Abstract
Autophagy is a cell-survival pathway with dual role in tumorigenesis, promoting either tumor survival or tumor death. WNK2 gene, a member of the WNK (with no lysine (K)) subfamily, acts as a tumor suppressor gene in gliomas, regulating cell migration and invasion; however, its role in autophagy process is poorly explored. The WNK2-methylated human glioblastoma cell line A172 WT (wild type) was compared to transfected clones A172 EV (empty vector), and A172 WNK2 (WNK2 overexpression) for the evaluation of autophagy using an inhibitor (bafilomycin A1—baf A1) and an inducer (everolimus) of autophagic flux. Western blot and immunofluorescence approaches were used to monitor autophagic markers, LC3A/B and SQSTM1/p62. A172 WNK2 cells presented a significant decrease in LC3B and p62 protein levels, and in LC3A/B ratio when compared with control cells, after treatment with baf A1 + everolimus, suggesting that WNK2 overexpression inhibits the autophagic flux in gliomas. The mTOR pathway was also evaluated under the same conditions, and the observed results suggest that the inhibition of autophagy mediated by WNK2 occurs through a mTOR-independent pathway. In conclusion, the evaluation of the autophagic process demonstrated that WNK2 inhibits the autophagic flux in glioblastoma cell line.
Highlights
Gliomas are the most common adult primary brain tumors [1]
To investigate the potential effect of WNK2 overexpression on autophagy, the A172-derived cell lines (A172 WT, A172 EV, and A172 WNK2) were evaluated for the main markers recommended by the guidelines for the use and interpretation of assays for autophagy monitoring (3rd edition) [26]
The cells were treated with bafilomycin A1 (baf A1) as an autophagy flux inhibitor, as well as with starvation (EBSS) and everolimus as autophagy inducers
Summary
Gliomas are the most common adult primary brain tumors [1]. Glioblastoma (GBM) is the highest-grade form of glioma (WHO grade IV), as well as one of the most aggressive types of cancer with rapid cellular growth, and highly invasive behavior, with a median overall survival time of. The current therapeutic approach is surgery followed by concomitant radiotherapy and temozolomide-based chemotherapy [3,4,5]; despite significant advances in diagnosis and therapy in recent decades, the outcomes for high grade gliomas (WHO grade III-IV) remains unfavorable [6]. Since autophagy can be viewed as pro- or anti-tumor, depending on the context [14], the dissection of its role in gliomas, as well as the associated molecular mechanisms is of critical importance. This is relevant because glioma cells usually respond to therapeutic agents that induce the autophagic process such as, TMZ and rapamycin in a clinical setting [15]. The present study aimed to explore the in vitro role of WNK2 in autophagic process in gliomas
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