乳腺上皮特异性基因敲除研究Dach1拮抗ErbB2的致瘤作用及其分子机理

About 25% of patients with breast cancer are diagnosed as ErbB2-amplified status associated with a high risk of relapse. Targeting ErbB2 signaling prolongs survival time, and therefore it is the standard therapy for breast cancer ErbB2+ subtype. However, primary resistance in some patients and secondary resistance raised in majority of patients after long time treatment limit its efficiency. Recent studies demonstrate that ErbB2 interacts with many signaling pathways and therapies toward multiple targets improve treatment outcome. The molecular mechanisms underlying the aggressive nature of ErbB2 tumors remain to be further elucidated. ..ErbB2 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas and recapitulates clinical characteristics of breast cancer patients, thus ErbB2-driven transgenic tumor model provides an idea tool to explore novel molecules underlying ErbB2 signaling and develop effective therapeutics. ...The works by us and others indicate that DACH1/EYA signaling also interacts with ErbB2 signaling and contributes to the breast cancer initiation and progression. Our previous study demonstrated that loss of DACH1 expression correlated with enhanced properties of breast cancer stem cells and increased migration and invasion. Moreover, Nuclear expression of DACH1 is negatively associated with aggressive growth paterns and prognosis. Effects of DACH1 on growth arrest in vitro and tumor inhibitory on xenograft model have been well documented, how faithfully these cultutred cell based assays mirror in vivo function remains an open question...Since genetic ablation of Dach1 leads to lethality within 24 hours, we will employ an inducible, mammary-specific Dach1 knock out mouse model to compare mammary progenitor cell pool and mammary gland morphogenesis in early postnatal mammary development and mammary gland differentiation during pregnancy. To evaluate Dach1's function in early ErbB2-induced tumor initiation and progression, we will cross Dach1flox/MMTV-CRE double transgenic mice with MMTV-ErbB2 to create triple transgenic mice. Then, the incidence of mammary gland tumor and metastasis process will be monitored. For better understanding the effect of DACH1 as a tumor suppressor, orthotropic mammary pad injection of Erbb2-positive mammary tumor cell lines with silence of DACH1 in accompany with or without over-expression of EYA1 will be coupled with in vivo analysis in complementary to transgenic mouse model, confirming the role of DACH1/EYA1 pathway in ErbB2 positve breast cancers...Together, our study will create genetic models to examine function of Dach1 in mammary gland development and ErbB2-induced mammary tumor at a condition similar to human breast development. Interaction between Dach1/Eya1 pathway and ErbB2 signaling may determine outcome of ErbB2 targeting therapy; therefore, our model is of great value for developing novel personalized anti-ErbB2 strategy.

ErbB2扩增和过表达驱动原发性乳腺癌的发生和进展。因而，靶向ErbB2的药物是此类患者的标准治疗方案。但是，耐药性限制其疗效。ErbB2能诱发鼠乳腺肿瘤，且重现人乳腺癌的生物学特征。利用此模型探索与ErbB2交叉的信号分子和基因网络，将有助于发现ErbB2靶向药物的耐药机理。近年发现DACH1决定细胞命运与多种器官的发育。乳腺癌组织DACH1丢失导致肿瘤干细胞扩增、上皮-间质转换，促进乳癌细胞侵袭和转移；是影响乳腺癌预后的关键基因之一。但细胞学的研究结果转化于临床应用之前，还有待合适动物模型来验证。因此，我们将建立乳腺上皮特异性Dach1敲除模型，分析Dach1对乳腺发育及干细胞分化的影响；结合ErbB2致瘤模型，明确Dach1体内抑瘤作用；利用ErbB2阳性的细胞系和乳腺癌标本，探索DACH/EYA信号对干细胞群和ErbB2靶向药物敏感性的作用。预期成果为ErbB2靶向治疗提供新依据。

通过Dach1flox与ROSA 26creERT2小鼠，获得乳腺上皮特异的Dach1基因敲除模型。结果表明Dach1缺失导致乳腺导管分支减少，基底细胞的比例下降，乳腺组织p-Smad活性增加。证明了Dach1抑制TGF-b信号通路，调控乳腺导管发育。在ErbB2阳性的乳腺癌细胞系SKBR3和MDA-MB-453建立稳定表达DACH1的细胞群，发现DACH1抑制两种细胞的体外增殖、迁移，降低SKBR3细胞在小鼠体内的致瘤作用，并增加靶向ErbB2的单克隆抗体Herceptin的敏感性。此外，过表达EYA1可减低DACH1的体外抑制效应。利用公共数据库和组织芯片，分析DACH1、EYA和SIX基因家族与ErbB2、乳腺癌分子分型的关系，发现三阴性乳腺癌组织DACH1 mRNA和蛋白水平均显著下降，ErbB2阳性的乳腺癌组织DACH1轻度减少。进一步分析显示DACH1 mRNA与CD44、Vimentin、Fibronectin及EGFR负相关，与FOXA1呈正相关。外源性表达DACH1抑制肿瘤生长，同时降低干细胞相关基因、 EMT基因、EGFR的表达和Ki-67阳性细胞。DACH1 mRNA高表达的乳腺癌患者总生存时间(OS)、无复发时间(RFS)和无转移时间(MFS)均延长。我们发现EYA2在分化差和ErbB2阳性的乳腺癌组织高表达，EYA2与乳腺导管上皮标志ESR1、PGR、FOXA1呈反比，而与KRT5、EGFR、SNAIL2、YBX1和KLF5呈正相关。外源性导入EYA2增加乳腺癌细胞增殖，上调YBX1、EGFR、Cyclin E和PCNA的表达,EYA2高表达的乳腺癌患者OS、RFS和MFS减少。综合分析乳腺癌患者肿瘤组织mRNA表达谱，发现SIX1在管腔型乳腺癌组织表达高，而SIX2在三阴性乳腺癌组织表达高；SIX1高表达的患者OS、RFS减少。生物信息分析发现MAT1与SIX1的表达成正相关，MAT1 mRNA及蛋白表达均与ER、PR正相关，在管腔型和ErbB2阳性的乳腺癌组织富集。外源性表达MAT1促进细胞增殖，临床资料分析表明MAT1高表达缩短乳腺癌患者RFS、MFS。在项目的资助下，明确了DACH1通过TGF-b信号调控乳腺上皮的分化和发育，DACH1抑制ErbB2的促增殖和致瘤作用。DACH、EYA和SIX与乳腺癌分子分型的关系及临床意义。