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

描述(由申请人提供)： 胆管细胞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。我们课题组和其他人的研究发现，调节细胞死亡/凋亡途径为胆管癌细胞的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配体可保护细胞免受凋亡并促进其肿瘤的发生。此外，Fas信号通路中的几个抗凋亡介质，包括蛋白激酶B/AKT和FLICE样抑制蛋白(FLIP)在胆管癌细胞中表达上调，使肿瘤细胞对凋亡具有更强的抵抗力，并将Fas诱导的信号转移到生存和增殖途径。以人胆管癌细胞为例，我们证明了Fas的表达与肿瘤的分化程度有关。我们发现钙调素(CaM)拮抗剂通过Fas介导的细胞凋亡途径诱导胆管癌细胞凋亡，抑制小鼠胆管癌细胞的形成。机制研究进一步证实了钙依赖的CaM与Fas之间的直接结合，这种结合受Fas激活的调节。此外，CaM还被招募到Fas激活的死亡诱导信号复合体(DISC)中。CaM拮抗剂和钙络合剂可抑制CaM在Fas诱导的视盘中的募集，并阻断FADD在视盘中的募集，提示CaM/Fas结合有助于Fas激活的视盘的形成。最近，我们发现CaM与另一种在胆管癌细胞中升高的盘蛋白Flio结合，并在Fas激活后激活生存信号。因此，我们假设CaM是Fas-Death受体信号通路的关键调节因子，是胆管癌细胞潜在的治疗靶点。在这一应用中，我们将继续追求我们的长期目标，通过确定CaM在调节Fas激活的盘中的作用，以及CaM/Fas和CaM/Flip结合在动物模型中调控胆管癌细胞增殖和凋亡以及肿瘤形成中的作用，来了解Fas死亡受体信号通路在胆管癌细胞发病机制中的作用。其具体目的是：1)研究CaM在胆管癌细胞Fas信号通路中的调控作用；2)研究CaM/Fas/Flip相互作用在小鼠胆管癌细胞发生中的作用。本研究将确定CaM通过Fas途径调节信号的基本机制，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治疗。考虑到癌症是退伍军人群体中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.