Dz13: c-Jun Downregulation and Tumour Cell Death

Authors


Corresponding author: Crispin R. Dass,crispin.dass@vu.edu.au

Abstract

DNAzymes (DNA enzymes and deoxyribozymes) are synthetic, single-stranded DNA-based catalysts engineered to bind to their complementary sequence in a target messenger RNA (mRNA) through Watson–Crick rules for base-pairing and cleave the mRNA at predetermined phosphodiester linkages. Dz13, a DNAzyme that cleaves c-Jun mRNA, has been found to have efficacious effects against tumours directly, activity against tumour-induced angiogenesis, inhibition of neointima formation after arterial injury and control of inflammatory responses. Recent studies in endothelial cells demonstrate that the off-target effects of Dz13 may in fact be driving some of these potentially therapeutic effects, although no mechanisms have been clearly defined in tumour cells. Recent data show that Dz13 is capable of inhibiting more types of tumours and potently induces apoptosis in a panel of tumour cell lines. Hand-in-hand with in vivo testing, Dz13 has been formulated into a biocompatible nanoparticle, enabling its full potential to be realized. Its chemistry is partly responsible for its activity against tumour cells, but it is safe to use in vivo and surprisingly shows little harmful effects against normal cells. These findings provide hope that Dz13 may be useful clinically for the treatment of a variety of cancers.

The DNAzyme molecule in its most reported form is the ‘10–23’ subtype comprising a cation-dependent catalytic core of 15 deoxyribonucleotides (1) which binds to and cleaves its target RNA between an unpaired purine and paired pyrimidine through a de-esterification reaction. The core is flanked by complementary binding arms of 6–12 nucleotides in length which, via Watson–Crick base-pairing, confer specificity for the targeted mRNA. Once the target is bound, catalysis proceeds and the mRNA strand is cleaved, rendering it untranscribable.

The first report that presented data on the c-Jun downregulating DNAzyme Dz13, Zhang et al. (2), demonstrated that it blocked endothelial cell proliferation, migration, chemoinvasion and tubule formation. Dz13 was coinjected with tumour cells and was found to inhibit VEGF-induced neovascularization in the rat cornea and B16 melanoma growth in mice. There was a 2.5-fold decrease in tumour volume in the Dz13 cohort of animals. This was accompanied by a 60% decrease in the number of tumour-associated blood vessels in the B16-bearing mice. The decrease in corneal vascularization owing to VEGF stimulus was 75% in the Dz13 group of rats.

We have since found that Dz13 is also a potent inducer of caspase-2 (3), which may explain how its other major feature, an off-target effect (OTE) causing dramatic levels of apoptosis in tumour cells occurs. In various tumour cells, Dz13 also upregulates E2F1. The E2F class of transcription factors determines the timely expression of genes involved in the S phase of the cell cycle (4). More recently, it has been reported that E2Fs function in mitosis, DNA replication, DNA damage checkpoints, DNA repair, development and differentiation. One member of this transcription factor family, E2F1, is capable of inducing apoptosis via both p53-dependent and p53-independent pathways (5). Cell death via E2F1 is attributed to upregulation of pro-apoptotic genes.

The issue of OTE by Dz13 was first reported when it was found that in human dermal microvascular endothelial cells (HMEC-1) and rat SV40-transformed rat smooth muscle cells, Dz13 kills cells at doses as low as 100 nm (6). While that study had some flaws, such as the fact that RT-PCR was used to rule out c-Jun downregulation by Dz13, and no attempt to examine levels of protein was made, the authors stated that the cell growth inhibitory effect of Dz13 could be due to the G-rich 5′ sequence, although the formation of a G-quadruplex was ruled out. The importance of a G-rich 5′ sequence was confirmed later in HMEC-1 cells (7), which also demonstrated the requirement for a high proliferative index of human retinal pigmented epithelium (ARPE-19) cells for Dz13 activity to be exhibited at the low-treatment dose. In a third study, the cell growth inhibitory effect of Dz13 on HMEC-1 cells was attributed to non-apoptotic programmed cell death, although no specific pathway was elucidated (8).

Anticancer Evidence for Dz13

Previously, the deoxyribozyme Dz13 has been shown to reduce the growth of melanoma indirectly via anti-angiogenesis (2) and squamous cell carcinoma directly (9). Dz13 downregulates c-jun (its specific target) levels in rapidly proliferating cells, for instance, when cells are stimulated postquiescence and when c-Jun is elevated. More recently, Dz13 has been shown to inhibit the growth of both osteosarcoma, OS (10–12), and liposarcoma, LS (13), in orthotopic tumour models (13,14). Dz13 effects tumour growth inhibition for orthotopic growth of prostate cancer, breast cancer and OS in bone of mice when tumour cells are mixed with the oligonucleotide prior to tumour inoculation (15). Rather interestingly and promisingly, normal bone-residing cells such as osteoblasts and chondrocytes are left relatively unharmed (16), indicating a tumour cell-selective mode of action for Dz13.

