Clinical Science (2013) 124, 203–214 (Printed in Great Britain) doi: 10.1042/CS20120270
Autophagy negatively regulates cancer cell proliferation via selectively targeting VPRBP Bo-Shi WANG*, Yi-Zhen LIU*, Yang YANG*†, Yu ZHANG*, Jia-Jie HAO*, Hai YANG*, Xiao-Min WANG*, Zi-Qiang ZHANG*, Qi-Min ZHAN* and Ming-Rong WANG* *State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People’s Republic of China †Department of Histology and Embryology, Anhui Medical University, Hefei, People’s Republic of China
Abstract There have been multiple lines of evidence suggesting that autophagy selectively targets signalling proteins and regulates cancer cell signalling in addition to bulk clearance of long-lived proteins and organelles. Protein degradation through autophagy requires receptor protein LC3B to sequester the substrates into the autophagosome. In the present study, we screened LC3B (light-chain 3B)-binding partners and identified autophagic substrates in cancer cells. With lung cancer NCI-H1975 and oesophageal cancer KYSE30 cell lines as models, we found that VPRBP (viral protein R-binding protein) was a novel LC3B-binding protein through GST (glutathione transferase)–LC3B pull-down combined with LC–MS/MS (liquid chromatography–tandem MS) methods. Co-immunoprecipitation assay showed that VPRBP–LC3/p62 were in the same protein complex as the two cell lines. Induction of autophagy led to a down-regulation of VPRPB, which could be rescued by the inhibition of autophagy degradation by BFA1 (bafilomycin A1) and by the disruption of autophagy through ATG5-knockdown. We also found that induction of autophagy promotes VPRBP–LC3/p62 interaction. Immunohistochemical examination of human NSCLC (non-small cell lung cancer) tissues showed that VPRBP was positively correlated with p62 and negatively correlated with LC3B. Moreover, p62 and VPRBP were associated with poor prognosis in lung ADC (adenocarcinoma) (p62, P = 0.019; VPRBP, P = 0.005). Patients with low expression of both p62 and VPRBP showed the best prognosis. Key words: autophagy, lung cancer, p62, prognosis, viral protein R-binding protein (VPRBP)
INTRODUCTION Autophagy is considered as a protein degradation mechanism in addition to the ubiquitin–proteasome pathway. It is essential in the self-digestion of long-lived proteins and organelles. Autophagy plays an important role in both the response to metabolic stresses and the regulation of cell signal transduction [1–4]. Dysfunction of autophagy has been reported to be associated with multiple types of human diseases including cancers [3,5,6]. The autophagic protein degradation pathway consists of macroautophagy (referred to as autophagy), microautophagy and CMA (chaperone-mediated autophagy) [7]. The selective forms of autophagic degradation are autophagy and CMA. They are similar in degrading substrates through lysosomes but differed in using different mechanisms to deliver substrates to lysosomes [8]. The CMA pathway employs the protein chaperone Hsc70
(heat-shock cognate 70 stress protein) to selectively transport substrates to the lysosome via the receptor protein LAMP2a (lysosome-associated membrane protein 2a). Autophagy selectively recognizes its substrates by specific adaptor–receptor proteins [p62/NBR1 (neighbour of BRCA1 gene)–LC3B (lightchain 3B)], which help sequester cytosolic proteins or organelles into the autophagosome. [9]. The regulatory role of autophagy in cell signalling transduction has emerged, because there is evidence that autophagy can selectively target signalling mediators for degradation, such as Dvl [10] and Src [11]. Currently, the precise role of autophagy in tumorigenesis remains unclear. However, selective targeting at signal proteins and the regulation of cancer cell signalling by autophagy may partially explain how autophagy affects cancer cells. For instance, autophagy negatively regulates Wnt signalling by targeting the Dvl protein [10], and the accumulation of the autophagic
Abbreviations: ADC, adenocarcinoma; BFA1, bafilomycin A1; CCK-8, cell counting kit-8; CMA, chaperone-mediated autophagy; Cy3, indocarbocyanine; DAPI, 4 ,6-diamidino-2-phenylinodole; GST, glutathione transferase; IHC, immunohistochemistry; LC3B, light-chain 3B; MS/MS, tandem MS; nano-LC–ESI-CID collision-induced dissociation, nano-liquid chromoatography–electrospray ionization-collision-induced dissociation; NSCLC, non-small cell lung cancer; PI, propidium iodide; SCC, squamous cell carcinoma; siRNA, small interfering RNA; TMA, tissue microarrays; Vpr, viral protein R; VPRBP, viral protein R-binding protein. Correspondence: Professor Ming-Rong Wang (email
[email protected]).
