Plac8‐mediated autophagy regulates nasopharyngeal carcinoma cell function via AKT/mTOR pathway

Abstract To explore the relationship between autophagy and cell function, we investigated how PLAC8‐mediated autophagy influences proliferation, apoptosis and epithelial‐mesenchymal transition (EMT) in NPC. Colony formation analyses and CCK8 assays were used to assess the proliferative capacity of NPC cells. Transmission electron microscopy (TEM) was used to identify autophagosomes. Autophagic flux was monitored using the tandem monomeric RFP‐GFP‐tagged LC3 (tfLC3) assay. The rate of apoptosis in NPC cells was analysed by flow cytometry. Western blot analysis was used to evaluate the activation of autophagy and the signalling status of the AKT/mTOR pathway. Our study reveals that knocking out PLAC8 (koPLAC8) induces autophagy and apoptosis, while suppressing NPC cell proliferation and EMT. However, inhibition of autophagy with 3‐methyladenine or by knocking down Beclin‐1 reverses the cell proliferation, apoptosis and EMT influenced by koPLAC8. We find that koPLAC8 inhibits the phosphorylation of AKT and its downstream target, mTOR. Moreover, immunofluorescence and co‐immunoprecipitation reveal complete PLAC8/AKT colocalization and PLAC8/AKT interaction, respectively. Furthermore, knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway of NPC xenografts. Overall, our findings demonstrate that koPLAC8 induces autophagy via the AKT/mTOR pathway, thereby inhibiting cell proliferation and EMT, and promoting apoptosis in NPC cells.

all undifferentiated NPC have evidence of EBV infection. 4 EBV infection is the most widely and deeply studied pathogenic factor of NPC.
Available options for the treatment of NPC include concurrent chemoradiotherapy, adjuvant chemotherapy, induction chemotherapy and immunotherapy. 1 While NPC responds to concurrent chemoradiotherapy, local recurrence and distant metastasis remain as the main causes of mortality in NPC patients. 5 Additionally, development of resistance to both chemotherapy and radiotherapy further limits treatment options for some NPC patients. 6 These challenges raise an urgent need to elucidate the molecular basis of NPC and to leverage this knowledge for the development novel therapeutic strategies against NPC.
The 16kDa placenta specific gene 8 (PLAC8), also known as onzin, was first characterized to be highly expressed in the mouse placenta. 7,8 This gene was later found to be expressed in various cell types, including myeloid cells, lymphoid cells and epithelial cells of the lungs and intestines. 9-11 PLAC8 appears to play an important role in normal tissue homeostasis. Its deficiency has been associated with congenital immune deficiency in mice, 12 and its function has been reported to modulate the cell cycle in pancreatic cancer cells 13 and cell proliferation in hepatocellular carcinoma cells. 14 This gene has also been reported to regulate the expression of genes involved in cell growth, autophagy and epithelial-mesenchymal transition (EMT). 10,13,15 Autophagy (macro-autophagy) is the process by which a cell digests its cellular components, including organelles and recycles them. 16 Autophagy has been shown to modulate a wide range of cellular processes, including cellular responses to oxidation and nutritional stress, and tumorigenesis. 17 The role of autophagy in cancer is complex, and its function may vary depending on a number of biological factors, including tumour type, stage of progression and genetic background, as well as invasion and metastasis. 18 EMT refers to the process through which certain physiologic and pathologic conditions trigger the transformation of epithelial cells into mesenchymal cells. It is characterized by the loss of polarity by epithelial cells and their acquisition of migratory and invasive capacity, properties strongly associated with cancer cells. 19 Moreover, it has been shown that the relationship between autophagy and EMT varies between different tumour types. For instance, it has been reported that while inhibiting autophagy favours EMT in papillary thyroid carcinomas, 20 suppression of autophagy inhibits EMT in hepatocellular. 21 Considering our previous findings that PLAC8 influences EMT via the TGF-β/SMAD pathway 22 and that the TGF-β/SMAD pathway is reported to be involved in autophagy, 23 we wondered whether as part of this EMT-regulating network, PLAC8 might also modulate the autophagic pathway in NPC cells. To address this question, we investigated whether PLAC8 knockout in NPC cells affects the autophagic pathway and how this alters cell proliferation, apoptosis and EMT.

