Overexpression of urokinase plasminogen activator receptor (uPAR) or HER2 (erbB-2) in breast cancer is associated with a poor prognosis. We previously reported that gene amplification and overexpression of HER2 and uPAR occur in 70% of HER2-amplified tumor cells from blood or tissue of patients with breast cancer. In this study, we first examined whether depletion of HER2 and uPAR synergized in suppression of the growth of breast cancer cells that overexpress both HER2 and uPAR (SKBR3 and ZR 751). The results showed that depletion of either HER2 or uPAR by RNA interference suppressed cell growth and induced cell apoptosis, but that these effects were significantly enhanced in cells depleted of both HER2 and uPAR. Mechanistic analysis demonstrated that silencing of HER2 and uPAR caused suppression of MAPK signal pathways, resulting in decrease of ERK activity and prompting a high p38/ERK activity ratio. The level of the phosphorylated form of ERK was decreased in cells depleted of HER2, uPAR or both, and the effect in cells depleted of both is the most evident. Moreover, downregulation of uPAR synergized with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells. uPAR RNAi significantly enhanced the effect of trastuzumab on inhibition of MAPK signal pathways. In conclusion, targeting HER2 and uPAR has a synergistic inhibitory effect on breast cancer cells. Our results provide evidence that simultaneous downregulation of HER2 and uPAR may offer an effective tool for breast cancer therapy.
The HER2/neu oncogene (also called erbB-2) encodes a transmembrane receptor tyrosine kinase. The HER receptor family consists of 4 members: epidermal growth factor receptor (EGFR, also known as Her-1 or erbB-1), erbB-2 (Her-2), erbB-3 (Her-3) and erbB-4 (Her-4).1, 2 HER2 seems to be the preferred heterodimerization partner for other family members, and the HER2-containing heterodimers are characterized by extremely high signaling potency because HER2 markedly reduces the rate of ligand dissociation, allowing strong and prolonged activation of downstream signaling pathways.3 HER2 is amplified or overexpressed in approximately 30% of human breast cancers and its overexpression is related to the number of lymph node metastases and a poor prognosis in patients.3, 4 A humanized monoclonal antibody targeting HER2, trastuzumab (Herceptin, Genentech), can effectively treat tumors with HER2 gene amplification in 25% of patients as monotherapy and 50% when given with taxane or other chemotherapy.5, 6
Urokinase plasminogen activator receptor (uPAR) (CD87) is a single-chain, highly glycosylated, membrane-bound protein. The urokinase plasminogen activator (uPA) is its specific and only known ligand. The uPA–uPAR interaction plays an essential role in the proteolytical degradation of extracellular matrix and the basement membrane surrounding the primary tumor, which may favor cancer invasion and metastasis. uPAR also affects migration, adhesion, angiogenesis, differentiation and proliferation through intracellular signaling pathways.5–7 uPAR overexpression in breast cancer and many other cancers is associated with a poor prognosis.8, 9 uPAR is also expressed in the tumor stroma.10 Giannopoulou et al. demonstrated that uPAR was expressed in both malignant and stromal cells in 68.8 and 74.6% of the 173 paraffin-embedded samples of invasive breast carcinomas, respectively, and that stromal uPAR expression is related to an aggressive tumor phenotype and patients' poor relapse-free survival.11
HER2 may upregulate uPAR expression through protein kinase C (PKCα) and Src.12 Our previous studies indicated that the overexpression and gene amplification of HER2 and uPAR occurred most frequently in the same individual tumor cells (TCs) by analysis of uPAR and HER-2 expression and gene status in primary breast carcinomas and circulating tumor cells (CTCs) of patients with advanced breast carcinomas.13 Pierga et al. recently found similar results in disseminated tumor cells (DTCs). They analyzed uPAR mRNA expression in DTCs from bone marrow of patients with breast cancer. High levels of uPAR mRNA in micrometastatic cells are associated with a more aggressive primary tumor phenotype, and patients with overexpression of HER2 also had high levels of uPAR in DTCs.14 The serum uPAR has been assayed in patients with breast cancer, and its concentration was much higher in HER2-positive patients than in HER2-negative patients.15 Moreover, a positive association between HER2 and uPA (ligand of uPAR) was found in breast cancer tissues.16 All these studies indicate that there may be crosstalk and cooperativity between the HER2 and uPAR signaling pathways. Targeting HER2 and uPAR together may provide a more efficient therapy for patients with breast cancer.
