Heterozygous Knockout of ARID4B Using CRISPR/Cas9 Attenuates Some Aggressive Phenotypes in a Breast Cancer Cell Line.

Abstract

Breast cancer is one of the leading causes of death in women around the world. Over time, many genes and mutations that are associated with the development of this disease have been identified. However, the specific role of many genes has not yet been fully elucidated. Higher ARID4B expression has been identified as a risk factor for diverse cancer types. Silencing experiments also showed that ARID4B is associated with developing cancer-associated characteristics. However, no transcriptomic studies have shown the overall cellular effect of loss of function in breast cancer in humans. This study addresses the impact of loss-of-function mutations in breast cancer MCF-7 cells. Using the CRISPR/Cas9 system, we generated mutations that caused heterozygous truncated proteins, isolating three monoclonal lines carrying insertions and deletions in ARID4B. We observed reduced proliferation and migration in in vitro experiments. In addition, from RNA-seq assays, a differential expression analysis shows known and novel deregulated cancer-associate pathways in mutated cells supporting the impact of ARID4B. For example, we found the AKT-PI3K pathway to be altered at the transcript level but through different genes than those reported for ARID4B. Our transcriptomic results also suggest new insights into the role of ARID4B in aggressiveness by the epithelial-to-mesenchymal transition and TGF-β pathways and in metabolism through cholesterol and mevalonate pathways. We also performed exome sequencing to show that no off-target effects were apparent. In conclusion, the ARID4B gene is associated with some aggressive phenotypes in breast cancer cells.