Among breast cancer patients, 10-20 % have triple-negative breast tumors (TNBC) and bear the worst prognosis due to the lack of targetable proteins: ER, PR, and Her2. CD73 is a nucleotidase, which converts AMP to adenosine and involved in many processes, such as promotion of cancer cell proliferation, migration and tumor immunoescape. We and others have shown previously that TNBC patients with low tumor TLR9 expression had significantly worse breast cancer-specific survival, as compared with TNBC patients whose tumors had high TLR9 expression upon diagnosis. TLR9 and CD73 expression have demonstrated mutual regulation in immune cells in certain disease conditions, such as diabetes. However, the mutual relationship of these proteins has not been investigated in cancer. The aim of this study was to investigate whether CD73 and TLR9 have mutual effects in TNBC.
The effect of hypoxia (1% O2) on viability and migration of murine 4T1 control and CD73 shRNA murine tumor cells was analyzed with WST-8 and scratch wound experiments. CD73, four adenosine receptors and TLR9 gene expressions was studied with qPCR. Immunofluorescence was used to study TLR9 localization and actin organization. Additionally, expression of TLR9 and cytoskeletal proteins involved in cell migration, such as paxillin, vimentin and cofilin was determined by Western blotting.
In the present study, we show that CD73 suppression induces significant inhibition on cell viability and migration. Lower CD73 gene expression correlated with increased TLR9 mRNA levels in normoxia. On other hand, there was no significant correlation of CD73 and TLR9 on protein level in hypoxic conditions. Cytoskeletal proteins vimentin, cofilin and paxillin had lower protein expression in the CD73 shRNA cells compared to the control shRNA cells in normoxia, but not in hypoxia. Additionally, CD73 shRNA cells showed morphological changes and had less cell protrusions.
The data indicate that hypoxia upregulates TLR9 mRNA expression, but does not affect CD73 or cytoskeletal proteins expression. In addition, CD73 might promote migration and viability of cancer cells. Our findings suggest a CD73-TLR9 dependent pathway.
University of Turku.
Jane and Aatos Erkko Foundation.
All authors have declared no conflicts of interest.