Pharmaceutical composition for preventing or treating cancer

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating thyroid cancer, the composition comprising a substance that increases the level of the SIRT4 (sirtuin 4) gene or mRNA, or the level or activity of the SIRT4 protein.

Description

Pharmaceutical composition for the prevention or treatment of cancer

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer.

[0002]

[0003] Papillary thyroid carcinoma (PTC) is the most common subtype of thyroid cancer, accounting for over 90% of all thyroid cancers. PTC is generally a slow-growing tumor with a relatively good prognosis. In fact, the 10-year survival rate reaches approximately 93%. In most cases, it is effectively treated with radioactive iodine therapy following surgical resection; however, some patients exhibit aggressive disease characteristics and do not respond to conventional treatment. Recurrence occurs in about 10% of patients and is primarily associated with distant metastasis. Factors influencing the prognosis and recurrence of PTC include age, stage, lymph node metastasis, extrathyroidal expansion, vascular invasion, and distant metastasis. Additionally, reactive oxygen species (ROS) play a significant role in inhibiting the progression of thyroid cancer, and factors related to glutamine metabolism are associated with tumor formation. Nevertheless, the precise factors contributing to the progression of PTC are not yet clearly understood. Therefore, it is important to understand the molecular mechanisms of PTC proliferation and progression.

[0004] Sirtuins are a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases involved in various cellular processes, including energy metabolism, chromosomal stability, DNA repair, cell cycle progression, and apoptosis. Seven sirtuin isoforms (SIRT1 to SIRT7) have been identified in mammals, each possessing a unique intracellular localization and target protein. SIRT4 is a member of the sirtuin family, primarily located in mitochondria, and is involved in various biological processes such as glucose and lipid metabolism, mitochondrial function, and oxidative stress responses. Recent studies have suggested a possible link between SIRT4 and cancer progression. SIRT4 is characterized as a tumor suppressor and is downregulated in various malignancies, including thyroid cancer. However, the precise function of SIRT4 in cancer is unclear, although it is suggested to be associated with cancer development.

[0005] There are only a few studies focusing on SIRT4 and thyroid cancer, and the role of SIRT4 in this disease has not been sufficiently elucidated.

[0006]

[0007] The present invention aims to identify the role of SIRT4 in thyroid cancer and to provide a pharmaceutical composition capable of exhibiting an improvement effect on thyroid cancer by regulating it.

[0008] The present invention aims to provide a method for providing information necessary for predicting the prognosis of thyroid cancer using SIRT4 as a marker.

[0009] The present invention aims to provide a method for screening candidate substances for the prevention or treatment of thyroid cancer.

[0010]

[0011] 1. A pharmaceutical composition for the prevention or treatment of thyroid cancer comprising a substance that increases the level or activity of the SIRT4 (sirtuin 4) gene or mRNA; or the level or activity of the SIRT4 protein.

[0012] 2. The above-mentioned substance is a pharmaceutical composition for the prevention or treatment of thyroid cancer, wherein the substance is a SIRT4 gene or a carrier carrying the same.

[0013] 3. A pharmaceutical composition for the prevention or treatment of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with 1 above.

[0014] 4. A method for providing information necessary for predicting the prognosis of thyroid cancer, which provides information that if the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells of a thyroid cancer patient; or the level or activity of SIRT4 protein is lower than that of a control group, there is a high likelihood of a poor prognosis compared to a control group.

[0015] 5. A method for providing information necessary for predicting the prognosis of thyroid cancer, which provides information that if the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein is lower than that of the control group, there is a high likelihood of a poor prognosis compared to the control group.

[0016] 6. A method for providing information necessary for predicting the prognosis of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with 5 above.

[0017] 7. A screening method for candidate substances for the prevention or treatment of thyroid cancer, which screens substances that increase the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein.

[0018] 8. A screening method for candidate substances for the prevention or treatment of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with 7 above.

[0019]

[0020] The present invention exhibits pharmacological effects against thyroid cancer, such as inhibiting cancer cell proliferation, promoting apoptosis, and inhibiting invasion and metastasis.

[0021] The present invention can provide useful information for prognosis prediction by using SIRT4 as a marker.

[0022] The present invention can contribute to the development of thyroid cancer treatments by screening substances that increase SIRT4 levels.

