Dysregulation of Notch signaling has been implicated in cellular transformation and tumorigenesis in a variety of cancers while potential roles of MIB1, an E3 ubiquitin ligase required for efficient Notch activation, remains to be investigated. We analyzed MIB1 expression levels in tumor samples and performed gain-of-function and loss-of-function studies in cell lines to investigate potential roles of MIB1 in epithelial-to-mesenchymal transition (EMT), cell migration, and cell survival. We found that overexpression of MIB1 is detected in a subset of lung squamous carcinoma and adenocarcinoma samples and negative correlation is observed between MIB1 expression and overall patient survival. Ectopic expression of MIB1 in A549 cells induces EMT and stimulates cell migration via a Notch-dependent pathway. Meanwhile, MIB1 stimulates the degradation of nuclear factor erythroid 2-related factor 2 (NRF2) in a Notch-independent manner and disrupts the antioxidant capacity of cells, rendering them more sensitive to inducers of ferroptosis. On the other hand, MIB1 knockout induces accumulation of NRF2 and resistance to ferroptosis. Collectively, these results indicate that MIB1 may function as a positive regulator of ferroptosis through targeted degradation of the master antioxidant transcription factor NRF2.

Implications:

This study identifies a MIB1-induced proteasomal degradation pathway for NRF2 and reveals elevated ferroptosis sensitivity in MIB1-overexpressing cells which may provide novel insights into the treatment of MIB1-overexpressing cancers.

This article is featured in Highlights of This Issue, p. 181

Ferroptosis is a recently identified form of regulated cell death that is characterized by the accumulation of lipid reactive oxygen species (ROS) and membrane damage (1). The lipid peroxides are generated from membrane polyunsaturated fatty acid (PUFA) by lipoxygenases or in a non-enzymatic manner thus elevated level of PUFA sensitizes cells to ferroptosis (2). Meanwhile, lipid peroxides can be removed by several metabolic pathways including GPX4/GSH pathway (3), FSP1/CoQ10 pathway (4, 5), and GCH1/BH4 pathway (6, 7). These lipid peroxide removal pathways are essential for cell survival because their inhibition leads to ferroptotic cell death in various cell lines. Nuclear factor erythroid 2-related factor 2 (NRF2), a master transcription factor involved in cellular antioxidant response, stimulates the expression of GPX4 as well as genes involved in iron metabolism (i.e., FTL, FTH1, SLC40A1) or GSH synthesis pathway (SLC7A11, GSS, GCLC/GCLM) thus prevents the accumulation of lipid peroxides and ferroptotic cell death (8). Under normal condition, the intracellular protein level of NRF2 is usually kept low through the KEAP1-CUL3-RBX1–mediated ubiquitination and proteasome mediated degradation (9). Deficiency in this regulatory pathway in certain cancer cells leads to constituent activation of NRF2 signaling which favors tumorigenesis (10, 11). On the other hand, inhibition of NRF2 is able to resensitize cancer cells to ferroptosis inducing reagents and inhibit tumor formation in vivo (12). Changes of sensitivity to ferroptosis inducing reagents were also observed in other circumstances, for example, dedifferentiated melanoma cells (13) or cancer cells in a high mesenchymal state (14) or drug-resistant persister cells (15) appear more dependent on the GPX4 pathway for their survival so they are generally more vulnerable to GPX4 inhibition–induced ferroptosis. Thus, targeting ferroptosis-related pathways may provide novel opportunities in cancer treatment.

MIB1 was first identified as an E3 ubiquitin ligase essential for efficient Notch signaling from the zebrafish mind bomb (mib) mutant (16). Notch-related developmental defects were confirmed in Mib1 knockout mice (17, 18) or human patients with germline mutations in MIB1 gene (19), indicating a conserved function of MIB1 in vertebrate Notch signaling. Other studies revealed that MIB1 is also involved in Wnt signaling (20), NFκB activation (21), cell proliferation (22), directional cell migration (23), or cellular senescence (24). In addition to these signaling events, MIB1 is essential for centriole dynamics in normal cell division as well as stressed conditions. For example, MIB1 ubiquitination of PLK4 is required for proper centriole duplication (25). Centriole component proteins including PCM1, GABARAP, and Talpid3 are substrates of MIB1 and overactivation of MIB1 mediated ubiquitination disrupts the homeostasis of these proteins and results in defects in serum starvation–induced autophagy (26) or ciliogenesis (27–29). Interestingly, the Zika virus infection induced degradation of PCM1 and dispersion of PCM granule is also dependent on MIB1 (30). Thus, MIB1 regulates a variety of intracellular processes and mis-regulation of MIB1 occurs in many diseased conditions.

In this study, we report that MIB1 overexpression correlates with impaired overall survival in patients with lung cancer. Ectopic expression of MIB1 in lung cancer cell line A549 induces epithelial-to-mesenchymal transition (EMT) and stimulates cell migration. Meanwhile, MIB1 induces ubiquitination and proteasome degradation of the master antioxidant transcription factor NRF2 and sensitizes cells to ferroptosis. Thus, targeting ferroptosis pathway might provide novel opportunity in treating subtypes of cancers with high MIB1 expression.

Constructs

Molecular cloning was performed according to standard protocols. cDNAs were cloned by reverse transcription-PCR using mRNAs isolated from human H1 ES cells. For pSIN-hMIB1-Flag-Puro, hMIB1 cDNA was inserted into the SnaBI site of pSIN-EF1-alpha-IRES-puro. For pCS2+-hMIB1-HA and pCS2+-hMIB1-dR-HA and pCS2+-hMIB1-dC-HA, full-length or truncated versions of hMIB1 cDNAs were inserted into the StuI site of pCS2+-HA. For pKD-Flag-hNRF2, hNRF2 cDNAs were inserted into the BsrGI and EcoRI sites of pKD-Flag-IRES-Puro. For pKD-Flag-hNRF2-dNEH2 and pKD-Flag-hNRF2-dNEH4–5 and pKD-Flag-hNRF2-dNEH6, deletions were performed according to standard mutation protocol. pCR3.1-Myc-His-Ubiquitin was kindly provided by Xiaofei Zhang (GIBH, Guangzhou, P.R. China). All constructs were confirmed by DNA sequencing. PCR primers used in this study are listed in Supplementary Table S1.

qRT-PCR

Total RNA was extracted using the TRIzol reagent (MRC, TR118) and cDNA synthesis was performed by reverse transcription of 5 μg of total RNA using the ReverTra Ace qPCR RT Kit (TOYOBO, TRT-101) according to the manufacturer's instructions. RT-PCR was performed using SYBR Green Supermix (Bio-Rad, 172-5274) on a CFX96 Real Time PCR system (Bio-Rad). GAPDH was used as an internal control in all experiments. PCR primers used are listed in Supplementary Table S1.

