Antisense gapmers selectively suppress individual oncogenic p73 splice isoforms and inhibit tumor growth in vivo
© Emmrich et al; licensee BioMed Central Ltd. 2009
Received: 22 June 2009
Accepted: 11 August 2009
Published: 11 August 2009
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 (ΔTAp73) and ΔNp73 (summarized as DNp73) in the tumorigenic process, we designed locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers against individual species that were complementary to ΔEx2 and ΔEx2/3 splice junctions and a region in exon 3B unique for ΔN' and ΔN.
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 ΔEx2/3 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 ΔEx2/3 towards apoptotic full-length p73.
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.
The human p73 is a member of the p53 tumor suppressor family, based on substantial structural and functional homologies. Unlike p53, p73 is rarely mutated in human cancers . As an early-recognized feature, the TP73 gene produces multiple transcripts with opposing functions. A detailed analysis of p73 in tumor cells revealed the presence of multiple N-terminally truncated isoforms (ΔN, ΔN', ΔEx2, ΔEx2/3; collectively called DNp73), which lack all or most of the transactivation domain that accounts for the tumor suppressor function of the full-length TAp73 protein. While the ΔNp73 transcript is generated from a cryptic promoter in intron 3, the majority of the amino-truncated p73 variants is produced by alternative exon splicing from the E2F1 responsive promoter in the 5'UTR upstream of the non-coding exon 1. All isoforms fail to induce cell cycle arrest and apoptosis. In addition, they act as dominant-negative competitors for DNA binding and/or heteroduplex formation with p53 and wild-type TAp73, and confer drug resistance to tumor cells harbouring wild-type p53 and/or TAp73 [2, 3]. We also demonstrated that the ΔEx2/3 isoform inactivates functions of the retinoblastoma (RB) tumor suppressor protein in cell cycle and differentiation control [4, 5]. By inactivating both major tumor suppressor pathways in human cells they are functionally analogous to several viral oncoproteins. In this sense, overexpression of ΔEx2/3 and ΔN has been shown to transform fibroblasts to tumorigenicity in nude mice [6, 7], and drive carcinogenesis in transgenic mice in vivo .
NH2-truncated p73 forms were found frequently elevated in various types of tumor cells and primary malignancies from patients, but not in the surrounding normal tissues . Beyond, increased DNp73 expression levels are strongly associated with a worse recurrence-free and overall survival of patients, and poor prognosis features such as lymph node metastasis and vascular invasion . Specifically, overexpression of ΔEx2/3p73 and ΔNp73 was associated with advanced pathologic tumor stages. Likewise, alterations in the relative levels of TAp73 and the apparently tumor-specific aberrant expression of individual oncogenic p73 species, that might account for a shift in the net function of p73 from proapoptotic to prosurvival, have been shown to correlate with prognosis in some cancers. All this strongly underscores that the expression pattern of NH2-truncated p73 is an important determinant of tumor development and the cellular response to treatments, making it a biological relevant target for cancer prevention and therapy.
Although there is increasing evidence for an equally important role of individual amino-terminal p73 splice variants and ΔNp73 in the tumorigenic process, we are currently unable to evaluate their oncogenic potency under physiological conditions in vivo. The aim of our study was to develop mono-specific inhibitors for the majority of NH2-truncated alternative-spliced p73 isoforms that allow differential knockdown of all closely related oncoproteins in growing human tumors.
Cell Culture, Viruses and Fluorescence Microscopy
Human H1299 lung cancer cells, human embryonic kidney HEK 293 cells, and human WI-38 lung fibroblasts were maintained in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany). Human SK-Mel-29 melanoma cells were cultured in DMEM containing sodium pyruvate. Medium contained 2 mM L-glutamine, 100 μg/ml penicillin, 100 U/ml streptomycin, and 1,25 μg/ml amphotericin B. The adenoviral (Ad) vectors Ad-shGFP and Ad-shp73 have been described elsewhere . Adenovirus infection was carried out at a multiplicity of infection (MOI) that allows 100% transduction of target cells. FITC and GFP imaging was performed by fluorescence microscopy at indicated time points.
