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.

Graphical abstract

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.

中文翻译：

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USP7 介导的 JUND 抑制 RCAN2 转录并升高 NFATC1 以增强结直肠癌干细胞特性

癌症干细胞（CSC）包含参与肿瘤发生和进展的高度侵袭性肿瘤细胞的子集。本研究探讨了钙调磷酸酶 2 (RCAN2) 调节因子在结直肠癌 (CRC) 干细胞特性中的功能。通过分析四个GEO数据集，我们获得了RCAN2作为CRC中的干性相关基因。RCAN2 在 CRC 组织和细胞中表达较差，尤其是在 CSC 中。RCAN2 恢复降低了钙调神经磷酸酶活性，促进活化 T 细胞核因子 1 (NFATC1) 蛋白的磷酸化和降解，导致 CSC 干性降低。JunD 原癌基因 (JUND) 的蛋白水平在 CRC 样本和 CRC 干细胞中增加，与 RCAN2 结合并抑制其转录。CSC 中丰富的泛素特异性肽酶 7 (USP7) 通过去泛素化修饰增强了 JUND 蛋白的稳定性。慢病毒介导的 USP7 或 JUND 敲低也阻断了钙调神经磷酸酶-NFATC1 信号传导并降低了干性相关蛋白的蛋白水平。此外，USP7敲低削弱了CSC的集落/球体形成能力以及致瘤性，并降低了异种移植肿瘤中CSC的含量。然而，JUND 的进一步恢复挽救了 CSC 的干性。总体而言，本研究表明 USP7 介导的 JUND 抑制 RCAN2 转录并激活 NFATC1 以增强 CRC 中的干细胞特性。

图形概要

1. RCAN2在CRC组织和细胞中表达较差，尤其是在CSC中。

2. RCAN2通过阻断钙调磷酸酶-NFATC1信号转导来降低CSC的干性。

3. JUND与RCAN2启动子结合抑制RCAN2转录。

4. USP7通过去泛素化修饰增强JUND蛋白稳定性。

5. USP7或JUND的下调可恢复RCAN2水平并抑制CSC的干性。