Proteolytic Pathways in Progression of Pre-Malignant Breast Disease

描述（由申请人提供）：乳房x线摄影筛查导致许多女性被诊断为导管原位癌（DCIS），但我们无法准确预测哪些病变会恶性发展为浸润性导管癌（IDC），也无法有效阻止这种转变。人类乳腺活检的研究表明，肿瘤细胞和巨噬细胞中的半胱氨酸组织蛋白酶V/L2和B以及肌上皮细胞/[肌]成纤维细胞中的组织蛋白酶F、K和L参与了这一过程。异常的信号转导，例如通过p21激活的激酶1 [PAK1]，可能导致细胞周围蛋白水解增加。我们的工作假设是，从浸润前DCIS到浸润性癌的转变以及一些DCIS病变的快速进展是通过DCIS细胞和DCIS相关细胞中蛋白水解途径的改变介导的，而PAK1的失调有助于诱导这些异常的蛋白水解途径。为了验证这一假设，我们将使用体外和体内进展模型概括从浸润性前DCIS到浸润性癌的转变，我们为乳腺结构微环境工程指定了MAME。在这些模型中，我们将使用等基因MCF10细胞系[AT1， DCIS1和CA1d]和两种人类DCIS细胞系[SUM-102和SUM-225]。我们的具体目标是：1。通过直接靶向和PAK1水平的干预，调节半胱氨酸组织蛋白酶V或B在等基因和SUM DCIS细胞系中的表达和活性，并利用体外MAME模型确定侵袭性表型是否改变；2. 使用体外MAME模型，确定Aim 1修饰细胞与肌上皮细胞、[myo]成纤维细胞或两种细胞类型共培养，使用野生型细胞和半胱氨酸组织蛋白酶F、K或L的表达和活性被调节的细胞，是否可以改变侵袭性表型；3. 使用体内MAME模型，确定通过同时植入肌上皮细胞、[肌]成纤维细胞或两种细胞类型，使用野生型细胞和半胱氨酸组织蛋白酶F、K或L的表达和活性被调节的细胞，是否可以改变Aim 1修饰细胞的异种移植物的恶性表型；和4。[通过我们的Hu/Mu ProtIn芯片]筛选体内MAME模型，以寻找可能有助于从DCIS向IDC过渡的蛋白水解途径，并使用体外MAME模型与蛋白水解途径中心的试剂库定义功能变化。在肿瘤及其微环境的背景下，验证对DCIS发展为IDC至关重要的蛋白酶，以及调节它们的激酶途径，应该确定治疗干预的潜在靶点，以及区分将迅速发展为IDC的DCIS病变的生物标志物。公共卫生相关性：蛋白酶和激酶是制药工业为开发人类疾病（包括癌症）的新治疗策略而加紧努力的主题。我们提出的研究将发现并验证在肿瘤微环境中活跃并介导向全面恶性肿瘤转变的蛋白酶途径，以及调节这些蛋白酶途径的激酶途径。我们预计我们的研究将确定生物标志物，以区分将发展为侵袭性癌症的癌前病变，并确定将消除这种进展的靶点。

DESCRIPTION (provided by applicant): Mammographic screening leads to many women being diagnosed with ductal carcinoma in situ [DCIS], yet we cannot accurately predict which lesions will undergo malignant progression to invasive ductal carcinomas [IDC] or effectively block this transition. Studies of human breast biopsies have implicated in this process cysteine cathepsins V/L2 and B in tumor cells and macrophages and cathepsins F, K and L in myoepithelial cells/[myo]fibroblasts. Aberrant signal transduction, for example through p21-activated kinase 1 [PAK1], may contribute to increased pericellular proteolysis. Our working hypothesis is that the transition from pre-invasive DCIS to invasive carcinomas and the rapid progression of some DCIS lesions are mediated through alterations in proteolytic pathways in DCIS cells and DCIS-associated cells, and that dysregulated PAK1 contributes to the induction of these aberrant proteolytic pathways. To test this hypothesis, we will recapitulate the transition from pre-invasive DCIS to invasive carcinoma using in vitro and in vivo progression models that we have designated MAME for mammary architecture microenvironment engineering. In these models, we will use isogenic MCF10 cell lines [AT1, DCIS1 and CA1d] and two human DCIS cell lines [SUM-102 and SUM-225]. Our specific aims are to: 1. Modulate expression and activity of cysteine cathepsin V or B in the isogenic and SUM DCIS cell lines, both by direct targeting and through intervention at the level of PAK1, and determine using the in vitro MAME model whether the invasive phenotype is altered; 2. Determine using the in vitro MAME model whether the invasive phenotype can be altered by co-culturing modified cells from Aim 1 with myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; 3. Determine using the in vivo MAME model whether the malignant phenotype of xenografts of modified cells from Aim 1 can be altered by simultaneous implantation of myoepithelial cells, [myo]fibroblasts or both cell types, using wild-type cells and ones in which expression and activity of cysteine cathepsin F, K or L have been modulated; and 4. Screen [via our Hu/Mu ProtIn chip] the in vivo MAME model for proteolytic pathways that may contribute to the transition from DCIS to IDC and use the in vitro MAME model to define functional changes with libraries of reagents from the Center on Proteolytic Pathways. Validating, in the context both of the tumor and its microenvironment, proteases key to progression of DCIS to IDC, and kinase pathways that regulate them, should identify potential targets for therapeutic intervention as well as biomarkers to distinguish DCIS lesions that will rapidly progress to IDC. PUBLIC HEALTH RELEVANCE: Proteases and kinases are the subject of intensive efforts by the pharmaceutical industry to develop new treatment strategies for human diseases, including cancer. Our proposed studies will discover and validate protease pathways that are active in the tumor microenvironment and that mediate the transition to a full-blown malignancy, and kinase pathways that regulate these protease pathways. We anticipate that our studies will identify biomarkers to distinguish premalignant lesions that will progress to invasive cancers and define targets that will abrogate that progression.