ATXN3 Deubiquitinates KLF4 To Promote Breast Tumorigenesis And Tumor Progression

Kruppel like factor 4(KLF4)is a member of the zinc finger transcription 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 U20S 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.