As opposed to conventional therapy, traditionally, that is, in the case of loss of function, gene therapy aims at permanent correction of a defected or missing gene by replacing with or providing, respectively, the corrected version—for example, by the introduction of plasmid DNA (pDNA). Ideally, this approach translates into a single treatment, or few initial treatments, rather than several (or life long) required to provide the patients with the functional form of the protein. However, this permanent correction treatment has proven very challenging.
In the last twenty years, new nucleic acids with attractive therapeutic properties were discovered, notably, siRNA and microRNAs. Small interference RNA (siRNA) has the ability to specifically silence protein expression—an asset particularly valuable for antiviral and cancer regimens. In general, also miRNA negatively regulates gene expression, although via two different mechanism depending on the degree of complementarity towards its mRNA target. Nucleic acid-based approaches offer several advantages when compared to treatment with small molecules or proteins. They can be seen as mostly inactive prodrugs, which are activated at the tumor site producing a therapeutically active protein or knocking down a specific target gene. Importantly, nucleic acid targeted delivery systems, preferably also relying in transcriptional targeting, decreasing off-target effects and toxicity, and permitting a systemic administration otherwise not feasible with a therapeutic agent with toxic properties.
In parallel with new therapeutic nucleic acid tools, the last two decades brought insight into tumorgenesis in general and unveiled a plethora of therapeutic concepts against cancer (). The following paragraphs will deal with different antimelanoma approaches based on nucleic acids.
Figure 3 Different strategies used in antitumor nucleic acid approaches. RNA-based strategies are commonly used to downregulate agents that are upregulated to favor cell proliferation or migration, such as Bcl-2. Alternatively, double stranded RNA (dsRNA) mimic (more ...)
Despite the apparent tumor tolerance, humoral and cellular immune responses are naturally generated against tumor antigens. Hence, whether the tumor grows as a result of stealth and nonrecognition or as the result of escape and immunological shaping [128
], its recognition by the immune system can still be prompted. Indeed, at a later stage, during the progressive growth phase, tumors may become more immune-activating for varies reasons: damage or disruption of surrounding tissue, generation of reactive oxygen species, upregulation of stress protective factors, or death by necrosis or apoptosis. However, at this stage, it is not known whether the tumor still needs to escape immune recognition, as it is unclear that these immune responses can cause tumor destruction [128
]. Therefore, a number of studies have focused in eliciting earlier and suitable tumor recognition by the immune system. In a nucleic acid therapy context, this transliterates into genetic immunization or DNA vaccination: the delivery and transcription of a gene encoding antigens or immunestimulatory molecules that elicit an immune response. As an example, interleukine-12 (IL-12) has been used and studied in different animal models [104
]. IL-12 is originally produced by mononuclear phagocytes and dendritic cells and is responsible for activating NK and CD4+
T cells and inducing the production of high levels of interferon gamma (INF-γ
). Interestingly, IL-12 has been described to increase antitumor immune responses [129
], and later studies investigated its suitability for a DNA vaccine approach against melanoma [106
]. IL-12 effects appeared to be long lasting and efficient against tumor metastases, although not mainly mediated by INF-γ
]. The murine studies also revealed moderate toxicity caused by IL-12, and while lower IL-12-encoding pDNA doses can be administered, ideally the gene expression should be controlled, regarding the tissue and the durability of the expression. Although DNA vaccination against a strong melanoma tumor antigen should be possible, the authors have not seen an effect on lung metastases when using melanoma-associated glycoprotein 100 (gp 100)/pmel17 pDNA alone. Adjuvants appear to be necessary for a successful DNA vaccination: the authors have seen an effect when the gp 100-pDNA was administered together with IL-12, similar to other murine study where granulocyte-macrophage colony-stimulating factor was used [107
]. Alternatively, in a canine study, the developed vaccine was based on the human (rather than canine) gp 100 protein [108
], where the human form of the antigen acted as adjuvant. Together with gp 100, and for the case of melanoma, two more tumor genes have been described for DNA vaccination: MART-1 and tyrosinase [108
Also, the expression of chemokines, such as monocyte chemoattractant protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10), can mediate an immune response. In particular, IP-10 as been described by Sgadari et al. as an antitumor agent and found to promote damage in established tumor vasculature as well as tissue necrosis in a murine model for the human Burkitt lymphomas [131
]. Based on this, and after their studies with IL-12, Keyser and collaborators have investigated the efficiency of IP-10-encoding pDNA therapy in murine melanoma models [110
]. The authors have used two murine tumor models, whereupon cells have been injected subcutaneously (originating a solid tumor) or intravenously, inducing lung metastases. When administered alone, and intramuscularly (resulting in systemic circulation), IP-10-encoding pDNA showed an antimetastatic effect, reducing the number of lung metastases as compared to the control-pDNA treated group. When administered with IL-12-encoding pDNA, IP-10 pDNA enhanced the IL-12 effect, and decreased its earlier observed toxicity. This anti-neoplastic effect of IP-10 has been attributed to the engagement of NK cells and the inhibition of angiogenesis and cell proliferation.
