Overexpression of Ecto-5'-nucleotidase (CD73) promotes T-47D human breast cancer cells invasion and adhesion to extracellular matrix.

Ecto-5'-nucleotidase (CD73) was overexpressed in malignancies of epithelial origin and was involved in a variety of cellular processes such as cytoprotection and anti-inflammation. In the present study, human mammary T-47D cells were transfected with pcDNA-NT5E to establish a CD73 overexpressed cell model. Short small interfering RNA (siRNA) was used to silence the epidermal growth factor receptor (EGFR) gene. Real-time PCR, RT-PCR and western-blot were used to study CD73 and EGFR expression. Surface CD73 activity was assessed by quantifying the conversion of etheno-AMP to ethenoadenosine via HPLC. Interleukin (IL)-8 mRNA and protein expression were analyzed by RT-PCR and ELISA. Cell motility, invasiveness and adhesion to extracellular matrix (ECM) were measured by in vitro invasion and adhesion assay before and after transfection. We demonstrated that abilities of migration, invasions and adhesion to ECM in pcDNA-NT5E transfected T-47D cells increased significantly, which can be blocked by CD73 inhibitor alpha, beta-methylene ADP(APCP). Addition of adenosine reversed the effects of APCP. The mRNA and protein expression of EGFR and IL-8 increased in pcDNA-NT5E transfected T-47D cells. EGFR siRNA down-regulated EGFR expression dramatically, leading to migration and invasion activities inhibition in pcDNA-NT5E transfected T-47D cells. Our results suggest that up-regulated adenosine production, EGFR and IL-8 expression due to overexpressed CD73 may involved in CD73-promoted breast cancer metastasis.