Menin maintains lysosomal and mitochondrial homeostasis through epigenetic mechanisms in lung cancer

Abstract

Lysosome-mediated autophagy (including mitophagy) is crucial for cell survival and homeostasis. Although the mechanisms of lysosome activation during stress are well recognized, the epigenetic regulation of lysosomal gene expression remains largely unexplored. Menin, encoded by the MEN1 gene, is a chromatin-related protein that is widely involved in gene transcription via histone modifications. Here, we report that menin regulates the transcription of specific lysosomal genes, such as CTSB, CTSE, and TFE3, through MLL-mediated H3K4me3 reprogramming, which is necessary for maintaining lysosomal homeostasis. Menin also directly controls the expression of SQSTM1 and MAP1LC3B to maintain autophagic flux in a manner independent of AMPK/mTORC1 pathways. Furthermore, loss of menin led to mitochondrial dysfunction, elevated levels of reactive oxygen species (ROS), and genome instability. In genetically engineered mouse models, Men1 deficiency resulted in severe lysosomal and mitochondrial dysfunction and an impaired self-clearance ability, which further led to metabolite accumulation. SP2509, a histone demethylase inhibitor, effectively reversed the downregulation of lysosomal and mitochondrial genes caused by loss of Men1. Our study confirms the previously unrecognized biological and mechanistic importance of menin-mediated H3K4me3 in maintaining organelle homeostasis.

Fig. 1. Menin maintains lysosomal enzyme expression and function.

A Volcano plot showing 4685 downregulated and 4689 upregulated genes in the MEN1-Low group compared to the MEN1-High group. Some relevant genes were labeled in the plot. The selection criteria were an adjusted P-value < 0.01 and a fold-change <2/3 or >3/2. B Bar chart showing KEGG analysis of the downregulated genes in (A). C Volcano plot showing 1961 downregulated and 1842 upregulated genes in the MEN1-KD A549 cells compared to the control cells. Some relevant genes were labeled in the plot. The selection criteria were an adjusted P-value < 0.01 and a fold-change <2/3 or >3/2. D Bar chart showing KEGG analysis of the downregulated genes in (C). E RT-qPCR was performed to analyze the mRNA levels of indicated genes in A549 and 16HBE cells. All RT-qPCR results were normalized to ACTB mRNA levels. Fold-change values were calculated relative to the first column. The bar graph data represent the mean ± SD of three independent experiments. F Western blotting analysis of protein levels of menin, CTSB-p and CTSE-p in lung cancer tissues isolated from KS and KMS mice. G Transmission electron microscopy (TEM) was used to visualize the lysosomes in A549 cells, and their quantity was plotted as mean ± SD. The scale bar represents 2 μm, and a local area was magnified by 10 times. H Immunofluorescence (IF) staining of the LAMP1 (Red) and CTSB (Green) in A549 cells. DAPI visualizing nuclei. Scale bar, 20 μm. All the fluorescence intensity was quantified using ImageJ software. The bar graph represents the mean ± SEM. I CTSB enzymatic activity was determined by Magic Red staining (Red) in A549 cells. Hoechst visualizing nuclei. Scale bar, 20 μm. BafA1 (20 nM, 12 h) treatment serves as positive control. J Cellular endocytic activity was detected by Texas Red Dextran staining in A549 cells. Hoechst visualizing nuclei. Scale bar, 20 μm. BafA1 (20 nM, 12 h) treatment serves as positive control. K The pH of lysosomes was determined by calculating the fluorescence ratio of LysoSensor staining in A549 cells, and the results were plotted as the mean ± SD. L LysoTracker fluorescence intensity was used to detect intact lysosomes at different time points after withdrawing LLOMe. A549 cells were transfected with siRNA 3 days prior and then treated with 1 mM LLOMe for 2 h to disrupt lysosomal membranes.

Fig. 2. MEN1 deletion inhibits autophagic flux.

