Overexpression of Sirt7 Exhibits Oncogenic Property and Serves As a Prognostic Factor in Colorectal Cancer

Published OnlineFirst April 25, 2014; DOI: 10.1158/1078-0432.CCR-13-2952

Clinical Cancer Human Cancer Biology Research

Overexpression of Sirt7 Exhibits Oncogenic Property and Serves as a Prognostic Factor in Colorectal Cancer

Hongyan Yu1, Wen Ye1, Jiangxue Wu1, Xiangqi Meng1, Ran-yi Liu1, Xiaofang Ying1, Yi Zhou1, Hui Wang1, Changchuan Pan2, and Wenlin Huang1,3

Abstract Purpose: Sirtuins play an important role in cancer development. Sirt7, as a member of this family, is frequently overexpressed in certain carcinomas, but the oncogenic mechanism is seldom reported. In this study, Sirt7 was characterized for its role in colorectal cancer aggressiveness and underlying molecular mechanisms. Experimental Design: Quantitative PCR, Western blotting, and immunohistochemistry were performed to study Sirt7 expression in a cohort of colorectal cancer tissues and non-tumor tissues and cells. A series of in vitro and in vivo assays was performed to elucidate the function of Sirt7 in colorectal cancer and its underlying mechanisms. Association between the Sirt7 signature and survival was examined using Kaplan–Meier analysis and log-rank tests. Results: The Sirt7 protein level significantly correlated with tumor stage (P ¼ 0.029), lymph node metastasis (P ¼ 0.046), and poor patient survival (P < 0.05). Sirt7 knockdown significantly inhibited colorectal cancer cell proliferation, colony formation, and motility. Ectopic Sirt7 expression promoted colony formation, induced a more invasive phenotype, and accelerated cell growth both in vitro and in vivo. Moreover, Sirt7 enhanced MAPK pathway activity concomitantly with p-ERK and p-MEK upregulation. In Sirt7-overexpressing cells, the mesenchymal markers vimentin and fibronectin were upregulated, and the epithelial markers E-cadherin and b-catenin were downregulated, which was linked to enhanced invasion by colorectal cancer cells. Conclusion: Our findings suggest that Sirt7 plays an important role in the development and progression of human colorectal cancer and functions as a valuable marker of colorectal cancer prognosis. Clin Cancer Res; 1–12. 2014 AACR.

Introduction Colorectal cancer is one of the three leading causes of cancer mortality worldwide (1). Despite the innovative fi 1 Authors' Af liations: Sun Yat-sen University Cancer Center; State Key therapeutic strategies applied in colorectal cancer, the prog- Laboratory of Oncology in South China; Collaborative Innovation Center for Cancer Medicine, Guangzhou; 2Medical Oncology, Sichuan Cancer Hos- nosis has not significantly changed in the last 20 years. In pital and Institute, Second People's Hospital of Sichuan Province, past decades, studies have reported alternative factors Chengdu; and 3CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Science, involved in colorectal cancer pathogenesis, including genet- Beijing, PR China ic mutations in certain oncogenes or tumor suppressor Note: Supplementary data for this article are available at Clinical Cancer genes (KRAS, APC, DCC, Smad-2, and Smad-4) and changes Research Online (http://clincancerres.aacrjournals.org/). in the p53, b-catenin, TGF-b, and WNT transduction pathways (2, 3). These findings suggest that colorectal carcino- Current address for X. Ying: Department of Radiotherapy, Hubei Cancer Hospital, Wuhan 430000, PR China; and current address for H. Wang: State genesis results from an accumulation of genetic alterations. Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer How these genetic changes precisely cause the clinical Institute, Shanghai Jiao Tong University School of Medicine, Shanghai characteristics observed in individual patients with colorec- 200032, PR China. tal cancer is unclear; consequently, the mechanisms under- Corresponding Authors: Wenlin Huang, State Key Laboratory of Oncol- ogy in South China, Sun Yat-sen University Cancer Center, No. 651 lying colorectal cancer development and progression Dongfeng East Road, Guangzhou 510060, PR China. Phone: 86-20- remain poorly understood. Sirtuins are homologous with 8734-3146; Fax: 862087343146; E-mail: [email protected]; and yeast sirtuin silent information regulator 2 (Sir2), which Changchuan Pan, Medical Oncology, Sichuan Cancer Hospital and Insti- tute, Second People's Hospital of Sichuan Province, Chengdu 610000, PR was originally described as a transcriptional silencing reg- China. Phone: 862885420660; Fax: 862885420660; E-mail: ulator of mating-type loci, telomeres, and ribosomal DNA [email protected] (4, 5) and extends the yeast lifespan (6). Sir2 was soon doi: 10.1158/1078-0432.CCR-13-2952 found to be an NAD-dependent histone deacetylase 2014 American Association for Cancer Research. (HDAC)(7). Sirtuins, also designated as class III histone

