Abstract
Invadopodia are actin- and protease-rich membrane structures present on metastatic cancer cells. We have previously shown that invadopodia-mediated ECM degradation is cell cycle-dependent. In mesenchymal human breast cancer cells, invadopodia degrade predominantly in G1 phase of the cell cycle. More recently, we have discovered that the hybrid epithelial/mesenchymal (E/M) metastatic cells also have the ability to assemble invadopodia. Here, we investigated how different EMT states affect the cell cycle regulation of invadopodia and invasion of breast cancer cells. Starting with the Early E/M cell line 4T1, we used TGFβ1 treatment to transition cells to a Late E/M state, or the induced Slug overexpression to achieve a fully mesenchymal (M) state, characterized by the complete loss of E-cadherin. We demonstrated that in Early E/M cells, invadopodia-mediated ECM degradation and invasion occur predominantly during the G2 phase, while Late E/M preferentially degrade ECM and invade during the G1, similarly to M cells. Moreover, when E cells are treated with TGFβ1, they transition to Early E/M state and degrade in G2 phase. To identify invadopodia components responsible for EMT- and cell cycle-dependent ECM degradation, we performed bulk mRNA sequencing of Early-versus Late-E/M cells in G1-or G2-phase of the cell cycle. The top candidate of our RNASeq analysis, FILIP1L, was expressed at significantly higher levels during G2 phase in Early E/M cells and during G1 phase in Late E/M cells, matching the invasive phase of each cell population. Expression of FILIP1L is upregulated in cells transitioning from E/M to M state. We next demonstrate FILIP1L is a novel invadopodia component, which colocalizes with Tks5 and cortactin. In the absence of FILIP1L expression, ECM degradation is significantly increased. Meanwhile, cell migration is significantly decreased, resulting in an overall decrease in the 3D spheroid invasion. Finally, we demonstrate that the high expression of FILIP1L is associated with poor prognosis in breast cancer patients. In summary, FILIP1L is a novel invadopodia component which regulates breast cancer invasion and tumor progression in a cell cycle- and EMT-dependent manner by controlling invadopodia and motility of cancer cells.