EZH2的差异表达在皮肤CD30+淋巴细胞增生性疾病中的功能研究

Primary cutaneous CD30+ lymphoproliferative diseases are comprised of a spectrum of diseases ranging from lymphomatoid papulosis (LyP) to primary cutaneous anaplastic large cell lymphoma (PCALCL), which demonstrates more aggressive clinical behavior. In a previous study done by our group, we identified that overexpression of nuclear matrix protein SATB1 promotes malignant T-cell proliferation in cutaneous CD30+ lymphoproliferative disease by modulating cell cycle related genes. However, 28% PCALCL samples are negative for SATB1, according to our study; the pathogenesis and mechanism of SATB1 suppression in this group of patients are largely unknown. We recently found that histone methyltransferase EZH2 specifically overexpressed in SATB1 negative patients, and that EZH2 can inhibit SATB1 expression in CD30+ tumorous T cells, which suggested new clues for the disease progression mechanism. As an important epigenetic regulator, EZH2 plays important roles in multiple tumors. This proposed study will focus on the role of EZH2 in SATB1 expression and in the pathogenesis of SATB1 negative PCALCL. By restoring/knocking down EZH2 and its mutants in PCALCL cell models, the effects on SATB1 expression, and the effect on CD30+ tumorous T cell biological behavior will be observed. Further, the epigenetic mechanism of SATB1 regulation, the molecular pathway downstream SATB1, as well as the mechanism of EZH2 differential expression will be investigated. This study will contribute to develop specific targeted therapy for PCALCL, which currently does not have a cure.

皮肤CD30+淋巴细胞增生性疾病是由淋巴瘤样丘疹病（LyP）以及侵袭性的皮肤间变性大细胞淋巴瘤（PCALCL）组成的病谱。本课题组既往的研究证明了核基质蛋白SATB1在CD30+肿瘤性T细胞中的高表达通过调控细胞周期促进肿瘤进展。然而，28%的PCALCL标本不表达SATB1，这一部分患者疾病发生发展的机制及其SATB1不表达的原因尚不清楚。我们新近发现SATB1阴性的患者特异性高表达组蛋白甲基转移酶EZH2，且EZH2可以抑制SATB1的表达，为研究提供了新的线索。EZH2是重要的表观遗传调控基因，参与多种肿瘤的发病。本研究拟以PCALCL细胞系为研究模型，通过过表达/敲减EZH2及其突变体，从体内、体外及临床标本三个层面证明EZH2对SATB1的调控作用、阐明EZH2在PCALCL疾病进展中的作用及机制，并明确EZH2差异表达的原因，为开发特异性的靶向治疗提供理论基础。

原发皮肤间变大细胞淋巴瘤（Primary cutaneous anaplastic large cell lymphoma, PCALCL）是原发皮肤 CD30+淋巴细胞增生性疾病（Primary cutaneous CD30 positive lymphoproliferative disorders, CD30+LPDs）的一种主要亚型，属于第二大类原发皮肤T细胞淋巴瘤（Primary cutaneous T cell lymphoma）。该病主要以肿瘤细胞表达CD30抗原为特点，总体预后较好，但20%的患者会出现系统侵犯并导致死亡。Zeste同源物2增强子（Enhancer of zeste homolog 2，EZH2）能够介导组蛋白H3第27位赖氨酸三甲基化（H3K27me3），参与了多种肿瘤的发生发展。本项目发现EZH2在PCALCL中表达升高，在PCALCL细胞系中敲减EZH2后，发现细胞增殖下降，细胞周期发生阻滞，且细胞凋亡增加。通过EZH2抑制剂处理后可以得到类似的结果。对比EZH2敲减前后的基因表达谱后，发现EZH2可以下调硫氧还原蛋白结合蛋白（Thioredoxin-interacting protein，TXNIP）及C-X-C白细胞介素10（C-X-C motif chemokine ligand 10，CXCL10）的基因表达。通过免疫共沉淀技术测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq）证实了EZH2通过H3K27me3调控了该两个基因的启动子区域。通过流式检测活性氧ROS实验，发现EZH2可以通过抑制TXNIP降低细胞内活性氧的产生从而减少肿瘤细胞凋亡；同时，通过细胞迁移实验验证了EZH2可以通过抑制CXCL10导致肿瘤微环境中T细胞浸润减少使得肿瘤可以逃避机体免疫。最后，通过异种移植肿瘤实验验证了EZH2抑制剂对肿瘤治疗的效果。该项目阐述了 EZH2促进了PCALCL发展的机制，并提出EZH2可以作为PCALCL的治疗靶点，为常规治疗抵抗的病人提供新的治疗希望。