EVALUATION OF TUMOR CELL FREE DNA IN THE TUMORIGENESIS OF NON-MALIGNANT CELLS

The presence of cell-free DNA in biological fluids may offer an opportunity to discover new biomarkers for different diseases. In addition, cell-free DNA when released by tumor cells may contribute to trigger pro-tumor events such as malignant transformation in adjacent cells to the tumor and metastasis. This study was conducted to determine the potential of cfDNA, isolated from prostate cancer (PCa) patients, in the malignant transformation of non-tumor cells on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, fragments of cfDNA were isolated in the plasma samples of cancer patients (n = 22) and from healthy donors (n = 14). For the functional assays 3 pools de cfDNA were used. The first pool contained cfDNA obtained from 14 healthy donors, used as controls (cfDNA healthy), the second pool contained cfDNA from 15 PCa patients staged at T1 and T2 (T1/T2 cfDNA), and the last pool contained cfDNA from seven PCa patients staged at T3 and T4 (T3/T4 cfDNA). RWPE-1 and PNT-2, non-tumor prostate cell lines, were cultured and treated with 20 ug of each cfDNA (cfDNA Healthy, T1/T2 or T3/T4) to evaluate EMT-linked genes expression by qPCR (VIM, ECAD, NCAD, EPCAM, MMP9, CD44 and CD133). We also evaluated differential microRNA expression profiles employing the nCounter Human v.3 miRNA expression assay, and the cellular migration and proliferation upon cells treatment. The qPCR data shows alterations in the expression of MMP9 and CD44 genes in PNT-2, and MMP9 gene in RWPE-1 cells after treatment with cfDNA (T1/T2 or T3/T4). This treatment also promoted deregulation of nine miRNAs, in the treatment with cfDNA T1/T2, hsa-miR-539-5p, hsa-miR-493-3p were upregulated, while hsa-miR-32-5p, hsa-miR-27b-3p, hsa-miR-125a-5p, hsa-miR-99b-5p, hsa-let-7f-5p and hsa-miR-16-5p were down-regulated. However, in the treatment with T3/T4 cfDNA only hsa-miR-125b-5p was up-regulated. Cell migration was enhanced after treatment with T3/T4 cfDNA in both cell lines (RWPE-1 and PNT-2). The treatment with T1/T2 cfDNA also increased the migration of PNT-2 cells. However, the cell proliferation did not show statistical differences in any of the treatments with cfDNA. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.