Polymeric Nanomedicines of Hedgehog Inhibitor and miRNA for Treating Pancreatic Cancer

 描述：胰腺癌(PDAC)的成功治疗仍然是一个挑战，因为促结缔组织增生性微环境促进了肿瘤的生长和转移，并形成了对化疗的障碍。Hhedgehog(HH)信号在PDAC的进展中起着关键作用，并有助于结缔组织发育。HH抑制剂GDC-0449可克服促结缔组织反应。虽然胰腺癌干细胞(CSCs)中的HH水平增加，但肿瘤抑制基因miR-let7b针对与PDAC发病相关的几个基因。因此，抑制HH通路，恢复miR-let7b基因表达可有效治疗PDAC。在我们的初步研究中，miR-let7b在胰腺癌细胞系、人类患者的癌症组织以及基因工程KPC小鼠的自发性胰腺癌进展过程中下调。我们的电子分析和体外实验表明，miR-let7b针对在PDAC中上调的几个基因，如MUC4、NCOA3、Kras、HMGA2和TGF？R1。MiR-let7b和GDC-0449对人胰腺癌细胞(CAPAN-1、HPAF-II和T3M4)的生长均有抑制作用，并与聚乙二醇-嵌段-聚(2-甲基-2-羧基-丙烯-carbonate-graft-dodecanol-graft-tetraethylenepentamine))共聚物形成胶束具有协同作用。这种共聚物自组装成胶束，将疏水的GDC-0449包裹到其核心中，并允许miR-let7b和阳离子吊链之间形成络合物。与单独携带GDC-0449或miR-let7b的胶束相比，这种联合疗法在注射到异位荷瘤小鼠时有效地抑制了肿瘤的生长。将抗EGFR受体的单抗西妥昔单抗与纳米药物偶联，实现了对PDAC的主动靶向。因此，我们推测利用西妥昔单抗结合纳米药物联合应用miR-let7b和GDC-0449可以有效地通过减少结缔组织增生来增加PDAC细胞的化疗敏感性，并通过降低其致瘤性来治疗PDAC细胞。我们的总体目标是开发利用纳米制剂通过miR-let7b靶向多个信号通路和通过GDC-0449靶向HH通路的PDAC的新的联合疗法。我们的具体目的是：i)确定miR-let7b和GDC-0449联合治疗PC细胞的疗效和分子机制；ii)在西妥昔单抗共轭聚合物胶束中共形成miR-let7b和GDC-0449，并评估它们对胰腺癌细胞的协同抑制作用；iii)确定miR-let7b和GDC-0449胶束在原位NSG和基因工程KPC小鼠PDAC模型中的治疗效果。长期意义在于开发miR-let7b和GDC-0449的纳米药物，增加这些肿瘤纤维化间质中的局部药物浓度，并绕过允许肿瘤生长和抑制当前标准化疗疗效的耐药机制。

 DESCRIPTION: Successful treatment of pancreatic adenocarcinoma (PDAC) remains a challenge due to the desmoplastic microenvironment that promotes both tumor growth and metastasis and forms a barrier to chemotherapy. Hedgehog (Hh) signaling plays a crucial role in PDAC progression and contributes to desmoplasia. Hh inhibitor GDC-0449 can overcome desmoplastic reaction. While Hh levels are increased in pancreatic cancer stem cells (CSCs), tumor suppressor miR-let7b targets several genes involved in PDAC pathogenesis. Therefore, inhibition of Hh pathway and restoration of miR-let7b could effectively treat PDAC. In our preliminary studies, miR-let7b was downregulated in pancreatic cancer cell lines, human patient's cancer tissues and during progression of spontaneous pancreatic cancer in genetically engineered KPC mice. Our in-silico analysis and in vitro experiments indicate that miR-let7b targets several genes like MUC4, NCOA3, Kras, HMGA2 and TGFßR1, which are upregulated in PDAC. miR-let7b and GDC-0449 could inhibit the proliferation of human pancreatic cancer cells (Capan-1, HPAFII and T3M4) and there was synergistic effect when miR-let7b and GDC- 0449 were co-formulated into micelles using poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl- propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (PEG-b-PCC-g-DC-g-TEPA) copolymer. This copolymer self-assembles into micelles and encapsulates hydrophobic GDC-0449 into its core and allows complex formation between miR-let7b and cationic pendant chains. This combination therapy effectively inhibited tumor growth when injected to ectopic tumor bearing mice compared to micelles carrying GDC-0449 or miR-let7b alone. Cetuximab, a monoclonal antibody for EGFR receptors, was conjugated to the nanomedicine to achieve active targeting to PDAC. Therefore, we hypothesize that combination therapy of miR-let7b with GDC-0449 using cetuximab conjugated nanomedicines can effectively chemosensitize PDAC cells by reducing desmoplasia and treat PDAC by reducing their tumorigenicity. Our overall objective is to develop novel combination therapy for PDAC using nanoformulations targeting multiple signaling pathways by miR-let7b and Hh pathway by GDC-0449. Our specific aims are to: i) determine the efficacy and molecular mechanisms of miR-let7b and GDC-0449 combination therapy in PC cells; ii) co-formulate miR-let7b and GDC- 0449 in cetuximab conjugated polymeric micelles and assess their synergistic effect on the inhibition of pancreatic cancer cells, iii) determine the therapeutic efficacy of micelles encapsulating miR-let7b and GDC-0449 in orthotopic NSG and genetically engineered KPC mouse models of PDAC. Long- term significance is to develop nanomedicines of miR-let7b and GDC-0449 that increase the local drug concentrations within the fibrotic stroma of these tumors and bypass the resistance mechanisms that allow tumor growth and inhibit the efficacy of current standard chemotherapies.