Tumor progression is a multifaceted process, which is initiated through activation and/or modulation of various proteolytic systems. These proteolytic cascades, once modified, assist several aspects of tumorigenesis. Irrespective of histological type of origin, invasion is one of the inherent properties acquired by transformed brain cells. During invasion, the tumor cells detach from the primary tumor mass and reattach to the extracellular matrix or surrounding tissue. This process involves excessive secretion of several proteases, which degrade the extracellular matrix (ECM), thereby creating momentary access for the tumor cells to invade into surrounding tissue. Previous studies performed by our group have established cathepsin B and MMP-9 as potential targets for therapeutic treatment of glioblastoma. We found that downregulation of these proteinases through antisense or RNAi-mediated approach reduced tumor growth, invasion and angiogenesis (28
). Meningiomas are the most commonly occurring brain tumors, but very little is known about the underlying molecular mechanism. Moreover, depending on their position in the brain, meningiomas can pose difficulty during surgical resection. At present, apart from surgical resection of the tumor, radiation therapy is generally used for treatment of meningiomas and other alternative treatments are under trial. In recent years, the RNAi-mediated approach has emerged as a promising schema for the treatment of various in vivo
disease models (38
). Our investigation uses this approach to examine the therapeutic significance of RNAi-mediated downregulation of cathepsin B and MMP-9 in malignant meningiomas.
Overexpression of cathepsin B by the tumor cells and adjacent stromal cells have been reported in human breast carcinoma and fibroadenoma (39
). Recent studies in highly invasive melanoma cells reported an increased release of mature cathepsin B upon contact of the cells with collagen I and affirmed an active role of cathepsin B in extracellular matrix degradation (40
). Augmentation of both cathepsin B and MMP-9 expression and their cumulative role in attributing aggressiveness to budding tumors has been reported in colorectal cancer (4
). Several earlier studies on various cancers have revealed that matrix metalloproteinases act in concert with serine and cysteine proteinases resulting in tissue remodeling and ECM degradation, which in turn, enable tumor growth, invasion and angiogenesis (42
). Enhanced expression of both MMP-2 and MMP-9 was reported in human colorectal cancer (43
). In the present study, administration of shRNA plasmids into IOMM-Lee cells targeting cathepsin B and MMP-9 efficiently downregulated expression of these proteins, both at the mRNA and protein levels, when compared to the parental cells by western blotting, gelatin zymography and immunohistochemical analysis.
Aggressive proliferation of cells is a characteristic feature of malignant tumors irrespective of source of origin. Once transformed, tumor cells divide aggressively and thus, aid in the propagation of the tumor. Several proteolytic systems, apart from proteolysis also send mitogenic signals to cellular machinery. Enhanced expression of cathepsin B and active participation in tumor progression was reported in lung cancer (44
). Elevated MMP-9 expression has also been observed in several cancer scenarios where there is increased cell proliferation (45
). Because cell proliferation is an essential feature of tumor progression, we evaluated the proliferative ability of IOMM-Lee cells following downregulation of cathepsin B and MMP-9 through RNA interference. Downregulation of cathepsin B and MMP-9 hampered cell proliferation significantly in the IOMM-Lee cells transfected with pCB, pM and pMC when compared to either untreated cells (mock) or cells transfected with pSV. The ability to invade surrounding tissue is a hallmark of malignant tumors and this characteristic allows the tumor to establish and propagate in an altered environment. Migration of tumor cells not only helps the tumor to establish in the primary site, but is also required for metastasis to secondary sites. In the present study, the migratory capacity of tumor cells was evaluated following knockdown of cathepsin B and MMP-9. Interestingly, knockdown of cathepsin B and MMP-9 reduced migration of tumor spheroids significantly when compared to parental and pSV-treated spheroids.
Several previous studies have established the roles played by both cathepsin B and MMP-9 during tumor invasion and metastasis. Elevated secretion of both precursor and active forms of cathepsin B followed by altered trafficking in the cell (i.e., the redistribution from perinuclear lysosomes to peripheral vesicles and association with plasma membrane) has been reported to occur in several forms of cancer (46
). Intracellular and extracellular cathepsin B activity contributes to the in vitro
invasiveness of MCF 10AT cells (49
). Synthetic cysteine proteinase inhibitors, selective for cathepsin B, have been shown to significantly reduce the invasiveness of MCF 10AT cells (50
). There was a reduction in the number of lung colonies formed in an experimental metastasis in MMP-9 null mice (51
) as well as inhibition of MMP-9 expression (52
). MMP-9 has been shown to actively degrade triple helix type IV collagen of basal lamina of basement membrane and thus aid in tumor invasion (53
). Studies on cell adhesion, migration and invasion of the prostrate cancer cell line LNCap revealed that high expression levels of MMP-2 and MMP-9 correlate with increased tumor migration and invasion (54
). Our results also demonstrated that the invasive potential of meningioma cells treated with the pMC vector was significantly inhibited. Together, these studies establish the significance of MMP-9 and cathepsin B on tumor invasion.
