PinX1-Induced Autophagy Inhibits Cell Proliferation and Induces Cell Apoptosis by Inhibiting the NF-κB/p65 Signaling Pathway in Nasopharyngeal Carcinoma

Abstract

reversed these outcomes. Mechanistic investigations clari ed that PinX1 overexpression signi cantly reduced the expression of p-AKT, p-mTOR, p65, and p-p65. Chloroquine treatment in PinX1-overexpressing cells did not signi cantly alter p-AKT and p-mTOR levels, whereas 3-MA treatment in PinX1overexpressing cells resulted in increased p65 and p-p65 expression, relative to untreated PinX1overexpressing cells.

Conclusion
These ndings indicate that PinX1 promotes autophagy by inhibiting the AKT/mTOR signaling pathway; this, in turn, inhibits the NF-κB/p65 signaling pathway, thereby inhibiting cell proliferation and induces cell apoptosis in NPC cells.

Background
Nasopharyngeal carcinoma (NPC) is a highly malignant tumor, originating from the nasopharyngeal mucous membrane, which metastasizes easily throughout the body via the lymph nodes; it is common in South China and Southeast Asia 1, 2 . Patients with early-stage NPC often lack symptoms or have nonspeci c symptoms. Approximately 75% of NPC patients are at an advanced stage when they rst seek medical attention, and about 10% exhibit distant organ metastasis 3 . The locoregional control rate of NPC has improved signi cantly in the past decade following therapeutic improvements, including the development of comprehensive treatment strategies such as intensity-modulated radiotherapy and chemotherapy, concurrent radiotherapy and chemotherapy 4 , surgical treatments under nasal endoscopy 5 , and PD-1 antibody immunosuppressive therapy [6][7][8] . However, the long-term survival rate of patients with NPC remains poor due to recurrence and/or distant metastasis 9 . Further the side-effects of atreatments used in NPC can lead to poor outcomes, especially in patients with locoregionally advanced NPC 10,11 . Current research is therefore focused on clarifying the molecular mechanisms underlying tumor invasiveness and metastasis in NPC, and to investigate new treatment methods to improve prognosis and prolong survival in NPC patients.
Autophagy is a highly conserved cyclical degradation process, regulated by lysosomes, that is stably present in eukaryotes 12 . Abnormal or inhibited autophagy may induce various diseases 12 , including cancer and neurodegeneration. Autophagy can make tumor cells more resistant to apoptosis 13 . In contrast, autophagy and apoptosis can act on cancer cells to promote their death 14 . Autophagy plays important roles in NPC cell proliferation and differentiation, and in chemo-and radioresistance 15 ; it promotes both survival and apoptosis 16 . The effects of autophagy on tumor growth in NPC remain to be clari ed; this lack of clarity may be due to differences in research targets or drugs investigated.
This study therefore aimed to clarify the regulation of autophagy in NPC cells, on the basis of our prior investigations. Preliminary research from our group indicates that transfecting NPC cells with PinX1 inhibited their telomerase activity and proliferation, and signi cantly increased their apoptosis rate 17 . Our objective was therefore to examine how PinX1 affects autophagy in NPC cells, and to elucidate the molecular mechanisms involved. Further, we aimed to elucidate the interaction between autophagy and apoptosis. Finally, the effect of PinX1 on the oncogenesis of NPC was evaluated in vivo. This work will provide new and precise treatment strategies for NPC.

Materials And Methods
Cell lines and cell culture The nasopharyngeal cancer cell lines CNE2 and 6-10B were purchased from the Beijing Concord Cell Resource Center (Beijing, China), and were cultured in RPMI-1640 (HyClone, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/mL streptomycin, and 100 U/mL penicillin, in a humidi ed incubator with 5% CO 2 at 37 °C. The culture medium was replaced after 24 h, and cells were passaged after 72 h. The third passage of nasopharyngeal cancer cell lines, at the logarithmic phase of growth, were used for further experiments.

MTT assay
The proliferative capacity of the transfected and non-transfected CNE2 cells was measured via MTT Assay Kit (ab211091, USA). Brie y, after culturing for 24, 48, and 72 h, the cells were seeded onto 96-well plates at 1 × 10 5 cells per well. MTT was added to the plates, which were then incubated for 2 h. Absorbance at 570 nm was determined using a microplate reader. The experiment was repeated three times to obtain the mean values. The cell viability curves were plotted using the culturing time as the abscissa and the OD value as the ordinate.

