Targeting tumor and its microenvironment using nanotherapeutics for pancreatic cancer

摘要 胰腺癌(PC)是致命的，五年生存率不到9.2%，中位生存期为5-6 月份。单一疗法的有限疗效导致了对有限的联合疗法的探索。 成功是因为与剂量限制副作用、药物相关毒性、药物 耐药性和较差的药代动力学。重要的是，这些过去的方法并没有尝试同时进行 靶向胰腺肿瘤及其间质和PC干细胞。我们提出的工作解决了这些问题 通过确定miR-345的功能和临床病理意义以及开发双重 用于联合输送miR-345和GEM的纳米级输送装置(DDND)，用于治疗PC。我们的 初步研究表明，miR-345针对几个重要的基因，包括sonic hedgehog(Shh)， KRAS、MUC4粘蛋白及其下游靶点、与肿瘤干细胞相关的基因(ALDH1、ESA、HIF1α、 和Oct/3/4)，并导致裂解的caspase-3、-7和PARP上调。Kras、Shh和MUC4-信号 通过促进上皮间充质转化(EMT)在肿瘤生长和转移中发挥关键作用 干细胞、血管生成、结缔组织增生症，这些都限制了化疗的传递和疗效。MIR-345瞄准 KRAS、Shh和MUC4使miR-345成为诊断/预后和治疗的极佳候选者 PC中的目标。我们假设miR-345的下调通过上调参与了PC的发病。 Kras、SHH和MUC4；它的修复，通过DDND与GEM相结合，增强了GEM的敏感性 在PC中通过调节SHH/Kras/MUC4通路，导致抑制促结缔组织增生、胰腺星状 细胞和PC干细胞通过改善PC的肿瘤而改善GEM对PC的治疗效果 灌流。DDND是基于温度和pH响应型五嵌段共聚物的静电 与miR-345络合，然后与宝石包覆层自组装。DDND设计 允许miRNA/GEM组合的有效共掺；促进细胞进入；增强稳定性 与脂质体载体相比；提供miRNA保护；通过选择性地促进内涵体而允许靶向 通过利用细胞内pH差异在癌细胞中逃逸，而不是正常细胞；并允许剂量- 省去了细胞毒性药物。目标1将重点确定功能角色和临床病理意义 高侵袭性和转移性PC中miR-345/Shh/Kras/MUC4轴的突变。目标2将专注于开发 DDND负载miR-345/GEM作为治疗致死性PC的新型药物的疗效评价 体外药效。在最终目标3中，我们将评估单独负载DDND的miR-345/GEM或 在小鼠模型中结合使用。总之，这项拟议的工作破译了该病的临床病理意义。 MIR-345，预计将大大推进将GEM和miR-345结合起来用于治疗的目标 加强对PC患者的协同作用机制的了解，并将提供一种新的DDND 为未来其他疗法的交付而设计。

ABSTRACT Pancreatic cancer (PC) is lethal with a five-year survival rate of less than 9.2 % and a median survival of 5-6 months. The limited efficacy of mono-therapies has led to the exploration of combination therapies with limited success because of challenges associated with dose-limiting side effects, drug-associated toxicities, drug resistance, and poor pharmacokinetics. Importantly, these past approaches have not attempted the concurrent targeting of the pancreatic tumor and its stroma and PC stem cells. Our proposed work addresses these challenges by determining functional and clinic-pathological significance of miR-345 as well as developing a dual delivery nanoscale device (DDND) for combined delivery of miR-345 and GEM for the treatment of PC. Our preliminary studies have shown that miR-345 targets several important genes, including sonic hedgehog (Shh), Kras, MUC4 mucin and its downstream targets, genes-associated with cancer stem cells (ALDH1, ESA, Hif1α, and Oct/3/4), and causes up regulation of cleaved caspase-3, -7, and PARP. The Kras, Shh and MUC4-signaling play critical roles in tumor growth and metastasis by promoting epithelial to mesenchymal transition (EMT), PC stem cells, angiogenesis, desmoplasia, which limit the delivery and efficacy of chemotherapy. MiR-345 targeting Kras, Shh and MUC4, which makes miR-345 is an excellent candidate for diagnostic/prognostic and therapeutic targets in PC. We hypothesize that downregulation of miR-345 contributes to PC pathogenesis by upregulation of Kras, SHH, and MUC4; Its restoration, combination with GEM through the DDND, enhances GEM sensitivity in PC through modulation of SHH/Kras/MUC4 pathways, resulting in inhibition of desmoplasia, pancreatic stellate cells, and PC stem cells leading to an improved therapeutic outcome of GEM in PC through improving its tumor perfusion. The DDND is based on temperature and pH responsive pentablock copolymers electrostatically complexed with miR-345 and subsequently self-assembled with GEM encapsulated layers. The DDND design allows effective co-incorporation of miRNA/GEM combination; facilitates cellular entry; enhances stability compared to liposomal carriers; provides miRNA protection; allows targeting by selectively facilitating endosomal escape in cancer cells as opposed to normal cells by exploiting intracellular pH differences; and allows dose- sparing of the cytotoxic drugs. Aim 1 will focus to determine functional role and clinico-pathological significance of miR-345/Shh/Kras/MUC4 axis in highly aggressive and metastatic PC. Aim 2 will focus on the development of DDND loaded miR-345/GEM as a novel therapeutic agent against lethal PC by evaluating their therapeutic efficacy in vitro. In the final Aim 3, we will evaluate therapeutic efficacy of DDND loaded miR-345/GEM alone or in combination in mouse models. Altogether, the proposed work decipher the clinic-pathological significance of miR-345 and expected to significantly advance the goal of combining GEM and miR-345 delivery for treatment of PC patients, enhance understanding of the synergistic mechanisms involved, and will provide a novel DDND design for delivery of other therapeutics as well in the future.