A zebrafish xenotransplantation model of oncogene-induced senescence in breast cancer

Cells are equipped with a natural surveillance system that detects cancer-initiating events and shuts down growth, effectively putting the cells in a state of suspended animation known as senescence. Senescence effectively blocks many breast cancer-initiating events. However, the accumulation of senescent cells in breast tissues, via aging and chemotherapy, can cause unhelpful local inflammation that actually promotes invasive behavior of breast cancer cells. Using innovative new approaches, including the development of a new zebrafish model of oncogene-induced senescence, this study will determine the efficacy of existing and newly identified anti-inflammatory drugs in disrupting the cancer-promoting effects of senescent cells. 1. Developing a novel zebrafish xenotransplantation model of SASPWe have created hTERT-immortalized microvascular endothelial cells that bear a drug-inducible H-RasV12 oncogene (iTIME-H-RasV12 cells); exposure to doxycycline in vitro triggers rapid engagement of OIS and recapitulates all features of the senescence program. Zebrafish embryos will be injected with iTIME-H-RasV12 cells and treated with doxycycline to trigger senescence and elaboration of the SASP, measured by ELISA and IHC. To model effects of SASP cytokines on bystander cancer cells, we will co-inject differentially labeled pre-senescent or senescent iTIME-H-RasV12 cells with MDA-MB-231 breast cancer cells, and monitor effects on bystander cell proliferation and tissue invasion.2. Identification of candidate SASP-inhibitor drugsUsing senescent iTIME-H-RasV12 cells, a small molecule library will be screened in vitro for (i) blockade of SASP production by ELISA, and (ii) disruption of TASCC structure by the loss of co-localization of fluorescent mTOR and LAMP1 fusion proteins. Controls include mTORC1/2 inhibitor Torin and validated TASCC-inhibitor Brefeldin A. TASCC/SASP disrupting drugs will be further screened to ensure that they do not disrupt senescence-associated growth arrest. For each candidate TASCC/SASP inhibitor, mechanism of action will be determined via established OIS assays.3. Validation of drug-mediated SASP disruption in vivoDrug candidates identified in Specific Aim 2 will be validated in the zebrafish xenotransplantation model, permitting in vivo measurements of drug inhibition on OIS, SASP production and bystander cell proliferation and tissue invasion.