Small molecule amiloride modulates oncogenic RNA alternative splicing to devitalize human cancer cells

• Published in: PloS one 2011-06, Vol.6 (6), p.e18643
• Main Authors: Chang, Jan-Gowth, Yang, Den-Mei, Chang, Wen-Hsin, Chow, Lu-Ping, Chan, Wen-Ling, Lin, Hui-Hua, Huang, Hsien-Da, Chang, Ya-Sian, Hung, Cheng-Hao, Yang, Wen-Kuang
• Format: Article
• Language: English
• Subjects: Acids; AKT protein; Alternative splicing; Alternative Splicing - drug effects; Alternative Splicing - genetics; Amiloride; Amiloride - chemistry; Amiloride - pharmacology; Apoptosis; Apoptosis - drug effects; Bcl-x protein; Biochemistry; Bioinformatics; Biology; Brain cancer; Cancer; Cancer cells; Cancer therapies; Cell cycle; Cell Line, Tumor; Cell migration; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Change detection; Chemotherapy; Clinical medicine; Colorectal cancer; Cytokines; Cytokinesis; Cytokinesis - drug effects; Cytoskeleton; Cytoskeleton - metabolism; Deoxyribonucleic acid; DNA; DNA Fragmentation - drug effects; DNA Repair - drug effects; Drug screening; Enzyme Activation - drug effects; Exons; Exons - genetics; Extracellular signal-regulated kinase; Gene expression; Gene Expression Regulation, Neoplastic - drug effects; Gene therapy; Genes; Genes, Neoplasm - genetics; Genome, Human - genetics; Genomes; Genomics; Helicobacter pylori; Hepatocellular carcinoma; Humans; Information retrieval; Invasiveness; Ion transport; Isoforms; JNK protein; Kinases; Laboratories; Leukemia; Liver cancer; Medical research; Medicine; Metastasis; Mitosis - drug effects; Neoplasm Invasiveness; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Neoplasms - genetics; Neoplasms - pathology; Nuclear Proteins - metabolism; Ontology; Open source software; Phosphatases; Phosphorylation; Phosphorylation - drug effects; Phosphotransferases; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Phosphatase 1 - metabolism; Proteins; Proteomics; Proto-Oncogene Proteins c-akt - metabolism; Ribonucleic acid; RNA; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Neoplasm - genetics; RNA, Neoplasm - metabolism; RNA-Binding Proteins - metabolism; Ron protein; Serine-Arginine Splicing Factors; Solid tumors; Splicing factors; Stem cells; Structure-function relationships; Transcription
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0018643

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.