SIRT1 Is Downregulated in Gastric Cancer and Leads to G1-Phase Arrest Via NF- Κb/Cyclin D1 Signaling

Published OnlineFirst October 9, 2013; DOI: 10.1158/1541-7786.MCR-13-0214

Molecular Cancer Cell Cycle and Senescence Research

SIRT1 Is Downregulated in Gastric Cancer and Leads to

G1-phase Arrest via NF-kB/Cyclin D1 Signaling

Qing Yang1, Bo Wang3, Wei Gao5, Shanying Huang4, Zhifang Liu2, Wenjuan Li1, and Jihui Jia1

Abstract Sirtuin 1 (SIRT1) is a class III histone/protein deacetylase, and its activation status has been well documented to have physiologic beneﬁts in human health. However, the function of SIRT1 in cancer remains controversial. Here, the expression and role of SIRT1 in gastric cancer is delineated. SIRT1 was present in all normal gastric mucosa specimens; however, it was only present in a portion of the matched gastric cancer tumor specimens. In SIRT1- positive tumors, both mRNA and protein levels were downregulated as compared with the corresponding nonneoplastic tissue. Ectopic expression of SIRT1 inhibited cell proliferation, diminished clonogenic potential,

and induced a G1-phase cell-cycle arrest, the effects of which were not apparent when a catalytic-domain mutant form of SIRT1 was introduced, suggesting that SIRT1 functions in gastric cancer are dependent on its deacetylase activity. Further evidence was obtained from depletion of SIRT1. At the molecular level, SIRT1 inhibited the transcription of Cyclin D1 (CCND1), and inhibition of NF-kB in SIRT1-depleted cells rescued Cyclin D1 expression. Furthermore, inhibition of either NF-kB or Cyclin D1 in SIRT1-depleted cells reversed the inhibitory effects of SIRT1. The inhibitory role of SIRT1 was also veriﬁed in vivo using xenografts. This work characterizes SIRT1 status and demonstrates its inhibitory function in gastric cancer development, which involves NF-kB/ Cyclin D1 signaling, offering a therapeutic role for SIRT1 activators. Implications: The inhibitory functions of SIRT1, which involve NF-kB/Cyclin D1 signaling, suggest the utility of SIRT1 activators in the prevention and therapy of gastric cancer. Mol Cancer Res; 11(12); 1497–507. 2013 AACR.

Introduction curable disease with a high 5-year overall survival rate (3). On the basis of incidence and mortality rate, gastric cancer Therefore, studies about new diagnostic or prognostic mar- is among the top five leading causes of cancer worldwide. In kers and therapeutic targets for gastric cancer are urgently needed. 2008, there were approximately 12.7 million cancer cases þ worldwide, and gastric cancer accounted for 8% of all Sirtuin 1 (SIRT1), a class III NAD -dependent histone cancers (1). The majority of new gastric cancer cases occur deacetylase, is the mammalian homolog of yeast silent in Eastern Asia, Eastern Europe, and South America. information regulator 2. SIRT1 substrates include not only Although gastric cancer rates have decreased globally, it histones but also nonhistone proteins. Through these tar- remains high in developing countries, especially China (1, gets, SIRT1 plays a crucial role in multiple pathways such as 2). Most of the patients with gastric cancer are not diagnosed cellular metabolism, stress response, calorie restriction, and aging (4, 5). An abundance of recent data has demonstrated until they have reached advanced stages of the disease when, fi despite the use of conventional therapies such as surgery, that activation of SIRT1 has physiologic bene ts, including increased health and longevity, and assists in certain meta- chemotherapy, and radiotherapy, patients have a poor prog- – nosis. However, early-stage gastric cancer is a potentially bolic disorders (4 6). However, the function of SIRT1 in cancer is controversial. SIRT1 was initially regarded as a potential oncogene because of its first nonhistone substrate,

1 2 p53, a well-known tumor suppressor. SIRT1 deacetylates Authors' Affiliations: Institute of Pathogen Biology, Department of Biochemistry, Shandong University School of Medicine; 3Department of p53 with specificity for the C-terminal Lys382 residue and Traditional Chinese Medicine, 4Key Laboratory of Cardiovascular Re- silences p53 activity as a transcription factor (7, 8). In modeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital, Shandong University; and addition, two tumor suppressors, hypermethylated in cancer 5Department of Pathology, Jinan Central Hospital, Jinan, China (HIC1) and deleted in breast cancer (DBC1), have been fi Note: Supplementary data for this article are available at Molecular Cancer identi ed as negative regulators of SIRT1 (9, 10). A recently Research Online (http://mcr.aacrjournals.org/). published study has provided new evidence for the role of Corresponding Author: Jihui Jia, Institute of Pathogen Biology, Shandong SIRT1 as a tumor promoter by describing a positive feedback University School of Medicine, Jinan 250012, China. Phone: 86-531-8838- loop between C-Myc and SIRT1 in hepatocellular carcino- 2672; Fax: 86-531-8838-2502; E-mail: [email protected] mas (11). However, studies from other laboratories have doi: 10.1158/1541-7786.MCR-13-0214 indicated that SIRT1 acts as a tumor suppressor. First, 2013 American Association for Cancer Research. acetylation of H3K56, a substrate for SIRT1, is increased

www.aacrjournals.org 1497

Yang et al.

