RNAi-mediated abrogation of cathepsin B and MMP-9 gene expression in a malignant meningioma cell line leads to decreased tumor growth, invasion and angiogenesis

Abstract

Malignant meningiomas are highly aggressive and frequently recur after surgical resection of the tumor. Earlier studies have reported that the cysteine protease cathepsin B and the matrix metalloproteinase MMP-9 play important roles in tumor progression. In the present study, we made an attempt to evaluate the roles of these proteases in the malignant meningioma tumor microenvironment and determined the effectiveness of using single or bicistronic siRNA constructs for cathepsin B and MMP-9, in both in vitro and in vivo models. Transfection of a plasmid vector expressing double-stranded RNA for cathepsin B and MMP-9 significantly inhibited mRNA and protein levels of cathepsin B and MMP-9. The migration and invasion of meningioma cells were decreased after treatment with single or bicistronic siRNA constructs for cathepsin B and MMP-9 compared to controls and vector controls. Inhibition of angiogenesis was observed when the cells were transfected with single or bicistronic constructs for cathepsin B and MMP-9, when compared to controls or empty vector controls. Our study revealed that abrogation of cathepsin B and MMP-9 expression decreased the activation of major proteins involved in MAP kinase and PI3 kinase pathways indicating that targeting these proteases may hinder intracellular signaling and thus decrease cell survival and proliferation in malignant meningiomas. In addition to the in vitro evidence, we observed a significant regression of pre-established orthotopic tumors after treatment with RNAi plasmid vectors targeting cathepsin B and MMP-9. Furthermore, these observations demonstrate that the simultaneous RNAi-mediated targeting of cathepsin B and MMP-9 has potential application for the treatment of human meningiomas.

Figure 1. RNAi-mediated downregulation of cathepsin B and MMP-9 expression in IOMM-Lee cells.

(A) Total RNA was extracted from IOMM-Lee parental cells (mock) and cells transfected with pSV, pCB, pM and pMC. Semi-quantitative RT-PCR was performed as per standard protocol. GAPDH expression was also analyzed in all the samples to serve as a loading control. (B) Cell lysates were collected from cells transfected with pSV, pCB, pM and pMC and also from parental cells (mock). Immunoblot analysis was performed using the cell lysates to detect expression of cathepsin B in the parental cells and the cells transfected with shRNA plasmids. GAPDH was analyzed as a loading control. (C) Enzymatic activity of MMP-9 was analyzed in the conditioned media collected from cells transfected with pSV, pCB, pM, pMC and parental cells. 30 μg of protein were loaded onto 10% SDS-PAGE containing gelatin.

Figure 2. Detection of in situ cathepsin B and MMP-9 gene expression.

IOMM-Lee cells were cultured in 8-well chamber slides at a concentration of 5×10⁴ and transfected with pSV, pCB, pM and pMC. Parental cells (mock) were simultaneously maintained. 72 h after transfection, the cells were fixed in formaldehyde. Expression of cathepsin B and MMP-9 was detected in intact cells (see Materials & Methods). The cells were mounted with 4′, 6-diamidino-2-phenylindole (DAPI) to visualize the nucleus.

Figure 3. RNAi-mediated abrogation of cathepsin B and MMP-9 gene expression negates IOMM-Lee cell proliferation.

5×10³ IOMM-Lee cells were maintained in triplicate in vitronectin-coated 96-well plates and transfected with pSV, pCB, pM and pMC as described in Materials & Methods. Viable cell mass was measured in both parental and treated cells at different time intervals (1−5 days) 48 h after transfection. A₅₇₀ was plotted against the respective time intervals. Mean ± S.D. values from 3 different experiments are shown (p<0.001).

Figure 4. Knockdown of cathepsin B and MMP-9 through RNAi treatment reduces spheroid migration and invasion.

