CXCR3 in Pancreatic Cancer Progression and Metastasis

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47，000例癌症相关死亡。PDAC预后不佳的根本原因是， 进展为局部晚期和转移性疾病。最终，这种快速的进展导致了 晚期疾病，从而限制了适合根治性切除的患者数量， 那些适合切除局部和转移性复发的患者。我们的癌前病变细胞因子筛查 侵袭性（KPC）和惰性（KC）本地PDAC小鼠模型损伤鉴定了两种CXCR 3配体 (PF4和IP 10）仅在KPC模型中过表达。随后的工作确定了CXCR 3A和 B（分别为IP 10和PF 4的高亲和力受体）在PDAC细胞中异常表达，而IP 10 PF 4来源于胰腺星状细胞（PSC），PF 4来源于血小板和内皮。体外 研究表明，IP 10/CXCR 3介导的信号传导导致EMT标志物包括MMP的上调， PDAC细胞系Capan 1，而PF 4/CXCR 3信号转导导致Capan 1细胞在低浓度下存活的能力增加。 附着条件和粘附于内皮。这些结果表明，CXCR 3在细胞凋亡中起作用。 PDAC细胞在转移过程中的多点播散。在目前的研究中，我 本研究旨在探讨CXCR 3在PDAC侵袭转移中的作用及其机制， 体外和体内模型，以检验“微环境来源的PF 4和IP 10信号传导介导的”这一假设。 通过CXCR 3的表达有助于PDAC转移过程的侵袭和血管内阶段。为了验证这一 在这个假设中，我设计了两个独立的具体目标。目的1，将阐明PDAC的机制， 细胞诱导癌症相关（CA）PSC中IP 10的表达和CA-PSC生物学的改变 允许IP 10表达。随后，我将探讨IP 10/CXCR 3信号转导的整体影响， 对PDAC行为的影响以及IP 10在体外和体内发挥细胞作用的机制， 基于qRT-PCR的筛选加上发现的生物化学验证。目标2，侧重于机制， 其中PF 4影响血行性PDAC转移的血管内期。在这里，体外工作将扩大 基于初步数据阐明血小板背景下PF 4/CXCR 3信号传导激活的信号级联反应 脱粒并将这些途径的激活与功能测定的结果相关联。最后，影响 将使用尾静脉注射在体内测试体外发现的PF 4功能的机制 PDAC转移模型结合CXCR 3B和总CXCR 3表达的遗传抑制。 重要的是，将在每个目标中做出具体努力以区分CXCR 3A和B的相对贡献 异构体的问题的现象，从而提供清晰的时空重要性，每个异构体 PDAC进展。通过这些目标，我希望最终阐明生物化学途径， CXCR 3A和B的功能以及CXCR 3A和B信号传导轴在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.