SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma

SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma

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Available online at www.sciencedirect.com

ScienceDirect Journal of the Chinese Medical Association xx (2017) 1e7 www.jcma-online.com

Original Article

SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma Yuan-Yuan Kang a,*, Fu-Li Sun a, Ying Zhang a, Zhe Wang b a

Department of Emergency and Oral Medicine, The School of Stomatology, China Medical University & Liaoning Institute of Dental Research & Liaoning Province Key Laboratory of Oral Diseases & Liaoning Province Translational Medicine Research Center of Oral Diseases, Shenyang, Liaoning, China b Department of Pathology, The Second Affiliated Hospital and College of Basic Medical Sciences, China Medical University, Shenyang, Liaoning, China Received April 4, 2017; accepted September 29, 2017

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in oral cancer, however, the mechanism underlying OSCC tumorigenesis is unknown. SIRT1, has been considered a prominent tumor-suppressing/promoting gene in various solid tumors, although the precise role of SIRT1 in OSCC progression remains unknown. Methods: SIRT1 expression was assessed in surgically resected specimens from patients with OSCC for histopathologic factors. SIRT1 levels in OSCC were determined, SIRT1 overexpression was achieved on transfecting OSCC cells with a SIRT1-containing plasmid, followed by evaluation of proliferative ability and invasiveness of these cells. Results: SIRT1 levels were significantly lower in patients with OSCC than in controls ( p < 0.05). Moreover, SIRT1 levels in patients with OSCC were significantly associated with the lymphovascular permeation but not with the sex, age, stage and location. Furthermore, SIRT1 overexpression inhibited proliferation and invasion in OSCC cells. Conclusion: The present results suggest that SIRT1 is a potential tumor suppressor in OSCC. Copyright © 2017, the Chinese Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Invasion; Oral cancer; Proliferation; SIRT1

1. Introduction Oral squamous cell carcinoma (OSCC) is the most common head and neck squamous cell carcinoma, with more than 3,00,000 new cases diagnosed annually worldwide.1 The high mortality associated with OSCC mainly results from cervical lymph node metastasis and occasionally distant organ metastasis.2 The pathogenesis of OSCC depends on the rates of cell proliferation and apoptosis.3 The 5-year survival rate of patients with OSCC has not significantly improved, despite progress in OSCC treatment.4 Therefore, an understanding of

Conflicts of interest: The authors declare that they have no conflicts of interest related to the subject matter or materials discussed in this article. * Corresponding author. Dr. Yuanyuan-Kang, Department of Emergency and oral medicine, school of Stomatology, China Medical University, 117, Nanjing North Street, Shenyang, Liaoning 110002, China. E-mail address: [email protected] (Y.-Y. Kang).

the molecular mechanisms underlying OSCC tumorigenesis is required to identify tumor-specific biomarkers and therapeutic targets for early diagnosis and treatment. Sirtuins (SIRT1-7) are a family of NADþ e dependent deacetylases that belong to the class III histone deacetylases and are implicated in many cellular processes including metabolism, the cell cycle, and aging.5 The best studied sirtuin is SIRT1 (silent mating type information regulation 2 homolog 1), a mammalian homolog of yeast Sir2.6 SIRT1 promotes or inhibits many biological processes, including regulation of gene expression, cellular metabolism, stress response, aging, and chemo-resistance,7 however, its role in tumorigenesis is ambiguous. SIRT1 upregulation has been reported in many solid tumors.8e10 SIRT1 has been shown to silence tumor suppressors, e.g, p53, or activate tumor drivers, e.g., the PTEN/PI3K/AKT pathway, thereby promoting tumorigenesis.11,12 However, numerous studies have reported downregulation of SIRT1 in some tumors, suggesting its role as a

https://doi.org/10.1016/j.jcma.2017.09.004 1726-4901/Copyright © 2017, the Chinese Medical Association. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Kang Y-Y, et al., SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma, Journal of the Chinese Medical Association (2017), https://doi.org/10.1016/j.jcma.2017.09.004