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study also revealed the key functions of Sirt7 in activating Translational Relevance and promoting colorectal cancer growth, epithelial–mes- Colorectal cancer is one of the three leading causes of enchymal transition (EMT), which may have implica- cancer mortality worldwide. Recurrence and metastasis tions for colorectal cancer metastasis. Thus, we propose are common lethal outcomes of colorectal cancer that Sirt7 is a promising target in colorectal cancer curative resection. Thus, a better understanding of the therapy. molecular mechanisms underlying colorectal cancer progression is conducive to the development of targeted Materials and Methods therapy. In this study, we reveal a novel role of Sirtuin7 Patients (Sirt7) in promoting colorectal cancer growth by acti- Tumorous and adjacent non-tumorous colorectal tissues vating the MAPK signal pathway and enhancing colo- were collected from 131 patients (median age, 56 years; rectal cancer invasiveness and metastasis by promoting 55.6% male) at the Cancer Center of Sun Yat-sen University epithelial–mesenchymal transition for the first time. We (Guangzhou, China) between September 1999 and Decem- also find that overexpression of Sirt7 in colorectal cancer ber 2005. The samples contained matched tumors (per- is associated with lymph node metastasis and TNM stage centage of tumor cells 70%) and corresponding normal in patients with colorectal cancer. Taken together, these mucosal tissue (>5 cm laterally from the edge of the cancer- results implicate Sirt7 as a new effective therapeutic ous region); all the patients who had a single primary lesion target for intervention of the progression of colorectal and no neoadjuvant therapy before operation were includ- cancer. ed in the study. The patients were followed-up once every 3 months during the first 2 years, once every 6 months during

þ the third and fourth year, and once a year from the fifth year deacetylases, are NAD -dependent deacetylases that target postoperatively, respectively. All patients were contacted by non-histone and histone proteins and are implicated in the phone or with questionnaire to check upon their health control of a wide range of biologic processes, including status and the last censor date was May 1, 2012. The apoptosis, stress responses, cell cycle, DNA repair, metab- patients who did not have the followed-up information olism, and senescence (8). were excluded from this study. Informed consent was The importance of sirtuins is demonstrated by their role obtained from each patient, and the study protocol was in several major human pathologies, including cancer, approved by the Ethics Committee of the Sun Yat-sen cardiovascular disease, diabetes, and neurodegeneration University Cancer Center Institutional Board. Colorectal (9). In mammals, the sirtuin family comprises 7 proteins cancer diagnosis was confirmed by histologic examina- (Sirt1–Sirt7) with considerably different functions and cat- tion. The TNM classification of our study is according to alytic activities (10). The most studied sirtuin that is most the sixth edition of AJCC/UICC; 15 patients were graded closely related to yeast Sir2 is Sirt1. Little is known about the as stage I, 59 patients as stage II, 42 patients as stage III, biologic function of the other 6 sirtuins (4, 11–13). and 15 patients as stage IV. Sirt7, which is primarily located in the nucleus, binds to the ribosomal RNA (rRNA) gene and is relevant to the Cell culture reactivation of rDNA transcription following mitosis The human colorectal cancer cell lines HT29, SW480, (14). Sirt7 participates in the activation of RNA polymer- DLD-1, SW620, and HCT116; the human embryonic kid- ase I transcription and may play a role in controlling ney cell lines 293T; and the normal colon epithelial cell line rRNA expression (15). Furthermore, certain studies have FHC were obtained from the American Type Culture Col- reported that Sirt7 is overexpressed in thyroid and breast lection. THC8307 and GP293 cells were obtained from our cancer tissues compared with normal tissues (16, 17), own laboratory collection. The colorectal cancer cell lines, suggesting that Sirt7 has an oncogenic role. In addition, the GP293 and 293T cells, were grown in DMEM, and the Sirt7 is located on chromosome 17q25.3, which is FHC cells were grown in DMEM:F12 (containing 10 ng/mL involved in various malignancies, including colorectal cholera toxin, 0.005 mg/mL insulin, 0.005 mg/mL trans- cancer (18). Barber and colleagues also found that Sirt7 ferrin, and 100 ng/mL hydrocortisone) supplemented with mRNA expression was higher in colon tumors than in 10% FBS (Invitrogen), 100 U/mL penicillin, and 100 mg/mL non-tumor tissues (19). streptomycin. All cell lines were cultured in a humidified No study has assessed Sirt7 exact activity in colorectal chamber with 5% CO2 at 37 C. The cells were tested cancer, which prompted our interest in investigating its regularly for mycoplasma (New MycoProbe Mycoplasma biologic role in human colorectal cancer. In the present Detection Kit, R&D Systems). study, to elucidate the biologic roles of Sirt7 in colorectal tumorigenesis, we evaluated Sirt7 expression in a subset Quantitative reverse transcription PCR of human colorectal cancers using immunohistochemis- Total RNA was extracted using TRIzol reagent (DSBIO) try (IHC) and analyzed its mRNA levels in 18 patients according to the manufacturer’s instructions. cDNA was with colorectal cancer. We showed that the frequent Sirt7 synthesized from 2 mg total RNA using M-MLV Reverse overexpression in primary colorectal cancer tumors cor- Transcriptase (Promega) and amplified with AmpliTaq Gold related with shorter survival in early-stage patients. Our DNA Polymerase (Applied Biosystems) and the following

