Studying the role of DNA-PK in transformation of breast epithelial cells

Abstract

DNA alkylating agents are one of the most common DNA damaging agents that we encounter and widely used in cancer chemotherapy. The greatest limitation of such drugs is they can often induce carcinogenic mutations. Methylnitrosourea (MNU) is a monofunctional DNA methylating agent that acts as a mutagen as well as a potential carcinogen by forming O6-methyl Guanine (O6MeG) adducts in the DNA. Studies in the lab demonstrated that MNU triggers transformation and Golgi dispersal in non-tumorigenic breast epithelial cells (MCF10A) through activation of DNA-dependent protein kinase (DNA-PK). DNA-PK is a major player involved in double-strand DNA break repair through Non-homologous End Joining (NHEJ), it is known to be upregulated in most of the cancers. DNA-PK transcript analysis in breast cancer patients using The Cancer Genome Atlas (TCGA) database also showed an increased RNA expression in tumors compared to adjacent normal. Moreover, Survival analysis of breast cancer patients expressing high level of DNA-PK showed a low survival chance implicating that DNA-PK may have a role in breast cancer. We have also observed DNA-PK activation and aberrant Golgi phenotypes in various breast cancer cell lines such as MCF10AT1, MCF10CA1a, MCF7 and T47D. Here we are investigating the role of DNA-PK in breast epithelial cell transformation and Golgi dispersal. To check for MNU induced transformation of MCF10A on a DNA-PK null background, a stable knockdown cell line will be generated which will help to investigate the role of DNA-PK in transformation. Also, we will be knocking down DNA-PK in already transformed breast epithelial cells to identify whether the cellular transformation and aberrant Golgi phenotypes are DNA-PK dependent.