Cell：PAR1蛋白促进乳腺癌细胞的侵润和转移
生物谷报道：本期国际著名刊物Cell封面文章报道了PAR1蛋白与乳腺癌的关系。PAR蛋白全称为蛋白酶体活化受体（Protease-activated receptors），是独立家族的G蛋白偶联受体 ，以往研究认为与炎症，实体瘤等密切相关，但与肿瘤的具体的关系并不是十分清楚。
英国曼彻斯特大学Kuliopulos教授发现，PAR1蛋白是乳腺癌细胞侵润和迁移的必需因子，它是作为基质金属蛋白酶体1（MMP-1）的受体关键性作用，这一研究结果为研制新的抗癌药物提供新的靶点。
Figure 1. PAR1 Is an Invasogenic Factor in Breast Cancer Cells
(A) Migration of MCF-7 cells transiently transfected with either wt PAR1, a PAR1 mutant (F43A) with a deficient tethered ligand, or vector alone (pcDEF3). MCF-7 cells (50,000) were seeded onto an 8 µm pore membrane in the upper well of a transwell apparatus and allowed to migrate for 5 hr toward conditioned media from NIH-3T3 fibroblasts. Cells that migrated to the lower side of the membrane were counted as described in the Experimental Procedures.
(B) Invasion of transiently transfected MCF-7 cells through 10 µg matrigel per well using NIH-3T3 medium as the chemoattractant source in the bottom well.
(C) Tumor formation of MCF-7 cells (PAR1 null) versus MCF-7 cells stably expressing PAR1 (MCF7-PAR1/N55) inoculated into mammary fat pads of NCR nu/nu mice. Representative tumors shown are from the fourth week postinoculation.
(D) Tumorigenicity of MCF7-PAR1/N55 and MDA-MB-231 breast cancer xenografts.
(E) Silencing of PAR1 expression in MDA-MB-231 cells with PAR1 siRNA versus firefly luciferase siRNA control. Surface expression of PAR1 and PAR4 after 48 hr treatment with siRNA was determined by flow cytometry. Gray traces: secondary antibody alone, Black traces: primary plus secondary antibodies.
(F) Effect of PAR1 siRNA transfection (48 hr) on migration of MDA-MB-231 cells (50,000) toward NIH-3T3 media.
(G) Effect of PAR1 siRNA transfection (48 hr) on invasion of MDA-MB-231 cells (25,000) through matrigel.
Figure 3. Effect of Inhibition or Stimulation of PAR1 on Migration of MDA-MB-231 Cells
(A) MDA-MB-231 cells migration toward NIH-3T3 CM supplemented with PAR1 antagonists based on either the extracellular ligand (RWJ-56110, BMS-200261) or cell-penetrating pepducins derived from the third intracellular loop of PAR1 (P1pal-12, P1pal-7).
(B) MDA-MB-231 cells migration toward NIH-3T3 CM supplemented with 0–500 pM thrombin in the presence of either MMP-200 (200 nM), BMS-200261 (100 µM), or 0.2% DMSO vehicle (open circle).
Figure 4. Fibroblast MMP-1 Mediates PAR1-Dependent Migration of Breast Cancer Cells
(A) MDA-MB-231 cell migration toward MDA-MB-231 CM in the presence of 5 nM of APMA-activated MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, or dialysis buffer alone.
(B) MDA-MB-231 migration toward RPMI-1640/0.1% BSA in the presence or absence of MMP-1 and/or the PAR1 antagonist BMS-200261.
(C) MDA-MB-231 cells were treated for 48 hr with PAR1 siRNA or luciferase siRNA control (−) and allowed to migrate toward MDA-MB-231 CM in the presence or absence of MMP-1.
(D) MDA-MB-231 cell migration toward CRL-2076 CM that was immunodepleted for individual MMPs. Monoclonal antibodies against the indicated MMPs or IgG control were coupled to Protein A Sepharose beads and used to immunodeplete CRL-2076 CM. The immunodepleted media served as chemoattractant in a standard migration assay.
(E) MDA-MB-231 cell migration toward NIH-3T3 CM that was supplemented with selective MMP inhibitors used at concentrations ≥3-fold above relevant IC50: 3 μM MMP Inh-1 (IC50 = 1 μM [MMP-1], 30 μM [MMP-3], 1 μM [MMP-8], 150 μM [MMP-9]), 5 μM MMP2-Inh (IC50 = 1.7 μM [MMP-2]), 50 μM MMP2/3-Inh (IC50 = 17 μM [MMP-2], 150 nM [MMP-3]). # p < 0.05, * p < 0.01.
Figure 5. Primary Human CRL-2076 Fibroblasts Secrete MMP-1 into Their Media
(A) CRL-2076 fibroblasts but not MDA-MB-231 breast cancer cells express MMP-1 mRNA. Total RNA was extracted from CRL-2076 or MDA-MB-231 cells. cDNA was prepared and portions of the cDNA corresponding to MMP-1, MMP-2, MMP-9 or β-actin messages were amplified by reverse transcriptase PCR and separated on a 1.5% agarose gel.
(B) Serum-free CRL-2076 CM was concentrated 20-fold and 200 µl applied onto a Superose-12 column equilibrated with TBS at 4°C. MMP-1 concentration (mean ± 1 SD; n = 3) was determined in 1 ml fractions by Quantikine ELISA (R&D Systems, Minneapolis). Chemotactic activity of each fraction (dashed line) was determined in duplicate using a standard MDA-MB-231 cell migration assay. This fractionation experiment was repeated three times and gave similar results.
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