Gene expression changes caused by KRAS in MCF-10A

Tumor formation hinges on the acquisition of two or more driver genes that cause normal cells to progress from proliferation to abnormal expansion and malignancy. In order to understand the minimum genetic alterations involved in this process, we compared the transcriptomes of an isogenic set of breast epithelial cell lines that are non-transformed, or contain a single or double knock-in (DKI) of mutant PIK3CA (H1047R) or KRAS (G12V). Gene set enrichment analysis revealed that DKI cells were enriched over single mutant cells for genes that characterize a MYC- target gene signature. This gene signature was mediated in part by the bromodomain-containing protein, 9 (BRD9) that was found in the SWI-SNF chromatin-remodeling complex, bound to the MYC super-enhancer locus. Small molecule inhibition of BRD9 reduced the MYC transcript and increased the MYC-negatively regulated target, NDRG1. Critically, only DKI cells had the capacity for anchorage-independent growth in semi-solid medium, and CRISPR/Cas9 manipulations showed that PIK3CA and BRD9 expression were essential for this phenotype. In contrast, KRAS was necessary for DKI cell migration, and overexpression of BRD9 partially rescued the growth of KRAS single mutant cells in semi-solid medium. These results provide new insight into the earliest transforming events driven by oncogene cooperation, and suggest BRD9 is an important mediator of mutant PIK3CA/KRAS- driven transformation. MCF-10A cells were modified by knock-in of KRAS-G12V mutation in a single endogenous locus or by the combination of PIK3CA-H1047R and KRAS-G12V. Total RNA was extracted from these cell lines and compared with existing RNAseq data of MCF-10A and MCF-10A PIK3CA-H1047R.