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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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Phosphorylation of Akt kinase (activation) and PP1 phosphatase (inactivation) in relation to amiloride-altered AS mechanisms.(Panel A). Western blots show significant decreases of cytoplasmic p-Akt, nuclear p-Akt and nuclear p-PP1 in Huh-7 cells treated with amiloride at 0.3 mM and above. (Panel B) RT-PCR and western blot analyses show that pre-treatment with a PP1 inhibitor, okadaic acid, abrogated the amiloride effects on the BCL-X and HIPK3 oncogenic RNA splicing, the phosphorylation of ASF2/ASF splicing factor and the level of SRp20 in Huh-7 cells.
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pone-0018643-g004: Phosphorylation of Akt kinase (activation) and PP1 phosphatase (inactivation) in relation to amiloride-altered AS mechanisms.(Panel A). Western blots show significant decreases of cytoplasmic p-Akt, nuclear p-Akt and nuclear p-PP1 in Huh-7 cells treated with amiloride at 0.3 mM and above. (Panel B) RT-PCR and western blot analyses show that pre-treatment with a PP1 inhibitor, okadaic acid, abrogated the amiloride effects on the BCL-X and HIPK3 oncogenic RNA splicing, the phosphorylation of ASF2/ASF splicing factor and the level of SRp20 in Huh-7 cells.

Mentions: Recent studies have shown that amiloride inhibits phosphorylation of kinases and phosphatases associated with the PI3K-AKT pathway [21], [22], [23], and that AKT kinase and PP1 phosphatase may regulate the activity of SR proteins [23], [24], [25], [26], [27], [28]. We therefore determined the effects of amiloride on the phosphorylated forms of Akt kinase and related phosphatases in Huh-7 cells. Decreased levels of the Ser(473) p-AKT and the Thr(320) p-PP1 were observed in the cytoplasm and nucleus, suggesting inhibition of AKT kinase activity and activation of PP1 phosphatase activity (Figure 4A). To determine whether inactivation of Akt kinase per se would play a role in regulating the alternative splicing, Huh7 cells were treated with PI3K-AKT pathway inhibitors, including LY294002, wortmannin and triciribine; 1 µM triciribine but not LY294002 or wortmannin decreased the level of p-Akt. However, triciribine at this concentration exerted no effect on the alternative splicing of BCL-X and HIPK3 transcripts (Figure S1). Interestingly, pre-treatment of the cells with okadaic acid to inhibit PP1 phosphatase activity abrogated the effects of amiloride on BCL-X and HIPK3 splicing, as well as effects of amiloride on the dephosphorylation of SF2/ASF and decreasing of the SRp20 level, but okadaic acid exerted no definite effect on the expression of hnRNP A1 and hnRNP A2B1 (Figure 4B). Taken together, these results suggest that PP1 plays an important role in modulating phosporylation of SR splicing factors and hence the modulation of oncogenic AS in Huh-7 cells after amiloride treatment.


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)

Phosphorylation of Akt kinase (activation) and PP1 phosphatase (inactivation) in relation to amiloride-altered AS mechanisms.(Panel A). Western blots show significant decreases of cytoplasmic p-Akt, nuclear p-Akt and nuclear p-PP1 in Huh-7 cells treated with amiloride at 0.3 mM and above. (Panel B) RT-PCR and western blot analyses show that pre-treatment with a PP1 inhibitor, okadaic acid, abrogated the amiloride effects on the BCL-X and HIPK3 oncogenic RNA splicing, the phosphorylation of ASF2/ASF splicing factor and the level of SRp20 in Huh-7 cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0018643-g004: Phosphorylation of Akt kinase (activation) and PP1 phosphatase (inactivation) in relation to amiloride-altered AS mechanisms.(Panel A). Western blots show significant decreases of cytoplasmic p-Akt, nuclear p-Akt and nuclear p-PP1 in Huh-7 cells treated with amiloride at 0.3 mM and above. (Panel B) RT-PCR and western blot analyses show that pre-treatment with a PP1 inhibitor, okadaic acid, abrogated the amiloride effects on the BCL-X and HIPK3 oncogenic RNA splicing, the phosphorylation of ASF2/ASF splicing factor and the level of SRp20 in Huh-7 cells.
Mentions: Recent studies have shown that amiloride inhibits phosphorylation of kinases and phosphatases associated with the PI3K-AKT pathway [21], [22], [23], and that AKT kinase and PP1 phosphatase may regulate the activity of SR proteins [23], [24], [25], [26], [27], [28]. We therefore determined the effects of amiloride on the phosphorylated forms of Akt kinase and related phosphatases in Huh-7 cells. Decreased levels of the Ser(473) p-AKT and the Thr(320) p-PP1 were observed in the cytoplasm and nucleus, suggesting inhibition of AKT kinase activity and activation of PP1 phosphatase activity (Figure 4A). To determine whether inactivation of Akt kinase per se would play a role in regulating the alternative splicing, Huh7 cells were treated with PI3K-AKT pathway inhibitors, including LY294002, wortmannin and triciribine; 1 µM triciribine but not LY294002 or wortmannin decreased the level of p-Akt. However, triciribine at this concentration exerted no effect on the alternative splicing of BCL-X and HIPK3 transcripts (Figure S1). Interestingly, pre-treatment of the cells with okadaic acid to inhibit PP1 phosphatase activity abrogated the effects of amiloride on BCL-X and HIPK3 splicing, as well as effects of amiloride on the dephosphorylation of SF2/ASF and decreasing of the SRp20 level, but okadaic acid exerted no definite effect on the expression of hnRNP A1 and hnRNP A2B1 (Figure 4B). Taken together, these results suggest that PP1 plays an important role in modulating phosporylation of SR splicing factors and hence the modulation of oncogenic AS in Huh-7 cells after amiloride treatment.

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