Cell culture

A549 (RRID: CVCL_0023) and MDA-MB-231 (RRID: CVCL_0062) cells were cultured in RPMI1640 (Gibco, C11875500BT) supplemented with 10% FBS (Gibco, 12657-029) and 100 U/mL penicillin-streptomycin (Hyclone, SV30010). HEK293T cells were cultured in DMEM (Hyclone, SH30022.01) supplemented with 10% FBS and 100 U/mL penicillin-streptomycin. All cell lines were cultured under humidified conditions in 5% CO2 at 37°C. A549 and HEK293T cells were sourced from Duanqing Pei's Laboratory (GIBH, Guangzhou, P.R. China);MDA-MB-231 cells were purchased from GuangZhou Jennio Biotech Co., Ltd. All of the cell lines were Mycoplasma free as determined using a kit obtained from Lonza (LT07-318). Cell line identities were confirmed by short tandem repeat authentication.

Generation of A549-MIB1 stable cell line

Lentivirus was produced by transfecting expression vector (pSIN-hMIB1-Flag-Puro) together with packaging plasmid (psPAX2; RRID: Addgene_12260), envelop plasmid (pMD2.G; RRID: Addgene_12259) into HEK293T cells. Cell supernatants were harvested 48 hours after transfection. To obtain stable cell line, A549 cells were infected at low confluence (20%) for 24 hours with lentiviral supernatants in the presence of 8 μg/mL Polybrene (Sigma, H9268). A total of 72 hours after infection, cells were placed under puromycin selection (5 μg/mL; Selleck, S7417) for 48 hours and then passaged before use.

Generation of MIB1 knockout MDA-MB-231 cell lines

MIB1 knockout MDA-MB-231 cell lines were generated using the lentiCRISPRV2 system with the targeting sequence (5′-GTTATAGAAGTACTACATCGAGG-3′) located at exon 11 of human MIB1 gene. Lentivirus was produced by transfecting lentiCRISPRV2 system together with packaging plasmid (psPAX2), envelop plasmid (pMD2.G) into HEK293T cells. Cell supernatants were harvested 48 hours after transfection. To obtain stable cell lines, MDA-MB-231 cells were infected at low confluence (20%) for 24 hours with lentiviral supernatants in the presence of 8 μg/mL Polybrene. A total of 72 hours after infection, cells were placed under puromycin selection (5 μg/mL) for 48 hours. Cells were then transferred into 96-well plates at approximately 0.8 cell per well and expanded as clones. Genomic DNA was extracted from all clones with the TIANamp Genomic DNA Kit (TIANGEN, DP304). PCR products spanning the single-guide RNA (sgRNA) target site were generated with the LA Taq DNA Polymerase (TaKaRa, RR52A) with primers (5′-CTTCTGCCTTGTCCTCCC-3′ and 5′-TAGGCTGTGATTTCTTTGAT-3′) and sequenced. Depletion of MIB1 protein in MDA-MB-231 MIB1 knockout clone 1 and 2 was confirmed by Western blot analysis.

Short hairpin RNA knockdown assay

Short hairpin RNA (shRNA) knockdown of NOTCH1/2/3 in A549 was performed in the lentiviral pLKO.1-TRC cloning vector (RRID: Addgene_10878). Cells were infected with a combination of shRNA expression constructs to NOTCH1 (Sigma, TRCN0000350330), NOTCH2 (Sigma, TRCN0000262587), and NOTCH3 (Sigma, TRCN0000020234) and stable cell lines were generated as described above. Knockdown efficiency was determined by qRT-PCR.

Cell viability assay

Cells were seeded on 96-well plates (5,000 cells per well) one day before treating with the indicated concentrations of RSL3 (Selleck, S8155), Staurosporine (Selleck, S1421), H2O2 (Sigma, 1.06097), cisplatin (Selleck, S1166), doxorubicin (Selleck, S1208), taxol (Selleck, S1150), or DMSO (Sigma, W387520). Cell viability was assessed 24 hours after treatment using the CCK8 Kit (KeyGEN BioTECH, KGA317) according to the manufacturer's instructions. At least three biological repeats were assayed and analyzed for each compound.

Transwell assay

For cell migration assay, 2.5 × 104 cells were suspended to the top chambers (24-well inserts, 8.0 μmol/L; Corning, 3422) in 200 μL of serum-free RPMI1640 media. A total of 600 μL of RPMI1640 containing 10% FBS was added into the bottom compartment. After 24 hours of incubation, cells on the upper surface of the filters were removed, and cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet. Six fields per filter were counted at 100× magnification and assays were repeated three times.

Western blot analysis

Cells were washed twice with cold PBS (HyClone, SH30028.02) then lysed in buffer (50 mmol/L Tris–HCl pH 6.8, 80 mmol/L NaCl, 2% SDS, 0.4% NP40, 0.8 mmol/L EDTA, 10% glycerol, 1% β-mercaptoethanol, and 0.02% bromophenol blue) containing protease inhibitor cocktail (Roche, 04693132001). Samples were boiled for 10 minutes then centrifuged at 13,000 × g. Supernatants were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, WBKL S0500). Membranes were incubated in blocking buffer [5% (w/v) non-fat dry milk (Sangon, NB0669) dissolved in Tris Buffered Saline with Tween-20 (TBST) buffer (10 mmol/L Tris–HCl, pH 8.0, 150 mmol/L NaCl, and 0.05% Tween-20] for 1 hour at room temperature. Membranes were then washed three times (10 minutes each time) in TBST and incubated with primary antibodies overnight at 4°C. After washed three times (10 minutes each time) in TBST, membranes were incubated with an appropriate secondary antibody for 1 hour at room temperature. GAPDH was the loading control. At least two biological repeats were performed for all Western blot analysis with consistent results. Antibodies used in this study are listed in Supplementary Table S2.

Immunofluorescence staining

Cells grown on coverslips were first washed twice with PBS, fixed in 4% paraformaldehyde solution (Genelc, WJ0012) for 30 minutes then permeabilized in 0.2% Triton X-100 (Sigma, T8787) in PBS for 15 minutes at room temperature. For F-Actin staining, cells were incubated with 1 unit per mL Alexa Fluor 568 phalloidin (Invitrogen, A12380) for 60 minutes at room temperature. For antibody staining, samples were washed three times (5 minutes each time) with PBS and blocked with 5% FBS in PBS for 1 hour at room temperature. Samples were then incubated with primary antibodies diluted in blocking buffer overnight at 4°C. After three washes with PBS (5 minutes each time), samples were incubated with a secondary antibody in blocking buffer for 1 hour at room temperature. Finally, samples were washed three times in PBS and counterstained with DAPI (10 μg/mL) (Thermo Fisher Scientific, D1306) for 1 minute and mounted onto glass slides with anti-fade solution (VECTOR, H-1000) at room temperature and visualized using a Zeiss 710 NLO confocal microscope.

