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

6.项目概要/摘要。胆管癌是一种高度恶性的肿瘤， 肝内和肝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/凋亡途径为胆管癌提供了潜在的新途径 疗法胆管癌细胞死亡受体（Fas）表达下调及Fas配体表达上调 保护细胞免于凋亡并增强其在小鼠中的肿瘤发生。此外，几种抗细胞凋亡 Fas信号通路中的介质，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，这使得癌细胞对细胞凋亡更具抗性 并将Fas诱导的信号传导转移到存活和增殖途径。人胆管癌 我们证实Fas的表达与肿瘤分化有关。我们发现 钙调素拮抗剂通过与以下相关的机制诱导胆管癌细胞凋亡： Fas介导的细胞凋亡途径并抑制小鼠肿瘤发生。进一步确定的机制研究 Ca 2+依赖性的CaM和Fas之间的直接结合，其在Fas活化后被调节。此外，本发明还提供了一种方法， 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CaM募集到Fas诱导的DISC中，并阻断FADD募集到Fas诱导的DISC中。 DISC，表明CaM/Fas结合有助于Fas激活的DISC形成。最近，我们发现 CaM与FLIP结合，FLIP是另一种在胆管癌细胞中升高的DISC蛋白， Fas激活后的生存信号。因此，我们假设CaM是Fas的关键调节因子， 死亡受体信号通路，并代表了一个潜在的治疗靶点， 胆管癌在本申请中，我们将继续追求理解 Fas死亡受体信号通路在胆管癌发病中的作用 CaM在Fas激活的DISC中的调节作用以及CaM/Fas和CaM/FLIP结合在DISC中的作用。 调节培养胆管癌的增殖和凋亡以及动物模型中的肿瘤发生。 本研究的具体目的是：1）研究钙调素在Fas信号通路中的作用， 胆管癌细胞;和2）表征CaM/Fas/FLIP相互作用在调节胆管癌细胞中的作用。 小鼠胆管癌肿瘤发生。目前的研究将确定基本机制 CaM通过Fas途径调节信号传导，从而促进进一步的研究， 将发现转化为患者护理的策略和疗法。考虑到癌症是重要的健康之一， 退伍军人人口的问题，我们的研究将导致退伍军人的健康改善。

6. Project Summary/Abstract. 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. Downregulating the death receptor (Fas) and upregulating 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 upregulated 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.