Antisense Oligonucleotides, shRNA and Transient Transfections
Oligonucleotide (ON) LNA modifications and FITC-labeling was conducted by BioTeZ GmbH. ASO sequences were the following: ASO-115 5'-CTGT CTGGTTCCCTGCAGCC-3', ASO-116 5'-GATT GAACTGGGCCTGCAGC-3', and ASO-185/451 5'-GGAA CTGGTGTCCCGTGGGA-3' (LNA bases are bold). The non-sense control ON (nsc) with 5'-TAAC CGTTTCTTCCTCGTCC-3' serving as a negative control was verified by NCBI blastn search. For in vivo experiments a scrambled ON (sc) to ASO-116 with the same base content was generated using the siRNA Wizard™ platform at http://www.sirnawizard.com (ASO-sc: 5'-GCCC AGAAGGTGCCTAGGTT-3'). ASO transfections were performed with Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany). Cotransfection experiments were carried out with 2 μg plasmid mixed with respective amounts of antisense molecules. Plasmid encoding shRNA was transfected using Effectene (Qiagen, Hilden, Germany). The RNA interference target sequence was 5'-GGCCATGCCTGTTTACAAG-3'.
Quantitative Real-Time PCR (qPCR)
Total RNA was extracted with the NucleoSpin Kit (Macherey-Nagel, Düren, Germany) and reverse transcribed with Omniscript RT (Qiagen, Hilden, Germany) using Oligo(dT)18 primer. The cDNA sample was mixed with iQ SYBR Green Supermix (Biorad, München, Germany). QRT-PCR was performed on iQ5 Multicolor Real-Time PCR Detection System (Biorad, München, Germany) using 1/10 volume of RT reaction. Relative gene expression was calculated using iQ5 Optical System Software. Primer sequences are available upon request.
Cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 0.1% Triton X-100, 5 mM EDTA) supplemented with protease inhibitor mixture Complete Mini (Roche Applied Science, Mannheim, Germany) and total protein concentration was quantified by a modified Bradford assay. Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-p73 ER-15 (BD Biosciences, Heidelberg, Germany), anti-ΔNp73 (Imgenex, San Diego, CA, USA) or anti-β-Actin (Sigma-Aldrich, Taufkirchen, Germany). Primary antibodies were detected with appropriate secondary antibody-horseradish peroxidase conjugates according to the enhanced chemoluminescence protocol.
Hoechst 33342 Staining and Caspase-3 Activity
After treatment, cells were incubated with Hoechst 33342 at 1 μg/ml for 15 min and subjected to fluorescence microscopy. Hoechst-stained cells were harvested, counterstained with 10% trypan blue solution, and counted in a hämcytometer. Caspase-3 activity was assayed using the ApoAlert kit (Takara Bio, Saint-Germain-en-Laye, France) as described by the manufacturer. Absorbance was measured at 405 nm in a spectrophotometer.
Cell Proliferation Assay
Cells were seeded at a density of 5 × 104, serum-starved for 24 h, and transfected with PEI-ASO complexes at day 1 and 3 after plating. DNA synthesis was measured by Cell Proliferation BrdU (5-bromo-2'-deoxyuridine)-ELISA (Roche Applied Science, Mannheim, Germany) according to the instructions.
Magnetic Nanobead (MNB)/Polyethyleneimine (PEI)/DNA Complex Formation
Branched PEI (average MW 25 000) (Sigma-Aldrich, Taufkirchen, Germany) was purified through dialysis in three changes of a 100-fold volume excess of water, lyophilized and re-hydrated before use. PEI/ASO complexes were prepared at the N/P ratio of 8. The PEI solution and DNA solution at a given N/P ratio were diluted in 5% glucose to ensure iso-osmolarity for transfection experiments and in vivo application. PEI was added to DNA and immediately mixed and incubated for 30 min at RT. For intratumoral injections, PEI/ASO complexes were conjugated with streptavidin-coated magnetic nanobeads (MNBs) (Promega, Mannheim, Germany) by vortexing for 30 s followed by incubation at RT for 30 min. The resulting MNB/polymer/ASO complexes are stable in aqueous solution and can be stored at 4°C for several days.
