title: Acquisition of epithelial-mesenchymal transition phenotype in the tamoxifen-resistant breast cancer cell: a new role for G protein-coupled estrogen receptor in mediating tamoxifen resistance through cancer-associated fibroblast-derived fibronectin and β1-integrin signaling pathway in tumor cells.
abstract: BACKGROUND
  Acquired tamoxifen resistance remains the major obstacle to breast cancer endocrine therapy. β1-integrin was identified as one of the target genes of G protein-coupled estrogen receptor (GPER), a novel estrogen receptor recognized as an initiator of tamoxifen resistance. Here, we investigated the role of β1-integrin in GPER-mediated tamoxifen resistance in breast cancer.
  
  
  METHODS
  The expression of β1-integrin and biomarkers of epithelial-mesenchymal transition were evaluated immunohistochemically in 53 specimens of metastases and paired primary tumors. The function of β1-integrin was investigated in tamoxifen-resistant (MCF-7R) subclones, derived from parental MCF-7 cells, and MCF-7R β1-integrin-silenced subclones in MTT and Transwell assays. Involved signaling pathways were identified using specific inhibitors and Western blotting analysis.
  
  
  RESULTS
  GPER, β1-integrin and mesenchymal biomarkers (vimentin and fibronectin) expression in metastases increased compared to the corresponding primary tumors; a close expression pattern of β1-integrin and GPER were in metastases. Increased β1-integrin expression was also confirmed in MCF-7R cells compared with MCF-7 cells. This upregulation of β1-integrin was induced by agonists of GPER and blocked by both antagonist and knockdown of it in MCF-7R cells. Moreover, the epidermal growth factor receptor/extracellular regulated protein kinase (EGFR/ERK) signaling pathway was involved in this transcriptional regulation since specific inhibitors of these kinases also reduced the GPER-induced upregulation of β1-integrin. Interestingly, silencing of β1-integrin partially rescued the sensitivity of MCF-7R cells to tamoxifen and the α5β1-integrin subunit is probably responsible for this phenomenon. Importantly, the cell migration and epithelial-mesenchymal transition induced by cancer-associated fibroblasts, or the product of cancer-associated fibroblasts, fibronectin, were reduced by knockdown of β1-integrin in MCF-7R cells. In addition, the downstream kinases of β1-integrin including focal adhesion kinase, Src and AKT were activated in MCF-7R cells and may be involved in the interaction between cancer cells and cancer-associated fibroblasts.
  
  
  CONCLUSIONS
  GPER/EGFR/ERK signaling upregulates β1-integrin expression and activates downstream kinases, which contributes to cancer-associated fibroblast-induced cell migration and epithelial-mesenchymal transition, in MCF-7R cells. GPER probably contributes to tamoxifen resistance via interaction with the tumor microenvironment in a β1-integrin-dependent pattern. Thus, β1-integrin may be a potential target to improve anti-hormone therapy responses in breast cancer patients.
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. yuanjie630805104@163.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing, 400016, China. mliu-hncq@hotmail.com.
authors.forename: Manran
authors.initials: M
authors.lastname: Liu
authors.affiliation: Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing, 400016, China. sunny_6103@163.com.
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. tugang68@126.com.
authors.forename: Gang
authors.initials: G
authors.lastname: Tu
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. zhuqing198897@163.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhu
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. maoshanchen@126.com.
authors.forename: Maoshan
authors.initials: M
authors.lastname: Chen
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. eschenghong@126.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. luo_asa@163.com.
authors.forename: Haojun
authors.initials: H
authors.lastname: Luo
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. 895916380@qq.com.
authors.forename: Weijie
authors.initials: W
authors.lastname: Fu
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. Lizhenhua_35@126.com.
authors.forename: Zhenhua
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Endocrine and Breast Surgery, the First Affiliated Hospital of Chongqing Medical University, #1 You-Yi Rd, Yu-zhong District, Chongqing, 400016, China. guanglunyang@163.com.
authors.forename: Guanglun
authors.initials: G
authors.lastname: Yang
mesh_terms: D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology* / Q000627:therapeutic use; D015415:Biomarkers; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000378:metabolism; D061986:MCF-7 Cells; D011960:Receptors, Estrogen / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology* / Q000627:therapeutic use; D015398:Signal Transduction / Q000187:drug effects; D013629:Tamoxifen / Q000494:pharmacology* / Q000627:therapeutic use; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D018931:Antineoplastic Agents, Hormonal; D015415:Biomarkers; D005353:Fibronectins; D019012:Integrin beta1; D011960:Receptors, Estrogen; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; D066246:ErbB Receptors; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases