Stabilized microtubule protrusions in detached mammary epithelial cells

描述(申请人提供)：乳腺肿瘤细胞可以扩散并在远处组织中休眠较长时间。这种休眠的、播散的肿瘤细胞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，抗凋亡的乳腺上皮细胞可以通过在扩散的挑战中存活下来，但不能立即生长成肿瘤，从而促进肿瘤休眠。然而，我们最近的结果表明，这些细胞并不像之前怀疑的那样处于休眠状态，因为它们在脱离后持续产生变性的微管突起。这些突起促进再附着，在转移性乳腺肿瘤细胞系中发现的频率更高。肌动蛋白解聚强烈增加突起的形成。这种脱离诱导的突起以前没有报道过，不同于以肌动蛋白为基础的通过细胞外基质调节细胞侵袭的侵袭。最近的体内研究表明，转移性肿瘤细胞与毛细血管壁的黏附依赖于微管蛋白，并通过肌动蛋白解聚来增强。虽然这种微管蛋白依赖的黏附机制尚不清楚，但这与我们关于分离细胞中突起的证据是一致的。我们将验证这一假设，即α-微管蛋白的降解和肌动蛋白皮质微管捕获的减少会导致稳定的细胞突起，从而增强乳腺肿瘤的转移。这一假说的预测将在以下特定目标中得到验证：1)阐明微管蛋白降解调节突起形成的分子机制。2)用已知的肿瘤蛋白(胸腺肽-B4、皮质酮和ARC)干扰肌动蛋白皮质微管的捕获，确定突起是如何受到影响的。3)测定肿瘤细胞与肺毛细血管内皮细胞黏附的影响。活体小鼠肺部捕获的肿瘤细胞的生物发光成像将使用最近资助的Xenogen IVIS-200动物成像仪进行。PI在细胞骨架信号转导和凋亡抵抗在乳腺肿瘤休眠中的作用的经验得到了在肺微血管内皮细胞和肌动蛋白皮质组织方面具有专业知识的合作研究人员的补充。我们的长期目标是确定乳腺上皮细胞中这些新的微管突起的分子机制，并确定它们在乳腺肿瘤转移扩散中的作用。

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.