MECHANISM OF BREAST CANCER CELL INVASION

该建议的目的是研究侵袭足细胞局部基质降解的机制，并确定它们在乳腺癌侵袭和转移中的意义。侵袭足是侵袭性细胞的长膜突起，它们接触并降解细胞基质。我们的目的是：1)证明c-Src及其底物Cortactin在侵袭足细胞介导的局部基质降解中的作用；2)使用显性负性c-Src和Cortactin稳定的转染体来测试这些分子在裸鼠模型侵袭和转移中的作用；以及3)确定内翻修复与人类恶性乳腺癌的相关性。在目标1中，c-Src酪氨酸激酶及其底物皮质肌动蛋白的突变体将被引入乳腺癌细胞。结构上激活的c-Src激酶的转导促进内翻足的形成，反之，转导失活的c-Src激酶则抑制内翻足的形成。皮质素对酪氨酸磷酸化的要求将通过将皮质素的点突变和缺失突变导入细胞，并确定对内陷形成的显性负面影响来测试。带有SH2结构域改变的c-Src变异体将被导入侵袭细胞，以测试在侵袭足细胞形成过程中对c-Src SH2/Cortactin结合的需求。C-Src SH2结构域的磷酸肽抑制剂将被测试其阻断c-Src/Cortactin结合的能力。在目标2中，当识别出阻止内翻的c-Src或Cortactin变体时，表达这些变体的细胞系将在裸鼠体内作为异种移植肿瘤生长，以测量肿瘤生长并可视化内翻。肿瘤细胞将被注入心脏内，以测量肺和骨转移的发生率，以检验当内翻被抑制时，转移也被抑制的假设。最后，在目标3中，将使用已在分离细胞(目标1)和在异种摄影的肿瘤(目标2)中表征的抗内隐孢子虫抗体的组合，在快速冷冻和存档的人类乳腺肿瘤中识别出内隐孢子虫。总之，这些研究结果将为理解促进肿瘤细胞侵袭的信号转导途径及其与人类侵袭性乳腺癌生长的相关性提供基础。将开发用于测试内陷足类抑制物的模型系统，它们将对未来侵袭和转移的研究具有极其重要的价值。

The objective of this proposal is to study the mechanisms of localized matrix degradation by invadopodia and to determine their significance for breast cancer invasion and metastasis. Invadopodia are long membrane protrusions of invasive cells, they contact and degrade the cellular matrix. Our aims are to: 1) demonstrate the role of c-Src and its substrate cortactin in localized matrix degradation mediated by invadopodia, 2) use stable transfectants of dominant negative c-Src and cortactin to test the role of these molecules in invasion and metastasis in the nude mouse model, and 3) determine the relevance of invadopodia to malignant breast cancer in humans. In Aim 1, mutants of c-Src tyrosine kinase and its substrate cortactin will be introduced in breast cancer cells. Transfection of constitutively activated c-Src kinase promotes the formation of invadopodia, and conversely, transfection of the inactive c-Src kinase inhibits invadopodia. A requirement for phosphorylation of cortactin on tyrosine will be tested by transfecting cells with point and deletion mutants of cortactin and determining dominant negative effects upon upon invadopodia formation. c-Src variants with altered SH2 domains will be transfected into invasive cells to test the requirement for c-Src SH2/cortactin association during invadopodia formation. Phosphopeptide inhibitors of c-Src SH2 domain will be tested for their ability to block c-Src/cortactin association. In Aim 2, when c-Src or cortactin variants that block invadopodia are identified; the cell lines expressing these variants will be grown as xenographed tumors in the nude mouse to measure tumor growth and to visualize invadopodia. Tumor cells will be injected intracardially to measure the incidence of metastases to the lung and bone to test the hypothesis that when invadopodia are inhibited, metastasis is suppressed. Finally, in Aim 3, invadopodia will be identified in snap frozen and archival human breast tumors using combinations of anti-invadopodia antibodies that have been characterized in isolated cells (Aim 1) and in xenographed tumors (Aim 2). In conclusion, the results of these studies will provide the basis for understanding the signal transduction pathways that promote tumor cell invasion and their relevance for aggressive breast cancer growth in humans. Model systems for testing inhibitors of invadopodia will be developed, and they will be extremely valuable for future studies on invasion and metastasis.