[0023]

[0024] Fig. 1. Clinical significance of SIRT4 expression in thyroid cancer. (A) Analysis of the GSE33630 database showed that SIRT4 mRNA expression levels in thyroid cancer tissue (n = 45) were significantly lower than in adjacent normal thyroid tissue (n = 60) (P < 0.0001, t-test). (B) Based on the TCGA dataset, overall survival rates of patients with high and low SIRT4 expression were analyzed using Kaplan-Meier (KM) plots (P = 0.0155, log-rank test). (C) Representative IHC staining images of PTC tissues with varying degrees of SIRT4 expression. Number of positive cells: () <10%; (+) >10%. ****P < 0.0001. PTC stands for papillary thyroid cancer, and TCGA stands for The Cancer Genome Atlas.

[0025] Fig. 2. SIRT4 inhibits B-CPAP cell proliferation. (A) Western blot analysis of sirtuin expression levels in B-CPAP cells. (B) Comparison of SIRT4 overexpressing and shSIRT4 transcribed B-CPAP cells with the control group in CCK-8 analysis. B-CPAP cells were treated with CCK-8 solution 72 hours after seeding in a 24-well plate. SIRT4 significantly inhibited the viability of B-CPAP cells. (C) Results of clonal survival analysis of SIRT4 overexpressing and shSIRT4 transcribed B-CPAP cells compared with the control group. Surviving clones decreased in SIRT4 overexpressing B-CPAP cells, but increased in SIRT4 downregulated B-CPAP cells. *P < 0.05; **P < 0.01; ***P < 0.001. CCK-8 refers to the cell viability measurement kit.

[0026] Fig. 3. SIRT4 regulates the cell cycle of B-CPAP cells and modulates ROS levels to promote apoptosis. (A) Proportion of cells in the sub-G1 phase in control and SIRT4-overexpressing B-CPAP cells. B-CPAP cells were treated for 24, 48, and 72 hours and analyzed via flow cytometry. Histograms represent PI fluorescence intensity. SIRT4 overexpression significantly increased the number of sub-G1 cells. (B) Proportion of sub-G1 phase cells in control and shSIRT4-transcribed B-CPAP cells. (C) Analysis of the apoptosis rate in control and SIRT4-overexpressing B-CPAP cells via flow cytometry using the Annexin V (FITC)-PI apoptosis detection kit. SIRT4 overexpression significantly increased the apoptosis rate after 72 hours. (D) Apoptosis rate in control and SIRT4-downregulated B-CPAP cells. (E) Difference in total ROS levels according to SIRT4 expression regulation. (F) Increased SIRT4 expression significantly increased MitoSOX Red levels and decreased MTDR intensity over time, while SIRT4 downregulation showed the opposite effect. MTDR refers to MitoTracker Deep Red, and ROS refers to reactive oxygen species.

[0027] Fig. 4. SIRT4 inhibits the invasion and migration of B-CPAP cells by regulating the expression of EMT-related proteins. (A) Transwell analysis results comparing the relative invasiveness and (B) migration ability of SIRT4-overexpressing B-CPAP cells with a control group. Cells were cultured for 24 hours, and invading and migrating cells were observed at ×100 magnification in five randomly selected fields using a fluorescence microscope. (C) Transwell analysis results comparing the relative invasiveness and (D) migration ability of shSIRT4-transcribed B-CPAP cells with a control group. (E) Wound healing analysis results of SIRT4-overexpressing cells and (F) SIRT4-downregulated cells. Representative images at specified times are shown on the right. (G) Regulation of EMT-related proteins according to SIRT4 expression. The expression of E-cadherin, N-cadherin, and other EMT markers was analyzed and quantified. *P < 0.05, **P < 0.01, ***P < 0.001. EMT stands for epithelial-mesenchymal transition.

[0028] Fig. 5. SIRT4 inhibits tumor growth in a B-CPAP xenograft mouse model. (A) 42 days after tumor cell injection, mice received intratumoral injections of Ad-GFP or Ad-SIRT4. Images of tumors excised from each group show the control group at the top and the SIRT4 expression group at the bottom. (B) Tumor diameter was measured every 3 days using digital calipers, and tumor volume was calculated. This plot shows the tumor growth curves of 14 mice at specified times after intratumoral injections of Ad-GFP or Ad-SIRT4. (C) Tumor weights were compared between the SIRT4 expression group and the control group. (D) SIRT4 overexpression induces apoptosis and superoxide production in mouse tumors. FFPE tumor sections were stained with DAPI (0.5 μg / mL) to visualize nuclei, and apoptosis and superoxide production were detected using TdT enzyme (200 μg / mL) and DHE (10 μmol / L), respectively. Green fluorescence of apoptosis, red fluorescence of oxidation, and blue fluorescence of the nucleus were analyzed under a fluorescence microscope at 400× magnification. The graph below shows the results of quantifying cell signals stained positive for TdT and DHE in five random fields. n = 8. *P < 0.05, **P < 0.01, ***P < 0.001. DAPI stands for 4',6-diamidino-2-phenylindole, DHE for dihydroethidium, FFPE for formalin-fixed paraffin-embedded fragments, and TdT for terminal deoxynucleotide transferase. TUNEL stands for TdT-mediated dUTP nick end labeling.

