Targeting stromal reaction in pancreatic cancer to overcome chemo-resistance

描述（由申请人提供）： 美国癌症协会的最新估计显示，2010年有43，140例胰腺导管腺癌（PDAC）新发病例和36，800例PDAC死亡病例。这种令人沮丧的预后是由于胰腺癌不仅具有侵袭性，而且是一种高度耐药的恶性肿瘤。具体而言，原发性肿瘤和远处转移瘤中存在的显著纤维化反应限制了化疗的递送和疗效。PDAC肿瘤表现出胶原酶膜1型基质金属蛋白酶（MT 1-MMP，MMP-14）和高迁移率族A2（HMGA 2）的表达增加，HMGA 2是一种参与调节染色质结构的结构DNA结合蛋白。因此，本申请的目的是描述纤维化反应导致化疗耐药性的机制。我们的中心假设是胶原微环境通过诱导常染色质形成和促进DNA修复来促进化学抗性。我们进一步假设体内靶向MT 1-MMP将因此不仅通过改变染色质结构而且通过减弱纤维化进展本身来增加化学敏感性。这一假设是基于我们的初步数据，这些数据表明，在3D胶原凝胶中生长的PDAC细胞增加了组蛋白H3乙酰化;降低了异染色质蛋白11（HP 11）的表达;并保护其免受吉西他滨诱导的检查点阻滞。我们的初步数据还显示，胶原蛋白增加PDAC细胞中的HMGA 2和MT 1-MMP，并且用siRNA降低HMGA 2或MT 1-MMP水平减弱了胶原蛋白微环境中观察到的吉西他滨抗性。令人惊讶的是，人MT 1-MMP在胰腺癌的转基因和异种移植小鼠模型中的表达矛盾地导致与星状细胞活化相关的显著纤维化。目的1中提出的实验已被设计用于确定3D胶原诱导常染色质以促进化学抗性的机制。具体来说，我们将研究HMGA 2在增强3D胶原蛋白中的常染色质和促进DNA修复中的作用。目的2中提出的实验已被设计用于测试靶向MT 1-MMP将通过诱导异染色质和减少DNA修复来增加化学敏感性的假设。MT 1-MMP将被功能阻断抗体和MT 1-MMP特异性siRNA功能化的金纳米颗粒靶向。目的3中的实验已被设计用于测试靶向MT 1-MMP除了增强化学敏感性之外还将减弱纤维化的假设。该方法是创新性的，因为它将利用胰腺癌的体外器官型和体内原位和转基因小鼠模型，以及用siRNA功能化的金纳米颗粒，以证明靶向MT 1-MMP使PDAC细胞对化疗药物的作用敏感。拟议的研究是重要的，因为它是连续研究的关键第一步，不仅解决了明显的纤维化反应如何导致化疗耐药性，而且还将导致MT 1-MMP作为治疗靶点的鉴定，以增加PDAC肿瘤的化疗反应。

DESCRIPTION (provided by applicant): The American Cancer Society's most recent estimates showed that there were 43,140 new cases of pancreatic ductal adenocarcinoma (PDAC) and 36,800 deaths from PDAC in 2010. This dismal prognosis results from the fact that pancreatic cancer is not only aggressive but also a highly chemo-resistant malignancy. Specifically, the pronounced fibrotic reaction present in both the primary tumor and distant metastases limits the delivery and efficacy of chemotherapy. PDAC tumors demonstrate increased expression of the collagenase membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) and high mobility group A2 (HMGA2), an architectural DNA-binding protein involved in regulating chromatin structure. The objective of this application is thus to de- lineate the mechanisms by which the fibrotic reaction contributes to chemotherapy resistance. Our central hy- pothesis is that the collagen microenvironment promotes chemo-resistance by inducing euchromatin formation and facilitating DNA repair. We further hypothesize that targeting MT1-MMP in vivo will consequently increase chemo-sensitivity not only through altering the chromatin structure but also by attenuating fibrosis progression itself. This hypothesis is based on our preliminary data demonstrating that PDAC cels grown in 3D collagen gels have increased histone H3 acetylation; decreased expression of heterochromatin protein 11 (HP11); and are protected from gemcitabine-induced checkpoint arrest. Our preliminary data also show that collagen in- creases HMGA2 and MT1-MMP in PDAC cells, and decreasing HMGA2 or MT1-MMP levels with siRNA at- tenuates the gemcitabine resistance seen in the collagen microenvironment. Surprisingly, expression of human MT1-MMP in transgenic and xenograft mouse models of pancreatic cancer paradoxically results in pronounced fibrosis associated with stellate cell activation. Experiments proposed in Aim 1 have been designed to deter- mine the mechanism by which 3D collagen induces euchromatin to promote chemo-resistance. Specifically, we will examine the role of HMGA2 in enhancing euchromatin in 3D collagen and in facilitating DNA repair. Ex- periments proposed in Aim 2 have been designed to test the hypothesis that targeting MT1-MMP will increase chemo-sensitivity by inducing heterochromatin and reducing DNA repair. MT1-MMP will be targeted with func- tion blocking antibody and gold nanoparticles functionalized with MT1-MMP-specific siRNA. Experiments in Aim 3 have been designed to test the hypothesis that targeting MT1-MMP will also attenuate fibrosis in addi- tion to enhancing chemo-sensitivity. The approach is innovative because it will utilize in vitro organotypic and in vivo orthotopic and transgenic mouse models of pancreatic cancer, together with gold nanoparticles functional- ized with siRNA, to demonstrate that targeting MT1-MMP sensitizes PDAC cells to the effects of chemother- apy. The proposed research is significant because it is a critical first step in the continuum of research that will not only address how the pronounced fibrotic reaction contributes to chemo-resistance but will also lead to identification of MT1-MMP as a therapeutic target to increase chemotherapy response of PDAC tumors.