MECHANISM OF BREAST CANCER CELL INVASION

乳腺癌细胞侵袭机制

• 批准号: 6476214
• 负责人: SUSETTE C MUELLER
• 金额: $ 23.8万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476214
• 关键词: biological signal transduction; breast neoplasms; cell line; enzyme activity; enzyme substrate; human tissue; laboratory mouse; metastasis; mutant; neoplasm /cancer invasiveness; phosphorylation; protein degradation; protein structure function; protein tyrosine kinase; tissue /cell culture; transfection

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.

本研究的目的是研究侵袭伪足局部基质降解的机制，并确定其对乳腺癌侵袭和转移的意义。入侵伪足是入侵细胞的长膜突起，它们接触并降解细胞基质。 我们的目标是：1）证明c-Src及其底物corneumen在由侵袭伪足介导的局部基质降解中的作用，2）使用显性阴性c-Src和corneumen的稳定转染子来测试这些分子在裸鼠模型中的侵袭和转移中的作用，和3）确定侵袭伪足与人类恶性乳腺癌的相关性。 在目标1中，将c-Src酪氨酸激酶及其底物corpine的突变体引入乳腺癌细胞中。 组成型激活的c-Src激酶的转染促进侵袭伪足的形成，并且相反地，失活的c-Src激酶的转染抑制侵袭伪足。 将通过用corneumn的点突变体和缺失突变体转染细胞并确定对侵袭伪足形成的显性负效应来测试corneumn对酪氨酸磷酸化的要求。 将具有改变的SH 2结构域的c-Src变体转染到侵袭性细胞中，以测试侵袭伪足形成期间c-Src SH 2/corneum结合的需要。 将测试c-Src SH 2结构域的磷酸肽抑制剂阻断c-Src/corpine结合的能力。 在目的2中，当鉴定出阻断侵袭伪足的c-Src或corpegrin变体时，表达这些变体的细胞系将在裸鼠中作为异种移植肿瘤生长，以测量肿瘤生长并使侵袭伪足可视化。 将心内注射肿瘤细胞以测量肺和骨转移的发生率，以检验当侵袭伪足被抑制时，转移被抑制的假设。 最后，在目标3中，将使用已在分离的细胞（目标1）和异种移植的肿瘤（目标2）中表征的抗侵袭伪足抗体的组合在速冻和存档的人乳腺肿瘤中鉴定侵袭伪足。 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。 将开发用于检测侵袭伪足抑制剂的模型系统，它们将对未来的侵袭和转移研究非常有价值。