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substrate p62 in autophagy-deficient cells was related to tumorigenesis [12,13]. The aim of the present study was to find novel autophagy-targeting signal proteins, so as to shed light on how autophagy regulates tumorigenesis. Since the delivery of autophagic substrates to the autophagosome robustly required the autophagosome membrane receptor LC3B [12,14], we screened LC3B-binding partners and identified that VPRBP (viral protein R-binding protein) was one of the autophagy-targeting proteins. We demonstrated that autophagy negatively regulated cell proliferation via VPRBP. Furthermore, we studied the prognostic impact of VPRBP in lung cancer patients.
MATERIALS AND METHODS Cell culture and transfection The human lung cancer cell line NCI-H1975 was purchased from the A.T.C.C. The human ESCC cell line KYSE30 was generously provided by Dr Y. Shimada (Faculty of Medicine, Kyoto University, Kyoto, Japan) [15]. The cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10 % FBS (fetal bovine serum). Cell transfection was performed with LipofectamineTM 2000 (Invitrogen), according to the manufacturer’s instructions.
Table 1 Basic clinicopathological data of the 168 NSCLC tissue samples The age of the subjects was 59 (32–84) years [value is the median (range)]. Tumour stage was classified according to the 7th edition of the International Union against Cancer (UICC) Tumour Node Metastasis (TNM) classification of malignant tumours. Grade was based on the criteria of the World Health Organization Classification of Tumours. Parameter
Number of bronchial brushings
Sex Male Female
116 (69.0 %) 52 (31.0 %)
Tumour type SCC
87 (51.8 %)
ADC
81 (48.2 %)
NSCLC stage III
159 (94.6 %)
IV
9 (5.4 %)
T status T2
78 (46.4 %)
T3
52 (40.0 %)
T4
38 (22.6 %)
N status N0 N1–3
11 (6.5 %) 157 (93.5 %)
M status
Patients and samples
M0
157 (93.5 %)
A total of 168 surgically resected NSCLC (non-small cell lung cancer) tissues were collected at the Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC) in Beijing, China from 2006 to 2009. The tissue samples were collected shortly after radical surgery of NSCLC patients. Primary tumour regions were excised by experienced pathologists and fixed in neutral buffered formalin (pH 7.4). Paraffin-embedded tumour tissues were routinely prepared. All the samples used in this study were residual specimens after diagnosis sampling. All the patients received no treatment before surgery and signed separate informed consent forms for sampling and research. The study has been approved by the Ethics Committee of Cancer Institute (Hospital), Chinese Academy of Medical Sciences and Peking Union Medical College. Survival data were available with a median follow-up of 725 (range, 63 – 1597) days. The clinicopathological characteristics of the patients are summarized in Table 1.
M1
11 (6.5 %)
Reagents and antibodies BFA1 (bafilomycin A1) and rapamycin were purchased from Sigma. Rapamycin was diluted in RPMI 1640 medium without serum. H2 O2 was purchased from Zhongshan Goldenbridge Biotechnology. H2 O2 was diluted in RPMI 1640 medium without serum. The primary antibodies include an anti-LC3B antibody (1:1000 dilution for Western blotting; PM036; MBL International), an anti-LC3B antibody (1:100 dilution for immunofluorescence; M152-3; MBL International), an anti-β-actin antibody (1:5000 dilution for Western blotting; A5316; Sigma), an antiVPRBP antibody (1:1000 dilution for Western blotting; 11612-1AP; Proteintech Group), an anti-VPRBP antibody (1:100 dilution for immunofluorescence; ab75458; Abcamn) and an anti-p62 an-
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Grade G1 + G2 G3
66 (39.3 %) 102 (60.7 %)
Gross pathology Central type Peripheral type
106 (63.1 %) 62 (36.9 %)
Smoking status Current or former smoker Non-smoker
110 (65.5 %) 58 (34.5 %)
tibody (1:1000 dilution for Western blotting; M162-3; MBL International).
GST (glutathione transferase) pull-down The recombinant GST-tagged LC3B fusion protein expressed in Escherichia coli was extracted using the BugBuster@ Protein Extraction Reagent (Merck), affinity-purified and subsequently immobilized on glutathione affinity resin (Thermo Fisher Scientific). The NCI-H1975 or KYSE30 cells were lysed using the cell lysis buffer in the immunoprecipitation kit (Roche Applied Science) on ice for 30 min. GST-tagged LC3B resin or the control (GST resin) was incubated for 3 h at 4 ◦ C with 5 mg of the total protein from NCI-H1975 or KYSE30 cells. The bound proteins were isolated by centrifugation of Sepharose beads at 15 000 g for 1 min. After washing the resin five times, the bound proteins were eluted by boiling with 5× SDS gel-loading buffer, subjected to SDS/PAGE and visualized by zinc staining using a zinc reversible stain kit (Thermo Fisher Scientific). The bands present only in
Autophagy regulates VPRPB
the GST–LC3B resin elutes were excised, digested with trypsin and then subjected to nano-LC–ESI-CID collision-induced dissociation (nano-liquid chromoatography–electrospray ionizationcollision-induced dissociation)–MS/MS (tandem MS).
