Targeting USP22 Suppresses Tumorigenicity And Enhances Sensitivity To Cisplatin In Lung Cancer Stem Cells Through Downregulating ALDH1A3

Purpose: Lung cancer is the most common cause of cancer death worldwide.Non-small cell lung cancer(NSCLC)accounts for 85% of lung cancers.Although most lung cancer are relatively sensitive to chemotherapy at the initial stage,and there are great advances in target therapy and immunotherapy,drug resistance is still difficult to avoid.We need some new ideas to develop more effective treatments.Cancer stem cells(CSCs)are subpopulations of cancer cells with self-renewal and differentiation potential.Recent studies have shown that they are involved in tumor progression and drug resistance.In previous work,we found that H2 B monoubiquitination(H2Bub1)was extremely low or absent in lung cancer cells.This phenomenon is closely related with the progression and drug resistance of lung cancer.With RNA sequencing analysis(RNA sequencing),we found that ubiquitin-specific protease 22(USP22),the most critical deubiquitinase of H2Bub1,may regulate ALDH1A3,a member of the ALDH superfamily.In this study,we investigated the expression and regulatory effect of USP22 in lung cancer stem cells(LCSCs)with self-renewal and differentiation potential.We also observed the impact and mechanisms of different levels of USP22 on cisplatin resistance of LCSCs.Method: 1.Collect 3 specimens of non-small cell lung cancer tissue from City of Hope National Medical Center and isolate the cells population containing LCSCs.Study the correlation between USP22/H2Bub1 and stem cell marker CD133 by double-labeled immunofluorescence imaging analysis.Sorting the cells containing LCSCs by Fluorescence-Activated Cell Sorting(FACS)labeled by CD133 antibody.Identify the stem-like characteristics of CD133+ cell populations by sphere formation,ALDH activity assay,in vitro differentiation induction assay and NOD-SCID gamma(NSG)mouse xenograft tumorigenesis experiment.We compared the expression levels of USP22 and related genes between CSCs and non-CSCs by Western Blot analysis.Observe the changes of USP22 and related genes in LCSCs treated by cisplatin.2.Establish the USP22-KD-LCSCs cell line by knocking down USP22 with sh RNA lentivirus.Evaluate the impact to CD133,ALDH1A3,H2Bub1 and RNF20 of USP22knockdown by Western Blot.Sphere formation,NSG mouse xenograft tumorigenesis experiment and cisplatin-induced apoptosis assay were used to study the impact to the self-renewal ability and cisplatin sensitivity of LCSCs of USP22.3.Knocking down ALDH1A3 by ALDH1A3-si RNA transfection.Western Blot was used to verify the expression of USP22 after ALDH1A3 knockdown and the relationship between them.The impact to the sensitivity of cisplatin in LCSCs of ALDH1A3 knockdown was verified by cisplatin-induced apoptosis assay.After that,we transfered ALDH1A3 ORF c DNA clone to restore the expression of ALDH1A3 in USP22-KD-LCSCs.Then we observed the changes of cisplatin sensitivity in LCSCs by cisplatin-induced apoptosis assay.Finally,we observed whether the monoubiquitination of histone H2 B in the vicinity of ALDH1A3 promoter was significantly increased in USP22-KD-LCSCs by Ch IP and Quantitative real-time PCR.Thus we can clarify the relationship between them.Results: 1.The immunofluorescence showed that USP22 was highly expressed and H2Bub1 expression was reduced or absent in the isolated cells with high CD133 expression.After FACS,we found that CD133+ cells grew as tumorspheres.They could be induced differentiation by fetal bovine serum(FBS).A small number of CD133+ cells had the ability to form tumors in NSG mice.These results could verify the stem-like characteristics of CD133+ cells: the isolated CD133+ cells were LCSCs.Western Blot showed that the expression of USP22 and ALDH1A3 in LCSCs were significantly higher than CD133-lung cancer cells.The ALDH avtivity and the expression of USP22,ALDH1A3 and CD133 were markedly increased in LCSCs by cisplatin treatment.2.The ability of sphere formation was significantly reduced by knocking down the expression of USP22 in LCSCs(USP22-KD).Western Blot showed that the expression of ALDH1A3 and CD133 were markedly decreased,while H2Bub1 was significantly increased in USP22-KD LCSCs.We found that the tumorigenicity of USP22-KD LCSCs was significantly decreased by NSC mice xenograft experiments,while the cisplatin sensitivity was significantly increased.3.There was no significant change of the expression of USP22 by ALDH1A3knockdown in LCSCs while the cisplatin sensitivity was markedly increased.The cisplatin sensitivity was restored by restoring the expression of ALDH1A3 in USP22-KD LCSCs.H2Bub1 level in the vicinity of the ALDH1A3 promoter was significantly elevated in USP22-KD LCSCs.Conclusion: 1.The isolated CD133+ cell population can be confirmed as LCSCs.The expression of USP22 and ALDH1A3 were high,and the expression of H2Bub1 was low in LCSCs.The cisplatin resistance of LCSCs with higher expression of USCP22 and ALDH1A3 was observably higher.2.Knockdown of USP22 in LCSCs significantly reduced tumorigenicity and cisplatin resistance.The expression of ALDH1A3 was also decreased.The results demonstrated the regulatory relationship between USP22 and ALDH1A3.3.ALDH1A3 may locate at the downstream of USP22,which may be the key role in cisplatin resistance of LCSCs.USP22 may indirectly affect the transcription and expression of ALDH1A3 by regulating the ubiquitination level of histone H2 B in the vicinity of the ALDH1A3 promoter,thereby regulate the cisplatin sensitivity of LCSCs.