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Identification of Novel Gene Expression Targets for the Ras Association Domain Family 1 (RASSF1A) Tumor Suppressor Gene in Non-Small Cell Lung Cancer and Neuroblastoma¹

Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom [A. A., J. A-C., E. R. M., F. L.]; Laboratoire d’Oncogénétique-INSERM E0017, Centre René Huguenin, F-92210 St-Cloud, France [I. B.]; Signal Transduction Laboratory, R222, Cancer Research United Kingdom London Research Institute, London WC2A 3PX, United Kingdom [B. N., S. B., J. D.]; AG Tumorgenetik der Medizinischen Fakultät, Martin-Luther Universität Halle-Wittenberg, D-06097 Halle (Saale), Germany [R. D.]; Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center at Dallas, Texas 75390-8593 [B. G., J. D. M.]; and Cancer Research United Kingdom Renal Molecular Oncology Research Group, University of Birmingham, Birmingham B15 2TG, United Kingdom [E. R. M., F. L.]

RASSF1A is a recently identified 3p21.3 tumor suppressor gene. The high frequency of epigenetic inactivation of this gene in a wide range of human sporadic cancers including non-small cell lung cancer (NSCLC) and neuroblastoma suggests that RASSF1A inactivation is important for tumor development. Although little is known about the function of RASSF1A, preliminary data suggests that it may have multiple functions. To gain insight into RASSF1A functions in an unbiased manner, we have characterized the expression profile of a lung cancer cell line (A549) transfected with RASSF1A. Initially we demonstrated that transient expression of RASSF1A into the NSCLC cell line A549 induced G₁ cell cycle arrest, as measured by propidium iodide staining. Furthermore, annexin-V staining showed that RASSF1A-expressing cells had an increased sensitivity to staurosporine-induced apoptosis. We then screened a cDNA microarray containing more than 6000 probes to identify genes differentially regulated by RASSF1A. Sixty-six genes showed at least a 2-fold change in expression. Among these were many genes with relevance to tumorigenesis involved in transcription, cytoskeleton, signaling, cell cycle, cell adhesion, and apoptosis. For 22 genes we confirmed the microarray results by real-time RT-PCR and/or Northern blotting. In silico, we were able to confirm the majority of these genes in other NSCLC cell lines using published data on gene expression profiles. Furthermore, we confirmed 10 genes at the RNA level in two neuroblastoma cell lines, indicating that these RASSF1A target genes have relevance in non-lung cell backgrounds. Protein analysis of six genes (ETS2, Cyclin D3, CDH2, DAPK1, TXN, and CTSL) showed that the changes induced by RASSF1A at the RNA level correlated with changes in protein expression in both non-small cell lung cancer and neuroblastoma cell lines. Finally, we have used a transient assay to demonstrate the induction of CDH2 and TGM2 by RASSF1A in NSCLC cell lines. We have identified several novel targets for RASSF1A tumor suppressor gene both at the RNA and the protein levels in two different cellular backgrounds. The identified targets are involved in diverse cellular processes; this should help toward understanding mechanisms that contribute to RASSF1A biological activity.

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