AREG mediates the epithelial‑mesenchymal transition in pancreatic cancer cells via the EGFR/ERK/NF‑κB signalling pathway

Wang,Li; Wang,Lili; Zhang,Hui; Lu,Junliang; Zhang,Zhiwen; Wu,Huanwen; Liang,Zhiyong

doi:10.3892/or.2020.7523

AREG mediates the epithelial‑mesenchymal transition in pancreatic cancer cells via the EGFR/ERK/NF‑κB signalling pathway

Authors:
- Li Wang
- Lili Wang
- Hui Zhang
- Junliang Lu
- Zhiwen Zhang
- Huanwen Wu
- Zhiyong Liang
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Affiliations: Department of Pathology, Peking Union Medical College Hospital, Research Center for Molecular Pathology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, P.R. China
Published online on: February 27, 2020 https://doi.org/10.3892/or.2020.7523
Pages: 1558-1568
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Amphiregulin (AREG) is a member of the epidermal growth factor (EGF) family and is expressed in a plethora of cancers. The biological roles of AREG in the regulation of the epithelial‑mesenchymal transition (EMT) in pancreatic cancer remain unclear. To investigate the expression of epidermal growth factor receptor (EGFR) and AREG in pancreatic cancer cell lines, RT‑qPCR, western blot analysis, and ELISA were performed. RNAi and exogenous AREG treatment were used to alter AREG expression. Wound‑healing and Transwell assays were performed to evaluate cell migration and invasion abilities. Western blot analysis and immunofluorescence staining were utilized to detect the expression of EMT markers. The protein expression of potential key factors involved in EMT, as well as those of the ERK, AKT, STAT3 and NF‑κB pathways, were analysed by western blotting. The role of AREG in tumour growth in vivo was further determined using an orthotopic model of pancreatic cancer. Knockdown of AREG inhibited AsPC‑1 cell migration and invasion. AREG knockdown upregulated E‑cadherin but downregulated vimentin, Snail and Slug expression in AsPC‑1 cells. In addition, AREG stimulation increased cell migration, invasion and EMT in PANC‑1 cells, and an NF‑κB inhibitor decreased AREG‑induced cell migration, invasion and EMT in PANC‑1 cells. AREG stimulation increased the nuclear accumulation of NF‑κB through the EGFR/ERK signalling pathway to induce EMT. Tumour growth and metastasis were decreased by AREG silencing in an orthotopic model of pancreatic cancer. AREG may play a critical role in cell migration, invasion, and EMT by activating the EGFR/ERK/NF‑κB signalling pathway in pancreatic cancer cells.

