Small interfering RNA directed reversal of urokinase plasminogen activator demethylation inhibits prostate tumor growth and metastasis

Abstract

Recent studies have shown that small interfering RNA (siRNA) silences genes at the transcriptional level in human cells. However, the therapeutic potential of siRNA-mediated transcriptional gene silencing remains unclear. Here, we show that siRNA targeted to the urokinase plasminogen activator (uPA) promoter induced epigenetic transcriptional silencing in human prostate cancer cells. This silencing resulted in a dramatic reduction of tumor cell invasion and angiogenesis in vitro. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the silencing of uPA significantly inhibits prostate tumor growth and the incidence of lung metastasis. Our findings represent a potentially powerful new approach to not only epigenetic silencing of metastasis or growth-promoting genes as a cancer therapy, but also as a means to shed light on how aberrant de novo methylation during cancer progression might be targeted to specific sequences.

Figure 1. siRNA targeting the uPA promoter induced DNA methylation and transcriptional silencing in PC3 prostate cancer cells

(A) Design of uPA siRNA. Locations of uPA siRNA nos. 1, 2, 3 and 4 are shown relative to the transcriptional start site (0).(B) MSP analysis of the CpG island in the uPA promoter in mock-transfected cells and in cells transfected with siuPA or siMM (left). Bisulfite-based DNA methylation analysis using quantitative real-time PCR (right). Using bisulfite-treated genomic DNAs from siRNA-transfected and mock cells, the CpG island in the uPA promoter region was PCR-amplified with methylation- or unmethylation-specific primers. Real-time PCR was used to determine the relative levels of methylated and unmethylated uPA. The PCR product amplified with methylation-insensitive primers was used for normalization. uPA-U, unmethylated PCR product; uPA-M, methylated PCR product. Positive and negative controls were described in Methods.(C) Bisulfite sequencing analysis of the CpG island in the uPA promoter in mock-transfected cells and in cells transfected with siuPA or siMM. CpG positions are indicated relative to the transcriptional start site (0); each circle in the figure represents a single CpG site. A filled circle represents a methylated CG dinucleotide, and an empty circle represents an unmethylated CG dinucleotide. Bar diagram showing the percentage of methylation in cells transfected with mock, siMM or siuPA.(D) RT-PCR (top) and immunoblot (bottom) analysis of cells transfected with mock, siMM or siuPA were collected on the indicated days (D2-D14). GAPDH was used as a loading control for RNA and protein analysis.

Figure 2. uPA silencing suppressed invasion and angiogenesis in PC3 prostate cancer cells

(A) Comparison of the in vitro invasive potentials of cells transfected with mock, siMM or siuPA in the presence or absence of 5-aza and TSA. Data are mean ± SD three independent experiments (p<0.01).(B) Representative invasion photographs from cells transfected with mock, siMM or siuPA as described in (A).(C) The PC3-derived conditioned medium significantly increased EC tube-like network formation in culture, an effect nearly completely blocked by neutralizing antibodies to either uPA or VEGF.(D) Angiogenic activity in media conditioned by uPA knockdown or control cell culture using the EC tube-like formation assay. Data are mean ± SD three independent experiments (p<0.01). Representative angiogenic activity photographs in media conditioned by uPA knockdown or control cell cultures are shown below.

Figure 3. siRNA targeting the uPA promoter induced DNA methylation and transcriptional silencing in DU145 prostate cancer cells

(A) MSP analysis of the CpG island in the uPA promoter in mock-transfected cells and in cells transfected with siuPA or siMM. Positive and negative controls are described in Methods.(B) RT-PCR (top) and immunoblot (bottom) analysis on day 4 of DU145 cells transfected with mock, siMM or siuPA. GAPDH was used as a loading control for RNA and protein analysis.(C) Expression levels of uPA mRNA as determined by real-time RT-PCR. DU145 cells were transfected with mock, siMM or siuPA in the presence or absence of 5-aza and TSA (p<0.01). Results shown are the mean ± SD of three independent experiments (p<0.01).(D) Fibrin zymography of cells transfected with mock, siMM or siuPA in the presence or absence of 5-aza and TSA. Fibrinolytic activity was detected as clear lysis bands after Amido Black staining and subsequent destaining with methanol/acetic acid.

Figure 4. Silencing of uPA expression inhibited tumor growth in an orthotopic mouse prostate tumor model

(A) Photon counts of orthotopic prostate tumors on days 10, 20 and 40 (left). Nude mice carrying an orthotopic prostate tumor, established from either mock or siMM-transfected PC3-luc cells, showed substantial bioluminescence signal as indicated by photon counts on day 40. In contrast, the orthotopic prostate tumors developed from PC3-luc cells transfected with siuPA showed significantly low bioluminescence signal (right).(B) Comparison of dissected prostate tumors in (A) from mice 40 days after cell implantation (p<0.01).(C) We excised and photographed prostate tumors in (A) from mice 40 days after cell implantation.(D) RNA samples extracted from PC3-luc prostate tumors (6 animals per group) were analyzed using RT-PCR for uPA expression levels. The siuPA group showed the most prominent and specific knockdown of uPA. GAPDH was used as a control and remained unchanged.

Figure 5. Inhibition of metastatic tumor growth in lungs by promoter-directed uPA siRNA

(A) Bioluminescence signal in the lung, representative of lung metastasis, was recorded for each mouse. Lung images from different mice (top). Lung metastasis signals were analyzed by photon counts (bottom, p<0.01). We observed significant inhibition of lung metastasis in PC3-luc cells transfected with siuPA relative to the control groups.(B) Scatter plot of the lung surface metastasis counts of mice implanted with control and uPA knockdown cells.(C) Effect of uPA silencing on the metastatic potential of PC3-luc cells.(D) Tumors metastasized to lung in (A) from mice were examined for uPA expression by RT-PCR. GAPDH was used as a control and remained unchanged.

Figure 6. Histopathology and von Willebrand Factor staining of paraffin-embedded tissue sections from orthotopic prostate tumors

(A) Tumors were harvested from all PC3-luc prostate tumor groups as described in Figure 4(C). Tissue sections (5 μm) were prepared and stained with hematoxylin and eosin (H&E) for histopathological analysis (left column) and with an anti-vWF for immunohistochemistry (middle and right columns). Following blinded procedures, a pathologist examined the H&E and vWF-stained sections (n = 12−15) from each tumor (n = 6 tumors from each treatment group) and determined the morphology, invasive edge and vascularity.(B) Cells positive for vWF counted in five high-power fields in tumor sections with indicated groups. The siuPA group has a significantly lower mean blood vessel density (p<0.01).(C) Tumor angiogenesis was monitored in the dorsal skin-fold chamber of an immunodeficient mouse (left). The photographs show changes in microvessel density of mice skin-folds in response to the secretions of PC3 cells either expressing uPA (mock and siMM) or experiencing uPA knockdown (siuPA). Arrowheads indicate newly formed blood vessels in the skin-folds. Microvessels were counted under a microscope in five random fields (right). The results represent mean ± SD from six implants per group (p<0.01).(D) Schematic model of RNAi-directed alteration of uPA methylation status as a key regulatory switch for metastatic potential.