Analysis of ROS

The day before experiment, 200,000 cells per well were seeded into 6-well plates. Cells were then treated with test compounds for 24 hours, harvested by trypsinizaiton and washed twice with PBS. Samples were then resuspended in 200 μL RPMI1640 containing DCFH-DA (10 μmol/L; Beyotime, S0033), C11-BODIPY (2 μmol/L; Invitrogen, D3861), or MitoSOX (5 μmol/L; Invitrogen, M36008) and incubated in a tissue culture incubator for 20 minutes at 37°C. Cells were washed twice with PBS and resuspended in 200 μL of fresh PBS and strained through a 40-μmol/L cell strainer then analyzed using the BD LSR Fortessa FACScanner and FlowJo software (RRID: SCR_008520). A minimum of 10,000 cells were analyzed per condition.

Ubiquitination assay

HEK293T cells in 60 mm plate were transfected with the indicated plasmids using the Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) transfection reagent according to the manufacturer's instructions. After 36 hours, cells were washed twice in cold PBS with 10 mmol/L N-Ethylmaleimide (NEM; Sigma, E3876) and lysed in 8 mol/L urea buffer (8 mol/L urea, 0.1 mol/L Na2HPO4, 0.1 mol/L NaH2PO4, 10 mmol/L Tris-HCl pH 8.0, 10 mmol/L imidazole) supplemented with 10 mmol/L β-mercaptoethanol (Gibco, 21985) and protease inhibitor cocktail. Lysates were centrifuged at 13,000 rpm for 10 minutes at room temperature and incubated with nickel resins (Qiagen, 30210) at room temperature for 2 hours, beads were then washed three times with 8 mol/L urea buffer, and proteins were eluted with 2× SDS loading buffer at 42°C for 15 minutes then analyzed by SDS-PAGE. For ubiquitination analysis by Flag Resin, cells were washed twice with cold PBS containing 10 mmol/L NEM, and lysed in radioimmunoassay buffer [20 mmol/L NaH2PO4, Na2HPO4 (pH 7.4), 150 mmol/L NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, and 1% SDS] supplemented with cocktail and 10 mmol/L NEM. Lysates were sonicated and boiled for 5 minutes, diluted to 0.1% SDS, and centrifuged at 13,200 rpm at 4°C for 10 minutes. The supernatant was incubated with Flag-Resin for 2.5 hours at 4°C. After three times washing, bound proteins were eluted with 2× SDS loading buffer at 42°C (1,000 rpm for 15 minutes), and then separated by SDS-PAGE and analyzed by Western blot analysis.

IHC

IHC analysis of MIB1 was performed according to standard protocols for IHC. In brief, heat-induced epitope retrieval was achieved using citrate buffer. Endogenous peroxidases were blocked with hydrogen peroxide block buffer. Sections were incubated with a primary antibody overnight at 4°C, followed by incubation with a secondary antibody for 30 minutes at room temperature. Finally, sections were counterstained with hematoxylin. The survival curves were estimated using the Kaplan–Meier method, and the difference was tested using the log-rank test. Associations between VIMENTIN (VIM) and MIB1 expression were tested using the Fisher exact test, all tests were two sided. Statistical analysis was performed using IBM SPSS Statistics 25 (RRID: SCR_019096). P < 0.05 was considered statistically significant.

Statistical analysis

Experiments were performed with three biological repeats when possible. Data were presented as mean ± SD, which were calculated using Graphpad Prism 8.0 software (RRID: SCR_002798). For pairwise comparisons, P values were calculated using the two tailed t test. For comparison across multiple experimental groups, P values were determined by ordinary one-way ANOVA with Tukey multiple comparisons as a post hoc test for comparing every mean to every other mean or ordinary one-way ANOVA with Dunnett multiple comparisons as a post hoc test for comparing every mean to a control mean. For comparison between any of the two groups, P values were calculated using two-way ANOVA with Tukey multiple comparison. P < 0.05 was considered statistically significant. No statistical methods were used to predetermine sample size.

Data availability statement

The data generated in this study are available within the article and its Supplementary Data files.

MIB1 protein level correlates negatively with survival of patients with lung cancer

To evaluate possible roles of MIB1 in human tumorigenesis, we first determined the protein levels of MIB1 in several tumor samples by immunohistochemistry and analyzed the relationship of MIB1 expression level with the outcome of patients. Among the 89 lung squamous carcinoma samples analyzed, 14 samples show MIB1 expression levels similar to nearby normal tissue while 75 samples have elevated MIB1 expression (Fig. 1A). Kaplan–Meier survival analysis revealed that the cumulative survival is significantly reduced in patients with high MIB1 expression (P = 0.004, log-rank test; Fig. 1B). A negative correlation was also observed in lung adenocarcinoma samples (Fig. 1C and D). A total of 35 samples show elevated MIB1 expression and 59 samples show low MIB1 expression in 94 lung adenocarcinoma sections analyzed and patients with high MIB1 expression have significantly reduced overall survival (P = 0.009).

Figure 1.

MIB1 expression is negatively associated with cumulative survival in patients with lung cancer. A, Representative results for MIB1 immunohistochemistry staining in human lung squamous carcinoma and para-cancerous tissue sections. A total of 14 of 89 (15.7%) tumor samples have basal MIB1 expression levels (low MIB1) comparable with nearby non-cancerous tissues and 75/89 (84.3%) tumor samples have elevated expression of MIB1 (high MIB1). B, Kaplan–Meier survival curves were generated with IBM SPSS Statistics 25 for A. P value (log-rank test) <0.05, indicating that high MIB1 correlates with poor patient survival. C, MIB1 expression in representative lung adenocarcinoma and para-cancerous tissue sections (n = 94). D, Kaplan–Meier curves for lung adenocarcinoma patients (59/94 (62.8%) for low MIB1 and 35/94 (37.2%) for high MIB1). Negative correlation is also observed between MIB1 expression and patient survival. E and F, Representative results for VIM expression by IHC in low MIB1 expression (n = 14) or high MIB1 expression (n = 72) lung squamous carcinoma samples used in A. G and H, VIM expression in lung adenocarcinoma samples used in C. P value in F and H is determined by two-tailed Fisher exact test. Scale bar: 50 μm.

Figure 1.

