MECHANISM OF BREAST CANCER CELL INVASION

本研究的目的是研究浸润细胞局部降解基质的机制，并确定其在乳腺癌侵袭和转移中的意义。侵足是侵入细胞的长膜突起，它们与细胞基质接触并降解。我们的目标是：1)证明c-Src及其底物接触在invadopodia介导的局部基质降解中的作用；2)在裸鼠模型中使用显性阴性c-Src和接触的稳定转染物来测试这些分子在侵袭和转移中的作用；3)确定invadopodia与人类恶性乳腺癌的相关性。在Aim 1中，将在乳腺癌细胞中引入c-Src酪氨酸激酶及其底物接触的突变体。转染组成型活化的c-Src激酶促进invadopodia的形成，相反，转染无活性的c-Src激酶抑制invadopodia。对酪氨酸磷酸化的需求将通过转染细胞的点和缺失突变体来测试，并确定对入侵虫形成的主要负面影响。将SH2结构域改变的c-Src变异体转染到侵袭性细胞中，以测试在侵殖细胞形成过程中c-Src SH2/接触关联的需求。c-Src SH2结构域的磷酸肽抑制剂将测试其阻断c-Src/接触关联的能力。在Aim 2中，当c-Src或接触蛋白变异阻断侵过性椎体时；表达这些变异的细胞系将在裸鼠体内培养成异种肿瘤，以测量肿瘤的生长并可视化侵过足。将肿瘤细胞注入心内，测量肺和骨转移的发生率，以验证当侵入性被抑制时，转移被抑制的假设。最后，在aims 3中，将使用抗invadopodia抗体的组合在快速冷冻和存档的人类乳腺肿瘤中进行鉴定，这些抗体已在分离细胞（Aim 1）和异种肿瘤（Aim 2）中得到表征。总之，这些研究的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。研究人员还将开发出用于检测入侵过继细胞抑制剂的模型系统，这些模型系统将对未来的侵袭和转移研究具有重要价值。

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.