title: Defining NOTCH3 target genes in ovarian cancer.
abstract: NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here, we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell-cycle regulation and nucleotide metabolism. Chromatin immunoprecipitation (ChIP)-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data showed that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified, we showed that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G(2)-M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacologic or RNA interference-mediated NOTCH inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight the role of DLGAP5 in mediating the function of NOTCH3.
authors.affiliation: Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Chen
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Thiaville
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Alexander
authors.initials: A
authors.lastname: Stoeck
authors.forename: Jianhua
authors.initials: J
authors.lastname: Xuan
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.forename: Ie-Ming
authors.initials: IeM
authors.lastname: Shih
authors.forename: Tian-Li
authors.initials: TL
authors.lastname: Wang
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D005260:Female; D005784:Gene Amplification; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010051:Ovarian Neoplasms / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics*; D059467:Transcriptome
chemical_list: C465041:DLGAP5 protein, human; C499374:NOTCH3 protein, human; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000071656:Receptor, Notch3; D051880:Receptors, Notch