siRNA-mediated simultaneous downregulation of uPA and its receptor inhibits angiogenesis and invasiveness triggering apoptosis in breast cancer cells

Abstract

A wide variety of tumor cells exhibit overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR). In breast cancer, expression of uPA and uPAR is essential for tumor cell invasion and metastasis. It is also known that uPA binds to uPAR and activates the RAS extracellular signal regulated kinase (ERK) signaling pathway. In our study, small interfering RNA (siRNA) was introduced to downregulate the expression of uPA and uPAR in two breast cancer cell lines (MDA MB 231 and ZR 75 1). uPA and uPAR were downregulated individually using single constructs, and in combination using a bicistronic construct driven by a CMV promoter in a pcDNA-3 mammalian expression vector. Reverse transcription PCR (RT-PCR) and Western blot analyses indicated downregulation at both the mRNA and protein levels. In vitro angiogenesis studies using conditioned medium in HMEC-1 cells indicated a decrease in the angiogenic potential of conditioned media from treated cells when compared to the controls. This decrease in angiogenic potential was remarkably higher with the bicistronic construct. Similarly, the invasive potential of these cells decreased dramatically when treated with the bicistronic construct, thereby suggesting a synergistic effect from the downregulation of both uPA and uPAR. Furthermore, when uPA and uPAR were downregulated simultaneously, the apoptotic cascade was triggered as indicated by the upregulation of both initiator and effector caspases as well as other pro-apoptotic molecules. A mitochondrial permeability assay and FACS analysis revealed an increase in apoptotic cells in the uPA/uPAR treatment as compared to the other treatments. This overexpression of pro-apoptotic caspases in relation to the RNAi-induced downregulation of uPA and uPAR clearly suggests the involvement of the uPA-uPAR system in cell survival and proliferation in addition to their role in tumor progression.

Figure 1.

Reverse transcription PCR analysis of RNAI transfected breast cancer cells. 1×10⁶ cells were plated on 100 mm petri plate for each transfection experiment. Cells were transfected with CO (control with no plasmid). Scrambled vector (pSV), uPAR (pU), pUPA and pU2. After 48 hrs., total RNA was isolated from the cells to prepare first strand cDNA followed by PCR analysis using the primers for uPA and uPAR. GAPDH was used as an internal control.

Figure 2.

Western blot analysis and fibrin zymography. Transfection was carried out as described earlier. 1×10⁶ cells were plated on 100 mm petri plate for each transfection experiment. Cells were transfected with CO (control with no plasmid). Scrambled vector (pSV), uPAR (pU), pUPA and pU2. After 36 hours, serum containing media from the plate was replaced with serum-free media. After 48 hrs., cells were harvested for protein extraction and conditioned media was collected 20μg of total protein from each of the samples was loaded on SDS-PAGE and blotted on nitrocellulose membranes. The membrane was probed with anti-uPAR antibody (A). 0.2μg of conditioned media was loaded for fibrin zymography (C). Quantification of uPAR protein (B) and uPA enzymatic activity (D) was obtained by scanning the auto-radiograms with a densitometer. Data are shown mean values ± S.D. of three different experiments in each group.

Figure 3.

In vitro angiogenesis. Network formation by human microvascular endothelial cells in conditioned media from MDA MB 231/ZR 751 cells. Breast cancer cells were transfected with control (CO), scrambled vector (pSV) or RNAi constructs for uPAR (pU), uPA (pUPA), uPAR-uPA (pU2) and conditioned media was collected after 48 hrs. as described earlier. HMEC-1 cells grown in conditioned media were stained with H&E after 72 hours and examined under a confocal scanning laser microscope (A). Quantification of angiogenesis in co-cultures transfected with pSV, pU, pUPA and pU2 as described in Methods. Values are mean ± S.D. from three different experiments.

Figure 4:

Matrigel invasion assay. Breast cancer cells transfected with control (CO), scrambled vector (pSV), or RNAi constructs for uPAR (pU), uPA (pUPA), uPAR-uPA (pU2) were transferred to 12μm transwell chambers coated with matrigel. After 48 hours, the cells were scraped from the chamber and stained to check for invasion of cells to the lower surface (A). The invasion was quantified as described in Methods (B). Values are mean ± S.D. of four experiments.

Figure 5:

MITO-PT 100 and FACS analysis. MDA MB 231 cells were transfected with control (CO), scrambled vector (pSV), or RNAi constructs for uPAR (pU), uPA (pUPA), uPAR-uPA (pU2). After 48 hrs, the cells were resuspended in MitoPT dye diluted in serum-free media. After incubating at 37° C for 15 min, 100 μl of the suspension was visualized under a fluorescent microscope (A). MitoPT kit allows the distinction between non-apoptotic red fluorescent cells and apoptotic green fluorescent cells. FACS analysis was carried out according to manufacturer's instructions and 10,000 cells were recorded for each transfection. When cells stained with MitoPT are run through a flow cytometer, the instrument will measure apoptosis by monitoring the amount of red fluorescence in each region (B).

Figure 6

: Simultaneous inhibition of uPAR-uPA induces apoptotic cascade. Breast cancer cells were transfected with control (CO), scrambled vector (pSV), or RNAi constructs for uPAR (pU), uPA (pUPA), uPAR-uPA (pU2). After 48 hrs, cell lysates were prepared as per standard protocol and 15 μg of total protein was loaded on SDS-PAGE and blotted on nitrocellulose membrane. The membrane was probed with antibodies specific for ERK, pERK, p38, pP38, Caspases, 3,6,8 and 9 and APAF-1.