Kim Ji Won, Park Miso, Kim Suntae, Lim Sung Chul, Kim Hyung Shik, Kang Keon Wook
Division of Hematology and Medical Oncology, University of California, San Francisco, CA, 94143, USA.
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
Cell Biosci. 2021 Nov 7;11(1):191. doi: 10.1186/s13578-021-00704-3.
Gonadotropin-releasing hormone receptor (GnRHR) transmits its signal via two major Gα-proteins, primarily Gαq and Gαi. However, the precise mechanism underlying the functions of Gαs signal in prostate cancer cells is still unclear. We have previously identified that GV1001, a fragment of the human telomerase reverse transcriptase, functions as a biased GnRHR ligand to selectively stimulate the Gαs/cAMP pathway. Here, we tried to reveal the potential mechanisms of which GV1001-stimulated Gαs-cAMP signaling pathway reduces the migration and metastasis of prostate cancer (PCa) cells.
The expression of epithelial-mesenchymal transition (EMT)-related genes was measured by western-blotting and spheroid formation on ultra-low attachment plate was detected after GV1001 treatment. In vivo Spleen-liver metastasis mouse model was used to explore the inhibitory effect of GV1001 on metastatic ability of PCa and the transwell migration assay was performed to identify whether GV1001 had a suppressive effect on cell migration in vitro. In order to demonstrate the interaction between androgen receptor (AR) and YAP1, co-immunoprecipitation (co-IP), immunofluorescence (IF) staining, chromatin immunoprecipitation (ChIP) were performed in LNCaP cells with and without GV1001 treatment.
GV1001 inhibited expression of EMT-related genes and spheroid formation. GV1001 also suppressed in vivo spleen-liver metastasis of LNCaP cells as well as cell migration in vitro. GV1001 enhanced the phosphorylation of AR and transcription activity of androgen response element reporter gene through cAMP/protein kinase A pathway. Moreover, GV1001 increased Ser-127 phosphorylation of YAP1 and its ubiquitination, and subsequently decreased the levels of AR-YAP1 binding in the promoter region of the CTGF gene. In contrast, both protein and mRNA levels of NKX3.1 known for tumor suppressor gene and AR-coregulator were upregulated by GV1001 in LNCaP cells. YAP1 knockout using CRISPR/Cas9 significantly suppressed the migration ability of LNCaP cells, and GV1001 did not affect the cell migration of YAP1-deficient LNCaP cells. On the contrary, cell migration was more potentiated in LNCaP cells overexpressing YAP5SA, a constitutively active form of YAP1, which was not changed by GV1001 treatment.
Overall, this study reveals an essential role of AR-YAP1 in the regulation of PCa cell migration, and provides evidence that GV1001 could be a novel GnRHR ligand to inhibit metastasis of PCa via the Gαs/cAMP pathway.
促性腺激素释放激素受体（GnRHR）主要通过两种主要的Gα蛋白，即Gαq和Gαi传递信号。然而，Gαs信号在前列腺癌细胞中发挥功能的精确机制仍不清楚。我们之前已经确定，人端粒酶逆转录酶的一个片段GV1001作为一种偏向性GnRHR配体，可选择性地刺激Gαs/环磷酸腺苷（cAMP）途径。在此，我们试图揭示GV1001刺激的Gαs-cAMP信号通路降低前列腺癌（PCa）细胞迁移和转移的潜在机制。
通过蛋白质印迹法检测上皮-间质转化（EMT）相关基因的表达，并在GV1001处理后检测超低附着板上的球体形成情况。采用体内脾-肝转移小鼠模型探讨GV1001对PCa转移能力的抑制作用，并进行Transwell迁移试验以确定GV1001在体外是否对细胞迁移有抑制作用。为了证明雄激素受体（AR）与Yes相关蛋白1（YAP1）之间的相互作用，在有无GV1001处理的LNCaP细胞中进行了免疫共沉淀（co-IP）、免疫荧光（IF）染色和染色质免疫沉淀（ChIP）。
GV1001抑制EMT相关基因的表达和球体形成。GV1001还抑制了LNCaP细胞在体内的脾-肝转移以及体外细胞迁移。GV1001通过cAMP/蛋白激酶A途径增强AR的磷酸化和雄激素反应元件报告基因的转录活性。此外，GV1001增加了YAP1的Ser-127磷酸化及其泛素化，随后降低了CTGF基因启动子区域中AR-YAP1的结合水平。相反，在LNCaP细胞中，作为肿瘤抑制基因和AR共调节因子的NKX3.1的蛋白质和mRNA水平均被GV1001上调。使用CRISPR/Cas9敲除YAP1可显著抑制LNCaP细胞的迁移能力，且GV1001不影响YAP1缺陷型LNCaP细胞的迁移。相反，在过表达YAP1组成型活性形式YAP5SA的LNCaP细胞中，细胞迁移更明显增强，且GV1001处理对此无影响。
总体而言，本研究揭示了AR-YAP1在调节PCa细胞迁移中的重要作用，并提供了证据表明GV1001可能是一种新型的GnRHR配体，可通过Gαs/cAMP途径抑制PCa的转移。