Based on the observations above, the aims of this study were to determine whether simultaneous RNAi-mediated downregulation of HER2 and uPAR has additive or synergistic inhibition of proliferation/apoptosis of breast cancer cells, and whether downregulation of uPAR enhances their sensitivity to the HER2-directed humanized antibody trastuzumab. We studied the effect of knocking down HER2 and uPAR through siRNA on HER2 and uPAR overexpressing breast cancer cells, and effects of uPAR depletion on cancer inhibition by trastuzumab. The results could shed light for more effective treatment for breast cancer.
Material and Methods
Cell lines and culture conditions
The 3 breast cancer cell lines (SKBR3, ZR 751 and MDA MB 231) obtained from ATCC (Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS).
Plasmid vector construction
Two short interfering RNA(siRNA) sequences targeting the genes of HER2 and uPAR were selected according to published studies.17, 18 GFP shRNA was selected as control. All oligonucleotides were synthesized by Takara Biotechnology Co. Ltd and annealed. HER2ShRNA, uPARshRNA1 and GFPshRNA were inserted into the SalI and XbaI sites of plasmid pAVU6+27 and they were designated as pHER2, puPAR and pGFP, respectively. uPAR shRNA2 was inserted into BglII and XhoI sites of plasmid pSUPER and called pH1-uAPR. After pH1-uAPR was digested by XhoI and XbaI, the H1 promoter and uPARshRNA expression cassette were cloned into pHER2. The reconstructed plasmid was called pHu, which expressed both shHER2 and shuPAR.
Transfections were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. The transfection efficiency was measured by GFP fluorescence, which was about 70–80% in SKBR3 and ZR 751 cells.
Direct immunofluorescence (IF) was performed as described in Ref. 13.
Western blot assay
Cell lysis and determination of protein concentration were performed as previously described.19 Equal amounts of protein (25 μg/lane) from cell lysates were immunoblotted with antibodies against HER2 (Cell Signaling Biotech), uPAR (R&D Systems), total and phosphorylated forms of ERK (Cell Signaling Biotech), p38 MAPK (Cell signaling Biotech) and actin (Santa Cruz Biotech), respectively. Finally protein was visualized using the ECL™ detection kit with appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotech) according to the manufacturer's instructions. Actin served as the control.
3H-TdR incorporation in vitro
Twelve hours after transfection, the cells on 6-well plates were trypsinized and seeded in triplicate into 96-well microtiter plates (2 × 104 cells/well) and allowed to grow for 56 hr. Subsequently, [3H]thymidine (GE) (1 μCi/well) was added and the cells were incubated for an additional 16 hr. The cells were harvested with micromate 96-well harvester, and incorporation of [3H]thymidine into DNA was measured by liquid scintillation counter (Beckmen). Data were adjusted to equal amount of cells among different treatments. Inhibition of proliferation was determined by comparing the level of [3H] thymidine uptake with controls.
At 24, 48, and 72 hr after transfection, 10 μl of the Cell-Counting Kit (CCK)-8 (Dojindo, Kumamoto, Japan) was added into the triplicate wells and incubated for 1.5 hr. Subsequently, the absorbance at 450 nm was measured to calculate the numbers of vital cells in each well. Additional untransfected cells were trypsinized and seeded in triplicate into 96-well plates (2 × 104 cells/well). Then, CCK-8 solution was immediately added into wells, and the absorbance was measured as the value of zero hours after transfection.
Apoptosis assay was performed using Vybrant® Apoptosis Assay kit (Invitrogen).