| Cell culture
CNE-2, a human nasopharyngeal squamous cell carcinoma cell line, was obtained from the China Center for Type Culture Collection (CCTCC) at Wuhan University. The cells were cultured in RPMI 1640 medium (Biosun) supplemented with 10% FBS (Gibco, Thermo Fisher), in a humidified incubator at 37°C, 5% CO2.

| Generation of koPLAC8 CNE-2 cells
Services for the generation of koPLAC8 CNE-2 cells (PLAC8-knockout CNE-2 cells) were procured from Biocytogen, a Beijing-based biotechnology company (www.bbctg.com.cn). 24 To this end, the PLAC8 intron between exons 2 and 3 was targeted through CRISPR/Cas9 and substituted with the puromycin resistance gene via homologous recombination. Puromycin resistance screening identified koPLAC8 clones, which were confirmed using PCR, sequencing and Western blotting.

| Cell migration and invasion assay
5 × 10 4 cells were seeded onto a Transwell chambers. The cells were then placed in culture for 48 hours, following which we analysed their migration and invasion capacity using 0.1% crystal violet. Cells on the lower chambers were stained. Five fields were randomly selected for cell counting, and cell images were taken under a microscope at 200× magnification. We next counted the number of cells that migrated through the membrane, and computed the migration and invasion scores as previously described. 23

| Tandem mRFP-GFP fluorescence microscopy
To monitor the autophagic flux, we used a tandem monomeric RFP-GFP-tagged LC3 (tfLC3) reporter to as previously described. 25 To evaluate tandem fluorescent LC3 puncta, cells were transfected with tfLC3 alone or co-transfected with siRNA (anti-Beclin-1 or nontargeting siRNA) and tfLC3. 48 hours post-transfection, the cells were washed with 1X PBS and immediately analysed via confocal microscopy using a Zeiss LSM 710 confocal microscope system (Carl Zeiss). Images analysis was performed by the ZENLE confocal analysis software (Carl Zeiss).

| Transmission electron microscopy
For transmission electron microscopy, cells were grown on 6-well plates and divided into two groups, the control group and koPLAC8 group and cultured for 24 hours. They were trypsinized and rinsed with 1X PBS. The cells were then collected by centrifugation at 1000 g for 5 minutes and fixed by resuspension in 2.5% glutaraldehyde and incubation for 2 hour. After fixation, samples were treated with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer and dehydrated through a graded series of acetone before embedment in resin. Finally, the samples were sectioned at a thickness of 65 nm and processed for TEM. TEM analysis was done with a Hitachi electron microscope H-600 (Hitachi, Ltd.). 25

| Western blot analysis
For Western blot analysis, cells were plated onto 6-well plates at a seeding density of 3 × 10 5 cells per well. The cells were then cultured overnight as described in section 2.1. The cells were then harvested and lysed on ice with ice-cold RIPA buffer supplemented with protease inhibitor cocktail (Cell Biolabs). Protein concentrations were then determined using the BCA assay method (Thermo Fisher Scientific). Prior to SDS-PAGE, the protein samples were denatured. SDS-PAGE was then used to resolve equal amounts of total protein (30-50 μg) on precast 10% polyacrylamide gels (Bio-Rad Laboratories). Next, the proteins were transferred onto a PVDF membrane (Thermo Fisher Scientific). Non-specific antibody binding was blocked by incubating membranes (with rocking) in 5% non-fat milk at room temperature for 1 hour. Next, membranes were incubated with the following primary antibodies overnight at the indicated dilutions: rabbit polyclonal p-AKT antibody, dilution 1:500 (Abcam, cat.

| Data analysis
All statistical data analyses were done using SPSS software, version 22.0 (SPSS Inc). All quantitative data are presented as mean ± standard deviation. Chi-square test and student t tests were applied as deemed appropriate different types of data analyses. P values of P < .05 are considered to indicate statistically a difference.