[0029] Fig. 6. Schematic diagram of the role of SIRT4 in papillary thyroid cancer cells. This figure was created with BioRender.com.

[0030] Fig. 7. Sirt4 inhibits cell proliferation in TPC-1 (AC) and SNU-790 (DF) cells.

[0031] (A) Sirtuin expression levels in TPC-1 cells. (B) CCK-8 analysis of Sirt4 overexpressing and shSirt4 transcribed TPC-1 cells and (C) results of cloning analysis compared with the control group. (D) Sirtuin expression levels in SNU-790 cells. (E) CCK-8 analysis of Sirt4 overexpressing and shSirt4 transcribed SNU-790 cells and (F) results of cloning analysis compared with the control group. CCK-8 refers to Cell Counting Kit-8.

[0032] Fig. 8. Sirt4 promotes apoptosis by regulating ROS levels in TPC-1 (AD) and SNU-790 (EH) cells.

[0033] (A,B) Cell death rates in TPC-1 cells with up-regulated or down-regulated Sirt4. (C) Differences in total ROS levels according to Sirt4 expression regulation. (D) Changes in MitoSox Red levels and MTDR intensity in TPC-1 cells according to Sirt4 expression regulation. (EH) These results were consistently observed in SNU-790 cells as well.

[0034] Fig. 9. Sirt4 regulates the invasion and migration of TPC-1 and SNU-790 cells.

[0035] (AD) Results of analyzing invasion, migration, and wound healing according to Sirt4 upregulation and downregulation in TPC-1 cells compared with the control group. (EH) These results were consistently observed in SNU-790 cells as well. (I) Results of confirming the regulation of EMT-related proteins according to Sirt4 expression in B-CPAP, TPC-1, and SNU-790 cell lines.

[0036]

[0037]

[0038] The present invention will be described in detail below.

[0039]

[0040] The present invention relates to a pharmaceutical composition for the prevention or treatment of thyroid cancer.

[0041] The pharmaceutical composition of the present invention comprises a substance that increases the level of SIRT4 (sirtuin 4) gene or mRNA; or the level or activity of SIRT4 protein.

[0042] SIRT4 is a member of the Sirtuin family and is a NAD+-dependent enzyme found in mammals, including humans.

[0043] The inventors conceived the present invention upon confirming that when the expression of SIRT4 is reduced in cancer cells, the progression and prognosis of the tumor worsen, and when it is overexpressed, the tumor can be improved.

[0044] A substance that increases the level or activity of the SIRT4 (sirtuin 4) gene or mRNA; or the level or activity of the SIRT4 protein, may be a substance that increases the said level or activity in thyroid cancer cells. This is not limited to substances that exhibit the effect. For example, it may be nucleic acids, proteins, compounds, macromolecules, natural products, etc.

[0045] Nucleic acids can be used in the form of DNA, RNA, or siRNA to promote the expression of the SIRT4 gene. For example, the SIRT4 gene, mRNA, or a carrier containing it can increase the level of SIRT4 by inducing the expression of the said gene.

[0046] The protein can be a recombinant SIRT4 protein or an enzyme activity regulatory protein that directly increases the activity of the SIRT4 protein. These proteins can bind to the active site of SIRT4 to enhance enzyme activity.

[0047] The compound can be a small molecule compound or a synthetic compound that promotes SIRT4 activity. For example, it may include a compound that promotes the NAD+-dependent response of SIRT4 or a pharmacologically active ingredient that induces SIRT4 expression.

[0048] The polymer may consist of polymeric materials or biopolymers that promote SIRT4 gene expression or increase protein stability. For example, it may be used as an RNA nanostructure or a gene delivery vehicle.

[0049] Natural products may be natural extracts, such as plant extracts, substances derived from marine organisms, or mushroom extracts, that increase SIRT4 expression or promote protein activity. For example, certain plant polyphenols or flavonoids can promote SIRT4 expression.

[0050] Thyroid cancer can be, for example, papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), anaplastic thyroid carcinoma (ATC), etc.