MIB1 expression is negatively associated with cumulative survival in patients with lung cancer. A, Representative results for MIB1 immunohistochemistry staining in human lung squamous carcinoma and para-cancerous tissue sections. A total of 14 of 89 (15.7%) tumor samples have basal MIB1 expression levels (low MIB1) comparable with nearby non-cancerous tissues and 75/89 (84.3%) tumor samples have elevated expression of MIB1 (high MIB1). B, Kaplan–Meier survival curves were generated with IBM SPSS Statistics 25 for A. P value (log-rank test) <0.05, indicating that high MIB1 correlates with poor patient survival. C, MIB1 expression in representative lung adenocarcinoma and para-cancerous tissue sections (n = 94). D, Kaplan–Meier curves for lung adenocarcinoma patients (59/94 (62.8%) for low MIB1 and 35/94 (37.2%) for high MIB1). Negative correlation is also observed between MIB1 expression and patient survival. E and F, Representative results for VIM expression by IHC in low MIB1 expression (n = 14) or high MIB1 expression (n = 72) lung squamous carcinoma samples used in A. G and H, VIM expression in lung adenocarcinoma samples used in C. P value in F and H is determined by two-tailed Fisher exact test. Scale bar: 50 μm.

Close modal

MIB1 is a positive regulator of Notch signaling which is well established to promote EMT and tumorigenesis, so we analyzed whether MIB1 overexpression is associated with more mesenchymal state in above-mentioned samples. We found that all lung squamous carcinoma with low MIB1 expression (n = 14) show low expression of the mesenchymal marker VIM. Among the 72 samples with high MIB1 expression, 21 (29.2%) show elevated VIM expression (P = 0.018 by two-tailed Fisher exact test; Fig. 1E and F), indicating high MIB1 tumors are more mesenchymal. Similar correlation is also observed in lung adenocarcinoma samples (P = 0.034, n = 94; Fig. 1G and H). Together, these results suggest that high MIB1 expression correlates with a more mesenchymal signature and impaired patient survival in lung cancers.

MIB1 overexpression induces EMT in A549 cells

The above results suggest an oncogenic function of MIB1 while direct evidence for MIB1 in tumorigenesis is still missing. So we performed gain-of-function assay to determine whether MIB1 overexpression leads to cellular changes that favor tumorigenesis. We first analyzed MIB1 expression level by Western blot analysis in a panel of cell lines (Fig. 2A). MIB1 protein can be detected in all cell lines examined while the expression level varies. We chose A549 which is a lung adenocarcinoma–derived cell line with low endogenous MIB1 for gain-of-function studies. We established a stable A549 cell line with high expression of ectopic MIB1 (Fig. 2B) and noticed that MIB1 overexpression induces morphologic changes of A549 from an epithelial-like to a mesenchymal-like morphology (Fig. 2C, left). Immunofluorescence (IF) staining revealed that MIB1 overexpression induces the formation of intracellular stress fibers (middle) and the upregulation of mesenchymal marker N-Cadherin (right). Western blot analysis confirmed the upregulation of mesenchymal marker N-Cadherin and downregulation of epithelial marker E-Cadherin in MIB1 overexpression A549 cells (Fig. 2D). We then determined the expression levels of several EMT-related genes by qRT-PCR and found that the epithelial gene CDH1 (which encodes E-Cadherin) is downregulated while mesenchymal genes such as VIM, SLUG, and ZEB2 are upregulated in MIB1 overexpression cells (Fig. 2E). These observations indicate that MIB1 promotes EMT in A549 cells. Because EMT is well known to promote cell migration and tumor metastasis, we tested whether MIB1 is able to stimulate cell migration by the transwell assay and found that MIB1 overexpression clearly enhances the transwell migration of A549 cells (Fig. 2F and G). We then investigated the regulatory mechanism of MIB1 involved in EMT and cell migration. Among the known downstream pathways regulated by MIB1, Notch is well established to be a positive regulator of EMT and cell migration so we investigated whether MIB1 stimulates A549 EMT and migration in a Notch-dependent manner. We established cells line expressing shRNAs to NOTCH1–3 and confirmed the knockdown of NOTCH1/2 and the inhibition of Notch target gene HES1 by qRT-PCR (Supplementary Fig. S1A). HES1 expression is also effectively inhibited by a Notch pathway small chemical inhibitor LY411575 (Supplementary Fig. S1B). We performed qRT-PCR analysis for EMT-related genes in shNOTCH or LY411575-treated cells and found Notch inhibition abolishes the MIB1-induced downregulation of CDH1 and upregulation of VIM, SLUG, and ZEB2 (Supplementary Fig. S1C). Western blot analysis confirmed the involvement of Notch pathway in MIB1-induced upregulation of N-Cadherin and downregulation of E-Cadherin at protein level (Fig. 2H). Furthermore, MIB1-stimulated cell migration is also blocked by shNOTCH or LY411575 treatment (Fig. 2I). Collectively, these results indicate that MIB1 induces EMT and promotes cell migration in a Notch-dependent manner.

Figure 2.

MIB1 overexpression promotes EMT and cell migration in A549. A, Western blot analysis for MIB1 protein in a panel of cell lines. GAPDH is loading control. SK: SK-MES-1; MDA: MDA-MB-231. B, Relative expression level of MIB1 in control A549 (WT) and a stable MIB1-expressing A549 cell line (MIB1). C, MIB1 overexpression in A549 induces EMT-like morphologic changes (left, scale bar is 100 μm), formation of F-Actin stress fibers (middle, scale bar is 20 μm) and induction of mesenchymal marker N-Cadherin (right, scale bar is 20 μm). D, Western blot analysis for N-Cadherin and E-Cadherin in WT and MIB1 overexpression A549 cells. E, qRT-PCR analysis for EMT-related genes in WT and MIB1 overexpression A549 cells. Data represent mean ± SD from three independent repeats and P value is calculated using unpaired t test. **, P < 0.01; ***, P < 0.001. F, Transwell assay for the migration activity of WT and MIB1 overexpression A549 cells. Scale bar is 200 μm. G, Statistical results for F. Data represent mean ± SD from three biological repeats and P value is calculated using unpaired t test. **, P < 0.01. H, Western blot analysis for E-Cadherin and N-Cadherin in shRNAs to NOTCH1/2/3 (shNOTCH) or Notch pathway inhibitor (LY411575) treated cells. I, Transwell assay for shNOTCH or LY411575-treated cells. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Tukey multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Figure 2.