in multiple cancers (12). Second, SIRT1 has been reported performed for genes, including SIRT1, cyclin D1, and to play a key role in repairing DNA-breaks and maintaining b-actin as previously described (18). The sequences of the genome stability (13, 14). Moreover, SIRT1 has been shown amplification primers are listed in Supplementary Table S1. to suppress the growth of colon cancer by inhibiting E2F1 or The levels of SIRT1 mRNA in clinical specimens were b C -catenin (15, 16). The role of SIRT1 in oncogenic pro- calculated on the basis of the threshold cycle ( t) values gression seems to be specific for tumor type and signaling and normalization of b-actin expression using the 2DDCt pathway involved. method (19). The mRNA expression of cyclin D1 was In this study, we investigated the expression and function normalized to b-actin relative to the control using the of SIRT1 in gastric cancer. We compared the expression of 2DDCt method. Experiments were performed in triplicate SIRT1 between normal and malignant gastric tissues. Then and repeated three times. we analyzed cell viability, clonogenic potential, cell-cycle distribution, and apoptosis and investigated the mechanism Western blot analysis for how SIRT1 suppresses tumorigenesis. The inhibitory Total protein from the tumor specimens or cells was activity of SIRT1 on gastric cancer development was also extracted using RIPA lysis buffer (Beyotime) as previously evaluated in vivo. described (18). The protein concentration was determined using a BCA Protein Assay Kit (Pierce). The membrane was Materials and Methods probed with antibodies against SIRT1 and b-catenin Patients and tissue specimens (Abcam), cyclin D1, cyclin D2, cyclin D3, cyclin E1, Forty-four patients with primary gastric cancer were cyclin-dependent kinase (CDK)4, bcl-2, bax, caspase-3, included in this study, which was approved by the local NF-kB p65, and c-Jun (Cell Signaling Technology), cyclin ethics committee (No. 2011008 for Ethics Approval). The D2, p21, and p16 (Santa Cruz Biotechnology). Horseradish patients underwent gastrectomy at Jinan Central Hospital peroxidase (HRP)–conjugated anti-rabbit or -mouse anti- between 2011 and 2012. The tissue specimens were col- body was used as the secondary antibody. The protein bands lected and stored as previously described (17). There were 11 were visualized using an ECL system (Pierce). b-actin (Cell female patients and 33 male patients, with a median age of 60 Signaling Technology) served as a loading control. (range, 35–82). Details of patient and disease characteristics are documented in Table 1. Immunohistochemistry The tumor specimens from patients with gastric cancer and Cell lines, plasmids, and siRNA the nude mice were deparaffinized, rehydrated, and antigen Human gastric cancer cell lines AGS, MGC-803 (Cell retrieved. The antibodies against SIRT1 (1:200; Santa Cruz Resource Center, Shanghai Institute of Biochemistry and Biotechnology) and Ki67 (1:500; Abcam) were added to the Cell Biology at the Chinese Academy of Sciences, Shanghai, sections and then incubated overnight at 4C. HRP-conju- China), BGC-823, and SGC-7901 (China Center for Type gated anti-rabbit immunoglobulin G (IgG) and 3,30-diami- Culture Collection, Wuhan, China) were cultured in F12 nobenzidine (DAB) staining were used to visualize the (AGS) or RPMI 1640 (BGC-823, SGC-7901, and MGC- primary antibody. Then, the slides were counterstained with 803) containing 10% FBS, 100 U/mL penicillin and 2 hematoxylin and imaged by an Olympus light microscope mmol/L L-glutamine at 37 C in a humidified atmosphere using cellSens Dimension software. The percentages of Ki67- fi containing 5% CO2. Both of the cell banks routinely positive cells were counted under microscope (magni cation, perform cell line authentication by short tandem repeat 400) and five fields were counted for each section. (STR) profiling and all of the cell lines were passaged in our laboratory for no more than 6 months after receipt. High- MTS assay purity cycloheximide (CHX; 3-[2-(3,5-dimethyl-2-oxocy- The CellTiter96 AQueous One Solution Cell Prolifera- clohexyl)2-hydroxyethyl-]-glutarimide) was purchased tion Assay (Promega) was performed to indicate cell prolif- from Beyotime, dissolved in dimethyl sulfoxide (DMSO), eration. Briefly, 2 103 cells were seeded in a 96-well plate and added into the culture medium at a concentration of 20 and were allowed to grow for 24 hours. Twenty-four hours mg/mL. The vectors expressing wild-type SIRT1 (pECE- later, 20 mL MTS was added to each well. After incubation SIRT1) and the catalytic-domain mutant form of SIRT1 for 3 hours at 37 C, the absorbance at 490 nm was recorded (pECE-SIRT1-H363Y) were purchased from Addgene on a Varioskan Flash Multiplate Reader (Thermo Scientific). and recombined into pcDNA3.1 (Invitrogen). Chemically Percentage of vehicle was calculated by the following for- modified siRNA targeting SIRT1, NF-kB, cyclin D1, and mula: [(average absorbance of treated group average control siRNA were purchased from GenePharma. The absorbance of blank)/(average absorbance of control group sequences of the earlier siRNA are listed in Supplementary average absorbance of blank)] 100%. Assays were Table S1. performed in triplicate and repeated three times.

RNA extraction and quantitative real-time PCR Colony formation assay Total RNA in tissue specimens or cells was isolated using Cells were seeded into 6-well plates (300 cells per well) and TRIzol reagent (Invitrogen) and converted into cDNA using incubated for 10 days until the colonies were large enough to the PrimeScript RT reagent kit (Takara). qRT-PCR was be clearly discerned. The cells were ﬁxed with methanol and

1498 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

SIRT1 Inhibits Gastric Cancer via G1 Arrest

Table 1. Patients and tumor characteristics, SIRT1 mRNA and protein expression in gastric cancer and the matched normal mucosa

