The current study demonstrates the in vitro
and in vivo
efficacy of adenovirus-mediated transfer of siRNA against the MMP-2 gene in a novel spinal metastatic melanoma model. The use of adenoviral vectors for gene therapy in cancer is the delivery method of choice due to high infectivity and lack of integration into the host genome, which limits the risk of insertional mutagenesis. Additionally, expression of the adenoviral genome is of short duration without integration, which is all that is necessary for tumor therapy (as compared with chronic genetic disorders that require long-term expression) (22
). From this perspective, our results substantiate previous claims as infectivity and expression were high as shown by the in vitro
results, lack of mutagenic effects, and retention of neurological function in all treated animals. Furthermore, few studies exist which have used systemic (intravenous) delivery of adenoviral siRNA sequences rather than local intratumoral injection (20
). Our results demonstrate that it is possible to treat neoplasia to the degree of preventing neurological deficit via the intravenous delivery method, and illustrate the potential of systemic adenoviral delivery of siRNA. This novel and reproducible animal model that we developed to study metastatic spinal melanoma can be applied to practically any tumor and allows for the observation of physical behavior correlating with tumor growth.
RNAi has already been demonstrated as a potent therapy for targeting a wide variety of diseases (24
). Much of the potential of RNAi is derived from a considerable post-transcriptional silencing effect even at a low concentration; the concentrations required are usually several magnitudes lower than that necessary for antisense oligonucleotide therapy (27
). Although not integrated into the genome, RNAi activity has been shown to last for more than three weeks via the stability afforded by the RNA interfering silencing complex (RISC) (29
). Phase I and II human trials are already underway to determine the safety and effectiveness of local delivery of siRNA for treatment of respiratory synctial virus (RSV) and macular degeneration, and future studies for the treatment of hepatitis, amyotrophic lateral sclerosis (ALS), and HIV are anticipated (18
Cancer cell invasion and metastasis are complex, multi-faceted and multi-step processes, requiring the interaction of cancer cells with the host environment. Within this milieu, there exist many enzymes necessary for the controlled digestion of the basement membrane and extracellular domain. This includes the plasminogen activator system, aspartyl proteinases, cysteine proteinases, serine proteinases, and matrix metalloproteinases. Of these enzymes, general inhibition of MMPs has shown a benefit in melanoma. For example, the synthetic non-specific hydroxamate-type MMP inhibitor batimastat had a 68% inhibition of lung tumor deposits in a B16 murine metastatic melanoma model (31
). The support for MMP-2 as a pivotal enzyme in metastasis is strong as this zinc-dependent gelatinase (also known as 72 kDa type IV collagenase) is highly expressed in malignant phenotypes characterized by increasing architectural disorder, atypia, and progression (11
). MMP-2-deficient mice also showed reduced angiogenesis and tumor progression in a metastatic model (34
). Furthermore, the data support a correlation between MMP-2 expression and the prognosis of a melanoma patient (35
). Because MMP-2 is upregulated in the vertical growth phase of melanoma, and thus, may represent a state of increased invasion and proliferation potential, we hypothesized that specifically targeting MMP-2 would result in significant growth inhibition.
The adenoviral delivery of siRNA against MMP-2 has already been described in the treatment of cancer. For example, in a study involving lung cancer metastasis, siRNA against MMP-2 resulted in decreased invasion, migration, and angiogenesis with a resultant 60% reduction in tumor volume in treated animals (20
). Other methods of downregulation have validated MMP-2 as a practical target. Montgomery et al.
, showed that cDNA transfection with tissue inhibitor of metalloproteinase-2 (TIMP-2) markedly reduced melanoma growth in a cutaneous immunodeficient mouse model although metastasis still ensued (36
). The reasons for the observed growth inhibition seen with MMP-2 downregulation are likely multi-factorial. In the initial phase of metastasis, invasion and migration are key steps. In the present study, the matrigel assay results show that MMP-2 downregulation resulted in a greater than 80% inhibition of invasion as compared to the control. This method mimics the basement membrane barrier to invasion, thus illustrating the interaction between tumor cells and the microenvironment. Melanoma cells utilize precise methods of digestion occurring in conjunction with simultaneous adhesion to the stroma. It has been demonstrated that at the invasion front of melanoma, this is characterized by upregulation of MMP-2 only in the aggressive cell lines while MMP-9, TIMP-1 and MT1-MMP were expressed in all cells at the tumor-stromal interface (37
). This illustrates the specific actions of MMP-2 not only in the context of its function, but also in its localization of function during metastatic tumor formation.
