Upregulation of AGR2vH facilitates cholangiocarcinoma cell survival under endoplasmic reticulum stress via the activation of the unfolded protein response pathway

Suwanmanee,Gunticha; Yosudjai,Juthamas; Phimsen,Suchada; Wongkham,Sopit; Jirawatnotai,Siwanon; Kaewkong,Worasak

doi:10.3892/ijmm.2019.4432

Upregulation of AGR2vH facilitates cholangiocarcinoma cell survival under endoplasmic reticulum stress via the activation of the unfolded protein response pathway

Authors:
- Gunticha Suwanmanee
- Juthamas Yosudjai
- Suchada Phimsen
- Sopit Wongkham
- Siwanon Jirawatnotai
- Worasak Kaewkong
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Affiliations: Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand, Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand, Siriraj Center of Research for Excellence for Systems Pharmacology, Department of Pharmacology, Faculty of Medicine, Siriraj Medical School, Mahidol University, Bangkok 10700, Thailand
Published online on: December 18, 2019 https://doi.org/10.3892/ijmm.2019.4432
Pages: 669-677

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Abstract

Cholangiocarcinoma (CCA) is an epithelial cell malignancy arising within the biliary tree in the liver. CCA is usually diagnosed at an advanced stage, subsequent to developing with metastasis. Recently, anterior gradient‑2 (AGR2) was characterized as one of the most highly upregulated genes among all metastasis‑associated genes in highly metastatic CCA cell lines. Previous reports have demonstrated that AGR2 is required for triggering the unfolded protein response (UPR) pathway to support cancer cell survival, particularly under endoplasmic reticulum (ER) stress conditions. A previous study identified an AGR2 short isoform generated by aberrant splicing, AGR2vH, which contributed to the metastatic phenotype of CCA cells. The aim of the present study was to determine the function of AGR2vH in UPR pathway activation to support cancer cell survivability and apoptosis evasion. Subsequent to experimentally inducing ER stress in AGR2vH‑overexpressing CCA cells using tunicamycin, the UPR pathway was activated by the upregulation of UPR marker genes (activating transcription factor 6, eukaryotic initiation factor 2a and spliced X‑box binding protein 1), UPR proteins [binding immunoglobulin protein/glucose‑regulated protein (GRP)78 kDa and phosphorylated eukaryotic translation initiation factor 2a] and UPR downstream targets (GRP94). In addition, the results were verified by AGR2vH knockdown using specific small interfering RNAs. Under ER stress conditions, the overexpression of AGR2vH reduced the number of apoptotic cells by decreasing caspase‑3/7 activity and downregulating C/EBP homologous protein mRNA and B‑cell lymphoma‑2 (Bcl‑2)‑associated X protein expression, whereas the Bcl‑2 protein was upregulated, resulting in a higher number of viable cells. The results of the present study support the previous data that indicate that an oncogenic AGR2vH isoform may not only promote metastasis‑associated phenotypes, but also CCA cell survival and apoptosis evasion, thereby favoring cancer progression.