Patients and tumor characteristics SIRT1 in tumors SIRT1 in normal mucosa

Patient no. Age (y)/sex Size (cm) TNMa IHSb qRT-PCRc IHS RT-PCR

1 53/M 8 7T3N0M0 þ 0.64 þ 1.01

2 49/M 6 4.5 T3N3aM1 þþ

3 35/M 5 4T2N2M0 þ 0.0049 þþ 8.22

4 66/M 5 5T3N0M0 þþ

5 64/M 5 2T3N3aM0 þþ

6 74/F 8 7T3N3aM0 þþ

7 82/M 7.5 6T3N3aM0 þþ

8 49/M 3.5 3.5 T3N0M0 þ 0.07 þþ 4.94

9 74/M 5.5 5T3N2M0 þþ

10 39/F 6.5 3T3N3bM1 þþ

11 65/M 9.3 7.5 T3N1M0 þ 0.029 þþ 13.83

12 76/M 4.8 3.8 T3N0M0 þþ

13 60/F 5 3T3N0M0 þ 0.33 þþ 6.53

14 51/M 4 3.5 T3N2M0 þþ

15 54/F 6 5T3N0M0 þ 0.052 þþ 9.86

16 68/M 5.5 5T3N0M0 þ 0.23 þþ 4.61

17 61/M 5 3.8 T3N0M0 þ 0.093 þþ 5.19

18 70/M 6.6 6T3N2M0 þþ

19 60/M 4.5 3.5 T3N2M0 þþ

20 64/F 4 2.5 T3N0M0 þ 0.036 þ 2.98

21 75/M 4.5 4T3N3aM0 þþ

22 57/M 7 3T3N3aM0 þþ

23 75/F 5 3.5 T3N0M0 þþþ

24 59/F 6 5T3N3bM0 þþ

25 47/M 6 4T3N3aM0 þ

26 72/M 8 5T3N1M0 þ 0.027 þþ 12.77

27 64/M 7 6.7 T3N3aM0 þþ

28 73/M 6 4T2N1M0 þ 0.036 þ 1.74

29 76/M 6 6T3N0M0 þþþ

30 66/F 10 10 T3N0M0 þ 0.033 þþ 9.5

31 74/M 4 3T3N3bM1 þ

32 72/M 5.3 5T3N1M0 þ 0.026 þþ 4.93

33 61/F 6 7T3N2M0 þ

34 67/M 6 5T3N3aM0 þ

35 71/M 13 8T3N3aM0 þ

36 73/M 3.5 3.5 T3N0M0 þþþ

37 64/F 3.3 1.7 T3N0M0 þ 0.048 þþ 5.07

38 55/M 9.5 7T3N3bM0 þ

39 55/M 3 3T2N0M0 þ 0.8 þ 3.15

40 79/M 5.5 5T3N1M0 þ 0.011 þþ 6.54

41 62/F 6 6T3N1M0 þ 0.0098 þþ 5.05

42 58/M 3.5 3T3N0M0 þþþ

43 50/M 2.5 2T3N1M0 þ

44 53/M 5 3.5 T3N1M0 þ 0.009 þþ 8.33 aTNM (tumor–node–metastasis) stages according to the American Joint Committee on Cancer (AJCC) Cancer Staging Manual (7th ed., 2010). bIHS, immunohistochemistry; , negative, no visible speciﬁc SIRT1 stain; þ, positive with weak to medium signals; þþ, positive with strong signals. cqRT-PCR, quantitative real-time PCR. The relative level of SIRT1 mRNA was calculated as described in Materials and Methods. , failure to detect the SIRT1 mRNA.

www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 1499

Yang et al.

