There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells.

Department of Pathophysiology Faculty of Medicine Masaryk University 625 00 Brno Czech Republic

Regional Centre for Applied Molecular Oncology Masaryk Memorial Cancer Institute 656 53 Brno Czech Republic

Zobrazit více v PubMed

Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, Znaor A, Bray F. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144:1941–1953. PubMed

de Kort S, Masclee AAM, Sanduleanu S, Weijenberg MP, van Herk-Sukel MPP, Oldenhof NJJ, van den Bergh JPW, Haak HR, Janssen-Heijnen ML. Higher risk of colorectal cancer in patients with newly diagnosed diabetes mellitus before the age of colorectal cancer screening initiation. Sci Rep. 2017;7:46527. doi: 10.1038/srep46527. PubMed DOI PMC

Sun L, Yu S. Diabetes mellitus is an independent risk factor for colorectal cancer. Dig Dis Sci. 2012;57:1586–1597. doi: 10.1007/s10620-012-2059-x. PubMed DOI

van de Poll-Franse LV, Haak HR, Coebergh JW, Janssen-Heijnen ML, Lemmens VE. Disease-specific mortality among stage I–III colorectal cancer patients with diabetes: A large population-based analysis. Diabetologia. 2012;55:2163–2172. doi: 10.1007/s00125-012-2555-8. PubMed DOI PMC

Giovannucci E, Harlan DM, Archer MC, Bergenstal RM, Gapstur SM, Habel LA, Pollak M, Regensteiner JG, Yee D. Diabetes and cancer: A consensus report. Diabetes Care. 2010;33:1674–1685. doi: 10.2337/dc10-0666. PubMed DOI PMC

Mills KT, Bellows CF, Hoffman AE, Kelly TN, Gagliardi G. Diabetes mellitus and colorectal cancer prognosis: A meta-analysis. Dis Colon Rectum. 2013;56:1304–1319. doi: 10.1097/DCR.0b013e3182a479f9. PubMed DOI PMC

Sharma A, Ng H, Kumar A, Teli K, Randhawa J, Record J, Maroules M. Colorectal cancer: Histopathologic differences in tumor characteristics between patients with and without diabetes. Clin Colorectal Cancer. 2014;13:54–61. doi: 10.1016/j.clcc.2013.10.002. PubMed DOI

Yang J, Nishihara R, Zhang X, Ogino S, Qian ZR. Energy sensing pathways: Bridging type 2 diabetes and colorectal cancer? J Diabetes Complications. 2017;31:1228–1236. doi: 10.1016/j.jdiacomp.2017.04.012. PubMed DOI PMC

Ariyasu D, Yoshida H, Hasegawa Y. Endoplasmic Reticulum (ER) stress and endocrine disorders. Int J Mol Sci. 2017;18 doi: 10.3390/ijms18020382. PubMed DOI PMC

Dumartin L, Alrawashdeh W, Trabulo SM, Radon TP, Steiger K, Feakins RM, di Magliano MP, Heeschen C, Esposito I, Lemoine NR, Crnogorac-Jurcevic T. ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation. Oncogene. 2017;36:3094–3103. doi: 10.1038/onc.2016.459. PubMed DOI PMC

Brychtova V, Vojtesek B, Hrstka R. Anterior gradient 2: A novel player in tumor cell biology. Cancer Lett. 2011;304:1–7. doi: 10.1016/j.canlet.2010.12.023. PubMed DOI

Tian S, Hu J, Tao K, Wang J, Chu Y, Li J, Liu Z, Ding X, Xu L, Li Q, et al. Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling. Exp Cell Res. 2018;364:198–207. doi: 10.1016/j.yexcr.2018.02.004. PubMed DOI

Higa A, Mulot A, Delom F, Bouchecareilh M, Nguyên DT, Boismenu D, Wise MJ, Chevet E. Role of pro-oncogenic protein disulfide isomerase (PDI) family member anterior gradient 2 (AGR2) in the control of endoplasmic reticulum homeostasis. J Biol Chem. 2011;286:44855–44868. doi: 10.1074/jbc.M111.275529. PubMed DOI PMC

Chevet E, Fessart D, Delom F, Mulot A, Vojtesek B, Hrstka R, Murray E, Gray T, Hupp T. Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development. Oncogene. 2013;32:2499–2509. doi: 10.1038/onc.2012.346. PubMed DOI

Inzucchi SE, Bergenstal RM, Buse JB, Diamant M, Ferrannini E, Nauck M, Peters AL, Tsapas A, Wender R, Matthews DR, American Diabetes Association (ADA); European Association for the Study of Diabetes (EASD) Management of hyperglycemia in type 2 diabetes: A patient-centered approach: Position statement of the American Diabetes Association (ADA) and the European Association for the Study of Diabetes (EASD) Diabetes Care. 2012;35:1364–1379. doi: 10.2337/dc12-0413. PubMed DOI PMC