Cell cycle analysis
Cell cycle analysis was performed using a previously described method with minor modification.18 Twenty-four hours after transfection, cells were serum starved for 24 hr and then 10% FBS medium was added to cultures. After 24 hr, cells were harvested and fixed with ice-cold 70% ethanol overnight and then stained with 50 μg/ml of propidium iodide (PI) (Sigma) and 50 μg/ml of RNAse A (TaKaRa). Fluorescence was quantitated by a FACSCAN flow cytometer (BD Systems) and analyzed using CellQuest software 6.0.
Each experiment was performed in triplicate, and all experiments were repeated 2–3 times. Student's t test or Mann–Whitney nonparametric U test was used for comparison between groups. A p value <0.05 is considered a significant difference. The combination index (CI) method according to Chou and Talalay20 was used to analyze the synergistic effect. The antiproliferation effect and apoptosis-inducing effect of trastuzumab and uPAR RNAi were analyzed using the software CalcuSyn (Biosoft, Ferguson, MO), which applies the median-effect equation of Chou and the CI equation of Chou and Talalay.20, 21 Calculated CIs were used to determine the presence of strong synergism (<0.3), moderate synergism (between 0.3 and 1.0), additive effect (equal to 1.0), or antagonism (>1.0) between trastuzumab and uPAR RNAi treatment.
Inhibition of HER2 and uPAR by RNA interference suppresses the growth of HER2 and uPAR overexpressing breast cancer cell lines and induces apoptosis
In this study, we selected 3 breast cancer cell lines for analysis of HER2 and uPAR expression. SKBR3 and ZR 751 cells are both HER2 and uPAR highly expressed as determined by IF staining and Western blot (Figs. 1a and 1d). We used MDA MB 231 cells that is only uPAR overexpressed as a HER-2 low expressing control cell line. HER2 and uPAR expression levels were subjectively quantified into category 1–3+.13 As seen in Figure 1a, HER2 expression in SKBR3 and ZR 751 cells was 3+, and uPAR expression was 2–3+. In control cell line MDA MB 231, HER2 expression was 1+, and uPAR expression was 2–3+. HER2 and uPAR gene status was further determined by fluorescent in situ hybridization analysis.13 The average ratios for HER2 and uPAR were 3.2 and 2.1 in SKBR3 cells and 2.0 and 2.0 in ZR 751 cells, respectively. Both genes are amplified in these 2 cell lines.
We first examined the effects of knocking down HER2 and uPAR through siRNA. SKBR3 and ZR 751 cells were transfected with reagent control (with no plasmid), and the RNAi vectors targeting GFP (pGFP as mock), HER2 (pHER2), uPAR (puPAR) or both HER2 and uPAR (pHu). Seventy-two hours after transfection, mRNA levels of HER2 and uPAR were determined by real-time PCR (Figs. 1b and 1c), and protein levels (Fig. 1d) were analyzed by Western blot. Compared with control and mock, transfection with pHER2 led to decrease of HER2 and slight decrease of uPAR in both mRNA and protein levels. Similar results were observed for puPAR. Transfection with uPAR RNAi not only greatly decreased uPAR levels but also caused a slight decrease of HER2 expression. Of note, pHu transfection caused the most evident downregulation of HER2 and uPAR compared with pHER2 or puPAR alone, which indicates that the siRNA construct targeting both HER2 and uPAR is efficient, and that crosstalk might exist between the HER2 and uPAR signaling pathways. Downregulation of HER2 and uPAR expression by siRNA constructs was confirmed by IF staining (Fig. 1e). After transfection with pHER2, HER2 expression decreased from 3+ to 1+, and uPAR expression decreased from 2–3+ to 2+. uPAR RNAi knocked down uPAR expression from 2–3+ to 1+, and HER2 expression from 3+ to 2+. pHu caused significant decrease of both HER2 and uPAR expression to 1+.
To assess the potential effects of RNAi-mediated HER2 and uPAR silencing on cell proliferation and survival, CCK-8 and 3H-thymidine incorporation assays were performed. Cell proliferations were assessed by CCK-8 assay at 24, 48, and 72 hr after siRNA transfection. As shown in Figures 2a and 2b, a significant decrease of cell proliferation was detected in SKBR3 and ZR 751 cells at 48 or 72 hr after treatment with pHER2, puPAR or pHu (p < 0.05 vs. mock at 48 hr, and p < 0.01 at 72 hr). pHu caused the most evident decrease of cell proliferation in SKBR3 cells (p = 0.036091 vs. pHER2, or p = 0.013984 vs. puPAR at 72 hr), whereas there was no significant difference between pHu and pHER2 or puPAR-treated ZR 751 cells in proliferation (p = 0.141093 vs. pHER2, or p = 0.073198 vs. puPAR at 72 hr).