stained with crystal violet and the number of colonies with different SIRT1-lentiviral shRNAs were injected respective- more than 50 cells was counted manually. Experiments was ly (regarded as LV-sh-1 and LV-sh-2; 8 mice for each group). performed in triplicate and repeated three times. The subcutaneous tumor size was measured with a caliper and the tumor volume was calculated by the formula (length) Cell-cycle analysis (width2)/2. Mice were sacrificed 4 weeks after implanta- Cells were harvested, fixed with precooled 70% ethanol at tion. Tumors were harvested and processed for Western blot 4C overnight, and then stained with propidium iodide (PI) analysis and immunohistochemistry studies. containing RNase A at 37C for 30 minutes in the dark. Cell-cycle distribution was determined using a flow cyt- Statistical analysis ometer (BD Biosciences). Each experiment was carried out The difference in SIRT1 mRNA levels between cancerous in triplicate and the data were analyzed with MultiCycle and matched normal gastric tissues was analyzed using software (Phoenix Flow Systems). Experiments were per- Student t test. The comparison of cell proliferation, foci formed independently three times. number, and cell-cycle distribution was performed using one-way ANOVA with a Tukey post-hoc test or a Student Apoptosis assay t test. The data were expressed as mean SD. The statistical Detection and quantitation of apoptosis were performed analyses were performed using Statistical Package for the by labeling of DNA strand breaks using an In Situ Cell Death Social Sciences (SPSS, version 16.0). P values less than 0.05 Detection Kit, TMR red (Roche Applied Science). After were considered statistically significant. transfection, cells were seeded onto coverslips and labeled with terminal deoxynucleotidyl transferase–mediated dUTP Results nick end labeling (TUNEL) according to the manufacturer's SIRT1 is downregulated in human gastric cancer instructions. Treatment with 0.2 mmol/L H2O2 for 12 To explore the role of SIRT1 in gastric cancer, we first hours served as the positive control. The nuclei were coun- determined the protein levels of SIRT1 using immunohis- 0 terstained with 4 ,6-diamidino-2-phenylindole (DAPI; tochemistry in gastric tissues from patients with gastric Beyotime) and the slides were imaged by a fluorescence cancer. The cancerous and matched normal gastric speci- microscopy (Olympus) using cellSens Dimension software. mens from 44 patients were analyzed. The SIRT1 protein Experiments were performed independently three times. was detected in all the normal gastric tissues with positive cell fractions ranging from 78% to 100% (Fig. 1A and Table 1). Stable lentivirus–infected gastric cancer cells The SIRT1 protein was present in both cytoplasmic and Lentiviral vectors containing SIRT1 cDNA, SIRT1 short nuclear compartments of the epithelial cells, and most of the hairpin (sh)RNAs, or their controls were constructed by positive cells exhibited intermediate to strong signals (Fig. GenePharma and used to transfect BGC-823 cells. For 1A and Table 1). However, the expression of SIRT1 was shRNAs, the same targeting sequences were used as the detected in 59% (24 of 44) of the cancerous samples. The siRNAs. For stable infection, BGC-823 cells infected with SIRT1-positive gastric cancer samples exhibited weak to different lentiviral vectors were cultured in complete medi- intermediate staining with similar distribution patterns (Fig. um supplied with 2 mg/mL puromycin (Acros Organics) for 1B and C and Table 1). 4 weeks. We further analyzed the SIRT1 mRNA levels in 20 available gastric cancer and the corresponding normal tissue Nude mice xenograft model samples by qRT-PCR, including two specimens that lacked Female athymic BALB/c nude mice (6–8 weeks) were SIRT1 protein detection in the cancerous region. Consistent purchased from Peking University (Beijing, China) and with the protein profile, the presence of SIRT1 transcripts maintained under specific pathogen-free conditions at the was not observed in these two cancer specimens. Among the Key Laboratory of Cardiovascular Remodeling and Function other 18 pairs of samples, the cancerous samples had a Research, Qilu Hospital, Shandong University. The study significantly lower level of SIRT1 mRNA expression com- was approved by the local ethics committee (No. 001 in pared with the matched normal tissues (Fig. 1D and Table 2011 for Animal Ethics Approval). The mice were randomly 1). These data indicate that SIRT1 is downregulated in divided into six groups and BGC-823 cells infected with or gastric cancer, which inspired us to further investigate the without different lentiviral vectors (1 106 cells in 0.1 mL details of SIRT1 in gastric cancer progression. PBS) were subcutaneously injected into the flank region of each group. In detail, group I, BGC-823 cells without SIRT1 inhibits cell proliferation and colony formation of infection were injected (regarded as control; n ¼ 8); group gastric cancer cells II, BGC-823 cells infected with the control-lentiviral expres- To determine the function of SIRT1 in gastric cancer cells, sion vector were injected (regarded as LV-C; n ¼ 8); group we separately transfected the mock vector (pcDNA), the III, BGC-823 cells infected with the SIRT1-lentiviral vectors expressing wild-type SIRT1 (pcDNA-SIRT1), and expression vector were injected (regarded as LV-S; n ¼ catalytic-domain mutant form of SIRT1 (pcDNA-SIRT1- 8); group IV, BGC-823 cells infected with the control- H363Y) into the gastric cancer cell lines AGS, BGC-823, lentiviral shRNA were injected (regarded as LV-Ci; n ¼ 14), and SGC-7901. Furthermore, we knocked down SIRT1 group V and VI, BGC-823 cells infected with one of the two expression using SIRT1-specific siRNAs in the gastric cancer

1500 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

SIRT1 Inhibits Gastric Cancer via G1 Arrest

Figure 1. SIRT1 is downregulated in human gastric cancer (GC). A–C, normal and GC tissue specimens were analyzed for SIRT1 expression using immunohistochemistry. SIRT1 expression is stained in brown and the cell nuclei were counterstained with hematoxylin (blue). Original magniﬁcation, 400. Scale bars, 20 mm. D, qRT-PCR analysis of SIRT1 mRNA in normal and the matched GC tissues. Data, mean SD.

– cell lines AGS, BGC-823, SGC-7901, and MGC-803. The lations in S and G2 M phases (Fig. 3A). The induction of G1 efficient transfection was verified by Western blot analysis phase arrest by SIRT1 was also dependent on its deacetylase (Fig. 2A and Supplementary Fig. S1). The MTS assay activity because transfection of the mutant SIRT1 did not showed that overexpression of SIRT1 significantly inhibited have an effect on the cell-cycle distribution (Fig. 3A). Further cell proliferation, which was absent when the catalytic- evidence was obtained from the RNA interference experi- domain mutant form of SIRT1 was transfected (Fig. 2B). ments. Depletion of SIRT1 significantly decreased the Moreover, there was a clear increase in cell proliferation in fractions of cells in G1 phase, whereas the number of cells – SIRT1-depleted cells (Fig. 2B). Thus, SIRT1 exerts an in S and G2 M phases increased obviously (Fig. 3A). To inhibitory activity on cell proliferation in gastric cancer validate these results, we examined the expression of cell- dependent on its deacetylase activity. cycle regulators operative in G1 phase, including cyclin D1, Next, we determined the effect of SIRT1 on colony cyclin D2, cyclin D3, cyclin E1, CDK4, p21, and p16. As formation of gastric cancer cells. Upregulation of SIRT1 shown in Fig. 3B, when wild-type SIRT1 was upregulated, caused a significant reduction in foci number as well as sizes expression of cyclin D1 was obviously inhibited, which was in gastric cancer cells. This inhibitory effect of SIRT1 was absent when the mutant SIRT1 was transfected. In contrast, dependent on its deacetylase activity because transfection of when SIRT1 was efficiently decreased by a specific siRNA, the mutant SIRT1 did not have an observable effect on expression of cyclin D1 was clearly increased (Fig. 3B and colony formation (Fig. 2C and E). However, knockdown of Supplementary Fig. S1). However, no significant changes SIRT1 significantly increased the foci number compared were observed in the expression of other regulators, includ- – with the corresponding control group (Fig. 2D and E). ing the activators of G1 S transition (cyclin D3, cyclin E1, These results indicate that SIRT1 inhibits colony formation and CDK4) and the inhibitors of CDKs (CDKI; p21 and of gastric cancer cells. p16). However, we failed to detect expression of cyclin D2 in AGS and BGC-823 cells. These data indicate that SIRT1 SIRT1 induces G1 phase arrest in gastric cancer cells induces G1 phase arrest in gastric cancer cells by inhibiting involving NF-kB/cyclin D1 signaling cyclin D1. To examine the inhibitory effects of SIRT1 on gastric Previous studies have shown that deacetylation by SIRTs cancer cells, we performed cell-cycle analysis. We observed can decrease the stability of proteins by ubiquitination and that overexpression of SIRT1 increased the proportion of subsequent proteasomal degradation (20, 21). Here, we cells in G1 phase. This increase of cell fractions in G1 phase detected the stability of cyclin D1 using CHX to inhibit was accompanied with a concomitant decrease of cell popu- protein synthesis. Overexpression of SIRT1 in BGC-823

