Besides its carcinogenic role WTAP was also discovered to
Besides its carcinogenic role, WTAP was also discovered to participate in several physical processes in normal cells. As previously reported, WTAP was involved in cell proliferation, survival, apoptosis [7,29,30], eye development  and embryonic development  owing to its complex function in transcriptional and post-transcriptional regulation. Firstly, as a WT1 associated protein, WTAP could bind to WT1 to inhibit its transcription factor activity which in turn suppressed TBL1 , amphiregulin and Bcl-2  transcription. Secondly, as an important component of spliceosome , WTAP was reported to regulate the alternative splicing of survivin pre-mRNA in vascular smooth muscle Amitriptyline HCl australia . Thirdly, WTAP could enhance the stability of cyclin A2 and CDK2 mRNA stability by binding to their 3′-UTR [7,16]. However, on the contrary, WTAP might also accelerate the degradation of some of its target mRNAs by promoting m6A formation [32,33]. Fak, a non-receptor protein tyrosine kinase, can be dimerized and activated by overexpression . Several studies have confirmed its pro-metastatic role and the increased chemo-resistance to gemcitabine induced by Fak in PC [, , , , , ]. Moreover, Fak expression can be subject to transcriptional regulation [34,35], alternative splicing [36,37], and mRNA stability . Our preliminary data showed that WTAP overexpression could increase Fak and Fak mRNA in PC cells, while WTAP knockdown revealed an opposite effect. Taking together, we presumed that Fak was very likely to be the downstream target of WTAP in PC. In order to determine whether WTAP regulate Fak expression through a transcriptional or post-transcriptional manner, we conducted the Fak mRNA stability assay using actinomycin D. And the results indicated that Fak mRNA could be stabilized by WTAP. Further RIP assay demonstrated that WTAP could bind to WTAP mRNA specifically. Previous studies found WTAP could stabilize cyclin A2 and CDK2 mRNA by binding to their 3′-UTR [7,16]. And it has been proved that ACAAAUUAU, which corresponds to the 3′-UTR 1526–1534 in cyclin A2 mRNA, was the specific binding sequence for WTAP . Then the sequence of Fak mRNA was obtained from NCBI (https://www.ncbi.nlm.nih.gov) (Supplementary file. 2) and a similar sequence, ACAAAGAAU (3′-UTR 4025–4033) was identified. It is very likely that ACAAAGAAU (3′-UTR 4025–4033) is an essential element required for the WTAP-mediated stabilization of Fak mRNA which needs to be confirmed by further study. Next, with the concern that whether WTAP-induced Fak overexpression could result in the activation of itself and its downstream signaling pathways, western blot was performed. Not surprisingly, the western blot results confirmed that the WTAP could activate both Fak-PI3K-AKT and Fak-Src-GRB2-Erk1/2 signaling pathways.
GSK2256098 has been demonstrated to effectively inhibit Fak-PI3K-AKT and Fak-Src-GRB2-Erk1/2 signaling pathways in PC . Our study revealed a similar result using western blot. In addition, we also found that after GSK2256098 treatment, the chemo-resistance to gemcitabine and pro-metastatic effect induced by WTAP overexpression could be completely reversed. Thus, we presumed that Fak inhibitor might be a promising option for PC patients with WTAP overexpression.
Conflicts of interest
Acknowledgements The authors acknowledge the supporting from Dr. Bo-Ju Pan, Dr. Cheng-Cheng Wang and Dr. Jun-Ze Pang. This study was supported by grants from the CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-3-019) and Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (No. 2018PT32014).
Introduction Radiotherapy (RT) is a common treatment method for pelvic malignancies combined with surgery or chemotherapy (Hong et al., 2001). In pelvic and abdominal radiation therapy, the intestine and rectum are dose-limiting organs. The small intestine may be partially or completely removed from the radiation field via patient positioning and absorbable mesh (Deutsch and Stern, 1989; Das et al., 1997). In contrast, the rectum is inevitably exposed to pelvic tumor irradiation due to its fixed position. Radiation proctitis refers to the rectal injury caused by radiation therapy in pelvic cancer (Andreyev et al., 2011; Huang et al., 2016). Predictably, due to the popularity of RT, the number of people suffering from gastrointestinal complications is also increasing (Denton et al., 2002). Radiation proctitis not only affects quality of life but also limits the dose of radiation; above all, severe acute injury may lead to interruption of therapy and limit the curative effect of RT (Sher, 2010). Radiation proctitis associated with pelvic RT is scored based on the Common Toxicity Criteria (CTC) of the National Cancer Institute and it is identified by assessing clinical symptoms (Trotti et al., 2000). However, patient-reported symptoms are subjective by nature, so an objective index is needed for management and making clinical decisions such as reducing dosage or interrupting RT. Although several methods have been applied, there are still no effective non-invasive or objective markers to measure RT intestinal and rectal injury (Larsen et al., 2007; Hille et al., 2009). It is essential to determine the mechanism of radiation toxicity and search for a specific therapeutic target in order to develop a therapeutic strategy for preventing or treating acute and late radiation damage.