While Dz13 does downregulate its target gene (c-jun) potently at doses of 400 nm, it is its OTEs that have drawn more attention recently. Dz13-mutated oligonucleotides were designed and tested in a proliferation assay, and Dz13 was also tested for its safety in vivo when administered intravenously in a bolus dose or systemically in an in utero assay (17). While Dz13 downregulated target gene (c-Jun) expression in human tumour cells, c-Jun siRNA failed to cause cell growth inhibition. Furthermore, alteration of contiguous G motifs in Dz13 flanking arms inhibits cell death activity, but removal of the same from the catalytic core can increase cell death activity. A 20mer (truncated) derivative Dz13 exhibited similar activity. Dz13 was not toxic to blood and solid tissues in adult or foetal mice, although slight hepatotoxicity was noted with histology. It was also void of adverse effects to the physiological processes of angiogenesis and apoptosis. Collectively, thus, the data support the safety of Dz13 and its activity attributed to OTEs.

When Dz13-treated tumour cells were observed with electron microscopy (EM), death of cells occurred exhibiting the classical signs of apoptosis (18) – nuclear fragmentation, chromatin condensation and membrane blebbing. In addition, mitochondrial structure was perturbed, with septae destroyed. Apoptosis was confirmed with poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, TUNEL assay, annexin-5 staining and nuclear chromatin condensation as evidenced by DAPI staining (3). Structures resembling transfection complexes were observed within cells, specifically within endosome-like organelles. Death occurred via the mitochondrial pathway as shown by cytochrome c release into the cytosol. These results put beyond doubt that the major cause for cancer cell growth inhibition by Dz13 was apoptosis induction.

Dz13-Mediated Apoptosis in Panel of Cancer Cells is Via Caspase-2 and E2F1

The next logical step for Dz13 research was how the molecule was causing apoptosis. The first examination was into finding which caspase was behind this cell kill function. After evaluation, it was found that caspase-2 was the one caspase that was activated by Dz13 in a clear majority of tumour cells (3). This set up an exploitable situation. Signalling pathways for caspase-2-mediated apoptosis are poorly defined, and one reason for this is the lack of a reproducible stimulus to trigger caspase-2 activation. Against this backdrop, Dz13 was presented as a stimulus for caspase-2 activation.

Dz13-mediated cell death occurred even in the absence of known caspase-2 molecular partners in PIDD, RAIDD or DNA-PKcs, or other caspases in cell lines of breast cancer, prostate cancer, osteosarcoma and liposarcoma. Z-VDVAD-fmk, caspase-2 −/− MEFs and siRNA silencing of caspase-2 in tumour cells abrogated Dz13-mediated cell death. When further analysis of Dz13 activity on cancer cells was undertaken, there was an upregulation of ERK, Akt and p38 (19). Most striking was the increase in expression levels of E2F1, a tumour suppressor protein (20). The tumour suppressor role played by E2F1 was observed by a rapid and sharp response (in 30–60 min) in E2F1 expression levels posttransfection of Dz13.

Dz13 Activity is Linked to Metastasis

In previous studies using orthotopic models of disease, the inhibitory effects of Dz13 on secondary growth were shown to be a direct result of growth inhibition at the primary lesion site (2,9–13). Thus, the direct and genuine effects on metastasis were not gauged. In a more recent study (21), Dz13 was able to inhibit both locoregional and distal metastasis of tumour cells in mice, in studies where the primary tumours were unaffected because of the late and clinically mimicking nature of treatment commencement. In addition, the effect of Dz13 against tumours has now been extended to encompass breast and prostate cancer. Dz13 upregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 and decreased the expression of MT1-MMP (MMP-14) in cultured tumour cells (21). While data in vitro with MMP-9 and MMP-2 were not supportive of previous studies (2,9) and of Dz13 activity against metastasis, when tested in vivo, in sections of ectopic tumours treated with Dz13, both MMP-2 and MMP-9 were downregulated. Thus, this study proved that Dz13 is also able to inhibit tumour growth at the secondary site. These findings further highlight the growing potential of Dz13 as an antineoplastic agent. There are other oligonucleotides derived from Dz13 synthetically like DzM14. The efficacy of this derivative of Dz13 is similar to that of the parent molecule (Dz13) but the oligonucleotide sequence is different and shorter (Figure 1).