A TEM was used to visualize the Avi and Avd in A549 cells. The quantity was plotted as mean ± SEM. The scale bar represents 2 μm, and a local area was magnified by 10 times. B The A549 cells were treated with or without BafA1 (50 nM for 4 h), and IF staining were used to detect the expression of LAMP1 and LC3B. DAPI visualizing nuclei. Scale bar, 20 μm. C Western blotting was performed to assess the expression of indicated proteins under amino acid starvation (AA-star, 2 h) or glucose starvation (glu-star, 4 h) in A549 cells. D GSEA showing correlation between MEN1 expression and the autophagic gene signature in the transcriptomic data of A549 cells. E RT-qPCR was performed to analyze the mRNA levels of indicated genes in A549 and 16HBE cells with MEN1-KD. F Western blotting was performed to assess the impact of MEN1-KD on the expression of indicated proteins in A549 and 16HBE cells. G Western blotting was performed to dynamically assess the impact of MEN1-KD on the expression of indicated proteins in A549 cells after AA-star and BafA1 treatment. H IHC staining was performed for the indicated proteins in the mouse lung tissues from WT (n = 6), MS (n = 6), KS (n = 8), and KMS (n = 8). The scale bar represents 100 μm. The positive area was quantified using ImageJ software and plotted as mean ± SD. I RT-qPCR was performed to analyze the mRNA levels of indicated genes in MEFs isolated from WT, Men1^∆/∆, Kras^G12D/+ and Kras^G12D/+; Men1^∆/∆ mice.

Fig. 3. Menin maintains lysosomal gene expression through H3K4me3.

A RT-qPCR was performed to analyze the mRNA levels of the indicated genes in A549 cells. The cells treated with different doses of MI-3 for 24 h or treated with 1 μM MI-3 for 12-48 h. B RT-qPCR was performed to analyze the mRNA levels of indicated genes in A549 cells with MLL1-KD or MLL4-KD. C RT-qPCR was performed to analyze the mRNA levels of indicated genes in A549 cells with simultaneous KD of MLL1 and MLL4 by siRNA. D Heatmap showing the coverage profiles for H3K4me3 within a 5 kb range of the transcriptional start site (TSS). E Venn diagram showing the overlap between genes with reduced H3K4me3 at TSSs upon MI-3 exposure and genes downregulated by MEN1-KD. F Bar chart showing KEGG analysis of the overlapping genes in (E). G Diagrams presenting the genome browser view of normalized ChIP-seq signals of H3K4me3, menin, and MLL1 for target genes. H, I Diagrams showing the primer pairs (PPs) designed for ChIP targeting the promoter regions of CTSB and SQSTM1. ChIP-qPCR was performed with antibodies of anti-menin, anti-H3K4me3, anti-MLL1 and anti-RNA polymerase II in A549 cells. IgG served as the negative control. J The bar graph representing the fluorescence quantification of Magic Red staining for CTSB enzymatic activity. The A549 cells were treated with indicated doses of MI-3 for 48 h. K The bar graph representing the fluorescence quantification of LysoTracker staining. The A549 cells were treated with siRNAs simultaneously targeting MLL1 and MLL4 for 3 days.

Fig. 4. Menin epigenetically regulates TFE3.

A Western blotting was used to assess the expression of indicated proteins in A549 and 16HBE cells (left). RT-qPCR was employed to measure mRNA levels of the corresponding genes in MEN1-KD A549 cells (right). B The A549 cells were exposed to AA-star for 2 h, and the nuclear and cytoplasmic proteins were extracted. Western blotting was used to examine the localization of TFE3 and TFEB in the nucleus and cytoplasm. H3 was used as the loading control. C Diagrams showing the genome browser view of normalized menin and MLL1 ChIP-seq signals for target genes. D Diagrams showing the PPs designed for ChIP targeting the promoter regions of TFEB and TFE3 genes. E The ChIP-qPCR for TFE3 was performed with antibodies of anti-menin, anti-H3K4me3, anti-MLL1 and anti-RNA polymerase II in control and MEN1-KD A549 cells. IgG served as the negative control. F Western blotting and RT-qPCR were used to examine the TFE3 expression in A549 cells exposure to MI-3 for 3 days. G The A549 cells were transfected with siRNAs simultaneously targeting MLL1 and MLL4. After 3 days, western blotting and RT-qPCR were used to examine the expression of TFE3. H, I The MEN1-KD A549 cells were transfected with either siRNA-TFE3 or full-length TFE3. RT-qPCR was used to measure the mRNA levels of the indicated genes. J The MEN1-KD A549 cells were transfected with full-length TFE3. Western blotting was used to examine the expression of indicated proteins. K The stable MEN1-overexpression A549 cells were transfected with siRNA-TFE3. RT-qPCR was used to measure the mRNA levels of the corresponding genes. L The MEN1-KD A549 cells were transfected with full-length TFE3. Bar graph was generated to quantitatively analyze the fluorescence intensity of LAMP1 and LC3B in IF experiments. Values were presented as mean ± SEM.

Fig. 5. Loss of menin causes mitochondrial dysfunction.

