Sirt6-mediated cell death associated with Sirt1 suppression in gastric cancer

doi:10.21203/rs.3.rs-3346699/v1

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Sirt6-mediated cell death associated with Sirt1 suppression in gastric cancer

https://doi.org/10.21203/rs.3.rs-3346699/v1

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Background

Gastric cancer, one of the leading causes of cancer-related death, is strongly associated with H. pylori infection, although other risk factors have been identified. The sirtuin (Sirt) family is involved in the tumorigenesis of gastric cancer, and sirtuins can have pro-tumorigenic or anti-tumorigenic effects.

Methods

After determining the overall survival rate of gastric cancer patients with or without Sirt6 expression was determined, the effect of Sirt6 upregulation was also tested using an in vivo xenograft mouse model. The regulation of Sirt6 and Sirt1, leading to the induction of MDM2 and reactive oxygen species (ROS), was mainly analyzed with western blot and immunofluorescence staining, and gastric cancer cell (SNU-638) death associated with these proteins were measured using flow cytometric analysis.

Results

Sirt6 overexpression lead to Sirt1 suppression of gastric cancer cell, resulted in a higher level of gastric cancer cell death in vitro and a reduced tumor volume in vivo. The ROS and MDM2 expression levels were upregulated by Sirt6 overexpression and/or Sirt1 suppression on western blot analysis. The upregulated ROS ultimately led to gastric cancer cell death on western blot and flow cytometric analysis.

Conclusion

We found that upregulation of Sirt6 suppressed Sirt1, and Sirt6- and Sirt1-induced gastric cancer cell death was mediated by ROS production. These findings highlight the potential of Sirt6 and Sirt1 as therapeutic targets in gastric cancer.

Sirtuins

Sirtuin1

Sirtuin6

stomach neoplasm

Sirtuins are NAD + dependent signaling proteins that regulate various metabolic processes such as energy metabolism, inflammation, and apoptosis in a wide range of organisms from yeast to mammals [1]. They play essential roles in ageing-related pathological conditions including diabetes, cardiovascular diseases, neurodegenerative diseases, and cancer [2]. Sirtuins play a dual role in cancer, and some sirtuins suppress tumorigenesis whereas others promote it [3, 4]. Sirtuins have anti-tumorigenic activity because they inhibit senescence and allow unchecked cell division [4, 5]. However, sirtuin family members can protect DNA from unfavorable environmental conditions such as oxidative stress, maintain genomic stability, and regulate replicative lifespan as pro-tumorigenic proteins [4, 5]. Although the role of sirtuins in tumorigenesis have been studied extensively, the underlying mechanisms are complex [3, 4].

Among seven mammalian sirtuin family proteins (Sirt1-Sirt7), Sirt6 is the most studied tumor suppressor protein [3]. The anti-tumorigenic activity of Sirt6 is mediated by various mechanisms, and interaction between Sirt6 and Sirt1 may underlie its role in malignant transformation [6, 7]. Sirt1 has a dual function as a pro-tumorigenic and anti-tumorigenic factor according to the environment and cell type [8, 9, 10]. Sirt1 is overexpressed in many solid tumors, and its dual effect has been demonstrated, especially in gastric cancer (Table 1).

The anti-tumorigenic effect of Sirt6 associated with Sirt1 suppression was previously reported in several malignancies but not in gastric cancer [6]. Sirt6 and Sirt1 are involved in signaling networks such as those mediated by the Forkhead-box transcription factor family, nuclear factor kappa B (NF-κB), and the tumor suppressor p53, as well as in the generation of ROS [6]. ROS production is increased by Sirt6-mediated Sirt1 suppression through the regulation of MDM2, and the relation between Sirt6 and Sirt1 under oxidative stress conditions is essential for carcinogenesis [11]. In this study, we examined the anti-tumorigenic effect of Sirt6 on inducing ROS-related gastric cancer cell death by suppressing Sirt1 expression.

Patient samples and data collection

The study was approved by the Institutional Review Board of the Gyeongsang national university hospital. Tissue samples were obtained from patients with stage I–IV gastric cancer who had been diagnosed at Gyeongsang national university hospital between 2001 and 2010. For immunohistochemical (IHC) studies, tissue microarrays were incubated overnight at 4°C with anti-Sirt6 antibodies (Abcam, Cambridge, UK). Sirt6 gene expression and clinical data was obtained from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/), specifically datasets GSE29272, and The Cancer Genome Atlas (TCGA) dataset. These data were used to compare Sirt6 gene expression between normal gastric tissue and gastric cancer samples, and the association between Sirt6 expression and survival rates was evaluated.

