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, 27 (32), 4434-45

Persistent Transactivation of EGFR and ErbB2/HER2 by Protease-Activated receptor-1 Promotes Breast Carcinoma Cell Invasion

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Persistent Transactivation of EGFR and ErbB2/HER2 by Protease-Activated receptor-1 Promotes Breast Carcinoma Cell Invasion

P Arora et al. Oncogene.

Abstract

Hyperactivation of ErbB signaling is implicated in metastatic breast cancer. However, the mechanisms that cause dysregulated ErbB signaling and promote breast carcinoma cell invasion remain poorly understood. One pathway leading to ErbB activation that remains unexplored in breast carcinoma cell invasion involves transactivation by G-protein-coupled receptors (GPCRs). Protease-activated receptor-1 (PAR1), a GPCR activated by extracellular proteases, is overexpressed in invasive breast cancer. PAR1 is also proposed to function in breast cancer invasion and metastasis, but how PAR1 contributes to these processes is not known. In this study, we report that proteolytic activation of PAR1 by thrombin induces persistent transactivation of EGFR and ErbB2/HER2 in invasive breast carcinoma, but not in normal mammary epithelial cells. PAR1-stimulated EGFR and ErbB2 transactivation leads to prolonged extracellular signal-regulated kinase-1 and -2 signaling and promotes breast carcinoma cell invasion. We also show that PAR1 signaling through Galpha(i/o) and metalloprotease activity is critical for ErbB transactivation and cellular invasion. Finally, we demonstrate that PAR1 expression in invasive breast carcinoma is essential for tumor growth in vivo assessed by mammary fat pad xenografts. These studies reveal a critical role for PAR1, a receptor activated by tumor-generated proteases, in hyperactivation of ErbB signaling that promotes breast carcinoma cell invasion.