MIB1 overexpression promotes EMT and cell migration in A549. A, Western blot analysis for MIB1 protein in a panel of cell lines. GAPDH is loading control. SK: SK-MES-1; MDA: MDA-MB-231. B, Relative expression level of MIB1 in control A549 (WT) and a stable MIB1-expressing A549 cell line (MIB1). C, MIB1 overexpression in A549 induces EMT-like morphologic changes (left, scale bar is 100 μm), formation of F-Actin stress fibers (middle, scale bar is 20 μm) and induction of mesenchymal marker N-Cadherin (right, scale bar is 20 μm). D, Western blot analysis for N-Cadherin and E-Cadherin in WT and MIB1 overexpression A549 cells. E, qRT-PCR analysis for EMT-related genes in WT and MIB1 overexpression A549 cells. Data represent mean ± SD from three independent repeats and P value is calculated using unpaired t test. **, P < 0.01; ***, P < 0.001. F, Transwell assay for the migration activity of WT and MIB1 overexpression A549 cells. Scale bar is 200 μm. G, Statistical results for F. Data represent mean ± SD from three biological repeats and P value is calculated using unpaired t test. **, P < 0.01. H, Western blot analysis for E-Cadherin and N-Cadherin in shRNAs to NOTCH1/2/3 (shNOTCH) or Notch pathway inhibitor (LY411575) treated cells. I, Transwell assay for shNOTCH or LY411575-treated cells. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Tukey multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Close modal

MIB1 overexpression sensitizes A549 cells to ferroptosis

In addition to induce morphologic changes and stimulate cell migration, EMT is known to play roles in drug resistance. Interestingly, recent studies reported that mesenchymal state tumor cells and drug-resistant persister cells are indeed more sensitive to ferroptosis inducers (14, 15). So we tested whether MIB1 overexpression changes the sensitivity of A549 to commonly used chemotherapeutic reagents and inducers of various cell death pathways. We found that MIB1 does not change the sensitivity of A549 to chemotherapeutic reagents such as doxorubicin, taxol, or cisplatin (Fig. 3A). Sensitivity to Staurosporine (apoptosis inducer) and H2O2 (necrosis inducer) remain unchanged in MIB1 overexpression A549; however, MIB1 overexpression clearly sensitizes A549 to ferroptosis inducer RSL3 (IC50 = 2.5 μmol/L for A549-MIB1 and 14.4 μmol/L for A549; Fig. 3A). To further prove that the RSL3-induced cell death is ferroptotic cell death, we performed rescue experiments and found the RSL3-induced cell death in MIB1 overexpression cells can be effectively rescued only by ferroptosis inhibitor Fer-1 but not necroptosis inhibitor Nec-1s or apoptosis inhibitor zVAD-fmk (Fig. 3B). As a control, the H2O2-induced cell death in wild-type (WT) or MIB1 overexpression A549 can be rescued by antioxidant N-acetyl-L-cysteine (NAC) or Nec-1s but not Fer-1 (Supplementary Fig. S2). These data indicate that MIB1 overexpression mainly affect ferroptosis but not necrosis pathway. We then tested whether MIB1 enhances sensitivity to ferroptosis inducer in mouse xenograft model. WT and MIB1 overexpression cells were subcutaneously injected into immune-deficient mice then RSL3 was injected intratumorally and tumors were analyzed after 2 weeks. We found that tumor weights are comparable between WT and MIB1 overexpression cells in the absence of RSL3; however, MIB1-derived tumors are significantly smaller than WT after RSL3 treatment (Supplementary Fig. S3). These data suggest that MIB1 stimulates ferroptosis sensitivity both in vitro and in vivo.

Figure 3.

MIB1 overexpression sensitizes A549 to ferroptosis. A, CCK8 cell viability assay for A549 and A549-MIB1 cells treated with the indicated small chemicals. Doxorubicin, taxol, and cisplatin are commonly used chemotherapeutic reagents. Staurosporine is an apoptosis inducer and H2O2 induces necrosis. RSL3 is a ferroptosis inducer. At least three biological repeats were performed and data represent mean ± SD. P value is determined by two-way ANOVA with Sidak multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. B, Ferroptosis inhibitor rescues RSL3-induced cell death in A549-MIB1 cells. Cells were treated with RSL3 together with ferroptosis specific inhibitor (Fer-1), necroptosis inhibitor (Nec-1s) or apoptosis inhibitor (zVAD-fmk) and cell viability determined as describe in A. Data represent mean ± SD from three biological repeats and P value is determined by ordinary one-way ANOVA with Dunnet multiple comparisons test. ns, no significance; ****, P < 0.0001. C, Representative FACS analysis results for various intracellular ROS species in A549 and A549-MIB1 cells treated with RSL3. DCFH-DA is a probe for cytosol ROS, C11-BODIPY is a lipid ROS specific probe and MitoSox detects mitochondrial ROS. D, Statistical results for C. Data represent mean ± SD of three repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. ns, no significance; **, P < 0.01; ***, P < 0.001. E, Notch inhibition by shNOTCH or LY411575 does not change ferroptosis sensitivity in A549 and A549-MIB1 cells. Assays were performed as described in A and data represent mean ± SD from three biological repeats. P value is determined by two-way ANOVA with Tukey multiple comparisons test. ns, no significance.

Figure 3.

MIB1 overexpression sensitizes A549 to ferroptosis. A, CCK8 cell viability assay for A549 and A549-MIB1 cells treated with the indicated small chemicals. Doxorubicin, taxol, and cisplatin are commonly used chemotherapeutic reagents. Staurosporine is an apoptosis inducer and H2O2 induces necrosis. RSL3 is a ferroptosis inducer. At least three biological repeats were performed and data represent mean ± SD. P value is determined by two-way ANOVA with Sidak multiple comparisons test. ***, P < 0.001; ****, P < 0.0001. B, Ferroptosis inhibitor rescues RSL3-induced cell death in A549-MIB1 cells. Cells were treated with RSL3 together with ferroptosis specific inhibitor (Fer-1), necroptosis inhibitor (Nec-1s) or apoptosis inhibitor (zVAD-fmk) and cell viability determined as describe in A. Data represent mean ± SD from three biological repeats and P value is determined by ordinary one-way ANOVA with Dunnet multiple comparisons test. ns, no significance; ****, P < 0.0001. C, Representative FACS analysis results for various intracellular ROS species in A549 and A549-MIB1 cells treated with RSL3. DCFH-DA is a probe for cytosol ROS, C11-BODIPY is a lipid ROS specific probe and MitoSox detects mitochondrial ROS. D, Statistical results for C. Data represent mean ± SD of three repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. ns, no significance; **, P < 0.01; ***, P < 0.001. E, Notch inhibition by shNOTCH or LY411575 does not change ferroptosis sensitivity in A549 and A549-MIB1 cells. Assays were performed as described in A and data represent mean ± SD from three biological repeats. P value is determined by two-way ANOVA with Tukey multiple comparisons test. ns, no significance.

Close modal

Ferroptosis is characterized by the accumulation of lipid ROS, so we determined whether RSL3 stimulates ROS accumulation by FACS analysis. We found that RSL3 treatment induces accumulation of cytosol ROS (DCFH-DA staining) and lipid ROS (C11-BODIPY staining) in MIB1 overexpression cells while mitochondrial ROS (MitoSox staining) is not induced in the same assay (Fig. 3C and D). Thus, MIB1 overexpression appears to disrupt the homeostasis of lipid ROS but not mitochondrial ROS. Because MIB1 can stimulate EMT in a Notch-dependent manner and EMT is associated with ferroptosis sensitivity, we tested whether Notch activation is required for the MIB1-stimulated ferroptosis. We found that shNOTCH knockdown of NOTCHs or pretreatment of cells with LY411575 does not change ferroptosis sensitivity in A549 or A549-MIB1 cells (Fig. 3E), indicating MIB1-stimulated ferroptosis is Notch independent.