Higurashi T, Nakajima A. Metformin and colorectal cancer. Front Endocrinol. 2018;9:622. doi: 10.3389/fendo.2018.00622. PubMed DOI PMC

Meng F, Song L, Wang W. Metformin improves overall survival of colorectal cancer patients with diabetes: A meta-analysis. J Diabetes Res. 2017;2017:5063239. doi: 10.1155/2017/5063239. PubMed DOI PMC

Mogavero A, Maiorana MV, Zanutto S, Varinelli L, Bozzi F, Belfiore A, Volpi CC, Gloghini A, Pierotti MA, Gariboldi M. Metformin transiently inhibits colorectal cancer cell proliferation as a result of either AMPK activation or increased ROS production. Sci Rep. 2017;7:15992. doi: 10.1038/s41598-017-16149-z. PubMed DOI PMC

Diaz-Morales N, Iannantuoni F, Escribano-Lopez I, Bañuls C, Rovira-Llopis S, Sola E, Rocha M, Hernandez-Mijares A, Victor VM. Does metformin modulate endoplasmic reticulum stress and autophagy in type 2 diabetic peripheral blood mononuclear cells? Antioxid Redox Signal. 2018;28:1562–1569. doi: 10.1089/ars.2017.7409. PubMed DOI

Kobiela J, Dobrzycka M, Jędrusik P, Kobiela P, Spychalski P, Śledziński Z, Zdrojewski T. Metformin and colorectal cancer-a systematic review. Exp Clin Endocrinol Diabetes. 2019;127:445–454. doi: 10.1055/a-0621-8830. PubMed DOI

Yue W, Wang T, Zachariah E, Lin Y, Yang CS, Xu Q, DiPaola RS, Tan XL. Transcriptomic analysis of pancreatic cancer cells in response to metformin and aspirin: An implication of synergy. Sci Rep. 2015;5:13390. doi: 10.1038/srep13390. PubMed DOI PMC

Arumugam T, Deng D, Bover L, Wang H, Logsdon CD, Ramachandran V. New blocking antibodies against novel AGR2-C4.4A pathway reduce growth and metastasis of pancreatic tumors and increase survival in mice. Mol Cancer Ther. 2015;14:941–951. doi: 10.1158/1535-7163.MCT-14-0470. PubMed DOI PMC

Hrstka R, Bouchalova P, Michalova E, Matoulkova E, Muller P, Coates PJ, Vojtesek B. AGR2 oncoprotein inhibits p38 MAPK and p53 activation through a DUSP10-mediated regulatory pathway. Mol Oncol. 2016;10:652–662. doi: 10.1016/j.molonc.2015.12.003. PubMed DOI PMC

Hrstka R, Brychtova V, Fabian P, Vojtesek B, Svoboda M. AGR2 predicts tamoxifen resistance in postmenopausal breast cancer patients. Dis Markers. 2013;35:207–212. doi: 10.1155/2013/761537. PubMed DOI PMC

Zhang Q, Gu J, Li L, Liu J, Luo B, Cheung HW, Boehm JS, Ni M, Geisen C, Root DE, et al. Control of cyclin D1 and breast tumorigenesis by the EglN2 prolyl hydroxylase. Cancer Cell. 2009;16:413–424. doi: 10.1016/j.ccr.2009.09.029. PubMed DOI PMC

Hrstka R, Nenutil R, Fourtouna A, Maslon MM, Naughton C, Langdon S, Murray E, Larionov A, Petrakova K, Muller P, et al. The pro-metastatic protein anterior gradient-2 predicts poor prognosis in tamoxifen-treated breast cancers. Oncogene. 2010;29:4838–4847. doi: 10.1038/onc.2010.228. PubMed DOI

Ostatná V, Vargová V, Hrstka R, Ďurech M, Vojtěšek B, Paleček E. Effect of His6-tagging of anterior gradient 2 protein on its electro-oxidation. Electrochim Acta. 2014;150:218–222. doi: 10.1016/j.electacta.2014.10.125. DOI

Ma SR, Mao L, Deng WW, Li YC, Bu LL, Yu GT, Zhang WF, Sun ZJ. AGR2 promotes the proliferation, migration and regulates epithelial-mesenchymal transition in salivary adenoid cystic carcinoma. Am J Transl Res. 2017;9:507–519. PubMed PMC

Park K, Chung YJ, So H, Kim K, Park J, Oh M, Jo M, Choi K, Lee EJ, Choi YL, et al. AGR2, a mucinous ovarian cancer marker, promotes cell proliferation and migration. Exp Mol Med. 2011;43:91–100. doi: 10.3858/emm.2011.43.2.011. PubMed DOI PMC

Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: Mechanisms of action and physiological activities. Exp Mol Med. 2016;48:e224. doi: 10.1038/emm.2016.16. PubMed DOI PMC

Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 2007;3:542–545. doi: 10.4161/auto.4600. PubMed DOI

Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) Autophagy. 2016;12:1–222. doi: 10.1080/15548627.2015.1100356. PubMed DOI PMC

Andrzejewski S, Gravel SP, Pollak M, St-Pierre J. Metformin directly acts on mitochondria to alter cellular bioenergetics. Cancer Metab. 2014;2:12. doi: 10.1186/2049-3002-2-12. PubMed DOI PMC

Owen MR, Doran E, Halestrap AP. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 348 Pt. 2000;3:607–614. doi: 10.1042/0264-6021:3480607. PubMed DOI PMC

Larsson SC, Orsini N, Wolk A. Diabetes mellitus and risk of colorectal cancer: A meta-analysis. J Natl Cancer Inst. 2005;97:1679–1687. doi: 10.1093/jnci/dji375. PubMed DOI

Zanders MM, Vissers PA, Haak HR, van de Poll-Franse LV. Colorectal cancer, diabetes and survival: Epidemiological insights. Diabetes Metab. 2014;40:120–127. doi: 10.1016/j.diabet.2013.12.007. PubMed DOI

Kim JH, Lee KJ, Seo Y, Kwon JH, Yoon JP, Kang JY, Lee HJ, Park SJ, Hong SP, Cheon JH, et al. Effects of metformin on colorectal cancer stem cells depend on alterations in glutamine metabolism. Sci Rep. 2018;8:409. doi: 10.1038/s41598-017-18762-4. PubMed DOI PMC

Nangia-Makker P, Yu Y, Vasudevan A, Farhana L, Rajendra SG, Levi E, Majumdar AP. Metformin: A potential therapeutic agent for recurrent colon cancer. PLoS One. 2014;9:e84369. doi: 10.1371/journal.pone.0084369. PubMed DOI PMC

Herzig S, Shaw RJ. AMPK: Guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 2018;19:121–135. doi: 10.1038/nrm.2017.95. PubMed DOI PMC

Emami Riedmaier A, Fisel P, Nies AT, Schaeffeler E, Schwab M. Metformin and cancer: From the old medicine cabinet to pharmacological pitfalls and prospects. Trends Pharmacol Sci. 2013;34:126–135. doi: 10.1016/j.tips.2012.11.005. PubMed DOI

Hrstka R, Murray E, Brychtova V, Fabian P, Hupp TR, Vojtesek B. Identification of an AKT-dependent signalling pathway that mediates tamoxifen-dependent induction of the pro-metastatic protein anterior gradient-2. Cancer Lett. 2013;333:187–193. doi: 10.1016/j.canlet.2013.01.034. PubMed DOI

Li Z, Wu Z, Chen H, Zhu Q, Gao G, Hu L, Negi H, Kamle S, Li D. Induction of anterior gradient 2 (AGR2) plays a key role in insulin-like growth factor-1 (IGF-1)-induced breast cancer cell proliferation and migration. Med Oncol. 2015;32:577. doi: 10.1007/s12032-015-0577-z. PubMed DOI PMC

Matoulkova E, Sommerova L, Pastorek M, Vojtesek B, Hrstka R. Regulation of AGR2 expression via 3′UTR shortening. Exp Cell Res. 2017;356:40–47. PubMed

Pandurangan AK. Potential targets for prevention of colorectal cancer: A focus on PI3K/Akt/mTOR and Wnt pathways. Asian Pac J Cancer Prev. 2013;14:2201–2205. doi: 10.7314/APJCP.2013.14.4.2201. PubMed DOI

Tian SB, Tao KX, Hu J, Liu ZB, Ding XL, Chu YN, Cui JY, Shuai XM, Gao JB, Cai KL, et al. The prognostic value of AGR2 expression in solid tumours: A systematic review and meta-analysis. Sci Rep. 2017;7:15500. doi: 10.1038/s41598-017-15757-z. PubMed DOI PMC

Alves MR, E Melo NC, Barros-Filho MC, do Amaral NS, Silva FIB, Baiocchi Neto G, Soares FA, de Brot Andrade L, Rocha RM. Downregulation of AGR2, p21, and cyclin D and alterations in p53 function were associated with tumor progression and chemotherapy resistance in epithelial ovarian carcinoma. Cancer Med. 2018 doi: 10.1002/cam4.1530. PubMed DOI PMC

Riener MO, Thiesler T, Hellerbrand C, Amann T, Cathomas G, Fritzsche FR, Dahl E, Bahra M, Weichert W, Terracciano L, Kristiansen G. Loss of anterior gradient-2 expression is an independent prognostic factor in colorectal carcinomas. Eur J Cancer. 2014;50:1722–1730. doi: 10.1016/j.ejca.2014.04.012. PubMed DOI

