Molecular targeting of DCLK1 signaling in hepatocellular carcinoma

肝细胞癌中 DCLK1 信号传导的分子靶向

• 批准号: 10590489
• 负责人: Courtney Wayne Houchen
• 金额: --
• 依托单位: OKLAHOMA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-10-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10590489
• 关键词: Alcoholic Liver Diseases; Apoptosis; Automobile Driving; Binding; Biological Assay; Carbon Tetrachloride; Cells; Chronic viral hepatitis; Cirrhosis; Clinical; Complex; Cytoskeleton; Data; Deletion Mutation; Development; Diabetes Mellitus; Diethylnitrosamine; Disease; Disease Progression; Etiology; Fibrosis; Flow Cytometry; General Population; Goals; Healthcare; Healthcare Systems; Hepatic; Hepatitis B Vaccination; Hepatitis C Therapy; Hepatocyte; Heterogeneity; Histology; Immune; Immune response; Immunosuppression; Immunotherapy; Incidence; Inflammation; Inflammatory; Inflammatory Response Pathway; Injury; Innate Immune Response; Interleukin-1 beta; Interleukin-10; Interruption; Intracellular Transport; Kupffer Cells; Liver; Liver Cirrhosis; Liver Fibrosis; Liver diseases; Liver neoplasms; Macrophage; Malignant Neoplasms; Malignant neoplasm of liver; Mediating; Mediator; Medical; Metabolic Diseases; Methods; Microtubules; Molecular Target; Mus; Mutation; Natural Immunity; Nude Mice; Obesity; Oncogenic; Organ; Parents; Pathway interactions; Patient-Focused Outcomes; Patients; Phosphorylation; Phosphotransferases; Point Mutation; Prevalence; Prevention approach; Primary carcinoma of the liver cells; Process; Property; Proteins; Publishing; Radiation; Regulation; Reporting; Risk Factors; Role; Signal Pathway; Signal Transduction; Site; Solid Neoplasm; TCF7L2 gene; TNF gene; Testing; Tissues; Transplantation; Tumor Markers; Tumorigenicity; Unresectable; Veterans; Viral; Work; Xenograft Model; acute liver injury; beta catenin; cancer cell; chemotherapy; chronic liver disease; cytokine; effective therapy; hepatoma cell; immunoregulation; improved; in vivo; inhibitor; liver cancer model; liver injury; monocyte; mortality; mouse model; neoplastic cell; nonalcoholic steatohepatitis; overexpression; phosphoproteomics; polarized cell; prevent; screening; stemness; treatment effect; tumor; tumor growth; tumor heterogeneity; tumor microenvironment; tumor xenograft; tumorigenesis; tumorigenic

Chronic liver diseases (CLDs) including cirrhosis of any etiology are the major risk factors for the development of hepatocellular carcinoma (HCC). The national prevalence and mortality due to HCC in the US VA healthcare systems have increased by 3-fold during the last decade. Despite HBV vaccination, improved screening methods, and successful antiviral treatment for HCV, the incidence of CLD and cirrhosis has substantially (13%) increased since 2000. The existing treatment options (radiation, chemotherapy, and immunotherapy) of unresectable and metastatic HCC have not been successful in the majority of cases. Thus, HCC remains a significant healthcare challenge. The fundamental goal of this proposal is to determine mechanisms driving the transformation process and tumor growth in the liver, and develop strategies to improve clinical outcomes for these patients. In this quest, we found that doublecortin-like kinase 1 (DCLK1) protein is highly expressed in chronic viral hepatitis, cirrhosis, and HCC but not in normal liver. Elevated DCLK1 is associated with decreased survival of HCC patients. Recently, we demonstrated that DCLK1 activates an atypical β-catenin(48 kDa)/TCF4 signaling in hepatoma cells. However, its significance in the CLDs and HCC and DCLK1/β-catenin signaling axis in hepatic cell stemness is completely unknown. Using a mouse model of HCC, we demonstrated that DCLK1 is heavily induced in hepatocytes at sites of injury and likely contributes to the HCC-like tumor growth. Our preliminary data revealed that DCLK1-expressing hepatoma cells can polarize Kupffer cells (KCs) into immunosuppressive M2-like macrophages. IL-10 secreted by M2-like KCs has been reported to promote the selective apoptosis of M1 pro-inflammatory macrophages. Thus, DCLK1-dependent M2 polarization has the potential to promote immunosuppression in the hepatic tumor microenvironment (TME). Furthermore, inhibition of DCLK1 kinase activity resulted in a dramatic reduction in key pro-inflammatory (TNFα, IL-1β) and immunosuppressive IL-10. These observations suggested that DCLK1 is a central regulator of the complex interconnection of fibrogenic, immunoregulatory, and tumorigenic processes in CLDs. Our central hypothesis is that DCLK1 is a key mediator of oncogenic signaling and immune dysregulation in the tissue microenvironment in CLDs, which collectively stimulates the transformation of hepatocytes. We will test this hypothesis with the following interrelated yet independent specific aims. Aim 1 will determine the mechanisms by which DCLK1 mediates immunoregulatory and tumorigenic processes in the liver by introducing deletion and point mutation in DCLK1 and assay for the functional impacts. We will use inhibitors to DCLK1 kinase (DCLK1-IN- 1) and β-catenin/TCF interaction (FH535) to verify DCLK1-regulated clonogenicity, CSC properties, and monocyte/KCs polarization. Aim 2 will determine the effects of DCLK1 kinase inhibition on the DCLK1/β-catenin regulated signaling axis, hepatic transformation process, and immune dysregulation in the HCC TME in a murine model of cirrhosis/liver cancer. Finally, Aim 3 will define the role of the DCLK1/β-catenin axis in the regulation of liver cancer cell stemness and tumor cell heterogeneity in a patient-derived tumor xenografts (PDX) model. The results will be confirmed by the treatment of PDX tumors with DCLK1-IN-1, FH535, and in combination. In addition, we will perform scRNAseq and phosphoproteomic analysis of the tumors to determine differential phosphorylation in DCLK1+ tumor cells and DCLK1 signaling network mediated by phosphorylation. These studies will deepen our understanding of DCLK1 regulation of a complex signaling axis contributing to hepatic tumorigenicity and suppression of innate immune response in the liver tissues, which together drive HCC initiation and tumor growth. In addition, the project will deliver potential anti-DCLK1-based approaches for the prevention and treatment of HCC and cancers of other organs that rely upon DCLK1 functions.