Figure 1.

 The DNA-based sequence of Dz13 (top) and that of the shorter version, DzM14. The box indicates the region where the change in sequence between the long and short version is located.

Dz13 Plus Doxorubicin (Dox) as a Potential Therapeutic Strategy for Cancer

As Dz13-activation of caspase-2 caused significant tumour cell death in vitro (3), we evaluated Dz13 in orthotopic models of various tumours. Tumours (in this case, of the bone) were allowed to establish before treatment was initiated at a stage of tumour growth which resembled tumours observed when patients present to the clinic (including ours) with initial complaints such as joint tenderness and/or swelling and are scheduled for an initial X-ray and/or magnetic resonance imaging (MRI). Both primary and secondary tumours were inhibited with chitosan-based nanoparticles encapsulating Dz13 (16,22), but were further inhibited when Dz13-NP was combined with intraperitoneally administered Dox, highlighting the usefulness of Dz13-NP plus Dox for tumour death (13). However, one problem encountered was a typical toxicity to both the heart and skin when free Dox is administered systemically at even these relatively low doses to mice (15,23), an event unfortunately mimicking the human response to this cytotoxic agent.

Thus, a method for limiting Dox activity to the lesion site and avoiding normal tissue exposure was deemed necessary. To this end, a drug delivery system (DDS) for Dox was concurrently developed (16). The NP DDS for Dz13 was developed in our laboratory to overcome reliance on commercial transfection reagents, which have been used for more than a decade (24–26) prior to development of our DDS. These commercial reagents are not recommended for in vivo use mainly because of their cytotoxicity in general against mammalian cells (27,28). One other DDS commonly used for oligonucleotide administration in vivo is the commercial osmotic pump (Alzet®) which can deliver such potentially therapeutic agents such as antisense (29,30), but when used to administer Dz13, slight hepatotoxicity in mice was noted (17). Thus, as was predicted (31,32), the development of a chitosan-based DDS for Dz13 has permitted a more thorough investigation of the efficacy attached to this caspase-2-inducing molecule, and we anticipate that in future, this naturally abundant (‘green’, as chitosan is sourced from the exoskeleton of crustaceans and insects), easily formulated, stable, biocompatible and biodegradable DDS will be used for siRNAs as well.

We demonstrated that Dz13, a DNA enzyme that cleaves c-Jun mRNA, is capable of inhibiting cancer cell growth in vitro, can be encapsulated into chitosan nanoparticles. For optimization of a prototypical chitosan-based formulation (22), pH 6, 0.02% chitosan concentration and temperature of 55 °C were found to be best among the variables tested (15). Particles were 50–300 nm in diameter, and encapsulated Dz13 was active when particles were exposed to cancer cells (15). These nanoparticles were stable during storage even for a month, but were not stable in mouse and human serum. In two different clinically relevant disease models, and using a clinically adoptable dosing regimen, these Dz13-nanoparticles were shown to be efficacious against a bone tumour (osteosarcoma), for which no real cure exists currently. However, no toxicity against other bone-dwelling cells was observed with the formulation, and no side effects were noted in vivo in lymphatic and reticuloendothelial tissues proximal and distal to the administration site.

Future Directions

As listed in Table 1, Dz13 has a variety of functions against tumour cells in vitro and tumours in vivo. Some of these are attributed to its ability to downregulate c-Jun, its intended target. More recent data show that Dz13 possesses potent OTEs that give it better anticancer activity. In addition, this molecule is capable of inhibiting endothelial cell growth, a key requirement for molecules that are antiangiogenic. Summarily, Dz13 is one DNAzyme that has shown great promise as an agent that can inhibit tumour growth. Apart from the panel of tumours tested against this molecule to date, other tumour varieties should also be evaluated.

Table 1.   Major findings with Dz13 in oncological research
FindingsReference
Dz13 downregulates c-Jun(2)
Antiangiogenesis activity(2)
Anticancer activity against melanoma, squamous cell carcinoma(9)
Dz13 is safe in adult and in utero mice – 4-day continuous delivery(17)
Dz13 activates caspase-2(3)
Dz13 upregulates E2F1 (a tumour suppressor)(19)
Dz13 modulates levels of matrix metalloproteinases(21)
Antimetastatic activity(21)
Dz13 can be encapsulated into a nanoparticulate drug delivery system(14,16,22)
Efficacious against osteosarcoma, liposarcoma, prostate cancer, breast cancer(12,14,16,19,21)
Off-target effects just as important as direct anti-c-Jun effects(17)
DzM14 just as good, but shorter and hence cheaper(17)

Acknowledgments

Nil.

Conflict of interest

The authors report no declarations of interest.

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