TIFAB Negatively Regulates Nrf2 Pathway By Competitively Binding To P62 With Keap1 To Inhibit Non-small Cell Lung Cancer Progression

Objective: Lung cancer has become a global problem and a public health problem.Lung cancer cases and deaths are on the rise globally.According to histopathological classification,lung cancer can be divided into small-cell lung cancer and non-small-cell lung cancer.Non-small cell lung cancer(NSCLC)accounts for about 85% of total lung cancer,including squamous cell carcinoma,adenocarcinoma,and large cell carcinoma.In the past,the main treatment for lung cancer was surgery,radiation,and chemotherapy.With the in-depth understanding of mutant genes,targeted therapy has become a hot spot,among which EGFR mutation accounts for 40% of lung cancer cases in China.However,due to the complex genetic background of lung cancer,the emergence of drug resistance makes the prognosis of patients still unsatisfactory.In recent years,immunotherapy has gradually come to the public’s attention.Although we have made remarkable breakthroughs in the treatment of lung cancer,the five-year survival rate of patients with advanced non-small cell lung cancer increased from less than 5% to 16% with the immunotherapy represented by PD-L1,immunotherapy also has its limitations.Therefore,it is important to study new therapeutic targets in non-small cell lung cancer.TRAF interacting protein(TIFA)with fork-head associated domains and its structural homologous TIFAB are members of the protein family containing fork-head associated domains(FHA)and can recognize and bind phosphothreonine motifs on interacting proteins.Studies have shown that TIFA and TIFAB regulate various signaling pathways involved in hematopoietic and immune cells.TIFA promotes NF-κB and JNK signaling in spleen B cells by binding to TRAF6 and promoting TLR signaling,ultimately leading to cell cycle progression and cell survival.TIFAB is a gene that has homologous sequence with TIFA and can bind to TRAF6 by homologous search.Unlike TIFA,TIFAB binds to TRAF6 and inhibits its mediated NF-κ B signaling pathway.The difference in function between TIFA and TIFAB may be due to the N-terminal threonine residues at the 9th position of TIFA,where TIFAB has no homologous residues.Although TIFA and TIFAB are of small molecular weight and have no catalytic function,they are important vectors of innate immune signaling and have implications for immune diseases,cancers and hematologic malignancies.TIFAB is located on human chromosome 5,partial deletion of which is closely associated with hematologic malignancies,so TIFAB is involved in the development of myelodysplastic syndrome(MDS)and acute myeloid leukemia(AML).It has been found that TIFAB maintains effective hematopoietic function in mice by regulating TRAF6 protein levels.In addition,TIFAB deficient hematopoietic cells exhibit characteristics of human MDS,including progressive cytopenia,changes in bone marrow differentiation,and a tendency to develop bone marrow(BM)failure.There is growing evidence that abnormal innate immune signals can be observed in TIFAB deficient models and induce MDS phenotypes.Moreover,through the combination of proteomics and genetics,TIFAB has been found to bind and regulate the deubiquitination activity of USP15 and the downstream p53 signaling pathway,thus playing a role in stress and malignant tumors.In this study,we explored a novel mechanism by which TIFAB regulates the Nrf2 signaling pathway.By analyzing clinical tissue samples from patients with non-small cell lung cancer,we found that TIFAB was underexpressed and closely correlated with the clinicopathologic factors of the patients.In addition,TIFAB significantly inhibited the proliferation and promoted apoptosis of non-small cell lung cancer by inhibiting the Keap1-Nrf2 signaling pathway.TIFAB and Keap1 competitively bind to p62,leading to the upregulation of Keap1 and inhibition of the Nrf2 signaling pathway in non-small cell lung cancer.In conclusion,we suggest that the absence of TIFAB promotes the progression of non-small cell lung cancer by up-regulating the Nrf2 signaling pathway,suggesting that TIFAB may be a promising therapeutic target for non-small cell lung cancer.Methods: We collected 197 lung cancer tissue samples from a hospital.Firstly,immunohistochemical staining was used to detect the expression and localization of TIFAB in lung cancer and its adjacent tissues,and then SPSS software was used to analyze the correlation between the expression of TIFAB and clinicopathologic factors.Kaplan-Meier plotter was used to predict the correlation between TIFAB and clinical outcomes.The protein expression of TIFAB in human normal bronchial epithelial cells and non-small cell lung cancer cell lines was detected by Western blot assay.After transfection of TIFAB and shTIFAB and detection of their transfection efficiency,Edu,colony formation,CCK-8 and flow cytometry were used to detect the changes