Calmodulin Regulates Fas-Mediated Apoptosis: A Target for Cancer Therapy

描述（由申请人提供）： 胆管癌是一种高度恶性的肿瘤，起源于肝内外胆管系统的胆管细胞。我们小组和其他人的研究发现，调节细胞死亡/凋亡途径为胆管癌治疗提供了潜在的新途径。在小鼠胆管癌细胞中下调死亡受体（Fas）和上调Fas配体可保护细胞免于凋亡并促进其成瘤。此外，Fas信号传导途径中的几种抗凋亡介质，包括蛋白激酶B/AKT和FLICE样抑制蛋白（FLIP）在胆管癌细胞中上调，这使得癌细胞对凋亡更具抗性，并将Fas诱导的信号传导转移到存活和增殖途径。我们用人胆管癌标本证明Fas的表达与肿瘤分化有关。我们发现钙调素（CaM）拮抗剂通过Fas介导的凋亡途径诱导胆管癌细胞凋亡并抑制小鼠肿瘤的发生。机制研究进一步确定了Ca 2+依赖性的直接结合之间的钙调素和Fas，这是调节Fas激活。此外，CaM被募集到Fas激活的死亡诱导信号复合物（DISC）中。钙调素拮抗剂和钙螯合剂抑制招聘的钙调素到Fas诱导的DISC和阻断招聘的FADD到DISC，表明钙调素/Fas结合有助于Fas激活的DISC的形成。最近，我们发现钙调素结合到FLIP，另一种DISC蛋白，在胆管癌细胞中升高，并激活Fas激活后的生存信号。因此，我们假设钙调素是Fas-死亡受体信号通路的关键调节因子，并代表胆管癌的潜在治疗靶点。在本申请中，我们将继续追求我们的长期目标，了解Fas死亡受体信号通路在胆管癌的发病机制，重点是确定钙调素在调节Fas激活的DISC的作用和功能的钙调素/Fas和钙调素/FLIP结合在调节增殖和细胞凋亡的胆管癌在文化和肿瘤发生的动物模型。具体目标是：1）表征CaM在胆管癌细胞中调节Fas信号通路的功能;和2）表征CaM/Fas/FLIP相互作用在调节小鼠胆管癌肿瘤发生中的作用。目前的研究将定义CaM通过Fas通路调节信号传导的基本机制，从而促进进一步的研究，将这些发现转化为患者护理的策略和疗法。考虑到癌症是退伍军人群体的重要健康问题之一，我们的研究将有助于改善退伍军人的健康状况。

DESCRIPTION (provided by applicant): Cholangiocarcinoma is a highly malignant tumor that arises from cholangiocytes of the intra- and extra-hepatic biliary system. Studies from our group and others have found that modulating cell death/apoptosis pathways provide potential novel avenues for cholangiocarcinoma therapy. Down-regulating the death receptor (Fas) and up-regulating Fas Ligand in cholangiocarcinoma cells protect cells from apoptosis and enhance their tumorigenesis in mice. Further, several anti-apoptotic mediators in the Fas signaling pathways, including protein kinase B/AKT and FLICE like Inhibitory Protein (FLIP) are up-regulated in cholangiocarcinoma cells, which render the cancer cells more resistant to apoptosis and divert Fas-induced signaling to survival and proliferative pathways. With human cholangiocarcinoma samples we demonstrated that the expression of Fas is associated with tumor differentiation. We have found that antagonists of calmodulin (CaM) induce apoptosis of cholangiocarcinoma cells via a mechanism related to the Fas-mediated apoptosis pathways and inhibit tumorigenesis in mice. Mechanistic studies further identified Ca2+dependent direct binding between CaM and Fas, which is regulated upon Fas-activation. In addition, CaM is recruited into the Fas-activated death inducing signaling complex (DISC). A CaM antagonist and a calcium chelator inhibit recruitment of CaM into the Fas-induced DISC and block the recruitment of FADD into the DISC, suggesting that CaM/Fas binding contributes to Fas-activated DISC formation. Recently, we found that CaM binds to FLIP, another DISC protein that is elevated in cholangiocarcinoma cells and activates survival signals upon Fas activation. Therefore, we hypothesize that CaM is a critical regulator of the Fas- death receptor signaling pathway and represents a potential therapeutic target for cholangiocarcinoma. In this application, we will continue pursuing our long-term goal of understanding the Fas death receptor signaling pathways in the pathogenesis of cholangiocarcinoma by focusing on determining the role of CaM in regulating the Fas-activated DISC and the function of CaM/Fas and CaM/FLIP binding in regulating proliferation and apoptosis of cholangiocarcinoma in culture and tumorigenesis in animal models. The Specific Aims are: 1) characterize the function of CaM in regulating Fas signaling pathways in cholangiocarcinoma cells; and 2) characterize the role of the CaM/Fas/FLIP interaction in regulating cholangiocarcinoma tumorigenesis in mice. The present studies will define the fundamental mechanisms by which CaM regulates signaling through the Fas pathway, thus facilitating further studies to translate these findings into strategies and therapies for patient care. Considering that cancer is one of the important health problems in the Veteran's population, our studies will lead to improvement of the health of veterans.