Transglutaminase Modulated Tumor-Stroma Interaction in Pancreatic Cancer

描述(申请人提供)：癌细胞和胰腺间质之间的相互作用与胰腺癌的进展以及对化疗和放射的抵抗有关。基于我们的初步结果显示组织转谷氨酰胺酶(TG2)在胰腺肿瘤间质中的显著表达和活性，我们假设胰腺癌细胞分泌的TG2调节间质，促进癌细胞生存，并损害化疗的输送。我们建议通过三个特定的目标来检验这一假设：目的1：测量胰腺癌细胞分泌的TG2对胰腺基质细胞的增殖和激活的反应。目的2：证明TG2基因被敲除或其酶抑制作用调节胰腺肿瘤的间质成分。目的3：证明TG2基因被敲除或其酶抑制可增加吉西他滨在胰腺环境中的转运。目的1利用共培养实验检测分泌型TG2对成纤维细胞和内皮细胞增殖、存活和激活的影响。成纤维细胞分泌的细胞因子和生长因子将在基质细胞和癌细胞的条件培养液中用多重酶联免疫吸附试验进行检测。AIMS 2和AIMS 3将使用胰腺癌细胞产生的原位异种胰腺移植，其中TG2通过稳定的sh-RNA或针对TG2的反义构建而被击倒，或者TG2将被特异性的酶抑制剂KCC009抑制。实验的终点是测量和比较：纤维化(胶原和三色染色)，间质细胞(肌纤维母细胞和胰星状细胞)的数量和增殖，血管生成(微血管密度)，胰腺癌细胞的存活和增殖，以及化疗药物对胰腺组织的渗透性。对于后一个终点，我们将使用高效液相色谱质谱来测量来自表达正常或低TG2水平的癌细胞的胰腺组织中吉西他滨的浓度。总之，组织转谷氨酰胺酶在调节胰腺间质中的作用的成功证明将为进一步研究抑制TG2作为一种抗癌策略或作为使胰腺肿瘤对化疗敏感的手段提供有力的理论基础。这些研究对胰腺癌患者有直接的临床意义。

DESCRIPTION (provided by applicant): The interaction between cancer cells and the pancreatic stroma has been implicated in pancreatic cancer progression and resistance to chemotherapy and radiation. Based on our preliminary results demonstrating prominent tissue transglutaminase (TG2) expression and activity in the pancreatic tumor millieu, we hypothesized that TG2 secreted by pancreatic cancer cells modulates stroma, promotes cancer cell survival, and impairs chemotherapy delivery. We propose to test the hypothesis by three specific aims: Aim 1: Measure proliferation and activation of pancreatic stromal cells in response to TG2 secreted by pancreatic cancer cells. Aim 2: Demonstrate that TG2 knockdown or its enzymatic inhibition regulate stroma composition in pancreatic tumors. Aim 3: Demonstrate that TG2 knockdown or its enzymatic inhibition increases gemcitabine delivery into the pancreatic milieu. Aim 1 will use co-culture experiments to measure the effects of secreted TG2 on proliferation, survival and activation of fibroblasts and endothelial cells. Cytokines and growth factors secreted by fibroblasts will be measured in media conditioned by stromal and cancer cells by using Multiplex ELISA. Aims 2 and 3 will use orthotopic pancreatic xenografts generated from pancreatic cancer cells in which TG2 was knocked down by using stable transfection of sh-RNA or antisense construct targeting TG2, or TG2 will be inhibited by using KCC009, a specific enzymatic inhibitor. Endpoints of the experiments are measurement and comparison of: fibrosis (immunostaining for collagen and trichrome stain), number and proliferation of stromal cells (myofibroblasts and pancreatic stellate cells), angiogenesis (microvessel density), survival and proliferation of pancreatic cancer cells, and penetrance of chemotherapy into pancreatic tissue. For the latter endpoint, we will measure the concentration of gemcitabine within pancreatic tissue derived from cancer cells expressing normal or low TG2 levels by using HPLC mass spectrometry. In summary, successful demonstration of the role of tissue transglutaminase in modulation of the pancreatic stroma will provide a strong rationale for investigating further TG2 inhibition as an anti-cancer strategy or as means to sensitize pancreatic tumors to chemotherapy. These studies have direct clinical relevance to pancreatic cancer patients.