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Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo.

Emmrich S, Wang W, John K, Li W, Pützer BM - Mol. Cancer (2009)

Bottom Line: Differential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, D-18057 Rostock, Germany. emmrichs@med.uni-rostock.de

ABSTRACT

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.

Conclusion: 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.

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Site-directed targeting of NH2-truncated p73 mRNAs by LNA gapmers. (A) Structure of the human TP73 gene. Exons are shown as boxes and structured according to the domain: transactivation domain (blue); exon 3B-derived coding sequence (purple); DNA-binding domain (red); oligomerization domain (yellow); COOH terminus (green). C-terminal splice variations are indicated. The transcriptional start sites of the two promoter regions (TA-promoter, ΔN-promoter) are marked by arrows. Aberrantly spliced transcripts regulated by the TA-promoter are labeled ΔEx2, ΔEx2/3, and ΔN'. The unique 5' untranslated region of the ΔN transcript is colored grey. (B) Binding sites of gapmer ASOs in different DNp73 transcripts. ASO-115/ΔEx2p73, ASO-116/ΔEx2/3, and ASO-185/451 directed against ΔN'p73 and ΔNp73 mRNA. LNA bases are in bold.
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Figure 1: Site-directed targeting of NH2-truncated p73 mRNAs by LNA gapmers. (A) Structure of the human TP73 gene. Exons are shown as boxes and structured according to the domain: transactivation domain (blue); exon 3B-derived coding sequence (purple); DNA-binding domain (red); oligomerization domain (yellow); COOH terminus (green). C-terminal splice variations are indicated. The transcriptional start sites of the two promoter regions (TA-promoter, ΔN-promoter) are marked by arrows. Aberrantly spliced transcripts regulated by the TA-promoter are labeled ΔEx2, ΔEx2/3, and ΔN'. The unique 5' untranslated region of the ΔN transcript is colored grey. (B) Binding sites of gapmer ASOs in different DNp73 transcripts. ASO-115/ΔEx2p73, ASO-116/ΔEx2/3, and ASO-185/451 directed against ΔN'p73 and ΔNp73 mRNA. LNA bases are in bold.

Mentions: Differential mRNA splicing and alternative promoter usage of the human TP73 gene results in the expression of multiple N-terminally truncated forms (ΔEx2, ΔEx2/3, ΔN', ΔN; Figure 1A) that act as oncogenes. Current evidence suggests an equally important role for individual p73 splice variants and ΔNp73 in the tumorigenic process, but there is no attempt so far for their selective silencing without concomitant reduction of the apoptotic TAp73 form. In order to develop mono-specific inhibitors for each amino-terminal isoform, we used a modified antisense technology. According to previous reports indicating that ONs with locked nucleic acids (LNA) at their 3'- and 5'-ends efficiently recruit RNase H and promote degradation of target RNA [12,13], we designed 20-mer LNA-DNA gapmers with a central DNA region of 12 nucleotides and 3'-5' ends of four LNA monomers. LNA bases have been shown to confer resistance to nucleases when incorporated at the 5' and 3' ends of oligomers [14]. Antisense specificity for ΔEx2p73 and ΔEx2/3p73 was achieved by using the splice junction of each exon deletion variant (Figure 1B). The ASO targeting ΔN' and ΔN was directed against a region in the intron-derived exon 3B, which is unique to both isoforms.


Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo.

Emmrich S, Wang W, John K, Li W, Pützer BM - Mol. Cancer (2009)

Site-directed targeting of NH2-truncated p73 mRNAs by LNA gapmers. (A) Structure of the human TP73 gene. Exons are shown as boxes and structured according to the domain: transactivation domain (blue); exon 3B-derived coding sequence (purple); DNA-binding domain (red); oligomerization domain (yellow); COOH terminus (green). C-terminal splice variations are indicated. The transcriptional start sites of the two promoter regions (TA-promoter, ΔN-promoter) are marked by arrows. Aberrantly spliced transcripts regulated by the TA-promoter are labeled ΔEx2, ΔEx2/3, and ΔN'. The unique 5' untranslated region of the ΔN transcript is colored grey. (B) Binding sites of gapmer ASOs in different DNp73 transcripts. ASO-115/ΔEx2p73, ASO-116/ΔEx2/3, and ASO-185/451 directed against ΔN'p73 and ΔNp73 mRNA. LNA bases are in bold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734544&req=5

Figure 1: Site-directed targeting of NH2-truncated p73 mRNAs by LNA gapmers. (A) Structure of the human TP73 gene. Exons are shown as boxes and structured according to the domain: transactivation domain (blue); exon 3B-derived coding sequence (purple); DNA-binding domain (red); oligomerization domain (yellow); COOH terminus (green). C-terminal splice variations are indicated. The transcriptional start sites of the two promoter regions (TA-promoter, ΔN-promoter) are marked by arrows. Aberrantly spliced transcripts regulated by the TA-promoter are labeled ΔEx2, ΔEx2/3, and ΔN'. The unique 5' untranslated region of the ΔN transcript is colored grey. (B) Binding sites of gapmer ASOs in different DNp73 transcripts. ASO-115/ΔEx2p73, ASO-116/ΔEx2/3, and ASO-185/451 directed against ΔN'p73 and ΔNp73 mRNA. LNA bases are in bold.
Mentions: Differential mRNA splicing and alternative promoter usage of the human TP73 gene results in the expression of multiple N-terminally truncated forms (ΔEx2, ΔEx2/3, ΔN', ΔN; Figure 1A) that act as oncogenes. Current evidence suggests an equally important role for individual p73 splice variants and ΔNp73 in the tumorigenic process, but there is no attempt so far for their selective silencing without concomitant reduction of the apoptotic TAp73 form. In order to develop mono-specific inhibitors for each amino-terminal isoform, we used a modified antisense technology. According to previous reports indicating that ONs with locked nucleic acids (LNA) at their 3'- and 5'-ends efficiently recruit RNase H and promote degradation of target RNA [12,13], we designed 20-mer LNA-DNA gapmers with a central DNA region of 12 nucleotides and 3'-5' ends of four LNA monomers. LNA bases have been shown to confer resistance to nucleases when incorporated at the 5' and 3' ends of oligomers [14]. Antisense specificity for ΔEx2p73 and ΔEx2/3p73 was achieved by using the splice junction of each exon deletion variant (Figure 1B). The ASO targeting ΔN' and ΔN was directed against a region in the intron-derived exon 3B, which is unique to both isoforms.

Bottom Line: Differential mRNA splicing and alternative promoter usage of the TP73 gene results in the expression of multiple NH2-truncated isoforms that act as oncogenes.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Vectorology and Experimental Gene Therapy, Biomedical Research Center, University of Rostock, D-18057 Rostock, Germany. emmrichs@med.uni-rostock.de

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus