Fig 1: ENO2 K394 acetylation represses glycolysis and invasion of PDAC cells in vitro and in vivo. a WT ENO2 and its K394R and K394Q mutants were reintroduced into ENO2 knockdown Colo357 cells. The extracellular acidification rate (ECAR) of the indicated cells was detected by using a Seahorse XF96 Extracellular Flux Analyzer, and the maximum glycolytic rates were summarized, n = 9 per group. b Lactate secretion in the culture medium of the indicated cells for 12 h; n = 4 per group. c, d Colony assays (c) and transwell assays (d) were performed with the indicated Colo357 cells; n = 4 per group (scale bar = 100 μm). e, f Colo357 cells with ENO2 knockdown and Colo357 cells with ENO2 knockdown plus reintroduced with wild-type ENO2 or K394Q mutants were injected into the flanks of nude mice (n = 5 for each group). The tumor volumes (e, left), tumor weight (e, right) and tumor growth curves (f) of subcutaneous implantation models of PDAC at day 32 are shown. g, h The effect of ENO2 K394 acetylation on metastasis of PDAC in metastatic mouse models (n = 5 per group). g Livers were harvested after mice died, and metastatic nodules were counted by H&E staining (See Supplementary Fig. S2e). The total numbers of liver metastatic lesions were summarized. h Kaplan–Meier survival curve of mice after intrasplenic injection of Colo357 cells with ENO2 knockdown and Colo357 cells with ENO2 knockdown plus reintroduced with wild-type ENO2 or K394Q mutants. i, j The K394 acetylation levels of ENO2 in PDAC samples. Twelve pairs of tumor (T) and adjacent normal tissues (N) (n = 12 per group) (i) and eight tumor samples with or without liver metastasis (j) were subjected to IP with ENO2 antibody and western blot to detect K394 acetylation of ENO2. MFPs, metastasis-free patients (n = 4); LMPs, liver metastasis patients (n = 4). Error bars represent the mean ± SD, and the dots represent the value of each experiment; **P < 0.01, ***P < 0.001, ns: no significance. An unpaired t test was employed in (a–d, j), one-way ANOVA followed by Bonferroni’s post hoc test was employed in (e) and (g), two-way ANOVA followed by Bonferroni’s post hoc test was employed in (f), the log-rank test was employed in (h), and a paired t test was employed in (i)
Fig 2: Inhibition of IGF-1R restrains the K394 deacetylation and enzymatic activation of ENO2-induced by IGF-1. a Flag-tagged ENO2 was stably overexpressed in Colo357 cells, followed by separate incubation with insulin (100 ng/mL, 12 h), IGF-1 (100 ng/mL, 12 h), EGF (30 ng/mL, 24 h), TGFβ (10 ng/mL, 72 h), CSF1 (10 ng/mL, 30 min) or HGF (50 ng/mL, 30 min) stimulation. ENO2 protein was immunopurified with Flag beads, and ENO2 K394 acetylation was analyzed by western blot with anti-AcK394 antibody. b IGF-1 induces ENO2 K394 deacetylation. Flag-tagged ENO2 was stably overexpressed in Colo357 cells treated with 0, 5, 10, or 100 ng/mL IGF-1 for 2 h (left) or 10 ng/mL IGF-1 for 0, 0.5, 1, 2, or 4 h (right). ENO2 was immunopurified with Flag beads, and K394 acetylation levels were determined by western blot and normalized against Flag. The protein levels of HDAC3 and HDAC3 S424 phosphorylation were detected. c HDAC3 knockdown inhibits the ENO2 K394 deacetylation induced by IGF-1. HDAC3 was stably knocked down in Colo357 cells following treatment with 0 or 10 ng/mL IGF-1 for 2 h. Immunoprecipitation and western blot were performed to validate the knockdown of HDAC3 and to detect the change in K394 acetylation levels. K394 acetylation levels were normalized against ENO2. d IGF-1 activates the PI3K/AKT/mTOR and ERK pathways. Colo357 cells treated with 0, 2.5, 5, or 10 ng/mL IGF-1 for 2 h (left) or treated with 10 ng/mL IGF-1 for 0, 0.5, 1, 2, or 4 h (right) were then harvested and subjected to western blot to evaluate the activation of the PI3K/AKT/mTOR and ERK pathways with the indicated antibodies. e Inhibition of IGF-1R increased ENO2 K394 acetylation. Colo357 cells stably overexpressing Flag-tagged ENO2 were treated with 0, 5, or 10 μM linsitinib for 6 h. The phosphorylation of IGF-1R and ENO2 K394 acetylation were assessed by IP and western blot using the indicated antibodies. f Linsitinib restored the K394 acetylation level in cells with wild-type ENO2 reintroduction but not in K394Q-mutant