www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 1501

Yang et al.

Figure 2. SIRT1 inhibits cell viability and colony formation of gastric cancer cells. C, the mock vector; S, the vector expressing wild-type SIRT1; H, the vector expressing the mutant SIRT1; Ci, the control siRNA; Si, the SIRT1 siRNA. A, cells were harvested 48 hours after the vector transfection or 72 hours after siRNA transfection. The transfection efﬁciency was veriﬁed by Western blot analysis. B, cell proliferation was then measured by MTS assays and shown as percentage of vehicle. C and D, the representative graphs of colony formation experiments were illustrated. The quantitative analysis was demonstrated as histogram in E. Data, mean SD; statistical results are indicated by asterisks. , P < 0.01; , P < 0.001.

cells did not affect the stability of cyclin D1 protein (Fig. 4A). mRNA to a level comparable with the controls (Fig. 4D and Furthermore, depletion of SIRT1 did not exert signiﬁcant E). Moreover, The changes in cell proliferation, colony effects on the stability of cyclin D1 protein either (data not formation, and cell-cycle distribution were also reversed by shown). The qRT-PCR results indicated that expression of downregulation of NF-kBorcyclinD1inSIRT1-depleted – þ cyclin D1 mRNA was negatively regulated by SIRT1 (Fig. cells (Fig. 4F H; the percentages of cells in G1 phase: SIRT1 4B), which suggests that SIRT1 transcriptionally inhibits the control siRNA vs. SIRT1 þ NF-kBsiRNA,47.5 0.89 vs. cyclin D1 gene. Among the three common transcription 54.53 2.75, P ¼ 0.007; SIRT1 þ control siRNA vs. SIRT1 factors that regulate the mRNA levels of cyclin D1, NF-kB, þ cyclin D1 siRNA, 47.5 0.89 vs. 59.33 1.1, P < 0.001; b-catenin, and AP-1, expression of NF-kB p65 was inhib- SIRT1 þ NF-kB siRNA vs. control siRNA, 54.53 2.75 vs. ited by SIRT1, whereas expression of the other two tran- 56.3 2.1, P ¼ 0.683; SIRT1 þ cyclin D1 siRNA vs. control scription factors did not change with SIRT1 (Fig. 4C). These siRNA, 59.33 1.1 vs. 56.3 2.1, P ¼ 0.405). Taken data suggest that SIRT1 may inhibit the transcription of together, our results indicate that SIRT1 induces G1 phase cyclin D1 through downregulation of NF-kB. arrest in gastric cancer cells via NF-kB/cyclin D1 signaling. Next, RNA interference experiments were performed. Inhi- Lack of sub-G1 and results of the TUNEL labeling bition of NF-kB in SIRT1-depleted cells rescued cyclin D1 experiments indicated that SIRT1 did not trigger apoptosis

1502 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

SIRT1 Inhibits Gastric Cancer via G1 Arrest

Figure 3. SIRT1 induces G1 phase arrest in gastric cancer cells. A, the distribution of cell cycle was analyzed by a flow cytometer. The quantitative analysis was demonstrated as histogram. B, expression of cell-cycle regulators was detected by Western blot analysis. Data, mean SD; statistical results were indicated by asterisks. , P < 0.05; , P < 0.01; and , P < 0.001. in gastric cancer cells (Fig. 4H and Supplementary Fig. S2). moted tumor progression (LV-Ci vs. LV-sh-1: 1.2898 Moreover, the protein levels of apoptosis-related molecules 0.1947 vs. 2.2344 0.1935 cm3, P < 0.001; LV-Ci vs. LV- (bcl-2 and bax) from each group were comparable and no sh-2: 1.2898 0.1947 vs. 2.0456 0.18 cm3, P < cleaved caspase-3 was detected in the gastric cancer cells 0.001; Fig. 5A and B). The inhibitory effects of SIRT1 on (Supplementary Fig. S2). BGC-823 xenografts were then corroborated in the tumor samples by detecting the protein levels of Ki67 using SIRT1 inhibits tumor growth in vivo immunohistochemistry. The percentages of Ki67-positive Next, the effects of SIRT1 on gastric cancer development cells in groups of control, LV-C, LV-S, LV-Ci, LV-sh-1, and were verified using a nude mouse xenograft model. For in LV-sh-2 were 46.1 1.44, 45.7 0.95, 6 1.13, 45.56 vivo study, stable lentivirus–infected BGC-823 cells were 1.56, 87.59 1.93, and 82.23 3.21, respectively (LV-C used. Efficient infection was verified using a fluorescence vs. LV-S, P < 0.001; LV-Ci vs. LV-sh-1, P < 0.001; LV-Ci microscope (data not shown). All of the nude mice survived vs. LV-sh-2, P < 0.001; Fig. 5B). The changes in the to the end of our experiments and there were no apparent expression of cyclin D1 and NF-kB were similar to what differences in their body weights (data not shown). Four we observed in vitro (Fig. 5C). Our results suggest that weeks after implantation, there were no observable differ- SIRT1 inhibits the gastric cancer development in vivo, which ences in the tumor volumes among the three groups of involves NF-kB/cyclin D1 signaling. control, LV-C, and LV-Ci. The tumor volumes for the earlier three groups were 1.4297 0.1821, 1.369 0.1443, and 1.2898 0.1947 cm3, respectively (P > 0.05; Fig. 5A Discussion and B). Overexpression of SIRT1 significantly inhibited SIRT1 was initially concerned because its homolog in the tumor development (LV-C vs. LV-S: 1.369 0.1443 vs. budding yeast, silent information regulator 2, has been 0.5988 0.1123 cm3; P ¼ 0.001; Fig. 5A and B). shown to extend the life span of the yeast (22). Subsequent Interestingly, tumor had not developed in one of the nude studies have revealed that SIRT1 plays an essential role in mice in the LV-S group at the end of the xenograft experi- metabolic pathways and is linked to health, longevity, ments. However, stable depletion of SIRT1 obviously pro- and diseases such as cancer (5, 6). The role of SIRT1 in

www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 1503

Yang et al.

Figure 4. SIRT1 exerts inhibitory effects on gastric cancer cells via NF-kB/cyclin D1 signaling. A, the stability of cyclin D1 protein was detected after BGC-823 cells were transfected with the vector expressing wild-type SIRT1. CHX was used to inhibit the synthesis of protein. B, the mRNA levels of cyclin D1 were detected in BGC- 823 cells using qRT-PCR. C, the three common transcription factors of cyclin D1 were detected by Western blot analysis in BGC- 823 cells. D, interference efﬁciency of NF-kB or cyclin D1 was veriﬁed by Western blot analysis in BGC- 823 cells. Ni, the NF-kB siRNA; Di, the cyclin D1 siRNA. E, after inhibition of NF-kB in SIRT1- depleted BGC-823 cells, the mRNA levels of cyclin D1 were determined by qRT-PCR. F, cell proliferation of BGC-823 cells with different treatment was measured by MTS assays. G, representative graphs of colony formation of BGC-823 cells with different treatment were illustrated. The quantitative analysis was demonstrated as histogram. H, representative graphs of cell-cycle distribution of BGC-823 cells with different treatment are shown. Data, mean SD; statistical results were indicated by asterisks. , P < 0.001.

tumorigenesis is an area of considerable debate. First, the expression of differentiation markers in vitro, and has been expression levels of SIRT1 in different types of cancers are shown to be beneficial in blocking tumor formation in vivo conflicting. The expression of SIRT1 is relatively higher in (11, 29). However, the opposite is true in colon cancer. Two hepatocellular carcinoma, breast cancer, and thyroid cancer different groups have reported that SIRT1 inhibited prolif- (10, 23, 24) but lower in colon and lung cancer (15, 16, 25) eration and intestinal tumorigenesis both in vitro and in vivo compared with their corresponding normal tissues. Even in (15, 16). These contrary data suggest that the role of SIRT1 the same types of cancer, such as prostate cancer, contrary in oncogenic progression is cancer-type specific. In our results have been reported (26, 27). Here, we detected the experiments, SIRT1 exerted inhibitory effects on cell pro- expression of SIRT1 in gastric cancer and found that both liferation and colony formation in gastric cancer cells. This the mRNA and the protein levels of SIRT1 decreased inhibitory activity was verified using a nude mouse xenograft compared with the matched normal tissues. This result model. Our results indicate that SIRT1 is an inhibitor of conflicts with previous results reported by Cha and col- gastric cancer and that downregulation of SIRT1 promotes leagues (28). Because the same antibody was used to detect gastric tumorigenesis. the expression of SIRT1, the conflicting data may be due The regulation of cell cycle is an important antiprolifera- to the different race from which the tumor samples were tion mechanism in cancer (30, 31). Among the seven obtained. members of the mammalian sirtuin family, SIRT2, a tubulin Second, the role of SIRT1 in different types of cancers has deacetylase required for normal mitotic progression, has also been disputed. Results from the hepatocellular carci- been frequently linked to cell-cycle transition (4). Few noma indicate that inhibition of SIRT1 in hepatocellular studies have focused on SIRT1 and cell-cycle regulation. carcinoma cells impairs their proliferation, increases the A study by Wang and colleagues indicates that SIRT1 binds

1504 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

SIRT1 Inhibits Gastric Cancer via G1 Arrest

Figure 5. SIRT1 inhibits tumor growth in vivo. A, after implantation, the volumes of the subcutaneous tumors were measured weekly with a caliper. B, the representative images of the dissected tumors are shown on the left. A ruler is used to indicate the size of the tumor. The results of Ki67 immunohistochemistry of the corresponding tumor specimens are shown on the right. Original magniﬁcation, 400. Scale bars, 20 mm. C, total protein was extracted from tumor samples from groups of control, LV-S, LV-Ci, LV-sh-1, and LV-sh-2. The protein levels of SIRT1, cyclin D1, and NF-kB in subcutaneous tumors were examined by Western blot analysis.