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gene-specific primers for Sirt7: 50-CAGGGAGTACGTG- treated with antibody dilution solution was used as a CGGGTGT-30 (forward) and 50-TCGGTCGCCGCTTCCC- negative control. All controls yielded satisfactory results. AGTT-30 (reverse). GAPDH was used as an internal control. Sirt7 expression was evaluated using H-scores and dichot- omized according to overall survival (OS) by an ROC curve. Cell transfection The positive rate’s cut-off value was the maximized sum of siSirt7 and negative control oligonucleotides were pur- the sensitivity and specificity points (Supplementary Mate- chased from Santa Cruz Biotechnology. The coding rials and Methods). sequences of the Sirt7 and Sirt7 mutations m1 (H187!Y) The H-scores consisted of an assessment of staining and m3 (H187!Y, N249!S) were amplified and cloned intensity and the percentage of the stained area with a given into the EcoRI and XhoI sites of pcDNA3.1 to generate intensity. Only stained nuclei of malignant cells were pcDNA3.1-Sirt7–, pcDNA3.1-Sirt7 m1–, and pcDNA3.1- assessed. Sirt7m3–expressing vectors; each resulting construct was confirmed by sequencing. For transient transfections, Cell viability assay HCT116 and THC8307 cells were seeded into 24-well plates The 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazo- (at 4 104 and 3 104 cells per well, respectively) at 50% to lium bromide (MTT) assay was used to detect cell prolifer- 60% confluence. After 24 hours, the cells were transfected ation. HCT116 and THC8307 cells were plated in 96-well with oligonucleotides at a concentration of 20 nmol/L or plates at 1 103 cells per well. The absorbance of each with 0.8 mg plasmid DNA using Lipofectamine 2000 (Invi- sample was measured at 490 nm. The average of 3 repeated trogen) according to the manufacturer’s instructions. After 6 experiments was calculated. hours of incubation at 37C, the transfection medium was For the colony formation assay, HT29 cells (8 102 cells replaced with 1 mL complete medium containing 10% FBS. per 6-well plate) overexpressing Sirt7 or control GFP and The cells were collected for real-time PCR, Western blotting, HCT116 cells (5 102 cells per 6-well plate) with siRNA- migration, and invasion assays at the indicated times. downregulated Sirt7 expression or a negative control were seeded in complete medium. The cells were cultured for 14 Western blot analysis days at 37 Cin5%CO2 humidified air. Colony formation Protein lysates were resolved on 8% and 12% SDS-PAGE and growth were visualized using crystal violet staining. The gels and transferred electrophoretically onto a polyvinyli- numbers of colonies containing more than 50 cells were dene fluoride membrane. determined, and 12 fields were counted. After blocking for nonspecific binding, the blots were incubated with specific antibodies against Sirt7 (1:1,000; Migration and invasion assays Sigma-Aldrich); b-catenin, E-cadherin, vimentin, p-AKT, Transfected HCT116 and THC8307 cells (1 105 each) phospho-MEK1/2, and MEK1/2 (1:1,000; Cell Signaling were serum starved in medium overnight and then seeded Technology); Raf-1 (1:1,000; Epitomic); and p-ERK into 24-well plates with or without Matrigel-coated inserts (1:1,000), ERK (1:2,000), fibronectin (1:1,000), and (8-mm pore; BD Falcon). After 18 to 24 hours, the cells GAPDH (1:2,000; Santa Cruz Biotechnology). After incu- attached to the lower surface of the insert filter were counted bation with a horseradish peroxidase–conjugated second- after crystal violet staining. ary antibody, protein bands were visualized using enhanced chemiluminescence detection (Perkin Elmer). Overexpression retrovirus packaging and transduction Immunohistochemical assay Sirt7 and control sequences were amplified from human Sirt7 expression in primary tumors and adjacent noncan- gDNA and cloned into the BglII and SalI sites of the cerous colorectal mucosa was examined using IHC. Paraf- retroviral vector pLNCX2Virus (Clontech). Packaging was fin-embedded blocks containing adjacent noncancerous performed in GP293 cells cultured in DMEM with 10% FBS colorectal mucosa and more than 70% primary tumor tissue in a 37 C incubator with 5% CO2. Forty-eight hours after were selected for immunohistochemical staining. Paraffin transfection, the supernatant was harvested and cleared by sections were cut to a thickness of 4 mm and mounted on centrifugation at 1,000 g for 10 minutes. HT29 cells were silanized slides and the slides were preserved at 4C. The then transduced with the retrovirus containing Sirt7 or paraffin sections were dewaxed, rehydrated, and blocked control plasmids. Forty-eight hours after infection, 1000 with 0.3% H2O2. Tissue antigens were retrieved with a mg/mL G418 was added to the medium for 2 weeks to select microwave oven at 95C for 25 minutes and cooled to retrovirus-infected cells. Western blotting was performed to room temperature in 10 mmol/L sodium citrate buffer (pH evaluate Sirt7 expression in the 2 stable cell lines. 6.0). Each slide was washed with PBS and incubated overnight at 4C with Sirt7 (1:2,000, clone ABE103, Millipore). Sirt7 shRNA retrovirus packaging and transduction Primary antibodies were diluted with background-reducing Virus packaging was performed in 293T cells. p-SUPER- components (S2022, Dako). Secondary antibody was Sirt7 shRNAs or p-SUPER-control and the p-SUPER PIK applied using the Envision Detection Kit (Dako). Slides Expression Packaging plasmids (OligoEngine) were were stained for 2 minutes with 3,30-diaminobenzidine cotransfected with the transfection reagent according to the (DAB) and then counterstained with hematoxylin. Tissue manufacturer’s instructions. Viral particles were harvested