(A) Fluorescent-labeled IOMM-Lee cells (IOMM-Lee GFP) were cultured in 96-well low attachment plates at a concentration of 7×10⁴ and spheroids were allowed to grow for 3−4 days at 37°C with shaking at 40−60 rpm. The spheroids were then transfected with pSV, pCB, pM and pMC. Untreated spheroids were also maintained to serve as the control (mock). 48 h after transfection, the spheroids were transferred to 8-well chamber slides and maintained for another 72 h in serum-free media. Spheroid migration was analyzed by taking pictures using fluorescence microscope. (B) The migration of the spheroid cells was quantified as the distance cells migrated from the spheroids. Values are mean ± S.D. from three different experiments (p<0.001). (C) IOMM-Lee cells were transfected with pSV, pCB, pM and pMC. Untransfected cells (mock) were also maintained to serve as the control. 48 h later, the cells were collected through trypsinization and resuspended in DMEM serum-free media. 1×10⁵ cells were counted and cultured in the upper chamber of a Transwell insert coated with matrigel (1 mg/mL) and processed. (D) Number of cells was counted in three different fields for each sample and the percentage invasion of cells treated with shRNA plasmids was analyzed and compared with the untreated (mock) cells. The graph represents the percentage invasion shown by cells transfected with pSV, pCB, pM and pMC in comparison with untreated cells (mock). Values are mean ± S.D. from three different experiments (p<0.001). (E) IOMM-Lee cells were cultured in agar-coated 96-well plates at a concentration of 7×10⁴ and allowed to grow for 3−4 days in a 37°C incubator with shaking at 40−60 rpm. The spheroids were later transfected with pSV, pCB, pM and pMC. 48 h after transfection, the cells were labeled with the red fluorescent dye Dil. Untreated spheroids (mock) were maintained as the control under similar conditions. Simultaneously, fetal rat brain aggregates (FRBA) were grown from 16−17-day-old fetal rat brain cells and labeled with the green fluorescent dye DiO. Later, both the IOMM-Lee spheroids and spheroids from fetal rat brain cells were co-cultured and maintained in serum-free media. Invasion of fetal rat brain spheroids by tumor cell spheroids was recorded at periodic intervals of 24, 48 and 72 h using a fluorescent microscope. (F) The percentage of invasion in the untransfected spheroids and spheroids treated with shRNA plasmids was quantified using image analysis software and plotted as the fetal rat brain aggregates which remained uninvaded against different time intervals. Values shown are the mean ± S.D. from three different experiments (p<0.001).

Figure 5. RNAi-mediated targeting of cathepsin B and MMP-9 inhibits angiogenesis initiated by IOMM-Lee cells.

(A) 4×10⁴ IOMM-Lee cells were cultured in 8-well chamber slides and transfected with pSV, pCB, pM and pMC and grown for 24 h. Untreated cells (mock) were maintained as a control. After 24 h, the media was replaced with serum-free media and maintained for another 24 h. Simultaneously, HMEC cells (3×10⁴) were maintained in 8-well chamber slides. The conditioned media was collected from transfected cells 24 h after adding serum-free media. HMEC were grown for another 72 h in the presence of conditioned media collected from parental (mock) and transfected IOMM-Lee cells. Then, HMEC were stained with H&E and capillary-like network formation was analyzed under light microscopy. (B) The ability of capillary network formation was analyzed as number of branch points and number of branches per branch point and plotted against the respective cells. Values represent mean ± S.D. from three different experiments (p<0.001). (C) The dorsal skin fold chamber model results revealed inhibition of in vivo angiogenesis, most likely due to RNAi-mediated abrogation of cathepsin B and MMP-9. Diffusion chambers holding 2×10⁶ IOMM-lee parental cells and cells transfected with pSV, pCB, pM and pMC were introduced into dorsal air sacs of athymic nude mice as described in Materials & Methods. Ten days after introduction of the diffusion chambers, the animals were sacrificed. The skin around the chamber was carefully removed and observed under a light microscope. Delicate, zigzag-shaped microvessels with irregular arrangement was recorded as tumor-induced neovasculature (TN) and were compared to the more organized pre-existing vasculature (PV).

Figure 6. RNAi-mediated downregulation of cathepsin B and MMP-9 gene expression effects intracellular signaling events.

IOMM-Lee cells were transfected with pSV, pCB, pM and pMC; untreated cells (mock) were maintained to serve as the control. The cells were lysed after 48 h and cell lysates were analyzed for various proteins participating in the MAPK and PI3 kinase intracellular signaling pathway molecules by western blotting as described in Materials and Methods.

Figure 7. Downregulation of cathepsin B and MMP-9 through RNAi treatment leads to regression of pre-established orthotopic tumors.

(A) Orthotopic intracranial tumors were established in nude mice and treated with shRNA plasmids. Following extraction, the brains were paraffin-embedded, sectioned and stained with H&E. Photomicrographs of tumor sections revealing total tumor (40X) and rapidly dividing tumor cells (400X) are shown in the figure. (B) Semi-quantification of tumor volume was performed as described in Materials & Methods. Values represent mean ± S.D. from five different animals. (C) Immunofluorescence detection of cathepsin B and MMP-9 expression in tumor sections. Paraffin-embedded tumor sections were subjected to immunofluorescence detection of cathepsin B and MMP-9 expression. Following incubation in appropriate secondary antibody, slides were mounted and observed under a fluorescent microscope. Fields with intense fluorescence were scored for protein expression.