to E2F1, a cell-cycle and apoptosis regulator, and inhibits its cyclin D1. Unlike FOXO3 (21), the stability of cyclin D1 activity (32), which suggests that SIRT1 may participate in protein was not affected by SIRT1. Detection of the mRNA the regulation of cell cycle. A subsequent study reveals that in levels of cyclin D1 revealed that SIRT1 inhibited the addition to E2F1, SIRT1 interacts with another transcrip- expression of cyclin D1 at the transcriptional level. Several tion factor, c-Myc. SIRT1 deacetylates c-Myc and activates transcription factors that bind the promoter of cyclin D1 its target genes, including cyclin D2, thus plays an essential gene have been proven to be inhibited by SIRT1, including role in cell proliferation and cell-cycle regulation (33). In our NF-kB, b-catenin, and AP-1 (16, 34–37). Among these k study, overexpression of SIRT1 induced G1 phase arrest in three transcription factors, only NF- B was inhibited by gastric cancer cells. Detection of regulators of the G1 phase, SIRT1 at the protein level. Although our results do not including the activators (cyclin D1, cyclin D2, cyclin D3, eliminate the possibilities that other transcription factors cyclin E1, and CDK4) and the inhibitors (p21 and p16), may participate in the regulation of cyclin D1 by SIRT1, indicated that SIRT1 negatively regulated the expression of inhibition of NF-kB in SIRT1-depleted cells rescued the

www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 1505

Yang et al.

mRNA levels of cyclin D1. These results suggest that NF-kB Disclosure of Potential Conflicts of Interest is responsible for SIRT1 inhibiting the transcription of No potential conflicts of interest were disclosed. cyclin D1. Moreover, the changes in cell proliferation, colony formation, and cell-cycle distribution caused by Authors' Contributions SIRT1 depletion were reversed by downregulation of NF- Conception and design: Q. Yang, J. Jia Development of methodology: Q. Yang, B. Wang, W. Gao, S. Huang kB or cyclin D1, indicating that NF-kB/cyclin D1 signaling Acquisition of data (provided animals, acquired and managed patients, provided accounts for the inhibitory effects on gastric cancer cells facilities, etc.): B. Wang, W. Gao Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- induced by SIRT1. The inhibitory effect of SIRT1 on NF- tational analysis): Q. Yang, Z. Liu, W. Li kB/cyclin D1 pathway was also verified by Western blot Writing, review, and/or revision of the manuscript: Q. Yang, J. Jia analysis using xenograft tumor tissues. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Li Taken together, using clinical gastric cancer specimens, Study supervision: B. Wang, J. Jia gastric cancer cells, and nude mouse xenograft models, our study shows that SIRT1, a histone/protein deacetylase, Grant Support exerts tumor-suppressive function in gastric cancer. This work was supported by the National Basic Research Program of China (973 Through the NF-kB/cyclin D1 pathway, SIRT1 induces Program; No. 2012CB911202), the National Natural Science Foundation of China (No. 81101869, 81100103, 81171536, 81000868, and 81371781), and the Inde- G1 phase arrest in gastric cancer and leads to the inhibition of pendent Innovation Foundation of Shandong University (2012TS108). gastric cancer both in vitro and in vivo. Results from our work The costs of publication of this article were defrayed in part by the payment of page demonstrate the inhibitory function of SIRT1 in gastric charges. This article must therefore be hereby marked advertisement in accordance with cancer development and suggest a potential therapeutic 18 U.S.C. Section 1734 solely to indicate this fact. effect for SIRT1 activators in the prevention and therapy Received May 3, 2013; revised September 16, 2013; accepted September 23, 2013; of gastric cancer. published OnlineFirst October 9, 2013.

References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer 16. Firestein R, Blander G, Michan S, Oberdoerffer P, Ogino S, Campbell J, statistics. CA Cancer J Clin 2011;61:69–90. et al. The SIRT1 deacetylase suppresses intestinal tumorigenesis and 2. Bertuccio P, Chatenoud L, Levi F, Praud D, Ferlay J, Negri E, et al. colon cancer growth. PLoS ONE 2008;3:e2020. Recent patterns in gastric cancer: a global overview. Int J Cancer 17. Li W, Ge Z, Liu C, Liu Z, Bjorkholm€ M, Jia J, et al. CIP2A is over- 2009;125:666–73. expressed in gastric cancer and its depletion leads to impaired clo- 3. Roukos DH, Kappas AM. Perspectives in the treatment of gastric nogenicity, senescence, or differentiation of tumor cells. Clin Cancer cancer. Nat Clin Pract Oncol 2005;2:98–107. Res 2008;14:3722–8. 4. Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and 18. Yang Q, Xu S, Li X, Wang B, Wang X, Ma D, et al. Pathway of toll-like disease relevance. Annu Rev Pathol 2010;5:253–95. receptor 7/b cell activating factor/ b cell activating factor receptor 5. Brooks CL, Gu W. How does SIRT1 affect metabolism, senescence plays a role in immune thrombocytopenia in vivo. PLoS ONE 2011;6: and cancer? Nat Rev Cancer 2009;9:123–8. e22708. 6. Bordone L, Guarente L. Calorie restriction, SIRT1 and metabo- 19. Huang L, Chen S, Zha X, Yang L, Li B, Yu Z, et al. Expression feature of lism: understanding longevity. Nat Rev Mol Cell Biol 2005;6: CD3, FceRIg, and Zap-70 in patients with chronic lymphocytic leuke- 298–305. mia. Hematology 2012;17:71–5. 7. Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, 20. Inuzuka H, Gao D, Finley LW, Yang W, Wan L, Fukushima H, et al. Guarente L, et al. hSIR2(SIRT1) functions as an NAD-dependent Acetylation-dependent regulation of Skp2 function. Cell 2012;150: p53 deacetylase. Cell 2001;107:149–59. 179–93. 8. Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, et al. 21. Wang F, Chan CH, Chen K, Guan X, Lin HK, Tong Q. Deacetylation of Discovery, in vivo activity, and mechanism of action of a small-mol- FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitina- ecule p53 activator. Cancer Cell 2008;13:454–63. tion and degradation. Oncogene 2012;31:1546–57. 9. Chen WY, Wang DH, Yen RC, Luo J, Gu W, Baylin SB. Tumor 22. Kaeberlein M, McVey M, Guarente L. The SIR2/3/4 complex and SIR2 suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent alone promote longevity in Saccharomyces cerevisiae by two different DNA-damage responses. Cell 2005;123:437–48. mechanisms. Genes Dev 1999;13:2570–80. 10. Kim JE, Chen J, Lou Z. DBC1 is a negative regulator of SIRT1. Nature 23. Eades G, Yao Y, Yang M, Zhang Y, Chumsri S, Zhou Q. miR-200a 2008;451:583–6. regulates SIRT1 expression and epithelial to mesenchymal transition 11. JangKY,NohSJ,LehwaldN,TaoGZ,BellovinDI,ParkHS,etal. (EMT)-like transformation in mammary epithelial cells. J Biol Chem SIRT1 and c-Myc promote liver tumor cell survival and predict poor 2011;286:25992–6002. survival of human hepatocellular carcinomas. PLoS ONE 2012;7: 24. Herranz D, Maraver A, Canamero~ M, Gomez-L opez G, Inglada-Perez L, e45119. Robledo M, et al. SIRT1 promotes thyroid carcinogenesis driven by 12. Das C, Lucia MS, Hansen KC, Tyler JK. CBP/p300-mediated acety- PTEN deﬁciency. Oncogene 2013;32:4052–6. lation of histone H3 on lysine 56. Nature 2009;459:113–7. 25. Beane J, Cheng L, Soldi R, Zhang X, Liu G, Anderlind C, et al. SIRT1 13. Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, Park pathway dysregulation in the smoke-exposed airway epithelium and SK,etal.SIRT1redistributiononchromatinpromotesgenomic lung tumor tissue. Cancer Res 2012;72:5702–11. stability but alters gene expression during aging. Cell 2008;135: 26. Wang RH, Sengupta K, Li C, Kim HS, Cao L, Xiao C, et al. Impaired DNA 907–18. damage response, genome instability, and tumorigenesis in SIRT1 14. Li K, Casta A, Wang R, Lozada E, Fan W, Kane S, et al. Regulation of mutant mice. Cancer Cell 2008;14:312–23. WRN protein cellular localization and enzymatic activities by SIRT1- 27. Huffman DM, Grizzle WE, Bamman MM, Kim JS, Eltoum IA, Elgavish A, mediated deacetylation. J Biol Chem 2008;283:7590–8. et al. SIRT1 is signiﬁcantly elevated in mouse and human prostate 15. Kabra N, Li Z, Chen L, Li B, Zhang X, Wang C, et al. SirT1 is an inhibitor cancer. Cancer Res 2007;67:6612–8. of proliferation and tumor formation in colon cancer. J Biol Chem 28. Cha EJ, Noh SJ, Kwon KS, Kim CY, Park BH, Park HS, Lee H, 2009;284:18210–7. et al. Expression of DBC1 and SIRT1 is associated with poor

