Role of splice- and translation-variants of the cysteine protease cathepsin L in the progression and metastasis of mammary cancer

蛋白水解酶在恶性肿瘤的生长和扩散中发挥着广泛的作用。我们最近发现，在转移乳腺癌的MMTV-PYMT小鼠模型中，组织蛋白酶L的过表达促进了肺转移的形成。此外，我们还证实，即使在与肿瘤相关的应激条件下，组织蛋白酶L的生物合成也保持在高水平。在未来，我们想要阐明组织蛋白L在细胞内的促肿瘤功能，特别感兴趣的是溶酶体蛋白水解酶与细胞生长的一个中心关卡，即mTOR通路的可能联系。因此，本项目旨在通过对小鼠乳腺癌模型和人乳腺癌细胞系的分析，阐明溶酶体蛋白组织蛋白酶L是如何影响肿瘤的生长和转移的。作为准备工作，我们产生了一只转基因小鼠，允许选择性地消融驻留在癌细胞中的各种类型的组织蛋白酶L。这一小鼠品系已经被交叉到MMTV-PYMT小鼠转移乳腺癌的模型中，并将对肿瘤的进展和转移转移进行系统的研究。对来自小鼠模型的原代和永生化细胞培养的功能分析以及在患者原发乳腺癌标本中的表达研究是对遗传动物研究的补充。以下关键问题将被解决：1)组织蛋白酶L在MMTV-PYMT乳腺癌模型中的细胞类型选择性消融；2)mTOR活性、溶酶体生物发生和自噬将在肿瘤相关细胞培养条件下组织蛋白酶抑制的情况下进行研究。3)纯化的组织蛋白酶L缺陷溶酶体的生化分析。4)新的分子连接在体内的功能验证。

Proteolytic enzymes are versatile players in the growth and dissemination of malignant tumors. We have recently shown that overexpression of cathepsin L promotes formation of lung metastasis in the MMTV-PyMT mouse model for metastasizing breast cancer. In addition we established that cathepsin L biosynthesis is maintained at high level even under tumor-relevant stress conditions. In the future we want to elucidate the intracellular tumor promoting functions of cathepsin L. Of special interest is the possible link of the lysosomal protease to a central ceck point of cell growth, i.e. the mTOR pathway. Thus, this project aims to elucidate how the lysosomal protease cathepsin L affects tumor growth and metastasis by analysis of breast cancers in mouse models and human breast cancer cell lines. As preparatory work we generated a transgenic mouse allowing for selective ablation of cathepsin L in various cell types residing in the cancer. This mouse line has been crossed to the MMTV-PyMT mouse model of metastasizing breast cancer and tumor progression and metastatic dissemination will be systematically studied. Functional analysis of primary and immortalized cell cultures derived from the mouse models and expression studies in primary breast cancer specimens of patients complement the genetic animal studies.The following key questions will be addressed: 1) Cell type selective ablation of cathepsin L in the cancer cells of the MMTV-PyMT breast cancer model 2) mTOR activity, lysosomal biogenesis and autophagy will be studied upon cathepsin inhibition in tumor-relevant cell culture conditions. 3) Biochemical analysis of purified cathepsin L-deficient lysosomes. 4) Functional validation of the novel molecular links in vivo.