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Fig. 1. SIRT1 expression in OSCC (A) SIRT1 mRNA levels in OSCC and OSCC adjacent normal tissues from 9 patients, determined through RT-qPCR analysis. (B) SIRT1 mRNA and protein levels examined using RT-qPCR and western blot analyses in OSCC cell lines and in a keratinocyte cell line(HaCaT); (C) Representative images of SIRT1 protein levels assessed through immunohistochemical staining in OSCC tissue samples and adjacent normal tissues. *P < 0.05, vs. controls.

tumor suppressor.13e15 Currently, the role of SIRT1 in OSCC progression and metastasis is unknown. In this study, we examined SIRT1 expression in OSCC, investigated the association between SIRT1 expression and clinicopathological factors of OSCC, and explored its putative in OSCC.

2. Methods 2.1. Ethics statement Experiments using human tissue samples were approved by the ethics committee of the School of Stomatology, China

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Medical University, and written informed consent was obtained from the donors.

Table 1 The integral optical density (IOD) of SIRT1 in OSCC tissue specimens on immunohistochemical staining.

2.2. Tissue samples

Tissue

IOD (median ± interquartile)

OSCC OSCC adjacent

76.22 ± 11.54* 138.07 ± 10.30

Tissue specimens, including nine fresh-frozen OSCC specimens and nine OSCC adjacent normal epithelium specimens, from nine patients with OSCC who underwent surgical resection at the Department of Oral and Maxillofacial Surgery at the School of Stomatology, China Medical University between January 2015 and September 2016. Another batch of specimens, including 90 formalin-fixed and paraffin-embedded OSCC tissue specimens and 20 OSCC adjacent normal epithelium specimens were acquired from the Department of Oral Pathology at the School of Stomatology, China Medical University from January 2012 to March 2016. No patients were administered radiotherapy or chemotherapy before biopsy. Clinicopathological data, including sex, age, T classification, N classification, histological grade, stage, location, lymphovascular permeation, and distant metastasis, were obtained from of 90 patients from their pathological and clinical records. 2.3. Cell culture Human OSCC cell lines SCC-9 and SCC-25 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). HaCaT cells, the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). OSCC cells were maintained in Dulbecco modified Eagle medium (DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, HaCaT cells, plain DMEM(Gibco). Cells cultures were maintained in a humidifying incubator at 5% CO2 and 37  C. 2.4. Real-time quantitative polymerase chain reaction Total RNA was isolated from the fresh-frozen specimens and cultured cells, using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. After quantification using NanoDrop (Nyxor Biotech, France), 2 mg of DNase-treated RNA was used for cDNA synthesis with M-MLV Reverse transcriptase (Promega, WI, USA), The cDNA amplification reactions were performed using Ex_Taq™ polymerase (TaKaRa, Japan). Gene expression was normalized with GAPDH as an internal control, and the mean relative change was determined in triplicate or quintuplicate through relative quantification and application of the deltaedelta cycle threshold method. The following primer pairs were used: SIRT1 Forward primers 50 -TAGCCTTGTCAGATAAGGAAGGA-30 Reverse primers. 50 -ACAGCTTCACAGTCAACTTTGT-30 GAPDH: Forward primers 50 -ACCACAGTCCATGCCATCAC-30 , Reverse primers. 50 -TCCACCACCCTGTTGCTGTA-30

*P < 0.05, vs controls.

2.5. Western blot analysis OSCC cells were lysed directly in RIPA buffer containing 50 mM TriseHCl (pH 7.8), 150 mM NaCl, 5 mM EDTA, 5 mL/mL TritonX-100, 5 mL/mL Nonidet-P40, and 1 mL/mL sodium deoxycholate on ice for 20 min. The lysates were centrifuged and supernatants were used for SDS-PAGE. Protein concentrations were determined using the bicinchoninic acid method. In total, 20e50 mg of protein samples were loaded in each well and were separated using a 10% polyacrylamide resolving gels and then electroblotted onto polyvinylidene difluoride membranes. After blocking with 5% non-fat milk, the membranes were probed with anti-SIRT1 and anti-GAPDH antibodies (Abcam, Cambridge, MA, USA) and further probed with secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were visualized

Table 2 The association between clinicopathological characteristics of OSCC patients and SIRT1 expression. Variables

Sex Male Female Age(y) 55 >55 T classification T1,2 T3,4 N classification N0 N0þ Histological grade Well Poor/moderate Stage Ⅰ and Ⅱ Ⅲ and Ⅳ Location Buccal mucosa Tongue Mouth floor Others Lymphovascular permeation Present Absent Distant metastasis Present Absent

n

P Univariate analysis

60 30

0.462

47 43

0.812

32 58

0.644

39 51

0.704

41 49

0.312

36 54

0.235

24 35 20 11

0.732

70 20

0.004

54 36

0.052

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with an enhanced chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA, USA). 2.6. Immunohistochemical staining Immunohistochemical staining was performed to detect protein localization and expression in paraffin-embedded OSCC specimens. The sections were stained with rabbit anti-SIRT1 polyclonal antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and goat anti-rabbit polyclonal antibody (1:5000; Santa Cruz Biotechnology), using an automatic slide stainer BenchMark XT (Ventana Medical Systems, Tucson, AZ, USA). Hematoxylin was used as the counterstain. Sections were evaluated using a microscope (Nikon, Tokyo, Japan) by one of the authors. At higher magnification (400), five visual fields were selected randomly, the expression positive signal was analyzed using Image-proplus software. SIRT1 protein levels in OSCC tissue and adjacent normal epithelium specimens were compared in accordance with the integral optical density (IOD) as a parameter for semi-quantitative detection.

free medium with 0.1% BSA (Sigma) and seeded onto the upper chamber and 0.5 mL of media containing 10% FBS was added in the lower chamber to act as a chemoattractant. Cells in the transwell plates were incubated at 37  C in a humidified incubator with 5% CO2 for 48 h. The adherent cells on the upper surface of the insert membrane were carefully removed with cotton swabs. Cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100, and stained with 0.1% crystal violet for 30 min. The invading cells were visualized and quantified using a light microscope in five separate fields per membrane. 2.10. Statistical analysis Statistical analysis was performed using SPSS17.0 software. The ManneWhitney rank sum test was used to compare SIRT1 mRNA and SIRT1 protein expression between the OSCC cells and the controls. In addition, correlations between the levels of IOD of SIRT1 protein with clinicopathological factors were assessed using univariate analysis. A p-value less than 0.05 indicated a statistically significant difference.

2.7. Transfection assay

3. Results

On reaching 70e80% confluence, OSCC cells were trypsinized and transfected with M02-SIRT1 plasmids and control (M02-con) plasmids (RiboBio, Guangzhou, China)by using Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Transfection efficiency was assessed through western blot and RT-qPCR analyses.

3.1. Low expression of SIRT1 in OSCC

2.8. MTS assay Proliferation of plasmid-transfected OSCC cells was evaluated using a Cell Proliferation Assay kit (Promega, Madison, WI, USA) per the manufacturer's. After being trypsinized and counted, the cells were seeded into 96-well plates, at a density of 2  103 cells/well. At 0, 24, 48, 72, 96 and 120 h in culture, 20 mL of MTS was added into every well, and the cells were incubated at a temperature of 37  C for 2 h. Finally, the absorbance of each well at 490 nm which represented cell quantity, was recorded using a microplate reader (BioTek Synergy2, Winooski, VT, USA) for plotting growth curves. 2.9. Transwell assay A Cell Invasion Assay Kit (BD Biosciences, Billerica, MA, USA) was used to evaluate invasion of the plasmid-transfected cells in accordance with the manufacturer's instructions. The upper chamber(Corning Costar, Tewksbury, MA, USA) was precoated with 50 mL of 20% growth factor-reduced Matrigel for the invasion assay. The cells (5  104) were resuspended in serum-

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To determine the SIRT1 mRNA expression level, the specimen from the OSCC and adjacent tissues groups were separated. The total mRNA was isolated and the mRNA expression level of SIRT1 was investigated with RT-qPCR. Using SPSS17.0 software, RT-qPCR data are presented as fold change in gene expression normalized against GAPDH. SIRT1 mRNA levels were significantly lower in OSCC tissue samples than in adjacent normal tissue samples ( p < 0.05; Fig. 1A). We also used RT-qPCR and western blot to measure the expression of SIRT1 in OSCC cell lines (SCC9, SCC25) and a normal epithelium cell line (HaCaT). Similarly, SIRT1 mRNA and protein levels were significantly lower in OSCC cells than in normal epithelium cell lines ( p < 0.05, Fig. 1B). On immunohistochemical analysis, SIRT1 was detected in both the OSCC and adjacent tissues groups (including 20 cases of OSCC adjacent tissues and 90 cases of OSCC tissues) (Fig. 1C). SIRT1 was mainly localized in the cytoplasm and occasionally present in nuclei of OSCC cells. Furthermore, the OSCC group exhibited significantly lower SIRT1 levels than the adjacent tissues controls ( p < 0.05; Table 1). 3.2. Association between SIRT1 and clinicopathological factors The association between SIRT1 expression level and clinicopathological factors of OSCC patients was also analyzed.

Fig. 2. Tumor-suppressing functions of SIRT1 in OSCC cells. (A) The mRNA levels of SIRT1 determined through RT-qPCR after transfection. (B) The protein levels of SIRT1 determined through western blot analyses after transfection. (C) The inhibitory effect of SIRT1 on cell proliferation in SCC9 cells and SCC25 cells, as evaluated using the MTS assay; (D) The inhibitory effect of SIRT1 on cell invasion in SCC9 cells and SCC25 cells was evaluated by transwell assay. Please cite this article in press as: Kang Y-Y, et al., SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma, Journal of the Chinese Medical Association (2017), https://doi.org/10.1016/j.jcma.2017.09.004

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SIRT1 expression was estimated on the basis of the IOD on immunohistochemical staining. Univariate analysis revealed a notable association between lymphovascular permeation and SIRT1 protein levels ( p < 0.05, Table 2). Distant metastasis was borderline significant ( p ¼ 0.052). Other factors, including sex, age, stage, location, etc, were not associated with SIRT1 expression. 3.3. SIRT1 suppressed the proliferation and migration of OSCC cells On investigating the biological function of SIRT1 in OSCC, transiently transfected SCC9 and SCC25 cell lines expressing SIRT1 had SIRT1 mRNA levels greater than 5 times that of cells with empty vectors, on RT-qPCR and western blot analyses ( p < 0.05, Fig. 2A and B). To determine the role of SIRT1 in proliferation of OSCC cells after transfection, MTS cell proliferation assays were performed (Fig. 2C). We found that proliferation of SIRT1transfected SCC9 cells was slightly reduced at 24 h ( p > 0.05) and 48 h ( p > 0.05), and significantly at 72 h ( p < 0.05), 96 h ( p < 0.05), and 120 h ( p < 0.05), compared to the vector control. Proliferation of SIRT1-transfected SCC25 cells decreased slightly at 24 h ( p > 0.05), and significantly at 48 h ( p < 0.05), 72 h ( p < 0.05), 96 h ( p < 0.05) and 120 h ( p < 0.05), compared to the vector control. These results show that SIRT1 has a significant effect on OSCC cell proliferation. Random cell counting in the transwell assay revealed that the invasiveness of 48 h SIRT1-transfected OSCC cells was significantly lower than that of the vector control( p < 0.05, Fig. 2D). The number of invasive cells in SCC9-control and SCC9-SIRTI groups was 716 ± 86 and 241 ± 45, respectively, and that in SCC25-con and SCC25-SIRTI groups was 621 ± 34 and 236 ± 82, respectively. A significant difference was observed between the number of OSCC cells and the control cells(HaCaT) ( p < 0.05). The transwell assays showed that SIRT1 overexpression reduced the invasiveness of SCC9 and SCC25 cell lines. 4. Discussion SIRT1, a class Ⅲ histone deacetylase, is associated with multiple age-related diseases owing to its ability to deacetylate histones and non-histone proteins.16 Although several studies have described the functional localization of SIRT1 in various cells, including those of the tumor microenvironment, the role of SIRT1 in tumorigenesis remains controversial and may depend on the tumor type. A recent study reported that enhanced SIRT1 expression inhibited intestinal-tumor formation in a b-catenindependent mouse model of colon cancer, thereby indicating that the effects of SIRT1 might vary in different tumor models and may depend on the presence of appropriate downstream targets.17 Wang et al. reported that develop tumors develop in multiple tissues in Sirt1þ/p53þ/ mice, and activation of SIRT1 by resveratrol reduces tumorigenesis. Moreover, several independent investigations have revealed lower levels of SIRT1 in Sirt1þ/p53þ/ mice than in healthy controls and suggested that