[0051] The composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients, and diluents that may be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the product is prepared using diluents or excipients such as commonly used fillers, volume expanders, binders, humectants, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules; these solid dosage forms are prepared by mixing at least one excipient with the above compounds, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa oil, laurin oil, glycerogelatin, etc. may be used.

[0052] The amount of the composition of the present invention used may vary depending on the patient's age, gender, and weight, but may be administered in an amount of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg, once or several times a day. Additionally, the dosage may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0053]

[0054] In addition, the present invention relates to a method for providing information necessary for predicting the prognosis of thyroid cancer.

[0055] The method of the present invention provides information that if the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells of a thyroid cancer patient; or the level or activity of SIRT4 protein is lower than that of a control group, there is a high likelihood of a poor prognosis compared to a control group.

[0056] The level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein can be used as a prognostic prediction marker.

[0057] The subjects for prognosis prediction and comparison may be thyroid cancer patients.

[0058] Thyroid cancer can be, for example, papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), anaplastic thyroid carcinoma (ATC), etc.

[0059] If the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells of patients with thyroid cancer; or if the level or activity of SIRT4 protein is lower than that of the control group, it may provide information that the prognosis is likely to be poor compared to the control group.

[0060] A poor prognosis may mean a low survival rate. The survival rate can be the overall survival (OS). This can be the 5-year or 10-year survival rate.

[0061]

[0062] In addition, the present invention relates to a method for screening candidate substances for the prevention or treatment of thyroid cancer.

[0063] The method of the present invention includes the step of selecting a substance that increases the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein.

[0064] This allows for the selection of substances that increase the level of SIRT4 (sirtuin 4) gene or mRNA, or the level or activity of SIRT4 protein, in thyroid cancer cells by treating candidate substances in vitro.

[0065] The types of candidate substances are not limited. For example, they may be nucleic acids, proteins, compounds, polymers, natural products, etc.

[0066] Thyroid cancer can be, for example, papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), anaplastic thyroid carcinoma (ATC), etc.

[0067]

[0068] The present invention will be explained in more detail with reference to the following examples.

[0069]

[0070] Materials and Methods

[0071] Patient Sample and Data Collection

[0072] This study was approved by the Institutional Review Board of Gyeongsang National University (GNUH 2023-07-027). All procedures were performed in accordance with the 1975 Helsinki Declaration. A total of 205 tissue samples were obtained from patients with papillary thyroid carcinoma (PTC) who underwent surgery at our hospital between 2011 and 2018. Patient medical records were reviewed retrospectively, and clinical and pathological parameters were recorded. Immunohistochemistry (IHC) was performed on all 205 tissues, and tissue microarrays were incubated overnight at 4°C using the anti-SIRT4 antibody (Abcam). The IHC results were independently reviewed by two pathologists who were unaware of the patient data. Because the reaction intensity and distribution were heterogeneous among the samples, SIRT4 immunostaining was considered positive if more than 10% of the tumor cells were stained by the antibody. This followed the criteria used in previous studies on other SIRT proteins (17). This study utilized gene expression data from the GSE33630 and GPL570 datasets, which include thyroid cancer and normal thyroid tissue samples, from the GEO database (http: / www.ncbi.nlm.nih.gov / geo / ). Raw data files were preprocessed to ensure accuracy. The relationship between SIRT4 expression and thyroid cancer prognosis was investigated via Kaplan-Meier analysis using patient survival data and SIRT4 expression levels from the TCGA data. SIRT4 expression was classified into 'low' and 'high' based on the median. Data processing and analysis were performed using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA).

[0073]

[0074] Cell culture and maintenance

[0075] In this study, three human thyroid cancer cell lines—B-CPAP, TPC-1, and SNU-790—were used. The B-CPAP cell line was provided by Dr. Soon-Hyun Ahn of Seoul National University, and the TPC-1 cell line (Sigma SCC147) was purchased from Sigma-Aldrich. The SNU-790 cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured in high-grade RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% v / v fetal calf serum (FBS; supplied by GenDEPOT). The medium was additionally supplemented with 1% v / v penicillin / streptomycin and 200 mM l-glutamine (Thermo Fisher Scientific). Cells were maintained under optimal growth conditions in a humid environment containing 5% CO2 at 37°C.

