Investigation of novel signaling protein in 3D and in vivo PDAC models using second generation Ref-1 inhibitors

项目摘要/摘要 胰腺导管腺癌(PDAC)的五年生存率仍然保持在令人沮丧的10%。这个可怜的人 结果归因于PDAC复杂的肿瘤微环境(TME)包含促结缔组织间质， 它由癌症相关成纤维细胞(CAF)和致密的细胞外基质(ECM)组成。这层基质 影响胰腺癌细胞(PCCs)的增殖、侵袭和耐药性。出乎意料的是，消融 CAF或靶向间质中的某些ECM蛋白导致的变化实际上加速了肿瘤的生长 和损害治疗结果。为了应对这一挑战，我们正在追捕有选择性地杀死PCCs的目标 而不是CAF。氧化还原效应因子-1(Ref-1)是几个关键信号通路的上游调控因子 转录因子(TF)。用我们的第一代抑制剂阻断Ref-1氧化还原功能 包括HIF-1α、NFkB和AP-1在内的关键转录因子的激活，所有这些都与生长和转移有关 肿瘤中的信号。然而，需要更有效的第二代化合物来有效治疗 PDAC。我们最近合成了新的类似物，并获得了数据，表明这些类似物在 肿瘤细胞存活率显著降低，CAF存活率变化不大。这种选择性的癌细胞杀伤 是我们新类似物的一个新特征，因为吉西他滨既能杀死PCCs，也能杀死CAF。潜在的 这些化合物的作用机制和治疗效果是本提案的重点。我们的首要目标 目的是通过比较Ref-1的作用机制来评价第二代Ref-1抑制剂治疗PDAC的效果 PCCs和CAF中的抑制作用。我们的假设是，通过抑制Ref-1，多种癌症相关 同时有效地靶向通路导致PCCs中生存信号的抑制，而 免了CAF的麻烦。PDAC的异质性和复杂性对筛选和 评估新化合物对肿瘤和间质成分的疗效。要克服这些技术问题 挑战并最终导致新的治疗方法，我们将使用以下技术对Ref-1抑制剂进行机械筛选 基于患者来源的PCCs和CAFS的新的体外肿瘤模型--3D共培养球体 用于筛查和肿瘤-间质相互作用；以及2)微流控肿瘤微环境芯片(T-MOC) 机械性评价模型。使用这些新的体外系统的研究结果将通过原位实验进行验证 PDAC型号。具体目标是：目标1.研究Ref-1信号的选择性及其影响 用T-MOC模型研究PCCs和CAF的抑制作用；目的2.表征Ref-1对PCCs和CAF的抑制机制 第二代化合物；以及目标3.验证Ref-1类似物的疗效和药效学特征 在原位PDAC模型中。这项提案的目的将提供关于两个方面的宝贵信息 新的Ref-1类似物在复杂的体内和体外PDAC模型中的有效性及体外验证 在需要治疗选择的癌症中进行新靶点和化合物筛选的共培养模型。

PROJECT SUMMARY/ABSTRACT Five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) still remains at a dismal 10%. This poor outcome is attributed to PDAC’s complex tumor microenvironment (TME) containing a desmoplastic stroma, which consists of cancer-associated fibroblasts (CAFs) and a dense extracellular matrix (ECM). This stroma affects proliferation, invasion and drug resistance of pancreatic cancer cells (PCCs). Unexpectedly, the ablation of CAFs or targeting of certain ECM proteins in the stroma led to changes that actually accelerated tumor growth and impaired treatment outcome. To address this challenge, we are pursuing targets that selectively kill PCCs rather than CAFs. Redox effector factor-1 (Ref-1) is upstream of several key signaling pathways as a regulator of transcription factors (TFs). Blockade of Ref-1 redox function with our first-generation inhibitor blocks the activation of key TFs including HIF-1α, NFkB, and AP-1, all of which are implicated in growth and metastasis signaling in the tumor. However more potent second-generation compounds are necessary to effectively treat PDAC. We recently synthesized new analogs and obtained data showing that these are more potent with a significant decrease in tumor cell survival with minimal changes in CAF survival. This selective cancer cell killing is a novel characteristic of our new analogs, as gemcitabine kills both PCCs and CAFs. The underlying mechanism and therapeutic efficacy of these compounds are the focus of this proposal. Our overarching goal is to evaluate second-generation Ref-1 inhibitors for treatment of PDAC by comparing the mechanisms of Ref-1 inhibition in PCCs and CAFs. Our hypothesis is that through inhibition of Ref-1, multiple cancer-associated pathways are simultaneously and effectively targeted leading to suppression of survival signaling in PCCs while sparing the CAFs. The heterogeneity and complexity of PDAC poses a significant challenge in screening and evaluating the efficacy of new compounds on tumors and on stromal components. To overcome these technical challenges and ultimately lead to new treatments, we will perform mechanistic screening of Ref-1 inhibitors using newly developed in vitro tumor models based on patient-derived PCCs and CAFs - 1) 3D co-culture spheroids for screening and tumor-stroma interaction; and 2) a microfluidic tumor-microenvironment-on-chip (T-MOC) model for mechanistic evaluation. Findings using these novel in vitro systems will be validated with orthotopic PDAC models. Specific aims are: Aim 1. Investigate the selectivity of Ref-1 signaling and the effects of its inhibition in PCCs and CAFs using T-MOC models; Aim 2. Characterize the mechanism of Ref-1 inhibition for second-generation compounds; and Aim 3. Validate the efficacy and pharmacodynamic profiles of Ref-1 analogs in orthotopic PDAC models. The aims of this proposal will provide invaluable information regarding both the effectiveness of new Ref-1 analogs in sophisticated in vivo and in vitro PDAC models and validate our in vitro co-culture models for novel target and compound screening in a cancer that is in need of therapeutic options.