Inhibition of PDE4D prevents migration of HCC cells through modulation of IGF2/H19 cluster

Cyclic nucleotide phosphodiesterases (PDEs) play major roles in several different signalling pathways and thus control key cellular events, including cell proliferation, differentiation, and survival. Among PDEs, PDE4 is abundantly expressed in liver and appears deregulated in many tumours. The PDE4 family is encoded by four genes, namely PDE4A, PDE4B, PDE4C and PDE4D. Our previous studies showed that PDE4D is a major regulator of cAMP expression in hepatocarcinoma (HCC) cells and tissues. Its silencing or inhibition reduced cell proliferation and increased apoptosis of HCC cell lines by interfering with the expression of key cell cycle effectors. In addition, PDE4D silencing, or inhibition, affected the expression of several cancer-related genes, with a significant down regulation of the pro-oncogenic insulin growth factor 2 (IGF2), an imprinted gene whose transcription is regulated in a cluster with the region encoding for the H19 long non-coding RNA deeply involved in HCC proliferation, migration and invasion. Specific objectives of this research were to investigate the possible correlation between PDE4D overexpression, epithelial-mesenchymal transition (EMT) and uncontrolled cell migration, as well as to verify whether pharmacological inhibition of PDE4D can reverse EMT by acting on the IGF2/H19 cluster. The correlation between PDE4D and IGF2 expression patterns were confirmed by the CancerLivER database. Low tumorigenic (HepG2) and highly tumorigenic (Hep3B and Huh7) cell lines were used as HCC in vitro models. PDE4D activity was selectively inhibited using Gebr-7b. Western blotting experiments were performed to analyze the expression of proteins involved in epithelial-mesenchymal transition and cell migration, while qRT-PCR was employed to analyse the expression of IGF2 and H19 mRNA. Finally, Real-Time cell migration was evaluated using the IncuCyte video microscopy system in cell treated and nontreated with Gebr 7b. Selective pharmacological inhibition of PDE4D, using Gebr-7b, induced an upregulation of the epithelial marker E-cadherin and a down-regulation of the mesenchymal markers Twist and Snail. Gebr-7b treatment also significantly reduced HCC cell migration and induced upregulation of H19 gene expression, thus reducing the expression of IGF2 protein. We hypothesize that an aberrant up-regulation of PDE4D might impact the transcription of the IGF2/H19 cluster by disrupting its epigenetic regulation, potentially involving various already identified H19lnc regulators such as cAMP, PKA, and paxillin. Therefore, targeting the PDE4D/IGF2/H19 cluster with pharmacological selective inhibitors of PDE4D (e.g. Gebr-7b) may prevent metastatic dissemination and increase the efficacy of current HCC treatments while reducing toxicity. However, since the upstream signals governing the regulation of the IGF2/H19 cluster are only partially understood during hepatocarcinogenesis, further exploration is warranted to corroborate our hypothesis.