Metformin inhibits TGF‑β1‑induced epithelial‑mesenchymal transition and liver metastasis of pancreatic cancer cells

Yoshida,Juichiro; Ishikawa,Takeshi; Endo,Yuki; Matsumura,Shinya; Ota,Takayuki; Mizushima,Katsura; Hirai,Yasuko; Oka,Kaname; Okayama,Tetsuya; Sakamoto,Naoyuki; Inoue,Ken; Kamada,Kazuhiro; Uchiyama,Kazuhiko; Takagi,Tomohisa; Naito,Yuji; Itoh,Yoshito

doi:10.3892/or.2020.7595

Metformin inhibits TGF‑β1‑induced epithelial‑mesenchymal transition and liver metastasis of pancreatic cancer cells

Authors:
- Juichiro Yoshida
- Takeshi Ishikawa
- Yuki Endo
- Shinya Matsumura
- Takayuki Ota
- Katsura Mizushima
- Yasuko Hirai
- Kaname Oka
- Tetsuya Okayama
- Naoyuki Sakamoto
- Ken Inoue
- Kazuhiro Kamada
- Kazuhiko Uchiyama
- Tomohisa Takagi
- Yuji Naito
- Yoshito Itoh
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Affiliations: Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto 602‑8566, Japan
Published online on: April 23, 2020 https://doi.org/10.3892/or.2020.7595
Pages: 371-381

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Abstract

Epithelial‑mesenchymal transition (EMT) is considered a crucial event in the development of cancer metastasis. Metformin is a drug used in the treatment of type 2 diabetes. Recently, increasing evidence has indicated that metformin possesses anti‑tumor activities. However, the effects of metformin on EMT and metastases in pancreatic cancer remain unknown. Thus, the present study investigated whether metformin inhibits EMT of human pancreatic cancer cell lines. Pancreatic cancer cells were stimulated with transforming growth factor β1 (TGF‑β1), an activator of EMT signaling, with or without metformin. After 48 h, the levels of epithelial and mesenchymal markers were evaluated by western blot analysis, immunocytochemistry and RT‑qPCR. Cancer cell migration was evaluated by an in vitro wound healing assay. The cells stimulated with TGF‑β1 acquired an elongated and fusiform morphology, which was inhibited by metformin. The wound healing assay revealed that metformin significantly suppressed the TGF‑β1‑stimulated migration of pancreatic cancer cells. Following treatment with metformin, E‑cadherin expression (epithelial marker) was upregulated, and the levels of mesenchymal markers were downregulated, which had been increased by TGF‑β1 in these cells. Exposure of the cells to TGF‑β1 activated the Smad2/3 and Akt/mammalian target of rapamycin (mTOR) pathways, and this effect was inhibited by metformin, suggesting that metformin inhibits TGF‑β1‑induced‑EMT through the down‑regulation of the Smad pathway in PANC‑1 cells and the downregulation of the Akt/mTOR pathway in BxPC‑3 cells. In an animal model of surgical orthotopic implantation, metformin inhibited liver metastasis without a significant reduction in the size of the primary pancreatic tumor. On the whole, the findings of the present study suggest that metformin inhibits EMT and cancer metastasis through the Smad or Akt/mTOR pathway.