Cell lines and chemicals

The gastric cancer cell line SNU-638 and human primary stomach epithelial cells (HPSECs) were obtained from the Korean Cell Line Bank (Seoul, Korea) and Cell Biologics, Inc. (Chicago, IL, USA). Cells were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (GenDEPOT) and 1% penicillin/streptomycin (Thermo Fisher Scientific); HPSECs were cultured at 37°C with 5% CO2 in a humidified incubator using Epithelial Cell Medium /w Kit (H6621). The chemicals used in this study were nutlin-3 (MDM2 inhibitor, Sigma-Aldrich (St. Louis, MO, USA).,) and MG-132 (Calbiochem, EMD Millipore Corp. Billerica, MA USA).

Western blot analysis

Total proteins were extracted from SNU-638 cells and HPSECs using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (GenDEPOT). Cells were sonicated for 2 min and centrifuged at 14,000 g for 10 min at 4°C to remove insoluble cell debris. Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA). Total protein lysates (30 µg) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with 5% BSA, membranes were incubated with primary antibodies against Sirt1, α-tubulin, lamin a/c (Santa Cruz, CA, USA, MDM2 (Cell Signaling Technology, Inc.), and Sirt6 (Abcam). The blots were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Thermo Fisher Scientific). Signals were visualized using the Clarity Western blot ECL Substrate (Bio-Rad, Hercules, CA, USA). and imaged using the ChemiDoc Touch Imaging System (Bio-Rad).

Flow cytometric DNA analysis

Collected cells were washed twice with cold PBS, fixed with 70% ethanol for 1 h at 4°C, treated with 1 mg/ml RNase A (Sigma-Aldrich, St. Louis, MO, USA), and then stained with 50 µg/ml PI (Sigma-Aldrich). Data were acquired on a Cytomics FC500 Flow Cytometer equipped with two laser sources (Beckman-Coulter). Results were analyzed using CXP Software (Beckman-Coulter).

ROS measurement

Intracellular generation of ROS was measured using 2’,7’-dichlorodihydrofluorescene diacetate (DCF-DA; Molecular Probes, Eugene, OR, USA). Cells were stained with 5 µM DCF-DA in serum-free medium for 15 min and removed from the plate with trypLE-Express (Gibco). Fluorescence intensity was measured on a Cytomics FC500 flow cytometry (Beckman-Coulter) with an excitation wavelength of 480 nm and an emission wavelength of 525 nm. Data were analyzed using CXP Software (Beckman-Coulter).

IHC analysis of Sirt6 and evaluation of IHC reactions

Formalin-fixed, paraffin-embedded tissue samples from nude mice injected with SNU-638 cells were subjected to IHC. Tissue blocks were cut into 5 µm slices, which were de-paraffinized and rehydrated. The slides were incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, and then heated for 20 min in 10 mM citrate buffer (pH 6.0) in a microwave oven (700 W). The sections were incubated overnight at 4°C with anti-Sirt6 antibodies (Abcam).

ICC-Immunofluorescence (IF) for Sirt1 and Sirt6 double staining

HPSECs and SNU-638 cells were seeded on coverslips and treated with Ad-Lac Z or Ad-Sirt6 for 72 h. The cells were fixed using paraformaldehyde (3.7%) for 15 min at RT, washed with 1 × PBS + 0.1 M glycine, and permeabilized with 1 × PBS + 0.2% Triton-X. Cells were then blocked with 1% BSA for 30 min at RT and stained with primary antibodies (Ad-Sirt1, Ad-Sirt6; Abcam) and the corresponding secondary antibodies. Nuclei were stained with DAPI. Stained cells were analyzed using a laser scanning confocal microscope (Olympus).

Immunoprecipitation (IP) assay

The interaction between proteins was examined by immunoprecipitation (IP) assays. Cell lysates were first incubated with Dynabeads with Sirt6 (1:50) antibody for 3 h at 4°C. The immunocomplexes were washed three times with 1× wash buffer, extracted with elution buffer, and analyzed by western blotting using antibodies against Sirt1 and Sirt6.

