Trefoil factor 3 promotes metastatic seeding and predicts poor survival outcome of patients with mammary carcinoma

Abstract

Introduction: Recurrence or early metastasis remains the predominant cause of mortality in patients with estrogen receptor positive (ER+) mammary carcinoma (MC). However, the molecular mechanisms underlying the initial progression of ER+ MC to metastasis remains poorly understood. Trefoil factor 3 (TFF3) is an estrogen-responsive oncogene in MC. Herein, we provide evidence for a functional role of TFF3 in metastatic progression of ER+ MC. Methods: The association of TFF3 expression with clinicopathological parameters and survival outcome in a cohort of MC patients was assessed by immunohistochemistry. The expression of TFF3 in MCF7 and T47D cells was modulated by forced expression or siRNA-mediated depletion of TFF3. mRNA and protein levels were determined using qPCR and western blot. The functional effect of modulation of TFF3 expression in MC cells was determined in vitro and in vivo. Mechanistic analyses were performed using reporter constructs, modulation of signal transducer and activator of transcription 3 (STAT3) expression, and pharmacological inhibitors against c-SRC and STAT3 activity. Results: TFF3 protein expression was positively associated with larger tumour size, lymph node metastasis, higher stage, and poor survival outcome. Forced expression of TFF3 in ER+ MC cells stimulated colony scattering, cell adhesion to a Collagen I-coated matrix, colony formation on a Collagen I- or Matrigel-coated matrix, endothelial cell adhesion, and transmigration through an endothelial cell barrier. In vivo, forced expression of TFF3 in MCF7 cells stimulated the formation of metastatic nodules in animal lungs. TFF3 regulation of the mRNA levels of epithelial, mesenchymal, and metastatic-related genes in ER+ MC cells were consistent with the altered cell behaviour. Forced expression of TFF3 in ER+ MC cells stimulated phosphorylation of c-SRC that subsequently increased STAT3 activity, which lead to the downregulation of E-cadherin. siRNA-mediated depletion of TFF3 reduced the invasiveness of ER+ MC cells. Conclusions: TFF3 expression predicts metastasis and poor survival outcome of patients with MC and functionally stimulates cellular invasion and metastasis of ER+ MC cells. Adjuvant functional inhibition of TFF3 may therefore be considered to ameliorate outcome of ER+ MC patients.