BACKGROUND CGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated. METHODS To investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer. RESULTS We show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment. CONCLUSION We demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.

中文翻译：

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CGRRF1是一种生长抑制剂，可调节乳腺癌中的EGFR泛素化。

背景技术CGRRF1是生长抑制剂，并且由跨膜结构域和RING-指结构域组成。它起与内质网相关降解作用的RING域E3泛素连接酶的作用。CGRRF1的表达在癌组织中降低；然而，CGRRF1在乳腺癌中的作用及其生长抑制功能的机制尚待阐明。方法为了研究CGRRF1是否抑制乳腺癌的生长，我们进行了MTT分析和异种移植实验。通过反相蛋白质阵列（RPPA）分析进一步分析从小鼠收获的肿瘤，以鉴定CGRRF1的潜在底物。使用共免疫沉淀测定法来验证CGRRF1及其底物之间的相互作用，然后进行体内泛素化测定。蛋白质印迹，亚细胞分级分离，进行了逆转录定量聚合酶链反应（qRT-PCR），以了解CGRRF1在乳腺癌中的作用机制。分析了可公开获得的乳腺癌数据集，以检查CGRRF1与乳腺癌之间的关联。结果我们表明，CGRRF1在体外和体内均能抑制乳腺癌的生长，而RING-手指结构域对于其生长抑制活性很重要。为了阐明CGRRF1的机制，我们鉴定了EGFR作为CGRRF1的新底物。CGRRF1通过K48连接的泛素化使EGFR泛素化，从而导致蛋白酶体降解。除了调节EGFR的稳定性外，敲除CGRRF1还可以增强EGF刺激后的AKT磷酸化。通过分析乳腺癌数据库，我们发现低CGRRF1表达的患者生存期较短。与正常乳腺组织相比，CGRRF1的mRNA水平在乳腺癌中较低，尤其是在HER2阳性和基底样乳腺癌中。我们还注意到，与正常乳腺组织相比，CGRRF1启动子甲基化在乳腺癌中增加，这表明CGRRF1在乳腺癌中是表观遗传学修饰的。5-氮丙啶和panobinostat的治疗可恢复CGRRF1表达，支持CGRRF1的启动子在乳腺癌中被表观遗传修饰。由于5-氮杂act啶和Panobinostat可以增加CGRRF1表达，因此它们可能是乳腺癌治疗的潜在疗法。结论我们证明了CGRRF1在乳腺癌中具有肿瘤抑制功能，并确定了EGFR作为其靶标。特别是在HER2阳性和基底样乳腺癌中。我们还注意到，与正常乳腺组织相比，CGRRF1启动子甲基化在乳腺癌中有所增加，这表明CGRRF1在乳腺癌中是表观遗传学修饰的。5-氮丙啶和panobinostat的治疗可恢复CGRRF1表达，支持CGRRF1的启动子在乳腺癌中被表观遗传修饰。由于5-氮杂act啶和Panobinostat可以增加CGRRF1表达，因此它们可能是乳腺癌治疗的潜在疗法。结论我们证明了CGRRF1在乳腺癌中的肿瘤抑制功能，并确定了EGFR作为其靶标。特别是在HER2阳性和基底样乳腺癌中。我们还注意到，与正常乳腺组织相比，CGRRF1启动子甲基化在乳腺癌中有所增加，这表明CGRRF1在乳腺癌中是表观遗传学修饰的。5-氮丙啶和panobinostat的治疗可恢复CGRRF1表达，支持CGRRF1的启动子在乳腺癌中被表观遗传修饰。由于5-氮杂act啶和Panobinostat可以增加CGRRF1表达，因此它们可能是乳腺癌治疗的潜在疗法。结论我们证明了CGRRF1在乳腺癌中具有肿瘤抑制功能，并确定了EGFR作为其靶标。提示CGRRF1在乳腺癌中是表观遗传修饰的。5-氮丙啶和panobinostat的治疗可恢复CGRRF1表达，支持CGRRF1的启动子在乳腺癌中被表观遗传修饰。由于5-氮杂act啶和Panobinostat可以增加CGRRF1表达，因此它们可能是乳腺癌治疗的潜在疗法。结论我们证明了CGRRF1在乳腺癌中具有肿瘤抑制功能，并确定了EGFR作为其靶标。提示CGRRF1在乳腺癌中是表观遗传修饰的。5-氮丙啶和panobinostat的治疗可恢复CGRRF1表达，支持CGRRF1的启动子在乳腺癌中被表观遗传修饰。由于5-氮杂act啶和Panobinostat可以增加CGRRF1表达，因此它们可能是乳腺癌治疗的潜在疗法。结论我们证明了CGRRF1在乳腺癌中具有肿瘤抑制功能，并确定了EGFR作为其靶标。