Chen N, Karantza-Wadsworth V. Role and regulation of autophagy in cancer. Biochim Biophys Acta. 2009;1793:1516–1523. doi: 10.1016/j.bbamcr.2008.12.013. PubMed DOI PMC

Madia F, Grossi V, Peserico A, Simone C. Updates from the intestinal front line: Autophagic weapons against inflammation and cancer. Cells. 2012;1:535–557. doi: 10.3390/cells1030535. PubMed DOI PMC

Yang HZ, Ma Y, Zhou Y, Xu LM, Chen XJ, Ding WB, Zou HB. Autophagy contributes to the enrichment and survival of colorectal cancer stem cells under oxaliplatin treatment. Cancer Lett. 2015;361:128–136. doi: 10.1016/j.canlet.2015.02.045. PubMed DOI

Li J, Hou N, Faried A, Tsutsumi S, Kuwano H. Inhibition of autophagy augments 5-fluorouracil chemotherapy in human colon cancer in vitro and in vivo model. Eur J Cancer. 2010;46:1900–1909. doi: 10.1016/j.ejca.2010.02.021. PubMed DOI

Abdelsatir AA, Husain NE, Hassan AT, Elmadhoun WM, Almobarak AO, Ahmed MH. Potential benefit of metformin as treatment for colon cancer: The evidence so far. Asian Pac J Cancer Prev. 2015;16:8053–8058. doi: 10.7314/APJCP.2015.16.18.8053. PubMed DOI

Hur KY, Lee MS. New mechanisms of metformin action: Focusing on mitochondria and the gut. J Diabetes Investig. 2015;6:600–609. doi: 10.1111/jdi.12328. PubMed DOI PMC

Wang L, Wang X, Wang CC. Protein disulfide-isomerase, a folding catalyst and a redox-regulated chaperone. Free Radic Biol Med. 2015;83:305–313. doi: 10.1016/j.freeradbiomed.2015.02.007. PubMed DOI

Schexnayder C, Broussard K, Onuaguluchi D, Poché A, Ismail M, McAtee L, Llopis S, Keizerweerd A, McFerrin H, Williams C. Metformin inhibits migration and invasion by suppressing ROS production and COX2 expression in MDA-MB-231 breast cancer cells. Int J Mol Sci. 2018;19 doi: 10.3390/ijms19113692. PubMed DOI PMC

Queiroz EA, Puukila S, Eichler R, Sampaio SC, Forsyth HL, Lees SJ, Barbosa AM, Dekker RF, Fortes ZB, Khaper N. Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells. PLoS One. 2014;9:e98207. doi: 10.1371/journal.pone.0098207. PubMed DOI PMC

Ramachandran V, Arumugam T, Wang H, Logsdon CD. Anterior gradient 2 is expressed and secreted during the development of pancreatic cancer and promotes cancer cell survival. Cancer Res. 2008;68:7811–7818. doi: 10.1158/0008-5472.CAN-08-1320. PubMed DOI PMC

Tiemann K, Garri C, Lee SB, Malihi PD, Park M, Alvarez RM, Yap LP, Mallick P, Katz JE, Gross ME, Kani K. Loss of ER retention motif of AGR2 can impact mTORC signaling and promote cancer metastasis. Oncogene. 2019;38:3003–3018. doi: 10.1038/s41388-018-0638-9. PubMed DOI PMC

Maurel M, Obacz J, Avril T, Ding YP, Papadodima O, Treton X, Daniel F, Pilalis E, Hörberg J, Hou W, et al. Control of anterior GRadient 2 (AGR2) dimerization links endoplasmic reticulum proteostasis to inflammation. EMBO Mol Med. 2019;11 doi: 10.15252/emmm.201810120. PubMed DOI PMC

Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med. 2005;352:476–487. doi: 10.1056/NEJMra040958. PubMed DOI

Miranda VC, Braghiroli MI, Faria LD, Bariani G, Alex A, Bezerra Neto JE, Capareli FC, Sabbaga J, Lobo Dos Santos JF, Hoff PM, Riechelmann RP. Phase 2 trial of metformin combined with 5-Fluorouracil in patients with refractory metastatic colorectal cancer. Clin Colorectal Cancer. 2016;15:321.e1–328.e1. doi: 10.1016/j.clcc.2016.04.011. PubMed DOI

Richard SM, Martinez Marignac VL. Sensitization to oxaliplatin in HCT116 and HT29 cell lines by metformin and ribavirin and differences in response to mitochondrial glutaminase inhibition. J Cancer Res Ther. 2015;11:336–340. doi: 10.4103/0973-1482.157317. PubMed DOI

Characterization of the AGR2-NPM3 axis uncovers the AGR2 involvement in PD-L1 regulation in colorectal cancer