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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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Modification of splicing patterns of BCL-X, HIPK3 and RON gene transcripts by amiloride.RNA was extracted from Huh-7 cells exposed to growth medium containing amiloride or other pHi modulator amiloride derivatives and examined for the oncogenic RNA alternative splicing by RT-PCR, as described in Materials and Methods. Panel (A) shows that amiloride increased the use of upstream alternative 5′-splice site within exon 2 that yields the apoptotic BCL-XS isoform (upper row), increased the exon 11 (U exon) skipping of HIPK3 that increases the apoptotic isoform (middle row), and slightly increased the exon 11 skipping of RON at 0.5 mM concentration (lower row). Panel (B) shows that splicing patterns of the tested genes were not significantly affected by four amiloride derivatives, including 5-(N-ethyl-N-isopropyl)-amiloride or EIPA; 5-(N-methyl-N-isobutyl)amiloride or MIBA; 5-(N,N-dimethyl)amiloride or DMA; and 5-(N,N-hexamethylene)amiloride or HMA, at equivalent concentrations of pHi modulation.
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pone-0018643-g001: Modification of splicing patterns of BCL-X, HIPK3 and RON gene transcripts by amiloride.RNA was extracted from Huh-7 cells exposed to growth medium containing amiloride or other pHi modulator amiloride derivatives and examined for the oncogenic RNA alternative splicing by RT-PCR, as described in Materials and Methods. Panel (A) shows that amiloride increased the use of upstream alternative 5′-splice site within exon 2 that yields the apoptotic BCL-XS isoform (upper row), increased the exon 11 (U exon) skipping of HIPK3 that increases the apoptotic isoform (middle row), and slightly increased the exon 11 skipping of RON at 0.5 mM concentration (lower row). Panel (B) shows that splicing patterns of the tested genes were not significantly affected by four amiloride derivatives, including 5-(N-ethyl-N-isopropyl)-amiloride or EIPA; 5-(N-methyl-N-isobutyl)amiloride or MIBA; 5-(N,N-dimethyl)amiloride or DMA; and 5-(N,N-hexamethylene)amiloride or HMA, at equivalent concentrations of pHi modulation.

Mentions: It has recently become clear that unbalanced RNA splicing of certain genes is associated with malignant properties of human cancer cells [11], [12], [13], e.g., resistance to chemotherapy and radiotherapy with BCL-X, onco-developmentally undifferentiated state with HIPK3, and invasive metastatic potentials with RON/MISTR1. In hepatocellular carcinoma Huh-7 cells, BCL-X is alternatively spliced into anti-apoptotic large RNA isoform, BCL-XL, more than pro-apoptotic small RNA isoform, BCL-XS [15]; HIPK3 is spliced into exon 11-excluded U (-) as well as exon 11-included U (+) isoforms for interaction with Fas/FADD to modulate apoptosis in the developmental process [16]; and RON/MISTR1 is spliced into 5-kb full-length (flRON) RNA and 2-kb exon 11-excluded RON (sfRON) RNA isoforms, the latter of which has been correlated with epithelial to mesenchymal transition in cancer cell invasion and metastasis [17], [18]. After screening more than 500 small molecules and drugs, including those in clinical use, we found that amiloride exerted a potent effect on AS of these gene transcripts (Figure 1A). Huh-7 cells treated with amiloride showed: (a) relative decrease of BCL-XL and increase of BCL-XS RNA isoforms; (b) relative maintenance of HIPK3 U (-) and decrease of HIPK3 U (+) isoforms; and (c) decrease of both exon 11 (+) flRON and exon 11 (-) sfRON RNA isoforms. Such effects of amiloride were dose- and time-dependent, being detectable by 2 hours and marked after 24 hours of incubation with 0.5 mM of amiloride. These observations suggested increased utilization of the upstream alternative 5′-splice site within exon 2 of BCL-X, increased exon 11 (U) exclusion of HIPK3, and decreased splicing complexes formed for production of both RON/MISTR1 exon 11 (+) and exon 11 (-) isoforms. Thus, amiloride could modulate the AS of these three gene transcripts towards less malignant “normalized” patterns, similar to the normal ones in previous reports [15], [16], [17], [18].


