing Ras plasma membrane translocation and enhancing EGF-stimulated Ras signaling. Knockdown of NgBR decreases the survival and colony formation of MDA-MB-231 cells after irradiation and increases the cisplatin-induced apoptosis and Caspase-3 activity of MDA-MB-231 Cells. These results suggest that NgBR can increase MDA-MB-231 breast cancer cell sensitivity to chemotherapeutic drug and irradiation. Consequently, our preliminary results further demonstrated that inducible knockdown of NgBR in tumor xenograft of MDA-MB-231 cells in nude mice significantly retards tumor growth. Thus, these data demonstrate that NgBR is a unique receptor that recruits farnesylated Ras to the plasma membrane via its hydrophobic cytoplasmic domain and then activates both PI3K-Akt and Raf-MEK-ERK pathways in human breast cancer cells. Collectively, it is the first study to demonstrate that NgBR is a potential therapeutic target for inhibiting Ras-dependent growth of breast cancer and decreasing the sensitivity of breast cancer cells to chemotherapeutic drug and irradiation.

Oncogenic transformation induced by cell-free nucleic acids circulating in plasma (genometastasis) remains after the surgical resection of the primary tumor: a pilot study

Background: The oncogenic transformation by cell-free nucleic acids circulating in plasma has been named as genometastasis. The feasibility of this phenomenon has been demonstrated and now it is necessary to value the impact of this phenomenon and to determine what conditions could promote or inhibit it. The goal of this study was to examine the transforming ability of plasma from colorectal cancer patients in a long-term follow-up after the surgical excision of the primary tumor, and to try correlate it with the clinical picture of patients.Materials and methods: Blood samples were taken from eight patients with K-ras-mutated colorectal tumors, who were under surgical primary tumor resection at least 2 years before. Plasma was isolated by two centrifugations and added to cultures of NIH-3T3 cells and human adipose-derived stem cells (hASCs). In two cases, plasma was separated from cells by a membrane with 0.4-μm pores. The presence of mutated and non-mutated human K-ras sequences was tested by real-time PCR in cultured cells. After 30 days, cells were subcutaneously injected into athymic nude mice in order to test their ability to generate tumors.Results: In four of the eight patients analyzed after surgery, tumor DNA was detected in plasma. Plasmas from three of them were able to oncogenically transform NIH-3T3 cells in culture and, when those cells were injected in mice, carcinomas were generated. After a 2-year follow-up, metastases were found in two of the three patients whose plasmas were able to transform cells, and in two of the four in whom plasma tumor DNA was not detected. Thus, after a mean follow-up of 29.5 months, only four of 13 patients (30.8%) were alive and disease-free.Conclusion: Primary tumor resection does not assure a complete clean of blood of circulating oncogenes, in spite of a disease-free clinical picture. Moreover, in some cases plasma kept their oncogenic capabilities. The value of these findings as prognosis factor remains unclear and needs further investigations.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature