Sorafenib-Triggered Stress Granules Promote Resistance in Renal Cancer Cells by Recruiting Anti-apoptotic Cyclooxygenase 2
A significant proportion of RCC patients are inherently refractory to sorafenib, and most of the remaining patients develop resistance. The stress granules (SGs) induced by sorafenib contributing to renal cancer cells resistance is unknown.
In ACHN and 786O cells, SGs were shown by immunofluorescence using canonical SGs markers HuR, TIA-1 and G3BP1. The activation of GCN2 and the phosphorylation of its downstream eIF2α were detected by western blotting. Cells apoptosis percentages were measured by AnnexinV-PI staining. Cox2 mRNA level was evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). RNA-FISH was performed using specific Cox2 mRNA probes.
We found that sorafenib can trigger the formation of many small cytoplasmic foci in renal cancer cells. These particles-like foci were characterized as stress granules (SGs), because they were nicely co-localized with SG markers HuR, TIA-1 and G3BP1 (Fig.1A, 1B). These SGs have a protective role. The SGs triggered by sorafenib were found to be dependent on GCN2-eIF2α signaling. Sorafenib increased the phosphorylation of General control nonderepressible kinase 2 (GCN2) and its downstream eIF2α without influencing the total protein. Silencing of GCN2 expression also promoted sorafenib-induced cell apoptosis and reduced the formation of SGs (Fig.1C). We found anti-apoptotic gene cyclooxygenase 2 (cox2) was recruited into SGs. And the expression of cox2 was higher in sorafenib-resistant cells. RNA-based fluorescence in-situ hybridization (RNA-FISH) experiment was performed to observe the cellular distribution of cox2 mRNA. In sorafenib-treated cells, many particle-like distribution of cox2 mRNAs were found to be co-localized with SGs (Fig.2). To override sorafenib resistance, we inactivated cox2 using celecoxib, a clinical analgesic specific cox2 inhibitor. A combination treatment with low dosage of celecoxib and sorafenib induces ACHN cells death more efficient than sorafenib alone (Fig.3).
The SGs triggered by sorafenib were dependent on GCN2-eIF2α signaling. Our findings suggest that a combination therapy of cox2 inhibitor celecoxib may represent a novel therapeutic approach for resistant RCC treatment.
Funding: This work was supported by grants from the National Natural Science Foundation of China (81502203), the Natural Science Foundation of Jiangsu Province (BK20150097).