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1	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2019. CA Cancer J Clin. 69:7–34. 2019. View Article : Google Scholar : PubMed/NCBI
2	Amrutkar M and Gladhaug I: Pancreatic cancer chemoresistance to gemcitabine. Cancers. 9:1572017. View Article : Google Scholar :
3	Du B and Shim JS: Targeting Epithelial-mesenchymal transition (EMT) to overcome drug resistance in cancer. Molecules. 21(pii): E9652016. View Article : Google Scholar : PubMed/NCBI
4	Arumugam T, Ramachandran V, Fournier KF, Wang H, Marquis L, Abbruzzese JL, Gallick GE, Logsdon CD, McConkey DJ and Choi W: Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. Cancer Res. 69:5820–5828. 2009. View Article : Google Scholar : PubMed/NCBI
5	Bronsert P, Enderle-Ammour K, Bader M, Timme S, Kuehs M, Csanadi A, Kayser G, Kohler I, Bausch D, Hoeppner J, et al: Cancer cell invasion and EMT marker expression: A three-dimensional study of the human cancer-host interface. J Pathol. 234:410–422. 2014. View Article : Google Scholar : PubMed/NCBI
6	Wang L, Wu H, Wang L, Zhang H, Lu J, Liang Z and Liu T: Asporin promotes pancreatic cancer cell invasion and migration by regulating the epithelial-to-mesenchymal transition (EMT) through both autocrine and paracrine mechanisms. Cancer Lett. 398:24–36. 2017. View Article : Google Scholar : PubMed/NCBI
7	Rhim AD, Mirek ET, Aiello NM, Maitra A, Bailey JM, McAllister F, Reichert M, Beatty GL, Rustgi AK, Vonderheide RH, et al: EMT and dissemination precede pancreatic tumor formation. Cell. 148:349–361. 2012. View Article : Google Scholar : PubMed/NCBI
8	Fischer KR, Durrans A, Lee S, Sheng J, Li F, Wong ST, Choi H, El Rayes T, Ryu S, Troeger J, et al: Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature. 527:472–476. 2015. View Article : Google Scholar : PubMed/NCBI
9	Zheng X, Carstens JL, Kim J, Scheible M, Kaye J, Sugimoto H, Wu CC, LeBleu VS and Kalluri R: Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature. 527:525–530. 2015. View Article : Google Scholar : PubMed/NCBI
10	So WK, Fan Q, Lau MT, Qiu X, Cheng JC and Leung PC: Amphiregulin induces human ovarian cancer cell invasion by down-regulating E-cadherin expression. FEBS Lett. 588:3998–4007. 2014. View Article : Google Scholar : PubMed/NCBI
11	Willmarth NE and Ethier SP: Autocrine and juxtacrine effects of amphiregulin on the proliferative, invasive, and migratory properties of normal and neoplastic human mammary epithelial cells. J Biol Chem. 281:37728–37737. 2006. View Article : Google Scholar : PubMed/NCBI
12	Thøgersen VB, Sorensen BS, Poulsen SS, Orntoft TF, Wolf H and Nexø E: A subclass of HER1 ligands is a prognostic marker for survival in bladder cancer patients. Cancer Res. 61:6227–6233. 2001.PubMed/NCBI
13	Ishikawa N, Daigo Y, Takano A, Taniwaki M, Kato T, Hayama S, Murakami H, Takeshima Y, Inai K, Nishimura H, et al: Increases of amphiregulin and transforming growth factor-alpha in serum as predictors of poor response to gefitinib among patients with advanced non-small cell lung cancers. Cancer Res. 65:9176–9184. 2005. View Article : Google Scholar : PubMed/NCBI
14	Jing C, Jin YH, You Z, Qiong Q and Jun Z: Prognostic value of amphiregulin and epiregulin mRNA expression in metastatic colorectal cancer patients. Oncotarget. 7:55890–55899. 2016. View Article : Google Scholar : PubMed/NCBI
15	Shinomiya H, Ito Y, Kubo M, Yonezawa K, Otsuki N, Iwae S, Inagaki H and Nibu KI: Expression of amphiregulin in mucoepidermoid carcinoma of the major salivary glands: A molecular and clinicopathological study. Hum Pathol. 57:37–44. 2016. View Article : Google Scholar : PubMed/NCBI
16	Wang L, Wu H, Wang L, Lu J, Duan H, Liu X and Liang Z: Expression of amphiregulin predicts poor outcome in patients with pancreatic ductal adenocarcinoma. Diagn Pathol. 11:602016. View Article : Google Scholar : PubMed/NCBI
17	Zhang H, Wu H, Guan J, Wang L, Ren X, Shi X, Liang Z and Liu T: Paracrine SDF-1α signaling mediates the effects of PSCs on GEM chemoresistance through an IL-6 autocrine loop in pancreatic cancer cells. Oncotarget. 6:3085–3097. 2015.PubMed/NCBI
18	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 : PubMed/NCBI
