Vitamin D/Vitamin D receptor signaling suppresses gastric cancer metastasis through autophagy-related protein 13/Beclin1-mediated autophagy

Abstract

Introduction: Gastric cancer (GC) exhibits a poor prognosis with high metastasis rates. This study investigates Vitamin D receptor’s (VDRs) role in suppressing GC metastasis through autophagy.

Objective: To elucidate the molecular mechanism by which VDR suppresses GC invasion and metastasis through autophagy regulation.

Methods: VDR expression was assessed in 91 paired GC and adjacent normal tissues using immunohistochemistry. The effects of Vitamin D on GC cell proliferation, cell cycle, migration, and invasion were evaluated using Cell Counting Kit-8, flow cytometry, wound healing, and Transwell assays. The autophagic activity was examined through LysoTracker Red staining, monomeric red fluorescent protein-green fluorescent protein-LC3 fluorescence, and transmission electron microscopy. The roles of VDR, autophagy-related protein 13 (ATG13), and Beclin1 in autophagy regulation were investigated through gene knockdown and silencing. A nude mouse peritoneal metastasis model was used to validate the anti-metastatic effects of the Vitamin D/VDR pathway.

Results: VDR expression was significantly downregulated in GC tissues, correlating with lymph node metastasis and poor prognosis (p<0.001). Vitamin D treatment upregulated VDR, ATG13, and Beclin1 expression, enhancing autophagosome and autolysosome formation without altering cell proliferation or apoptosis, likely due to a cytostatic autophagic response. Silencing ATG13or BECN1abolished the inhibitory effects of Vitamin D/VDR signaling on cell migration and invasion. VDR knockdown promoted peritoneal metastasis, whereas Vitamin D treatment suppressed it.

Conclusion: This study provides novel evidence that Vitamin D/VDR signaling inhibits GC metastasis by influencing ATG13/Beclin1-mediated autophagy, offering potential therapeutic targets for GC management.

Keywords

Vitamin D signaling / Gastric cancer metastasis / Autophagy regulation / Vitamin D receptor / Autophagy-related protein 13 / Beclin1

References

[1] Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3):209-249. doi: 10.3322/caac.21660

[2] Cao W, Chen HD, Yu YW, Li N, Chen WQ. Changing profiles of cancer burden worldwide and in China: A secondary analysis of the global cancer statistics 2020. Chin Med J (Engl). 2021; 134(7):783-791. doi: 10.1097/cm9.0000000000001474

[3] He Y, Wang Y, Luan F, et al. Chinese and global burdens of gastric cancer from 1990 to 2019. Cancer Med. 2021; 10(10):3461-3473. doi: 10.1002/cam4.3892

[4] Seeneevassen L, Bessède E, Mégraud F, Lehours P, Dubus P, Varon C. Gastric cancer: Advances in carcinogenesis research and new therapeutic strategies. Int J Mol Sci. 2021; 22(7):3418. doi: 10.3390/ijms22073418

[5] Haussler MR, Whitfield GK, Kaneko I, et al. Molecular mechanisms of vitamin D action. Calcif Tissue Int. 2013; 92(2):77-98. doi: 10.1007/s00223-012-9619-0

[6] Pike JW, Meyer MB. Fundamentals of vitamin D hormone-regulated gene expression. J Steroid Biochem Mol Biol. 2014; 144 Pt A:5-11. doi: 10.1016/j.jsbmb.2013.11.004

[7] Zella LA, Kim S, Shevde NK, Pike JW. Enhancers located within two introns of the vitamin D receptor gene mediate transcriptional autoregulation by 1,25-dihydroxyvitamin D3. Mol Endocrinol. 2006; 20(6):1231-1247. doi: 10.1210/me.2006-0015

[8] Haussler MR, Whitfield GK, Haussler CA, et al. The nuclear vitamin D receptor: Biological and molecular regulatory properties revealed. J Bone Miner Res. 1998; 13(3):325-349. doi: 10.1359/jbmr.1998.13.3.325

[9] Li H, Stampfer MJ, Hollis JB, et al. A prospective study of plasma vitamin D metabolites, vitamin D receptor polymorphisms, and prostate cancer. PLoS Med. 2007; 4(3):e103. doi: 10.1371/journal.pmed.0040103

[10] Gross M, Kost SB, Ennis B, Stumpf W, Kumar R. Effect of 1,25-dihydroxyvitamin D3 on mouse mammary tumor (GR) cells: Evidence for receptors, cellular uptake, inhibition of growth and alteration in morphology at physiologic concentrations of hormone. J Bone Miner Res. 1986; 1(5):457-467. doi: 10.1002/jbmr.5650010510

