癌症干细胞（CSCs）是一类高度侵袭性的肿瘤细胞子集，参与肿瘤的起源和发展。本研究探讨了钙调神经磷酸酶调节蛋白2（RCAN2）在结直肠癌（CRC）干细胞特性中的功能。通过分析四个GEO数据集，我们确定了RCAN2是CRC中与干细胞性质相关的基因。RCAN2在CRC组织和细胞中的表达较低，特别是在CSCs中。RCAN2恢复能够降低钙调神经磷酸酶活性，促进核活化T细胞因子1（NFATC1）蛋白的磷酸化和降解，从而降低CSCs的干细胞性。在CRC样本和CRC干细胞中，蛋白水平增加的JunD原癌基因（JUND）结合到RCAN2并抑制其转录。CSCs中丰富的泛素特异性蛋白酶7（USP7）通过去泛素化修饰提高JUND蛋白的稳定性。通过慢病毒介导的USP7或JUND的沉默也阻断了钙调神经-NFATC1信号传导，并降低了与干细胞性相关的蛋白质含量。此外，USP7沉默削弱了CSCs的集落/球形体形成能力，以及在异种移植肿瘤中减少了CSCs的含量。然而，进一步恢复JUND可以恢复CSCs的干细胞性。总之，本研究证明了USP7介导的JUND抑制RCAN2的转录，并激活NFATC1以增强CRC中干细胞特性。1. RCAN2在CRC组织和细胞中，特别是在CSCs中表达较低。2. RCAN2通过阻断钙调神经-NFATC1信号传导降低CSCs的干细胞性。3. JUND结合在RCAN2启动子上抑制RCAN2的转录。4. USP7通过去泛素化修饰增强JUND蛋白的稳定性。5. USP7或JUND的下调恢复了RCAN2水平并降低CSCs的干细胞性。© 2023. 作者，独家授权给 Springer Nature B.V.

Cancer stem cells (CSCs) encompass a subset of highly aggressive tumor cells that are involved in tumor initiation and progression. This study investigates the function of regulator of calcineurin 2 (RCAN2) in the stem cell property in colorectal cancer (CRC). By analyzing four GEO datasets, we obtained RCAN2 as a stemness-related gene in CRC. RCAN2 was poorly expressed in CRC tissues and cells, especially in CSCs. RCAN2 restoration reduced calcineurin activity and promoted phosphorylation and degradation of nuclear factor of activated T cells 1 (NFATC1) protein, leading to reduced stemness of CSCs. JunD proto-oncogene (JUND), whose protein level was increased in CRC samples and CRC stem cells, bound to RCAN2 and suppressed its transcription. The abundant ubiquitin specific peptidase 7 (USP7) in CSCs enhanced JUND protein stability through deubiquitination modification. Lentivirus-mediated knockdown of USP7 or JUND also blocked the calcineurin-NFATC1 signaling and reduced the protein levels of stemness-related proteins. Moreover, the USP7 knockdown weakened the colony/sphere formation ability as well as the tumorigenicity of CSCs, and it reduced the CSC content in xenograft tumors. However, further restoration of JUND rescued the stemness of the CSCs. Overall, this study demonstrates that USP7-mediated JUND suppresses RCAN2 transcription and activates NFATC1 to enhance stem cell property in CRC. 1. RCAN2 is poorly expressed in CRC tissues and cells and especially in CSCs. 2. RCAN2 reduces stemness of CSCs by blocking calcineurin-NFATC1 signal transduction. 3. JUND binds to RCAN2 promoter to suppresses RCAN2 transcription. 4. USP7 enhances JUND protein stability via deubiquitination modification. 5. Downregulation of USP7 or JUND restores RCAN2 level and suppresses stemness of CSCs.© 2023. The Author(s), under exclusive licence to Springer Nature B.V.