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48 hours after transfection. HCT116 cells were transduced or anti-mouse IgG. The coverslips were counterstained with with the retrovirus containing Sirt7 shRNA or control 40,6-diamidino-2-phenylindole (DAPI) and imaged with a shRNA. Forty-eight hours after infection, 1 mg/mL puromy- confocal laser scanning microscope (Olympus FV1000). cin was added to the medium for 2 weeks to select retro- Data were processed with Adobe Photoshop 7.0 software. virus-infected cells. Western blotting was performed to detect Sirt7 expression in the 2 stable cell lines as described In vivo proliferation assays above. Female athymic 4- to 5-week-old BALB/c nude mice were purchased from the Medical Experimental Animal Center of PD98059 treatment Guangdong (PR China). All animal studies were conducted Cells were grown in medium containing different in accordance with the NIH animal use guidelines and PD98059 concentrations (Sigma-Aldrich) for the indicated current Chinese regulations and standards for laboratory times. Cellular proteins were then extracted and subjected animal use. to different analyses. To determine the impact of Sirt7 on colorectal cancer cell line proliferation in vivo,1 106 cells were injected sub- Immunofluorescence analysis cutaneously into nude mice (HCT116, n ¼ 9; HT29, n ¼ 7), Cells grown on glass coverslips were fixed in 4% para- and tumor progression was studied over time. Four weeks formaldehyde, permeabilized using 0.5% Triton X-100, and post-implantation, the mice were sacrificed, and the tumors incubated with primary antibody against E-cadherin (1:50) were removed. The tumor volume was evaluated using the or vimentin (1:100). The cells were then incubated with following formula: tumor volume ¼ 4p/3 (width/2)2 rhodamine-conjugated or FITC-conjugated goat anti-rabbit (length/2).

Figure 1. Sirt7 expression in colorectal cell lines and tissues and its prognostic value in patients with colorectal cancers. A, the mRNA expression levels of Sirt7 were examined using real-time PCR analysis of 18 paired colorectal cancer and normal mucosal tissues. Alterations in expression are shown as scatter diagrams, with the y-axis indicating Sirt7 expression. The mean level of Sirt7 expression in colorectal cancer tissues was signiﬁcantly higher than that in non- tumor tissues. GAPDH was used as a control (P ¼ 0.019, independent t test). B, Sirt7 protein expression levels in FHC cells and 5 colorectal cancer cell lines were examined using Western blotting. C, representative immunohistochemical images showing the normal expression of Sirt7 in colorectal cancer adjacent non-tumor tissue (left) and the overexpression of Sirt7 in colorectal cancer tissue (right). D, the protein expression levels of Sirt7 were examined by IHC in 105 paired colorectal cancer and adjacent noncancerous tissues (P < 0.0001, independent t test). E and F, Kaplan–Meier survival analysis according to Sirt7 expression in patients (stages I and II) with colorectal cancer (log-rank test). The probability of OS (74) and disease-free survival (73) for patients is shown.

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In vivo metastasis assays abdominal cavity was closed with staples. After 6 weeks, the We chose the HCT116 and HT29 cell lines for the lung mice were euthanized, and the spleens and livers were and hepatic metastasis models, respectively, based on pre- removed for pathologic examination. Transverse sections liminary experiments. (5 mm) of the liver were prepared at 10 different levels to cover the entire liver, and the sections were stained with Hepatic metastasis model hematoxylin and eosin (H&E). Metastatic nodules were Mice were randomly assigned to 2 groups (8 per group). counted in a double-blind manner under a microscope. Both groups were anesthetized with isoﬂurane and subjected to laparotomy. The spleen was removed through a 1- Lung metastasis model cm incision in the upper left lateral abdomen, and 1 106 Tumor formation and Sirt7-overexpressing and control cells (NC-HCT116 or siSirt7-HCT116) in 20 mL PBS were cancer cell metastasis were examined following intravenous injected into the spleen’s distal tip using an insulin syringe. injection into the tail veins of nude mice. A modiﬁed The spleen was then replaced in the abdomen, and the transplantation through subcutaneous injection was

Table 1. Clinicopathologic ﬁndings and correlation with Sirt7 expression

Variable n (%) Sirt7-low (%) Sirt7-high (%) P Total cases 131 27 (20.6) 104 (79.4) Age, y 65 96 (73.3) 21 (21.9) 75 (78.1) 0.633 >65 35 (26.7) 6 (17.1) 29 (82.9) Gender Male 74 (56.5) 15 (20.3) 59 (79.7) 1.000 Female 57 (43.5) 12 (21.1) 45 (78.9) Tumor location Colon 71 (54.2) 15 (21.1) 56 (78.9) 1.000 Rectum 60 (45.8) 12 (20.0) 48 (80.0) Tumor size,a cm 5 69 (53.1) 12 (17.4) 57 (82.6) 0.388 >5 61 (46.9) 15 (24.6) 46 (75.4) Histology Adenocarcinoma 117 (89.3) 23 (19.7) 94 (80.3) 0.486 Mucinous 14 (10.7) 4 (28.6) 10 (71.4) Tumor invasive depth T1–T2 18 (13.7) 4 (22.2) 14 (77.8) 1.000 T3–T4 113 (86.3) 23 (20.4) 90 (79.6) Lymph node status N0 (n ¼ 0) 80 (61.1) 13 (16.3) 67 (83.8) 0.046c N1 (n 3) 35 (26.7) 7 (20.0) 28 (80.0) N2 (n > 3) 16 (12.2) 7 (43.8) 9 (56.3) Preoperative CEA,b ng/mL <5 62 (56.4) 12 (19.4) 50 (80.6) 0.288 5 48 (43.6) 5 (10.4) 43 (89.6) AJCC/TNM stage I—II 74 (56.5) 10 (13.5) 64 (86.5) 0.029c III—IV 57 (43.5) 17 (29.8) 40 (70.2) Preoperative CA199,b ng/mL 35 90 (81.8) 14 (15.6) 76 (84.4) 1.000 >35 20 (18.2) 3 (15.0) 17 (85.0)

NOTE: The numbers in parentheses indicate the percentages of tumors with a speciﬁc clinical or pathologic feature for a given Sirt7 subtype. Abbreviation: CA199, carbohydrate antigen 19–9. aAnalysis of this parameter was available for 130 cases. bAnalysis of this parameter was available for 110 cases. cStatistically signiﬁcant.