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)

Modification of splicing patterns of BCL-X, HIPK3 and RON gene transcripts by amiloride.RNA was extracted from Huh-7 cells exposed to growth medium containing amiloride or other pHi modulator amiloride derivatives and examined for the oncogenic RNA alternative splicing by RT-PCR, as described in Materials and Methods. Panel (A) shows that amiloride increased the use of upstream alternative 5′-splice site within exon 2 that yields the apoptotic BCL-XS isoform (upper row), increased the exon 11 (U exon) skipping of HIPK3 that increases the apoptotic isoform (middle row), and slightly increased the exon 11 skipping of RON at 0.5 mM concentration (lower row). Panel (B) shows that splicing patterns of the tested genes were not significantly affected by four amiloride derivatives, including 5-(N-ethyl-N-isopropyl)-amiloride or EIPA; 5-(N-methyl-N-isobutyl)amiloride or MIBA; 5-(N,N-dimethyl)amiloride or DMA; and 5-(N,N-hexamethylene)amiloride or HMA, at equivalent concentrations of pHi modulation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0018643-g001: Modification of splicing patterns of BCL-X, HIPK3 and RON gene transcripts by amiloride.RNA was extracted from Huh-7 cells exposed to growth medium containing amiloride or other pHi modulator amiloride derivatives and examined for the oncogenic RNA alternative splicing by RT-PCR, as described in Materials and Methods. Panel (A) shows that amiloride increased the use of upstream alternative 5′-splice site within exon 2 that yields the apoptotic BCL-XS isoform (upper row), increased the exon 11 (U exon) skipping of HIPK3 that increases the apoptotic isoform (middle row), and slightly increased the exon 11 skipping of RON at 0.5 mM concentration (lower row). Panel (B) shows that splicing patterns of the tested genes were not significantly affected by four amiloride derivatives, including 5-(N-ethyl-N-isopropyl)-amiloride or EIPA; 5-(N-methyl-N-isobutyl)amiloride or MIBA; 5-(N,N-dimethyl)amiloride or DMA; and 5-(N,N-hexamethylene)amiloride or HMA, at equivalent concentrations of pHi modulation.
Mentions: It has recently become clear that unbalanced RNA splicing of certain genes is associated with malignant properties of human cancer cells [11], [12], [13], e.g., resistance to chemotherapy and radiotherapy with BCL-X, onco-developmentally undifferentiated state with HIPK3, and invasive metastatic potentials with RON/MISTR1. In hepatocellular carcinoma Huh-7 cells, BCL-X is alternatively spliced into anti-apoptotic large RNA isoform, BCL-XL, more than pro-apoptotic small RNA isoform, BCL-XS [15]; HIPK3 is spliced into exon 11-excluded U (-) as well as exon 11-included U (+) isoforms for interaction with Fas/FADD to modulate apoptosis in the developmental process [16]; and RON/MISTR1 is spliced into 5-kb full-length (flRON) RNA and 2-kb exon 11-excluded RON (sfRON) RNA isoforms, the latter of which has been correlated with epithelial to mesenchymal transition in cancer cell invasion and metastasis [17], [18]. After screening more than 500 small molecules and drugs, including those in clinical use, we found that amiloride exerted a potent effect on AS of these gene transcripts (Figure 1A). Huh-7 cells treated with amiloride showed: (a) relative decrease of BCL-XL and increase of BCL-XS RNA isoforms; (b) relative maintenance of HIPK3 U (-) and decrease of HIPK3 U (+) isoforms; and (c) decrease of both exon 11 (+) flRON and exon 11 (-) sfRON RNA isoforms. Such effects of amiloride were dose- and time-dependent, being detectable by 2 hours and marked after 24 hours of incubation with 0.5 mM of amiloride. These observations suggested increased utilization of the upstream alternative 5′-splice site within exon 2 of BCL-X, increased exon 11 (U) exclusion of HIPK3, and decreased splicing complexes formed for production of both RON/MISTR1 exon 11 (+) and exon 11 (-) isoforms. Thus, amiloride could modulate the AS of these three gene transcripts towards less malignant “normalized” patterns, similar to the normal ones in previous reports [15], [16], [17], [18].

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