PCIF1 Modulates Glioblastoma Cell Migration and Invasion by Altering PI(3,4)P2 Levels through the PI5-Phosphatase INPP5B

Abstract

Phosphorylated CTD Interacting Factor 1 (PCIF1) is the exclusive methyltransferase responsible for the N6,2-O-dimethyladenosine (m6Am) modification in mammalian mRNA. Our previous research identified PCIF1 as a potent tumor suppressor in glioma, demonstrating its ability to impair cell proliferation, induce G2/M phase arrest, and promote apoptosis. However, its role in glioma cell migration and invasion remains unclear. In this study, we investigate how PCIF1 regulates glioma cell migration and invasion. Overexpression of PCIF1 inhibits migration and invasion, whereas PCIF1 knockdown enhances these behaviors. Corresponding changes are observed in mesenchymal markers (Vimentin, β-catenin, Snail, Slug) and the epithelial marker T-cadherin, indicating that PCIF1 suppresses epithelial-to-mesenchymal transition (EMT)-mediated glioma invasion. Mechanistically, PCIF1 modulates the AKT pathway by promoting proteasomal degradation of AKT while increasing phosphorylated AKT (p-AKT) levels, revealing a complex regulatory mechanism. PCIF1 knockdown upregulates INPP5B, a lipid phosphatase, causing accumulation of PI(3,4)P2 and enhanced AKT activation. Conversely, PCIF1 overexpression increases PI(3,4,5)P3 production, elevating p-AKT levels. This bidirectional regulation suggests that PCIF1 influences phosphoinositide signaling and AKT activation. Our findings highlight PCIF1 as a key modulator of glioblastoma cell migration and invasion through phosphoinositide signaling, positioning it as a potential biomarker and therapeutic target in glioma.

1. Introduction

Gliomas represent the most common type of primary intracranial tumors, accounting for approximately 48% of all primary malignant central nervous system (CNS) tumors. Among gliomas, glioblastoma multiforme (GBM) is the most aggressive and lethal subtype, characterized by rapid proliferation, diffuse infiltration into surrounding tissues, and a high recurrence rate after surgical resection [1]. The highly invasive nature of GBM significantly hampers effective treatment and is a major contributor to its dismal clinical outcomes [2,3]. One of the key drivers of GBM progression is the dysregulation of critical signaling pathways, particularly AKT signaling, which is frequently activated in these tumors [4,5]. The aberrant activation of AKT signaling plays a central role in promoting tumor growth, survival, and resistance to therapies [6,7]. Although several drugs targeting AKT signaling have been developed, their clinical success has been limited due to challenges such as drug resistance, activation of compensatory signaling pathways, and the complexity of the tumor microenvironment [8]. The gap in knowledge remains in understanding the precise molecular mechanisms by which this pathway is regulated, and this study aims to bridge that gap, offering insights into potential novel therapeutic targets for GBM treatment.

PCIF1, also known as cap-specific adenosine methyltransferase (CAPAM), is a critical enzyme responsible for m6Am modification in mammalian mRNA [9–11]. Structurally, PCIF1 contains a conserved N-terminal WW domain, two nuclear localization sequences (NLS) at the amino- and carboxyl-termini, and a conserved NPPF motif near the carboxyl-terminus that is essential for its m6Am methyltransferase activity [9–11]. The WW domain can bind to the phosphorylated C-terminal domain (CTD) of RNA polymerase II, potentially influencing transcriptional regulation, signaling, and cytoskeletal dynamics. These structural features suggest that PCIF1 may exert complex regulatory effects beyond its methyltransferase activity [11]. The m6Am modification plays a key role in regulating mRNA stability and translation efficiency, which in turn influences gene expression profiles associated with tumor progression in various cancers, including head and neck squamous cell carcinoma and gastric, renal and colorectal cancers, where PCIF1 promotes tumor growth and invasion [12–15]. In contrast, it acts as a tumor suppressor in bladder cancer, neuroblastoma, and melanoma [16–18]. Specifically, in bladder cancer, loss of PCIF1 enhances colony formation and tumor growth [16]; in neuroblastoma, m6Am modification promotes differentiation and reduces oncogenic potential [17]; and in melanoma, PCIF1 loss alters mRNA methylation patterns, contributing to tumor progression [18]. These findings highlight the context-dependent roles of PCIF1 and the complexity of its function, underscoring the novelty of investigating its functional mechanisms in glioma. Our previous study demonstrated an inverse correlation between PCIF1 expression and glioma grade, along with an inhibitory effect of PCIF1 on cell survival and proliferation, both in vitro and in vivo [19]. However, the molecular mechanisms by which PCIF1 regulates glioma migration, invasion, EMT, and AKT signaling remain largely unexplored.