Transwell assays
Migration and invasion by transfected and non-transfected CNE2 cells were determined using Transwell assays. For the migration analysis, DMEM supplemented with 10% FBS (Hyclone) was added to the lower chamber, and 2 × 10 4 cells in serum-free medium were added to the upper chamber. A similar protocol was used for the invasion analysis, except that the chambers were covered with Matrigel matrix (BD Biosciences, Franklin Lakes, NJ, USA). During incubation, the cells migrated and invaded through the lower membrane. Cells in the lower chambers were stained and xed with 4% paraformaldehyde and 0.1% crystal violet, then counted under an OLYMPUS CX41 upright microscope. At least four elds of vision were randomly selected from each sample, to calculate the mean number of cells that had moved through the Matrigel, providing an index of cell invasiveness.

Apoptosis assay
The cell-cycle phases and apoptosis rates of the transfected and non-transfected CNE2 cells were analyzed via ow cytometry, using an Annexin V/propidium iodide (PI) apoptosis detection kit (Beyotime, Shanghai, China), following the manufacturer's instructions. Brie y, after 48 h of incubation in a 96-well plate, the CNE2 cells (blank, Vector, Over-PinX1, and Over-PinX1+3-MA) were collected and stained with Annexin V-uorescein isothiocyanate (FITC) and PI, then kept in the dark at room temperature for 15-20 min. Flow cytometry data were then acquired using a FACSCalibur HG ow cytometer (BD Biosciences). FlowJo 10 (Tree Star Software, San Carlos, CA, USA) was used to analyze the ow cytometry data.

Reverse-transcription quantitative PCR (RT-qPCR)
Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions; it was used as a template for the reverse-transcription reactions into cDNA, following the instructions of the Bestar qPCR RT Kit (Applied Biosystems, Grand Island, NY, USA). RT-qPCR was performed using the Agilent Stratagene Mx3000 real-time qPCR Thermocycle Instrument (Agilent Stratagene, CA, USA), with the cDNA as the template and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference. PCR ampli cations were performed using the DBI
Xenograft tumorigenicity assay in nude mice Eight 4-week-old female nude mice with a body weight of 17 g were numbered randomly using earrings. A total of 1×10 4 logarithmically growing cells (Blank, Vector, or Over-PinX1) in 0.1 ml RPMI-1640 medium without FBS were injected subcutaneously into the right side of each nude mouse (n = 5 per group).
Tumor size was measured once a week in the feeding environment. At four weeks after injection, nude mice were sacri ced and tumor grafts were isolated. The size of tumor grafts was calculated using the equation V = (a 2 *b)/2, where a is the length of short side and b is the length of the long side of the tumor graft. Differences in tumor-graft volume were compared among the groups of mice injected with the three cell groups. The animals were provided by the Animal Laboratory of Southern Medical University. The in vivo experiments were approved by the Laboratory Animal Committee and were conducted in accordance with the National Laboratory Animal Care and Maintenance Guide.
Immuno uorescence CNE2 cells in each group were seeded into 6-well plates, washed with phosphate buffer solution (PBS), and xed with 4% paraformaldehyde (PFA) at 4 °C overnight. Next, the cells were washed twice with PBS (3 min per wash), blocked with 10% goat serum for 15 min, and incubated with LC3B antibodies (dilution, 1:200) at 4°C for 1 h. After washing three times with PBS (3 min per wash), the cells were incubated with Alexa Fluor 488 conjugated secondary antibodies (dilution, 1:1000) at room temperature for 1 h. Next, the secondary antibodies were removed, and the cells were washed three times with PBS (5 min per wash). Finally, the cells were mounted with DAPI staining solution and incubated for 10 min at room temperature in the dark, and analyzed under a Bx51 inverted orescence microscope (,Olympus Corporation, Shinjuku, Japan).

Hematoxylin-Eosin (H&E) Staining
To observe changes in tumor tissue morphology, para n-embedded sections of tumor tissue were stained with H&E solution. The nude mice in each group were sacri ced and their tumors were removed. The tumor tissue was then dehydrated by exposure to decreasing concentrations of ethanol, embedded in para n wax, and cut into sections 5 mm thick. The para n sections were depara nized then rehydrated in decreasing concentrations of ethanol, then stained with hematoxylin and eosin (both from Servicebio, Wuhan, China), following the manufacturer's protocols.