Alternative antitumor strategies aim at a direct destruction of cancer cells, through the delivery of pDNA encoding for a toxic protein—DNA-based strategies. This is referred to as a suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT), when the nucleic acid sequence encodes for an enzyme, which is not directly toxic but instead converts a nontoxic prodrug into a cytotoxic metabolite. The first proof of principle of GDEPT was presented in the mid-eighties and involved the herpes simplex thymidine kinase (HSV-tk) and the prodrug ganciclovir (GCV) [132
]. Presently, HSV-tk as well as other approaches, such as Diptheria toxin A chain (DTA), have been employed in the clinics, the most successful cases being reported in ovarian and prostate cancers [67
]. As for melanoma treatments, HSV-tk has been the most commonly used [111
], although there is no human clinical trial yet. Suicide gene therapy has also been proven effective when used in combined approaches, such as with cytokine-enhanced vaccine in a clinical trial involving canine melanoma patients [134
]. Despite promising, this strategy is currently restrained by a poor delivery; most nanocarriers are not as target-specific and efficient as required, and the toxic gene does not reach the tumor cells in efficacious concentrations.
A number of studies have instead focused on mediators of cell proliferation and differentiation, which are upregulated during tumorgenesis, aiming at their downregulation by means of siRNA delivery [114
]—these are RNA-based approaches. As an example, based on the fact that in epithelial cells, N-cadherin induces changes in morphology of a fibroblastic phenotype (rendering the cells more motile and invasive), the laboratory of Laidler has investigated the outcome of N-cadherin silencing in human melanoma cell lines [114
]. Although the results suggest that N-cadherin positively affects the regulation of the cell cycle and proliferation through activation of the AKT kinase pathway, further investigations are needed to describe the mechanism. Similarly, Villares et al., upon the observation that thrombin receptor (or protease-activated receptor-1, PAR-1) is overexpressed in highly metastatic melanoma cell lines, has evaluated the therapeutic potential of siRNA against PAR-1 [115
]. The authors have observed a significant reduction of in vivo
tumor growth as well as in the number of metastatic lung colonies. This report showed that downregulation of PAR-1 decreased the expression of matrix metallopeptidase-2 (MMP-2), interleukin 8 (IL-8), and vascular endothelial growth factor (VEGF), resulting in an overall decrease in angiogenesis and blood vessels. In 2010, Davis et al. reported on the first human clinical trial (including three melanoma patients) on siRNA therapy against melanoma [92
]. The siRNA targeted the M2 subunit of ribonucleotide reductase (RRM2), and the protein knock down was confirmed at the mRNA level but not corroborated to the same extend by the protein analysis. Nevertheless, the fact that the authors used a delivery vector targeting the transferrin receptor without showing analysis of such receptor expression in melanoma cells was left to be explained [138
Of special interests are combinatorial strategies involving siRNA delivery as these, similar to other combinatorial therapies, cause the most significant outcomes. Particularly, Poeck and coauthors have used a simple and elegant siRNA design [116
]. The authors targeted Bcl2 (an apoptosis regulator protein), which was reported to play a central role in the resistance of melanoma cells to chemotherapy [7
]. By adding 5′-triphosphate ends to their siRNA, the authors also activated innate immune cells, induced the expression of interferons, and caused specific cell tumor apoptosis. These actions are a consequence of the recognition of 5′-triphosphate ends by the cytosolic retinoic acid-induced protein-1 (Rig-1) and synergized with the silencing effects originated from siRNA resulting in massive tumor destruction in the murine lung metastases. Two years earlier, aiming at RNA-based vaccination, Tormo et al. first reported on a promising double stranded RNA (dsRNA) mimic polyinisine-polycytidylic acid (pIC) [117
]. Importantly, the therapeutic effect of the dsRNA was significantly increased when delivered in the form of a complex, together with polyethyleneimine (PEI)-[pIC]PEI
. Initially, the dsRNA mimic was thought to engage toll-like receptors (TLR), hereby mediating cellular tumor immunity [117
]. In turn, further investigation studies showed that it mobilizes the endo/lysosomal machinery of melanoma cells, and through melanoma differentiation associated gene-5 (MDA-5) induces self-degradation by (macro) autophagy and apoptosis, following the MDA-5-mediated activation of proapoptotic factor NOXA [118
]. Interestingly, at the exact same time, MDA-5 and NOXA were also reported to play a role in interferon-independent apoptosis in human melanoma cells by Besch and collaborators [141
]. Not only were these findings meaningful, opening new windows for cancer therapy, but also, in particular in the Damía Tormo studies, was the murine model used very suited, whereupon mice overexpressing hepatocyte growth factor (HGF) and carrying an oncogenic mutation in the cyclin-dependent kinase-4 [(CDK4)R24C
] developed invasive melanomas in the skin following neonatal exposure to carcinogenics.
While a number of microRNA has been described to play relevant roles in melanoma progression [127
], only few in vitro
studies have reported on the miRNA potential for antimelanoma therapy [119
]. However, pertinent therapeutic approaches targeting miRNAs described for other tumor types [142
] foretell the potential and the therapeutic window opportunities entailing these nucleic acids in metastatic melanoma.
As an overview of this section, presents the therapeutic nucleic acids herein described, and schematically summarizes the different strategies in nucleic acid therapies.
Table 2 Different therapeutic strategies against melanoma based on nucleic acids. In the case of DNA-based approaches, a therapeutic gene is delivered to induce a beneficial effect, whereas with RNA based, generally the regimen, is based on silencing of a tumor-active (more ...)