SQSTM1/p62 loss reverses the inhibitory effect of sunitinib on autophagy independent of AMPK signaling

Bolin Hou,Huaiyi Yang,Gang Wang,Yange Wang,Caining Zhang,Yanjie Wei,Quan Gao,Zhijun Xi,Xuejun Jiang,Yuqing Huo

doi:10.1038/s41598-019-47597-4

Abstract

Sunitinib (ST), a multitargeted receptor tyrosine kinase inhibitor, has been demonstrated to be effective for the treatment of renal carcinoma. It has been reported that ST is involved in the mediation of autophagy; however, its regulatory role in the autophagic process remains controversial. Furthermore, the mechanism by which activated AMP-activated protein kinase (AMPK) negatively regulates autophagy remains nearly unexplored. In the present study, we revealed that ST inhibited AMPK activity and regulated autophagy in a cell type- and dose-dependent manner. In a number of cell lines, ST was demonstrated to inhibit H2O2-induced autophagy and the phosphorylation of acetyl-CoA carboxylase (ACC), whereas alone it could block the autophagic flux concurrent with increased expression of p62. An immunoprecipitation assay revealed that LC3 directly interacted with p62, whereas ST increased punctate LC3 staining, which was well colocalized with p62. Taken together, we reveal a previously unnoticed pathway for ST to regulate the autophagic process, and p62, although often utilized as a substrate in autophagy, plays a critical role in regulating the inhibition of ST in both basal and induced autophagy.

Highlights

Macroautophagy is a process involving the bulk degradation of cytosolic components by autophagolysosomes[1]
The cytotoxic effect of ST was further confirmed by the observation that the cleavage of PARP-1, which serves as a marker of cells undergoing apoptosis[25], was induced by ST (Fig. S1b)
The addition of 3-MA, a widely used inhibitor of early autophagy[26], increased PARP-1 cleavage, while the usage of chloroquine diphosphate salt (CQ), which inhibited the fusion between autophagosomes and lysosomes[27], further augmented ST/3-MA-induced caspase-dependent apoptosis (Fig. S1b)

Summary

Introduction

Macroautophagy ( referred to as autophagy) is a process involving the bulk degradation of cytosolic components by autophagolysosomes[1] It occurs in most eukaryotic cells at basal levels and is activated in response to the number of stimuli. AMPK was firstly found to be a kinase that directly inhibited ACC through increasing its phosphorylation; it functions in multiple ways to influence cellular metabolism, and its activation is upregulated responsive to various stress conditions with an increased ratio of AMP to ATP. Shang et al observed that nutrient starvation simulates the autophagic response mediated by ULK1 dephosphorylation and its dissociation from AMPK22 They further suggested that AMPK might have dual roles in the regulation of autophagy depending on the nutrient condition. While deprivation of p62 reversed the inhibitory effect of ST on basal autophagy, it no longer blocked the H2O2-activated autophagic flux in p62-depleted cells

Methods

Results

Conclusion