包括任何病因的慢性肝病（CLDS）是发展的主要风险因素 肝细胞癌（HCC）。美国VA Healthcare的HCC造成的全国患病率和死亡率 在过去的十年中，系统增加了3倍。尽管HBV疫苗接种，但改进了筛查方法， 以及成功的HCV抗病毒治疗，CLD和肝硬化的事件大大增加（13％） 自2000年以来。无法切除的现有治疗选择（放射，化学疗法和免疫疗法） 在大多数情况下，转移性HCC并未成功。那是HCC仍然是重要的医疗保健 挑战。该提议的基本目标是确定推动转型过程的机制 肝脏中的肿瘤生长，以及制定策略以改善这些患者的临床结果。在这个任务中， 我们发现双铁蛋白样激酶1（DCLK1）蛋白在慢性病毒肝炎，肝硬化，高度表达 和HCC，但不在正常肝脏中。 DCLK1升高与HCC患者的生存率提高有关。最近， 我们证明DCLK1激活了肝癌细胞中非典型的β-catenin（48 kDa）/TCF4信号传导。然而， 它在CLDS和HCC和DCLK1/β-catenin信号轴上的重要性是完全的 未知。使用HCC的鼠标模型，我们证明了DCLK1在位置的肝细胞中大量诱导 损伤和可能导致类似HCC的肿瘤生长。我们的初步数据表明，表达DCLK1 肝癌细胞可以将库普弗细胞（KC）偏振成免疫抑制M2样巨噬细胞。 IL-10由分泌 据报道，M2样的KCS可以促进M1促炎性巨噬细胞的选择性凋亡。那， DCLK1依赖性M2极化具有促进肝肿瘤中免疫抑制的潜力 微环境（TME）。此外，对DCLK1激酶活性的抑制导致钥匙急剧减少 促炎（TNFα，IL-1β）和免疫抑制IL-10。这些观察结果表明DCLK1是 纤维化，免疫调节和肿瘤过程中复杂互连的中央调节剂 Clds。我们的中心假设是DCLK1是致癌信号传导和免疫失调的关键介体 CLDS中的组织微环境统一刺激了肝细胞的转化。我们将测试 该假设具有以下相关但独立的特定目的。 AIM 1将确定机制 通过引入缺失和 DCLK1中的点突变和功能影响的测定。我们将使用抑制剂进行DCLK1激酶（dclk1-in- 1）和β-catenin/tcf相互作用（FH535）以验证DCLK1调节的Clogenition，CSC特性和 单核细胞/KCS极化。 AIM 2将确定DCLK1激酶抑制对DCLK1/β-catenin的影响 鼠类中HCC TME中的调节信号轴，肝转化过程和免疫失调 肝硬化/肝癌的模型。最后，AIM 3将定义DCLK1/β-catenin轴在调节中的作用 肝癌细胞干和肿瘤细胞异质性在患者衍生的肿瘤异种移植物（PDX）模型中。这 通过使用DCLK1-IN-1，FH535和组合治疗PDX肿瘤，将证实结果。此外， 我们将对肿瘤进行SCRNASEQ和磷蛋白质组学分析，以确定差磷酸化 在DCLK1+肿瘤细胞和DCLK1信号网络中，磷酸化介导。这些研究将加深我们 理解DCLK1调节复杂信号轴，导致肝肿瘤性和 在肝脏时机中抑制先天免疫反应，这加剧了HCC倡议和肿瘤生长。 此外，该项目将提供潜在的基于抗DCLK1的方法，以预防和治疗 依靠DCLK1功能的其他器官的HCC和癌症。