SFRP2 and NFAT are therapeutic targets in angiosarcoma

描述(申请人提供)：血管肉瘤是一种生物学侵袭性的血管恶性肿瘤，具有高转移潜能和随后的死亡率(1)。在诊断为血管肉瘤时，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年总存活率分别为50%和30%，总存活率中位数为24个月。对于不能切除的血管肉瘤，基于阿霉素的方案可获得5个月的无进展生存期，紫杉醇可获得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度致命疾病患者的存活率。我们实验室最近发现了一个与血管肉瘤生长有关的新的信号通路。在对乳腺肿瘤血管细胞进行基因组图谱分析时，与激光捕获显微切割获得的正常血管相比，我们发现分泌型卷曲相关蛋白2(SFRP2)是一个在肿瘤内皮细胞中表达增加的基因。SFRP2是一种33kd的分泌蛋白，参与Wnt信号转导。我们的初步数据显示，经免疫组化检测，9/9的人血管肉瘤中存在SFRP2蛋白，并刺激鸡绒毛膜尿囊膜上的血管生成，诱导内皮细胞迁移和管状形成，并对缺氧诱导的细胞凋亡具有保护作用。在SVR血管肉瘤细胞中，沉默SFRP2导致管状形成的抑制，而SFRP2刺激内皮细胞导致核NFATc3的增加。NFAT是一种转录因子，在介导血管生成反应中发挥关键作用(3；4)。NFAT的核定位依赖于钙/钙调蛋白依赖的磷酸酶、钙调神经磷酸酶和丝氨酸/苏氨酸激酶(20)活性之间的动态平衡。正常情况下，NFAT在去磷酸化之前不能进入细胞核，但可以被钙调神经磷酸酶激活。激活的钙调神经磷酸酶使NFAT去磷酸化，然后NFAT从细胞质转移到细胞核，导致参与细胞生长、分化和细胞周期进展的基因转录。他克莫司(FK506)是一种免疫抑制药，能与淋巴细胞中的免疫亲和素FKBP12结合。FK506-FKBP12复合体与钙调神经磷酸酶结合并抑制其磷酸酶活性，从而抑制NFAT(5)的核转位。我们的初步数据显示，他克莫司在体外抑制血管肉瘤管的形成，全身给药在20天时可抑制SVR血管肉瘤裸鼠移植瘤的生长46%。综上所述，我们的数据引导我们提出以下假设：阻断SFRP2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，将协同改善肿瘤的消退。本研究的目的是显示这些药物单独治疗血管肉瘤的疗效，以及联合化疗和抗血管生成治疗的疗效，并阐明它们抑制肿瘤生长的分子机制。该项目的成功完成将使这些药物在未来的研究中在血管肉瘤患者中进行临床试验。 公共卫生相关性：血管生成是新的毛细血管的生长，是实体肿瘤生长的关键组成部分。我们发现了一种新的蛋白质，分泌型卷曲相关蛋白2，在人类血管肉瘤中过表达，是一种有效的血管生成刺激因子。本研究的目的是研究该蛋白在血管肉瘤生物学中的分子和细胞机制，并评价阻断该信号通路对血管肉瘤异种移植瘤生长的影响。

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.