MIB1 knockout induces EMT and reduces ferroptosis sensitivity in MDA-MB-231 cells

To further investigate the role of MIB1 in EMT and ferroptosis, we performed loss-of-function studies. The strategy of CRISPR/Cas9-mediated MIB1 knockout is shown in Fig. 4A and we choose the breast cancer cell line MDA-MB-231 for this experiment because MIB1 expression is readily detectable by Western blot analysis in this cell line (Fig. 4B). We obtained two mutant MDA-MB-231 cell lines with both alleles of MIB1 disrupted as determined by DNA sequencing (Fig. 4A) and Western blot analysis (Fig. 4B). We found that MIB1 knockout in MDA-MB-231 induces MET-like changes such as disruption of stress fiber (Fig. 4C), downregulation of mesenchymal genes (Fig. 4D and E) and reduced cell migration (Fig. 4F and G). Together with MIB1 overexpression studies in A549 and lung cancer samples, these data suggest a pro-EMT function of MIB1 in vitro and in vivo.

Figure 4.

MIB1 knockout induces EMT in MDA-MB-231 cells. A, CRISPR/Cas9-mediated MIB1 knockout in MDA-MB-231 cells. A sequence in the ANK repeats of MIB1 was targeted by sgRNA and two independent mutant cell lines with both alleles of MIB1 disrupted were obtained and confirmed by DNA sequencing. Both of them are nonsense mutations. B, Western blot analysis confirmed the loss of MIB1 protein in mutant cells. C, IF staining of F-actin stress fibers in WT and MIB1 knockout cells. Scale bar: 20 μm. D, Western blot analysis for SNAIL1 and VIM in WT and MIB1 knockout cells. E, qRT-PCR analysis for the expression of several mesenchymal transcription factors. Data represent mean ± SD from three independent repeats and P value is determined by ordinary one-way ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. F, Transwell assay for the migration activity of WT and MIB1 knockout cells. Scale bar is 200 μm. G, Statistical results for F. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Dunnett multiple comparisons test. *, P < 0.05.

Figure 4.

MIB1 knockout induces EMT in MDA-MB-231 cells. A, CRISPR/Cas9-mediated MIB1 knockout in MDA-MB-231 cells. A sequence in the ANK repeats of MIB1 was targeted by sgRNA and two independent mutant cell lines with both alleles of MIB1 disrupted were obtained and confirmed by DNA sequencing. Both of them are nonsense mutations. B, Western blot analysis confirmed the loss of MIB1 protein in mutant cells. C, IF staining of F-actin stress fibers in WT and MIB1 knockout cells. Scale bar: 20 μm. D, Western blot analysis for SNAIL1 and VIM in WT and MIB1 knockout cells. E, qRT-PCR analysis for the expression of several mesenchymal transcription factors. Data represent mean ± SD from three independent repeats and P value is determined by ordinary one-way ANOVA with Dunnett multiple comparisons test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. F, Transwell assay for the migration activity of WT and MIB1 knockout cells. Scale bar is 200 μm. G, Statistical results for F. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Dunnett multiple comparisons test. *, P < 0.05.

Close modal

We then tested the sensitivity of MIB1 knockout MDA-MB-231 cells to various cell death inducers. We found MIB1 knockout does not change the sensitivity of MDA-MB-231 cell to Staurosporine, doxorubicin or taxol; however, MIB1 knockout cells are more resistant to RSL3 (IC50 = 0.3 μmol/L for WT, 4.1 μmol/L for MU1 and 1.4 μmol/L for MU2; Fig. 5A). We then analyzed changes of ROS level in these cells upon RSL3 treatment and found that 0.5 μmol/L RSL3 is sufficient to induce accumulation of cytosol and lipid ROS in WT cells but not MIB1 knockout cells (Fig. 5B and C), which is consistent with reduced ferroptosis sensitivity in these cells. To further prove the ferroptosis phenotype in mutant cells is due to loss of MIB1 function, we performed rescue experiments and found that ectopic expression of MIB1 can restore ferroptosis sensitivity in both MU1 and MU2 cells (Fig. 5D). Together, these data demonstrated that MIB1 knockout induces MET and reduces ferroptosis sensitivity in MDA-MB-231. Together with our MIB1 overexpression studies in A549, we concluded that MIB1 is a positive regulator of EMT and ferroptosis.

Figure 5.

MIB1 knockout reduces ferroptosis sensitivity in MDA-MB-231 cells. A, CCK8 cell viability assay for WT and mutant cells treated with the indicated small chemicals. Assays were performed as described in Fig. 3A. Data represent mean ± SD from three biological repeats and P value is determined by two-way ANOVA with Tukey multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. B, FACS analysis for various intracellular ROS species in WT and knockout cells treated with RSL3 (0.5 μmol/L). Assays were performed as in Fig. 3C. C, Statistical results for B. Data represent mean ± SD from three repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. ns, no significance; ***, P < 0.001; ****, P < 0.0001. D, Overexpression of MIB1 in mutant cells restores ferroptosis sensitivity. Data represent mean ± SD from three repeats and P value is determined by two-way ANOVA with Tukey multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

MIB1 knockout reduces ferroptosis sensitivity in MDA-MB-231 cells. A, CCK8 cell viability assay for WT and mutant cells treated with the indicated small chemicals. Assays were performed as described in Fig. 3A. Data represent mean ± SD from three biological repeats and P value is determined by two-way ANOVA with Tukey multiple comparisons test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. B, FACS analysis for various intracellular ROS species in WT and knockout cells treated with RSL3 (0.5 μmol/L). Assays were performed as in Fig. 3C. C, Statistical results for B. Data represent mean ± SD from three repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. ns, no significance; ***, P < 0.001; ****, P < 0.0001. D, Overexpression of MIB1 in mutant cells restores ferroptosis sensitivity. Data represent mean ± SD from three repeats and P value is determined by two-way ANOVA with Tukey multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