Animal Experiments and In Vivo Imaging
Tumors were established by subcutaneous (s.c.) injection of 107 SK-Mel-29 cells into the rear flanks of 6- to 8-week-old female athymic nu/nu mice. Animals with palpable tumors were randomized in three groups to ensure uniform distribution: Group A untreated control (n = 9 tumors), group B treated with PEI/ASO-sc (n = 8 tumors), and group C treated with PEI/ASO-116 (n = 13 tumors). For magnetic force transduction, animals with tumors of diameter >8 mm were randomized into two groups (group D: MNB/PEI/ASO-sc, n = 6 tumors; group E: MNB/PEI/ASO-116, n = 10). Mice were anaesthetized by intraperitoneal injection of body-weight adapted doses of 10% ketamine and 2% xylazin. Oligonucleotide complexes were intratumorally administered at 1.5 mg/kg/day. In magnetic bead guided experiments, a magnet sized 4 × 1 mm with 1.41–1.45 T was placed closely adjacent to the tumor. Tumor volumes were measured with calipers and calculated from the longest diameter and average width by assuming a prolate spheroid shape (tumor volume = π/6 × (large diameter × [shortdiameter]2). The relative tumor volume (RTV) was determined using RTV = V i /V0, where V i is the daily-measured tumor volume and V0 is the initial tumor volume. After treatment, mice were sacrificed and representative tumor specimen of each experimental group were dissected. Total RNA was extracted and quantities of ΔEx2/3p73 and TAp73 transcripts were determined by qPCR. All animal procedures were conducted in adherenceto ethical standards and with approval of the local Animal CareCommittee.
For fluorescence detection, PEI was labeled by Oregon green 488 Protein labeling kit (Invitrogen, Karlsruhe, Germany) and injected intratumorally into mice. In vivo imaging was performed at 1, 8 and 24 h after injection. Images were acquired using NightOwl LB981 imaging system (Berthold Technologies, Bad Wildbad, Germany) with an exposure time of 100 s. For colocalization of the fluorescent image on the animal body, gray scale and pseudocolor images were merged. Quantification of signal intensity in all animals was performed by WinLight32 Software.
Computational Analysis and Secondary Stucture Predictions
Human genomic sequences for the 5'UTRs of TP73 transcripts were obtained from ENSEMBL database. The binding energies as well as the propability matrices for single-stranded target sequences were calculated using the Sfold server application module Soligo. Each N-terminal p73 transcript was calculated with the C-terminal configurations α, β, γ and δ, and their propability diagrams were merged at the indicated nucleotide positions. In silico secondary structure prediction was carried out using the CLC RNA Workbench Software (CLC Bio, Aarhus N, Denmark). For each N-terminal p73 transcript C-terminal configurations α, β, γ, δ and ε were used to calculate the mRNA structure. The respective ASO epitopes at the N-termini of these mRNAs were then compared for structural identity in the ASO target sequence.
Statistical analysis of tumor growth in nude mice was performed with one-way analysis of variance (ANOVA). Variances in BrdU incorporation, caspase activation and viable cell counts were compared with an unpaired Student's t-test (two-sided).
Results and Discussion
Design of LNA-DNA gapmers against oncogenic NH2-truncated p73 isoforms
The optimal target sequence was determined in silico by three parameters: (a) a probability profile for single-stranded regions in the mRNA, which serve as docking stations for initial oligo annealing [15, 16], (b) a minimum free energy model of the secondary structure of the mRNA with a more recent compilation of Turner's free energy parameters , and (c) the binding energies for a given ASO sequence on the mRNA . The probability plot for ΔEx2p73 mRNAs indicates single-strands downstream of nucleotide (nt) position 120 and around position 130 [see Additional file 1A]. This was confirmed by secondary structure prediction for ΔEx2p73 transcripts, where two bulges of more than 4 bases were present at positions 121 and 130, exposing the splice junction for oligo binding [see Additional file 1E]. Since mRNA 3D-structure depends predominantly on nearest neighbour interactions, alterations in the C-terminus may affect folding of N-terminal domains . Therefore, structure predictions were carried out for several C-terminal isoform configurations. Binding energies for ASO-115 (number indicates starting base of ASO position on the mRNA) targeting ΔEx2p73 mRNA reached values around -8 kcal/mol [see Additional file 2]. According to the Soligo algorithm using ≤ -8 kcal/mol as default filter criteria for potent ASOs, the values for ASO-115 were considered sufficient. Similar results were obtained for ASO-116. Analysis for single-stranded sections revealed a high probability for unpaired mRNA at the splice junction of ΔEx2/3p73 transcripts, which was confirmed by the predicted structure model [see Additional file 1B, F]. In the ΔNp73 and ΔN'p73 ASO, where the target region is of different location in each transcript owing to distinct transcriptional and mRNA maturation processes, ASO-185/451 denotes the starting positions for the ON in both isoforms. The probability plots and secondary structures of ΔNp73 showed the minimum of 4 unpaired bases, which are sufficient for initial oligo annealing [see Additional file 1C, G] . Likewise the predicted epitopes and probability plots for ΔN'p73 transcripts suggest mRNA accessibility at the target region of ASO-185/451 [see Additional file 1D, H].
