ATG14 facilitated lipophagy in cancer cells induce ER stress mediated mitoptosis through a ROS dependent pathway

https://doi.org/10.1016/j.freeradbiomed.2017.01.007Get rights and content

Highlights

  • ATG14 is a potential mitoptosis inducer.

  • ATG14 and Ulk1 interact to induce lipophagy.

  • ATG14 triggers cytoprotective autophagy.

Abstract

Understanding the dynamics of autophagy and apoptosis crosstalk in cancer progression remains a challenging task. Here, we reported how the autophagy protein ATG14 induces lipophagy-mediated mitochondrial apoptosis. The overexpression of ATG14 in HeLa cells inhibited cell viability and increased mitochondrial apoptosis and endoplasmic reticulum (ER) stress. Furthermore, inhibition of this ATG14-induced autophagy promoted apoptosis. ATG14 overexpression resulted in the accumulation of free fatty acids (FFA), with a concomitant decrease in the number of lipid droplets. Our data showed that ER stress induced by ATG14 was due to the lipophagy-mediated FFA accumulation, which resulted in ROS-dependent mitochondrial stress leading to apoptosis. Inhibition of lipophagy in HeLa-ATG14 cells enhanced the cellular viability and rescued them from lipotoxicity. Mechanistically, we found that ATG14 interacted with Ulk1 and LC3, and knock down of Ulk1 prevented the lipidation of LC3 and autophagy in HeLa-ATG14 cells. We also identified a phosphatidylethanolamine (PE) binding region in ATG14, and the addition of Ulk1 to Hela-ATG14 cells decreased the ATG14-PE interaction. Lastly, confocal microscopy studies showed that the decrease in ATG14-PE binding was concomitant with the increase in LC3 lipidation over time, confirming the importance of Ulk1 to sort PE to LC3 during ATG14 mediated lipophagy induction. In conclusion, ATG14 and Ulk1 interact to induce lipophagy resulting in FFA accumulation leading to ER stress-mediated apoptosis.

Introduction

Oncophagy or autophagy in cancer is a highly dynamic area of research with immense therapeutic potential. Autophagy being a highly regulated catabolism-based homeostasis maintenance process is used by cells to manage stress by engulfing the damaged and degraded organelles. However in cancer, autophagy plays a dual role which make it very difficult to target for therapy. During tumorigenesis, autophagy plays a lethal role whereas during malignancy it becomes protective to shield the cancer cells. In particular, the crosstalk between autophagy, autophagy dependent cell death and apoptosis make the scenario extremely complicated.

Lipids form a very crucial component of cellular signaling processes. Lipids are metabolized not only to remain as vital energy reservoirs but they have a critical role in protein quality control, hormonal signaling, pathogenesis and maintenance of homeostasis [1]. Cellular stress induces lipid autophagy (lipophagy-a specialised form of autophagy), a process where energy stores in the form of lipids are engulfed within a double membrane autophagosome and degraded by lysosomal enzymes to release free fatty acids, which are generally shuttled to fuel the beta-oxidation in mitochondria. Advances in the field of lipid biology have revealed that it is involved in mobilization of lipid stores into free fatty acids in order to provide physiological endurance during stress.

Autophagic perturbations have led to dysfunctional lipo-homeostasis leading to disease conditions like genetic lysosomal storage disorders and hepatic steatosis [2]. Moreover, it is well reported that autophagy and cell death are intimately related and sometimes they may even share the same molecular machinery [3]. Interestingly, abrogation of the endocytic protein Rab7 was found to induce morphological changes resulting in hepatocellular lipophagy [4]. Another report clearly showed that the decline of MAP1S, an autophagy activator in clear cell renal cell carcinoma (ccRCC) patients, resulted in impairment of lipophagy [5]. The involvement of cytosolic lipase Adipose triglyceride lipase (ATGL) was shown to have a LC3-interacting region (LIR) motifs, such that mutating this LIR detaches ATGL from lipid droplets (LDs) and hinders lipophagy [7]. Although the role of LDs as a vital source of autophagic isolation membrane constituent is under scrutiny, but they serve a vital role in endoplasmic reticulum (ER) homeostasis linked to autophagy regulation [6]. In this context, the autophagy pre-initiator protein ATG14, which resides within ER, gets involved in autophagosome formation by tethering to the ER-mitochondria contact site by binding with SNARE protein syntaxin 17 (STX17) [8]. Further, ATG14 plays an essential role in STX17-SNAP29 mediated t-SNARE complex stabilization on autophagosomes to promote membrane tethering and autophagosome-endolysosome fusion [9]. Importantly, ATG14 is the most critical regulator gene reported to modulate autophagy initiation, and its knockdown results in an increase of triglyceride levels in the liver and serum of mice [10]. In this report, we have deciphered the mechanism by which the autophagy pre-initiator ATG14 and the autophagy initiator Ulk1 interact to induce lipophagy, resulting in free fatty acid (FFA) accumulation, leading to ER stress and mitoptosis.

Section snippets

ATG14 upregulation results in inhibition of cell proliferation

We investigated the effect of ATG14 on the cell proliferation in HeLa cells. Initially, we overexpressed the ATG14 gene (Fig. 1A) and found that there was a significant decline in the viability in comparison to mock-transfected cells when treated with a standard chemotherapeutic drug (cisplatin/cis-Diammineplatinum (II) dichloride (CDDP)) in a dose (1–100 μM) and time (24–72 h) dependent manner (Fig. 1B).

ATG14 overexpression induces apoptosis

Besides the autophagic activity of ATG14 as demonstrated through gain and loss of function

Discussion

Autophagy and apoptosis are known to have intersecting molecular pathways that modulate cellular stress [16]. Additionally, autophagy has a dual sided role in cancer, and can limit tumor initiation by decreasing inflammation and genome instability [17]. The crosstalk between autophagy and apoptosis has the potential to limit the tumor progression [18]. Lipophagy is a specific type of autophagy where cellular degradation of lipid droplets takes place within a double membrane called autophagosome

Reagents

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), dimethylsulfoxide (DMSO), Annexin-V-PE, propidium iodide (PI), N-acetyl-L-cysteine (NAC), tetramethylrhodamine methyl ester (TMRM), cisplatin/cis-Diammineplatinum (II) dichloride (CDDP), 3-methyl adenine (3MA), salubrinal (Sal) and dihydroethidium (DHE) were purchased from Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum (sterile-filtered, South American origin), minimal

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

We thank National Institute of Technology, Rourkela for providing a facility for this research work. Research support was partly provided by Department of Biotechnology [Grant Number: BT/PR7791/BRB/10/1187/2013 to SKB]; Science and Engineering Research Board (SERB), Department of Science and Technology [Grant Number: SR/SO/BB-0101/2012 to SKB]; Council of Scientific and Industrial Research (CSIR) [Grant Number: 37(1608)/13/EMR-II to SKB] Human Resource Development Group, Government of India and

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