19	Gao S, Sun Y, Zhang X, Hu L, Liu Y, Chua CY, Phillips LM, Ren H, Fleming JB, Wang H, et al: IGFBP2 Activates the NF-κB pathway to drive epithelial-mesenchymal transition and invasive character in pancreatic ductal adenocarcinoma. Cancer Res. 76:6543–6554. 2016. View Article : Google Scholar : PubMed/NCBI
20	Nomura A, Majumder K, Giri B, Dauer P, Dudeja V, Roy S, Banerjee S and Saluja AK: Inhibition of NF-kappa B pathway leads to deregulation of epithelial-mesenchymal transition and neural invasion in pancreatic cancer. Lab Invest. 96:1268–1278. 2016. View Article : Google Scholar : PubMed/NCBI
21	Nieto MA, Huang RY, Jackson RA and Thiery JP: EMT: 2016. Cell. 166:21–45. 2016. View Article : Google Scholar : PubMed/NCBI
22	Busser B, Sancey L, Brambilla E, Coll JL and Hurbin A: The multiple roles of amphiregulin in human cancer. Biochim Biophys Acta. 1816:119–131. 2011.PubMed/NCBI
23	Liu JF, Tsao YT and Hou CH: Amphiregulin enhances intercellular adhesion molecule-1 expression and promotes tumor metastasis in human osteosarcoma. Oncotarget. 6:40880–40895. 2015. View Article : Google Scholar : PubMed/NCBI
24	Chung E, Cook PW, Parkos CA, Park YK, Pittelkow MR and Coffey RJ: Amphiregulin causes functional downregulation of adherens junctions in psoriasis. J Invest Dermatol. 124:1134–1140. 2005. View Article : Google Scholar : PubMed/NCBI
25	Coradini D, Casarsa C and Oriana S: Epithelial cell polarity and tumorigenesis: New perspectives for cancer detection and treatment. Acta Pharmacol Sin. 32:552–564. 2011. View Article : Google Scholar : PubMed/NCBI
26	Kleeff J, Shi X, Bode HP, Hoover K, Shrikhande S, Bryant PJ, Korc M, Büchler MW and Friess H: Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer. Pancreas. 23:259–265. 2001. View Article : Google Scholar : PubMed/NCBI
27	Peinado H, Olmeda D and Cano A: Snail, Zeb and bHLH factors in tumour progression: An alliance against the epithelial phenotype? Nat Rev Cancer. 7:415–428. 2007. View Article : Google Scholar : PubMed/NCBI
28	Batlle E, Sancho E, Francí C, Domínguez D, Monfar M, Baulida J and García De Herreros A: The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol. 2:84–89. 2000. View Article : Google Scholar : PubMed/NCBI
29	Guaita S, Puig I, Francı́ C, Garrido M, Dominguez D, Batlle E, Sancho E, Dedhar S, De Herreros AG and Baulida J: Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression. J Biol Chem. 277:39209–39216. 2002. View Article : Google Scholar : PubMed/NCBI
30	Yang HW, Menon LG, Black PM, Carroll RS and Johnson MD: SNAI2/Slug promotes growth and invasion in human gliomas. BMC Cancer. 10:3012010. View Article : Google Scholar : PubMed/NCBI
31	Liang C, Wang Z, Li YY, Yu BH, Zhang F and Li HY: miR-33a suppresses the nuclear translocation of β-catenin to enhance gemcitabine sensitivity in human pancreatic cancer cells. Tumor Biol. 36:9395–9403. 2015. View Article : Google Scholar
32	von Ahrens D, Bhagat TD, Nagrath D, Maitra A and Verma A: The role of stromal cancer-associated fibroblasts in pancreatic cancer. J Hematol Oncol. 10:762017. View Article : Google Scholar : PubMed/NCBI
33	Duner S, Lopatko LJ, Ansari D, Gundewar C and Andersson R: Pancreatic cancer: The role of pancreatic stellate cells in tumor progression. Pancreatology. 10:673–681. 2010. View Article : Google Scholar : PubMed/NCBI
34	Vonlaufen A, Joshi S, Qu C, Phillips PA, Xu Z, Parker NR, Toi CS, Pirola RC, Wilson JS, Goldstein D and Apte MV: Pancreatic stellate cells: Partners in crime with pancreatic cancer cells. Cancer Res. 68:2085–2093. 2008. View Article : Google Scholar : PubMed/NCBI
35	Kikuta K, Masamune A, Watanabe T, Ariga H, Itoh H, Hamada S, Satoh K, Egawa S, Unno M and Shimosegawa T: Pancreatic stellate cells promote epithelial-mesenchymal transition in pancreatic cancer cells. Biochem Biophys Res Commun. 403:380–384. 2010. View Article : Google Scholar : PubMed/NCBI
36	Saito K, Sakaguchi M, Maruyama S, Iioka H, Putranto EW, Sumardika IW, Tomonobu N, Kawasaki T, Homma K and Kondo E: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction. J Cancer. 9:2916–2929. 2018. View Article : Google Scholar : PubMed/NCBI