Immunohistochemistry
Para n sections prepared from the in vivo experiments were used for immunohistochemistry assays, to detect PinX1 protein expression. The indirect streptavidin-peroxidase method was, following the manufacturer's instructions. Para n sections were rehydrated using Histo-Clear (National Diagnostics) followed by a 100% to 70% ethanol gradient. Endogenous peroxidase activity was quenched using H 2 O 2 .
Antigen retrieval was performed in a steamer for 30 min, in citrate antigen retrieval solution. The sections were then placed in avidin and biotin blocking solutions (Vector Labs), followed by addition of 2.5% normal horse serum (Vector Labs) and overnight incubation with rabbit polyclonal anti-PinX1 (dilution, 1:100). ImmPRESS HRP Anti-Rabbit Ig and ImmPACT DAB Peroxidase (Vector Labs) were used for detection. Detection was followed by dehydration of the tissue in a 70% to 100% ethanol gradient and in Histo-Clear, followed by mounting using Vectashield mounting medium.

Statistical analysis
Statistical analyses were performed using SPSS 24.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as the mean ± SD from at least three independent experiments. Comparisons between two groups were performed using Student's t-tests, one-way ANOVA for multiple groups, and a parametric generalized linear model with random effects for tumor growth and MTT assay. All statistical tests were two-sided.

PinX1suppresses cell growth in vitro and tumorigenesis in vivo
To identify the role of PinX1 in NPC development, we rst examined its expression in the 6-10B and CNE2 cell lines. RT-qPCR and western blot analysis revealed that PinX1 was strongly expressed in 6-10B cells but weakly expressed in CNE2 cells (Fig. 1a). Therefore, the PinX1-overexpression plasmid (pcDNA3.0-PinX1) was introduced into the CNE2 cell line, to further explore its biological role in NPC. PinX1 expression was more than twofold greater in CNE2 cells treated with pcDNA3.0-PinX1 than in the blank and vector groups, based on RT-qPCR and western blot analysis (Student's t-tests, P < 0.001; Fig. 1b).
Subsequently, we examined the effect of PinX1 expression on hTERT expression and NPC cell growth in vitro. hTERT expression in CNE2 cells treated with pcDNA3.0-PinX1 was signi cantly suppressed relative to that in the blank and vector groups, based on RT-qPCR (Student's t-tests, P < 0.001; Fig. 1c). MTT assay revealed that PinX1-overexpression signi cantly suppressed cell growth (P < 0.001; Fig. 1d). These results suggest that PinX1 substantially inhibits the growth of NPC cells by targeting telomerase.
Next, we conducted an in vivo tumor-formation experiment, by subcutaneously injecting CNE2 cells (untreated, treated with the empty vector, or pcDNA3.0-PinX1-transfected cells) into the nude mice. The tumor growth curve was obtained by calculating the volume of tumors in each group at 7, 14, 21, and 28 days after inoculation: the tumor growth rate in the PinX1-overexpressing mice was signi cantly lower than that in the blank and vector groups (Fig. 2a). At 28 d after implantation, the PinX1-overexpressing mice had smaller tumor burdens (Fig. 2b) and displayed higher PinX1 expression in tumor tissues than the controls, and their transplanted tumor tissues showed fewer obviously pathological mitotic cell nuclei and cellular atypia than the control groups (Fig. 2c). These results suggest that PinX1 signi cantly inhibits tumorigenesis in vivo.