[0076]

[0077] SIRT4-expressing adenovirus production

[0078] Recombinant adenoviruses expressing SIRT4 (Ad-SIRT4) were generated using the ViralPower adenovirus expression system (Invitrogen by Thermo Fisher Scientific). The cDNA corresponding to SIRT4 was first cloned into the pENTR vector. After verifying sequence integrity, the SIRT4 cDNA was recombined into the pAd / CMV / V5-DEST gateway vector using the LR Clonase II enzyme mixture (Invitrogen). The generated Ad-SIRT4 configuration was linearized with PacI (New England Biolabs) and then transfected into 293A cells using Lipofectamine 3000 (Invitrogen). Viral particles were amplified in the same cell line using the ViralPower adenovirus expression system (Invitrogen). Viral titers were determined by plaque formation experiments using serial dilution. This viral suspension was applied to thyroid cancer cell lines. A recombinant adenovirus expressing green fluorescent protein (Ad-GFP) and β-galactosidase (Ad-LacZ) was used as a control.

[0079]

[0080] shRNA-mediated RNA interference for SIRT4 silencing

[0081] Repression of SIRT4 expression was performed using a bacterial glycerol reservoir (MISSION shRNA) containing shRNA targeting SIRT4 and a non-targeted control plasmid (SHC002; provided by Sigma-Aldrich). This silencing mechanism was delivered via lentivirus. Lentiviral particles were synthesized by transfecting 293FT cell lines (Thermo Fisher Scientific) with either the SIRT4-targeted shRNA plasmid or the non-targeted control shRNA plasmid in combination with MISSION Lentiviral Packaging Mix (SHP001; Sigma-Aldrich). After transfection, cell culture supernatants were collected at 24- and 48-hour intervals, and the supernatant rich in lentiviral particles was filtered and used to infect all thyroid cancer cell lines. Successful repression of SIRT4 expression was confirmed by Western blot analysis of whole cell lysates.

[0082]

[0083] Evaluation of cell viability through CCK-8 analysis

[0084] Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8, Tojindo, Kumamoto, Japan). Cells were seeded into 24-well plates at an optimal density. After a 24-hour attachment period, cells were infected with Ad-GFP and Ad-SIRT6 adenoviruses or Lenti-shGFP and Lenti-shSIRT4 lentiviruses. Cells were cultured for 72 hours, after which 50 μL of CCK-8 solution was added to each well and incubated in the dark at 37°C for 2 hours. Optical density was measured at a wavelength of 450 nm using an ELISA reader.

[0085]

[0086] Clone formation survival analysis

[0087] The colony-forming ability of the cells was evaluated via clonal formation analysis. Cells were dissociated using TrypLE™ Express (Thermo Fisher Scientific) and then quantified using an automated cell counter. A total of 500 cells were seeded into each well of a 6-well plate. After attachment, the cells were infected with Ad-GFP and Ad-SIRT6 adenoviruses or Lenti-shGFP and Lenti-shSIRT4 lentiviruses. Plates were incubated for 10 or 14 days at 37°C in a humid environment containing 5% CO2, and colonies were fixed and stained with crystal violet in a 0.5% methanol solution. Colonies consisting of 50 or more cells were counted manually.

[0088]

[0089] DNA content analysis through flow cytometry

[0090] Cell cycle distribution and apoptotic cell population were determined by flow cytometry. Harvested cells were washed twice with cold PBS, fixed by adding 70% ethanol dropwise, and incubated at 4°C for 1 hour. Fixed cells were treated with 1 mg / mL RNase A (Sigma-Aldrich) and stained with 50 μg / mL PI (Sigma-Aldrich) in the dark for 30 minutes. Stained cells were analyzed using a Cytomics FC500 flow cytometer (Beckman-Coulter), and the data were processed using CXP software (Beckman-Coulter).

[0091]

[0092] Measurement of mitochondrial reactive oxygen species (ROS) levels

[0093] Oxidative stress in cells was monitored using MitoSOX™ red mitochondrial superoxide indicator. Cells were treated with 5 μM MitoSOX reagent while cultured at approximately 70–80% density. Mitochondrial mass was evaluated by incubating with 50 nM MitoTracker Deep Red in the dark at 37°C for 30 minutes. After incubation, cells were detached with trypsin, washed with warm PBS, and suspended in PBS. Fluorescence intensity representing mitochondrial mass was measured using a Cytomics FC500 flow cytometer (Beckman-Coulter). Quantitative and median fluorescence intensity analyses were performed using CXP software (Beckman-Coulter).

[0094]

[0095] Analysis of apoptosis in human thyroid cancer cells

[0096] After infecting B-CPAP, TPC-1, and SNU-790 cells with Ad-GFP, Ad-SIRT4, Lenti-shGFP, and Lenti-shSIRT4, apoptosis analysis was performed according to the manufacturer's instructions of the Annexin V(FITC)-PI apoptosis assay kit (BD Biosciences).