[11] Moossavi M, Parsamanesh N, Mohammadoo-Khorasani M, et al. Positive correlation between vitamin D receptor gene FokI polymorphism and colorectal cancer susceptibility in South-Khorasan of Iran. J Cell Biochem. 2018; 119(10):8190-8194. doi: 10.1002/jcb.26826

[12] Xu Y, He B, Pan Y, et al. Systematic review and meta-analysis on vitamin D receptor polymorphisms and cancer risk. Tumour Biology. 2014; 35(5):4153-4169. doi: 10.1007/s13277-013-1544-y

[13] Gnagnarella P, Pasquali E, Serrano D, Raimondi S, Disalvatore D, Gandini S. Vitamin D receptor polymorphism FokI and cancer risk: A comprehensive meta-analysis. Carcinogenesis. 2014; 35(9):1913-1619. doi: 10.1093/carcin/bgu150

[14] Chen WY, Bertone-Johnson ER, Hunter DJ, Willett WC, Hankinson SE. Associations between polymorphisms in the vitamin D receptor and breast cancer risk. Cancer Epidemiol Biomarkers Prev. 2005; 14(10):2335-2339. doi: 10.1158/1055-9965.Epi-05-0283

[15] Bid HK, Kumar A, Kapoor R, Mittal RD. Association of vitamin D receptor-gene (FokI) polymorphism with calcium oxalate nephrolithiasis. J Endourol. 2005; 19(1):111-115. doi: 10.1089/end.2005.19.111

[16] Jurutka PW, Whitfield GK, Hsieh JC, Thompson PD, Haussler CA, Haussler MR. Molecular nature of the vitamin D receptor and its role in regulation of gene expression. Rev Endocr Metab Disord. 2001; 2(2):203-216. doi: 10.1023/a:1010062929140

[17] Colin EM, Weel AE, Uitterlinden AG, et al. Consequences of vitamin D receptor gene polymorphisms for growth inhibition of cultured human peripheral blood mononuclear cells by 1, 25-dihydroxyvitamin D3. Clin Endocrinol (Oxf). 2000; 52(2):211-216. doi: 10.1046/j.1365-2265.2000.00909.x

[18] Galluzzi L, Pietrocola F, Bravo-San Pedro JM, et al. Autophagy in malignant transformation and cancer progression. EMBO J. 2015; 34(7):856-880. doi: 10.15252/embj.201490784

[19] Debnath J, Gammoh N, Ryan KM. Autophagy and autophagy-related pathways in cancer. Nat Rev Mol Cell Biol. 2023; 24(8):560-575. doi: 10.1038/s41580-023-00585-z

[20] Li W, Zhou C, Yu L, et al. Tumor-derived lactate promotes resistance to bevacizumab treatment by facilitating autophagy enhancer protein RUBCNL expression through histone H3 lysine 18 lactylation (H3K18la) in colorectal cancer. Autophagy. 2024; 20(1):114-130. doi: 10.1080/15548627.2023.2249762

[21] Dong Y, Jin Q, Sun M, et al. CLDN6 inhibits breast cancer metastasis through WIP-dependent actin cytoskeleton-mediated autophagy. J Exp Clin Cancer Res. 2023; 42(1):68. doi: 10.1186/s13046-023-02644-x

[22] Li Y, Cook KL, Yu W, et al. Inhibition of antiestrogen-promoted pro-survival autophagy and tamoxifen resistance in breast cancer through vitamin D receptor. Nutrients. 2021; 13(5):1715. doi: 10.3390/nu13051715

[23] Sharma K, Goehe RW, Di X, et al. A novel cytostatic form of autophagy in sensitization of non-small cell lung cancer cells to radiation by vitamin D and the vitamin D analog, EB 1089. Autophagy. 2014; 10(12):2346-2361. doi: 10.4161/15548627.2014.993283

[24] Amin MB, Greene FL, Edge SB, et al. The eighth edition AJCC cancer staging manual: Continuing to build a bridge from a population-based to a more “personalized” approach to cancer staging. CA Cancer J Clin. 2017; 67(2):93-99. doi: 10.3322/caac.21388

[25] Tavera-Mendoza LE, Westerling T, Libby E, et al. Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells. Proc Natl Acad Sci U S A. 2017; 114(11):E2186-E2194. doi: 10.1073/pnas.1615015114

[26] Gao JP, Xu W, Liu WT, Yan M, Zhu ZG. Tumor heterogeneity of gastric cancer: From the perspective of tumor-initiating cell. World J Gastroenterol. 2018; 24(24):2567-2581. doi: 10.3748/wjg.v24.i24.2567

