Amplifications and signaling of oncoproteins in cancer

Scripps Research Institute, The, La Jolla CA

Investigators

Abstract

Cancer cells often amplify signaling proteins, altering cell signaling networks and leading to protumor cellular behaviors. These signaling proteins can be copy-number amplified genetically via formation of circular extrachromosomal DNA (ecDNA). ecDNA is detected in a quarter of cancer samples and half of all cancer types, and is associated with poor patient outcomes. Despite the prevalence of ecDNA-mediated oncogene amplification in cancer, we have a limited understanding of how ecDNAs are maintained in cancer cells. While there have been extensive studies on how overexpression of these genes affects tumor growth and activities of signaling molecules, we have a poor understanding of how these genomic changes ultimately affect the logic of signaling that alter cellular behavior. The goal of this work is to elucidate the mechanism of retention of amplified genes encoding signaling proteins on ecDNA in cancer cells and to investigate the logic of biochemical and mechanical integration via amplified signaling proteins in cancer cell proliferation and migration. I hypothesize that specific genetic elements on ecDNA enable hitchhiking onto chromosomes in order to partition into daughter nuclei during cancer cell division. In Aim 1, I propose to identify DNA elements and protein mediators that enable retention of ecDNAs using a shotgun episome-based genetic screen, mitotic chromatin conformation capture, CRISPR screening and integration of epigenomic datasets. I also hypothesize that cancer cells with EGFR amplification integrate epidermal growth factor (EGF) signals and mechanical forces differently than wild-type-EGFR cells. In Aim 2, I propose to investigate the logic of biochemical and mechanical integration via amplified signaling proteins in cancer cell proliferation and migration. I will use Forster resonance energy transfer (FRET)-based biosensors, traction force microscopy, and fluorescence cell imaging to measure ERK signaling activity in EGFR-amplified cancer cells under mechanical stretch. I will also measure cell migration and cell fate changes of EGFR-amplified cells after ERK activation and cultured in substrate with various stiffness levels. Together, elucidating the mechanisms of extrachromosomal oncogene amplification and altered signaling logic due to oncogene amplification in cancer cells will reveal potential therapeutic opportunities. This work will also provide novel insights into how biochemical and mechanical signaling is altered in these oncogene-amplified cells to drive cancer cell behaviors.