Overexpression of PinX1 induces autophagy in NPC cells via the AKT/mTOR signaling pathway
To investigate the impact of PinX1 overexpression on autophagy, we rst took advantage of the DAPI-FITC-LC3 reporter, to monitor the effect of PinX1 on autophagic ux. PinX1-overexpressing CNE2 cells contained more blue-green puncta than the control groups, suggesting that PinX1-overexpression activated autophagy (Fig. 3a). To establish whether autophagy was indeed activated in PinX1overexpressing cells, the level of the autophagy marker, Beclin-1, was measured using western blotting. Beclin-1 protein levels were markedly elevated in PinX1-overexpressing cells, which also exhibited an elevated LC3-II/LC3-I protein ratio. In addition, p62 protein levels were reduced in PinX1-overexpressing cells, relative to the controls (Fig. 3b). Together, these observations suggest that PinX1 overexpression induces autophagy. To further investigate the role of PinX1 overexpression in cell invasion and migration, we inhibited autophagy pharmacologically using 3-MA, a widely used speci c inhibitor of autophagy 18 , and monitored its effects on autophagy. By monitoring autophagic ux using the DAPI-FITC-LC3 reporter, we found that 3-MA treatment of PinX1-overexpressing cells reduced the number of blue-green puncta (Fig. 3a). Western blot analysis revealed that 3-MA reduced the LC3-II/LC3-I ratio, and abolished the PinX1-overexpression-induced reduction of p62 expression (Fig. 3b). These results indicate that 3-MA is a potent inhibitor of PinX1-overexpression-induced autophagy in CNE2 cells. We then assessed the number of migrating and invading cells using the Transwell assay. This analysis showed that migration and invasion by PinX1-overexpressing cells was signi cantly reduced relative to the controls, while treatment with 3-MA markedly increased the number of migrating and invading PinX1-overexpressing cells (Fig. 3c).
We next investigated the mechanism whereby PinX1 overexpression activates autophagy in NPC cells. It has been reported that inhibiting the AKT/mTOR signaling pathway potently induces autophagy 29 . Therefore, we examined the state of AKT/mTOR signaling in CNE2 cells, by measuring the changes in the levels of phosphorylated AKT and mTOR: these were signi cantly lower in PinX1-overexpressing CNE2 cells than in the controls (Fig. 3d), indicating suppressed AKT/mTOR signaling in these cells. Adding chloroquine to PinX1-overexpressing cells did not cause signi cant differences in the levels of phosphorylated AKT and mTOR, relative to the untreated PinX1-overexpressing cells, although it did inhibit autophagic ux, as revealed by the immuno uorescence assay (Fig. 3e), indicating that PinX1 might directly modulate AKT phosphorylation. Together, these results suggest a mechanism whereby PinX1 overexpression inhibits the activation of AKT/mTOR signaling, thereby activating autophagy in NPC cells.

Autophagy inhibitor 3-MA reverses the effects of PinX1 overexpression on cell proliferation, apoptosis, and the cell cycle in NPC cells
To establish whether the inhibition of cell proliferation and apoptosis induced by PinX1 overexpression is caused by the induction of autophagy, we used 3-MA to block autophagy, and investigated its effect on these processes in PinX1-overexpressing cells, using a MTT assay. Relative to the controls, the CNE2 cell proliferation was signi cantly inhibited by PinX1 overexpression, and this effect was reversed by treating PinX1-overexpressing cells with 3-MA (Fig. 4a). We next investigated how inhibiting autophagy affected apoptosis in PinX1-overexpressing cells. Flow cytometry analysis revealed an obviously higher apoptosis rate in PinX1-overexpressing cells than in the control groups; further, treating PinX1-overexpressing cells with 3-MA markedly reduced the rate of apoptosis, relative to the untreated PinX1-overexpressing cells (Fig. 4b). Taken together, these results demonstrate that inhibiting autophagy reverses the inhibition of cell proliferation, and induction of apoptosis, that result from PinX1 overexpression. Furthermore, we monitored the effect of inhibiting autophagy on the cell cycle in PinX1-overexpressing cells. The percentage of cells in the G0/G1 phase was higher, and that of cells in G2/M phase was lower, in PinX1overexpressing cells than in the control groups (Fig. 4c). Further, treating PinX1-overexpressing cells with 3-MA signi cantly reduced the percentage of cells in the G0/G1 phase and increased that of cells in the G2/M phase. These ndings indicate that inhibiting autophagy reverses the deceleration of cell-cycle progression caused by PinX1 overexpression in CNE2 cells.
PinX1 overexpression induces cell apoptosis by promoting autophagy, via the NF-κB/p65 signaling pathway To further elucidate the signaling pathway involved in PinX1-overexpression-induced apoptosis, we assessed NF-κB/p65 signaling in CNE2 cells, by measuring changes in the levels of p65 and p-p65. Expression of p65 and p-p65 was signi cantly lower in PinX1-overexspressing CNE2 cells than in the controls (Fig. 5). In addition, 3-MA treatment of PinX1-overexpressing cells increased the expression of p65 and p-p65 relative to untreated PinX1-overexpressing cells. These results indicate that PinX1 overexpression promotes autophagy, thereby inhibiting the NF-κB/p65 signaling pathway, thus inducing apoptosis.

