Stabilized microtubule protrusions in detached mammary epithelial cells

描述（由申请人提供）：乳腺肿瘤细胞可以扩散并在远处组织中长时间保持休眠状态。这种休眠的、播散的肿瘤细胞的最终重新出现是乳腺癌患者死亡的主要原因。我们已经证明，抗肿瘤的乳腺上皮细胞可以通过在传播的挑战中存活而促进肿瘤休眠，但不能立即生长成肿瘤。然而，我们最近的研究结果表明，这些细胞并不像以前怀疑的那样处于休眠状态，因为它们持续产生脱酪氨酸微管突起以响应脱离。这些突起促进再附着，并且在转移性乳腺肿瘤细胞系中发现频率更高。肌动蛋白解聚强烈增加突起的形成。这种侵袭诱导的突起以前没有报道过，并且与通过细胞外基质调节细胞侵袭的基于肌动蛋白的侵袭伪足不同。最近的体内研究表明，转移性肿瘤细胞粘附到毛细血管壁依赖于微管蛋白，并通过肌动蛋白解聚增强。虽然这种微管蛋白依赖性粘附的机制尚不清楚，但它与我们关于分离细胞突起的证据一致。我们将检验以下假设：α-微管蛋白的去酪氨酸化和肌动蛋白皮质微管捕获的减少导致稳定的细胞突起，从而增强乳腺肿瘤转移。该假说的预测将在以下具体目标中进行测试：1）阐明微管蛋白酪氨酸脱调节突起形成的分子机制。2)确定突起是如何受到干扰捕获微管在肌动蛋白皮质与已知的肿瘤蛋白（胸腺素-B4，corneum和ARC）的影响。3)测量突起形成对肿瘤细胞与肺毛细血管内皮细胞粘附的影响。捕获在活小鼠肺中的肿瘤细胞的生物发光成像将用最近资助的Xenogen IVIS-200动物成像仪进行。PI在细胞骨架信号转导和抗凋亡在乳腺肿瘤休眠中的作用方面的经验得到了在肺微血管内皮和肌动蛋白皮质组织方面具有专业知识的共同研究者的补充。我们的长期目标是表征这些分离的乳腺上皮细胞中的新型微管突起的分子机制，并确定它们对乳腺肿瘤转移扩散的贡献。

DESCRIPTION (provided by applicant): 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.