Post-transcriptional gene control via intravenous injection of adenovirus is classified as non-specific, meaning that theoretically, the DNA sequence is delivered to all cells without tropism towards a specific tissue type other than the known adenoviral affinity for the liver. We propose that some of the growth inhibition observed in vivo
may have resulted from the intravenous delivery mechanism, as stromal cells of the bone and peri-spinal region are also theoretically exposed to siRNA. Others have already shown that cells other than the tumor itself can be the major source of MMP-2 in melanoma (38
) and tumor growth factors such as the extracellular matrix metalloproteinase inducer (EMMPRIN; CD 147) can induce surrounding fibroblasts to secrete MMP-2 (39
). In addition, the issue of tumor versus stroma as the source of MMPs is highly variable depending on the tissue studied (37
). This observation perhaps strengthens the argument for systemic delivery as one would not need to deliberate the efficacy between intratumoral and intravenous therapy.
Once tumor cells have spread through the vasculature and seeded elsewhere, angiogenesis is a necessary component for subsequent tumor growth as malignant cells with a volume of more than 1-2 mm3
exceed the maximum diffusability of required nutrients necessary to sustain tumor growth (41
). Thus, in the absence of neovascularization, solid tumor deposits could not exceed a certain, small dimension. Our results support this mechanism as the immunohistochemical data show the presence of tumor in the treated animals but failure of sustained growth. This may be due to the downregulation of MMP-2-induced angiogenesis. This assertion is corroborated by our in vitro
data, which demonstrate the vascular tubular network was repressed to 30% of control levels. Indeed, others have shown this same effect. Fang et al.
, demonstrated via gel electrophoresis that the switch to the angiogenic phenotype correlated with an increase in MMP-2 in a rat chondrosarcoma model. Specific inhibition of MMP-2 resulted in decreased angiogenic and proteolytic activity of tumor nodules and suppressed in vivo
tumor growth by 70% (42
). The vitronectin-binding integrin αv
, which is highly expressed in melanoma metastases, binds active MMP-2 on the cell surface of endothelial cells and apparently promotes vascular proliferation. Active blocking of this interaction results in reduced tumor vascularity and size (43
). Radiation-induced angiogenesis was also found to be partially mediated by MMP-2 in B16 melanoma cells. This effect was abrogated by the MMP inhibitor metastat (44
Our rationale for developing a novel model was the lack of established protocols in studying neoplasia and the spine. A previous model utilizes left ventricular intracardiac injection in a murine metastatic B16 melanoma model (45
). While the yield was high with tumor nodules forming in 100 percent of animals, there was widespread metastatic involvement from the onset. With this high tumor load, the animal may deteriorate physically which may limit survival, thus affecting final analysis of the treatment results. Furthermore, this model may significantly alter or lower the volume of distribution in systemically administered adenovirus as other studies have found clustering of siRNA in and around the tumor neovasculature (22
). More recent animal models describe anatomically correct but technically difficult procedures in rabbits, requiring drilling and surgical expertise (46
). In contrast, our model requires little training and materials, and isolates metastatic disease to the spine with physically observable effects of tumor progression, namely paraplegia. Furthermore, we sought to recreate the pathophysiological state of spinal cord compression from metastatic tumor involvement of the vertebral body. In humans, the vast majority of tumor deposits occur in the vertebral body due to the presence of large amounts of hematopoietic marrow (47
). Resultant growth and local spread of the tumor results in spinal cord compression from an anterior and lateral direction (48
). As evidenced by the immunohistochemistry results, we were able to successfully mimic this scenario. Our results also indicate a correlation between paraplegia and the amount of tumor burden. Those with intact function had little tumor deposit present within the vertebral body while those with paraplegia had widespread tumor involvement from the anterior and lateral directions with direct compressive forces resulting in significant distortion of the spinal cord.
The present study validates systemic adenoviral siRNA delivery and the targeting of MMP-2 in metastatic melanoma. We have demonstrated significantly diminished tumor cell invasion, migration, and angiogenesis. In vivo, these results correlated with retention of neurological function in a novel metastatic spinal model. While other metastatic models to date rely upon either intracardiac injection or technically more involved procedures, this model relies upon simple anatomical concepts with reproducible results and confirmed immunohistochemical mimicry of the human pathophysiological state. The fact that tumor was still observed within the vertebrae of treated animals imply that the RNAi approach, while valid, may require a multimodal strategy aimed at all the specific steps involved in metastasis. With foreseeable improvements in siRNA delivery and specificity, proteases such as MMP-2 may become attractive targets in the clinical therapy of cancer and metastatic disease.