CXCR3 in Pancreatic Cancer Progression and Metastasis

摘要 摘要胰腺导管腺癌(Pdac)是人类最致命的恶性肿瘤之一。 美国每年有4.7万人死于癌症。PDAC预后惨淡的根本原因是 进展为局部晚期和转移性疾病。最终，这种快速的进步导致了演示 晚期疾病，从而限制了有资格接受根治性切除的患者的数量，并容易 符合手术切除条件的患者局部复发和转移复发。我们对癌前病变的细胞因子进行筛查 损毁攻击性(KPC)和惰性(KC)自发性PDAC小鼠模型发现两种CXCR3配体 (PF4和IP10)仅在KPC模型中过度表达。随后的工作发现，CXCR3A和 B(IP10和PF4的高亲和力受体)在PDAC细胞中异常表达，而IP10 PF4来源于胰腺星状细胞(PSCs)，PF4来源于血小板和内皮细胞。体外培养 研究表明，IP10/CXCR3介导的信号转导导致包括MMPs在内的EMT标志物上调 PF4/CXCR3信号通路增强PDAC细胞株CAPAN-1的低生存能力 附着条件与内皮细胞黏附。这些结果表明，CXCR3在细胞周期调控中起着重要作用。 在转移过程中，PDAC细胞在多个点扩散。在目前建议的研究中，我 本研究旨在探讨CXCR3基因S在肺癌侵袭转移中的作用及其机制。 用体外和体内模型检验“微环境衍生的PF4和IP10信号传导机制”的假说 CXCR3参与了PDAC转移过程的侵袭和血管内阶段。“为了测试这一点 假设，我设计了两个独立的具体目标。目标1，将描述PDAC 细胞诱导肿瘤相关PSC中IP10的表达及其生物学修饰 允许表达IP10的人。随后，我将探讨IP10/CXCR3信号的整体影响 对PDAC行为及IP10在体内外诱导细胞效应机制的研究 基于qRT-PCR的筛查与结果的生化验证相结合。目标2，通过以下方式重点介绍该机制 哪种PF4影响PDAC血源性转移的血管内时相。在这里，试管工作将会扩大 在血小板背景下阐明PF4/CXCR3信号激活的信号级联的初步数据 脱颗粒并将这些通路的激活与功能分析的结果相关联。最后，它的影响 体外发现的PF4功能将通过尾静脉注射在体内进行测试的机制(S) 基因抑制CXCR3B和总CXCR3表达的PDAC转移模型。 重要的是，将在每个目标中作出具体努力，以区分CXCR3A和B的相对贡献 这些现象的异构体，从而使每个异构体的时空重要性变得清晰 在PDAC进展中。通过这些目标，我希望最终阐明生化途径，通过 CXCR3A和B的功能以及CXCR3A和B信号轴在PDAC进展中的作用。

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human malignancies accounting for over 47,000 cancer related deaths in the U.S. annually. Underlying the dismal prognosis of PDAC is the rapid progression to locally advanced and metastatic disease. Ultimately, this rapid progression results in presentation with late stage disease thereby limiting the number of patients eligible for curative resection, and predisposing those patients eligible for resection to local and metastatic recurrence. Our cytokine screen in premalignant lesion of aggressive (KPC) and indolent (KC) autochthonous PDAC mouse models identified two CXCR3 ligands (PF4 and IP10) as being overexpressed only in the KPC model. Subsequent work identified that CXCR3A and B (the high affinity receptors of IP10 and PF4 respectively) are aberrantly expressed in PDAC cells while IP10 is derived from pancreatic stellate cells (PSCs) and PF4 is derived from platelets and endothelium. in vitro studies show that IP10/CXCR3 mediated signaling results in upregulation of EMT markers including MMPs in PDAC cell line Capan 1 while PF4/CXCR3 signaling resulted in increased ability of Capan 1 cells to survive low attachment conditions and adhere to endothelium. These results suggest that CXCR3 plays a role in the dissemination of PDAC cells at multiple points in the metastatic process. In the presently proposed studies, I will explore the significance and mechanism of CXCR3's contribution to PDAC invasion and metastasis using in vitro and in vivo models to test the hypothesis that “microenvironment-derived PF4 and IP10 signaling mediated by CXCR3 contributes to the invasion and intravascular phases of the PDAC metastatic process.” To test this hypothesis, I have devised two independent specific aims. Aim 1, will delineate the mechanism by which PDAC cells induce the expression of IP10 in cancer-associated (CA) PSCs and the modifications in CA-PSC biology that are permissive of IP10 expression. Subsequently, I will explore the overall effects of IP10/CXCR3 signaling on PDAC behavior as well as the mechanism through which IP10 elicits cellular effects in vitro and in vivo using a qRT-PCR based screen coupled with biochemical validation of findings. Aim 2, focuses on the mechanism by which PF4 affects the intravascular phase of hematogenous PDAC metastasis. Here in vitro work will expand on preliminary data to elucidate signaling cascades activated by PF4/CXCR3 signaling in the context of platelet degranulation and correlate activation of these pathways with outcomes of functional assays. Finally, the effects of and mechanism(s) through which PF4 functions found in vitro will be tested in vivo using a tail vein injection model of PDAC metastasis combined with genetic inhibition of CXCR3B and total CXCR3 expression. Importantly, specific efforts will be made in each aim to distinguish the relative contributions of CXCR3A and B isoforms to the phenomena in question thereby providing clarity to the spatiotemporal importance of each isoform in PDAC progression. Through these aims, I expect to conclusively elucidate the biochemical pathways through which CXCR3A and B function and the role of CXCR3A and B signaling axes in PDAC progression.