Angiogenesis is an important aspect of tumor progression and metastasis. At present, several studies are currently underway to find methods to effectively prevent angiogenesis, which would result in a better prognosis for several cancers. A recent study in human melanoma cells revealed that acidic extracellular pH existing in the tumor microenvironment stimulates overexpression of various pro-angiogenic factors including cathepsin B and MMP-9 and thus promotes pulmonary metastasis and angiogenesis (24
). Cathepsins B and L are highly expressed in pancreatic islet cell carcinogenesis. In these cells, mutations in the cathepsin B gene aimed to inactivate the protein impaired tumor growth and angiogenesis (33
). In another recent study, Olsson et al. (55
) reported a strong association between MMP-9 and angiogenesis through stimulation of VEGF. Tumor-associated macrophages are reported to release pro-angiogenic factors such as MMP-9 and VEGF. Blocking the release of these factors from macrophages resulted in the suppression of angiogenesis (56
). In this study, we noticed a reduction in capillary-like network formation of both in vitro
and in vivo
models of HMEC (human endothelial cells) when IOMM-Lee cells transfected with pCB, pM and pMC compared to controls and pSV-treated cells.
It is well known that normal cells, upon transformation to malignant cells, modulate several intracellular signaling pathways in order to establish themselves in an altered environment. This modulation is essential because extracellular signals interface with various intracellular regulatory targets through these signaling cascades, which generally involve a sequential activation of protein kinases through phosphorylation. Having observed a reduction in various aspects of tumorigenesis such as migration, invasion and angiogenesis, we next studied the effect of the downregulation of these proteins on intracellular signaling pathways. Stable transfection of pTEN reduced MMP-9 secretion caused by hyaluronic acid-induced phosphorylation of focal adhesion kinase and ERK1/ERK2 signaling (57
). Both signal transduction pathways, MAPK/ERK and PI3-K pathways, are responsible for mediating increased MMP-9 levels following EGFR activation and appear to promote the association of pro-MMP-9 with the cell surface (58
). Inhibition of ERK specific inhibitors blocked MMP-9 expression in breast cancer cells (59
). Moreover, prolonged activation of ERK signaling is necessary to induce MMP-9 activity (60
). Our results demonstrated that mt-ERK transfected cells showed decreased levels of MMP-9 activity and are also less invasive in matrigel invasion assay (61
). It has been reported that hepatitis B viral HBx induces the MMP-9 gene expression through activation of ERKs (62
). It has been recently reported that, inhibition of cathepsin B prevents the appearance of multi-nucleated cells, an early characteristic of MSA-induced cell death, pointing to a central, proximal role for cathepsin B in the novel death pathway (63
). Several studies indicated that PI3-K and AKT pathways induce proliferation of glioma cell lines (64
). Several earlier studies established the significant roles of ERK1 and ERK 2 in cell proliferation (65
). In the present study, downregulation of cathepsin B and MMP-9 decreased phosphorylation of MAP kinases, ERK1, ERK2, PI3-K, and AKT levels.
As already stated, RNA interference has emerged as an extremely potent technology for the explicit downregulation of selected target genes. In recent years, scientists have upgraded the use of this technology from in vitro
culture studies to in vivo
administration in mammals. Based on the reductions that we observed in various aspects of tumor progression in the present study, we analysed the in vivo
efficacy of downregulation of cathepsin B and MMP-9 in regressing pre-established orthotopic tumors in nude mice. Whereas parental and scrambled vector (pSV)-treated mice presented with enormous tumors, we observed significant tumor regression in the mice that received pCB, pM and pMC. RNAi technology uses an intricate natural pathway for sequence-specific mRNA degradation and regulates gene expression at the post-transcriptional level. Delivery of small RNA is accomplished through several methods. While synthetic RNA duplexes are introduced into biological systems directly, the RNAi effect is only transient. However, recent studies indicate that introducing DNA-directed RNAi expression cassettes through plasmid or viral vectors serve as an excellent source of RNAi supply to the biological system under investigation. In this case, dsRNA is continuously expressed within the cells using the DNA templates that direct synthesis of RNA duplexes or short hairpin RNAs, and thus, depending on the vector employed, RNAi effect can be sustained long term. The size of the siRNA molecules (≈ 25bp) is small enough to be transmitted from cell to cell, which has been reported previously (66
). Several earlier studies have revealed that both the proteinases in the present study—cathepsin B and MMP-9—play affirmative roles in various aspects of tumorigenesis in different types of cancer. However, at present, details about the role of these proteinases in malignant meningioma are not available. Consequently, the role of both these proteins in meningiomas merits further investigation. Our study clearly demonstrates that the downregulation of cathepsin B and MMP-9 in the malignant meningioma cell line IOMM-Lee resulted in the reduction of various aspects of tumorigenesis such as invasion, angiogenesis and migration. Our in vivo
studies also revealed significant tumor regression, indicating that these proteinases might serve as potential candidates for therapeutic targets for the treatment of malignant meningioma.