Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo.
BACKGROUND
Differential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes. Abundant levels of these p73 variants in a variety of human cancers correlated with adverse clinical prognosis and response failure to conventional therapies, underscoring their relevance as marker for disease severity and target for cancer intervention. With respect to an equally important role for amino-truncated p73 splice forms (DeltaTAp73) and DeltaNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to DeltaEx2 and DeltaEx2/3 splice junctions and a region in exon 3B unique for DeltaN' and DeltaN.


RESULTS
Treatment of cancer cells with these ASOs resulted in a strong and specific reduction of tumorigenic p73 transcripts and proteins, importantly, without abolishing the wild-type p73 tumor suppressor form as observed with p73-shRNA. The specific antisense oligonucleotides rescued cells from apoptosis inhibition due to overexpression of their corresponding amino-truncated p73 isoform and decreased tumor cell proliferation. Furthermore, ASO-116 against DeltaEx2/3 coupled to magnetic nanobead polyethyleneimine (MNB/PEI) carriers significantly inhibited malignant melanoma growth, which correlated with a shift in the balance between endogenous TAp73 and DeltaEx2/3 towards apoptotic full-length p73.


CONCLUSIONS
Our study demonstrates the successful development of LNA-ASOs that selectively differentiate between the closely related p73 oncoproteins, and provide new tools to further delineate their biological properties in different human malignancies and for therapeutic cancer targeting.
Emmrich
Stephan
S
Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, D-18057 Rostock, Germany. emmrichs@med.uni-rostock.de
Wang
Weiwei
W
John
Katja
K
Li
Wenzhong
W
Pützer
Brigitte M
BM
D017398:Alternative Splicing; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D047368:Tumor Burden / Q000235:genetics; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C000606460:TP73 protein, human; C000606368:Trp73 protein, mouse; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; C000606369:delta Np73 protein, human; D049452:Green Fluorescent Proteins