[0097]

[0098] Evaluation of cell invasion and migration through Transwell analysis

[0099] Cell invasion and migration were analyzed using Transwell plates (Costar, Cambridge, MA, USA) equipped with polycarbonate filters having 8 μm pores. For the invasion assay, the upper chamber of the Transwell plate was pre-coated with BD Matrigel™ Basement Membrane Matrix (BD Biosciences, Bedford, MA, USA). The migration assay was performed without Matrigel coating to evaluate basic migration characteristics without cells passing through the extracellular matrix. Cell lines were prepared at a concentration of 1 × 10^5 cells / well and resuspended in high-grade RPMI 1640 medium supplemented with 10% FBS. After incubation at 37°C for 24 hours, cells that crossed the membrane via invasion or migration were fixed with 4% paraformaldehyde and visualized by staining with 4′,6-diamidino-2-phenylindole (DAPI). Quantification was performed using a fluorescence microscope in five randomly selected 100x magnification fields.

[0100]

[0101] Analysis of gap closure for cell migration

[0102] For the analysis of gap closure, cells were seeded into 35 mm μ-dish containing 2 wells of Ibidi Culture-Insert using serum-free medium at a concentration of 1 × 10^5 cells / well. After 24 hours, the 2 wells of Culture-Insert were carefully removed using sterile tweezers, and standard culture medium was added. Time-course imaging of the thyroid cancer cell line was performed for 8 hours, and images were taken at 2-hour intervals to observe cell migration patterns.

[0103]

[0104] Western blot analysis of protein expression

[0105] B-CPAP, TPC-1, and SNU-790 cells were lysed using RIPA buffer (Thermo Fisher Scientific) supplemented with a protease inhibitor cocktail (GenDEPOT). After sonication for 2 minutes, the cell lysate was centrifuged at 14,000 g at 4°C for 10 minutes to remove insoluble fragments. Protein concentration was measured using a BCA protein assay kit (Pierce). 30 μg of protein was loaded into each well, separated by SDS-PAGE, and then transferred to a nitrocellulose membrane (Millipore). The membrane was blocked with 5% BSA and incubated with primary antibodies against E-cadherin, N-cadherin, MMP9, MMP3, SIRT4, SNAIL, and vimentin. After treatment with an HRP-conjugated secondary antibody, the protein bands were visualized using Clarity Western blot ECL Substrate (Bio-Rad) and imaged using the ChemiDoc Touch Imaging System (Bio-Rad).

[0106]

[0107] Immunohistochemical analysis of SIRT4 expression

[0108] Tissue sections fixed in formalin and embedded in paraffin from nude mice with B-CPAP tumors were processed for immunohistochemical analysis. Tissue blocks were cut into 5 μm thick sections, and after paraffin removal and rehydration, treated in 3% hydrogen peroxide for 10 minutes to inhibit endogenous peroxidase activity. Antigen recovery was performed by heating in a 700W microwave for 20 minutes using 10 mM citric acid buffer solution (pH 6.0). For the specific detection of SIRT4, tissue sections were incubated overnight at 4°C with an anti-SIRT4 antibody (Abcam).

[0109]

[0110] Xenograft mouse model for in vivo evaluation

[0111] All animal experiments were performed after obtaining approval from the Institutional Animal Care and Use Committee (IACUC) of Gyeongsang National University. All procedures strictly adhered to the guidelines of the National Research Council. B-CPAP cell suspension (5 × 10^6 cells per mouse) was injected subcutaneously into 6-week-old male hairless nude mice (KOATECH corporation, Harlan, IN, USA). 42 days after cell inoculation, mice with xenograft tumors measuring 0.6–0.7 cm in diameter were injected into the tumors with either Ad-GFP or Ad-SIRT4. Tumor size was measured using a precision digital caliper on days 42, 45, 48, and 54, and tumor volume was calculated using the modified ellipsoidal equation (tumor volume = 1 / 2 (length × width^2)).

[0112]

[0113] Analysis of Apoptosis and Oxidative Stress in Mouse Tumors

[0114] Apoptosis and oxidative stress in mouse tumor tissues were evaluated via TUNEL and DHE-based assays. Briefly, samples were fixed overnight in 4% formaldehyde at room temperature, dehydrated with graded ethanol, treated with xylene, and embedded in paraffin. Sections cut to a thickness of 5 μm were removed from the paraffin and treated with 1% H2O2 (Sigma-Aldrich) for 10 minutes to neutralize endogenous peroxidase. After rinsing with PBS (Thermo Fisher Scientific), sections were treated with 10 μg / mL Proteinase K (Thermo Fisher Scientific) for 6 minutes and 0.1% Triton X-100 (Sigma-Aldrich) at 4°C for 6 minutes. Subsequently, the slides were rinsed with PBS and incubated with a 200 μg / mL TdT mixture containing FITC-dUTP (BD Biosciences) at room temperature for 1 hour. After rinsing with PBS, sections were stained with 10 μmol / L DHE (Thermo Fisher Scientific) for 1 hour and counterstained with 0.5 μg / mL DAPI (Sigma-Aldrich) solution for 1 hour. Analysis was performed using a study microscope equipped with Nikon software, and fluorescence intensity was quantified by measuring the percentage of positively stained area in five random fields.