SIRT1 is an important inhibitor of the epithelialemesenchymal transition in various cancers.18e20 Our data further contributes to the results of these previous studies by further verifying that SIRT1 is a critical regulator of cancer progression and an important therapeutic target. Our findings suggest that SIRT1 can act as a tumor suppressor in OSCC. Through RT-qPCR, western blot, and immunohistochemical analyses, we assessed SIRT1 expression at the mRNA and protein levels in OSCC cells and surgically resected specimens from patients. We found that SIRT1 expression levels were significantly higher in adjacent normal tissues than in OSCC specimens. In addition, SIRT1 levels were significantly lower in OSCC cell lines than in a normal epithelial cell line(HaCaT). On comparing OSCC cell lines with different levels of SIRT1 expression, using an MTS assay and a transwell assay, we found that high SIRT1 expression inhibited cell proliferation and strongly suppressed mobility. Previous studies have reported that SIRT1 is overexpressed in many solid tumors; which, in turn, is associated with tumor progression in thyroid papillary carcinoma,21 prostate cancer,22 and hepatocellular carcinoma.23 Therefore, SIRT1 seems to be a useful prognostic factor for various kinds of malignancies.24 In the present study, SIRT1 expression was associated with certain clinicopathological parameters, particularly lymphovascular permeation, in patients with OSCC. Consequently, analysis of SIRT1 expression may be particularly useful during prognostic evaluation of this patient cohort. Although we have comprehensively analyzed the clinicopathologic value of SIRT1 in OSCC, our study has some limitations. First, the sample size in the subgroup analysis was not large enough; thus, the statistical power was limited. Second, we did not investigate the function of SIRT1 in vitro. In future, we intend to recruit more healthy controls and patients with OSCC. Furthermore, a SIRT1-stable OSCC cell line will be injected directly into the anterior tongue of nude mice to assess tumor proliferation, local invasion, and regional metastasis. In conclusion, SIRT1 was significantly downregulated in OSCC tissues and cell lines. SIRT1 suppressed OSCC cell proliferation and invasion in vitro. Hence, as a tumor suppressor in OSCC, SIRT1 may thus serve as a potential biomarker for diagnosis and as a new therapeutic target for OSCC. Acknowledgments This study was supported by grants from the Natural Science Foundation of Liaoning Province (20170541012) and the Youth Research Fund of the school of Stomatology, China Medical University(K101593-16-05). References 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011;61:69e90. 2. Liu CJ, Liu TY, Kuo LT, Cheng HW, Chu TH, Chang KW, et al. Differential gene expression signature between primary and metastatic head and neck squamous cell carcinoma. J Pathol 2008;214:489e97.

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Please cite this article in press as: Kang Y-Y, et al., SIRT1 acts as a potential tumor suppressor in oral squamous cell carcinoma, Journal of the Chinese Medical Association (2017), https://doi.org/10.1016/j.jcma.2017.09.004