Preparation of adenovirus

Adenovirus encoding Sirt6 (Ad-Sirt6) was created using the ViraPower adenovirus expression system (Invitrogen by Thermo Fisher Scientific, USA). Briefly, cDNA encoding Sirt6 was subcloned into the pENTR vector. After sequence verification, the Sirt6 cDNA was transferred to the pAd/CMV/V5-DEST vector using the Gateway system with LR Clonase (Invitrogen). The verified clone (Ad-Sirt6) was linearized using PacI (New England Biolab), and then transfected into 293A cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Virus was prepared and amplified with the ViraPower adenoviral expression system (Invitrogen), and viral titers were determined by plaque-forming assay after serial dilution. Aliquots of viral suspension were used to infect SNU-638 cell lines. Recombinant replication-defective adenovirus encoding green fluorescent protein (Ad-GFP) or β-galactosidase (Ad-LacZ) was used as a control.

Short hairpin RNA (shRNA)-mediated silencing of Sirt6 and MDM2

For shRNA-mediated depletion of Sirt6 and MDM2, glycerol stocks of bacteria containing Sirt6- or MDM2-targeting shRNA plasmid DNA (MISSION shRNA), as well as a non-targeting control plasmid DNA (SHC002) were purchased from Sigma-Aldrich. Lentiviral particles were used to deliver and express shRNAs to knock down human Sirt6 and MDM2, and a scrambled shRNA was used as a control. Lentiviral particles were generated by co-transfection of a targeting set of shRNA plasmids (Sirt6 and MDM2) or non-targeting control shRNA plasmid along with MISSION Lentiviral Packaging Mix (SHP001; Sigma-Aldrich) into 293FT cells (Thermo Fisher Scientific) using Lipofectamine 3000 (Life Technologies, Germany). Cell culture supernatants containing lentiviral particles were collected at 24 and 48 h post-transfection, filtered, and used to infect SNU-638 cells. The efficiency of Sirt6 and MDM2 knockdown was evaluated by western blotting of whole-cell extracts.

Preparation of nuclear and cytosolic extracts

Nuclear and cytoplasmic cell fractions were prepared using the NE-PER Reagent (Pierce). Briefly, cells were harvested in trypsin-EDTA (MediaTek) and spun at 500 g for 3 min. Cell pellets were resuspended in Cytoplasmic Extraction Reagents (CERI and CERII), vortexed at high speed for 15 seconds, and centrifuged at 16,000 g for 5 min at 4°C. Cytoplasmic protein was recovered from the supernatant. The pellet was resuspended in Nuclear Extraction buffer (NER) by intermittent high speed vortexing over 40 min, and the sample was centrifuged at 16,000 g for 10 min at 4°C. Nuclear protein was recovered from the supernatant. The protein concentration of the nuclear and cytoplasmic fractions was determined using the BCA assay. Cytosolic and nuclear proteins or whole-cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) in a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). Membranes were incubated with primary antibodies against lamin A/C (Santa Cruz Biotechnology, Dallas, TX, USA) and α-tubulin (Sigma, St. Louis, MO, USA), followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Cell Signaling Technology, Beverly, MA, USA). Antibody binding was detected using an enhanced chemiluminescence detection reagent (Pierce). Images were acquired with the ChemiDoc Touch Imaging System (Bio-Rad).

In vivo xenograft mouse model

All animal experiments were approved by the Institutional Animal Care and Use Committee of Gyeongsang National University and conducted according to the National Research Council Guidelines. A cell suspension (5 × 106 cells/mouse) of SNU-638 was injected subcutaneously into 6-week-old male nude mice (athymic nude mice; KOATECH corporation, Harlan, Indianapolis, IN, USA). Nine days after inoculation of the cells, animals with xenograft tumors measuring 0.6–0.7 cm in diameter were subjected to intratumoral injections of Ad-GFP or Ad-Sirt6. Tumor diameters were measured with digital calipers on days 5, 10, 15, and 20, and tumor volume was determined using the modified ellipsoidal formula (tumor volume = 1⁄2[length × width2]).