[0115]

[0116] Statistical analysis

[0117] Statistical analysis was performed using SPSS software (version 20.0, IBM) and GraphPad Prism (version 8.0, GraphPad Software). The relationship between SIRT4 expression and clinicopathological parameters was evaluated using the chi-square test, and differences between groups were analyzed using Student's t-test. A P-value of less than 0.05 was considered statistically significant.

[0118]

[0119] result

[0120] SIRT4's GEO, TCGA Database, and Roles in Human PTC Organizations

[0121] Analysis of data from the GEO database revealed that SIRT4 expression was reduced in thyroid cancer compared to normal thyroid tissue. In the TCGA database, patients with high SIRT4 expression had significantly better overall survival than those with low expression (P = 0.016, Figs. 1A, B). The association between decreased SIRT4 expression and reduced overall survival in thyroid cancer suggests that SIRT4 may act as a prognostic factor for the disease. Next, we evaluated SIRT4 expression in PTC tissues. The demographic characteristics of the patients are shown in Table 2. According to the clinical data, SIRT4 expression varied in PTC tissue samples, and they were divided into SIRT4-negative and SIRT4-positive groups based on expression. Of the 205 tissue samples, 86 were SIRT4-negative and 119 were SIRT4-positive (Fig. 1C). When comparing patient clinical data based on SIRT4 expression, there were no statistically significant differences in most parameters, but extracapsular expansion was reduced in SIRT4-positive patients (P = 0.000) (Table 1). These results suggest an association between SIRT4 expression and the progression of thyroid cancer.

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] The effect of SIRT4 expression on thyroid cancer cell proliferation

[0128] To further investigate whether SIRT4 is involved in PTC, we evaluated SIRT4 expression in three thyroid cancer cell lines. Western blot analysis of basal sirtuin levels in B-CPAP cells revealed that SIRT1 showed the highest expression, SIRT5 the lowest, and other sirtuins, including SIRT4, showed intermediate expression (Fig. 2A). Next, we analyzed the effects of SIRT4 overexpression and silencing in B-CPAP cells. CCK-8 analysis showed that SIRT4 overexpression decreased cell viability, while SIRT4 silencing significantly increased cell viability (Fig. 2B). In cloning analysis, SIRT4 overexpression significantly reduced the number of viable clones in B-CPAP cells, while SIRT4 silencing increased the number of clones compared to the control group (Fig. 2C). The results of CCK-8 and cloning analyses on TPC-1 and SNU-790 cells were also consistent, showing that SIRT4 overexpression reduced cell viability and decreased the number of viable clones (Fig. 7).

[0129]

[0130] Effects of SIRT4 on Apoptosis and ROS Level Regulation

[0131] After treatment with Ad-SIRT4, the proportion of sub-G1 cells in B-CPAP cells gradually increased, reaching a peak at 72 hours. In contrast, there was no significant difference in the proportion of sub-G1 cells after SIRT4 silencing (Fig. 3A, B). SIRT4 overexpression increased apoptosis in B-CPAP cells compared to the control group, but SIRT4 silencing did not have a significant effect on the apoptosis rate (Fig. 3C, D). These results suggest that SIRT4 is involved in regulating the proportion of sub-G1 cells in B-CPAP cells and that overexpression may promote apoptosis. The fact that SIRT4 expression affects apoptosis led us to investigate the potential involvement of ROS. We found that SIRT4 overexpression increased total ROS levels, while inhibition of expression decreased ROS levels (Fig. 3E). To determine whether SIRT4 regulation controls mitochondrial ROS levels, cells were stained with MitoSOX red. B-CPAP cells overexpressing SIRT4 showed a significant increase in MitoSOX red fluorescence intensity over time compared to the control group, indicating an increase in mitochondrial ROS levels. SIRT4 overexpression significantly reduced MitoTracker Deep Red (MTDR) staining intensity compared to the control group, suggesting a link between mitochondrial mass reduction and apoptosis. Conversely, inhibition of SIRT4 expression reduced MitoSOX red fluorescence intensity and increased MTDR intensity (Fig. 3F). Similar results were observed in TPC-1 and SNU-790 cells (Fig. 8), indicating that SIRT4 overexpression induces apoptosis by increasing mitochondrial ROS levels.

