Cell aggregation prevents anoikis and induces CD44 cleavage by maintaining lipid raft integrity to promote triple negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, and metastasis is the major cause of cancer morbidity and mortality. Therefore, it is urgent to discover novel therapeutic targets and develop effective treatments for this lethal disease. Circulating tumor cells (CTCs) are considered "seeds of metastasis". Compared to single CTCs, our previous studies have demonstrated that CD44 homophilic interaction mediates CTC aggregation to enhance the stemness, survival and metastatic ability of aggregated cells. Importantly, the presence of CD44+ CTC clusters correlates with a poor prognosis in breast cancer patients. Here, we further investigated the underlying mechanism of how CD44-mediated cell aggregation promotes TNBC metastasis. We found that cell detachment, which mimics the condition when tumor cells detach from the extracellular matrix (ECM) to metastasize, induces lipid raft disruption in single cells, but lipid rafts integrity is maintained in aggregated cells. We further found that lipid rafts integrity in aggregated cells is required for Rac1 activation to prevent anoikis. In addition, CD44 and γ-secretase coexisted at lipid rafts in aggregated cells, which promotes CD44 cleavage and generates CD44 intracellular domain (CD44 ICD) to enhance stemness. Consequently, lipid rafts disruption inhibited Rac1 activation, CD44 ICD generation and metastasis. These data reveal a new mechanism of cell aggregation-mediated TNBC metastasis via maintaining lipid raft integrity after cell detachment. The finding provides a potential therapeutic strategy to prevent CTC cluster-initiated metastasis by disrupting lipid raft integrity and its-mediated downstream pathways.