3H-thymidine incorporation assay showed that compared with mock, pHER2, puPAR and pHu reduced the cell proliferation in SKBR3 (Fig. 2c) and ZR 751 cells (Fig. 2d) by 35, 26, and 52% and 20, 22, and 44%, respectively, and the inhibitory effect of RNAi targeting both genes was more efficient than either gene alone (p = 0.043488 vs. pHER2, or p = 0.006324 vs. puPAR for SKBR3; p = 0.021331 vs. pHER2, or p = 0.037884 vs. puPAR for ZR 751).
To evaluate whether silencing HER2 and/or uPAR in SKBR3 cells induces cell apoptosis, cells were stained with Annexin V/PI followed by FACS analysis of cellular apoptosis (Fig. 2e). The percentage of Annexin V-positive SKBR3 cells (apoptotic cells, including PI+ and PI−) in transfected cells with pHER2, puPAR and pHu was more prominent than control and mock. The induction of apoptosis was more pronounced for pHu than pHER2 or puPAR alone (p = 0.018821 or p = 0.014671, respectively). Similar results were observed in ZR 751 cells (Fig. 2f). The results indicated that knockdown of HER2 or uPAR induces proliferation inhibition and cellular apoptosis in SKBR3 and ZR 751 cells, and depletion of HER2 has synergistic effects with downregulation of uPAR.
We next analyzed cell cycle distribution by flow cytometry in SKBR3 cells treated with HER2 and uPAR RNAi (Fig. 2g). Silencing of HER2 or uPAR by siRNA brought about accumulation of cells in the G0/G1 phase compared with mock, and there was a trend of increase in G0/G1 phase when simultaneous knocking down both genes, although the additive effect was not significant.
Inhibition of HER2 and uPAR affect the MAPK signal pathway
ERK and p38 MAPK pathways play major roles in cell proliferation, survival and G0-G1-S phase transition. We then investigated whether RNA interference with HER2 and uPAR affected MAPK signal pathway. Western blot was performed to compare the levels of total and phosphorylated forms of ERK and p38 using antibodies specific for these molecules (Fig. 3a). ERK and p38 expression levels were quantified by scanning the bands and calculating the ratios of phosphorylated forms to total proteins (Figs. 3b and 3c). When cells were transfected with pGFP, pHER2, puPAR and pHu, we observed no significant changes in the amounts of total ERK and p38 in SKBR3 and ZR 751 cells. However, compared with control and mock, the levels of phosphorylated forms of ERK were greatly decreased by pHER2, puPAR and pHu (the ratios of P-ERK to ERK were 0.81, 0.72, 0.37 in SKBR3 cells and 0.47, 0.15, 0.09 in ZR 751 cells, respectively), and the effect of pHu is the most evident among the 3 RNAi vectors. Unlike ERK, the levels of phosphorylated forms of p38 in HER2- and/or uPAR siRNA-treated cells did not seem to change much compared with mock and control. Because ERK/p38 ratio was reported to predict whether breast cancer cells will proliferate or enter a state of dormancy, we calculated the ratios of P-ERK to P-p38. Consistent with the above results, the ratios of P-ERK to P-p38 in pHu treated SKBR3 and ZR 751 cells (0.36 and 0.16, respectively) were lowest compared with either pHER2 or puPAR.
