p21-Activated Kinase-1 Promotes Aggressive Phenotype,Cell Proliferation,and Invasion in Gestational Trophoblastic Disease

Gestational trophoblastic disease (GTD) includes hydatidiform mole (HM), which can develop persistent gestational trophoblastic neoplasia requiring chemotherapy; choriocarcinoma, which is a frankly malignant tumor; placental site trophoblastic tumor; and epithelioid trophoblastic tumor. p21-Activated kinases (PAKs) promote malignant tumor progression. Therefore, this study investigated PAK1, PAK2, and p-PAK2 Ser²⁰ in the pathogenesis of GTD. By real-time PCR, PAK1 mRNA was significantly higher in HMs, particularly metastatic HMs (P = 0.046) and HMs that developed persistent disease (P = 0.011), when compared with normal placentas. By immunohistochemistry, significantly increased cytoplasmic PAK1 immunoreactivity in cytotrophoblasts was also detected in HMs (P = 0.042) and choriocarcinomas (P = 0.003). In addition, HMs that developed persistent disease displayed higher PAK1 immunoreactivity than those that regressed (P = 0.016), and elevated PAK1 immunoreactivity was observed in placental site trophoblastic tumors. Indeed, there was significant positive correlation between PAK1 expression and the proliferative indices Ki-67 (P = 0.016) and MCM7 (P = 0.026). Moreover, higher PAK1 mRNA and protein expression was confirmed in the choriocarcinoma cell-lines JEG-3 and JAR; however, PAK2 mRNA and p-PAK2 immunoreactivity showed a similar expression pattern in normal first trimester placentas and GTD. Knockdown of PAK1 in JEG-3 and JAR reduced cell proliferation, migration, and invasion ability, up-regulated p16, and down-regulated vascular endothelial growth factor and MT1-MMP expression. This is the first report revealing the involvement of PAK1 in the pathogenesis and clinical progress of GTD.Gestational trophoblastic disease (GTD) is a disease of the placenta resulting from abnormal trophoblastic proliferation. It encompasses hydatidiform mole (HM), choriocarcinoma (CCA), placental site trophoblastic tumor (PSTT), and epithelioid trophoblastic tumor. The latter three are malignant tumors, whereas HMs are nonneoplastic lesions with an increased risk of malignant transformation. Furthermore, 8% to 30% of HMs may persist after suction evacuation and develop gestational trophoblastic neoplasia (GTN), hence requiring chemotherapy. Although the response of GTD to chemotherapy is in general very good, chemoresistant cases still exist despite advances in chemotherapy. While there is growing understanding in the molecular biology of GTD, the precise molecular pathways of its development should be further explored.^1,2,3p21-Activated kinases (PAKs) were initially discovered as effector molecules of Rho GTPases, Rac, and CDC42,⁴ and are increasingly recognized as important mediators for a wide variety of cellular functions, including cell morphogenesis, motility, mitosis, apoptosis, and angiogenesis.^5,6 PAKs have been divided into group I (PAKs1-3) and group II (PAKs4-6), bases on structural organization and mode of regulation.⁵ In group I PAKs, the p21-binding domain in the N-terminal domain binds small GTPases Rac and CDC42 to cause autophosphorylation of certain sites in the N-terminal inhibitory domain, leading to conformational change that releases the auto-inhibition and activates the kinase activity.⁵ Phosphorylation of PAK1 at Ser²¹ regulates the binding of PAK1 to Nck, resulting in PAK1 cycling between membrane and cytosol and finally facilitates cell migration.^7,8 Its corresponding phosphorylation site at PAK2 is Ser²⁰.Overexpression of PAK1 has been observed in various human cancers,⁶ such as breast, colorectal, and ovarian cancers.⁹ PAK1 was found to regulate the invasiveness of breast cancer cells and the malignant progression of colorectal carcinomas to metastasis.^10,11 PAK2 has also been found to be involved in Rac3-mediated breast cancer cell proliferation.¹² The recent development of kinase inhibitors for the PAK family molecules^13,14 is intriguing since they may be explored as potential therapeutic targets in chemoresistant GTD.In this study, we investigated the expression and localization of PAK1, PAK2, and p-PAK2 Ser²⁰ in normal human placenta and in GTD to determine whether PAK1 or PAK2 are involved in development and malignant progression of GTD. We also correlated their expression with clinical outcome and examined the in vitro effects and potential downstream targets of PAK1 on CCA cells.