Statistics

All statistical analysis was performed with SPSS software, version 20.0 (IBM, Armonk, NY, USA) and GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA). Chi-squared analysis was used to assess the relationship between Sirt6 expression and clinicopathological parameters. Student’s t-test was used to analyze the differences between groups and among groups, respectively. All tests used a p-value < 0.05 as the cut-off for statistical significance.

Sirt6 expression was significantly lower in gastric cancer than in normal tissues (Fig. 1A). Sirt6 expression levels varied among gastric cancer cells, and high expression levels were associated with higher overall survival rates (Fig. 1B and C). Sirt6 expression level was lower in SNU-638 cells (gastric cancer cell line) than in control cells (HPSECs) (Fig. 2A). Sirt6 overexpression using adenovirus induced gastric cancer cell death, as indicated by the percentage of Sub G1 cells, whereas no effect was observed in HPSECs (Fig. 2B). Knockdown of Sirt6 by shSirt6 treatment decreased gastric cancer cell death (Fig. 2C). In a SNU-638 xenograft mouse model, Sirt6 overexpression significantly decreased gastric cancer tumor volume, suggesting that Sirt6 exerts anti-tumorigenic effects (Fig. 2D and E).

Because oxidative stress is a key factor regulating the tumor-promoting effect of sirtuins, we measured ROS levels in gastric cancer cells. Sirt6 overexpression induced by Ad-Sirt6 increased ROS accumulation, whereas Sirt6 knockdown by shSirt6 decreased ROS production in gastric cancer cells (Fig. 3A and B). The ROS scavenger NAC significantly reduced Sirt6-mediated gastric cancer cell apoptosis (Fig. 3C).

Ad-Sirt6–induced Sirt6 overexpression downregulated Sirt1 in gastric cancer cells (Fig. 4A), whereas shSirt6-mediated Sirt6 silencing upregulated Sirt1 expression (Fig. 4B). Sirt6 overexpression in gastric cancer cells was predominantly detected in the nucleus and not in the cytoplasm, as indicated by western blot and immunofluorescence analyses (Fig. 4C and D).

Oxidatively damaged proteins are known to be degraded via the proteasome system, and treatment with MG-132, proteasome inhibitor, upregulated Sirt1 (Fig. 5A). The suppressed gastric cancer cell apoptosis was associated with Sir6 (Fig. 5B). Knockdown of MDM2 by treatment with sh-MDM2 upregulated Sirt1 leading to reduced apoptotic activity (Fig. 5C and D). Consistently, inhibition of the p53-MDM2 interaction by Nutlin-3 suppressed Sirt1- and Sirt6-mediated apoptotic activity (Fig. 5E and F). Additionally, Sirt1 downregulation and Sirt6 upregulation was detected in the presence of MDM2 in gastric cancer cells (Fig. 5G).

A schematic diagram describing Sirt6- and Sirt1-mediated gastric cancer cell death is shown in Fig. 6C. MDM2-modulated Sirt6 upregulation and Sirt1 downregulation promoted gastric cancer cell death by increasing ROS accumulation (Fig. 6).

Gastric cancer is among the leading causes of cancer-related death, although the underlying pathogenetic mechanism remains unclear [12]. H. pylori infection is associated with an increased risk of developing gastric cancer; however, H. pylori infection is not present in all gastric cancer cases, and other factors such as inflammatory conditions and alterations in hormone levels have been associated with gastric cancer development [13, 14]. Among several oncogenic factors, members of the sirtuin family play a crucial role in the immune response of gastric cancer [10]. This study is the first to examine the role of Sirt1 and Sirt6 in the regulation of gastric cancer cell death.

Sirt6 has anticancer effects in many malignancies; however, whether Sirt1 acts as a pro-tumorigenic or anti-tumorigenic factor remains unclear. The cancer-related effects of Sirt1 or Sirt6 have also been reported in gastric cancer, and the corresponding studies are listed in Table 1. Pathways and molecules involved in both, the tumorigenic and anti-tumorigenic effects of Sirt1, include Yap, FOXO, p53, and miRNAs, whereas the JAK2/STAT3 pathway and ferroptosis are involved in the anti-tumorigenic effect of Sirt6 in gastric cancer. This study focused on ROS-mediated cancer cell death because ROS are associated with a variety of cellular processes involving both Sirt1 and Sirt6 [15, 16].

