J Natl Cancer Inst. 2019 Sep 1;111(9):970-982. doi: 10.1093/jnci/djy209.
Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.
By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.
EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.
This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.
尤因肉瘤（EWS）表现出所有癌症中最低的体细胞突变率之一，导致可用药的突变和新抗原稀缺。针对差异表达的细胞表面抗原的免疫疗法可为这类肿瘤提供治疗益处。妊娠相关血浆蛋白 A（PAPP-A）是一种由胎盘产生的细胞膜相关蛋白酶，通过诱导胰岛素样生长因子（IGF）信号促进胎儿生长。
通过比较 EWS（n=120）与正常组织（n=42）中的细胞表面蛋白的 RNA 表达，我们全面描绘了 EWS 的表面组，以鉴定高度差异表达的分子。我们使用 CRISPR/Cas-9 和抗 PAPP-A 抗体，在 NSG 异种移植模型中研究了 PAPP-A 在 EWS 中的生物学作用，并对 PAPPA 敲除克隆（n=5）和对照（n=3）进行了 RNA 测序。所有统计检验均为双侧检验。
EWS 表面组分析鉴定了 11 个高度差异过表达的基因，其中 PAPPA 在差异表达中排名第二。在 EWS 细胞系中，PAPPA 的基因敲除和抗 PAPP-A 抗体的治疗揭示了其通过调节局部 IGF-1 生物利用度而发挥的重要生存作用。MAb 介导的 PAPPA 抑制作用可减少 EWS 在原位异种移植中的生长（第 49 天的 leg area mm2 IgG2a 对照（CTRL）[n=14]，平均值=397.0，SD=86.1 与抗-PAPP-A [n=14]，平均值=311.7，SD=155.0；P=0.03；中位 OS 抗-PAPP-A=52.5 天，95%CI=46.0 至 63.0 天 vs IgG2a=45.0 天，95%CI=42.0 至 52.0 天；P=0.02）并提高了抗 IGF-1R 治疗的疗效（第 49 天的 leg area mm2 抗-PAPP-A+抗-IGF-1R [n=15]，平均值=217.9，SD=148.5 与 IgG2a-CTRL；P<0.001；中位 OS 抗-PAPP-A+抗-IGF1R=63.0 天，95%CI=52.0 至 67.0 天 vs IgG2a-CTRL；P<0.001）。出乎意料的是，EWS 细胞系中的 PAPPA 敲除诱导了干扰素（IFN）反应基因，包括与抗原加工/呈递相关的蛋白。一致地，PAPPA 低表达的 EWS 肿瘤的基因表达谱富含免疫反应途径。
这项工作全面描绘了 EWS 的表面组，证实了 PAPP-A 是一个高度差异表达的治疗靶点，并发现了 IGF-1 信号与癌症中免疫逃逸之间的新联系，从而提示了 EWS 与胎盘之间免疫逃逸的共同机制。