in cell proliferation and apoptosis after transfection of TIFAB and shTIFAB,and Western blotting was used to detect the expression of proliferation and apoptosis related proteins.Immunofluorescence was used to detect changes in reactive oxygen species(ROS)after TIFAB was up-regulated.Antioxidant was added into H1299 cells while TIFAB was transfected,and the expressions of proteins related to proliferation and apoptosis were detected by Western blotting.Protein immunoblots and immunofluorescence techniques were used to detect the Nrf2 signaling pathway and the expression of downstream related molecule(HO-1)protein,the expression of Nrf2 nucleoprotein and the distribution of nucleoplasma in cells after bidirectional regulation of TIFAB.The proliferation and apoptosis of TIFAB transfected with Nrf2 activator were detected by a series of functional tests and Western blotting tests.The protein expression of Keap1 and p62 was down-regulated after transfection with TIFAB,and the protein levels of Nrf2 and HO-1were detected by Western blotting.Co-localization and conjugation dependent domains of TIFAB and p62 were detected by immunoprecipitation and immunofluorescence.Quantitative co-immunoprecipitation and Western blotting were used to detect the competitive binding relationship between TIFAB,Keap1 and p62 and the inhibition of Nrf2 pathway caused by their interaction.The ability of full-length transfected TIFAB and truncated mutants to detect proliferation and apoptosis was measured by a series of functional and Western blotting tests.The effect of TIFAB on the proliferation of non-small cell lung cancer in vitro was investigated by mouse tumor carrying experiment.Results: 1.We found that TIFAB was expressed in both lung cancer and adjacent tissues,mainly in the cytoplasm;Decreased or absent expression in non-small cell lung cancer tissues;The abnormal expression of TIFAB was correlated with lymph node metastasis,tumor size,and TNM stage(P<0.05).Meanwhile,the KM Plotter database showed that low TIFAB expression was associated with poor prognosis in lung cancer patients.2.TIFAB was expressed in normal bronchial epithelial cells and lung cancer cells.3.We selected A549 cells with high TIFAB expression and H1299 cells with low TIFAB expression for bidirectional regulation of TIFAB.Through a series of functional experiments(CCK-8,clonoformation,Edu,flow cytometry),it was found that up-regulation of TIFAB could inhibit proliferation and promote apoptosis of H1299 cells.The opposite result was obtained by down-regulating TIFAB.However,TIFAB did not affect the proliferation and apoptosis of A549 cells.The results were also verified by Western blotting assay to detect proliferative and apoptosis-related proteins.4.TIFAB can increase the reactive oxygen species level of H1299 cells without affecting the reactive oxygen species level of A549 cells,and antioxidants can reverse the effects of TIFAB on the proliferation and apoptosis of H1299 cells.5.We found that up-regulated TIFAB inhibited the protein expression of the Keap1/Nrf2 signaling pathway and its downstream target genes as well as the nuclear translocation of Nrf2 in H1299 cells,while down-regulated TIFAB produced the opposite result.However,bidirectional TIFAB regulation did not affect Nrf2 protein expression and nuclear translocation in A549 cells.Nrf2 activator can reverse the function of TIFAB in H1299 cells.6.After Keap1 and p62 were down-regulated while transfected with TIFAB,the Western blotting assay showed that TIFAB inhibited the Nrf2 signaling pathway in a p62/Keap1-dependent manner.7.Through co-immunoprecipitation and immunofluorescence experiments,we found that TIFAB interacts with the KIR domain of p62 via the C-terminal in non-small cell lung cancer.8.Through quantitative co-immunoprecipitation experiments,we found that up-regulated or down-regulated TIFAB could decrease or increase the binding ability of Keap1 and p62.9.Through functional and protein Western blotting,we found that TIFAB can bind to p62 through the C-terminal and inhibit the Nrf2 signaling pathway and the progression of non-small cell lung cancer.10.Tumor-carrying experiments in nude mice showed that silencing TIFAB could promote tumor formation in mice,and the effect could be reversed after silencing Nrf2.Conclusion: 1.TIFAB is down-regulated in non-small cell lung cancer and is associated with poor prognosis in lung cancer patients;2.TIFAB inhibits proliferation and promotes apoptosis by affecting REDOX levels in non-small cell lung cancer.3.TIFAB inhibits the biological effects of non-small cell lung cancer by inhibiting the Nrf2 signaling pathway;4.TIFAB relies on p62/Keap1 to inhibit the Nrf2 signaling pathway;5.TIFAB and Keap1 competitively bind to the KIR domain of p62;6.TIFAB plays a biological role depending on its C-terminal;7.Silencing TIFAB promotes the progression of non-small cell lung cancer in vivo by activating the Nrf2 signaling pathway.