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

骨桥蛋白(OPN)是一种具有多种功能的循环基质细胞蛋白，其表达上调在 炎症和癌症。OPN具有多个功能结构域和一个凝血酶裂解位点(人类Arg168位 和小鼠体内的Arg153)。凝血酶切割暴露了α4β1和α9β1整合素的隐蔽整合素结合位点 新的C端，SVVYGLR。我们已经证明Arg168是一个真正的凝血酶裂解位点，而凝血酶- 裂解的骨桥蛋白-精氨酸(OPN-R)可增强α4β1依赖的细胞结合活性，这种结合活性在C- 末端精氨酸被羧基肽酶N(CPN)或凝血酶激活的CPB2裂解，将其转化为OPN- Leu(OPN-L)。炎性关节液和脑脊液中OPN-R和OPN-L水平升高 胶质母细胞瘤(GBM)，基于我们实验室开发的特定ELISA，证明这些分裂发生 在活体内。OPN与促进许多癌症的侵袭和转移有关，包括 乳腺癌、肺癌、前列腺癌和卵巢癌、基底膜和黑色素瘤。然而，凝血酶在OPN中的作用 在癌症生物学中，体内的定义尚不明确。我们创造了一种抗凝血酶的OPN敲入(KI)小鼠，在其中Arg153 被丙氨酸(OPNR153A)取代。OPNR153A Ki小鼠是健康和有生育能力的。在初步研究中，我们表明： 1)抑制OPN KO和OPNR153A Ki小鼠B16黑色素瘤的生长和肺转移 与WT小鼠相比。2)OPN KO和OPNR153A Ki B16肿瘤的浸润性F4/80显著增加 巨噬细胞。3)体内巨噬细胞耗尽可消除OPNR153A Ki小鼠的肿瘤抑制作用 在免疫缺陷的NOG-WT、NOG-OPN KO和NOG-OPNR153A Ki小鼠中均未观察到。因此， OPNR153A Ki小鼠的肿瘤抑制是由F4/80巨噬细胞介导的。4)口服达比加兰； 一种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 OPNR153A KI表型。5)OPNR153A Ki小鼠对小鼠卵巢癌恶性腹水的抑制作用 模特。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 免疫反应，与肿瘤相关巨噬细胞(TAM)减少有关，因此有利于肿瘤 生长和转移。阻断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。特异性靶点1将决定B16肿瘤 OPNR153A Ki小鼠的抑制作用可推广到其他小鼠和人类癌症模型。我们将测试 小鼠卵巢癌和GBM模型中的OPNR153A Ki小鼠(Subaim 1.1)。我们将重组 免疫缺陷NOG小鼠与人免疫细胞共同创造人源化NOG-OPN KO和NOG-OPNR153A 基鼠。我们将验证在小鼠癌症模型中观察到的肿瘤抑制作用 人类癌症模型，并在这些“人源化”小鼠身上测试其他人类癌症模型。特定目标2将 测试达比卡特兰是否作为标准黑色素瘤治疗的辅助治疗。我们将测试一下 携带BRAFV600E的小鼠YUMM3.1黑色素瘤细胞对OPNR153A Ki小鼠的抑瘤作用 突变(Subaim 2.1)。然后我们将测试达比加特兰与维莫拉非尼(BRAF激酶)联合应用 抑制剂)或维莫拉非尼和钴美替尼(MEK激酶抑制剂)，延长WT小鼠的存活时间 YUMM3.1/BRAFV600E黑色素瘤(Subaims 2.2和2.3)，以及达比卡特兰是否在 携带B16黑色素瘤的WT小鼠的存活(Subaim 2.4)。在特定的目标3中，我们将过表达OPN-R，OPN-L， 和OPN-CTF(C-末端片段)，以确定OPN-KO和OPNR153A小鼠中OPN的哪种切割形式 逆转B16肿瘤抑制表型(Subaim 3.1)。我们将描述来自B16肿瘤的TAM的特征 Opn KO和OPNR153A Ki小鼠，并与WT B16肿瘤(Subaim 3.2)进行比较。我们假设 WT小鼠骨桥蛋白对凝血酶的裂解可减少的侵袭并诱导“促肿瘤”的M2样细胞 表型，而OPN KO和OPNR153A小鼠中的TAMS将有促炎“肿瘤抑制” M1样表型。我们的观察结果新颖，意义重大，对癌症研究和临床实践具有重要意义。

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.