MIB1 downregulates the protein level of NRF2

MIB1 stimulates EMT and ferroptosis; however, MIB1-induced EMT is Notch-dependent while MIB1-induced ferroptosis appears Notch independent (Fig. 3E). These observations suggest MIB1 might regulate ferroptosis through other pathways. MIB1 is an E3 ubiquitin ligase that are reported to induce the ubiquitination and degradation of diverse intracellular proteins, so we hypothesized that MIB1 regulates ferroptosis by disrupting the homeostasis of key regulators involved in ferroptosis pathway. To test this hypothesis, we analyzed protein levels of critical regulators of ferroptosis by Western blot analysis. We found that overexpression of MIB1 in A549 does not change homeostasis of GPX4 (the central ferroptosis regulator), SLC7A11 (cysteine transporter), TFR (transferrin receptor), FTH1 (intracellular iron storage protein), or ACSL4 (PUFA synthesis). On the other hand, the master antioxidant transcription factor NRF2 and its target HMOX1 are clearly downregulated in MIB1 overexpression cells (Fig. 6A). Consistent with this result, we found that MIB1 knockout in MDA-MB-231 results in accumulation of NRF2 and HMOX1 proteins but not GPX4, SLC7A11, TFR, FTH1, or ACSL4 (Fig. 6B). We then determined whether the mRNA levels of NRF2 and HMOX1 are affected by MIB1 by qRT-PCR and found that mRNA level of NRF2 is not affected by MIB1 overexpression or knockout, while HMOX-1 mRNA is downregulated upon MIB1 overexpression in A549 and upregulated upon MIB1 knockout in MDA-MB-231 (Fig. 6C and D). These results indicate that overexpression of MIB1 disrupts cellular antioxidant activity by downregulating NRF2 and its target genes and sensitizes cells to ROS accumulation–induced ferroptosis. On the other hand, MIB1 deficiency stabilizes NRF2 which leads to ferroptosis resistance.

Figure 6.

MIB1 regulates homeostasis of NRF2. A, Western blot analysis for key ferroptosis regulators in A549. B, Similar Western blot analysis in MDA-MB-231 cells. Assays in A and B were repeated twice and similar results were observed. MIB1 overexpression reduces NRF2 and HMOX1 protein levels while MIB1 knockout has the opposite effect. GPX4, SLC7A11, TFR, FTH1, and ACSL4 proteins are less affected by MIB1. C, qRT-PCR analysis for NRF2 or HMOX-1 mRNA levels in A549 and A549-MIB1 cells. GAPDH is the internal control. Data represent mean ± SD from three biological repeats and P value is determined by unpaired t test. ns: no significance; **, P < 0.01. D, qRT-PCR analysis for NRF2 or HMOX-1 in WT and mutant MDA-MB-231 cells. Data represent mean ±SD from three biological repeats and P value is determined by ordinary one-way ANOVA with Dunnett multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01.

Figure 6.

MIB1 regulates homeostasis of NRF2. A, Western blot analysis for key ferroptosis regulators in A549. B, Similar Western blot analysis in MDA-MB-231 cells. Assays in A and B were repeated twice and similar results were observed. MIB1 overexpression reduces NRF2 and HMOX1 protein levels while MIB1 knockout has the opposite effect. GPX4, SLC7A11, TFR, FTH1, and ACSL4 proteins are less affected by MIB1. C, qRT-PCR analysis for NRF2 or HMOX-1 mRNA levels in A549 and A549-MIB1 cells. GAPDH is the internal control. Data represent mean ± SD from three biological repeats and P value is determined by unpaired t test. ns: no significance; **, P < 0.01. D, qRT-PCR analysis for NRF2 or HMOX-1 in WT and mutant MDA-MB-231 cells. Data represent mean ±SD from three biological repeats and P value is determined by ordinary one-way ANOVA with Dunnett multiple comparisons test. ns, no significance; *, P < 0.05; **, P < 0.01.

Close modal

MIB1 induces ubiquitination and proteasome degradation of NRF2

Intracellular NRF2 protein is under strict posttranslational regulation, usually via the KEAP1-CUL3-RBX– mediated ubiquitination and proteasome degradation pathway. Our observation that MIB1 regulates NRF2 protein level but not mRNA level raises the possibility that MIB1 functions as additional E3 ubiquitin ligase that regulates ubiquitination and degradation of NRF2. To test this hypothesis, we cotransfected HA-tagged hMIB1, Flag-tagged NRF2, and His-tagged ubiquitin into 293T cells and determined the ubiquitination level of NRF2 after immunoprecipitation. We found that cotransfection of WT hMIB1 clearly promotes the ubiquitination of NRF2 while catalytically inactive mutants of hMIB1 (dR and dC, depicted in Supplementary Fig. S4A) are not able to stimulate the ubiquitination of NRF2 (Fig. 7A), indicating the E3 ubiquitin ligase activity is essential for the regulation. We next determined the domains in NRF2 involved in the MIB1-induced ubiquitination of NRF2. We constructed mutant versions of NRF2 with the NEH2, NEH4–5, or NEH6 domain removed (Supplementary Fig. S4B) and tested whether they can be ubiquitinated by MIB1 or not. We found that deletion of NEH2 domain reduces NRF2 ubiquitination by MIB1 while removal of NEH4–5 or NEH6 domain has no effect on MIB1-mediated ubiquitination of NRF2 (Fig. 7B). These data indicate that, like the KEAP1-CUL3-RBX1complex (9), MIB1 ubiquitinates the NEH2 domain of NRF2. We also examined NRF2 ubiquitination levels in WT and MIB1 knockout MDA-MB-231 cells and found that ubiquitinated NRF2 is clearly reduced in knockout cells (Supplementary Fig. S5), further supporting a role of MIB1 in stimulating NRF2 ubiquitination.

Figure 7.

MIB1 promotes ubiquitination and proteasome degradation of NRF2. A, Cotransfection of WT but not catalytically inactive forms of MIB1 promotes NRF2 ubiquitination in 293T cells. The dR and dC mutants of MIB1 are described in Supplementary Fig. S4A. Assays were repeated twice and similar results were obtained. B, Deletion of the NEH2 but not NEH4-5 or NEH6 domain abolishes the MIB1-induced ubiquitination of NRF2. The NRF2 mutants used in these experiments are described in Supplementary Fig. S4B. C, NRF2 is degraded via the proteasome pathway. Treatments of cells with the proteasome inhibitor MG132 induce accumulation of NRF2 in A549 and A549-MIB1; furthermore, the MIB1-stimulated degradation of NRF2 in A549-MIB1 is also rescued by MG132. The lysosome inhibitor bafilomycin A1 (Baf A1) has no effect on NRF2 homeostasis. D, Statistical result of C. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Tukey multiple comparisons test. ns, no significance; ***, P < 0.001; ****, P < 0.0001. E, MIB1 promotes NRF2 degradation. A549 and A549-MIB1 cells were pretreated with MG132 to increase the basal protein level of NRF2 then cells were treated with CHX (with MG132 removed) to block de novo NRF2 synthesis. NRF2 protein levels at various timepoints after CHX treatment were determined by Western blot analysis. Overexpression of MIB1 stimulates NRF2 degradation. F, Statistical result of E. Data represent mean ± SD from three biological repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. **, P < 0.01; ***, P < 0.001.

Figure 7.