ASOs induce selective suppression of target RNAs
The main goal of our study was to design ASOs that allow specific knockdown of individual oncogenic p73 forms. As demonstrated in Figure 2F, ASO-115 induced a complete decrease of ΔEx2p73 mRNA to 0.03% of the control, whereas expression of TAp73 and other non-targeted p73 variants remained largely unaffected. Similar data was obtained with the ASO-116 against ΔEx2/3p73 (Figure 2G). Interestingly, both ASOs targeting the splice junction of exon 1 have very little effect on ΔNp73 transcripts, which lack exons 1 to 3 due to alternative promoter usage. Analogous to this, ASO-185/451 revealed no silencing impact on the ΔEx2 and ΔEx2/3 transcripts because of the absence of exon 3B in these variants (Figure 2H). These results indicate that all developed ASOs exhibit a selectively high inhibitory efficiency against their target, resulting in no or low knockdown of other p73 isoforms. Comparable high isoform-specific knockdown activities of these ASOs were also observed in H1299 tumor cells with amino-truncated p73 transcripts upregulated [see Additional file 3].
Effect of site-directed targeting on DNp73 protein level
This is in sharp contrast to the effect of a p73-shRNA. The use of RNAi is restricted to sequences that fullfill special criteria, such as the GC content, A at position 19 or absence of internal repeats . According to these parameters, the target sequence of the p73-shRNA is located in exon 5 of the transcript. In contrast to the data observed with the NH2-truncated isoform-specific gapmers, transfection of HEK293 cells with a plasmid encoding shRNA against p73 resulted in a general silencing of TAp73 and oncogenic forms up to 80% of the scrambled-shRNA, with the strongest potency against the wildtype transcript (Figure 3C, upper panel). This unspecific inhibitory effect was also confirmed on protein level. In H1299 cells infected with an adenoviral vector expressing p73-shRNA, we found all N-terminal isoforms and TAp73 equally suppressed at 48 h after infection (Figure 3C, bottom panel) compared to the control virus Ad-shGFP. From these results, we conclude that site-directed targeting of ΔEx2 and ΔEx2/3 splice junctions and a unique region in Exon3B for ΔN' and ΔN by antisense ON is a feasible technique for selective knockdown of individual p73 oncogenes.
Antitumorigenic activity of DNp73 ASOs
In order to improve in vivo administration, magnetic nanobeads (MNBs) were used to prevent diffusion of the coupled PEI/ASO complex from the injection site, and PEI/ASO-116 coated to MNBs versus PEI/ASO-116 alone were tested for their antitumoral effect in presence of a magnet implanted near the tumors. Consistent with the inhibitory effect on cell proliferation illustrated in Figure 5C, tumor growth curves revealed a robust decrease in growth rates when treated with ASO-116 independent of the mode of delivery (Figure 5E). However, with increasing treatment tumors injected with ASO-116 under magnetic force-guidance were significantly smaller than those treated with PEI/ASO-116 alone. This demonstrates that by keeping the ASO concentrated in the tumor, we could enhance its specific therapeutic efficacy. Quantification of p73 transcripts in the tumors after final injection revealed an equally strong suppression of ΔEx2/3p73 for the PEI- and MNB/PEI-delivered antisense oligonucleotide (8.5-fold and 7-fold) compared to the control groups (Figure 5F). Of note, enforced antitumorigenic activity of ASO-116 directly correlated with the upregulation of the tumor suppressive TAp73 form, which showed a more than 2 times higher increase when tumors were subjected to magnetic force-guided ASO-116 treatment. These data suggest that inhibition of TAp73 in response to increased ΔEx2/3p73 expression in growing tumors is the mechanism responsible for ΔEx2/3p73-mediated tumor growth.