[27] Ho SWT, Tan P. Dissection of gastric cancer heterogeneity for precision oncology. Cancer Sci. 2019; 110(11):3405-3414. doi: 10.1111/cas.14191

[28] Zhang Y, Li Y, Wei Y, Cong L. Molecular mechanism of vitamin D receptor modulating wnt/β-catenin signaling pathway in gastric cancer. J Cancer. 2023; 14(17):3285-3294. doi: 10.7150/jca.81034

[29] Wen Y, Da M, Zhang Y, Peng L, Yao J, Duan Y. Alterations in vitamin D signaling pathway in gastric cancer progression: A study of vitamin D receptor expression in human normal, premalignant, and malignant gastric tissue. Int J Clin Exp Pathol. 2015; 8(10):13176-13184.

[30] Parsamanesh N, Moossavi M, Tavakkoli T, et al. Positive correlation between vitamin D receptor gene TaqI variant and gastric cancer predisposition in a sample of Iranian population. J Cell Physiol. 2019; 234(9):15044-15047. doi: 10.1002/jcp.28145

[31] Cong L, Wang WB, Liu Q, Du JJ. FokI polymorphism of the vitamin D receptor gene is associated with susceptibility to gastric cancer: A case-control study. Tohoku J Exp Med. 2015; 236(3):219-224. doi: 10.1620/tjem.236.219

[32] Chen Y, Du J, Zhang Z, et al. MicroRNA-346 mediates tumor necrosis factor α-induced downregulation of gut epithelial vitamin D receptor in inflammatory bowel diseases. Inflamm Bowel Dis. 2014; 20(11):1910-1918. doi: 10.1097/mib.0000000000000158

[33] Wang J, Gong M, Fan X, Huang D, Zhang J, Huang C. Autophagy-related signaling pathways in non-small cell lung cancer. Mol Cell Biochem. 2022; 477(2):385-393. doi: 10.1007/s11010-021-04280-5

[34] Pangilinan C, Klionsky DJ, Liang C. Emerging dimensions of autophagy in melanoma. Autophagy. 2024; 20(8):1700-1711. doi: 10.1080/15548627.2024.2330261

[35] Kimmelman AC, White E. Autophagy and tumor metabolism. Cell Metab. 2017; 25(5):1037-1043. doi: 10.1016/j.cmet.2017.04.004

[36] Kung CP, Budina A, Balaburski G, Bergenstock MK, Murphy M. Autophagy in tumor suppression and cancer therapy. Crit Rev Eukaryot Gene Expr. 2011; 21(1):71-100. doi: 10.1615/critreveukargeneexpr.v21.i1.50

[37] Zhang QY, Wu LQ, Zhang T, Han YF, Lin X. Autophagy-mediated HMGB1 release promotes gastric cancer cell survival via RAGE activation of extracellular signal-regulated kinases 1/2. Oncol Rep. 2015; 33(4):1630-1638. doi: 10.3892/or.2015.3782

[38] Qin W, Li C, Zheng W, et al. Inhibition of autophagy promotes metastasis and glycolysis by inducing ROS in gastric cancer cells. Oncotarget. 2015; 6(37):39839-39854. doi: 10.18632/oncotarget.5674

[39] Onorati AV, Dyczynski M, Ojha R, Amaravadi RK. Targeting autophagy in cancer. Cancer. 2018; 124(16):3307-3318. doi: 10.1002/cncr.31335

[40] Gugnoni M, Sancisi V, Manzotti G, Gandolfi G, Ciarrocchi A. Autophagy and epithelial-mesenchymal transition: An intricate interplay in cancer. Cell Death Dis. 2016; 7(12):e2520. doi: 10.1038/cddis.2016.415

[41] Zhang Z, Yu X, Cheng G. Vitamin D sensitizes cervical cancer to radiation-induced apoptosis by inhibiting autophagy through degradation of ambra1. Cell Death Discov. 2025; 11(1):1. doi: 10.1038/s41420-024-02279-7

[42] Seuter S, Neme A, Carlberg C. Epigenome-wide effects of vitamin D and their impact on the transcriptome of human monocytes involve CTCF. Nucleic Acids Res. 2016; 44(9):4090-4104. doi: 10.1093/nar/gkv1519

[43] DeSmet ML, Fleet JC. Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression. J Steroid Biochem Mol Biol. 2017; 173:194-201. doi: 10.1016/j.jsbmb.2017.01.008

[44] Manson JE, Cook NR, Lee IM, et al. Vitamin D supplements and prevention of cancer and cardiovascular disease. N Engl J Med. 2019; 380(1):33-44. doi: 10.1056/NEJMoa1809944