Krüppel Like Factor 4（KLF4）是锌指转录因子家族KLF中的一员，在多种生物学过程中发挥关键作用。KLF4在肿瘤的发生发展中由于所在组织类型不同而具有抑癌基因和癌基因的双重功能。KLF4在细胞内半衰期很短，其蛋白水平可受多种因素刺激而发生迅速变化，进而造成细胞内生理和病理反应。这些调控大多通过蛋白翻译后修饰介导。近年来，去泛素化也作为调节蛋白水平和功能的一项重要的翻译后修饰机制而被广泛研究，于是我们想要进一步探究去泛素化酶在KLF4蛋白调节中的作用。

本实验中，我们在一个含有65个去泛素化酶（Deubiquitinating enzyme，DUB）的文库中筛选出KLF4的首个去泛素化酶。首先我们将候选DUB分别在工具细胞U2OS和乳腺癌细胞MCF7中过表达，通过检测细胞内KLF4的含量，筛选出在这两种细胞中均影响KLF4蛋白水平的DUB。而后利用免疫共沉淀技术，分析出通过与KLF4直接相互作用而对其进行调节的DUB。采用以上的筛选策略，我们筛选出ATXN3可能是KLF4的一个去泛素化酶。而后，我们通过半衰期实验和泛素化实验验证了ATXN3可通过减弱KLF4的泛素化而抑制其降解，确定了我们的筛选结果。接下来，我们验证了ATXN3的14位半胱氨酸是其酶活位点，将ATXN3上第14位半胱氨酸位点突变后，ATXN3催化KLF4去泛素的作用减弱。另外，我们确定了ATXN3与KLF4的结合区域。将KLF4上与ATXN3相互作用的区域删除后，ATXN3对KLF4的去泛素化作用也降低，证明ATXN3作为去泛素化酶与KLF4结合发挥作用。

微球体形成实验和小鼠成瘤实验的结果表明，敲降ATXN3减弱乳腺癌细胞的干性，抑制乳腺肿瘤的发生，且ATXN3对乳腺癌细胞干性的影响是通过其对KLF4的调节作用的实现的。通过体外的Transwell实验和小鼠乳腺癌转移模型，我们发现ATXN3还可通过影响KLF4抑制乳腺肿瘤细胞的侵袭、转移。临床资料分析结果也显示ATXN3与乳腺癌淋巴结转移情况有关。此外，卡铂处理条件下的流式细胞术和小鼠模拟卡铂化疗的实验结果表明，ATXN3 通过调节KLF4增加了乳腺癌细胞对卡铂的耐药性。说明ATXN3在乳腺肿瘤的发展中同样发挥重要作用。另外，对乳腺癌标本分析的结果显示，ATXN3和KLF4的蛋白表达有显著相关性。且ATXN3和KLF4的高表达均可导致乳腺癌的不良预后。

综上，我们筛选出KLF4的首个去泛素化酶ATXN3，且发现在乳腺癌中ATXN3通过调节KLF4增强乳腺癌细胞干性和对卡铂的耐药性，并促进乳腺癌转移。这一结果提示ATXN3可能是乳腺癌临床诊断和治疗中的一个潜在的诊断标志和药物靶点。

krüppel like factor 4 (klf4) is a member of the zinc finger tranion factor family klf and plays a key role in a variety of cellular processes. klf4 dually functions as a tumor suppressor gene and oncogene in the development of tumors from different tissue types. the protein level of klf4 changes rapidly in response to various environmental factors, resulting in different physiological and pathological reactions. most of these regulations are mediated through posttranslational modifications of the protein. in recent years, deubiquitination has been widely studied as an important posttranslational modification mechanism regulating protein levels and functions. in this study, we focused on identifying the deubiquitinating enzyme (dub) of klf4 and exploring its role in regulating klf4 in breast cancer.

we identified the dubs that affect the protein level of klf4 in both u2os and mcf7 cells by screening a library containing 65 dubs. next, we conducted an immunoprecipitation assay to identify the dubs that directly interact with klf4. using the abovementioned strategy, we identified atxn3 as a potential dub of klf4. then, we performed a chx pulse-chase assay and ubiquitination assay to determine whether atxn3 inhibits klf4 degradation by attenuating its ubiquitination, which verified our screening results. moreover, we confirmed that the c14 ​​residue of atxn3 is the catalytic site, as an increased level of klf4 ubiquitination was observed after introducing the c14a mutant atxn3 compared with that observed after introducing wild-type atxn3. we also mapped the binding areas within ​​atxn3 and klf4. when the binding region was deleted, the deubiquitinating effect of atxn3 on klf4 was also inhibited, which demonstrated that atxn3 fulfills its deubiquitinating function through binding with klf4.

mammosphere formation assays showed that upregulation of atxn3 increases the proportion of breast cancer stem cells by mediating klf4. a mouse xenograft assay also confirmed the tumor initiator role of atxn3. results from flow cytometry and mouse experiments demonstrated that atxn3 inhibits breast cancer cell apoptosis and promotes tumor progression by regulating klf4 after carboplatin treatment. moreover, we found that downregulation of atxn3 inhibits breast cancer metastasis by regulating klf4. clinical data analysis provided evidence that atxn3 is associated with lymph node metastasis in breast cancer. these results indicate that atxn3 plays an important role in breast cancer progression. in addition, clinical data analysis showed that atxn3 is significantly correlated with klf4. the high expression levels of both klf4 and atxn3 are related to the poor prognosis of breast cancer patients.

taken together, we identified atxn3 as a novel deubiquitinating enzyme of klf4. in breast cancer, atxn3 maintains cancer cell stemness, leads to carboplatin resistance, and promotes metastasis by regulating klf4, suggesting that atxn3 is a potential diagnostic or therapeutic target for breast cancer.