Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

et al. (2012) Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model. PLoS ONE 7(12): e52754. doi:10.1371/journal.pone.0052754 Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model Catalina Trejo-Becerril 0 Enrique Pe rez-Ca rdenas 0 Luca Taja-Chayeb 0 Philippe Anker 0 Roberto Herrera-Goepfert 0 Luis A. Medina-Vela zquez 0 Alfredo Hidalgo-Miranda 0 Delia Pe rez-Montiel 0 Alma Cha vez-Blanco 0 Judith Cruz-Vela zquez 0 Jose Daz-Cha vez 0 Miguel Gaxiola 0 Alfonso Duen as- 0 Gonza lez 0 Brian Lichty, McMaster University, Canada 0 1 Division of Basic Research, Instituto Nacional de Cancerolog a , Mexico City, Mexico, 2 OncoXL, Geneva , Switzerland , 3 Department of Pathology, Instituto Nacional de Cancerolog a, Mexico City, Mexico, 4 Instituto de F sica, Universidad Nacional Auto noma de Me xico/Instituto Nacional de Cancerolog a, Mexico City, Mexico, 5 Cancer Genomics Laboratory, Instituto Nacional de Medicina Geno mica, Mexico City, Mexico, 6 Department of Cytogenetics, Instituto Nacional de Cancerolog a, Mexico City, Mexico, 7 Unidad de Investigacio n, Instituto Nacional de Enfermedades Respiratorias, Mexico City, Mexico, 8 Instituto de Investigaciones Biome dicas, Universidad Nacional Auto noma de Me xico UNAM/Instituto Nacional de Cancerolog a , Me xico City , Mexico It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy. - Funding: This work was supported by CONACyT grants 34649-M, 50699 and from PAPIIT UNAM IN214902. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interest. Co-author Phillipe Anker is affiliated to OncoXL. There are no patents, products in development or marketed products to declare. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. The current paradigm in cancer progression is that it occurs via vertical gene transfer; this means that the offspring of initiating tumor cell inherit the genetic and epigenetic alterations leading to tumor progression. This model, however, ignores that horizontal or lateral transfer of DNA connects and shapes nearly all living things [1] and that within an organism, circulating DNA, such as exosomes that contain transcriptionally active mRNA and microRNA, may potentially act as an endocrine or paracrine messenger, able to affect the functionality of recipients cells [2]. Accordingly, it has been proposed that cell-free DNA (circulating DNA) could participate in the development of metastases via passive transfection-like uptake of such nucleic acids by susceptible cells [3]. In 1994, Anker et al., first demonstrated that the supernatant of cultured colon cancer cell line SW480 was able to transform recipient immortal murine NIH3T3 cells, which acquire mutated human K-ras [4]. Transformation of these recipient cells by plasma of colon cancer patients has been reported as well [5]. The ability of genetic material to circulate in eukaryotes has been known since 1948 [6], and that this DNA can be released by bacteria and higher organisms and enter into recipient cells was demonstrated by Anker and Stroun [79]. These findings led to the concept that DNA could act as a messenger [1016]. This view has been supported by the ease with which administered bacterial and eukaryote DNA can circulate freely throughout animal and plant bodies and in its ability to enter individual cells naturally, where it can locate in the host cell nuclei [12,1718]. The uptake of circulating DNA by eukaryotes shows that the biology of the recipient cells/organisms could be modified regardless of whether it is integrated or not [1927]. It is noteworthy that such DNA administration does not require any special vehicles, e.g., liposomes, electroporation, and gene guns, to aid entry into cells in order for it to be biologically active. How genetic material circulates and transfers into somatic cells of higher organisms remains controversial. Although this is not mutually exclusive, some authors have demonstrated that this occurs via the uptake of apoptotic bodies [28], while others have characterized circulating DNA as a complex of DNA/RNAlipoprotein termed virtosome, which is spontaneously released by living cells [29]. Here we demonstrate that circulating DNA is able to drive horizontal tumor progression in an immunocompetent colon-carcinogenesis rat model. Materials and Methods Cell Lines SW480 (human colon cancer cell line that has the point mutation Gly to Val at codon 12 of exon 1 of the K-ras oncogene), HeLa (human cervical cancer cell line positive for HPV-18), and NIH3T3 (mouse immortalized fibroblasts) were purchased from the American Type Culture Collection. Primary culture of foreskin fibroblasts (BB1) was derived from circumcision foreskin of newborn boy under written consent from his father and used in the second passage. Supernatant Preparation When cell cultures had a confluence of 80%, supernatants were collected by pipetting and cleared of any remaining cells and cell debris by a centrifugation step at 4006g for 20 min (Biofuge primo R, Heraeus) and passed through a 0.45-mm filter (Sartorius, 16555) to remove potentially contaminating cells. Aliquots of each supernatant samples were seeded in a culture flask and incubated at 37uC for 120 h to verify the absence of living cells. For DNA analysis, 50 ml of supernatant were concentrated, first with an ultrafiltration system wit (...truncated)