CHK2 Promotes Anoikis and is Associated with the Progression of Papillary Thyroid Cancer
Cellular Physiology and Biochemistry
Background/Aims: Cell cycle checkpoint kinase 2 (CHK2) performs essential cellular functions and might be associated with tumorigenesis and tumor progression. Here, we explored the function and molecular mechanisms of CHK2 in the progression of papillary thyroid cancer (PTC). Methods: The expression levels of both total CHK2 and activated CHK2 (p-CHK2) in tissues from 100 PTC patients were detected and evaluated using immunohistochemistry. The roles of CHK2 on cell proliferation, invasion,
... ion, invasion, migration, apoptosis and cancer stem cell (CSC) markers were investigated by CCK-8, Transwell, flow cytometry, western blot and ALDEFLOUR assay. PTC cells cultured in suspension conditions were assayed for anoikis. The anchorageindependent condition was further detected by soft agar colony formation assay. Furthermore, anoikis associated regulatory proteins were explored by western blot and verified by forced downregulation experiment, respectively. Results: We found that the levels of both CHK2 and p-CHK2 were significantly upregulated in PTC cancer tissues compared with those in tumoradjacent tissues. The overexpression of p-CHK2 in primary tumor tissues was associated with tumor aggressiveness and metastatic potential. However, the levels of both CHK2 and p-CHK2 were decreased in metastatic lymph nodes. Our results showed that CHK2 upregulated the levels of CSC markers with no effect on cell proliferation, invasion and migration. Interestingly, we revealed a previously undescribed anoikis-promoting role for CHK2 in PTC. Specifically, the detachment of PTC cells from the extracellular matrix (ECM) triggers CHK2 degradation. Then, the forced downregulation of CHK2 rescued PTC cells from anoikis, but no effect was observed on the apoptosis of adherent PTC cells. Additionally, as a novel regulator of anoikis, CHK2 can induce cell death in a p53-independent manner via the regulation of PRAS40 activation. Conclusion: High expression levels of CHK2 and p-CHK2 were associated with the progression of PTC. Our results defined an unexpected role for CHK2 as a mediator of anoikis that functions through the regulation of PRAS40 activation, which may be associated with the survival of circulating tumor cells and metastatic behavior. from the extracellular matrix (ECM) was associated with increased ERK1/2 and p38 MAPK activation, whereas the level of AKT and GSK-3β activation was decreased (Fig. 4A) . Evidence from previous studies has supported the idea that activation of the anti-apoptotic P13K-AKT signaling pathway plays an important role in anoikis of PTC cells  . In addition, recent studies have reported that PRAS40 acts as a downstream regulator of the P13K-AKT pathway and that the Thr246 phosphorylation site can be induced by Akt  . Here, we observed that detachment reduced the activation of PRAS40, which reminded us that the level of phosphorylation at the Thr246 site was also dependent on Akt in PTC cells (Fig. 4A) . Moreover, we observed that the detachment of the cells from the ECM triggered CHK2 downregulation (Fig. 4B) . Fig. 2. The expression levels of CHK2 in human papillary thyroid cancer cell lines and the effect of CHK2 on the expression of CSC markers. (A) CHK2 protein expression in three human PTC cell lines. (B) PTC cells were transfected with CHK2-specific siRNAs and assayed for CHK2 expression by qRT-PCR and Western blot. TPC-1 cells were used to select the optimal siRNA for the silencing of CHK2. CHK2 levels were decreased between 80% and 90% when cells were transfected with CHK2-specific siRNA-2. CHK2 expression was significantly lower than when CHK2-specific siRNA-1 and CHK2-specific siRNA-1 were used, as demonstrated in BCPAP and KTC-1 cells. Thus, CHK2-specific siRNA-2 was used in our study (mean± SD; *** p<0.001). (C) The identification of clonal populations of BCPAP cells that stably overexpressed CHK2 by Western blot. From the 8 clones, clones 3 and 4 were identified as CHK2-OE, while clone 4 was selected for further study. (D) The effects of CHK2 silencing on PTC cell growth after 1, 2 and 3 days were assessed using a CCK-8 assay (mean± SD). (E) The migration and invasiveness of PTC cells were assessed after siRNA transfection. Cell migration was analyzed using Transwell membranes without Matrigel. Invasion was analyzed using Transwell membranes with Matrigel. (F) The effect of CHK2 on CD44 expression in BCPAP cells. (G) Representative dot plots of the ALDEFLOUR assay results. (H) Graph plot of ALDH-positive cells by ALDE-FLOUR assay (mean± SD; * p<0.05).