Use of Cystatin C to Combat TGF-Beta Tumorigenesis

描述(申请人提供)：乳腺癌是美国女性癌症死亡的第二大原因。侵袭和转移是乳腺癌最致命的特征，也是乳腺癌相关死亡的主要原因。转化生长因子-β通常通过阻止乳腺上皮细胞(MEC)增殖或通过诱导MEC凋亡来抑制乳腺癌的发展。乳腺肿瘤的发生否定了转化生长因子-β的抑瘤活性，从而促进转化生长因子-β刺激乳腺癌的侵袭和转移。对于恶性微血管内皮细胞如何克服转化生长因子-β的细胞抑制作用，以及转化生长因子-β如何刺激乳腺肿瘤的发生和发展，我们的认识存在着根本的空白。这些知识差距阻碍了科学和医学开发有效地对抗乳腺癌发生和进展中的转化生长因子-β致癌作用的治疗方法。我们发现半胱氨酸蛋白酶抑制剂CystC是一种新型的转化生长因子-βII型受体(TbetaR-II)拮抗剂，可抑制转化生长因子-β刺激的基因表达、细胞侵袭、上皮向间充质转化以及正常和癌细胞的形态转化。基于这些和其他的初步发现，我们假设，将CystC(或CystC多肽模拟物)转移到对转化生长因子-β的细胞抑制作用具有耐药性的乳腺癌中，将通过(I)阻止转化生长因子-β与TbetaR-II结合；(Ii)减少转化生长因子-β受体-II与TAR-III和TbetaR-I的结合；以及(Iii)选择性地抑制由转化生长因子-β激活的Smad2/3非依赖的信号通路，从而拮抗转化生长因子-β的致癌作用。这一假设将通过三个具体目标来解决。具体目标1将确定介导CystC：TbetaR-II复合体形成的CystC和TbetaR-II决定因素，缺乏这些功能的突变体将被用来确定CystC在选择性拮抗正常和恶性MEC中的转化生长因子-β亚型信号转导中的作用。特异性目标2将确定CystC选择性抑制的转化生长因子-β信号系统，这些抑制反应的机制，以及这些反应对正常和恶性微血管内皮细胞转化生长因子-β功能的影响。具体目标3将通过测量CystC在阻止转化生长因子-β刺激乳腺癌细胞生长、侵袭、血管生成和转移方面的作用，以及在裸鼠体内治疗这些肿瘤行为，来确定CystC是否在体内拮抗转化生长因子-β致癌和乳腺癌进展。这些研究将提供关于转化生长因子-β如何促进乳腺癌侵袭和转移的有价值的信息，更重要的是，关于如何通过开发和使用基于CystC的TbetaR-II拮抗剂来对抗转化生长因子-β的致瘤性来控制这些致命过程。

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer death in women in the United States. Invasion and metastasis are the most lethal characteristics of breast cancer and the leading cause of breast cancer-related death. TGF-beta normally inhibits breast cancer development by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Mammary tumorigenesis negates the tumor suppressing activities of TGF-beta, thus facilitating TGF-beta to stimulate breast cancer invasion and metastasis. Fundamental gaps exist in our knowledge of how malignant MECs overcome the cytostatic actions of TGF-beta, and of how TGF-beta stimulates the development and progression of mammary tumors. These knowledge gaps have prevented science and medicine from developing treatments effective in antagonizing TGF-beta oncogenicity in developing and progressing breast cancers. We identified the cysteine protease inhibitor cystatin C (CystC) as a novel TGF-beta type II receptor (TbetaR-II) antagonist that inhibits TGF-beta -stimulated gene expression, cell invasion, epithelial-to-mesenchymal transition, and morphological transformation in normal and cancer cells. Based on these and other preliminary findings, we hypothesize that delivering CystC (or CystC peptide mimetics) to developing breast cancers that are resistant to the cytostatic actions of TGF-beta will antagonize TGF-beta oncogenicity by (i) preventing TGF-beta binding to TbetaR-II; (ii) reducing TGF-beta: TbetaR-II binding to TaR-III and TbetaR-I; and (iii) selectively inhibiting Smad2/3-independent signaling pathways activated by TGF-beta. This hypothesis will be addressed by three Specific Aims. Specific Aim 1 will identify the CystC and TbetaR-II determinants that mediate CystC: TbetaR-II complex formation and mutants lacking these functions will be used to establish the role of CystC in selectively antagonizing TGF-beta isoform signaling in normal and malignant MECs. Specific Aim 2 will determine the TGF-beta signaling systems selectively inhibited by CystC, the mechanisms underlying these inhibitory reactions, and the impact of these reactions on TGF-beta function in normal and malignant MECs. Specific Aim 3 will determine whether CystC antagonizes TGF-beta oncogenicity and breast cancer progression in vivo by measuring the effects of CystC in preventing TGF-beta to stimulate breast cancer cell growth, invasion, angiogenesis, and metastasis, and to therapeutically combat these tumor behaviors in nude mice. These studies will provide valuable information on how TGF-beta promotes breast cancer invasion and metastasis, and, more importantly, on how to control these deadly processes by combating the oncogenicity of TGF-beta through the development and use of CystC-based TbetaR-II antagonists.