Taken together, these results suggest that simultaneous inhibition of HER2 and uPAR could decrease ERK activity, and thus may reduce cell proliferation and induce apoptosis in cells that overexpress both HER2 and uPAR.
uPAR RNA interference synergizes with trastuzumab to suppress the growth and induce apoptosis of SKBR3 and ZR 751 cells
Trastuzumab can effectively treat tumors with HER2 gene amplification and overexpression. As we found that gene amplification and overexpression of HER2 and uPAR are correlated in tumor cells from patients with breast cancer, we analyzed the effect of uPAR RNAi combined with trastuzumab on proliferation and apoptosis of cell lines with both HER2 and uPAR overexpression. SKBR3 and ZR 751 cells were tansfected with reagent control, pGFP (mock) or puPAR, then treated with 10 μg/ml of trastuzumab or control antibody (no trastuzumab). The cell viability was detected by CCK-8 assay. As shown in Figures 4a and 4b, uPAR siRNA or trastuzumab singly could suppress the proliferation of SKBR3 and ZR 751 cells. Significant differences in cell growth inhibition rate were observed between trastuzumab treatment with and without uPAR RNAi in SKBR3 (0.573 ± 0.05 vs 0.32 ± 0.04, p = 0.002827) or in ZR 751 cells (0.671 ± 0.063 vs 0.487 ± 0.031, p = 0.021295). To determine whether uPAR RNAi promotes trastuzumab-induced apoptosis, SKBR3 cells were stained with Annexin V/PI followed by FACS after 72 hr for cotreatment. As illustrated in Figure 4c, the percentage of apoptotic cells under uPAR siRNA and trastuzumab cotreatment was more prominent than uPAR siRNA (p = 0.026389) or trastuzumab (p = 0.01194) treatment only. Similar results were observed in ZR 751 cells (data not shown).
We further analyzed the effects of different doses of trastuzumab on proliferation and apoptosis of SKBR3 cells under uPAR RNAi treatment. Knockdown of uPAR expression decreased the proliferation (Fig. 4d) and increased cell apoptosis (Fig. 4e) over a wide range of trastuzumab concentrations (0.1–50 μg/ml). The synergistic effects on antiproliferation and apoptosis induction of trastuzumab and uPAR RNAi was evaluated as in Materials and Methods section. All the CI values were <1.0, which was indicative of synergism in antiproliferation between trastuzumab and uPAR RNAi treatment, except for treatment with 50 μg/ml of trastuzumab, which CI value was 1.0 (indicative of additive effect) (Table 1). Similar result was observed for synergistic apoptosis induction. All the CI values were <1.0, which showed the synergistic effect between trastuzumab and uPAR RNAi in inducing apoptosis (Table 2).
Table 1. Combination index values of growth inhibition for trastuzumab and uPAR RNAi treatment in SKBR3 cells
Table 2. Combination index values of apoptosis induction for trastuzumab and uPAR RNAi treatment in SKBR3 cells
Because different doses of trastuzumab and uPAR RNAi had a synergistic effect on proliferation inhibition and apoptosis induction of SKBR3 cells, we then investigated the effect of trastuzumab on MAPK signal pathway under uPAR RNAi treatment. As shown in Figure 5, compared with either alone, the combination of puPAR and trastuzumab treatment caused obvious loss of P-ERK expression. With uPAR knockdown, trastuzumab caused significant reduction of phosphorylated ERK, suggesting that the increased inhibition effects of trastuzumab under uPAR RNAi was, at least in part, attributable to downregulation of ERK signaling.
Previous studies demonstrated that both chemically synthetic and vector-based siRNA are effective for inhibiting targeted gene expression in mammalian cells, including malignant cells,22 and there is no significant competition between cotransfected hairpin siRNA vectors. In this study, we successfully constructed RNAi vectors targeting HER2 and/or uPAR as determined by real-time PCR, western blot and IF assay. Furthermore, compared with pHER2 and puPAR, the bicistronic construct pHu was even more effective in downregulating HER2 and uPAR protein levels in breast cancer cells, including SKBR3 and ZR 751. A previous report indicated that HER2-activated Src and PKC kinases play critical roles in the HER2-mediated upregulation of uPAR.14 Consistent with this report, our results demonstrated that downregulation of HER2 caused a slight decrease of uPAR. Similar results were obtained for uPAR (Fig. 1). The EGFR and uPAR activate similar downstream signaling pathways and regulate common cell physiologic progresses.8, 23 Studies demonstrate that uPAR, EGFR and c-Src are intimately correlated in determining breast cancer progression and prognosis.24–26 Therefore, there may be crosstalk between HER2 and uPAR pathways. HER2 is also regulated by uPAR by currently unknown mechanisms. Because uPAR mediates adhesion directly and indirectly through different integrins as well as fibronectin, it is possible that cell apoptosis by uPAR RNAi is the consequence of de-adhesion. Our results (Fig. 3) showed that knocking down uPAR greatly reduced P-ERK levels, indicating that uPAR RNAi induces cell apoptosis through the ERK pathway rather than cell de-adhesion.
