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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
10:06

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Published on: April 26, 2017

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

Jan-Gowth Chang1, Den-Mei Yang, Wen-Hsin Chang

  • 1Department of Medical Research, University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. jgchang@ms.kmuh.org.tw

Plos One
|June 23, 2011
PubMed

Insights

Amiloride normalizes cancer cell RNA splicing by affecting splicing factors and signaling pathways. This leads to reduced cancer cell invasion, migration, and survival, offering a potential new cancer therapeutic strategy.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Drug Discovery

Background:

  • Alternative splicing generates diverse protein isoforms, and its dysregulation is linked to cancer malignancy, including chemo-resistance and invasion.
  • Identifying small molecules that modulate RNA splicing is crucial for developing novel cancer therapeutics.

Purpose of the Study:

  • To screen for small molecules that can modulate RNA splicing in human hepatocellular carcinoma (HCC) cells.
  • To investigate the molecular mechanisms by which amiloride affects RNA splicing and cancer cell phenotypes.
  • To explore the therapeutic potential of amiloride in cancer treatment.

Main Methods:

  • Screening of small molecules for RNA splicing modulation in Huh-7 HCC cells.
  • Proteomic analysis to identify changes in splicing factors and signaling pathway proteins.
  • Global exon array analysis to detect splicing pattern alterations.
  • Cellular functional assays to assess invasion, migration, cell cycle, and DNA integrity.

Main Results:

  • Amiloride selectively "normalized" aberrant splicing of BCL-X, HIPK3, and RON/MISTR1 transcripts in Huh-7 cells.
  • Amiloride treatment led to hypo-phosphorylation of splicing factor SF2/ASF and altered levels of other SR proteins.
  • Proteomic analysis revealed amiloride-induced changes in kinase/phosphatase activity affecting splicing factor phosphorylation, which were reversible with a PP1 inhibitor.
  • Global exon array identified widespread splicing changes in 551 genes, impacting various cellular functions.
  • Amiloride treatment impaired cancer cell invasion, migration, cell cycle progression, and induced DNA degradation.
  • Similar splicing alterations and cytotoxic effects were observed in other cancer cells, but not significantly in normal cells.

Conclusions:

  • Amiloride acts as a novel RNA splicing modulator, distinct from its pH-altering effects.
  • The mechanism involves down-regulation of specific kinases and up-regulation of phosphatases impacting splicing factor phosphorylation.
  • Amiloride-induced splicing normalization and subsequent cellular defects present a promising avenue for cancer therapeutics.

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