Figures

Figure 1
Figure 1
Protease-activated receptor-1 (PAR1) is essential for thrombin-induced ErbB transactivation in breast carcinoma cells. Serum-deprived MDA-MB-231 cells were incubated with 10 nM thrombin (a and b) for various times at 37 °C. Cells were lysed, immunoprecipitated with anti-PY99 antibody and immunoblotted with anti-epidermal growth factor (EGF) receptor (EGFR) or anti-ErbB2 antibody. Total cell lysates were immunoblotted for EGFR or ErbB2 as a control. The time-course of thrombin-induced EGFR and ErbB2 transactivation shown is from a representative experiment. The data (mean ± s.e.) are expressed as fold increase over basal from three independent experiments. In reciprocal experiments, serum-deprived MDA-MB-231 cells were incubated with 10 nM thrombin for 4 min at 37 °C, lysed and immunoprecipitated with anti-EGFR or anti-ErbB2 antibodies and then immunoblotted with anti-PY99 antibody. (c) Control, P1 and P2 shRNA-expressing MDA-MB-231 cells were incubated with anti-PAR1 antibody or preimmune serum, fixed and the amount of antibody bound to the cell surface was measured by enzyme-linked immunosorbent assay (ELISA). The data (mean ± s.d., n = 3) shown are from a representative experiment repeated at least three times. MDA-MB-231 control and P1 shRNA cells labeled with myo-[3H]inositol were incubated in the absence or presence of 10 nM thrombin, 100 µM TFLLRNPNDK or 100 µM SLIGKV for 60 min at 37 °C and the amounts of [3H]inositol phosphates generated were then measured. The data (mean ± s.d., n = 3) are expressed as fold increase over untreated control and are representative of three different experiments. (d) Control and PAR1-deficient P1 shRNA-expressing MDA-MB-231 cells were incubated in the absence or presence of 10 nM thrombin or 16 nM EGF for 4 min at 37 °C, lysed and immunoprecipitated with anti-PY99 antibody and immunoblotted with anti-EGFR or anti-ErbB2 antibody. Cell lysates were immunoblotted for EGFR or ErbB2 as a control. The data are expressed as fold increase over untreated control and are representative of three independent experiments.
Figure 2
Figure 2
Thrombin-induced epidermal growth factor (EGF) receptor (EGFR) and ErbB2 transactivation requires EGFR kinase activity. (a and b) MDA-MB-231 cells were pretreated with 0.1% dimethyl sulfoxide (DMSO) or 2 µM AG1478 for 2 h at 37 °C. Cells were then incubated with or without 10 nM thrombin or 16 nM EGF for 4 min at 37 °C and EGFR and ErbB2 tyrosine phosphorylation was detected as described in Figure 1. The data are expressed as fold increase over untreated control and are representative of three independent experiments. (c) MDA-MB-231 cells labeled with myo-[3H]inositol were incubated with 0.1% DMSO, 2 µM AG1478 or 1 µM PD153035 for 2 h at 37 °C. Cells were then stimulated with 100 µM TFLLRNPNDK for 60 min at 37 °C and the amounts of [3H]inositol phosphates generated were then measured.
Figure 3
Figure 3
Persistent transactivation of epidermal growth factor (EGF) receptor (EGFR) and ErbB2 induced by thrombin in invasive breast carcinoma. Serum-starved MDA-MB-231 cells (a), BT549 cells (b) and normal human mammary epithelial cells (HMECs) (c) were incubated with 10 nM thrombin various times at 37 °C. Cells were lysed and EGFR and ErbB2 tyrosine phosphorylation were detected as described in Figure 1. (d) The data shown are from the thrombin treated 120 min time point and is expressed as the fold increases over untreated control. (e) MDA-MB-231 cells were incubated in the absence or presence of 10 nM thrombin or 16 nM EGF for 4 min at 37 °C and EGFR and ErbB2 tyrosine phosphorylation was examined. The data shown are representative of at least three independent experiments.
Figure 4
Figure 4
Protease-activated receptor-1 (PAR1)-stimulated epidermal growth factor (EGF) receptor (EGFR) and ErbB2 transactivation contributes to sustained extracellular signal-regulated kinase-1 and -2 (ERK1/2) signaling and promotes breast carcinoma cell invasion. (a) MDA-MB-231 cells were pretreated with 0.1% dimethyl sulfoxide (DMSO) or 2 µM AG1478 for 2 h at 37 °C and then incubated with 100 µM TFLLRNPNDK for various times at 37 °C. Cell lysates were immunoblotted with anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) (ERK1/2) antibody. Membranes were stripped and reprobed with anti-p44/42 MAPK (ERK1/2) to control for loading. The data (mean ± s.e.) shown are expressed as fold increase over basal from at least three independent experiments. (b) MDA-MB-231 cells were electroporated with 600 nM ErbB2-specific or nonspecific siRNA. After 48 h, serum-deprived cells were incubated with or without 100 µM TFLLRNPNDK for various times at 37 °C and ERK1/2 activity was measured and quantified as described above. The inset confirms the loss of ErbB expression in ErbB2 siRNA-treated cells as detected by immunoblotting. (c) Serum-starved MDA-MB-231 cells were left untreated or treated with varying concentrations of thrombin at 37 °C and then added to the upper well and cellular invasion toward NIH 3T3-conditioned medium (CM) was assessed. Representative data from one experiment is shown. (d) MDA-MB-231 cells were pretreated with 0.1% DMSO or 2 µM AG1478 for 30 min at 37 °C. After the treatment, cells were incubated in the absence or presence of 1 pM thrombin and then added the upper well and cellular invasion toward NIH 3T3 fibroblast-CM present in the lower well with or without AG1478 was assessed. The data (mean ± s.e.) shown are expressed as fold increase over untreated control from three independent experiments. The difference between thrombin-stimulated cellular invasion in control and AG1478-treated cells was significant (***P<0.005).
Figure 5
Figure 5
Protease-activated receptor-1 (PAR1) signaling through Gαi/o is critical for epidermal growth factor (EGF) receptor (EGFR) and ErbB2 transactivation. (a and b) Serum-starved MDA-MB-231 cells were pretreated with 100 ng/ml pertussis toxin for 18 h at 37 °C or left untreated and then incubated in the absence or presence of 10 nM thrombin for 5 or 30 min at 37 °C. Cells were processed and EGFR and ErbB2 tyrosine phosphorylation was measured as described in Figure 1.
Figure 6
Figure 6
Metalloprotease activity is required for thrombin-induced epidermal growth factor (EGF) receptor (EGFR) and ErbB2 transactivation and cellular invasion. (a and b) Serum-deprived MDA-MB-231 cells were preincubated with 0.1% dimethyl sulfoxide (DMSO) (vehicle control), 10 µM GM6001 active or inactive agent for 1 h at 37 °C and then treated with or without 10 nM thrombin for 5 min at 37 °C. EGFR and ErbB2 tyrosine phosphorylation was detected as described in Figure 1. The data are expressed as fold increase over control and are representative of three different experiments. (c) MDA-MB-231 cells were pretreated for 1 h with 10 mM GM6001 active or inactive control agent then incubated with 1 pM thrombin at 37 °C. Cells were then added to the upper well and cellular invasion toward conditioned medium (CM) containing inactive or active GM6001 was assessed. (d) Serum-starved MDA-MB-231 cells were preincubated with 20 µg/ml of CRM 197, 100 ng/ml heparin or vehicle control for 30 min at 37 °C and then treated in the absence (Ctrl) or presence of 10 nM Th or 16 nM EGF ligand for 5 min at 37 °C. ErbB tyrosine phosphorylation was then examined as described above.
Figure 7
Figure 7
Protease-activated receptor-1 (PAR10 mediates conditioned media-induced ErbB transactivation and cellular invasion in vitro, and tumor growth in vivo. (a) The basal invasion of control and PAR1-deficient P1 shRNA-expressing MDA-MB-231 cells toward NIH 3T3 fibroblast-conditioned medium (CM) was assessed. The data (mean ± s.d., n = 5) shown are the number of invaded cells representative of at least three independent experiments. The difference between invasion observed with control and PAR1-deficient MDA-MB-231 cells was significant (***P<0.005). (b and c) Serum-deprived control and P1 shRNA-expressing cells were incubated in the absence or presence of 10 nM thrombin, CM or 16 nM EGF for 8 min at 37 °C, and epidermal growth factor (EGF) receptor (EGFR) and ErbB2 tyrosine phosphorylation was measured and quantified as described in Figure 1. The data shown are expressed as fold increase over control and are representative of at least three independent experiments. (d) PAR1-deficient and control MDA-MB-231 cells were implanted into the left and right mammary fat pad of immunodeficient severe combined immunodeficiency (SCID) mice, respectively. Tumor growth was measured weekly and the data (mean ± s.e., n = 3) are representative of two independent experiments. The difference between tumor size measured in PAR1-deficient versus control MDA-MB-231 cell implants was significant (***P<0.005). Tumors were excised from the mammary fat pads at 6-weeks postimplantation, fixed and stained with hematoxylin and eosin (H&E) and imaged at ×1 and boxed areas at ×20 magnification and examined for morphology and the invasive edge.

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