SFRP2 and NFAT are therapeutic targets in angiosarcoma

描述（申请人提供）：血管肉瘤是一种具有生物侵袭性的血管恶性肿瘤，具有高转移潜力和随后的死亡率（1）。在诊断血管肉瘤时，10 - 25%的患者已经有转移性疾病。所有血管肉瘤患者的2年和5年总生存率分别为50%和30%，中位总生存期为24个月。对于不可切除的血管肉瘤，基于多柔比星的方案产生5个月的无进展生存期，紫杉醇实现7个月的无进展生存期（2）。因此，迫切需要新的治疗方法来提高这种高致命性疾病患者的生存率。我们的实验室最近发现了一个新的信号通路负责血管肉瘤的生长。在对乳腺肿瘤血管细胞与通过激光捕获显微切割获得的正常血管进行基因组分析时，我们发现分泌型卷曲相关蛋白2（SFRP 2）是一种在肿瘤内皮中表达增加的基因。SFRP 2是一个33 kd的分泌蛋白，参与Wnt信号转导。我们的初步数据显示，SFRP 2蛋白存在于9/9的人血管瘤中，通过IHC，并刺激鸡绒毛尿囊膜上的血管生成，诱导内皮细胞迁移和管形成，并保护免受缺氧诱导的细胞凋亡。在SVR血管肉瘤细胞中沉默SFRP 2导致管形成的抑制，并且用SFRP 2刺激内皮细胞导致核NFATc 3的增加。NFAT是一种转录因子，在介导血管生成反应中起关键作用（3;4）。NFAT核定位取决于Ca 2 +/钙调蛋白依赖性磷酸酶、钙调磷酸酶活性和丝氨酸/苏氨酸激酶活性之间的动态输入-输出平衡（20）。NFAT在去磷酸化之前不能正常进入细胞核，但可以被钙调神经磷酸酶激活。活化的钙调磷酸酶使NFAT去磷酸化，然后NFAT从细胞质易位到细胞核，并导致参与细胞生长、分化和细胞周期进程的基因的转录。他克莫司（FK 506）是一种免疫抑制药物，与淋巴细胞中的免疫球蛋白FKBP 12结合。FK 506-FKBP 12复合物与钙调磷酸酶结合并抑制其磷酸酶活性，从而抑制NFAT的核转位（5）。我们的初步数据显示，他克莫司在体外抑制血管肉瘤管形成，全身给药在20天时抑制裸鼠中SVR血管肉瘤异种移植物的生长达46%。综上所述，我们的数据使我们提出以下假设：阻断SFRP 2将减少血管生成和血管肉瘤生长，并与化疗和抗血管生成治疗相结合，协同改善肿瘤消退。本研究的目的是显示这些药物单独治疗血管肉瘤的疗效，以及与化疗和抗血管生成治疗联合治疗的疗效，并阐明其抑制肿瘤生长的分子机制。该项目的成功完成将导致这些药物在血管肉瘤患者的临床试验在未来的研究。 公共卫生相关性：血管生成是新毛细血管的生长，是实体瘤生长的关键组成部分。我们已经发现了一种新的蛋白质，分泌卷曲相关蛋白2，这是过度表达的人血管肉瘤，是一个强大的刺激血管生成。本提案的目的是表征该蛋白对血管肉瘤生物学的分子和细胞机制，并评估阻断该信号通路对血管肉瘤异种移植物生长的功效。

DESCRIPTION (provided by applicant): Angiosarcoma is a biologically aggressive vascular malignancy with a high metastatic potential and subsequent mortality(1). At the time of diagnosis of angiosarcoma, 10¿ 25% of patients already have metastatic disease. The 2- and 5-year overall survival for all patients with angiosarcoma is 50 and 30%, respectively, with a median overall survival of 24 months. For unresectable angiosarcoma, doxorubicin based regimens yield progression-free survival of 5 months, and paclitaxel achieves a progression-free survival of 7 months(2). Therefore there is a desperate need for novel therapies to improve survival in patients with this highly lethal disease. Our laboratory has recently discovered a novel signaling pathway responsible for angiosarcoma growth. While conducting genomic profiling of breast tumor vascular cells compared to normal vessels obtained by laser capture microdissection, we identified secreted frizzled related protein 2 (SFRP2) as a gene with increased expression in tumor endothelium. SFRP2 is a 33kd secreted protein involved in Wnt signaling. Our preliminary data shows that SFRP2 protein is present in 9/9 human angiosarcomas by IHC, and stimulates angiogenesis on the chick chorioallantoic membrane, induces endothelial cell migration and tube formation, and protects against hypoxia induced apoptosis. Silencing of SFRP2 in SVR angiosarcoma cells resulted in inhibition of tube formation, and stimulation of endothelial cells with SFRP2 resulted in an increase in nuclear NFATc3. NFAT is a transcription factor that plays a critical role in mediating angiogenic responses(3;4). NFAT nuclear localization is dependent on a dynamic import-export balance between the activity of the Ca2+/calmodulin-dependent phosphatase, calcineurin, and the activity of serine/threonine kinases(20). NFAT cannot normally enter the nucleus until it is dephosphorylated, but can be activated by calcineurin. Activated calcineurin dephosphorylates NFAT, which then translocates from the cytoplasm to the nucleus and results in transcription of genes involved in cell growth, differentiation, and cell cycle progression. Tacrolimus (FK506) is an immunosuppressive drug that binds to the immunophlin FKBP12 in lymphocytes. The FK506-FKBP12 complex associates with calcineurin and inhibits its phosphatase activity, which inhibits nuclear translocation of NFAT(5). Our preliminary data shows that tacrolimus inhibits angiosarcoma tube formation in vitro, and systemic administration inhibits the growth of the SVR angiosarcoma xenograft in nude mice by 46% at 20 days. Taken together, our data leads us to propose the following hypothesis: Blockade of SFRP2 will decrease angiogenesis and angiosarcoma growth and will synergistically improve tumor regression in combination with both chemotherapy and antiangiogenic therapy. The objective of this study is to show the efficacy of these drugs in angiosarcoma alone, and in combination with chemotherapy and antiangiogenic therapy, and to elucidate their molecular mechanism in inhibiting tumor growth. Successful completion of the project will lead to clinical trials of these agents in patients with angiosarcoma in future studies. PUBLIC HEALTH RELEVANCE: Angiogenesis is the growth of new capillary blood vessels, and is a critical component of solid tumor growth. We have discovered a novel protein, secreted-frizzled related protein 2, that is overexpressed in human angiosarcoma that is a potent stimulator of angiogenesis. The objective of this proposal is to characterize the molecular and cellular mechanism of this protein on angiosarcoma biology, and evaluate the efficacy of blockade of this signaling pathway on angiosarcoma xenograft growth.