[0132]

[0133] Effects of SIRT4 on Cell Invasion, Migration, and Epithelial-Mesenchymal Transition Related Protein Expression

[0134] Next, we investigated the effects of SIRT4 on cell invasion and migration capabilities. SIRT4 overexpression significantly reduced invasion and migration, while inhibition of expression increased them (Figs. 4A-D). Wound healing analysis revealed that the rate of gap closure was significantly slower in SIRT4-overexpressing cells and accelerated after SIRT4 silencing (Figs. 4E, F). These results suggest that increased SIRT4 expression inhibits the invasion and migration capabilities of B-CPAP cells. To identify potential mechanisms supporting the inhibitory effects of SIRT4, we examined the expression of epithelial-mesenchymal transition (EMT) related proteins. SIRT4 overexpression significantly increased E-cadherin expression and decreased the expression of N-cadherin and other EMT markers. Inhibition of SIRT4 expression reduced E-cadherin levels and had no significant effect on N-cadherin expression, but increased the expression of other EMT markers such as MMP9 and vimentin (Fig. 4G). Analysis of the invasion and migration of TPC-1 and SNU-790 cells also showed that, similar to B-CPAP cells, SIRT4 overexpression reduced the cells' ability to invade and migrate. In addition, a consistent trend was observed in EMT marker expression across three human thyroid cancer cell lines (Fig. 9).

[0135]

[0136] Tumor growth inhibition by SIRT4 in a B-CPAP xenograft mouse model

[0137] The results of this study were also confirmed in a xenograft mouse model in which Ad-GFP and Ad-SIRT4 were injected into the tumors following the injection of B-CPAP cells into mice. Tumor size in mice expressing SIRT4 was smaller compared to the control group (Fig. 5A). The volume and weight of resected tumors were significantly smaller in mice overexpressing SIRT4 compared to the control group, indicating that B-CPAP cells expressing SIRT4 delay tumor growth in vivo (Fig. 5B, C). The presence of tumors formed in B-CPAP cells was confirmed through hematoxylin and eosin staining, and SIRT4 expression was confirmed in tumors injected with Ad-SIRT4 through immunohistochemical staining of tumor cells. The degree of apoptosis was evaluated via TUNEL staining analysis, a quantitative analysis designed to confirm the anti-tumor effect of SIRT4-induced apoptosis in PTC tumors. SIRT4 expression increased the number of TUNEL-positive apoptotic cells fourfold (52.2% compared to 13% in control cells), which was visualized as bright green dots under a fluorescence microscope. DHE staining revealed that ROS production in PTCs increased significantly from 15.8% in the control group to 60.8% after SIRT4 expression, representing a 3.8-fold increase (Fig. 5D). Therefore, the TUNEL and DHE staining results indicate that SIRT4 expression induces severe apoptotic stress in PTCs by increasing ROS production. These results further support the inhibitory effect of SIRT4 on B-CPAP cells.

Claims

1. A pharmaceutical composition for the prevention or treatment of thyroid cancer comprising a substance that increases the level or activity of SIRT4 (sirtuin 4) gene or mRNA; or SIRT4 protein.

2. A pharmaceutical composition for the prevention or treatment of thyroid cancer, wherein the substance is a SIRT4 gene or a carrier carrying the same.

3. A pharmaceutical composition for the prevention or treatment of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with Claim 1.

4. A method for providing information necessary for predicting the prognosis of thyroid cancer, which provides information that if the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells of a thyroid cancer patient; or the level or activity of SIRT4 protein is lower than that of a control group, there is a high likelihood of a poor prognosis compared to a control group.

5. A method for providing information necessary for predicting the prognosis of thyroid cancer, which provides information that if the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein is lower than that of a control group, there is a high likelihood of a poor prognosis compared to a control group.

6. A method for providing information necessary for predicting the prognosis of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with claim 5.

7. A screening method for candidate substances for the prevention or treatment of thyroid cancer, which screens substances that increase the level of SIRT4 (sirtuin 4) gene or mRNA in thyroid cancer cells; or the level or activity of SIRT4 protein.

8. A screening method for candidate substances for the prevention or treatment of thyroid cancer, wherein the thyroid cancer is papillary thyroid cancer, in accordance with claim 7.

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