Use of Cystatin C to Combat TGF-Beta Tumorigenesis

描述（由申请人提供）：乳腺癌是美国女性癌症死亡的第二大原因。浸润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。TGF-β通常通过阻止乳腺上皮细胞（MEC）增殖或诱导MEC凋亡来抑制乳腺癌的发展。乳腺肿瘤发生否定了TGF-β的肿瘤抑制活性，从而促进TGF-β刺激乳腺癌侵袭和转移。我们对恶性MEC如何克服TGF-β的细胞抑制作用以及TGF-β如何刺激乳腺肿瘤的发展和进展的认识存在根本性的空白。这些知识差距阻碍了科学和医学开发有效拮抗TGF-β致癌性的治疗方法，从而导致乳腺癌的发展和进展。我们确定半胱氨酸蛋白酶抑制剂胱抑素C（CystC）作为一种新型的TGF-β II型受体（TbetaR-II）拮抗剂，抑制TGF-β刺激的基因表达，细胞侵袭，上皮细胞间质转化，并在正常和癌细胞的形态转化。基于这些和其他初步发现，我们假设，（或CystC肽模拟物）对发展中的对TGF-β的细胞抑制作用有抗性的乳腺癌的抑制作用将通过以下方式拮抗TGF-β致癌性：（i）阻止TGF-β与TbetaR-II结合;（ii）减少TGF-β：TbetaR-II与TaR-III和TbetaR-I的结合;和（iii）选择性抑制TGF-β激活的Smad 2/3非依赖性信号传导途径。这一假设将通过三个具体目标来解决。具体目标1将鉴定介导CystC：TbetaR-II复合物形成的CystC和TbetaR-II决定簇，缺乏这些功能的突变体将用于确定CystC在正常和恶性MEC中选择性拮抗TGF-β亚型信号传导的作用。具体目标2将确定CystC选择性抑制的TGF-β信号传导系统，这些抑制反应的机制，以及这些反应对正常和恶性MEC中TGF-β功能的影响。具体目标3将通过测量CystC在防止TGF-β刺激乳腺癌细胞生长、侵袭、血管生成和转移中的作用以及在裸鼠中治疗性地对抗这些肿瘤行为来确定CystC是否拮抗体内TGF-β致癌性和乳腺癌进展。这些研究将提供关于TGF-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TGF-β的致癌性来控制这些致命的过程。

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.