14‐3‐3ζ binds to and stabilizes phospho‐beclin 1S295 and induces autophagy in hepatocellular carcinoma cells

Abstract Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14‐3‐3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up‐regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14‐3‐3ζ’s role in regulating autophagy in HCC cells, with a focus on beclin 1. The co‐localization of 14‐3‐3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT‐2 and HCC‐LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14‐3‐3ζ and phospho‐beclin 1S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT‐2 and HCC‐LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS295VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14‐3‐3ζ binding motif. CO‐IP results confirmed that 14‐3‐3ζ bound to beclin 1, and this connection was markedly weakened when S295 was mutated into A295 (alanine). Further, 14‐3‐3ζ overexpression prevented phospho‐beclin 1S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14‐3‐3ζ enhanced the chemoresistance of HCC cells to cis‐diammined dichloridoplatium by activating autophagy. Our work reveals that 14‐3‐3ζ binds to and stabilizes phospho‐beclin 1S295 and induces autophagy in HCC cells to resist chemotherapy.


| INTRODUC TI ON
Primary liver cancer comprises an unfavourable prognosis and is the second major cause of cancer-associated death around the world. 1 Hepatocellular carcinoma (HCC) that arises from the malignant transformation of hepatocytes is the major type of primary liver cancer. 2 Portal vein tumour thrombosis (PVTT) is a severe complication that links to poor survival of patients with HCC. 3 14-3-3ζ (gene symbol YWHAZ) belongs to the highly conserved 14-3-3 protein family. 4 By comparing to non-tumour specimens, 14-3-3ζ expression was higher in tumour tissues derived from patiesnts with varied cancers, such as breast cancer and lung cancer. 5,6 Our group also found an up-regulation in 14-3-3ζ in HCC tumour tissues, and this elevation might be associated with PVTT formation. 7,8 On the basis of our prior work, we continue exploring the role of 14-3-3ζ in HCC progress, with an emphasis on autophagy.
Autophagy is a catabolic mechanism by which the dysfunctional cytoplasmic components are degraded and recycled. 9 It occurs to sustain the body homeostatic functions under basal conditions and displays both detrimental and protective capabilities upon the setting of pathological states. 10 Low oxygen concentration (hypoxia) is commonly observed in the microenvironment of many solid malignancies. 11 Cancer cells often activate pro-survival autophagy to resist hypoxia-induced death. [12][13][14] We prior found that the expression level of hypoxiainducible factor 1α (HIF-1α), a central hypoxia-induced transcription factor, was higher in PVTT (+) HCC tumour samples than that in PVTT (−) tumour samples. 8 In addition, we also demonstrated that 14-3-3ζ expression was increased in HCC cells exposed to hypoxia, and 14-3-3ζ itself could stabilize HIF-1α, thereby promoting the growth and lung metastasis of HCC cells. 15 Considering the important role of HIF-1α in hypoxia-induced autophagy, 16 we propose that 14-3-3ζ affects HCC progress and PVTT formation by regulating autophagy.
Autophagy is precisely orchestrated by multiple autophagy-related proteins (ATGs). 17 A study from Zhao et al 18 showed that ATG7 mRNA expression was up-regulated after 14-3-3ζ overexpression and down-regulated when 14-3-3ζ was knocked down in SMMC-7721 cells. Weerasekara and coworkers proved that, under hypoxia, the phosphorylation of Atg9A at S761 (S, serine) was enhanced by AMPK, which enabled its binding to 14-3-3ζ, thereby promoting Atg9A recruitment to LC3-positive autophagosomes. 19 These two previous studies suggest a regulatory effect of 14-3-3ζ on autophagy-associated molecules. It is worth noting that, once activated, autophagy proceeds through four sequential steps: initiation and nucleation of phagophore, autophagosome expansion, autolysosome maturation and autophagy execution. 10,17 Beclin 1 (gene symbol BECN1) that forms a complex with phosphatidylinositol-3-kinase (PI3K) is a vital molecule for triggering the very first step of autophagy. 10,17 To explore whether 14-3-3ζ affects autophagy by interacting beclin 1 will provide novel sights into 14-3-3ζ-mediated autophagy.
The above-mentioned hypothesis was tested in CSQT-2 cells (established from PVTT tissue) 23 and HCC-LM3 cells 24 , two cell lines with high metastatic potential. We found that 14-3-3ζ indeed bound to beclin 1 by docking to RLPpS 295 VP motif and induced autophagy in these cells. 14-3-3ζ also prevented the protein degradation of phosphorylated beclin 1 in HCC cells exposed to transcriptional inhibitor.