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1	Shin HR, Oh JK, Masuyer E, Curado MP, Bouvard V, Fang YY, Wiangnon S, Sripa B and Hong ST: Epidemiology of cholangio-carcinoma: An update focusing on risk factors. Cancer Sci. 101:579–585. 2010. View Article : Google Scholar : PubMed/NCBI
2	Sripa B and Pairojkul C: Cholangiocarcinoma: Lessons from thailand. Curr Opin Gastroenterol. 24:349–356. 2008. View Article : Google Scholar : PubMed/NCBI
3	Sripa B, Bethony JM, Sithithaworn P, Kaewkes S, Mairiang E, Loukas A, Mulvenna J, Laha T, Hotez PJ and Brindley PJ: Opisthorchiasis and opisthorchis-associated cholangiocarcinoma in Thailand and Laos. Acta Trop. 120(Suppl 1): S158–S168. 2011. View Article : Google Scholar
4	Uthaisar K, Vaeteewoottacharn K, Seubwai W, Talabnin C, Sawanyawisuth K, Obchoei S, Kraiklang R, Okada S and Wongkham S: Establishment and characterization of a novel human cholangiocarcinoma cell line with high metastatic activity. Oncol Rep. 36:1435–1446. 2016. View Article : Google Scholar : PubMed/NCBI
5	Aberger F, Weidinger G, Grunz H and Richter K: Anterior specification of embryonic ectoderm: The role of the Xenopus cement gland-specific gene XAG-2. Mech Dev. 72:115–130. 1998. View Article : Google Scholar : PubMed/NCBI
6	Petek E, Windpassinger C, Egger H, Kroisel PM and Wagner K: Localization of the human anterior gradient-2 gene (AGR2) to chromosome band 7p21.3 by radiation hybrid mapping and fluorescence in situ hybridisation. Cytogenet Cell Genet. 89:141–142. 2000. View Article : Google Scholar
7	Obacz J, Takacova M, Brychtova V, Dobes P, Pastorekova S, Vojtesek B and Hrstka R: The role of AGR2 and AGR3 in cancer: Similar but not identical. Eur J Cell Biol. 94:139–147. 2015. View Article : Google Scholar : PubMed/NCBI
8	Higa A, Mulot A, Delom F, Bouchecareilh M, Nguyên DT, Boismenu D, Wise MJ and 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. 286:44855–44868. 2011. View Article : Google Scholar : PubMed/NCBI
9	Ramachandran V, Arumugam T, Wang H and Logsdon CD: Anterior gradient 2 is expressed and secreted during the development of pancreatic cancer and promotes cancer cell survival. Cancer Res. 68:7811–7818. 2008. View Article : Google Scholar : PubMed/NCBI
10	Ryu J, Park SG, Lee PY, Cho S, Lee DH, Kim GH, Kim JH and Park BC: Dimerization of pro-oncogenic protein anterior gradient 2 is required for the interaction with BiP/GRP78. Biochem Biophys Res Commun. 430:610–615. 2013. View Article : Google Scholar
11	Oslowski CM and Urano F: Measuring ER stress and the unfolded protein response using mammalian tissue culture system. Methods Enzymol. 490:71–92. 2011. View Article : Google Scholar : PubMed/NCBI
12	Suh DH, Kim MK, Kim HS, Chung HH and Song YS: Unfolded protein response to autophagy as a promising druggable target for anticancer therapy. Ann N Y Acad Sci. 1271:20–32. 2012. View Article : Google Scholar : PubMed/NCBI
13	Eizirik DL, Miani M and Cardozo AK: Signalling danger: Endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation. Diabetologia. 56:234–241. 2013. View Article : Google Scholar
14	Zhu G and Lee AS: Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis. J Cell Physiol. 230:1413–1420. 2015. View Article : Google Scholar
15	Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M and Ron D: Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell. 6:1099–1108. 2000. View Article : Google Scholar : PubMed/NCBI
16	Neeb A, Hefele S, Bormann S, Parson W, Adams F, Wolf P, Miernik A, Schoenthaler M, Kroenig M, Wilhelm K, et al: Splice variant transcripts of the anterior gradient 2 gene as a marker of prostate cancer. Oncotarget. 5:8681–8689. 2014. View Article : Google Scholar : PubMed/NCBI
17	Yosudjai J, Inpad C, Chomwong S, Dana P, Sawanyawisuth K, Phimsen S, Wongkham S, Jirawatnotai S and Kaewkong W: An aberrantly spliced isoform of anterior gradient-2, AGR2vH promotes migration and invasion of cholangiocarcinoma cell. Biomed Pharmacother. 107:109–116. 2018. View Article : Google Scholar : PubMed/NCBI
18	Yosudjai J, Wongkham S, Jirawatnotai S and Kaewkong W: Aberrant mRNA splicing generates oncogenic RNA isoforms and contributes to the development and progression of cholan-giocarcinoma. Biomed Rep. 10:147–155. 2019.PubMed/NCBI
19	Thamrongwaranggoon U, Seubwai W, Phoomak C, Sangkhamanonz S, Cha'on U, Boonmars T and Wongkham S: Targeting hexokinase II as a possible therapy for cholangiocar-cinoma. Biochem Biophys Res Commun. 484:409–415. 2017. View Article : Google Scholar : PubMed/NCBI
20	Nami B, Donmez H and Kocak N: Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells. Exp Toxicol Pathol. 68:419–426. 2016. View Article : Google Scholar : PubMed/NCBI
21	Li Q, Liu Z, Guo J, Chen J, Yang P, Tian J, Sun J, Zong Y and Qu S: Cholesterol overloading leads to hepatic L02 cell damage through activation of the unfolded protein response. Int J Mol Med. 24:459–464. 2009.PubMed/NCBI
22	Dioufa N, Kassi E, Papavassiliou AG and Kiaris H: Atypical induction of the unfolded protein response by mifepristone. Endocrine. 38:167–173. 2010. View Article : Google Scholar : PubMed/NCBI
23	Van Schadewijk A, van't Wout EF, Stolk J and Hiemstra PS: A quantitative method for detection of spliced X-box binding protein-1 (XBP1) mRNA as a measure of endoplasmic reticulum (ER) stress. Cell Stress Chaperones. 17:275–279. 2012. View Article : Google Scholar :
24	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C (T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar
25	Khan A, Gillis K, Clor J and Tyagarajan K: Simplified evaluation of apoptosis using the muse cell analyzer. Postepy Biochem. 58:492–496. 2012.PubMed/NCBI
26	Sačková V, Kuliková L, Kello M, Uhrinová I and Fedoročko P: Enhanced antiproliferative and apoptotic response of HT-29 adenocarcinoma cells to combination of photoactivated hypericin and farnesyltransferase inhibitor manumycin A. Int J Mol Sci. 12:8388–8405. 2011. View Article : Google Scholar
27	Yip NK and Ho WS: Berberine induces apoptosis via the mitochondrial pathway in liver cancer cells. Oncol Rep. 30:1107–1112. 2013. View Article : Google Scholar : PubMed/NCBI
28	Zheng YM, Shen JZ, Wang Y, Lu AX and Ho WS: Anti-oxidant and anti-cancer activities of Angelica dahurica extract via induction of apoptosis in colon cancer cells. Phytomedicine. 23:1267–1274. 2016. View Article : Google Scholar : PubMed/NCBI
29	Wang M and Kaufman RJ: The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 14:581–597. 2014. View Article : Google Scholar : PubMed/NCBI
30	Dumartin L, Alrawashdeh W, Trabulo SM, Radon TP, Steiger K, Feakins RM, di Magliano MP, Heeschen C, Esposito I, Lemoine NR, et al: ER stress protein AGR2 precedes and is involved in the regulation of pancreatic cancer initiation. Oncogene. 36:3094–3103. 2017. View Article : Google Scholar :
31	Ron D and Walter P: Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol. 8:519–529. 2007. View Article : Google Scholar : PubMed/NCBI
32	Yamamoto K, Sato T, Matsui T, Sato M, Okada T, Yoshida H, Harada A and Mori K: Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1. Dev Cell. 13:365–376. 2007. View Article : Google Scholar : PubMed/NCBI
33	Nishitoh H: CHOP is a multifunctional transcription factor in the ER stress response. J Biochem. 151:217–219. 2012. View Article : Google Scholar : PubMed/NCBI