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reported by Quintana and colleagues (20). Briefly, mice the ROC curve). Multivariate survival analysis was per- were injected with cells (106 per mouse) and sacrificed 6 formed for all parameters that were significant in univariate weeks after injection. The lungs were then removed for analyses using the Cox regression model. The data were pathologic examination. Transverse sections (5 mm) of assessed with Student 2-tailed t test. P < 0.05 was considered whole lungs were prepared and stained with H&E. Meta- statistically significant. static nodules were counted as described previously. Results Statistical analysis Sirt7 overexpression is frequently detected in Statistical analysis was performed using SPSS standard colorectal cancer tissues and cell lines version 16.0 (SPSS Inc.). Significant associations between Sirt7 expression was first examined using real-time PCR in Sirt7 expression and clinicopathologic parameters were 18 pairs of colorectal cancers and adjacent non-tumor assessed using the c2 test. Kaplan–Meier and log-rank tests colorectal tissues and using Western blotting in 6 colorectal were used to compare patient survival and to create survival cell lines. Sirt7 mRNA was overexpressed in 15 of 18 pairs curves based on high and low Sirt7 IHC scores (as defined by (83.3%, Fig. 1A), and all 5 colorectal cancer cell lines (i.e.,

Figure 2. The effect of Sirt7 on colony formation and the cell motility of colorectal cancer cell lines. A, Western blotting revealed that Sirt7 was efﬁciently knocked down by siSirt7 treatment and overexpressed by treatment with pcDAN3.1-Sirt7/pLCNX2-Sirt7. B, MTT assays on HCT116 cells 48 and 72 hours after transfection with siSirt7 or negative control siRNA (left) and the Sirt7-expressing and control stable HT29 cell lines 4 days after implantation (right). The data are the means of 3 independent experiments. Bars, SE. , P ¼ 0.006. C, the effect of Sirt7 on colorectal cancer cell line colony formation. In total, 500 siSirt7-infected HCT116 or 800 Sirt7-infected HT29 cells were plated, and a colony formation assay was conducted. Representative results of the colony formation assay with NC-HCT116 and siSirt7-HCT116, Sirt7-HT29, and ctrl-HT29 cells are presented. D, the invasion and migration assays showed different cell motilities in siSirt7-HCT116, NC-HCT116, ctrl-THC8307, and Sirt7-THC8307 cells. The ectopic expression of siSirt7 clearly inhibited the invasion and migration of HCT116 cells. In addition, the ectopic expression of Sirt7 had a greater promoting effect on the cell invasion and migration of THC8307 cells. Error bars indicate SE. , P < 0.05; , P < 0.01 by Student t test. All the results were reproducible in 3 independent experiments.

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HCT116, DLD1, THC8307, HT29, and SW620) demon- Sirt7 overexpression is associated with decreased strated higher Sirt7 expression than the normal colorectal survival in colorectal cancer cell line FHC (Fig. 1B). We then determined the association between Sirt7 expres- To examine Sirt7 protein expression, IHC was performed. sion and the clinicopathologic characteristics of 131 colo- Sirt7 was upregulated in tumors compared with paired rectal cancer tumor tissue samples with the associated adjacent non-tumor tissues (Fig. 1C and D; Supplementary clinical details (Table 1). Regression–correlation analysis Fig. S1). showed that Sirt7 overexpression was signiﬁcantly

Figure 3. The effect of Sirt7 on colorectal cancer cell growth and metastasis in vivo. A, mice were injected subcutaneously with cells (106 per mouse). Tumor volumes were measured as described in Materials and Methods and plotted as the mean SEM. Left, tumor growth in nude mice injected with 1 of the 2 groups of cells (NC-HCT116 and siSirt7-HCT116). B, lung metastasis model. Left, representative lungs were subjected to the indicated treatments. The metastatic nodules are indicated with arrows. Middle, representative results of H&E staining of the metastatic nodules in the lung. The metastatic nodules are indicated by arrows. Right, the numbers of metastatic nodules in the lungs of mice. The nodules were examined under a microscope. n ¼ 8. , P < 0.05. C, hepatic metastasis model. Left, representative livers were subjected to the indicated treatments. The metastatic nodules are indicated by arrows. Middle, representative results of H&E staining of metastatic nodules in the liver. The metastatic nodules are indicated by arrows. Right, the numbers of metastatic nodules in the livers of mice. The nodules were examined under a microscope. n ¼ 8. , P < 0.05.

