TRIM26 Promotes Non-small Cell Lung Cancer Cell Proliferation By Inducing The Degradation Of The Transcription Factor PBX1

ObjectivesPBX1 is a member of the TALE class of homeodomain transcription factor.It plays an important role in the proliferation and migration of non-small cell lung cancer cell.We have found that PBX1 is degraded through the ubiquitin-proteasome pathway in prostate cancer.Further studies found that the deubiquitinase USP9x interacts with and stabilizes PBX1 protein by attenuating its K48-linked polyubiquitination.However,the ubiquitin enzyme mediates the ubiquitination of PBX1 is yet to know.The present study was aimed to screen the specifc ubiquitin ligase that mediats the ubiquitination of PBX1 thereby promoting its degradation.This study may provide a rationale for lung cancer therapy by targeting at PBX1 ubiquitination...Methods1.The NSCLC cell line A549 were treated with individual inhibitors of proteasomes or lysosomes,then cells were collected for the detection of protein levels of PBX1;and to measure whether PBX1 could be ubiquitinated in lung cancer cells,A549 was treated with a proteasome inhibitor followed by pulldown for the assay of PBX1 ubiquitination.2.The LC/MS/MS strategy was performed to screen the ubiquitin ligases of PBX1.HEK293T cells were transfected with Flag-PBXl,and PBX1 were precipitated with anti-Flag antibody for further assays.3.The interaction between TRIM26 and PBX1 were verified through Co-Immunoprecipitation(Co-IP),immunofluorescence(IF)and Western Blot(WB)methods.TRIM26 truncates were constructed by PCR and subcloning,and the interaction between TRIM26 truncates and PBX1 were explored by the Co-IP/WB assay.4.To overexpress or knock down TRIM26 in cells,the protein stability of PBX1 was measured by WB.After overexpressing TRIM26 in A549 cells,the mRNA level of PBX1 was detected with RT-PCR.5.After overexpressing TRIM26 in A549 cells,cells were treated with proteasomal or lysosomal inhibitors,the degradation pathway of PBX1 mediated by TRIM26 were determined.6.The half-life of PBX1 was analyzed with CHX chase experiment in the presence or absence of TRIM26.7.PBX1 protein was immunoprecipitated in the presence or absence of TRIM26,followed by IP/WB assay to examine the ubiquitination levels of PBX1.8.RNF6 is a target gene of PBX1 and pGL4-RNF6-Luciferase plasmids were constructed and the luciferase assay was used to analyze the transcriptional activity of PBX1 mediated by TRIM26.9.RT-PCR was applied to detect the mRNA levels of TRIM26 and WB was applied to measure the protein level of TRIM26 and PBX1 in patients’ samples.10.TRIM26 were overexpressed in or knocked down from NSCLC cells,followed by MTT and colony formation assays to analyze the proliferation of NSCLC cells.Results1.The protein level of PBX1 was stablized by the proteasomal inhibitors(MG 132 or Bortezomib),but not by the lysosomal inhibitors(Chloroquine or Bafilomycin A1),and the ubiquitination levels of PBX1 was enhanced by MG 132.2.The ubiquitin ligases TRIM26,HUWE1 and UBR4 were identified from the immunoprecipitates of PBX1 by the LC/MS/MS strategy.3.The IP/WB showed that TRIM26 interacted with PBX1 in HEK293T following the ecotopic expression manner.The endogenous co-immunoprecipitation confirmed the interaction between TRIM26 and PBX1 in NSCLC cells.The immunofluorescence indicated that TRIM26 and PBX1 were co-localized in nuclei.The co-immunoprecipitation assay revealed that the SPRY domain of TRIM26 was essential for the interaction between TRJM26 and PBX1.4.In the context of the overexpression of TRIM26 and PBX1,TRIM26 mediated the degradation of PBX1 in a time-and dose-dependent manner.When TRIM26 was overexpressed,the protein levels of PBX1 were downregulated in both NSCLC cell lines A549 and H226.Consistently,PBX1 was upregulated when TRIM26 was knockdown.In contrast,the RT-PCR results showed that TRIM26 did not exert dramatical effects on the mRNA levels of PBX1.5.The degradation of PBX1 mediated by TRIM26 could be turnover by the proteasomal but not by the lysosomal inhibitors.6.The CHX chase assay showed that TRIM26 decreased the protein level and shortened the half-life of PBX1.7.The ubiquitination assay showed that TRIM26 enhanced the K48 linked ubiquitination of PBX1.8.The luciferase assay revealed that TRIM26 decreased the transcriptional activity of PBX1.9.The mRNA levels of TRIM26 in lung cancer tissues were higher than those in para-carcinoma tissues by RT-PCR assay.Importantly,the protein levels of PBX1 were negatively correlated with the TRIM26 expression levels in lung cancer tissues.10.The MTT and colony formation assay indicated that TRIM26 promotes the proliferation of NSCLC cell lines.ConclusionTRIM26 is identified from the immunoprecipitates of PBX1 by the LC/MS/MS protocol.TRIM26 interacts with PBX1 dependent on the SPRY domain.TRIM26 medaites PBX1 with the K48-linked polyubiquitination.TRIM26 not only leads to PBX1 degradation but also prevents the nuclear localization of PBX1 thus downregulating the transcriptional activity of PBX1.By inhibiting the PBX1 function,TRIM26 promotes NSCLC cell proliferation.Given the pathophysiological function of TRIM26 and PBX1 in NSCLC cell prolilferation,the TRIM26/PBX1 axis could be a potent target for the treatment of non-small cell lung cancers.