A TEM was employed to visualize the morphology of mitochondria in MEN1-KD A549 cells. The number and diameter of mitochondria were quantified and presented as mean ± SD. The scale bar represents 2 μm, and a specific region was magnified 10 times. B Control and MEN1-KD A549 cells were treated with or without CQ (10 μM, 12 h). JC-1 staining was used to detect membrane potential status of mitochondria. The fluorescence intensity was quantified, and the ratio of green to red was presented as the mean ± SEM. Hoechst visualizing nuclei. C Control and MEN1-KD A549 cells were treated with or without CQ (10 μM, 12 h). RT-qPCR was used to measure the mitochondrial mass by quantifying the expression of mtDNA (ND1 gene) relative to nDNA (HB2M gene). The fold-change values were presented as mean ± SD. D Lung cancer tissues were isolated from KS and KMS mice. RT-qPCR was used to measure the mitochondrial mass by quantifying the expression of mtDNA (Atp6 gene) relative to nDNA (Tert gene). E Control and MEN1-KD A549 cells were treated with or without BafA1 (20 nM, 12 h). The Seahorse Mito Stress Test was used to detect oxygen consumption rate in the indicated cells with the indicated inhibitors. F RT-qPCR was used to measure the mRNA levels of indicated genes in control and MEN1-KD A549 cells. G Diagrams showing the genome browser view of ChIP-seq signals of normalized H3K4me3, menin, and MLL1 for TFAM. H Diagram showing the PPs designed for ChIP targeting the promoter region of the TFAM gene. ChIP-qPCR was performed with antibodies of anti-menin, anti-H3K4me3, anti-MLL1 and anti-RNA polymerase II in control and MEN1-KD A549 cells. I RT-qPCR was used to measure the mRNA levels of Tfam in MEFs isolated from WT and Men1^Δ/Δ mice. J The indicated MEFs were treated with or without CQ (10 μM, 12 h). JC-1 staining was used to detect membrane potential status of mitochondria. K, L The MEN1-KD A549 cells were exposed to 20 μM CCCP (DMSO as control) for 4 h. IF staining of TOMM20 (Green) and LC3B (Red) in A549 cells. DAPI visualizing nuclei. Scale bar, 20 μm. All the co-localized fluorescence puncta of TOMM20 and LC3B were quantified using ImageJ software. The bar graph data represent the mean ± SEM. M Western blotting was performed to dynamically assess the impact of MEN1-KD on the expression of indicated proteins in A549 after CCCP (20 μM) exposure.

Fig. 6. Loss of menin causes metabolite accumulation in lung cancer.

A TEM was employed to visualize the electron-dense particles in control and MEN1-KD A549 cells. The number of the electron-dense particles was quantified and presented as mean ± SD. The scale bar represents 1 μm, and a specific region was magnified 10 times. B PAS staining was performed to detect glycogen in control and MEN1-KD A549 cells. The staining was quantified and presented as mean ± SD. C PAS staining was performed to detect glycogen in mouse lung tissues from WT (n = 6), MS (n = 6), KS (n = 8), and KMS (n = 8) at 2 months after TAM injection. The staining was quantified and presented as mean ± SD. D The whole, soluble, and insoluble proteins in A549 cells were determined by using the BCA kit. E Control and MEN1-KD A549 cells were treated with or without CQ (10 nM, 12 h). Flow cytometry was utilized to assess cellular ROS levels using the DCFH-DA probe (left panel). The cells without probe loading served as the negative control. The proportion of positive cells was presented as the mean ± SD (right panel). F Control and MEN1-KD A549 cells were treated with or without BafA1 (20 nM, 12 h). IF was performed to detect γH2A.x in indicated cells. The bar graph represents the mean ± SEM. G IF was performed to detect γH2A.x in A549 cells exposed to MI-3 (3 days). The bar graph represents the mean ± SEM. H The Kaplan-Meier survival curves, and representative dissection images of WT (n = 20) and Men1^∆/∆ (n = 30) mice. I The quantification of IHC staining of indicated proteins in (J). The positive area was quantified and plotted as mean ± SD. J IHC staining was performed to detect the indicated proteins in the lung tissues from WT (n = 9) and Men1^∆/∆ (n = 9) mice. Scale bars, 50 μm. K–M IHC staining was performed to detect the indicated proteins in the lung tissues from KMS mice with (n = 14) or without (n = 8) SP2509-treatment. The scale bar represents 50 μm. The positive area was quantified and plotted as mean ± SD.

Fig. 7

A model of the cellular mechanisms by which menin regulates organelle homeostasis.