WT associated protein WTAP a nuclear protein is

WT1 associated protein (WTAP), a nuclear protein, is first identified by Little [6] who noticed its specific interaction with WT1. Besides several essential physiological processes, such as mRNA stabilization [7], eye development [8], m6A methylation [9], mRNA alternative splicing [10] and acetylcholine receptors regulation [11], WTAP is also involved in the carcinogenesis and progression of several malignant tumors, including glioblastoma [12], cholangiocarcinoma [13], acute myeloid leukemia (AML) [14], colorectal cancer (CRC) [15] and renal cell carcinoma (RCC) [16]. In our previous study, WTAP was identified as an independent prognostic indicator for PC, whose high expression in the nucleus had a significant relationship with advanced N stage and dismal prognosis [17]. It has been demonstrated that WTAP plays an oncogenic role by targeting WT1-TBL1 axis in CRC [15], while by stabilizing CDK2 mRNA in RCC [16]. However, the exact molecular mechanism in PC is still not unveiled. Focal adhesion kinase (Fak) is a cytoplasmic protein tyrosine kinase that is overexpressed and activated in several solid cancers [18]. Increased Fak dimerization induced by higher Fak levels contributes to its catalytic activation [19]. Its downstream signaling pathways are extremely complex and vary upon different tumor types, including Fak-RHOGEF-RHO signaling pathway; Fak-PI3K-AKT signaling pathway; Fak-Src-GRB2-Erk1/2 signaling pathway; Fak-JNK-JUN signaling pathway et al. [18]. Several studies also confirmed a strong relationship between Fak and PC. Firstly, Fak expression correlated significantly with the T and M stages in PC [20,21]. Secondly, activation of Fak was involved with the aggressive capability in PC [22], while Fak silencing could reverse its pro-metastatic effect [23]. Thirdly, Fak could increase intrinsic chemo-resistance to gemcitabine in PC [24], whereas RNA interference [25] or small molecular inhibitor [26,27] targeting Fak could enhance chemo-sensitivity to gemcitabine. Here, we provided the first evidence that overexpression of WTAP increased cell mobility and chemo-resistance to gemcitabine in both MIA PaCa-2 and BxPC-3 cells, while down-regulation of WTAP had an opposite effect. The positive correlation between WTAP and Fak expression was further confirmed in PC tissues by using GEPIA. Mechanically, WTAP could stabilize Fak mRNA and activate Fak signaling pathway. Moreover, a small molecular Fak inhibitor, GSK2256098, could reverse WTAP-induced chemo-resistance and metastasis which indicated that Fak might be a promising therapeutic target for PC.
Materials and method
Results
Discussion In our previous investigation, we found that WTAP was a novel prognostic factor in PC [17]. For one thing, the nuclear WTAP expression in tumor tissues was significantly higher than that of non-tumor tissues. For another, patients with high WTAP expression had a dismal prognosis when compared with patients who had low WTAP expression. In our present study, we demonstrated that WTAP could enhance PC cell migration/invasion and suppress its chemo-sensitivity to gemcitabine in a Fak-dependent manner. In detail, WTAP could bind to Fak mRNA and enhance its stability. Subsequent Fak overexpression then induced the activation of Fak signaling pathway, including Fak-PI3K-AKT and Fak-Src-GRB2-Erk1/2 pathways. GSK2256098, a small molecular Fak inhibitor, could reverse WTAP-induced tumor cell migration/invasion and chemo-resistance to gemcitabine in PC by suppressing the phosphorylation of AKT and Erk1/2. As such, we suggest that WTAP plays its carcinogenic role in PC by activating Fak signaling pathway and Fak inhibition might be a promising therapy for PC patients with WTAP over-expression. Since WTAP was first identified as a prognostic factor in glioblastoma [12], an increasing number of studies focused on its oncogenic role in various types of malignant tumors. For example, high WTAP expression was associated with advanced TNM staging in cholangiocarcinoma which was in accordance with in vitro assay result that WTAP could promote cholangiocarcinoma cells migration and invasion [13]. Further cDNA microarray analysis indicated that MMP7, MMP28, Cathepsin H and Muc1might be its downstream targets. In CRC, WTAP was overexpressed as a result of epigenetic inactivation of the carbonic anhydrase IV gene [15]. The transcription of TBL1, a target gene of WT1 [28], decreased as the overexpressed WTAP antagonized the transcriptional activity of WT1, which led to the activation of Wnt/β-catenin signaling pathway. Abnormal activation of Wnt/β-catenin signaling pathway then mediated the proliferation, invasion and migration of colon cancer cells. Moreover, WTAP was also a prognostic indicator for RCC patients and played its oncogenic role in RCC by binding to and stabilizing CDK2 transcript [16]. Besides solid tumors, the oncogenic role of WTAP was also observed in AML [14]. The mTOR pathway, identified as another WTAP downstream target, mediated its important impact on proliferation, survival and differentiation blockage in AML cells. Our laboratory previously found that WTAP was an independent prognostic factor in PC and its high expression usually predicted a poor prognosis in PC patients [17]. This study further demonstrated that WTAP could promote PC cell migration and invasion both in vitro and in vivo. In addition, we also found that WTAP decreased chemo-sensitivity to gemcitabine in vitro. However, the underlying molecular mechanism is still unknown.