| Database
The expression levels of YWHAZ and BECN1 gene and their expression correlation were analysed with Gene Expression Profiling Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/; using data from The Cancer Genome Atlas [TCGA]). In short, the transcripts per million (TPM) of these two genes were detected in HCC tumours and non-tumours. Further, their correlation coefficient in HCC samples was determined with Spearman's analysis. Positive R value indicated positive correlation. A P value <.05 was considered significant.

| Cell culture and treatment
CSQT-2 cells 23 were stored in our laboratory and used as in vitro model for PVTT. HCC-LM3 cells were kindly provided by Prof.
The fragment encoding wild-type beclin 1 S295 (identical to NM_003766) was inserted into c-Flag pcDNA3 vector between Hind III and BamH I sites. The beclin 1 S295A mutant was generated by specific primers that replaced AGT (a triplet codon of serine) with GCA (a triplet codon of alanine). The beclin 1 S295A was also constructed into c-Flag pcDNA3 vector. Plasmid transfection into CSQT-2 cells was mediated by Lipofectamine 2000 (Invitrogen) according to the manufactory's protocols.

| Co-immunoprecipitation (co-IP) and Western blot
For co-IP, cell proteins were first extracted from cancer cells, and then, protein (200 μg) was incubated with 1 μL anti-beclin 1 antibody or ant-flag antibody overnight. Then, these samples were incubated with 60 μL Protein A Agarose at 2°C for 2 hours. After centrifugation, the mixture was rinsed with 1 × PBS and then resuspended in 60 μL loading buffer (5×). After being boiled for 5 minutes, the sample was subjected for Western blot. F I G U R E 1 Positive expression correlation of YWHAZ and BCEN1 gene in HCC. GEPIA public database was used to analyse the transcripts per million of (A) YWHAZ and (B) BCEN1 gene in samples derived from patients with HCC. (C) The expression correlation of these two genes was analysed with Spearman. (D) Immunofluorescence was performed to probe 14-3-3ζ and beclin 1 expression in primary HCC tissue. Green arrows indicated 14-3-3ζ, whereas red arrows indicated beclin 1. Yellow arrows indicated the colocalization of 14-3-3ζand beclin 1. Positive R value indicated positive correlation. A *P value <.05 was considered significant. BCEN1, beclin 1; GEPIA, Gene Expression Profiling Interactive Analysis For some experiments, cells were pretreated with 20 mg/mL actinomycin D (MedChemExpress).

| Patients
Tissue samples were obtained from a patient diagnosed with HCC/ PVTT before neoadjuvant therapy. A written informed consent was provided by this patient. The research was carried out according to the World Medical Association Declaration of Helsinki.

| Immunofluorescence
Cells were fixed in 4% paraformaldehyde and permeabilized in tri-tonX-100 (0.1%) at room temperature for half an hour. Primary HCC tissue samples were cut into 5-μm sections and deparaffinized. After dehydration, tissue slices were incubated in boiled antigen retrieval buffer and soaked in 1× PBS for a total of 15 minutes. Both cells and tissue slices were incubated in goat serum at room temperature and then incubated with one of the following antibodies: LC3 (1:200, CST), anti-14-3-3ζ (1:100 for cells; 1:50 for tissue slices; Sangon Biotech) and anti-beclin 1 (1:200 for cells; 1:50 for tissue slices; ProteinTech) overnight. Then, cells were treated with FITC-or Cy3labelled secondary antibody. Cell nuclei were counterstained with DAPI. 14-3-3ζ-and beclin 1-positive cells were counted and shown as a percentage. To determine the apoptotic ratios, cells were stained with Annexin V-Light 650/PI (Wanleibio) and analysed on a flow cytometry.

