Fig. 7

RBM4 endows ESCC metabolic vulnerability. a The tissue microarray of ESCC patients was subjected to IHC staining for the specific RBM4 and LKB1 antibodies. Representative images were acquired with ×10 and ×40 objectives (scale bar = 50 μm). The results were analyzed by the pathologist who provided a value ranging from 0 to 3 for each sample. “0” indicates negative staining; “1” means weak positive staining; “2” denotes moderately positive staining and “3” indicates strongly positive staining. H-score is obtained by the formula: 3 × percentage of strongly staining + 2 × percentage of moderately staining + percentage of weakly staining. The correlation between RBM4 and LKB1 expression in ESCC and the corresponding normal tissues was calculated by Pearson’s chi-square test. b Correlation of RBM4 and LKB1 levels were analyzed using data obtained from mass spectrometry-based proteomic profiling of ESCC tumors and adjacent non-tumor tissues (PXD021701). (P = 0.012 by Pearson’s chi-square test). c Kaplan–Meier curve showing overall survival of ESCC patients (PXD021701) with high RBM4 expression and low LKB1 expression or low RBM4 expression and high LKB1 expression (P = 0.0086 by log-rank test, dotted red/blue indicates the upper and lower confidence limits.). d Protein levels of LKB1 and RBM4 were determined in RBM4-depleted or overexpression ESCC cells, including KYSE30, KYSE70, KYSE150, KYSE450, and KYSE510 cells. e Protein levels of LKB1 and RBM4 in fresh-frozen tumor and the corresponding normal tissue specimens from ESCC patients were examined by western blot assay. “N” stands for the adjacent non-tumor tissues, “T” represents ESCC tissues. SE, short exposure; LE, long exposure. f Dose-response curves of cell viability in normal esophageal epithelial cells (NE2 and NE3) and distinct ESCC cells (KYSE30, KYSE70, KYSE150, and KYSE450) treated with CB-839 (n = 3; mean of three technical replicates from a representative experiment was shown in the graph) for 72 h. g Dose-response curves of cell viability in RBM4-overexpression cells (KYSE70, KYSE150, and A549) treated with CB-839 (n = 3; mean of three technical replicates from a representative experiment was shown in the graph) for 72 h. h–j Nude mice were subcutaneously injected with 5 × 10⁶ KYSE30 cells with stable overexpression of RBM4 or control respectively. When the tumor reached 50–250 mm³, the animals were randomized to the respective treatment groups. CB-839 (200 mpk) was administered by oral gavage twice a day, tumor volumes were measured every 3 days (h). After 21 days of treatment, the mice were sacrificed to remove the xenografted tumors as shown in (i), and the tumors weights were measured (j). n = 7, error bars indicate mean ± SEM. P-values were determined by two-way repeated measures ANOVA in H or one-way ANOVA with Dunnett multiple comparison in J. *P < 0.05, ****P < 0.0001. k, l After 21 days of oral administration of CB-839 or vehicle (0.5% CMC-Na), the nude mice bearing RBM4-overexpressing or empty vector-expressing xenografts were sacrificed to remove tumors for immunohistochemistry analysis of the proliferation marker Ki67, LKB1 and phosphorylation of AMPK (k). Scale bar = 50 μm. Quantification of Ki67 positive cells after IHC analysis was performed using image J software and plotted as mean ± SD with P-value in ANOVA test (l). ****P < 0.0001. m 21-days after oral administration of CB-839 or vehicle (0.5% CMC-Na), the nude mice bearing RBM4-overexpressing or empty vector xenografts were sacrificed to remove tumors for western blot and gray value analysis of RBM4, LKB1 and phosphorylation of AMPK. The correlation between the levels of RBM4 and LKB1/p-AMPK was calculated by Pearson correlation coefficient calculation and plotted. Gray circle represents RBM4-overexpressed samples, red/orange circle represents control vector samples. R, Pearson correlation coefficient; P-value, two-tailed P-value of the Pearson correlation