MIB1 promotes ubiquitination and proteasome degradation of NRF2. A, Cotransfection of WT but not catalytically inactive forms of MIB1 promotes NRF2 ubiquitination in 293T cells. The dR and dC mutants of MIB1 are described in Supplementary Fig. S4A. Assays were repeated twice and similar results were obtained. B, Deletion of the NEH2 but not NEH4-5 or NEH6 domain abolishes the MIB1-induced ubiquitination of NRF2. The NRF2 mutants used in these experiments are described in Supplementary Fig. S4B. C, NRF2 is degraded via the proteasome pathway. Treatments of cells with the proteasome inhibitor MG132 induce accumulation of NRF2 in A549 and A549-MIB1; furthermore, the MIB1-stimulated degradation of NRF2 in A549-MIB1 is also rescued by MG132. The lysosome inhibitor bafilomycin A1 (Baf A1) has no effect on NRF2 homeostasis. D, Statistical result of C. Data represent mean ± SD from three biological repeats and P value is calculated using ordinary one-way ANOVA with Tukey multiple comparisons test. ns, no significance; ***, P < 0.001; ****, P < 0.0001. E, MIB1 promotes NRF2 degradation. A549 and A549-MIB1 cells were pretreated with MG132 to increase the basal protein level of NRF2 then cells were treated with CHX (with MG132 removed) to block de novo NRF2 synthesis. NRF2 protein levels at various timepoints after CHX treatment were determined by Western blot analysis. Overexpression of MIB1 stimulates NRF2 degradation. F, Statistical result of E. Data represent mean ± SD from three biological repeats and P value is determined by two-way ANOVA with Sidak multiple comparisons test. **, P < 0.01; ***, P < 0.001.

Close modal

Ubiquitinated proteins can be degraded by proteasome or lysosome degradation pathway, so we blocked either of these pathways and determined its effect on NRF2 homeostasis. We found that only the proteasome pathway inhibitor MG132 rescues the MIB1-induced degradation of NRF2 while the lysosome pathway inhibitor bafilomycin A1 (Baf A1) fails to do so (Fig. 7C and D). We then compared the degradation curves of NRF2 between WT and MIB1 overexpression A549 cells. Cells were pretreated with MG132 for 30 minutes to increase the basal level of NRF2, then treated with CHX to block new protein synthesis and NRF2 protein levels at various timepoints after CHX treatment were determined by Western blot analysis. We found that, consistent with its elevated ubiquitination level, NRF2 in MIB1 overexpression cells is degraded significantly faster (IC50≈0.3 hours) than that in WT A549 cells (IC50≈1.2 hours; Fig. 7E and F). Together, these results suggest that MIB1 induces the ubiquitination of NRF2 at NEH2 domain and the ubiquitinated NRF2 is degraded via the proteasome pathway.

MIB1 is an E3 ubiquitin ligase that is essential for efficient Notch signaling and many Notch-related developmental abnormalities are observed in MIB1-deficient animals. Notch signaling is also actively involved in multiple steps of tumorigenesis such as EMT, cancer stem cell formation, metastasis, drug resistance, etc. However, potential roles of MIB1 in tumorigenesis have not been directly investigated. In this study, we demonstrate that MIB1 expression is elevated in a subset of lung squamous carcinoma and adenocarcinoma samples and high MIB1 expression level correlates with an EMT signature and impaired patient survival, indicating an oncogenic function of MIB1. We further reveal that ectopic expression of MIB1 promotes EMT and migration of lung cancer cell line A549 in a Notch-dependent manner which could be the underlying mechanism for the oncogenic function of MIB1. Blocking MIB1 function or MIB1-induced Notch signaling could be a therapeutic strategy in MIB1 overexpression tumors; however, no FDA-approved therapeutic reagents that target MIB1 or Notch pathway are currently available. We report here that MIB1 overexpression reduces the antioxidant capacity of a cell by targeted degradation of the master antioxidant transcription factor NRF2 and MIB1 overexpression cells are much more sensitive to ferroptosis inducers. Because several FDA-approved drugs including salazosulfapyridine, sorafenib, statins, artemisinin and its derivatives are able to induce ferroptosis in various cell lines (31), it will be interesting to test whether these drugs have clinical value in treating MIB1 overexpression tumors.

Several studies reported that the epithelial/mesenchymal state of a cell is associated with ferroptosis sensitivity. On study revealed that TGFβ/ZEB1 signaling, a classic EMT inducer, promotes the synthesis of PUFAs and sensitizes cells to ferroptosis (14). Another recent study discovered that E-Cadherin mediated cell–cell interaction in epithelial cells is able to suppress the expression of ferroptosis modulators such as ACSL4 and TFRC and inhibition of this cell–cell interaction sensitizes cancer cells to ferroptosis (32). In both cases, ferroptosis sensitivity is a downstream event of EMT. In this study, we found that MIB1 induces EMT and promotes ferroptosis. However, the mechanism of MIB1-induced ferroptosis appears different from those induced by TGFβ/ZEB1 signaling or E-Cadherin signaling. We show that MIB1 does not disrupt the homeostasis of ACSL4 and TFRC, indicating the generation of lipid ROS is unlikely to be affected by MIB1. On the other hand, MIB1 overexpression cells are defective in antioxidant defense as indicated by the downregulation of NRF2 and HMOX1 which might compromise the clearance of lipid ROS and sensitizes cells to lipid ROS accumulation induced ferroptosis. Together, these studies indicate that the interconnections between EMT and ferroptosis sensitivity are context dependent and, at least in MIB1 overexpression cells, EMT and ferroptosis programs can be separated as EMT is Notch dependent and ferroptosis is Notch independent. We propose that MIB1 overexpression induces two parallel downstream signaling pathways: the Notch-EMT pathway which promotes tumorigenesis and the NRF2-ferroptosis pathway which may be the Achilles' heel for those MIB1 overexpression tumors. Targeting NRF2-ferroptosis pathway may provide novel opportunity in the treatment of MIB1 overexpression tumors.

J. Xia reports grants from Natural Science Foundation of Guangdong Province Grant outside the submitted work. X. Shu reports grants from Science and Technology Planning Project of Guangdong Province and Guangzhou Regenerative Medicine and Health Guangdong Laboratory Grant during the conduct of the study. No disclosures were reported by the other authors.

H. Wang: Formal analysis, validation, investigation, writing–original draft. Q. Huang: Investigation, methodology. J. Xia: Methodology. S. Cheng: Investigation. D. Pei: Resources. X. Zhang: Resources, methodology. X. Shu: Conceptualization, resources, formal analysis, supervision, funding acquisition, writing–original draft, writing–review and editing.

We thank Guangjin Pan and Baoming Qin (GIBH) for reagents and technical support. This work was partially supported by Science and Technology Planning Project of Guangdong Province (2020B1212060052, to X. Shu), Guangzhou Regenerative Medicine and Health Guangdong Laboratory Grant (2018GZR110104008, to X. Shu), and Natural Science Foundation of Guangdong Province Grant (2016A030313166, to J. Xia).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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