Thrombin Cleavage of Osteopontin Suppresses Host-Anti-Tumor Immune Response in Cancer

骨桥蛋白的凝血酶裂解抑制癌症中宿主抗肿瘤免疫反应

• 批准号: 10016591
• 负责人: LAWRENCE L LEUNG
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10016591
• 关键词: Alanine; Animals; Anticoagulants; Antitumor Response; Arginine; Binding; Binding Sites; Blood coagulation; C-terminal; Cancer Biology; Cancer Model; Carboxypeptidase; Cells; Cerebrospinal Fluid; Cleaved cell; Colon Carcinoma; Cutaneous Melanoma; Dacarbazine; Enzyme-Linked Immunosorbent Assay; Functional disorder; Glioblastoma; Growth; Health; Human; Immune; Immunocompetent; Infiltration; Inflammation; Inflammatory; Integrin Binding; Integrins; Joints; Knock-in; Knock-in Mouse; Link; Liquid substance; Lysine Carboxypeptidase; MAP3K1 gene; Macrophage Activation; Malignant Neoplasms; Malignant neoplasm of brain; Malignant neoplasm of lung; Malignant neoplasm of ovary; Malignant neoplasm of prostate; Mediating; Melanoma Cell; Metastatic Neoplasm to the Lung; Modality; Modeling; Mus; Mutation; Neoplasm Metastasis; Oral; Oral Administration; Phenotype; Play; Population; Proteins; Proto-Oncogene Proteins B-raf; Resistance; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Skin Cancer; Testing; Thrombin; Transcript; Tumor Suppression; Tumor-associated macrophages; Veterans; alpha-Thrombin; anti-tumor immune response; anticancer research; base; c new; chemokine; clinical practice; clotting enzyme; cytokine; humanized mouse; in vivo; inhibitor/antagonist; integrin alpha9 beta1; kinase inhibitor; macrophage; malignant ascites; malignant breast neoplasm; melanoma; novel; novel therapeutics; osteopontin; overexpression; reconstitution; tumor; tumor growth

Expression of osteopontin (OPN), a circulating matricellular protein with pleiotropic functions, is up-regulated in inflammation and cancer. OPN has multiple functional domains and a thrombin cleavage site (at Arg168 in human and Arg153 in mouse). Thrombin cleavage exposes a cryptic integrin-binding site for α4β1 and α9β1 integrins at the new C-terminus, SVVYGLR. We have shown that Arg168 is a bona fide thrombin cleavage site, and thrombin- cleaved OPN-Arg (OPN-R) has enhanced α4β1-dependent cell-binding activity which is abolished when the C- terminal arginine is cleaved by carboxypeptidase N (CPN) or thrombin-activatable CPB2, converting it to OPN- Leu (OPN-L). OPN-R and OPN-L levels are elevated in inflammatory joint fluid and cerebral spinal fluid in glioblastoma (GBM), based on specific ELISAs developed in our lab, demonstrating that these cleavages occur in vivo. OPN has been implicated in promoting invasive and metastatic progression of many cancers, including breast, lung, prostate and ovarian cancers, GBM and melanoma. However, the role of thrombin cleavage of OPN in cancer biology in vivo is undefined. We created a thrombin-resistant OPN knock-in (KI) mouse in which Arg153 is replaced by alanine (OPNR153A). OPNR153A KI mice are healthy and fertile. In preliminary studies, we showed: 1) decreased murine B16 melanoma growth and pulmonary metastasis in OPN KO and OPNR153A KI mice compared to WT mice. 2) OPN KO and OPNR153A KI B16 tumors had significantly increased infiltrating F4/80+ macrophages. 3) Tumor suppression in the OPNR153A KI mice was abolished by macrophage depletion in vivo and it was not observed in the immune deficient NOG-WT, NOG-OPN KO and NOG-OPNR153A KI mice. Thus, tumor suppression in OPNR153A KI mice is mediated by F4/80+ macrophages. 4) Oral administration of dabigatran, an orally active direct thrombin inhibitor, suppressed B16 tumor growth and metastasis in WT mice, replicating the OPNR153A KI phenotype. 5) Malignant ascites was suppressed in OPNR153A KI mice in a murine ovarian cancer model. Our overall hypothesis is that thrombin cleavage of OPN leads to suppression of the host-anti-tumor immune response, associated with a decrease in tumor-associated macrophages (TAMs), thus favoring tumor growth and metastasis. Blocking thrombin cleavage of OPN will lead to enhancement of the host-anti-tumor response, resulting in reduced tumor growth and metastasis. Specific Aim 1 will determine if B16 tumor suppression in the OPNR153A KI mouse is generalizable to other murine and human cancer models. We will test the OPNR153A KI mouse in the murine ovarian cancer and GBM models (Subaim 1.1). We will reconstitute the immune deficient NOG mice with human immune cells to create “humanized” NOG-OPN KO and NOG-OPNR153A KI mice. We will validate the tumor suppression observed in the murine cancer models with the corresponding human cancer models, and test additional human cancer models in these “humanized” mice. Specific Aim 2 will test whether dabigatran functions as an adjunctive therapy with standard melanoma therapy. We will test whether the OPNR153A KI mouse shows tumor suppression with murine YUMM3.1 melanoma cells carrying the BRAFV600E mutation (Subaim 2.1). We will then test whether dabigatran, in combination with vemurafenib (BRAF kinase inhibitor) or vemurafenib and cobimetinib (MEK kinase inhibitor), prolongs the survival of WT mice with YUMM3.1/BRAFV600E melanoma (Subaims 2.2 and 2.3), and whether dabigatran adds to dacarbazine in the survival of WT mice with B16 melanoma (Subaim 2.4). In Specific Aim 3, we will over-express OPN-R, OPN-L, and OPN-CTF (C-terminal fragment) in the OPN-KO and OPNR153A mice to determine which cleaved OPN form reverses the B16 tumor suppression phenotype (Subaim 3.1). We will characterize TAMs from B16 tumors in OPN KO and OPNR153A KI mice and compare them to that from WT B16 tumors (Subaim 3.2). We hypothesize that thrombin cleavage of OPN in WT mice reduces TAM infiltration and induces a “tumor promoting” M2-like phenotype, whereas TAMs in the OPN KO and OPNR153A mice will have a proinflammatory “tumor suppressing” M1-like phenotype. Our observations are novel, significant and relevant to cancer research and clinical practice.

