Identification of proteins associated to extracellular DNA (exDNA) and their role in malignant transformation

Introduction : Cancer-derived exDNA has the ability to induce cellular transformation of susceptible cells associated with horizontal transfer of genetic information. Although it has been shown that exDNA is protected by a protein complex, the exact identity of those proteins is still unknown. Therefore, this work focuses on the identification of the proteins composing this extracellular structure.

Material and methods: Murine cell line NIH3T3 of fibroblast origin, was cultured during 30 days in the presence of vesicle-free conditioned media derived from a human adenocarcinoma cell line SW480. Then we evaluated cell transformation features on NIHT3T cells including loss of contact inhibition, anchorage-independent growth potential, and in vivo tumor formation. Next, proteins from the conditioned media were identified by liquid LC-MS/MS analysis. Finally, in order to isolate the DNA-protein complex in the medium, we designed an immuno-precipitation assay using an anti-DNA antibody.

Results The vesicle-free conditioned media was enriched with DNA, as evidenced by transmission electron microscopy. This media, induced cell transformation in murine fibroblasts, allowing that fibroblast formed foci in culture plates, also fibroblast acquired the ability to grow in anchorage-independent and develop tumor in NOD/scid/gamma. Proteomic analysis of the conditioned media showed the presence of 271 proteins. Further bioinformatic evaluation revealed six specific colorectal cancer-associated proteins, as well as 40 proteins related with DNA interactions, including transcriptional factors and chromatin remodelers. The data showed an important presence of GO terms at nuclear, cytosolic and membrane regions. Reactome analysis demonstrated that the most representative pathways were related with gene transcription. The analysis of proteins obtained from immunoprecipitation is underway and will be presented.

Conclusions: An important number of proteins are in complex with exDNA, many of the them being transcription factors and chromatin remodelers, precluding further complete characterization, so far, our results suggest that these proteins could be important for horizontal transfer-mediated, cell transformation.