| Statistical analysis
All data were presented in mean plus standard deviation (SD) and analysed with SPSS version 20.0 (IBM). Comparison between two groups was performed with unpaired Student's t test (two tailed).
Comparison over multiple groups was performed with one-way ANOVA followed by Bonferroni's multiple comparison test. A P value <.05 was considered significant.

| Positive expression correlation of YWHAZ and BCEN1 gene in HCC
By analysing the TPM of YWHAZ and BCEN1 gene via GEPIA public database, we found that their expression levels were concomitantly higher in HCC tumours (n = 369) than that in non-tumours (n = 160; Figure 1A,B). Further, Spearman's results indicated that YWHAZ expression was positively correlated with BCEN1 expression (P value <.05; R = .5; Figure 1C). In addition, immunofluorescence was performed to determine 14-3-3ζ (green) and beclin 1 (red) expression.
14-3-3ζ and beclin 1 were detectable in the primary HCC tissue, and their co-localization could be observed ( Figure 1D).

| 14-3-3ζ promotes autophagy in HCC-LM3 and CSQT-2 cells
HCC-LM3 and CSQT-2 cells were infected with n.c. oe-LV or 14-3-3ζ oe-LV for 72 hours, and the expression of mRNA and protein levels of YWHAZ were determined with qRT-PCR and Western blot ( Figure 3A,C,E,G). A significant up-regulation of YWHAZ at both transcriptional and translational levels confirmed the efficiency of 14-3-3ζ oe-LV. For knockdown detection, cells were first infected with n.c. sh-LV or 14-3-3ζ sh-LV for 72 hours and then incubated under hypoxia for additional 12 hours. 14-3-3ζ sh-LV could effectively knock 14-3-3ζ down in these two cell lines ( Figure 3B,D,E,G).
The overexpression of 14-3-3ζ also up-regulated the mRNA expression of BECN1 gene in cancer cells ( Figure S2). Moreover, 14-3-3ζ overexpression did not alter the expression of VPS34 in HCC cells, but augmented its binding to beclin-1 ( Figure 3F,H).

CO-IP was conducted in both HCC-LM3 and CSQT-2 cells. A co-localization of 14-3-3ζ and beclin 1 was observed in both HCC-LM3
and CSQT-2 cells ( Figure 4A,B). The encoding fragments of BECN1 gene were amplified from CSQT-2 cells and HCC-LM3 cells and analysed with agar electrophoresis ( Figure 4C). The fragments with correct size were then sequenced. We found that the amplified fragments shared identical nucleic acid information with BECN 1-NM_003766. The predicted polypeptides encoded by this fragment contained RLPS 295 VP, a potential motif for 14-3-3ζ binding ( Figure 4D,E). Next, flag-labelled beclin 1 S295 and beclin 1 S295A were transfected into CSQT-2 cells, and CO-IP with anti-flag antibody for IP and anti-14-3-3ζ antibody for IB was performed. The results showed that phosphorylation of beclin 1 on S295 was responsible for 14-3-3ζ binding. These data identify beclin 1 as a novel protein that interacts with 14-3-3ζ.

| 14-3-3ζ contributes chemoresistance of HCC cells to CDDP by inducing autophagy
Cis-diammined dichloridoplatium of 100 μmol/L was used to treat HCC-LM3 and CSQT-2 cells for 24 hours. Apoptotic cells were stained with Annexin V/PI and analysed via flow cytometry assay. (E-H) Western blot was performed to determine the protein levels of 14-3-3ζ, p62, beclin 1 and phospho-beclin 1 S295 . (F,H) Co-IP with antibeclin 1 for IP, and anti-beclin 1 and anti-VPS34 for IB was performed. (I-L) Immunofluorescence was performed to analyse the formation of LC3 puncta. Data were shown in mean with SD (N = 3). A *P value <.05 was considered significant. nc, negative control; LV, lentivirus; oe, overexpression; shRNA, short hairpin RNA ( Figure 5D,H). These data confirm that 14-3-3ζ overexpression activates autophagy in HCC cells to induce their resistance to CDDP.

| D ISCUSS I ON
Data from GEPIA revealed a positive correlation between YWHAZ and BCEN1. Furthermore, our in vitro results demonstrated that 14-3-3ζ bound to and stabilized phospho-beclin 1 S295 in HCC-LM3 and CSQT2 cells ( Figure 6) and induced autophagy in these cells to resist CDDP cytotoxicity. The RLPS 295 VP of beclin 1 was confirmed to provide the docking site for 14-3-3ζ.
During autophagy, beclin 1 consists a complex with PI3K to induce the initiation/nucleation of autophagosome. Sequentially, soluble LC3I converses to autophagosome-associated LC3II. Finally, p62 shuttles the aggregates of intracellular proteins into autolysosome for degradation. 10,17 Herein, HCC-LM3 and CSQT-2 cells were exposed to hypoxia up to 24 hours to mimic the in vivo tumour microenvironment. The strongest autophagy flux was observed in these cells at 12 hours after hypoxia: LC3II was increased, and p62 was decreased markedly. Interestingly, we noted that 14-3-3ζ protein level in these cancer cells also peaked at 12 hours after hypoxia. This concurrent event suggests that 14-3-3ζ elevation may participate in hypoxia-induced autophagy.
Beclin 1 contains several sites that can be phosphorylated by kinases. For instance, death-associated protein kinase (DAPK) phosphorylates beclin 1 on T119, 25 whereas AMPK phosphorylates beclin 1 on S90 and S93 in an ATG14-dependent manner. 26 Such phosphorylation on beclin 1 is important for autophagy initiation. Notably, hypoxia can phosphorylate beclin 1 at certain site as well-beclin 1 S30 was phosphorylated in U87 cells under hypoxia. 27 In the present study, we first analysed the sequence information of BECN1 gene in both HCC-LM3 and CSQT-2 cells and found that the encoding region of BECN1 gene of the two cell lines were identical to BECN1-NM_003766 uploaded on protein database of National Center for Biotechnology Information (NCBI). The sequence information implied that S295, but not S30, may provide a binding site for 14-3-3ζ. Thus, we additionally detected the protein level of phospho-beclin 1 S295 in HCC-LM3 and CSQT-2 cells F I G U R E 4 14-3-3ζ binds to phosphobeclin 1 S295 in HCC-LM3 and CSQT-2 cells. Co-IP was conducted in both (A) HCC-LM3 and (B) CSQT-2 cells. (C) Fragments (1371 bp) containing BECN 1 gene CDS were amplified from these cells and analysed with agar electrophoresis. The sequence information closely to RLPS 295 VP (R, arginine; L, leucine; P, proline; S, serine; V, valine) was shown in D,E. (F) Flag-labelled beclin 1 S295 and beclin 1 S295A were transfected into CSQT-2 cells, and co-IP with anti-flag antibody for IP and anti-14-3-3ζ antibody for IB was performed. (G) HCC-LM3 and (I) CSQT-2 cells were infected with n.c. oe-LV or 14-3-3ζ oe-LV, and 72 h later, cells were treated with 20 mg/mL actinomycin D for additional 12 h. Protein levels of phosphobeclin 1 S295 were assessed with Western blot. In addition, (H) HCC-LM3 and (J) CSQT-2 cells were infected with n.c. sh-LV or 14-3-3ζ sh-LV, and 72 h later, cells were treated with 20 mg/mL actinomycin D for additional 12 h under hypoxia. Protein levels of phospho-beclin 1 S295 were also assessed with Western blot. β-Actin was the reference. nc, negative control; LV, lentivirus; oe, overexpression; shRNA, short hairpin RNA under normoxic and hypoxic conditions. Our data revealed a novel phenomenon that hypoxic exposure can induce phosphorylation of beclin 1 on S295.
Interestingly, both the BECN 1 mRNA and total protein expression levels were increased when 14-3-3ζ was overexpressed, implying that 14-3-3ζ promoted the transcription and translation of BECN 1 gene in HCC cells. Of note, in SMMC-7721 cells, 14-3-3ζ positively regulated the mRNA expression of ATG7 gene. 18 Such findings together with our current data suggest that 14-3-3ζ may F I G U R E 5 14-3-3ζ contributes chemoresistance of HCC cells to CDDP by inducing autophagy. HCC-LM3 and CSQT2 cells were infected with n.c. oe-LV, 14-3-3ζ oe-LV or beclin 1 sh-LV, and 48 h later, cells were treated with 100 μmol/L CDDP, 10 mmol/L 3-MA or 30 μmol/L