骨桥蛋白（OPN）的表达是一种具有多效性功能的循环基质蛋白，在 炎症和癌症。 OPN具有多个功能域和凝血酶裂解位点（在人类的Arg168处 和鼠标中的arg153）。凝血酶裂解暴露了α4β1和α9β1整联蛋白的加密蛋白结合位点 新的C末端，SvvyGlr。我们已经表明，Arg168是一个真正的凝血酶裂解部位，凝血酶 - 裂解的OPN-ARG（OPN-R）具有增强的α4β1依赖性细胞结合活性，当C- 末端精氨酸被羧肽酶N（CPN）或凝血酶激活CPB2裂解，将其转换为OPN- Leu（OPN-L）。 OPN-R和OPN-L水平在炎症关节液和脑脊髓液中升高 基于我们实验室中开发的特定ELISA的胶质母细胞瘤（GBM），表明这些裂解发生 体内。 OPN已暗示促进许多癌症的侵入性和转移性进展，包括 乳腺癌，肺，前列腺和卵巢癌，GBM和黑色素瘤。但是，OPN的凝血酶裂解的作用 在癌症生物学中，体内不确定。我们创建了一种抗凝血酶的OPN敲入（KI）鼠标，其中Arg153 被丙氨酸（OPNR153A）取代。 OPNR153A KI小鼠健康且肥沃。在初步研究中，我们表明： 1）在OPN KO和OPNR153A KI小鼠中降低鼠B16黑色素瘤生长和肺转移 与WT小鼠相比。 2）OPN KO和OPNR153A KI B16肿瘤显着增加了F4/80+ 巨噬细胞。 3）OPNR153A Ki小鼠中的肿瘤抑制作用被巨噬细胞部署消除 并且在免疫降落式NOG-WT，NOG-OPN KO和NOG-OPNR153A KI小鼠中没有观察到它。那， OPNR153A Ki小鼠中的肿瘤抑制是由F4/80+巨噬细胞介导的。 4）口服dabigatran， 口服活跃的直接凝血酶抑制剂，抑制WT小鼠的B16肿瘤生长和转移，复制 OPNR153A KI表型。 5）在鼠类卵巢癌中，OPNR153A ki小鼠抑制恶性腹水 模型。我们的总体假设是，OPN的凝血酶裂解会导致抑制宿主 - 抗肿瘤 免疫反应，与肿瘤相关巨噬细胞（TAM）的减少有关，因此有利于肿瘤 生长和转移。阻塞OPN的凝血酶裂解将导致宿主 - 抗肿瘤的增强 反应，导致肿瘤生长和转移减少。特定的目标1将确定B16肿瘤是否 OPNR153A Ki小鼠中的抑制作用可推广到其他鼠类和人类癌模型。我们将测试 鼠卵巢癌和GBM模型中的OPNR153A Ki小鼠（Subaim 1.1）。我们将重构 具有人类免疫细胞的免疫缺陷NOG小鼠创建“人性化” Nog-Opn KO和Nog-Opnr153a Ki小鼠。我们将验证与相应的鼠类癌模型中观察到的肿瘤抑制 人类癌症模型，并在这些“人源”小鼠中测试其他人类癌症模型。具体目标2将 测试Dabigatran是否可以用标准黑色素瘤治疗的辅助治疗。我们将测试是否 OPNR153A Ki小鼠用含Brafv600E的鼠类Yumm3.1黑色素瘤细胞显示肿瘤抑制 突变（Subaim 2.1）。然后，我们将测试Dabigatran是否与Vemurafenib（BRAF激酶）结合 抑制剂）或vemurafenib和cobimetinib（MEK激酶抑制剂），延长WT小鼠的存活率 Yumm3.1/Brafv600E黑色素瘤（Subiaims 2.2和2.3），以及Dabigatran是否增加了Dacarbazine WT小鼠与B16黑色素瘤的生存（Subaim 2.4）。在特定的目标3中，我们将过表达OPN-R，OPN-L， OPN-KO和OPNR153A小鼠中的OPN-CTF（C末端片段），以确定哪种切割OPN形式 逆转B16肿瘤抑制表型（Subaim 3.1）。我们将表征来自B16肿瘤的TAM OPN KO和OPNR153A KI小鼠，并将其与WT B16肿瘤进行比较（Subaim 3.2）。我们假设 WT小鼠中OPN的凝血酶切割减少了TAM浸润并诱导“肿瘤促进” M2样 表型，而OPN KO和OPNR153A小鼠中的TAM将具有促炎的“抑制肿瘤” M1样表型。我们的观察结果是新颖的，重要的，并且与癌症研究和临床实践有关。