1506 Mol Cancer Res; 11(12) December 2013 Molecular Cancer Research

SIRT1 Inhibits Gastric Cancer via G1 Arrest

prognosis of gastric carcinoma. Clin Cancer Res 2009;15: 34. Lee I, Lee SJ, Kang TM, Kang WK, Park C. Unconventional role of the 4453–9. inwardly rectifying potassium channel Kir2.2 as a constitutive activator 29. Portmann S, Fahrner R, Lechleiter A, Keogh A, Overney S, Laemmle A, of RelA in cancer. Cancer Res 2013;73:1056–62. et al. Antitumor effect of SIRT1 inhibition in human HCC tumor models 35. Schug TT, Xu Q, Gao H, Peres-da-Silva A, Draper DW, Fessler MB, in vitro and in vivo. Mol Cancer Ther 2013;12:499–508. et al. Myeloid deletion of SIRT1 induces inﬂammatory signaling 30. Chan KS, Koh CG, Li HY. Mitosis-targeted anti-cancer therapies: in response to environmental stress. Mol Cell Biol 2010;30: where they stand. Cell Death Dis 2012;3:e411. 4712–21. 31. Starostina NG, Kipreos ET. Multiple degradation pathways regulate 36. Cho IR, Koh SS, Malilas W, Srisuttee R, Moon J, Choi YW, et al. SIRT1 versatile CIP/KIP CDK inhibitors. Trends Cell Biol 2012;22:33–41. inhibits proliferation of pancreatic cancer cells expressing pancreatic 32. Wang C, Chen L, Hou X, Li Z, Kabra N, Ma Y, et al. Interactions between adenocarcinoma up-regulated factor (PAUF), a novel oncogene, by E2F1 and SirT1 regulate apoptotic response to DNA damage. Nat Cell suppression of b-catenin. Biochem Biophys Res Commun 2012;423: Biol 2006;8:1025–31. 270–5. 33. Mao B, Zhao G, Lv X, Chen HZ, Xue Z, Yang B, et al. Sirt1 deacetylates 37. Li L, Zhang HN, Chen HZ, Gao P, Zhu LH, Li HL, et al. SIRT1 acts as a c-Myc and promotes c-Myc/Max association. Int J Biochem Cell Biol modulator of neointima formation following vascular injury in mice. Circ 2011;43:1573–81. Res 2011;108:1180–9.

www.aacrjournals.org Mol Cancer Res; 11(12) December 2013 1507

SIRT1 Is Downregulated in Gastric Cancer and Leads to G1-phase Arrest via NF- κB/Cyclin D1 Signaling

Qing Yang, Bo Wang, Wei Gao, et al.

Mol Cancer Res 2013;11:1497-1507. Published OnlineFirst October 9, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-13-0214

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2021/03/16/1541-7786.MCR-13-0214.DC1

Cited articles This article cites 37 articles, 12 of which you can access for free at: http://mcr.aacrjournals.org/content/11/12/1497.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://mcr.aacrjournals.org/content/11/12/1497.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/11/12/1497. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.