Breast tumor cells can disseminate and remain dormant in distant tissues for extended periods of time. The eventual reemergence of such dormant, disseminated tumor cells is a primary cause of patient death from breast cancer. We have shown that apoptotically-resistant mammary epithelial cells can promote tumor dormancy by surviving the challenges of dissemination, but failing to grow immediately into tumors. However, our recent results indicate that these cells are not as dormant as previously suspected, since they persistently generate detyrosinated microtubule protrusions in response to detachment. These protrusions promote reattachment, and are found with higher frequency in metastatic breast tumor cell lines. Actin depolymerization strongly increases protrusion formation. Such detachment-induced protrusions have not been reported before, and are distinct from the actin-based invadopodia that regulate cell invasion through extracellular matrix. Recent in vivo studies show that the adhesion of metastatic tumor cells to capillary walls depends on tubulin and is enhanced by actin depolymerization. Although the mechanism for this tubulin- dependent adhesion is not yet known, it is consistent with our evidence on protrusions in detached cells. We will test the hypothesis that detyrosination of alpha-tubulin and reduced microtubule capture at the actin cortex cause stabilized cellular protrusions that enhance breast tumor metastasis. Predictions of this hypothesis will be tested in the following specific aims: 1) Clarify the molecular mechanism by which tubulin detyrosination regulates protrusion formation. 2) Determine how protrusions are affected by disrupting capture of microtubules at the actin cortex with known tumor proteins (thymosin-B4, cortactin and ARC). 3) Measure the effects of protrusion formation on tumor cell adhesion to the lung capillary endothelium. Bioluminescent imaging of tumor cells trapped in the lungs of living mice will be performed with a recently- funded Xenogen IVIS-200 animal imager. The experience of the PI in cytoskeletal signal transduction and the role of apoptotic resistance in breast tumor dormancy is complemented by co-investigators with expertise in the lung microvascular endothelium and the organization of the actin cortex. Our long-term goal is to characterize the molecular mechanisms underlying these novel microtubule protrusions in detached mammary epithelial cells and define their contribution to the metastatic spread of breast tumors. ? ? ?
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