Tumor cell invasion and metastasis formation are the major cause of death in cancer patients. These processes rely on extracellular matrix (ECM) degradation mediated by organelles termed invadopodia, to which the transmembrane matrix metalloproteinase MT1-MMP (also known as MMP14) is delivered from its reservoir, the RAB7-containing endolysosomes. How MT1-MMP is targeted to endolysosomes remains to be elucidated. Flotillin-1 and -2 are upregulated in many invasive cancers. Here, we show that flotillin upregulation triggers a general mechanism, common to carcinoma and sarcoma, which promotes RAB5-dependent MT1-MMP endocytosis and its delivery to RAB7-positive endolysosomal reservoirs. Conversely, flotillin knockdown in invasive cancer cells greatly reduces MT1-MMP accumulation in endolysosomes, its subsequent exocytosis at invadopodia, ECM degradation and cell invasion. Our results demonstrate that flotillin upregulation is necessary and sufficient to promote epithelial and mesenchymal cancer cell invasion and ECM degradation by controlling MT1-MMP endocytosis and delivery to the endolysosomal recycling compartment.

Altered endocytosis and vesicular trafficking are major players during tumorigenesis. Flotillin overexpression, a feature observed in many invasive tumors and identified as a marker of poor prognosis, induces a deregulated endocytic and trafficking pathway called upregulated flotillin-induced trafficking (UFIT). Here, we found that in non-tumoral mammary epithelial cells, induction of the UFIT pathway promotes epithelial-to-mesenchymal transition (EMT) and accelerates the endocytosis of several transmembrane receptors, including AXL, in flotillin-positive late endosomes. AXL overexpression, frequently observed in cancer cells, is linked to EMT and metastasis formation. In flotillin-overexpressing non-tumoral mammary epithelial cells and in invasive breast carcinoma cells, we found that the UFIT pathway-mediated AXL endocytosis allows its stabilization and depends on sphingosine kinase 2, a lipid kinase recruited in flotillin-rich plasma membrane domains and endosomes. Thus, the deregulation of vesicular trafficking following flotillin upregulation, and through sphingosine kinase 2, emerges as a new mechanism of AXL overexpression and EMT-inducing signaling pathway activation.

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Epithelial-to-mesenchymal transition (EMT) is critical for cancer cell dissemination and metastasis formation. Here, we found that flotillin 1 and 2 upregulation, which is observed in many aggressive cancers, is sufficient to induce EMT in non-tumoral mammary cells. We identified that the Upregulated Flotillin-Induced Trafficking (UFIT) pathway promotes the endocytosis of several transmembrane receptors in flotillin-positive late endosomes, thus modifying their destiny and leading to the the activation of oncogenic pathways that promotes EMT. The receptor tyrosine kinase AXL, a key actor during EMT and metastasis formation, is stabilized by the UFIT pathway and is required for EMT induced by flotillin upregulation. Moreover, sphingosine kinase 2, a lipid kinase recruited in flotillin-positive plasma membrane domains and vesicles, is required to stabilize AXL. These findings show that flotillin upregulation is a key EMT inducer. The UFIT pathway, through sphingosine kinase 2 activity, targets membrane receptors, such as AXL, to flotillinpositive vesicles, allowing their stabilization and the activation of EMT-linked signaling pathways.