Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):626-31. doi: 10.1073/pnas.1006886108. Epub 2010 Dec 27.
The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1-silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation.
凝血酶受体蛋白酶激活受体-1（PAR-1）在转移性黑色素瘤细胞系和肿瘤标本中过度表达。先前，我们通过全身递送包裹在纳米脂质体中的 siRNA 证明了 PAR-1 沉默后肿瘤生长和实验性肺转移显著减少。基因表达谱分析表明，PAR-1 沉默的转移性黑色素瘤细胞系中 Maspin 的表达增加了 40 倍。PAR-1 沉默后 Maspin 启动子活性显著增加，表明 PAR1 在转录水平上负调控 Maspin。ChIP 分析显示，PAR-1 减少了 Ets-1 和 c-Jun 转录因子与 Maspin 启动子的结合，这两种转录因子都已知能激活 Maspin 转录。PAR-1 沉默不影响 Ets-1 或 c-Jun 的表达；相反，它导致染色质重塑复合物 CBP/p300 的表达增加，以及 CBP/p300 抑制剂 p38 的活性降低，导致 Ets-1 和 c-Jun 与 Maspin 启动子的结合增加和更高的 Maspin 表达。功能上，PAR-1 沉默后 Maspin 表达降低了黑色素瘤细胞的侵袭能力，而在用 PAR-1 挽救后这种作用被消除。此外，在转移性黑色素瘤细胞系中表达 Maspin 后，肿瘤生长和实验性肺转移显著减少。此外，在 PAR-1 沉默细胞中沉默 Maspin 恢复了对肿瘤生长和实验性肺转移的抑制。在此，我们证明了 PAR-1 通过负调控 Maspin 肿瘤抑制基因的表达来获得转移性黑色素瘤表型的机制，从而为 PAR-1 赋予了除凝血以外的另一种功能。