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associated with both the TNM stage (P ¼ 0.029) and the on proliferation in the control group, whereas Sirt7 over- lymph node status (P ¼ 0.046). expression promoted proliferation in stable HT29 cell lines Kaplan–Meier survival analysis showed that patients at (Fig. 2B). The clonogenic assay showed that Sirt7 knock- TNM stages I and II with high Sirt7 expression had decreased down significantly reduced the number of HCT116 colonies OS (P ¼ 0.006, log-rank test; Fig. 1E) and disease-free formed after 14 days of culture compared with the controls survival (P ¼ 0.011, log-rank test; Fig. 1F). High Sirt7 (P < 0.0001), whereas Sirt7 upregulation increased the expression tended to be associated with decreased OS in number of HT29 colonies compared with the controls a multivariate Cox proportional hazards model adjusted for (Fig. 2C). age, carcinoembryonic antigen (CEA), and sex [HR, 6.358; The effects of Sirt7 on cell invasion were observed using 95% confidence interval (CI), 1.37–29.51; (Supplementary Transwell and Matrigel invasion assays. Sirt7 depletion in Tables S1 and S2]. However, Kaplan–Meier analysis showed both the HCT116 and the SW620 lines significantly reduced no significant association between stages I–IV regarding OS cell invasion and migration (Fig. 2D; Supplementary or disease-free survival and Sirt7 expression (P > 0.05, data Fig. S2). Supporting the role of Sirt7 in promoting cell not shown). motility, ectopic expression of pcDNA3.1-Sirt7 in THC8307 cells increased invasion and migration by >50% (P ¼ Sirt7 promotes colorectal cancer cell line growth, 0.017; Fig. 2D). migration, and invasion in vitro The above observations prompted us to explore the Sirt7 promotes colorectal cancer growth and potential biologic function of Sirt7 in colorectal cancer metastasis in vivo tumorigenesis and/or progression. First, we investigated the To investigate the role of Sirt7 in vivo, we constructed a effects of Sirt7 manipulation (by gain-of-function and loss- retroviral vector to mediate Sirt7 expression and established of-function) on cancer cell proliferation. We knocked down 4 stable cell lines named NC-HCT116, shSirt7-HCT116, Sirt7 in the HCT116 cell line, which has high endogenous ctrl-HT29, and Sirt7-HT29. These 4 cell lines were injected expression, and evaluated the impact of Sirt7 on cellular subcutaneously into the flanks of nude mice. The volume proliferation using MTT and clonogenic assays. Sirt7 knock- and weight of the tumors resulting from shSirt7-HCT116 down inhibited proliferation in transiently transfected cell injection were significantly lower than those from NC- HCT116 cells. Sirt7 knockdown had a greater negative effect HCT116 cell injection; conversely, the tumors resulting

Figure 4. Overexpression of Sirt7 promotes colorectal cancer cell growth through ERK1/2 activation. A, forty-eight-hour siRNA/negative control (NC) knockdown of Sirt7 and 48-hour overexpression of Sirt7/empty vector (ctrl). p-AKT, ERK, p-ERK, and cyclin D1 expression was analyzed by Western blotting and quantiﬁed. The graphs show data expressed as fold over NC or ctrl. GAPDH served as the loading control. B, ERK inhibition by PD98059 abrogated the growth advantage of Sirt7-overexpressing cells (mean SEM; n ¼ 3; , P < 0.001). C, expression of the Sirt7 mutants failed to induce ERK1/2.

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Sirt7 Promotes Colorectal Cancer Tumorigenesis

from Sirt7-HT29 cell injection were larger and heavier than addition, forced overexpression of mutant Sirt7 (m1 and those from ctrl-HT29 cell injection (Fig. 3A; Supplementary m3) did not induce ERK1/2 or p-ERK1/2 (Fig. 4C). To Fig. S3A). confirm that the effects were directly dependent on Sirt7 We next evaluated the in vivo effects of Sirt7 on metastasis expression, we treated Sirt7-overexpressing cells with plas- using 2 experimental metastasis assays. The ctrl-HT29 and mid-resistant versions of Sirt7 siRNA or negative control Sirt7-HT29 stable cell lines were implanted into the tail siRNA (Supplementary Fig. S4A). The viability increase veins of nude mice, and the number and size of metastatic induced by Sirt7 was reduced by the expression of Sirt7 tumor nodules in the lungs were examined by microscopy. siRNA (Supplementary Fig. S4B). This approach demon- The number of pulmonary metastatic nodules was clearly strated that Sirt7 directly induces proliferation. more in the Sirt7-HT29 group than in the ctrl-HT29 group The observation that Sirt7 induces ERK1/2 activation led (P ¼ 0.045; Fig. 3B). More interestingly, tumors from 5 mice us to evaluate the RAS–RAF–MEK–ERK pathway upon Sirt7 also metastasized to the skin in the Sirt7-HT29 group, but overexpression. We observed robust RAF–MEK–ERK path- not in the ctrl-HT29 group (Supplementary Fig. S3B). When way activation upon Sirt7 overexpression, whereas Sirt7 shSirt7-HCT116 and NC-HCT116 cells were injected into knockdown inhibited the RAF–MEK–ERK pathway the spleens of nude mice, most mice in the NC-HCT116 (Fig. 5A and B). group developed liver metastases, in contrast to the siSirt7- Immunohistochemical staining of tumor nodules origi- HCT116 group (Fig. 3C). Collectively, these findings are nating from the in vivo demonstrated that Sirt7-HT29 cells consistent with the in vitro results, indicating that Sirt7 had increased p-ERK1/2 level compared with that from ctrl- promotes colorectal cancer growth and metastasis in vivo. HT29 cells, whereas p-ERK1/2 level was downregulated in shSirt7-HCT116 group compared with the NC-HCT116 Sirt7 induces ERK1/2 phosphorylation and activates group (Fig. 5C). the Raf–MEK–ERK pathway Several reports have shown that the MAPK–ERK signaling Sirt7 promotes colorectal cancer cell motility through pathway enhances epithelial cancer cell growth, particularly EMT induction in lung and colon cancers (21). Therefore, we examined Given that upregulated Sirt7 correlated with enhanced whether Sirt7 expression influences ERK1/2 activation. Sirt7 colorectal cancer cell migration, we examined whether EMT overexpression was associated with increased phosphory- is an underlying mechanism. lated ERK1/2 and cyclin D1 levels (Fig. 4A). In contrast, In Sirt7 siRNA–treated cells, Western blotting revealed Sirt7 siRNA decreased p-ERK1/2 and cyclin D1 levels, but upregulation of the cohesive epithelial markers E-cadherin AKT and p-AKT levels were not significantly altered. and b-catenin and a corresponding downregulation of the Next, we postulated that if Sirt7 affects proliferation mesenchymal markers fibronectin and vimentin. In Sirt7- by activating MEK–ERK, MEK–ERK inhibition should abro- overexpressing cells, fibronectin and vimentin expression gate Sirt7-induced growth. Indeed, PD98059 treatment was elevated compared with expression in vector-trans- completely abrogated Sirt7-induced growth (Fig. 4B). In fected cells, whereas E-cadherin and b-catenin were

