Investigation of horizontal gene transfer in cancer cells: novel mechanism of therapeutic resistance
The development of resistance to therapeutics is a common phenomenon in both bacteria and human cancers. Acquired resistance to drug therapy is a primary cause of mortality in cancer. However, the mechanisms by which cancers develop resistance to therapeutics are poorly understood. Horizontal gene transfer (HGT), the direct transfer of genetic material between cells, occurs among bacteria to develop resistant populations. Having considered this principle of microbiology, we hypothesize that tumor cells undergo HGT to develop drug resistance. We assessed the capability of tumor cells to carry out HGT using the melanoma cell line, A375, as a model. A375 harbors a BRAF V600E mutation and is sensitive to Vemurafenib, a targeted kinase inhibitor commonly used to treat advanced melanomas. Therefore, Vemurafenib and Puromycin, a well-characterized drug, were chosen as therapeutic pressures. Drug dose response curves were performed on the parental A375 line for Vemurafenib and Puromycin and yielded IC50s of 312nM and 79ng/mL, respectively. Parental A375 cells were modified to create Puromycin and Vemurafenib resistant populations. Fluorescent tags were introduced for easy visualization. The NRAS Q61K mutation has been previously reported to cause resistance to Vemurafenib, therefore a commercially available NRAS Q61K mutant A375 line was acquired. Stable transfection of a GFP vector into the NRAS mutant line had 98% efficiency, while transfection of the RFP-Puromycin resistance vector into the parental line had 1% efficiency. Drug dose response of the GFP-mutant population gave an IC50 of 800nM, validating resistance to Vemurafenib. Future studies will continue the development of the RFP-Puromycin resistance population and test drug resistance of the successfully modified populations. Following validation of mutually exclusive drug resistance, modified populations will be co-cultured and exposed to various combinations and concentrations of the two drugs. Continued cell viability during exposure to both pressures will denote successful HGT.