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Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Hepatocellular Carcinoma Huh-7 Cells

ABSTRACT: Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation and“normalized”oncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. 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, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons 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. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics.

ORGANISM(S): Homo sapiens

PROVIDER: GSE24581 | GEO | 2011/03/31

SECONDARY ACCESSION(S): PRJNA132483

REPOSITORIES: GEO

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Project description:Screening small molecules and drugs for activity to modulate alternative splicing, we found that amiloride, distinct from four other intracellular pH-affecting analogues, could “normalize” the splicing of BCL-X, HIPK3 and RON/MISTR1 transcripts in human hepatocellular carcinoma Huh-7 cells. To elucidate the underlying mechanisms, our proteomic analyses of amiloride-treated cells detected hypo-phosphorylation of splicing factor SF2/ASF and also decreased levels of SRp20 and two un-identified SR proteins. We further observed decreased phosphorylation of AKT, ERK1/2 and PP1, while increased phosphorylation of p38 and JNK, suggesting that amiloride treatment down-regulated kinases and up-regulated phosphatases in the signal pathways known to affect the splicing factor protein phosphorylation. The amiloride effects of splicing factor protein hypo-phosphorylation and“normalized”oncogenic RNA splicing were both abrogated by pre-treatment with a PP1 inhibitor. We then performed global exon array analysis of Huh-7 cells treated with amiloride for 24 hours. 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, many of which play key roles in functional networks of ion transport, extracellular matrix, cytoskeletons 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. This study thus provides mechanistic underpinnings for exploiting small molecule modulation of abnormal RNA splicing for cancer therapeutics. Target was prepared from 3 biological replicates of 0.5mM amiloride-treatment for 24hr and 3 control untreatment from Huh7 cell line and hybridized to the Affymetrix Human Exon 1.0 ST Array. The values of the hybridized probesets were normalized and analyzed for gene expression using dChip2010 software.

Project description:Alternative splicing (AS) is a process that enables a mRNA to generate different protein isoforms that may have different biological functions or properties. Cancer cells often use this maneuverability to produce proteins that contribute to growth and survival. In previous study, we found that the antihypertensive drug amiloride has a novel biological function that regulates the AS on human cancer cells. However, it also showed that the effective concentration of amiloride in cancer treatment is too high to limit its use in future therapeutic application. In this study, we therefore used computational algorithms to predict the potential amiloride derivatives and found that BS008 is the most promising compound in all derivatives. The results demonstrated that BS008 can modulate the AS of apoptotic gene transcripts, including HIPK3, SMAC, and BCLX in cancer cells. Splicing regulatory involved a variety of histone modifications and splicing factors, and accompanied the changed of phosphorylation state of SR proteins during the splicing process. Inhibition of protein phosphatase-1 with okadaic acid pretreatment partially alleviated the effects of BS008 on the AS of HIPK3 and SMAC transcripts, as well as on the phosphorylation levels of SRSF3. RNA sequencing further discovered that the AS of many other apoptosis-related gene transcripts such as AATF, ATM, AIFM1, NFKB1, and API5 was modulated by BS008. We extended the study to apoptosis-associated molecules, and detected decreased levels of BCLX and increased levels of BAX and active CASPASE-3 in BS008-induced apoptosis. In vivo experiments indicated that the treatment of tumor-bearing mice with BS008 resulted in a marked decrease in tumor size. The combination BS008/sorafenib enhances anti-tumor activity of sorafenib and allows dose reduction of sorafenib without compromising its anti-tumor activity. In conclusion, these findings suggested that BS008, an amiloride derivative, may provide therapeutic potential for cancer treatment in future.

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Small Molecule Amiloride Modulates Oncogenic RNA Alternative Splicing to Devitalize Human Hepatocellular Carcinoma Huh-7 Cells

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