title: Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells.
abstract: Alternative splicing involves differential exon selection of a gene transcript to generate mRNA and protein isoforms with structural and functional diversity. Abnormal alternative splicing has been shown to be associated with malignant phenotypes of cancer cells, such as chemo-resistance and invasive activity. Screening small molecules and drugs for modulating RNA splicing in human hepatocellular carcinoma cell line Huh-7, we discovered that amiloride, distinct from four pH-affecting amiloride analogues, could "normalize" the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts. Our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF, and decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, and increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulates kinases and up-regulates phosphatases in the signal pathways known to affect splicing factor protein phosphorylation. These amiloride effects of "normalized" oncogenic RNA splicing and splicing factor hypo-phosphorylation were both abrogated by pre-treatment with a PP1 inhibitor. Global exon array of amiloride-treated Huh-7 cells detected splicing pattern changes involving 584 exons in 551 gene transcripts, many of which encode proteins playing key roles in ion transport, cellular matrix formation, cytoskeleton remodeling, and genome maintenance. Cellular functional analyses revealed subsequent invasion and migration defects, cell cycle disruption, cytokinesis impairment, and lethal DNA degradation in amiloride-treated Huh-7 cells. Other human solid tumor and leukemic cells, but not a few normal cells, showed similar amiloride-altered RNA splicing with devitalized consequence. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of RNA splicing for cancer therapeutics.
authors.affiliation: Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw
authors.forename: Jan-Gowth
authors.initials: JG
authors.lastname: Chang
authors.forename: Den-Mei
authors.initials: DM
authors.lastname: Yang
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Chang
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Chow
authors.forename: Wen-Ling
authors.initials: WL
authors.lastname: Chan
authors.forename: Hui-Hua
authors.initials: HH
authors.lastname: Lin
authors.forename: Hsien-Da
authors.initials: HD
authors.lastname: Huang
authors.forename: Ya-Sian
authors.initials: YS
authors.lastname: Chang
authors.forename: Cheng-Hao
authors.initials: CH
authors.lastname: Hung
authors.forename: Wen-Kuang
authors.initials: WK
authors.lastname: Yang
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects* / Q000235:genetics; D000584:Amiloride / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D048749:Cytokinesis / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005091:Exons / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D052138:Genes, Neoplasm / Q000235:genetics; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D008938:Mitosis / Q000187:drug effects; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D054645:Protein Phosphatase 1 / Q000378:metabolism; D040901:Proteomics; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors
chemical_list: D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D000584:Amiloride; D051057:Proto-Oncogene Proteins c-akt; D054645:Protein Phosphatase 1