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Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells
Published on: April 26, 2017
Alternative splicing of the FGF antisense gene: differential subcellular localization in human tissues and esophageal
Shuo Cheng Zhang1, Christie Barclay, Leigh Ann Alexander
1Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Canada.
Abstract:
Overexpression of FGF-2 is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer, and these risks are reduced in tumors co-expressing the FGF antisense (FGF-AS) RNA. The aim of this study was to characterize the expression of alternatively spliced FGF-AS transcripts and encoded nudix-motif proteins in normal human tissues and in esophageal adenocarcinoma, and to correlate their expression with clinicopathologic findings and outcome. Three alternatively spliced FGF-AS transcripts encoding GFG/NUDT6 isoforms with distinct N termini were detected in various human tissues including esophageal adenocarcinoma. Expression of each isoform as a fusion protein with enhanced green fluorescent protein revealed differential subcellular trafficking: hGFGa is localized to mitochondria by an N-terminal targeting sequence (MTS), whereas hGFGb and hGFGc were localized in the cytoplasm and nucleus. Mutation/deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization. The predominant FGF-AS mRNA expressed in esophageal tumors was splice variant b. GFG immunoreactivity was detected in the cytoplasm of all esophageal adenocarcinomas and in 88% of tumor cell nuclei. Although we found a trend towards reduced disease-free survival in patients with FGF-2 overexpressing esophageal adenocarcinomas, significantly worse disease-free survival was noted among patients whose tumors did not also overexpress the FGF-AS b isoform (p = 0.03). Tetracycline-inducible FGF-AS b expression in stably transfected human Seg-1 esophageal adenocarcinoma cells resulted in a significant suppression of steady state FGF-2 mRNA content and cell proliferation. Our data implicate the FGF-AS b isoform in modulation of FGF-2 expression and clinical outcome in esophageal adenocarcinoma.
Insights
FGF-AS b isoform overexpression in esophageal tumors correlates with better disease-free survival by modulating FGF-2 expression. This finding suggests FGF-AS b as a potential therapeutic target for esophageal adenocarcinoma.
Area of Science:
- Molecular biology
- Oncology
- Biochemistry
Background:
- Fibroblast Growth Factor 2 (FGF-2) overexpression is linked to esophageal cancer recurrence and poor survival.
- FGF antisense (FGF-AS) RNA co-expression reduces these risks.
- Alternative splicing of FGF-AS transcripts generates distinct protein isoforms with varying functions.
Purpose of the Study:
- To characterize alternatively spliced FGF-AS transcripts and their encoded proteins in normal tissues and esophageal adenocarcinoma.
- To correlate FGF-AS expression with clinicopathologic features and patient outcomes.
- To investigate the functional role of FGF-AS b in regulating FGF-2 expression and cell proliferation.
Main Methods:
- Detection and characterization of alternatively spliced FGF-AS transcripts using molecular techniques.
- Expression of FGF-AS isoforms as fusion proteins with enhanced green fluorescent protein (EGFP) to study subcellular localization.
- Mutation/deletion analysis to confirm the role of the N-terminal targeting sequence (MTS).
- Immunohistochemistry to assess GFG protein expression in esophageal tumors.
- Functional studies involving tetracycline-inducible FGF-AS b expression in esophageal adenocarcinoma cell lines.
Main Results:
- Three alternatively spliced FGF-AS transcripts encoding GFG/NUDT6 isoforms (hGFGa, hGFGb, hGFGc) were identified.
- hGFGa localized to mitochondria, while hGFGb and hGFGc were found in the cytoplasm and nucleus.
- The predominant FGF-AS mRNA in esophageal tumors was splice variant b.
- FGF-AS b overexpression was significantly associated with better disease-free survival in esophageal adenocarcinoma patients (p = 0.03).
- Inducible expression of FGF-AS b suppressed FGF-2 mRNA levels and cell proliferation in esophageal adenocarcinoma cells.
Conclusions:
- The FGF-AS b isoform plays a crucial role in modulating FGF-2 expression in esophageal adenocarcinoma.
- FGF-AS b expression is a significant predictor of improved disease-free survival.
- FGF-AS b represents a potential therapeutic target for esophageal cancer.
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