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1	Wolfgang CL, Herman JM, Laheru DA, Klein AP, Erdek MA, Fishman EK and Hruban RH: Recent progress in pancreatic cancer. CA Cancer J Clin. 63:318–348. 2013. View Article : Google Scholar : PubMed/NCBI
2	Andersen DK, Korc M, Petersen GM, Eibl G, Li D, Rickels MR, Chari ST and Abbruzzese J: Diabetes, pancreatogenic diabetes, and pancreatic cancer. Diabetes. 66:1103–1110. 2017. View Article : Google Scholar : PubMed/NCBI
3	He H, Ke R, Lin H, Ying Y, Liu D and Luo Z: Metformin, an old drug, brings a new era to cancer therapy. Cancer J. 21:70–74. 2015. View Article : Google Scholar : PubMed/NCBI
4	Franciosi M, Lucisano G, Lapice E, Strippoli GF, Pellegrini F and Nicolucci A: Metformin therapy and risk of cancer in patients with type 2 diabetes: Systematic review. PLoS One. 8:e715832013. View Article : Google Scholar : PubMed/NCBI
5	Zhang P, Li H, Tan X, Chen L and Wang S: Association of metformin use with cancer incidence and mortality: A meta-analysis. Cancer Epidemiol. 37:207–218. 2013. View Article : Google Scholar : PubMed/NCBI
6	Bayraktar S, Hernadez-Aya LF, Lei X, Meric-Bernstam F, Litton JK, Hsu L, Hortobagyi GN and Gonzalez-Angulo AM: Effect of metformin on survival outcomes in diabetic patients with triple receptor-negative breast cancer. Cancer. 118:1202–1211. 2012. View Article : Google Scholar : PubMed/NCBI
7	Wan G, Sun X, Li F, Wang X, Li C, Li H, Yu X and Cao F: Survival benefit of metformin adjuvant treatment for pancreatic cancer patients: A systematic review and meta-analysis. Cell Physiol Biochem. 49:837–847. 2018. View Article : Google Scholar : PubMed/NCBI
8	Broadhurst PJ and Hart AR: Metformin as an adjunctive therapy for pancreatic cancer: A review of the literature on its potential therapeutic use. Dig Dis Sci. 63:2840–2852. 2018. View Article : Google Scholar : PubMed/NCBI
9	Yang J and Weinberg RA: Epithelial-mesenchymal transition: At the crossroads of development and tumor metastasis. Dev Cell. 14:818–829. 2008. View Article : Google Scholar : PubMed/NCBI
10	Lamouille S, Xu J and Derynck R: Molecular mechanisms of epithelial-mesenchymal transition. Nat Rev Mol Cell Biol. 15:178–196. 2014. View Article : Google Scholar : PubMed/NCBI
11	Jie XX, Zhang XY and Xu CJ: Epithelial-to-mesenchymal transition, circulating tumor cells and cancer metastasis: Mechanisms and clinical applications. Oncotarget. 8:81558–81571. 2017. View Article : Google Scholar : PubMed/NCBI
12	Okajima M, Kokura S, Ishikawa T, Mizushima K, Tsuchiya R, Matsuyama T, Adachi S, Okayama T, Sakamoto N, Kamada K, et al: Anoxia/reoxygenation induces epithelial-mesenchymal transition in human colon cancer cell lines. Oncol Rep. 29:2311–2317. 2013. View Article : Google Scholar : PubMed/NCBI
13	Wang Y, Wu Z and Hu L: The regulatory effects of metformin on the [SNAIL/miR-34]:[ZEB/miR-200] system in the epithelial-mesenchymal transition(EMT) for colorectal cancer(CRC). Eur J Pharmacol. 834:45–53. 2018. View Article : Google Scholar : PubMed/NCBI
14	Valaee S, Yaghoobi MM and Shamsara M: Metformin inhibits gastric cancer cells metastatic traits through suppression of epithelial-mesenchymal transition in a glucose-independent manner. PLoS One. 12:e01744862017. View Article : Google Scholar : PubMed/NCBI
15	Tong D, Liu Q, Liu G, Xu J, Lan W, Jiang Y, Xiao H, Zhang D and Jiang J: Metformin inhibits castration-induced EMT in prostate cancer by repressing COX2/PGE2/STAT3 axis. Cancer Lett. 389:23–32. 2017. View Article : Google Scholar : PubMed/NCBI