Figure 5. Sirt7 expression activates the Raf–MEK–ERK1/2 pathway. A and B, Western blot analyses show that C-Raf and p-MEK activation was observed in HCT116 and HT29 cells after Sirt7 knockdown or overexpressed. C, IHC showed decreased expression of p-ERK in tumor tissues originating from siSirt7-HCT116 cells compared with tissues originating from NC- HCT116 cells and increased expression of p-ERK in tumor tissues originating from Sirt7-HT29 cells compared with tissues originating from ctrl-HT29 cells. Scale bar, 100 mm.

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downregulated (Fig. 6A). In addition, the involvement of patients with early-stage colorectal cancers. In functional EMT was further supported by immunofluorescence (Fig. studies, Sirt7 reintroduction dramatically promoted colo- 6C). rectal cancer cell colony formation, migration, and invasion In our SCID mouse metastasis model, further immuno- in vitro and tumor metastasis in vivo. Thus, Sirt7 plays a histochemical staining demonstrated that the metastatic critical role in colorectal cancer growth, invasive, and/or tumor nodules originating from shSirt7-HCT116 cells had metastatic potential, but the detailed mechanisms remain increased the expression of E-cadherin and b-catenin, com- unclear. pared with that from NC-HCT116 cells (Fig. 6B). MEK and ERK are central to cell growth because they increase the synthesis of pyrimidine nucleotides and Discussion increase the activity of transcription factors that enhance Sirtuins have received significant attention since the gene expression and cyclin D1 activity (22–26). We postu- discovery of yeast Sir2, described as a transcriptional silenc- lated that Sirt7 was important to the subsequent activation er of mating-type loci, telomeres, and ribosomal DNA (4, of this pathway. In overexpression and knockdown assays, 5). Sirt7 overexpression has been detected in several malig- Sirt7 enhanced proliferation, ERK activation, and cyclin D1 nancies, including thyroid, breast, and hepatocellular can- levels. These findings suggest that ERK activation is respon- cers (16, 17). A recent breakthrough showed that Sirt7 sible for the proliferative influence and downstream effects maintains human cancer cell oncogenic transformation by on cell growth of Sirt7. Beyond ERK, MEK directs growth deacetylating lysine 18 of histone H3 (19). However, no factor and G-protein–coupled receptor signals to their detailed analysis of the biologic roles of Sirt7 in colorectal intracellular targets, which regulate cellular processes, cancer has been conducted. In our study, Sirt7 upregulation including proliferation, differentiation, cell morphology, was frequently observed in colorectal cancer tissues. Sirt7 and oncogenesis. MEK1/2 activation in mitogen-stimulated overexpression was a strong independent predictor of OS in cells is directly mediated by MAPKs, including Raf-1 kinase,

Figure 6. Sirt7 promotes colorectal cancer motility through EMT. A, Western blot analyses showed a significant increase in the expression of the epithelial markers b-catenin and E-cadherin and a corresponding decrease in the levels of the mesenchymal markers fibronectin and vimentin in Sirt7- depleted cells relative to siSirt7- treated cells (HCT116). Ectopic expression of Sirt7 suppressed the expression of b-catenin and increased fibronectin and vimentin levels (HT29 and THC8307). The endogenous levels of vimentin and E-cadherin were too low in HCT116 and THC8307 cells, respectively, to achieve confident interpretation. B, IHC showed increased expression of E-cadherin and b-catenin in tumor tissues originating from siSirt7-HCT116 cells compared with tissues originating from NC-HCT116 cells. Scale bar, 50 mm. C, immunofluorescence was used to compare the expression levels/patterns of epithelial and mesenchymal markers in ctrl/NC and Sirt7/siSirt7 cells. Epithelial markers (red signal) were downregulated in Sirt7- overexpressing cells, whereas mesenchymal markers (green signal) were upregulated.

