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

Chang JG, Yang DM, Chang WH, Chow LP, Chan WL, Lin HH, Huang HD, Chang YS, Hung CH, Yang WK - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw

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.

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Gene ontology distribution of 495 protein-encoding gene transcripts with amiloride-modulated AS patterns in Huh-7 cells.Bioinformatic analyses of the 495 gene transcripts with amiloride-modified alternative splicing, detected by the genome-wide exon-array measurements, were performed according to gene ontology categories of Biological Process (A), Molecular Function (B) and Cellular Component (C). Amiloride-altered AS patterns of APAF1, CRK,MBNL2, MIZF,WAC, PAPD5 and SURVIVIN specific protein-encoding gene transcripts detected by the exon-array analysis were validated by RT-PCR of spliced RNA isoforms (Panel D).
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pone-0018643-g005: Gene ontology distribution of 495 protein-encoding gene transcripts with amiloride-modulated AS patterns in Huh-7 cells.Bioinformatic analyses of the 495 gene transcripts with amiloride-modified alternative splicing, detected by the genome-wide exon-array measurements, were performed according to gene ontology categories of Biological Process (A), Molecular Function (B) and Cellular Component (C). Amiloride-altered AS patterns of APAF1, CRK,MBNL2, MIZF,WAC, PAPD5 and SURVIVIN specific protein-encoding gene transcripts detected by the exon-array analysis were validated by RT-PCR of spliced RNA isoforms (Panel D).

Mentions: It is possible that the effects of amiloride on the phosphorylation and intracellular localization of splicing factors and mRNA export [29] could affect splicing of other gene transcripts in addition to BCL-X, HIPK3 and RON/MISTR1. Using gene array chips (Affymetrix GeneChip® Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient ≥0.7, splicing index ≤−1.585 , and log2 ratio ≤−1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons (Table S1; MIAME accession number #GSE24581). These 495 known protein-coding genes could be classified into functional categories involving cytoskeleton remodeling, cell adhesion, ion transport, transcription factors, immune response, and muscle contraction (Figure 5A-C and Table S2). To verify these exon array results, we selected seven (APF-1, CRK, MBNL2, MIZF, WAC, PAPDS and survivin) for analysis by RT-PCR and indeed confirmed distinct AS pattern alterations of these gene transcripts in amiloride-treated cells (Figure 5D).


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

Chang JG, Yang DM, Chang WH, Chow LP, Chan WL, Lin HH, Huang HD, Chang YS, Hung CH, Yang WK - PLoS ONE (2011)

Gene ontology distribution of 495 protein-encoding gene transcripts with amiloride-modulated AS patterns in Huh-7 cells.Bioinformatic analyses of the 495 gene transcripts with amiloride-modified alternative splicing, detected by the genome-wide exon-array measurements, were performed according to gene ontology categories of Biological Process (A), Molecular Function (B) and Cellular Component (C). Amiloride-altered AS patterns of APAF1, CRK,MBNL2, MIZF,WAC, PAPD5 and SURVIVIN specific protein-encoding gene transcripts detected by the exon-array analysis were validated by RT-PCR of spliced RNA isoforms (Panel D).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111415&req=5

pone-0018643-g005: Gene ontology distribution of 495 protein-encoding gene transcripts with amiloride-modulated AS patterns in Huh-7 cells.Bioinformatic analyses of the 495 gene transcripts with amiloride-modified alternative splicing, detected by the genome-wide exon-array measurements, were performed according to gene ontology categories of Biological Process (A), Molecular Function (B) and Cellular Component (C). Amiloride-altered AS patterns of APAF1, CRK,MBNL2, MIZF,WAC, PAPD5 and SURVIVIN specific protein-encoding gene transcripts detected by the exon-array analysis were validated by RT-PCR of spliced RNA isoforms (Panel D).
Mentions: It is possible that the effects of amiloride on the phosphorylation and intracellular localization of splicing factors and mRNA export [29] could affect splicing of other gene transcripts in addition to BCL-X, HIPK3 and RON/MISTR1. Using gene array chips (Affymetrix GeneChip® Human Exon 1.0 ST Array of >518000 exons of 42974 genes) for exon array analysis (set parameters of correlation coefficient ≥0.7, splicing index ≤−1.585 , and log2 ratio ≤−1.585), we found that amiloride influenced the splicing patterns of 551 genes involving at least 584 exons, which included 495 known protein-coding genes involving 526 exons (Table S1; MIAME accession number #GSE24581). These 495 known protein-coding genes could be classified into functional categories involving cytoskeleton remodeling, cell adhesion, ion transport, transcription factors, immune response, and muscle contraction (Figure 5A-C and Table S2). To verify these exon array results, we selected seven (APF-1, CRK, MBNL2, MIZF, WAC, PAPDS and survivin) for analysis by RT-PCR and indeed confirmed distinct AS pattern alterations of these gene transcripts in amiloride-treated cells (Figure 5D).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw

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.

Show MeSH
Related in: MedlinePlus