| PLAC8 knockout induces autophagy in NPC cells
To investigate the impact of PLAC8 knockout on autophagy, we employed TEM to examine the presence of autophagosomes in ko-

| Autophagy inhibitor 3-MA reverses the cell proliferation inhibited and apoptosis induced by PLAC8 knockout
3-MA is a widely used specific inhibitor of autophagy. 26 To establish whether the cell proliferation or colony formation inhibited and apoptosis induced by PLAC8 knockout emanate from autophagy induction, we used 3-MA to block autophagy and investigated its effect on these processes in koPLAC8 cells. To this end, we treated the cells with 3-MA for 24h, 48h and 72h and then carried out a CCK8 assay. This analysis revealed that relative to the controls, the proliferation of the CNE-2 cells was significantly inhibited by ko-PLAC8 and this effect could be reversed by treating the koPLAC8 cells with 3-MA (P < .05, Figure 2A,B). This observation was further confirmed by treating the cells with 3-MA and performing a colony formation assay. This analysis revealed that koPLAC8 cells treated with 3-MA formed more colonies relative to the untreated controls (P < .01) ( Figure 2C,D). We next monitored the effect of autophagy inhibition on apoptosis in koPLAC8 cells. This analysis also revealed a markedly reduced rate of apoptosis upon treatment of koPLAC8 cells with 3-MA treatment, relative to the untreated koPLAC8 cells (P < .01) ( Figure 2E,F). Taken together, these results demonstrate that inhibition of autophagy reverses the cell proliferation inhibited and apoptosis induced by knockout of PLAC8.

| Beclin-1 knockdown reverses the cell proliferation inhibited and apoptosis induced by knockout of PLAC8
Beclin-1 is a critical modulator of autophagy. 27 To further investigate the relationship between PLAC8 and autophagy, we knocked down with siRNA and interrogated its effect on proliferation, colony formation and apoptosis of koPLAC8 cells. To this end, we first successfully knocked down Beclin-1 by siRNA ( Figure 3A

| Autophagy induced by knockout of PLAC8 suppresses EMT in NPC cells
We next wondered whether the autophagy induction resulting from PLAC8 knockout affects EMT in NPC cells. To address this question, we used the koPLAC8 cells to perform the Transwell assay and quan-  Figure 4C). We next assessed the number of migrating and invading cells using the Transwell assay. This experiment indicated that that the number of migrating and invading ko-

PLAC8 cells was markedly reduced upon treatment with 3-MA or
Beclin-1 knockdown, relative to the negative controls ( Figure 4A).
We then decided to further explore the mechanisms through koPLAC8-induced autophagy promotes invasion and migration of NPC cells. Published studies have reported that EMT is closely related to invasion and migration in NPC. 28 To address this question, we explored the EMT marker proteins. This analysis revealed that relative to the control cells, the epithelial marker E-cadherin was strikingly elevated in koPLAC8 cells. On the contrary, we observed a significantly lower expression of the mesenchymal marker Vimentin, in the koPLAC8 cells relative to controls. We

| PLAC8 knockout inhibits AKT/mTOR signalling during autophagy
We next wondered about the mechanisms by which PLAC8 knockout activates autophagy in koPLAC8 cells. Inhibition of the AKT/ mTOR signalling pathway is known to potently induce autophagy. 29 We therefore wondered PLAC8 knockout might inhibit AKT activation and consequently, its downstream target, mTOR. To answer this question, we examined the state of AKT/mTOR signalling in CNE-2 cells. From this analysis, we did not observe any significant changes in the levels of AKT and mTOR ( Figure 5A,B). However, the levels of phosphorylated AKT and mTOR were reduced in the koPLAC8 cells, indicating suppressed AKT/mTOR signalling. To assess whether PLAC8 might directly modulate AKT activation, we performed a colocalization immunofluorescence assay. This experiment revealed complete colocalization between PLAC8 and AKT, suggesting a direct interaction between the two proteins. To explore whether the two proteins can directly interact, we carried out a co-immunoprecipitation experiment, which revealed interaction between PLAC8 and AKT ( Figure 5C,D). Together, these results indicate that PLAC8 regulates AKT activation and suggests a mechanism through which PLAC8 knockout inhibits the activation of the AKT/mTOR signalling, thereby activation autophagy in NPC cells.

| Knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway in vivo
Next, we used mouse xenograft models to study the effect of PLAC8 on NPC in vivo. Staining for the biomarkers of autophagy (Beclin-1, LC3-II and P62) using immunofluorescence and immunohistochemistry showed that the tumour cells in the koPLAC8 group had higher autophagy activity compared with that in the control group. Moreover, the Phosphorylation of AKT and mTOR in the koPLAC8 group had lower activity compared with that in the control group ( Figure 6A-C).
These results showed that knockout of PLAC8 induced autophagy and inactivated AKT/mTOR signalling pathway in vivo.