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Sirt7 Promotes Colorectal Cancer Tumorigenesis

which phosphorylates 2 serine residues (S218, S222) in the mal markers fibronectin and vimentin. The ERK signaling activation loop of MEK. In turn, MEK activates ERK1/2 (26). pathway has been shown to upregulate Snail and slug, Indeed, Sirt7 overexpression in colorectal cancer cells sig- leading to the downregulation of E-cadherin (29, 33). To nificantly increased MEK1/2 phosphorylation and Raf-1 test whether Sirt7 promotes EMT through the MAPK levels, whereas Sirt7 knockdown had the opposite effect. pathway, we used the ERK inhibitor PD98059 in migra- MEK activation is the seminal signaling event that activates tion assays. PD98059 dose independently inhibited the ERK and causes cell proliferation. Taken together, these Sirt7 overexpression-induced migration of colorectal can- findings demonstrate that Sirt7 is an integral mediator of cer cells (Supplementary Fig. S5). Although ERK activa- cell proliferation via MEK–ERK MAPK pathway activation, tion occurs, it is not the only mechanism responsible for which is known to be dysregulated in many cancers (27). EMT induction, the exact mechanisms remain unclear. We failed to identify the proteins directly activated by Sirt7 In summary, we investigated Sirt7 expression in human in the MAPK pathway; thus, further research using co- colorectal cancers and found that Sirt7 overexpression may immunoprecipitation and direct binding assays will be be important in the aggressive phenotype of colorectal necessary. cancers. Furthermore, a functional analysis showed that Patients with colorectal cancers frequently develop Sirt7 plays a critical role in colorectal cancer cell prolifer- lymph node metastases during the early stages of disease. ation and metastasis by regulating MAPK signaling and At the advanced stages, most patients also develop liver, EMT. Thus, our data suggest that Sirt7 could be used as a lung, or peritoneal metastases (28). Therefore, we used 2 new prognostic marker and/or an effective therapeutic models (hepatic and lung metastasis) to simulate colorectal target in colorectal cancers. cancer metastasis (29). Sirt7 was a significant metastasis facilitator in human colorectal cancers. Surprisingly, Sirt7 Disclosure of Potential Conflicts of Interest overexpression promoted HT29 metastasis not only to the No potential conflicts of interest were disclosed lungs but also to the skin. We hypothesized that Sirt7 promoted the colorectal cancer cells primarily via the lymph Authors' Contributions nodes metastasis to the skin. All of the authors planned and implemented the investigation. We found that Sirt7 was a prognostic factor only in early- Conception and design: H. Yu, J. Wu, C. Pan, W. Huang stage colorectal cancers and that Sirt7 has the lowest expres- Development of methodology: H. Yu, R.-y. Liu, Y. Zhou Acquisition of data (provided animals, acquired and managed patients, sion in stage VI. It is possible that Sirt7 plays an oncogenic provided facilities, etc.): H. Yu, W. Ye, J. Wu, Y. Zhou, W. Huang role in early colorectal cancer development and that during Analysis and interpretation of data (e.g., statistical analysis, biosta- tistics, computational analysis): H. Yu, J. Wu, R.-y. Liu, X. Ying, Y. Zhou, H. the advanced stage, Sirt7 is corrupted by other proteins, Wang although the pathways and molecules downstream of Sirt7 Writing, review, and or revision of the manuscript: H. Yu, J. Wu, X. Meng, are already activated, further promoting colorectal cancer H. Wang, C. Pan, W. Huang Administrative, technical, or material support (i.e., reporting or orga- development. nizing data, constructing databases): H. Yu, J. Wu, Y. Zhou, W. Huang The two most important hallmarks of malignant Study supervision: J. Wu, Y. Zhou, W. Huang tumors, invasion and metastasis, are most associated with Principal investigator, managed the research fund, designed project, organized experimental materials: W. Huang fatality in human cancer. Therefore, identifying the factors involved in invasion and/or metastasis and understanding the underlying molecular mechanisms involved in Acknowledgments The authors thank Jiemin Chen and Ling Zhou (Sun Yat-sen University) tumor progression are critical (30). Accumulating evi- for their technical assistance. dence indicates that EMT mediates tumor progression, including local invasion, dissemination from the primary tumor, intravasation into the circulation, and metastasis Grant Support This work was supported by grants from the National High Technology (31, 32). EMT is a complex process requiring extensive Research and Development Program of China (project 863, No. changes in cell adhesion and morphology and signaling 2012AA02A204, 2012AA020803) and the National Basic Research Program pathway activation, and our findings suggest that Sirt7 of China (973 Program, No.2011CB504805, 2010CB912201). The costs of publication of this article were defrayed in part by the represents an upstream molecule that induces this tran- payment of page charges. This article must therefore be hereby marked sition. We found that Sirt7 reintroduction into colorectal advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate cancer cells promoted EMT, as shown by the decreased this fact. expression of the epithelial markers E-cadherin and Received October 26, 2013; revised March 5, 2014; accepted April 5, 2014; b-catenin and the enhanced expression of the mesenchy- published OnlineFirst April 25, 2014.

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Overexpression of Sirt7 Exhibits Oncogenic Property and Serves as a Prognostic Factor in Colorectal Cancer

Hongyan Yu, Wen Ye, Jiangxue Wu, et al.

Clin Cancer Res Published OnlineFirst April 25, 2014.

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