Identification of age-regulating breast cancer dormancy genes using C. elegans dauers - The recurrent growth of breast cancer (BrCa) cells at distal sites years after initial therapies have removed all vestiges of detectable disease underlies the significant role played by dormancy and reawakening programs in cancer metastasis. Dormancy reawakening and metastatic relapse are a function of chronological aging of the dormant cancer cells (DCC), and as such governed by aging-related mechanisms. Although some signatures of DCCs, such as activated p38MAPK, suppressed ERK1/2 activity, and the expression of several p38MAPKregulated genes (e.g.- NR2F1, DEC2/BHLHE41, HBP1, or WNT5A) have been identified, we lack a sufficient understanding of the genes and pathways that influence tumor-intrinsic and extrinsic programs of metastatic relapse and the effects of continued dormancy maintenance (chronological aging) on these programs. This limits our ability to develop therapeutically efficacious interventions. We previously used a functional genomic shRNA screen to identify “Dormancy-Reactivation Suppressor Genes” (DRSGs), which are required to maintain the p38MAPK-dependent dormancy of BrCa cell lines in a 3D bone endosteal niche (EN) model containing primary human bone endothelial, fibroblast, and osteoblast cells in a type I collagen matrix. Of the 412 potential DRSGs, only a few, BHLHE41, WNT3A, and HBP1, were functionally validated, mainly because of limits and high costs to in vitro dormancy culture models and aging-related in vivo models. As a novel solution, we propose to use C. elegans, with its well-defined dormancy and aging pathways, as well as highly defined genetics and clonal isogenicity, as a model to quickly screen our BrCa DRSG. C. elegans dauers share many DCC pathways, including dependence on the p38 ortholog, PMK-1, but most importantly, 67.1% of DRSG orthologs are upregulated in dauers. We hypothesize that dauers and DCC share genes with conserved dormancy-regulating functions (“dauer/DRSG”). This will allow us to use RNAi screens to determine which of the 151 shared dauer/DRSGs are required to maintain the dauer state and then test that list of human orthologs for their ability to induce BrCa dormancy in 3D-EN cultures. In addition, we will use in vivo models, such as dormancy-selected mouse D2A1-d mammary carcinoma cells and spontaneously-arising DCC and reawakened DCC in MMTVHER2/ neu transgenic mice because they better reflect the microenvironmental parameters governing dormancy (SA1). In SA2, we will identify changes in the dauer/DRSG genes that occur with aging their genetic interactions with known, conserved aging pathways. Lastly, we will use genomic analyses, including single animal-RNA-seq, of naturally-occurring, spontaneous C. elegans dauer-exiters to either confirm dauer/DRSG function (by their downregulation) or to identify new up-regulated recurrence-inducing genes that can be validated in our BrCa 3DEN and in vivo models. Our long-term goal is to develop a powerful system for unbiased discovery of genes involved in metastatic dormancy to identify potentially targetable pathways that could increase BrCa patient survival by suppressing the dormancy reawakening and disease recurrence.
