NEUTROPHIL EXTRACELLULAR TRAPS (NETS) DRIVE AN ANTIAPOPTOTIC PHENOTYPE IN HUMAN BREAST CANCER CELLS

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  • Presentation type: DR - Doctoral Student
  • Track:
  • Keywords: Breast cancer; NETs; apoptosis; Chemoresistance;
  • 1 Universidade Federal do Rio de Janeiro
  • 2 Instituto Nacional de Câncer

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Abstract

INTRODUCTION AND OBJECTIVES: Numerous aspects of tumor progression have been linked to Neutrophil Extracellular Traps (NETs), such as primary growth, aggressiveness, and metastasis. We have recently demonstrated that NETs promote a pro-metastatic phenotype in non-aggressive breast cancer cells through a process called epithelial-mesenchymal transition (EMT). In this context, EMT’s association with resistance to canonical cancer treatments has been previously reported. In this study, we analyzed the ability of isolated NETs in modulating an antiapoptotic phenotype in human breast cancer cells. MATERIAL AND METHODS: Three breast cancer cell lines were used in this study: MCF7 and T47D (described as maintaining its epithelial characteristics, lower aggressiveness and sensitivity to canonical chemotherapeutic agents) and MDA-MB-231 (described as undifferentiated, known for its elevated level of aggressiveness, invasiveness, and resistance to chemotherapeutic agents). Neutrophils isolated from the blood of healthy donors were stimulated with PMA to produce NETs. To evaluate cytotoxicity, tumor cells were treated with NETs for 24 or 48 h and further submitted to the MTT assay. Cell morphology was recorded before or after treatment with NETs for 24 hours, 7 or 14 days. Samples were generated for real-time PCR at the same time points, and gene expression of BCL2 (which encodes the antiapoptotic protein, Bcl-2) and BAX (which encodes Bax, a pro-apoptotic protein) was further analyzed. RESULTS AND CONCLUSION: In vitro analysis showed that NETs were not cytotoxic for none of the three cell lines employed in this study. As previously reported, T47D and MCF7 acquired mesenchymal characteristics after 24h of NETs treatment. These changes were sustained for 7 days, and cells recovered their epithelial traits after 14 days of treatment with NETs. NETs upregulated the expression of BCL2, in all cell lines. On the other hand, we observed a significant downregulation in BAX expression. Changes in the gene expression were partially dependent on DNA integrity and elastase activity, as assessed by co-treatment of cells with NETs and DNase I or Elastase Inhibitor III. Most remarkable, changes in the gene expression remained significantly altered even after 14 days of treatment in all cell lines. In sum, we conclude that NETs promote a long-term antiapoptotic phenotype in human breast cancer cell lines. The functional impact of these modifications in terms of chemoresistance shall be evaluated in future experiments.

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