chloroquine for additional 24 h. (A, C, E,G) The protein levels of phosphobeclin 1 S295 , beclin 1 and LC3 were determined with Western blot analysis. (B, D, F,H) Apoptotic cells were stained with Annexin V/PI and analysed via flow cytometry assay. nc, negative control; LV, lentivirus; oe, overexpression; shRNA, short hairpin RNA; CDDP, cis-diammined dichloridoplatium act as a transcription activator. However, to our knowledge, 14-3-3ζ is not a transcription factor. Why the transcription of BECN1 gene was initiated by 14-3-3ζ is unclear. We previously demonstrated that 14-3-3ζ was able to stabilize HIF-1α, a well-known transcription factor. 15 Such results are supported by a study conducted in non-small-cell lung cancer cells. 30 14-3-3ζ itself may not be a transcription factor, but it may affect other transcription factors, such as HIF-1α, to indirectly regulate BECN1 gene transcription. We additionally knocked down 14-3-3ζ in hypoxic cancer cells and found that, under hypoxia, 14-3-3ζ barely affected BECN1 gene expression at translational level. These data suggest that once the hypoxic pathway is activated, 14-3-3ζ is not the predominant regulator of BECN1 transcription or translation.
Moreover, we also analysed the protein levels of phosphobeclin 1 S295 in HCC cells overexpressing 14-3-3ζ. Notably, we found that 14-3-3ζ overexpression markedly promoted the phosphorylation of beclin 1 on S295. No kinase activity of 14-3-3ζ has been reported before. We then ask why 14-3-3ζ overexpression promotes beclin 1 phosphorylation. Previous studies report that 14-3-3s affect the stabilization of their binding partners. We therefore proposed that the observed elevation in phosphorylated beclin 1 was because 14-3-3ζ could prevent its degradation. VPS34 is the only class III PI3K in mammals that forms a stable complex with p150, which further associates with beclin-1. The VPS34-p150-beclin-1 complex serves as a binding partner for several proteins, such as Atg14, to promote autophagy. 32,33 Enhancing the interaction between VPS34 and beclin-1 is capable of promoting autophagy. 34,35 Our data illustrated that 14-3-3ζ overexpression enhanced the binding of VPS34 and beclin-1 and further triggered autophagy in HCC cells. These findings suggest that 14-3-3ζ can augment the formation of PI3K complex.
By analysing the protein levels of autophagy markers, we also found that, in addition to regulating beclin 1, 14-3-3ζ overexpression induced effective autophagic flux in HCC-LM3 and CSQT-2 cells-p62 degradation was enhanced and the number of LC3II puncta was increased. Moreover, hypoxia-induced autophagy was suppressed in 14-3-3ζ-silenced cells. Chemoresistance represents a major obstacle in clinical oncology. It is commonly considered that chemoresistance developed in cancer cells is associated with autophagy initiation. 36 Suppression of 14-3-3ζ has been reported to promote chemosensitivity of several types of cancer cells, such as lung and breast cancer. 37,38 Choi and coworkers demonstrated that silencing of 14-3-3ζ enhanced the cytotoxicity of CDDP in many hepatoma cell lines. 39 These previous literatures depict 14-3-3ζ as a contributor to the chemoresistance of cancer cells.
Our data suggest that HCC cells with high 14-3-3ζ expression activate beclin 1-mediated autophagy to resist cytotoxicity induced by chemotherapeutic drug.

ACK N OWLED G EM ENTS
This study was supported by grants from the National Natural

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N
Yufu Tang designed and performed the research, analysed data and wrote the paper. Yibing Zhang, Shupeng Liu and Zhongyi Sun performed the research, analysed data and revised the paper. Chunhui Wang and Longfei Li analysed the data. Wenping Zhou and Shuqun F I G U R E 6 Summary of major findings. In hepatocellular carcinoma (HCC) tumour microenvironment (hypoxia), the levels of 14-3-3ζ and phospho-beclin 1 S295 are up-regulated. 14-3-3ζ can bind to and prevent the phosphorylated beclin 1 from degradation in HCC cancer cells, thus promoting autophagy