MECHANISMS OF COLLECTIVE EPITHELIAL INVASION AND METASTASIS IN BREAST CANCER

Metastasis is the major driver of cancer-related deaths. Yet, the cellular and molecular regulators of metastasis remain poorly defined. At the cellular level, metastasis is frequently conceptualized by single cancer cells detaching from the tumor bulk and metastases arising from the clonal outgrowth of these single cells. This model is challenged by pathological evidence for tumor cell clusters in the bloodstream of cancer patients and genomic evidence for the presence of multiple clones within metastases. We use a multicolor lineage tracing strategy to demonstrate that >97% of metastases in a mouse model of breast cancer arise from cancer cell clusters. We further demonstrated the presence of multiclonal clusters across stages of metastasis including local invasion, circulating tumor cell clusters, and micro-metastases. Specifically, we found that cancer cell clusters are associated with 15-fold greater survival and colony forming potential. Intriguingly, the metastatic seed was shown to be enriched for cancer cells expressing the basal epithelial marker, keratin-14 (K14). We find K14 expression to be strongly correlated with the invasive potential of human breast cancers, independent of their hormone status, tumor grade, or nodal status. In mouse models, we find K14+ cancer cells to be enriched in stages of metastasis that are most associated with systemic spread, while K14- cells are most associated with proliferative stages; this highlights the possibility of cooperation between the two cell states. The predominant models for metastasis have been based on the loss of cell-cell adhesion as a central molecular event. The loss of E-cadherin (E-cad) has been linked to increased invasion of cancer cell lines and has been inferred to promote metastasis, leading to its classification as an invasion, metastasis, and tumor suppressor. However, about 90% of breast cancers are invasive ductal carcinomas and these cancers retain E-cad expression in both the primary tumor and distant metastases. We, therefore, tested the requirement of E-cad for metastasis using mouse models of luminal and basal breast cancers that allow for an inducible deletion of E-cad. Despite the loss of E-cad, these cancer cells broadly retain an epithelial gene expression signature. We observe an increase in tumor invasion and dissemination due to the loss of E-cad, both in 3D organotypic ex vivo assays and orthotopic in vivo assays. However, E-cad loss in cancer cells strongly inhibits metastasis. Careful analysis of various intermediate stages of metastasis revealed that despite an increase in local invasion, E-cad loss is associated with a decrease in migratory persistence, decrease in circulating tumor cell counts, decrease in tumor seeding potential, collectively causing a strong decrease in metastasis. Transcriptomic and imaging-based analyses demonstrated a TGFb-induced enrichment of reactive oxygen species and subsequent apoptosis specifically within the disseminated E-cad- cancer cells. Significantly, colony formation of E-cad-negative cells was rescued by treatment with a TGFb-receptor inhibitor, an anti-oxidant, or an apoptosis inhibitor. These results suggest that E-cad acts as a survival factor during the detachment, systemic dissemination, and seeding phases of metastasis by limiting reactive oxygen-mediated apoptosis