Mechanisms of linkage of stem and invasive phenotypes during metastatic colonization

ABSTRACT Metastasis, the primary cause of breast cancer-related mortality, is a multistep process culminating with the formation of tumor foci within distant organs. However, only a subpopulation of cancer cells within the primary tumor microenvironment is capable of completing the entire metastatic cascade, which includes intravasation, survival in circulation, extravasation, and tumor growth at distant sites. We identified a population of highly invasive, non-proliferating, non-apoptotic, chemo-resistant cancer cells capable of intravasation (published) and extravasation (preliminary results). These cells express high levels of MenaINV, a pro-metastatic isoform of the actin-regulatory protein Mena required for maturation of invasive protrusions called invadopodia, which enable cancer cells to cross endothelium and disseminate. MenaINV localization to the cell membrane, which is required for its function, may be regulated by PI3Kβ through phosphoinositide signaling. We found that MenaINV expression (published) and a stem cell program (preliminary results) are induced in tumor cells by direct contact with tumor-associated macrophages. Interestingly, we also found that chemotherapy co-induces MenaINV and the stem program in a macrophage-dependent manner. The emergence of MenaINV expressing stem cells may be one of the crucial steps to metastasis because these cells are not only transendothelial migration-competent but also have tumor-initiating capability. In primary breast tumors, cancer cells expressing high levels of MenaINV are able to enter blood vessels through Tumor Microenvironments of Metastasis (TMEM) doorways. These tightly controlled transient openings in capillary walls were first described by our group and are composed of macrophages, endothelial cells and Mena-expressing tumor cells in direct physical contact. TMEM doorways and cancer cell re-dissemination from lung metastases are also observed in lung metastases, but it is currently unknown if stem program is required for cancer cell dissemination from this secondary site. Interestingly, we and others found that chemotherapy in a macrophage-dependent manner increases the proportion of cancer cells co-expressing high levels of MenaINV and stem cell transcription factor SOX9. Thus, we hypothesize that induction of the transendothelial migration-competent phenotype in cancer cells (identified by the expression of MenaINV) is mechanistically linked to the stem program, and that the induction of this transendothelial migration- competent stem phenotype is potentiated by chemotherapy. We will use high resolution intravital imaging of the lungs in combination with stem and Mena biosensors to determine in vivo how the interplay of stemness and MenaINV regulate extravasation and metastatic colonization of the lungs and determine the mechanism by which chemotherapy induced hypoxia and macrophage influx affect cancer phenotype in metastatic foci in the lungs. The successful completion of this project will increase our understanding of the mechanisms involved in development of distant metastases, and provide a base for development of anti-metastatic therapies needed for improvement of survival in patients with metastatic breast cancer.