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: 10477201
• 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/10477201
• 关键词: Alanine; Animals; Anticoagulants; Antitumor Response; Arginine; Binding; Binding Sites; Blood coagulation; C-terminal; Cancer Biology; Cancer Model; Carboxypeptidase; Cells; Cerebrospinal Fluid; 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; chemokine; clinical practice; clotting enzyme; cytokine; humanized mouse; in vivo; inhibitor; 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）是一种具有多效性功能的循环基质细胞蛋白，其表达上调， 炎症和癌症。骨桥蛋白具有多个功能结构域和凝血酶切割位点（在人骨桥蛋白中位于Arg 168）， 和小鼠中的Arg 153）。凝血酶裂解暴露了α4β1和α9β1整合素的隐蔽整合素结合位点， 新的C末端SVVYGLR。我们已经证明Arg 168是一个真正的凝血酶切割位点，而凝血酶- 切割的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）或凝血酶激活的CPB 2切割，将其转化为OPN- Leu（OPN-L）。OPN-R和OPN-L水平在炎性关节液和脑脊液中升高， 胶质母细胞瘤（GBM），基于我们实验室开发的特异性ELISA，证明这些裂解发生在 in vivo.骨桥蛋白参与促进许多癌症的侵袭性和转移性进展，包括 乳腺癌、肺癌、前列腺癌和卵巢癌、GBM和黑素瘤。然而，凝血酶裂解骨桥蛋白的作用， 在体内癌症生物学中是不确定的。我们建立了一个凝血酶抗性OPN基因敲入（KI）小鼠，其中Arg 153 被丙氨酸（OPNR 153 A）取代。OPNR 153 A KI小鼠是健康和可生育的。在初步研究中，我们发现： 1)在OPN KO和OPNR 153 A 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OPNR 153 A KI B16肿瘤的浸润F4/80+显着增加 巨噬细胞3)体内巨噬细胞耗竭可消除OPNR 153 A KI小鼠的肿瘤抑制作用 而在免疫缺陷型NOG-WT、NOG-OPN KO和NOG-OPNR 153 A KI小鼠中未观察到。因此，在本发明中， OPNR 153 A KI小鼠中的肿瘤抑制由F4/80+巨噬细胞介导。4)口服达比加群， 口服活性直接凝血酶抑制剂，抑制WT小鼠B16肿瘤生长和转移，复制 OPNR 153 A KI表型。5)在小鼠卵巢癌中，OPNR 153 A KI小鼠的恶性腹水得到抑制 模型我们的总体假设是，OPN的凝血酶裂解导致宿主抗肿瘤的抑制。 免疫应答，与肿瘤相关巨噬细胞（TAM）减少相关，因此有利于肿瘤 生长和转移。阻断凝血酶对骨桥蛋白的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。具体目标1将确定B16肿瘤 OPNR 153 A KI小鼠中的抑制可推广到其它鼠和人癌症模型。我们将测试 小鼠卵巢癌和GBM模型中的OPNR 153 A KI小鼠（Subaim 1.1）。我们将重建 用人免疫细胞产生“人源化”NOG-OPN KO和NOG-OPNR 153 A的免疫缺陷NOG小鼠 KI小鼠。我们将用相应的肿瘤抑制剂来验证在小鼠癌症模型中观察到的肿瘤抑制。 人类癌症模型，并在这些“人源化”小鼠中测试其他人类癌症模型。具体目标2将 测试达比加群是否作为标准黑色素瘤治疗的辅助治疗。我们将测试 OPNR 153 A KI小鼠显示携带BRAFV 600 E的鼠YUMM 3.1黑素瘤细胞的肿瘤抑制 突变（Subaim 2.1）。然后，我们将测试达比加群与维罗非尼（BRAF激酶）联合治疗 抑制剂）或维罗非尼和cobimetinib（MEK激酶抑制剂），降低了WT小鼠的存活率， YUMM3.1/BRAFV 600 E黑色素瘤（子章节2.2和2.3），以及达比加群是否在达卡巴嗪基础上添加 患有B16黑素瘤的WT小鼠的存活率（Subaim 2.4）。在具体目标3中，我们将过表达OPN-R，OPN-L， 和OPN-CTF（C-末端片段），以确定哪种切割的OPN形成 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OPNR 153 A KI小鼠的肿瘤细胞中，并将它们与WT B16肿瘤的细胞进行比较（Subaim 3.2）。我们假设 WT小鼠中OPN的凝血酶裂解减少TAM浸润并诱导“肿瘤促进”M2样细胞凋亡。 表型，而OPN KO和OPNR 153 A小鼠中的TAM将具有促炎“肿瘤抑制”作用 M1样表型。我们的观察结果是新颖的，有意义的和相关的癌症研究和临床实践。