Sirt6- and Sirt1-mediated gastric cancer cell death via ROS regulation was demonstrated previously in many types of cancer but not in gastric cancer [6]. We showed that Sirt6 upregulates MDM2, which suppresses Sirt1 expression and induces ROS production, thereby promoting gastric cancer cell death. Sirt1 suppression itself does not lead to cancer cell death, requiring Sirt6 and MDM2-mediated downregulation to induce an anticancer effect. The mechanisms and molecular pathways involved in Sirt1 suppression need to be examined further to develop Sirt-based anticancer therapeutics in the future.

In summary, we found that elevated Sirt6 expression leads to Sirt1 suppression via MDM2 to induce gastric cancer cell death. The activity of the three proteins, Sirt6, MDM2, and Sirt1, results in increased ROS production, which is responsible for the anti-tumorigenic effect, suggesting that these three molecules are potential targets for the treatment of gastric cancer.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Gyeongsang National University College of Medicine.

Consent for publication

Not applicable.

Availability of data and materials

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A3067325), the National Research Foundation of Korea (NRF-2020R1A2C1012038, NRF-2017R1A2B4010122, and NRF-2021R1F1A1056420); and the Biomedical Research Institute fund (GNUHBRIF-2018-0012) from the Gyeongsang National University Hospital.

Authors' contributions

JH Seo and JJ Park participated in determining the overall experimental design and analyzing the data, and S Ryu and YS Hah wrote the main manuscript text. SY Cheon, SJ Lee and SJ Won prepared the figures and analyzed the statistics, and CD Yim, and HJ Lee collected journals and made Table1. All authors reviewed the final version of the manuscript.

Acknowledgments

Not applicable.

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Table 1. Pro-tumorigenic and anti-tumorigenic effect of Sirt1 and Sirt6 in gastric cancer

Name	Function	Mechanism	References
Sirt1	Pro-tumorigenic	Yap signaling pathway	[17,18]
		Modulation of angiogenesis via FOXO1	[19]
		Deacetylation of histone substrates, transcription factors and cofactors	[20]
		FOXO1 and YAP signaling pathway modulated by USP22	[21]
		Regulating autophagy through the deacetylation of ATGs	[22]
		Targeting p53 to suppress ferroptosis	[23]
		Mediated by candidate oncogene circNOP10	[24]
		FoxO1-Rab7—autophagy axis	[25]
		Deacetylation of Beclin-1 and other autophagy mediators	[26]
		Inhibiting PI3K/AKT pathway	[27]
		Sirt1 downregulation by MiR-204	[28]
		By the results of DBC1, H4K16Ac, and H3K9Ac	[29]
		Sirt1 transactivation by ATF4	[30]
		Nampt/sirt1/c-myc positive feedback loop	[31]
	Anti-tumorigenic	Sirt1 targeted by micro-RNAs (miR-543, miR-132-3p/miR-212-3p, miRNA-12129, miR-1301-3p, Has-miR-34a-5p, miR-132, miR-183)	[21,32,33,34, 35,36,37]
		Regulating ARHGAP5 expression	[38]
		Initiating an AMPK/FOXO3 positive feedback loop	[39]
		Via STAT3/MMP-12 signaling	[40]
		G1-phase arrest via NF-κB/Cyclin D1 signaling	[25]
		Repression of activation of STAT3 and NF-κB proteins via deacetylation	[41]
		Inducing G1 phase arrest and senescence by Resveratrol	[42]
Sirt6	Anti-tumorigenic	Inhibition of JAK2/STAT3 pathway	[43]
Sirt6	Anti-tumorigenic	Promoting ferroptosis	[44]

No competing interests reported.

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Sirt6-mediated cell death associated with Sirt1 suppression in gastric cancer

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Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Patient samples and data collection

Cell lines and chemicals

Western blot analysis

Flow cytometric DNA analysis

ROS measurement

IHC analysis of Sirt6 and evaluation of IHC reactions

ICC-Immunofluorescence (IF) for Sirt1 and Sirt6 double staining

Immunoprecipitation (IP) assay

Preparation of adenovirus

Short hairpin RNA (shRNA)-mediated silencing of Sirt6 and MDM2

Preparation of nuclear and cytosolic extracts

In vivo xenograft mouse model

Statistics

Results

Discussion

Declarations

References

Table 1

Additional Declarations

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