Cellular heterogeneity in cancer represents a major challenge to the development of curative therapies, as even small subsets of cells can fuel relapse, dormancy, treatment failure, and metastasis. Cellular plasticity i.e. reversible state change, likely provides a key contributing mechanism. However, clinical approaches that specifically target molecular mediators of cellular plasticity are lacking. This proposal investigates the CR1 pathway as a critical plasticity mediator in breast cancer progression. CR1 is a cell surface/secreted stem cell factor and oncofetal protein that promotes proliferation, migration and epithelial-mesenchymal transition in human mammary epithelial cells in vitro and stem cell maintenance in primary mouse mammary epithelial cells cultured ex vivo. Recently, we showed that inhibition of CR1 with a candidate, engineered, peptide therapeutic (ALK4L75A-Fc a.k.a. A4Fc) reduced growth of human breast cancer cells under nutrient stress, and blocked metastasis when these were transplanted into mice. CR1 blockade in these tumor models is associated with reduced fibrosis -- consistent with the discovery of CR1 as a key regulator of fibrosis in wounding. We now seek to understand molecular mechanisms of CR1’s sensitivity to microenvironmental or therapy associated stress, to determine the role of novel signal mediators, and to uncover targetable molecular underpinnings of the reprogramming that CR1 coordinates between cancer cells and their neighbors during breast cancer progression. To this end in Aim 1, we utilize a series of mouse transplant models (including patient derived breast cancer cells) and bioengineered systems to examine CR1 dependent cell state change at different stages of metastasis, and to test the effect of blocking CR1 on aggressive breast cancer phenotypes. In Aim 2, we will use multicellular culture systems to uncover the mechanics of CR1 signaling including its dependence on stress related upstream and downstream mediators, and cellular cross-talk. Both aims leverage our experience in single cell RNA-sequencing to elucidate cell state change at high resolution. Impact: This work will not only bring new understanding to the molecular biology of breast cancer cell plasticity and heterogeneity, but will also set the stage for clinical translational studies of CR1 blocking agents with newly defined targets, readouts and biomarkers at single cell resolution.

This proposal advances mouse transplant models and multicellular culture systems to investigate the role of the CRIPTO protein in promoting breast cancer progression. It deconstructs CRIPTO signaling at the cellular level and molecularly, examining its sensitivity to stress, reliance on novel interacting proteins, and ability to fundamentally change cancer cell transcriptional states in coordination with those of neighboring fibroblasts. The proposed studies explore the therapeutic potential of targeting CRIPTO to block these changes at key transitions in the metastatic cascade.