Fig 2: Identification of MEN1-modulated genes in breast cancer cells. (A) RT-qPCR of MEN1 in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus (n = 3). (B) Quantitative Western immunoassays (WES) of menin expression in T47D or MCF-7 cells treated with vehicle or MEN1 shRNA lentivirus (n = 3). (C) Venn diagrams of differentially expressed genes (fold change =1.5 or =0.66) in T47D or MCF-7 cells after shMEN1 knockdown compared with vehicle controls (n = 2). (D) Pathway annotation analysis of MEN1-upregulated and MEN1-downregulated genes in T47D or MCF-7 cells using DAVID including cancer hallmark pathways. (E) Schematic illustration of five major metabolic pathways. (F) Expression heat maps of oxidative phosphorylation (OXPHOS) and glycolytic genes in both MEN1 knockdown T47D and MCF-7 cells (fold changes relative to vehicle controls). (G) Bar charts of the expression levels of representative OXPHOS and glycolytic genes affected by MEN1 knockdown in T47D or MCF-7 cells using RT-qPCR. Data are presented as mean ± S.D. Unpaired two-tailed Student’s t-test was used for statistics. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig 3: Identification of menin-associated proteins (MAPs) in breast cancer cells. (A) and (B) WES of BirA-Menin fusion proteins (A) and biotin-labeled proteins (B) in total lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after incubating with or without doxycycline and biotin. (C) Schematic purification and proteomic identification of MAPs using LC–MS/MS. (D) Heatmap of the quantification of 35 MAPs commonly shared in T47D and MCF-7 cells. MAPs further verified by WES immunoassays were indicated by arrows. (E) Network analysis of 35 MAPs in MCF-7 cells. The distance between menin and MAPs represented the quantitative ratio of each MAP and menin. MAPs marked in blue were further assayed by WES. (F) Nuclear or cytoplasmic lysates of BirA-MEN1 BioID engineered T47D or MCF-7 cells after streptavidin beads pull-down were detected by WES with antibodies against menin, KMT2A, MED12, WAPL, GATA3, LaminA/C, or GAPDH. FL, full length; SP, spliced form.

Fig 4: MEN1 and OXPHOS expression are increased in breast circulating tumor cells (CTCs). (A) t-SNE profile plots and cell clustering of 93 CTCs from 5 breast cancer patients based on the single cell RT-qPCR expression profiling of 11 OXPHOS genes (NDUFA7, NDUFA11, NDUFA13, NDUFB7, NDUFS7, NDUFS8, NDUFV1, SDHA, SDHB, SDHC, and SDHD). (B) Violin plots of MEN1 or selected MAPs expression, mean expression of 7 glycolytic genes (ALDOA, ALDOC, ENO1, PFKL, PFKP, PGK1, and TPI1) or mean expression of 11 OXPHOS genes (aforementioned) in the five cell clusters. Statistical significance among clusters was carried out using the Duncan multi-range test. (C) Mean expression of 7 glycolytic genes and 11 OXPHOS genes in these 93 breast CTCs or in the TCGA primary breast cancer cohort. (D) Glycolytic and OXPHOS ATP productions of T47D or MCF-7 cells after circulation (n = 6–10 technical replicates). (E,F) Mitochondrial (E) and glycolytic (F) functions of T47D or MCF-7 cells after circulation (n = 5–9 technical-replicate wells). Statistics represented the difference between no circulating control and each treatment. Data are presented as mean ± S.D. An unpaired two-tailed Student’s t-test was used for statistical significance determination. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Fig 5: Expression correlation relationship of menin/MAPs genes and OXPHOS/glycolytic genes. (A) Workflow of the in silico correlation analysis of gene expression in The Cancer Genome Atlas (TCGA) breast cancer cohort. (B) Heatmaps of the expression correlation between MEN1/selected 4 MAP genes and OXPHOS genes (upper) or glycolytic genes (lower) in normal (N) and tumor (T) samples. The genes are arranged from the highest to the lowest according to gene expression correlation coefficients of MEN1–OXPHOS genes or MEN1–glycolytic genes in breast tumors. (C) and (D) Scatter plots and linear regression analyses of MEN1/selected MAPs expression and mean expression of OXPHOS genes (C) or glycolytic genes (D) in normal and tumor samples. (E) Violin plots (lower panel) shows the average expressions of the genes of OXPHOS complexes I-V and glycolysis in the samples of each of the corresponding 4 groups are shown as violin plots. Based on the median values (where =median is “high” and KMT2A, MEN12, WAPL, and GATA3) and MEN1, the TCGA breast tumor samples were divided into 4 groups—1: high-low, 2: high-high, 3: low-low, and 4: low-high (upper panel). Letters on top of the violin plot denote statistical significance, where two groups with different letters are significantly different (p < 0.05) and those with the same letter are not.