16	Song Y, Chen Y, Li Y, Lyu X, Cui J, Cheng Y, Zhao L and Zhao G: Metformin inhibits TGF-beta1-induced epithelial-to-mesenchymal transition-like process and stem-like properties in GBM via AKT/mTOR/ZEB1 pathway. Oncotarget. 9:7023–7035. 2018. View Article : Google Scholar : PubMed/NCBI
17	Zhao Z, Cheng X, Wang Y, Han R, Li L, Xiang T, He L, Long H, Zhu B and He Y: Metformin inhibits the IL-6-induced epithelial-mesenchymal transition and lung adenocarcinoma growth and metastasis. PLoS One. 9:e958842014. View Article : Google Scholar : PubMed/NCBI
18	Nakayama A, Ninomiya I, Harada S, Tsukada T, Okamoto K, Nakanuma S, Sakai S, Makino I, Kinoshita J, Hayashi H, et al: Metformin inhibits the radiation-induced invasive phenotype of esophageal squamous cell carcinoma. Int J Oncol. 49:1890–1898. 2016. View Article : Google Scholar : PubMed/NCBI
19	Li NS, Zou JR, Lin H, Ke R, He XL, Xiao L, Huang D, Luo L, Lv N and Luo Z: LKB1/AMPK inhibits TGF-β1 production and the TGF-beta signaling pathway in breast cancer cells. Tumour Biol. 37:8249–8258. 2016. View Article : Google Scholar : PubMed/NCBI
20	Uchiyama K, Naito Y, Takagi T, Mizushima K, Hayashi N, Harusato A, Hirata I, Omatsu T, Handa O, Ishikawa T, et al: Carbon monoxide enhance colonic epithelial restitution via FGF15 derived from colonic myofibroblasts. Biochem Biophys Res Commun. 391:1122–1126. 2010. View Article : Google Scholar : PubMed/NCBI
21	Kimura-Tsuchiya R, Ishikawa T, Kokura S, Mizushima K, Adachi S, Okajima M, Matsuyama T, Okayama T, Sakamoto N, Katada K, et al: The inhibitory effect of heat treatment against epithelial-mesenchymal transition (EMT) in human pancreatic adenocarcinoma cell lines. J Clin Biochem Nutr. 55:56–61. 2014. View Article : Google Scholar : PubMed/NCBI
22	Hwang HK, Murakami T, Kiyuna T, Kim SH, Lee SH, Kang CM, Hoffman RM and Bouvet M: Splenectomy is associated with an aggressive tumor growth pattern and altered host immunity in an orthotopic syngeneic murine pancreatic cancer model. Oncotarget. 8:88827–88834. 2017. View Article : Google Scholar : PubMed/NCBI
23	Li D, Yeung SC, Hassan MM, Konopleva M and Abbruzzese JL: Antidiabetic therapies affect risk of pancreatic cancer. Gastroenterology. 137:482–488. 2009. View Article : Google Scholar : PubMed/NCBI
24	Shi Y, He Z, Jia Z and Xu C: Inhibitory effect of metformin combined with gemcitabine on pancreatic cancer cells in vitro and in vivo. Mol Med Rep. 14:2921–2928. 2016. View Article : Google Scholar : PubMed/NCBI
25	Kisfalvi K, Moro A, Sinnett-Smith J, Eibl G and Rozengurt E: Metformin inhibits the growth of human pancreatic cancer xenografts. Pancreas. 42:781–785. 2013. View Article : Google Scholar : PubMed/NCBI
26	Algire C, Amrein L, Bazile M, David S, Zakikhani M and Pollak M: Diet and tumor LKB1 expression interact to determine sensitivity to anti-neoplastic effects of metformin in vivo. Oncogene. 30:1174–1182. 2011. View Article : Google Scholar : PubMed/NCBI
27	Jalving M, Gietema JA, Lefrandt JD, de Jong S, Reyners AK, Gans RO and de Vries EG: Metformin: Taking away the candy for cancer? Eur J Cancer. 46:2369–2380. 2010. View Article : Google Scholar : PubMed/NCBI
28	Schneider MB, Matsuzaki H, Haorah J, Ulrich A, Standop J, Ding XZ, Adrian TE and Pour PM: Prevention of pancreatic cancer induction in hamsters by metformin. Gastroenterology. 120:1263–1270. 2001. View Article : Google Scholar : PubMed/NCBI
29	Muniraj T and Chari ST: Diabetes and pancreatic cancer. Minerva Gastroenterol Dietol. 58:331–345. 2012.PubMed/NCBI
30	Yue W, Yang CS, DiPaola RS and Tan XL: Repurposing of metformin and aspirin by targeting AMPK-mTOR and inflammation for pancreatic cancer prevention and treatment. Cancer Prev Res (Phila). 7:388–397. 2014. View Article : Google Scholar : PubMed/NCBI
31	Ben Sahra I, Laurent K, Loubat A, Giorgetti-Peraldi S, Colosetti P, Auberger P, Tanti JF, Le Marchand-Brustel Y and Bost F: The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene. 27:3576–3586. 2008. View Article : Google Scholar : PubMed/NCBI
32	Lund SS, Tarnow L, Stehouwer CD, Schalkwijk CG, Teerlink T, Gram J, Winther K, Frandsen M, Smidt UM, Pedersen O, et al: Impact of metformin versus repaglinide on non-glycaemic cardiovascular risk markers related to inflammation and endothelial dysfunction in non-obese patients with type 2 diabetes. Eur J Endocrinol. 158:631–641. 2008. View Article : Google Scholar : PubMed/NCBI
33	Buzzai M, Jones RG, Amaravadi RK, Lum JJ, DeBerardinis RJ, Zhao F, Viollet B and Thompson CB: Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Res. 67:6745–6752. 2007. View Article : Google Scholar : PubMed/NCBI
34	Zhang YE: Non-Smad pathways in TGF-beta signaling. Cell Res. 19:128–139. 2009. View Article : Google Scholar : PubMed/NCBI
35	Hoot KE, Lighthall J, Han G, Lu SL, Li A, Ju W, Kulesz-Martin M, Bottinger E and Wang XJ: Keratinocyte-specific Smad2 ablation results in increased epithelial-mesenchymal transition during skin cancer formation and progression. J Clin Invest. 118:2722–2732. 2008.PubMed/NCBI
36	Lamouille S and Derynck R: Cell size and invasion in TGF-beta-induced epithelial to mesenchymal transition is regulated by activation of the mTOR pathway. J Cell Biol. 178:437–451. 2007. View Article : Google Scholar : PubMed/NCBI
37	Lamouille S, Connolly E, Smyth JW, Akhurst RJ and Derynck R: TGF-β-induced activation of mTOR complex 2 drives epithelial-mesenchymal transition and cell invasion. J Cell Sci. 125:1259–1273. 2012. View Article : Google Scholar : PubMed/NCBI
38	Julien S, Puig I, Caretti E, Bonaventure J, Nelles L, van Roy F, Dargemont C, de Herreros AG, Bellacosa A and Larue L: Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition. Oncogene. 26:7445–7456. 2007. View Article : Google Scholar : PubMed/NCBI
39	Shin HS, Ko J, Kim DA, Ryu ES, Ryu HM, Park SH, Kim YL, Oh ES and Kang DH: Metformin ameliorates the phenotype transition of peritoneal mesothelial cells and peritoneal fibrosis via a modulation of oxidative stress. Sci Rep. 7:56902017. View Article : Google Scholar : PubMed/NCBI
40	Thakur S, Viswanadhapalli S, Kopp JB, Shi Q, Barnes JL, Block K, Gorin Y and Abboud HE: Activation of AMP-activated protein kinase prevents TGF-β1-induced epithelial-mesenchymal transition and myofibroblast activation. Am J Pathol. 185:2168–2180. 2015. View Article : Google Scholar : PubMed/NCBI
41	Duan W, Qian W, Zhou C, Cao J, Qin T, Xiao Y, Cheng L, Li J, Chen K, Li X, et al: Metformin suppresses the invasive ability of pancreatic cancer cells by blocking autocrine TGF-β1 signaling. Oncol Rep. 40:1495–1502. 2018.PubMed/NCBI
42	Tan XL, Bhattacharyya KK, Dutta SK, Bamlet WR, Rabe KG, Wang E, Smyrk TC, Oberg AL, Petersen GM and Mukhopadhyay D: Metformin suppresses pancreatic tumor growth with inhibition of NFkappaB/STAT3 inflammatory signaling. Pancreas. 44:636–647. 2015. View Article : Google Scholar : PubMed/NCBI
43	Odawara M, Kawamori R, Tajima N, Iwamoto Y, Kageyama S, Yodo Y, Ueki F and Hotta N: Long-term treatment study of global standard dose metformin in Japanese patients with type 2 diabetes mellitus. Diabetol Int. 8:286–295. 2017. View Article : Google Scholar : PubMed/NCBI