Inhibiting HER2 or uPAR expression by RNA interference has been shown to lead to enhanced apoptosis and decreased tumor growth.27, 28 Moreover, several studies have further explored the potential of knockdown of HER2 or uPAR using RNAi in combination with other targeted therapies to obtain additive effects for suppression of cancer. It is reported that HER2 RNAi synergizes with a chemotherapeutic drug epirubicin to suppress breast cancer growth both in vitro and in vivo.29 In addition, synergistic antitumor activity of insulin-like growth factor-I (IGF-IR) and EGFR/HER2 inhibitors has been observed as IGF-IR is an attractive target for cancer therapy because of its overexpression in diverse tumor types.30 Studies of Kunigal et al. and Subramanian19, 31 have indicated that simultaneous downregulation of uPA and uPAR, or uPAR and matrix metalloprotease-9 protease molecules has an additive effect in inhibiting breast tumor invasion and angiogenesis. Based on the crosstalk and cooperativity between the HER2 and uPAR signaling pathways, and more importantly, the prevalence of HER2 and uPAR coamplification and co-overexpression in breast cancer cells,13, 14 investigation of simultaneous downregulation of HER2 and uPAR was performed in this study. Our results provide evidence that silencing of HER2 and uPAR together have synergistic effects of inhibition of breast cancer cells. Clinical studies have established that trastuzumab is effective against HER2-overexpressing breast cancers.32 However, the cancers begin to progress again usually within 1 year of treatment with trastuzumab-containing regimens.33 Current treatment regimens combine trastuzumab with the taxane paclitaxel6, 34 or docetaxel,35 which increase response rates, time to progression, and survival. Meanwhile, there are already drugs in clinical trials targeting the uPA/uPAR system. The inhibitors of uPA, WX-UK1 and MESUPRON, are currently being studied in several clinical trials with patients with cancer, including breast, gastric, ovarian, colon and pancreatic cancer.36, 37 In this study, we observed that uPAR RNA interference had synergistic effects with trastuzumab, and inhibition of uPAR enhanced the antiproliferative action of trastuzumab through MAPK/ERK pathways (Fig. 5). These findings expand on our previous observations of gene coamplification and overexpression of HER2 and uPAR in individual cancer cells,13 and may have implications for treatment of breast cancer with a combination of these 2 targeted therapies.
Our previous studies indicate that CTCs can be isolated from the blood of patients with breast cancer showing no sign of disease many years after mastectomy.38 Blocking of uPAR, β1-integrins, FAK, EGFR or HER2 signaling resulted in tumour suppression in vivo, leading to tumor cell dormancy.39–41 In this study, we found that downregulation of HER2 or uPAR decreased ERK activity, and simultaneous inhibition of both genes led to the lowest ERK activity and ERK/p38 ratio and led to accumulation of G0/G1 phase in SKBR3 cells. In fact, we also observed that SKBR3 cell clones stably transfected with HER2 and uPAR siRNA entered a state of growth quiescence with almost complete lack of growth (unpublished data). Our studies therefore indicate that targeting HER2 and uPAR together may provide a therapeutic opportunity to convert a cell's phenotype from tumorigenic to dormant.42, 43
To conclude, our data suggest that simultaneous targeting of HER2 and uPAR may provide effective strategies for antibreast cancer therapy, and downregulation of uPAR could enhance trastuzumab sensitivity of breast cancer with HER2 overexpression.
The authors thank Fulian Liao for technical help and advices in cell culture and Dr. Jonathan Uhr and Nancy Lane for critical reading of the manuscript.