Based on present studies, NH2-truncated p73 isoforms are upregulated in the majority of tumors with a concomitant rise of TAp73 , suggesting that the specific ratio between the apoptotic TAp73 form and different dominant-negative p73 variants determines the functional outcome of p73 and the cell fate. In fact, alterations in the relative levels of truncated p73 and TAp73 and/or p53 correlate with therapy failure and poor prognosis in many cancers [10, 25, 26]. In this regard, downregulation of the ΔNp73 species by antisense technique has been shown to alleviate its suppressive action and to enhance p53/TAp73-mediated apoptosis in cancer cells in response to chemotherapy . In turn, mice harbouring a specific knockout of TAp73 are tumor-prone and sensitive to carcinogens. Tomasini et al. reported that 73% of TAp73-/- mice spontaneously develop malignancies, thereby establishing TAp73 isoforms as bona fide tumor suppressors . Furthermore, this group showed that TAp73 is regulating the spindle assembly checkpoint (SAC) complex, suggesting that SAC impairment leads to genomic instability and aneuploidity in TAp73-deficient cells. Hence, the ability to induce and maintain proper mitotic arrest could be a mechanism of TAp73-mediated anti-tumorigenicity . Both studies emphasize the functional importance of an imbalanced TAp73:DNp73 ratio. Taking into account that the expression levels of individual DNp73 species might be tumor-specific and particularly alternative-spliced isoforms ΔEx2/3, ΔEx2, and ΔN' that mimic the TAp73 knockout are predominantly expressed in many cancers including malignant melanoma [10, 29–33] rather than ΔNp73, specific inhibitors for these potential oncogenes are needed. In this study, we show that selective knockdown of a single p73 splice product leads to the inhibition of melanoma tumor growth, and at the same time to the induction of TAp73, underscoring that the ratio between TAp73 and ΔEx2/3p73 accounts for its oncogenic activity. Consistent with the induction of apoptosis after ΔNp73 knockdown, ASO-mediated suppression of ΔTAp73 isoforms was sufficient to abrogate apoptosis resistance to chemotherapy mediated by ectopically expressed ΔTAp73. Enhanced chemosensitivity by inhibition of ΔTAp73 spliced isoforms was recently demonstrated in neuroblastoma using the cyclooxygenase inhibitor celecoxib, which blocks both ΔEx2/3p73 and ΔEx2p73 without isoform selectivity .
One of the most required desirements for LNA-ASOs is their efficient delivery to target cell nuclei. Polymers of cationic polyethyleneimine are well-studied compounds that improve the in vitro and in vivo penetrance of ASOs to cells and tissues , yet PEI-mediated DNA transfer is not tumor directed. Thus, we aimed at constructing targeted non viral vectors based on magnetic force-guided polyplexes by coating PEI/ASO to nanoparticles that allow enforced cellular uptake by complex concentration at the tumor site. As a result, mice that received accumulating intratumoral injections of MNB/PEI-ASO complex against ΔEx2/3 showed a stronger degree of tumor growth inhibition than those treated only with PEI/ASO, indicating that increased antisense activity, substantiated by a marked induction of TAp73, correlates with higher transfection efficiency in tumor cells. Since the release of p73/p53 tumor suppressor function from ΔTAp73-mediated inhibition has been linked to chemosensitivity , the use of oncogenic p73 ASOs as a therapeutic strategy for cancer treatment could be further improved by combination with other genotoxic agents.
We have developed LNA-modified ASOs that downregulate individual aberrantly expressed ΔEx2, ΔEx2/3, ΔN', and ΔN p73 forms in neoplastic cells and growing tumors with high specificity. ASO-mediated modulation of endogenous isoform expression resulted in tumor growth inhibition in vivo accompanied by the induction of apoptotic TAp73. The data of this study support the application of NH2-truncated p73 inhibitors as valuable tools to delineate their biological role in human cancers and as anticancer agents.
This work was supported by the Medical Faculty FORUN program grant 889012 and SFB Transregio 37. We thank Anja Stoll for technical assistance and Kathrin Sievert-Küchenmeister for giving advice in carrying out magnet-guided mice experiments.
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