Death Receptor Signaling in Pancreatic Cancer: Mechanisms and Therapeutic Targets

描述(由申请人提供)： 胰腺癌是美国癌症死亡的第四大原因，仍然是我们退伍军人的一个重要健康问题。自1975年以来，胰腺癌患者的5年生存率仅从2%提高到6%。我们和其他人已经证明，调节死亡受体激活的外源性细胞凋亡途径为癌症治疗提供了新的途径。死亡受体4或5(DR4/5)的重组配体或激动型抗体，包括DR5激动型抗体TRA-8，在临床前研究中已被证明可诱导几种类型的癌症细胞凋亡，目前正在进行包括胰腺肿瘤在内的几个实体肿瘤的I-III期临床试验。然而，胰腺癌对TRA-8和其他DR配体或激动型抗体的耐药性是有效治疗的主要障碍。在过去的8年里，我们一直在寻找有效的试剂来克服癌细胞对DR诱导的细胞凋亡的抵抗。我们已经证明，调控死亡诱导信号复合体(DISC)中的成分决定了DRS传递的下游生存和凋亡信号，从而决定了癌细胞对DR诱导的凋亡的敏感性。最近，我们在DR5相关的视盘中发现了一种新的成分--聚(ADP-核糖)聚合酶1(PARP1)。PARP1表达水平与胰腺癌细胞对TRA-8诱导的细胞凋亡的抵抗力相关。PARP1在体外抑制耐药胰腺癌细胞对TRA-8诱导的细胞凋亡；并在体内增强TRA-8治疗耐药胰腺癌的疗效。相反，在对TRA-8敏感的细胞中过表达PARP1使它们对TRA-8更具抵抗力。已知PARP1主要在细胞核内起DNA修复作用。在人胰腺癌组织和细胞中，我们鉴定了PARP1的胞浆定位。我们证明了PARP1通过促进caspase-8的聚(ADP-核糖)(PADPr)修饰抑制caspase-8的激活，通过新的机制调控DR5诱导的细胞凋亡信号。另一方面，抑制PARP1减少了钙调蛋白(CaM)和Src的盘状募集，我们先前报道这两种物质介导了死亡受体诱导的胰腺癌细胞生存途径。因此，我们假设抑制PARP1促进死亡受体诱导的凋亡信号和抑制生存信号，从而增强胰腺癌对TRA-8治疗的敏感性。这些拟议的研究将描述PARP1在1)调节死亡受体介导的凋亡信号；2)调节死亡受体介导的生存信号；以及3)TRA-8治疗小鼠胰腺癌模型中的功能。本研究将确定PARP1调控死亡受体介导的凋亡和生存信号通路的新功能和分子机制，这不仅将阐明PARP1在基本的外在凋亡机制中的作用，而且还将提供新的分子洞察力，有助于进一步研究将这些发现转化为退伍军人患者护理的治疗策略。

DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth leading cause of cancer death in the United States, and remains an important health problem for our Veterans. Since 1975, the 5-year survival rate for pancreatic cancer patients has only improved from 2% to 6%. We and others have demonstrated that modulating death receptor-activated extrinsic apoptosis pathways provides novel avenues for cancer therapy. Recombinant ligands or agonistic antibodies for death receptor 4 or 5 (DR4/5), including DR5 agonistic antibody TRA-8, have been shown to induce apoptosis of several types of cancers in preclinical studies and are currently being tested in several phase I-III clinical trals for solid tumors, including pancreatic tumors. However, resistance of pancreatic cancer to TRA-8, and other DR ligands or agonistic antibodies, are major hurdles for effective therapy. Over the past 8 years, we have been seeking effective reagents that overcome the resistance of cancer cells to DR-induced apoptosis. We have demonstrated that modulating the components in the Death-Inducing Signaling Complex (DISC) determines the downstream survival and apoptosis signals conveyed by the DRs, and thus the sensitivity of cancer cells to DR-induced apoptosis. Recently, we identified a new component of the DR5-associated DISC, poly(ADP-ribose) polymerase 1 (PARP1). PARP1 expression levels correlated with the resistance of pancreatic cancer cells to TRA-8-induced apoptosis. Inhibition of PARP1 sensitized resistant pancreatic cancer cells to TRA-8-induced apoptosis in vitro; and enhanced the efficacy of TRA-8 therapy of resistant pancreatic tumors in vivo. In contrast, overexpression of PARP1 in TRA-8-sensitive cells rendered them more resistant to TRA-8. PARP1 is known to function mainly in the nucleus for DNA repair. In human pancreatic cancer tissues and cells, we identified cytoplasmic localization of PARP1. We demonstrated that PARP1 regulated DR5-induced apoptotic signals through novel mechanisms, by promoting poly(ADP-ribose) (pADPr) modification of caspase-8 that inhibited caspase-8 activation. On the other hand, PARP1 inhibition decreased DISC-recruitment of calmodulin (CaM) and Src, which we previously reported to mediate death receptor-induced survival pathways in pancreatic cancer cells. Therefore, we hypothesize that inhibition of PARP1 promotes death receptor-induced apoptotic signals and inhibits survival signals, thus enhancing the sensitivity of pancreatic cancers to TRA-8 therapy. The proposed studies will characterize the function of PARP1 in 1) regulating death receptor-mediated apoptotic signals; 2) regulating death receptor- mediated survival signals; and 3) TRA-8 therapy in mouse models of pancreatic cancer. The present studies will define the novel function and molecular mechanisms underlying PARP1 regulation of death receptor- mediated apoptotic and survival signaling pathways, which will not only elucidate the role of PARP1 in the fundamental extrinsic apoptotic machinery, but also provide novel molecular insights facilitating further studies to translate these findings into therapeutic strategies for patient care for our Veterans.