| D ISCUSS I ON
We have previously reported roles for PLAC8 in the modulation of proliferation, apoptosis, EMT and response to radiotherapy in NPC cells. 22,24 Here, we report findings from our investigation of the mechanisms through which PLAC8 influences proliferation, apoptosis and EMT in NPC cells. Notably, our results show that autophagy and apoptosis are activated CNE-2 cells upon PLAC8 knockout. Additionally, the increased rate of apoptosis resulting from PLAC8 knockout was strongly countered by inhibition of autophagy both pharmacological with 3-MA or by siRNA-mediated silencing of Beclin-1. Up to now, the association between apoptosis and autophagy in NPC has been unclear. We are aware of one study reporting the transformation of autophagy and apoptosis through the interaction between the anti-apoptotic protein Bcl-2 and the autophagic protein Beclin-1. 31 It is well established that apart from being and important factor in the formation of autophagosomes, 32 Beclin-1 is also an important component of the Beclin-1-Bcl-2 complex. 33 In this study, we found that PLAC8 potently suppresses autophagy and apoptosis in NPC cells and that F I G U R E 6 PLAC8 induces autophagy through AKT/mTOR pathway activation in vivo. (A) Beclin-1, LC3-II, P62, p-AKT and p-mTOR are stained by immunofluorescence assay. (B) Beclin-1, LC3-II, P62, p-AKT and p-mTOR are stained by immunohistochemistry assay. (C) The quantitative analysis of Beclin-1, LC3-II, P62, p-AKT and p-mTOR. Scale bar: 50 μm. *P < .05, **P < .01 the fraction of apoptotic cells increases upon PLAC8 knockout, increasing autophagy. We speculate that when Beclin-1 is increased, it leads to suppression of Bcl-2, which in turn increases the rate of apoptosis in NPC cells.
EMT refers to the process through which certain physiologic and pathologic conditions, including cancer trigger the transformation of epithelial cells into mesenchymal cells. This process is known to precedes invasion and migration by tumour cells. 34 The process of EMT can be monitored via various marker proteins, including the epithelial marker E-cadherin 35

and the mesenchymal marker
Vimentin. 36 EMT results in the down-regulation of E-cadherin and a corresponding up-regulation of Vimentin. 36 This process involves an intricate network of factors that include components of various cell signalling pathways like the Rac1/Cdc42-PAK pathway, 37 GSK-3β/β-catenin pathway 38 and the TGF-β/SMAD pathway 39 among others. However, in our study EMT was effectively reversed upon inhibition of autophagy both pharmacologically with 3-MA and siRNA-mediated knockdown of Beclin-1 in KoPLAC8 CNE-2 cells.
These observations confirmed that koPLAC8-induced autophagy is a potent inhibitor of EMT. Our group has previously found that knockout of PLAC8 inhibits EMT in NPC cells through inhibition of the TGF-β/SMAD pathway. This suggests that the observed promotion of autophagy upon PLAC8 knockout may lead to inhibition the TGF-β/SMAD pathway and thus inhibit EMT in NPC cells.
Inhibition of AKT/mTOR signalling is known to activate autophagy. 29,40 In this study, we observed that koPLAC8 inhibits the activation of AKT and its downstream target, mTOR. And xenograft model experiments showed that the Phosphorylation of AKT and mTOR in the koPLAC8 group had lower activity compared with that in the control group. Furthermore, using immunofluorescence we observed a complete colocalization between PLAC8 and AKT in CNE-2 cells. In addition, co-immunoprecipitation analysis demonstrated an interaction between PLAC8 and AKT. These results demonstrated that PLAC8 knockout inhibits activation of the AKT/mTOR pathway, thereby activating autophagy of NPC cells.
However, there are still some limitations in this work. We only used CNE-2 as the cell model of NPC. If we could add another cell models that are more native and of the NPC origin, such as c666-1 and NPC43, our results would be more representative, and the relationship between the expression of PLAC8 and the carcinogenesis of EBV might also be elucidated. Further studies should be performed to explore these problems.
Taken together, our results show that loss of PLAC8 promotes autophagy by inhibiting the AKT/mTOR pathway, thereby inhibiting proliferation and EMT, while promoting apoptosis in NPC cells ( Figure 7). These results suggest that some small molecule drugs targeting PLAC8 can be designed for the treatment of NPC in the future.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Shi-Ming Chen
https://orcid.org/0000-0002-1216-004X F I G U R E 7 A schematic depiction of the PLAC8/AKT/mTOR signalling axis and its influence on cell proliferation, apoptosis and EMT in NPC cells