Molecular studies on ASPP and FBI-1 in gestational trophoblastic disease

Abstract

Gestational trophoblastic disease (GTD) encompasses a heterogeneous group of trophoblastic lesions. It includes hydatidiform mole (HM), choriocarcinoma (CCA), placental site trophoblastic tumour (PSTT) and epithelioid trophoblastic tumour (ETT). While the latter three are classical malignancies, HM represents abnormal placenta harbouring malignant potential and may develop persistent gestational trophoblastic neoplasm (GTN) requiring therapy. Apoptotic activity and p53 are known to be important in pathogenesis of GTD. However, understanding on the underlying mechanisms is still limited.

ASPP1 and ASPP2 are the two tumour suppressor members of the apoptosis stimulating protein of p53 (ASPP) family that stimulate p53-dependent apoptotic pathway. In this study, a differential downregulation of ASPP1 and ASPP2 was observed in GTD, illustrating their distinct roles in the pathogenesis. Results showed that both ASPP1 mRNA and protein levels were significantly downregulated in HM and CCA, when compared with normal placentas by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Methylation-specific polymerase chain reaction (MS-PCR) in placenta and GTD samples demonstrated a significant correlation between ASPP1 mRNA level and their hypermethylation status (P = 0.024). Most importantly, lower ASPP1 immunoreactivity was found in HM that progressed into persistent GTN than HM that regressed (P = 0.045). Significant correlation was also found between expression of ASPP1 and apoptotic activity (M30 CytoDeath index), p53 and caspase-8 immunoreactivities. In CCA cell lines, namely JEG-3 and JAR, ectopic expression of ASPP1 triggered apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression, demonstrating a potent tumour suppressive function through its proapoptotic nature.

Downregulation of ASPP2 was mainly detected in choriocarcinoma, the most aggressive form of GTD. In vitro, results showed that ASPP2 was a multi-functional protein with activities not limited to apoptosis stimulation. While activated Src is important in tumour progression, transfection of ASPP2 but not ASPP1 was found to be related to decreased Src-pY416 phosphorylation. This suggested an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-induced inactivation of Src could be abolished by RNA interference (RNAi) with Csk small interfering RNA. Corresponding effect on aggressive behaviors such as cell migration was also confirmed. Hence, loss of ASPP2 in the choriocarcinoma implicates its crucial role in tumourigenesis.

FBI-1 is a transcription factor frequently overexpressed in human cancers. Recently, overexpression of FBI-1 in GTD in association with GTN development was also reported. However, its role in GTD pathogenesis remains elucidated at molecular basis. This study unveiled the ability of FBI-1 in contributing overt aggressiveness in GTD. Ectopic FBI-1 expression resulted in decrease in apoptosis and repression of pro-apoptotic genes such as Bak, Fas and caspase-8 at mRNA level in vitro. FBI-1 overexpression also promoted Akt activation as indicated by Akt-pS473 phosphorylation. Interestingly, effect of FBI-1 on cell motility and invasiveness was eradicated in the presence of specific PI3 kinase inhibitor LY294002. This demonstrated that FBI-1 could promote cell migration and invasion through PI3K/Akt signaling.

In summary, this study highlighted the effects of dysregulated ASPP and FBI-1 in apoptosis and aggressiveness, implicating their crucial roles in pathogenesis in GTD.