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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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Related in: MedlinePlus

Effects of amiloride treatment on phosphorylation of SR-related proteins.Western blot analysis of subcellular fractions of Huh-7 cells with and without 0.5 mM amiloride 24-hour treatment was performed using specific antibodies against various splicing protein factors and β-tubulin. (Panel A) The amiloride treatment increased the dephosphorylated SF2/ASF forms in both cytoplasmic and nuclear fractions. (Panel B) The amiloride treatment decreased the expression of SRp20 and two un-identified phosphorylated SR proteins in both C (cytoplasmic) and N (nuclear) cellular fractions.
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pone-0018643-g003: Effects of amiloride treatment on phosphorylation of SR-related proteins.Western blot analysis of subcellular fractions of Huh-7 cells with and without 0.5 mM amiloride 24-hour treatment was performed using specific antibodies against various splicing protein factors and β-tubulin. (Panel A) The amiloride treatment increased the dephosphorylated SF2/ASF forms in both cytoplasmic and nuclear fractions. (Panel B) The amiloride treatment decreased the expression of SRp20 and two un-identified phosphorylated SR proteins in both C (cytoplasmic) and N (nuclear) cellular fractions.

Mentions: As phosphorylation status of SR protein components in the AS complexes is known to affect protein-protein interaction for the splicing function, we next used 2D-gel electrophoresis to analyze differential expression of serine-phosphorylated proteins. We found more than ten proteins with quantitative changes after amiloride treatment (Figure 2). Choosing seven of these protein spots for proteomic identification by mass spectrometry (Table 1), we found one of them to be ASF/SF2, a splicing factor known to be involved in AS of the RON transcript [20]. To verify this finding, we performed western blots of ASF/SF2 and found markedly reduced ASF/SF2 phosphorylation in both the cytoplasm and nucleus of amiloride-treated Huh-7 cells (Figure 3A). Analysis of other protein constituents of the splicing complexes (such as SRp20, hnRNPA1, hnRNPA2/B1, Sam68 and common SR proteins) showed that SRp20 and two un-identified phosphorylated SR proteins were also reduced in the cytoplasm and nucleus after amiloride treatment (Figure 3B). These results imply that amiloride modulates the AS of BCL-X, HIPK3, and RON/MISTR1 through hypo-phosphorylation of ASF/SF2 and decreased expression of SRp20 and some other SR proteins.


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)

Effects of amiloride treatment on phosphorylation of SR-related proteins.Western blot analysis of subcellular fractions of Huh-7 cells with and without 0.5 mM amiloride 24-hour treatment was performed using specific antibodies against various splicing protein factors and β-tubulin. (Panel A) The amiloride treatment increased the dephosphorylated SF2/ASF forms in both cytoplasmic and nuclear fractions. (Panel B) The amiloride treatment decreased the expression of SRp20 and two un-identified phosphorylated SR proteins in both C (cytoplasmic) and N (nuclear) cellular fractions.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0018643-g003: Effects of amiloride treatment on phosphorylation of SR-related proteins.Western blot analysis of subcellular fractions of Huh-7 cells with and without 0.5 mM amiloride 24-hour treatment was performed using specific antibodies against various splicing protein factors and β-tubulin. (Panel A) The amiloride treatment increased the dephosphorylated SF2/ASF forms in both cytoplasmic and nuclear fractions. (Panel B) The amiloride treatment decreased the expression of SRp20 and two un-identified phosphorylated SR proteins in both C (cytoplasmic) and N (nuclear) cellular fractions.
Mentions: As phosphorylation status of SR protein components in the AS complexes is known to affect protein-protein interaction for the splicing function, we next used 2D-gel electrophoresis to analyze differential expression of serine-phosphorylated proteins. We found more than ten proteins with quantitative changes after amiloride treatment (Figure 2). Choosing seven of these protein spots for proteomic identification by mass spectrometry (Table 1), we found one of them to be ASF/SF2, a splicing factor known to be involved in AS of the RON transcript [20]. To verify this finding, we performed western blots of ASF/SF2 and found markedly reduced ASF/SF2 phosphorylation in both the cytoplasm and nucleus of amiloride-treated Huh-7 cells (Figure 3A). Analysis of other protein constituents of the splicing complexes (such as SRp20, hnRNPA1, hnRNPA2/B1, Sam68 and common SR proteins) showed that SRp20 and two un-identified phosphorylated SR proteins were also reduced in the cytoplasm and nucleus after amiloride treatment (Figure 3B). These results imply that amiloride modulates the AS of BCL-X, HIPK3, and RON/MISTR1 through hypo-phosphorylation of ASF/SF2 and decreased expression of SRp20 and some other SR proteins.

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