Impaired p65 degradation by decreased chaperone-mediated autophagy activity facilitates epithelial-to-mesenchymal transition

J Tang,Q-Q Yin,Q Zhao,M He,C-X Zhou,M-N Zhan,L-L Wo,C-L Wang,G-Q Chen

doi:10.1038/oncsis.2017.85

Abstract

Aberrant activation of nuclear factor-κB (NF-κB) has been observed in a wide range of human cancers and is thought to promote tumorigenesis and metastasis. As a central component of NF-κB pathway, p65 protein level is tightly regulated and could be subjected to proteasome degradation. Here we demonstrated that p65 can bind to HSC70 with four consensus recognition motif in its RHD domain and be constitutively transported to the lysosome membrane to bind with lysosome-associated membrane protein type 2A and degraded within the lysosome in two epithelial cell lines, proposing that p65 can be degraded by chaperone-mediated autophagy (CMA). Of great importance, there is a decreased CMA activity together with impaired degradation of p65 in a process of epithelial–mesenchymal transition (EMT). The resulted accumulation of p65 leads to higher NF-κB activity and contributes to the progression and maintenance of the EMT program. Taken together, our results define a novel regulatory mechanism for the important transcription factor p65, and these findings would shed new light on the inhibition of EMT, as well as metastasis of cancer cells.

Highlights

The nuclear factor-κB (NF-κB) signaling is a major transducer of external signals, controlling the expression of a broad range of genes involved in cell survival, proliferation, stress response and inflammation
To investigate whether chaperone-mediated autophagy (CMA) activity was altered in this Snailinduced Epithelial–mesenchymal transition (EMT) process, we first detected the level of lysosome-associated membrane protein type 2A (LAMP2A) and HSC70, the two major effectors of CMA in MCF-10A-NC and 6SA cells, and the results showed that the LAMP2A protein was significantly decreased in MCF-10A-6SA cells compared with MCF-10A-NC cells, whereas HSC70 showed no alteration (Figure 4e)
Immunofluorescence staining of LAMP2 was conducted, in consistent with the phenomenon observed in MCF-10A-6SA cells, decreased intensity and sporadic distribution of LAMP2 were observed in the mesenchymal MCF-10A cells under different inductions (Figure 5g). All these results showed that CMA activity was inhibited during EMT progressing, either by transcription factor Snail overexpression or by hypoxia/cytokines induction, indicating that the compromised activity of CMA could be a general event during EMT process

Summary

Introduction

The nuclear factor-κB (NF-κB) signaling is a major transducer of external signals, controlling the expression of a broad range of genes involved in cell survival, proliferation, stress response and inflammation. Canonical activation of the NF-κB pathway depends on degradation of its inhibitor, IκBα, which retains the cytosolic distribution of p65/p50 heterodimer by direct binding with them.[1] Hyper activation of NF-κB has been linked to abnormal proliferation, tumor initiation and progression in many types of cancer, including myeloma, leukemia and breast cancer.[2]. Activation of NF-κB is believed to be required for the induction and maintenance of EMT through a series of mechanisms including regulation of Twist and Snail.[7,8]

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Results

Conclusion