PO.MCB11.01 · 分子与细胞生物学
Ccn6缺失与EZH2过表达协同作用诱导化生性乳腺癌的肉瘤样分化
Ccn6 loss cooperates with EZH2 overexpression to induce sarcomatoid differentiation in metaplastic breast carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：三阴性乳腺癌（TNBC）已被证明对常规治疗和免疫治疗耐药，5年生存率为50%。其中最具侵袭性的是化生性乳腺癌（mBrCA），这是一种独特的组织病理学类型，具有梭形或肉瘤样（软骨样/骨样）成分，免疫细胞浸润低，对免疫治疗反应差。我们曾报道，乳腺上皮细胞特异性Ccn6敲除小鼠（MMTV-Cre；Ccn6 fl/fl）发生类似人类梭形mBrCA的mBrCA，将CCN6定义为梭形mBrCA的抑癌基因。近期我们发现CCN6拮抗Wnt/beta-catenin/EZH2信号的促侵袭效应。在此，我们检验了以下假设：CCN6与EZH2发生遗传学相互作用，诱导从梭形到肉瘤样癌细胞表型的表型转换，并限制CD8+ T细胞浸润。
方法：将MDA-MB-231（TNBC）和源自MMTV-Cre;Ccn6 fl/fl（Ccn6-KO）TNBC小鼠模型的Ccn6-KO细胞用载体或EZH2 shRNA敲低（KD）转导。用含EZH2野生型（WT）的慢病毒挽救Ccn6-KO EZH2 KD细胞。为过表达EZH2，我们用含载体或EZH2-MYC构建体的腺病毒感染癌细胞。我们通过在成软骨和成骨专用培养基中对乳腺癌细胞进行分化实验并与对照比较，研究Ccn6缺失和EZH2如何影响mBrCA肉瘤样分化。在体内，为重现骨微环境，我们将Ccn6-KO细胞注射到FVB小鼠的胫骨中，并用EZH2抑制剂EPZ-6438对其进行治疗。采用对来自Ccn6-KOshC、shEZH2和EZH2野生型挽救细胞的肿瘤进行流式细胞术分析，以及对经EZH2i治疗的Ccn6-KO肿瘤进行IHC，测量CD8+ gzmb+ IFNg+ T细胞的百分比。使用显性负性（dn）TCF4突变体与对照的ChIP测序研究，探讨Ccn6-KO细胞中双价基因的调控。
结果：在Ccn6-KO梭形mBrCA细胞中过表达EZH2可诱导在专用培养基中生长的细胞发生成软骨和成骨分化，而EZH2抑制在体内挽救了肉瘤样表型。我们发现EZH2 shRNA KD或药理学抑制增加了Ccn6-KO肿瘤中CD8+ T细胞的浸润，而这一效应被EZH2过表达所逆转。在Ccn6-KO细胞中使用dnTCF4阻断WNT/beta-catenin信号，可下调19个具有发育功能的双价基因靶点启动子区域的EZH2和H3K27me3标记。
结论：这些数据提供了CCN6缺陷与EZH2过表达作为肉瘤样、免疫细胞低的mBrCA诱导因素之间的明确联系。我们证明了药理学抑制EZH2甲基转移酶活性在逆转这些侵袭性表型方面的有效性。这些数据揭示了一种新的致癌协同作用，并提供证据支持恢复CCN6、抑制Wnt通路和EZH2作为mBrCA患者有前景的治疗策略的潜在治疗价值。
查看英文原文 English abstract
Background: Triple negative breast cancers (TNBC) have proven resistant to conventional and immune therapies with 5-year survival rates of 50%. Among the most aggressive of these is metaplastic breast carcinoma (mBrCA), a unique histopathology with spindle or sarcomatoid (chondroid/osseous) components and low immune cell infiltration with poor responses to immunotherapy. We have reported that mammary epithelial cell-specific Ccn6 knockout mice (MMTV-Cre; Ccn6 fl/fl ) develop mBrCAs like human spindle mBrCAs, defining CCN6 as a tumor suppressor for spindle mBrCA . Lately we showed CCN6 antagonizes the pro-invasive effects of Wnt/ beta-catenin/EZH2 signaling. Here, we tested the hypothesis that CCN6 genetically interacts with EZH2 to induce a phenotypic switch from spindle to sarcomatoid cancer cell phenotype and limits CD8 + T cell infiltration.
Methods: MDA-MB-231 (TNBC) and Ccn6-KO cells derived from MMTV-Cre;Ccn6 fl/fl (Ccn6-KO) TNBC mouse model were transduced with vector or EZH2 shRNA knockdown (KD). Ccn6-KO EZH2 KD cells were rescued with lentivirus containing EZH2-wild-type (WT). To overexpress EZH2 we infected cancer cells with adenovirus with vector or EZH2-MYC construct. We investigated how Ccn6 loss and EZH2 affect mBrCA sarcomatoid by differentiation assays of the breast cancer cells in chondrogenic and osteogenic specialized media compared to controls. In vivo, to recapitulate the bone microenvironment, we injected Ccn6-KO cells into the tibia of FVB mice and treat them with the EZH2 inhibitor EPZ-6438. Flow cytometry analyses of tumors derived from Ccn6-KOshC, shEZH2 and EZH2-wild type rescue cells and IHC of Ccn6-KO tumors treated with EZH2i were used to measure the percentage of CD8 + gzmb + IFNg + T cells. ChIP-sequencing studies in dominant negative (dn)TCF4 mutant vs. controls were used to study regulation of bivalent genes in Ccn6-KO cells.
Results : EZH2 overexpression in Ccn6-KO spindle mBrCA cells induces chondrogenic and osteogenic differentiation in cells grown in specialized media and EZH2 inhibition rescues the sarcomatoid phenotype in vivo. We found that EZH2 shRNA KD or pharmacological inhibition increases CD8+T cell infiltration in Ccn6-KO tumors, which is rescued by EZH2 overexpression. WNT/beta-catenin signaling blockade in Ccn6-KO cells using dnTCF4 downregulates EZH2 and H3K27me3 mark at the promoter regions of 19 bivalent gene targets with roles in development.
Conclusion : These data provide a clear link between CCN6 defects and EZH2 overexpression as inducers of sarcomatoid immune cell-low mBrCAs. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reversing these aggressive phenotypes. These data reveal a novel oncogenic cooperation and provide evidence to support the potential therapeutic value of CCN6 restoration, Wnt pathway and EZH2 inhibition as promising strategies for mBrCA patients
利益披露 Disclosure
M. E. Gonzalez, None..
A. Eido, None..
K. Miller, None.