Discussion
We have previously reported roles for PinX1 in modulating proliferation, apoptosis, EMT, and stemness in NPC cells 19 . Here, we investigated the mechanisms whereby PinX1 in uences proliferation, apoptosis, and autophagy in NPC cells.
There is substantial evidence that PinX1, which has crucial roles in the carcinogenesis of many cancers, is a potential novel human cancer diagnostic biomarker and therapeutic target 20,21 . Previous studies have shown that PinX1 can speci cally inhibit telomerase activity and induce tumor-cell apoptosis 22,23 . Here, we examined PinX1 expression in two NPC cell lines, CNE2 and 6-10B, using RT-qPCR and western blot analysis. Our ndings showed that PinX1 was strongly expressed in 6-10B cells but weakly expressed in CNE2 cells. We therefore chose the CNE2 cell line for subsequent experiments, to clarify the roles and mechanisms of action of PinX1 in cell proliferation and tumorigenesis. Not only did PinX1 signi cantly inhibit NPC cell proliferation in vitro, it also suppressed tumorigenicity in vivo. These results suggest that PinX1 functions as a potential tumor suppressor in NPC.
It is well established that autophagy is involved in tumor growth, proliferation, and apoptosis [24][25][26] . However, it remains unclear how autophagy affects carcinogenesis in NPC; this may be related to differences in research targets. For example, Liu et al. demonstrated that TIPE1 promoted NPC progression by inducing cell proliferation and inhibiting autophagy via the AMPK/mTOR signaling pathway. Zhu et al. 27 showed that Annexin A1 promotes NPC cell invasion and metastasis by suppressing autophagy via activation of PI3K/AKT signaling. Here, we found that PinX1 overexpression signi cantly increased the density of characteristic autophagosomes and increased LC3B expression in NPC cells. Further, the LC3-II/LC3-I ratio, and Beclin-1 expression, were higher in PinX1-overexpressing cells than in the controls. Conversely, PinX1 overexpression downregulated p62 levels. Together, these results con rm that PinX1 overexpression induces autophagy in NPC cells. In addition, pharmacological inhibition of autophagy using 3-MA signi cantly reversed these outcomes in PinX1-overexpressing cells, which signi cantly increased the number of migrating and invading cancer cells, enhanced their proliferative capacity, and reduced apoptosis. Our data strongly suggest that PinX1 inhibits cell proliferation and induces cell apoptosis by inducing autophagy in NPC cells.
The AKT/mTOR signaling pathway is a key pathway in regulating autophagy, and plays a vital role in tumorigenesis [28][29][30] . It is known that p-AKT and p-mTOR are highly expressed in various NPC cell lines, and activation of the AKT/mTOR signaling pathway is closely related to poor prognosis 31,32 . Here, PinX1 overexpression produced signi cantly lower levels of phosphorylated AKT and mTOR than in the control groups, suggesting that PinX1 inhibits AKT and its downstream target, mTOR. Further, adding chloroquine to PinX1-overexpressing cells inhibited autophagic ux but did not induce any signi cant changes in the levels of phosphorylated AKT and mTOR, relative to the untreated PinX1-overexpressing cells. Chloroquine inhibits autophagy mainly by reducing autophagosome-lysosome fusion, rather than by altering the acidity or degradation activity of organelles [49] . Therefore, chloroquine did not affect the signaling molecules that induce autophagosome formation. Our results con rm that PinX1 overexpression inhibits the activation of the AKT/mTOR pathway, thereby activating autophagy in NPC cells.
Cell-cycle progression is a predominant factor promoting tumor-cell proliferation and inducing apoptosis. Therefore, we examined the effects of PinX1 on the cell cycle. PinX1 overexpression decelerated cell-cycle progression and induced cell apoptosis by activating autophagy in CNE2 cells. Notably, activation of the NF-κB pathway is the main catalyst for the expression of anti-apoptotic genes in cells, which plays an important role in promoting tumor survival 33 . Therefore, we examined the state of NF-κB/p65 signaling in CNE2 cells, by measuring changes in the levels of p65 and p-p65, to elucidate the mechanism involved in PinX1-overexpression-induced cell apoptosis. PinX1 overexpression remarkably inhibited the NF-κB/p65 signaling pathway in CNE2 cells. Furthermore, inhibiting autophagy in PinX1-overexpressing cells remarkably rescued their p65 and p-p65 expression. These ndings show that PinX1, by promoting autophagy, inhibits the NF-κB/p65 signaling pathway, thereby inducing apoptosis.
Our ndings show that PinX1 promotes autophagy by inhibiting the AKT/mTOR signaling pathway. This, in turn, inhibits the NF-κB/p65 signaling pathway, thereby inhibiting cell proliferation and inducing cell apoptosis in NPC cells. As these processes play a vital role in NPC malignancy in humans, our results reveal that autophagy may be a pivotal target for NPC therapy.

Declarations
Ethics approval and consent to participate The in vivo experiments were approved by the Laboratory Animal Committee and were conducted in accordance with the National Laboratory Animal Care and Maintenance Guide.

Consent for publication
Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.