title: ARTEMIN synergizes with TWIST1 to promote metastasis and poor survival outcome in patients with ER negative mammary carcinoma.
abstract: BACKGROUND
  ARTEMIN (ARTN) is an estrogen regulated growth factor, the expression of which promotes resistance to antiestrogen therapies and predicts poorer survival outcome of patients with estrogen receptor (ER) positive mammary carcinoma (ER+MC) treated with tamoxifen. ARTN is also expressed in ER negative mammary carcinoma (ER-MC). Herein, we determined the role of ARTN in ER-MC and defined the mechanism of action producing poor patient prognosis.
  
  
  METHODS
  We modulated the expression of ARTN in two ER- (mesenchymal/claudin-low) mammary carcinoma cell lines (BT549 and MDA-MB-231) by forced expression or small interfering RNA (siRNA) mediated depletion. The effects of modulation of ARTN expression were examined by various in vitro measures of oncogenicity, including the expression of TWIST1 messenger RNA (mRNA) and protein. In vitro results were correlated to xenograft studies in immunodeficient mice. Co-expression of ARTN and TWIST1 and their association to poor survival outcome were examined in a cohort of patients with ER-MC. Pathway analysis was performed by pharmacological inhibition of phosphorylation of AKT (pAKT-Ser 473) or modulation of TWIST1 expression.
  
  
  RESULTS
  ARTN expression resulted in ER-MC cells with enhanced mesenchymal characteristics, including increased invasion and a gene expression profile consistent with enhanced mesenchymal phenotype. ARTN stimulated ER-MC cell anchorage independent and 3D matrigel growth, endothelial cell adhesion and transmigration of ER-MC cells through an endothelial cell barrier. Forced expression of ARTN produced a larger, locally invasive tumour mass with tumour emboli that produced distant metastasis. ARTN regulated TWIST1 expression in ER-MC cells and ARTN expression was significantly correlated to TWIST1 expression in a panel of mammary carcinoma cell lines and in a cohort of patients with ER-MC. Low expression of both ARTN and TWIST1 predicted 100% relapse free and overall survival in patients with ER-MC, whereas high expression of both ARTN and TWIST1 was associated with a poor survival outcome. ARTN stimulated an increase in TWIST1 expression via increased AKT activity. siRNA mediated depletion of TWIST1 abrogated ARTN stimulated cellular behaviour associated with metastasis, and forced expression of TWIST1 abrogated the functional effects of ARTN depletion.
  
  
  CONCLUSIONS
  ARTN and TWIST1 synergize to produce a worse outcome in ER-MC and combined inhibition of ARTN and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) may therefore provide a novel therapeutic strategy in this subtype of mammary carcinoma.
authors: Liggins Institute, University of Auckland, 2-6 Park Avenue, Auckland, 1023, New Zealand.
authors: Arindam
authors: A
authors: Banerjee
authors: Zheng-Sheng
authors: ZS
authors: Wu
authors: PengXu
authors: P
authors: Qian
authors: Jian
authors: J
authors: Kang
authors: Vijay
authors: V
authors: Pandey
authors: Dong-Xu
authors: DX
authors: Liu
authors: Tao
authors: T
authors: Zhu
authors: Peter E
authors: PE
authors: Lobie
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality*; D002448:Cell Adhesion / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D004789:Enzyme Activation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009362:Neoplasm Metastasis / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011379:Prognosis; D011485:Protein Binding; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D051879:Twist-Related Protein 1 / Q000235:genetics* / Q000378:metabolism
chemical_list: C490886:ARTN protein, human; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011960:Receptors, Estrogen; C103173:TWIST1 protein, human; D051879:Twist-Related Protein 1; D051057:Proto-Oncogene Proteins c-akt