Nano-LC–ESI-CID–MS/MS and peptide identification An Agilent 1100 HPLC system was connected to an LTQ Orbitrap linear ion trap mass spectrometer (Thermo Fisher Scientific). The instrument was equipped with a 20 mm×100 μm internal diameter Aqua C18 trap column (Phenomenex), and a 200 mm × 50 μm internal diameter Reprosil C18 reverse-phase analytical column (Ammerbuch-Entringen). The tryptic peptides were separated by using a 90 min 200 nl/min linear gradient from 0 to 60 % solvent B (0.1 M acetic acid in 80 % acetonitrile, in which solvent A was 0.1 M acetic acid. MS was performed in positiveion mode, and parent ions were isolated for fragmentation in data-dependent mode. The search parameters were set as follows: taxonomy; database, NCBI, IPI; enzyme, trypsin; fixed modifications, C (carbamidomethyl); variable modifications, oxidation (M); max missed cleavages, one/two; mass tolerances for MS and MS/MS were 10 p.p.m. and 0.5 Da.
Immunoprecipitation Cells were washed with ice-cold PBS and lysed with lysis buffer using a Roche immunoprecipitation kit. The total protein concentration was estimated using a Protein Assay Kit (Bio-Rad). Total protein (5 mg) was incubated with 50 μl of Protein G–agarose suspension (Roche Applied Science) for 3 h at 4 ◦ C on a rocking platform to reduce non-specific binding. After removing the beads, the supernatant was supplemented with 5 μg of anti-LC3B antibody (PM036; MBL International), 5 μg of anti-p62 antibody (M162-3; MBL International) or 5 μg of anti-VPRBP antibody (ab75458; Abcam), followed by incubation for an additional 3 h at 4 ◦ C. A total of 50 μl of Protein G–agarose was then added to each immunoprecipitation mixture, and the incubation was continued overnight at 4 ◦ C on a rocking platform. The immunoprecipitates were collected by centrifugation and washed three times with the wash buffer. After the loading buffer was added, the agarose was boiled and subjected to Western blot analysis.
Western blot analysis Cells were washed with ice-cold PBS and lysed in RIPA buffer [50 mmol/l Tris/HCl (pH 7.5), 150 mmol/l NaCl, 1 % Nonidet P40 (NP-40), 0.5 % sodium DOC (deoxycholate), 0.1 % SDS] containing 1 mmol/l PMSF and 2 g/ml protease inhibitors (protease inhibitor cocktail Set III; Calbiochem) on ice for 30 min. The lysates were clarified by centrifugation at 13 000 g for 30 min at 4 ◦ C. The total protein concentration was estimated using a protein assay kit (Bio-Rad). Protein samples (50–150 μg) were loaded on to and separated using SDS/PAGE, transferred on to PVDF membranes (Millipore), blocked and probed with the primary antibodies. After washing, the blots were incubated with HRP (horseradish peroxidase)-conjugated secondary antibodies and visualized using super-ECL (enhanced chemiluminescence) detection reagent (Applygen Technologies).
Immunofluorescence staining Cells were seeded on to coverslips, followed by a wash with PBS, fixation with 4 % PFA (paraformaldehyde) at room temperature (25 ◦ C) for 15 min and permeabilization with 0.2 % Triton X-100 in PBS for 10 min. After another wash with PBS, cells were subsequently incubated with a blocking solution (5 % goat serum) for 30 min and then incubated with primary antibodies overnight at 4 ◦ C. The cells were then washed three times and incubated with Cy3 (indocarbocyanine)-conjugated anti-(mouse IgG) antibodies or FITC-conjugated anti-(rabbit IgG) secondary antibodies (dilution 1:200; Jackson ImmunoResearch Laboratories) for 30 min at room temperature. DNA was counterstained using DAPI (4 ,6-diamidino-2-phenylinodole). The slides were observed under confocal microscope (Leica TSC SP2).
Plasmid construction and siRNA (small interfering RNA) synthesis The VPRBP expression vector was constructed by Proteintech Group. The duplex siRNAs were chemically synthesized by Genepharma with the following sequences: VPRBP siRNA-1: 5 -GCGACTCATTCTCCAATAT-3 , VPRBP siRNA-2: 5 -GGCAGCTGAAGCTCTATAA-3 , ATG5 siRNA: 5 -GACGTTGGTAACTGACAAA-3 . Non-silencing siRNA (5 -TTCTCCGAACGTGTCACGT-3 ) served as the control siRNA.
Flow cytometry assay Cells were harvested to assay apoptosis using an Annexin V– FITC apoptosis detection kit (Sigma) at 48 h after transfection. The cells were incubated with Annexin V for 30 min and with PI (propidium iodide) for 5 min, and then the cells were analysed using flow cytometry. The apoptotic cells were a combination of early apoptotic cells (Annexin V-positive and PI-negative) and late apoptotic cells (Annexin V-positive and PI-positive).
Cell proliferation assay Cell proliferation was assessed by CCK-8 (cell counting kit-8). Briefly, the cells were plated in 96-well plates. The 10 μl of CCK8 was added to each well at different time points and incubated at 37 ◦ C for 2 h. The absorbance (450 nm) was measured using a microplate spectrophotometer.
TMA (tissue microarray) construction and IHC (immunohistochemistry) The TMA was constructed as described previously [16]. For each case, three cancer tissue cores (diameter = 1 mm; height = 5 mm) were taken from the primary block. IHC was performed on the 4-μm sections of the resulting TMA block. The slides were deparaffinized, rehydrated, immersed in 3 % H2 O2 solution for 15 min, heated in citrate buffer (pH 6.0) for 25 min at 95 ◦ C and cooled for 60 min at room temperature. For immunohistochemical staining, the slides were incubated overnight at 4 ◦ C with anti-VPRBP (1:150 dilution; 11612-1-AP; Proteintech Group), anti-p62 (1:150 dilution; M162-3; MBL International) and anti-LC3B (1:150 diltion; PM036; MBL International) antibodies. After washing with PBS, the slides were visualized using the PV-9000 Polymer Detection
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System GBI), following the manufacturer’s instructions, and were counterstained with haematoxylin. The expression levels of VPRBP, p62 and LC3B were determined on the basis of staining intensity and the percentage of immunoreactive cells. Staining intensity was rated as 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive). The percentage of immunoreactive cells was graded as 0 (0 %), 0.5 (1–10 %), 1 (11–20 %), 2 (21–50 %), 3 (51–80 %) or 4 (81–100 %). The average of tumour cell staining intensity score multiplied by the percentage score of positive cells represented the final score of the sample. All cases were divided into two groups: a strongly positive group (score range 9–12) and a low/no expression group (score range 0–9). Assessment and imaging of IHC was performed using a Leica DM2000 microscope equipped with Leica DFC Cameras-Image Acquisition System (software version 3.5.0).
both NCI-H1975 and KYSE30 cells (Figure 1B). The interaction of autophagic substrates with LC3B usually requires p62 as an adaptor [10]. In order to examine the possibility whether VPRBP was a candidate autophagic substrate, we tested the interaction of VPRBP with the autophagic adaptor protein p62. The results showed that VPRBP was co-immunoprecipitated with p62 in NCI-H1975 and KYSE30 cell lines (Figure 1C). Immunoprecipitation assay with anti-VPRBP antibody indicated that VPRBP, LC3B and p62 proteins were co-immunoprecipitated in the same complex (Supplementary Figure S1A at http://www.clinsci.org/cs/124/cs1240203add.htm). Immunofluorescence imaging showed that the signals of VPRBP and LC3B covered a large area of cytoplasm and the two signals partially overlapped, which suggested the potential co-localization between endogenous VPRBP and LC3B in the absence or presence of BFA1 (Figure 1D and Supplementary Figure S1B).
Statistical analysis
Induction of autophagy down-regulates VPRBP and promotes the VPRBP–LC3/p62 interaction
The differences in the results between groups were compared using ANOVA or Student’s t test. Results are expressed as the means + − S.D. Analysis in tissue samples was performed using the PASW 18.0 Statistical program (SPSS). The correlation between protein expression levels was analysed using the Spearman correlation test. Associations between protein expression and clinicopathological parameters were assessed by the Mann–Whitney test and the Kruskal–Wallis test. For survival analyses, Kaplan–Meier survival curves were constructed, and differences were tested by the log-rank test. Overall survival was defined as the time between the date of surgery and the date of death from cancer or the date of last contact. The data of patients alive at the end of the study were censored. All P values