cells. Colo357 cells stably overexpressing Flag-tagged ENO2 were treated with IGF-1 (10 ng/mL, 2 h) stimulation, linsitinib (10 μM, 6 h) plus IGF-1 (10 ng/mL, 2 h) stimulation or IGF-1R knockdown plus IGF-1 (10 ng/mL, 2 h) stimulation. ENO2 proteins were immunoprecipitated, and K394 acetylation was examined with anti-AcK394 and normalized against Flag. g Inhibition of IGF-1R depressed the ENO2 K394 deacetylation and the activation of the PI3K/AKT/mTOR and ERK pathways induced by IGF-1. Colo357 cells treated as indicated in Fig. 5g were subjected to immunoprecipitation and western blot with the indicated antibodies to elucidate K394 acetylation levels (normalizing against Flag), the phosphorylation levels of HDAC3 S424 and the activity of the PI3K/AKT/mTOR and ERK pathways. h–i IGF-1R inhibition suppressed the enzymatic activity of ENO2 and invasion of tumor cells induced by IGF-1. Enzymatic activity assays (h, n = 3 per group) and transwell assays (i, n = 4 per group) were performed with the Colo357 cells indicated in Fig. 5g (scale bar = 100 μm). Error bars represent the mean ± SD, and the dots represent the value of each experiment; **P < 0.01, ***P < 0.001. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post hoc test
Fig 3: Expression levels of C-kit, NSE, and PGP9.5 in the mid colon of C3 KO mice. (a) Expression levels of C-kit, NSE, and PGP9.5 proteins. The expression level of the three proteins was determined by Western blot analysis using the specific primary antibody and HRP-labeled anti-rabbit IgG antibody. The band intensities were determined using an imaging densitometer, and protein expressions were calculated relative to the intensity of β-actin. Four mice per group were used to prepare the tissue lysates, and Western blots were assayed in duplicate for each sample. Data are reported as the mean ± SD. * p < 0.05 compared with the WT group. (b) Tissue distribution of PGP9.5 proteins. This level was detected in the mid colon using an immunofluorescence (IF) staining assay. Three mice per group were used in the slide section, and IF staining was assessed in duplicate in two different slides. Arrowheads indicate PGP9.5-stained subpopulations. Abbreviations: WT, wildtype; KO, knockout; C-kit, receptor tyrosine kinase; NSE, neuron-specific enolase; PGP9.5, protein gene product 9.5; HRP, horseradish peroxidase; IgG, immunoglobulin G.
Fig 4: Expression levels of NSE and GFAP in the hippocampal tissue. (A) NSE expression 1, 3 and 7 days after different treatments. KA administration decreased the NSE expression (D1 and D3), whilst BHB alleviated this reduction (D1). (B) GFAP expression 1, 3 and 7 days after different treatments. KA administration increased the GFAP expression but pretreatment with BHB significantly reduced GFAP expression (D3 and D7). aP<0.05 vs. NS; bP<0.05 vs. NS+KA; cP<0.05 vs. BHB+KA; #P<0.05 vs. D1 NS + KA; *P<0.05 vs. D3 NS + KA; OD, optical density; NSE, neuron specific enolase; GFAP, glial fibrillary acidic protein; BHB, β-hydroxybutyrate; KA, kainic acid; NS, normal saline.
Fig 5: RACGAP1 overexpression promoted neuroendocrine transformation in prostate cancer. (A) Western blot analysis showed that RACGAP1 was upregulated in NEPC-like cells. (B) Immunohistochemical study of different types of prostate cancer tissues showed that RACGAP1 was highly expressed in NEPC. (C) Data for quantified immunohistochemistry in adjacent normal tissues (n=10), tumor tissues (n=10) and NEPC (n=7) of prostate cancer are shown as mean + SD. (D) Enzalutamide (MDV3100) induced the production of RACGAP1, and DHT partially reversed this effect. (E) Western blot analysis showed the protein expression of RACGAP1, CHGA, and SYP in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days. (F) The mRNA level of RACGAP1, NCAM, CHGA, SYP, and NSE in cells treated with or without 10 μmol/L enzalutamide for 2, 4, or 7 days were determined by qRT-PCR analysis. (G) RACGAP1 and NE markers (CHGA, NCAM, NSE, and SYP) in C4-2 cells following transient transfection with control (shNC) or RACGAP1 shRNA (sh1, sh2), as detected by qRT-PCR. (H) RACGAP1 and NE markers (CHGA, SYP) in C4-2B cells following transient transfection with RACGAP1 or an empty vector, as detected by qRT-PCR. (I) Protein expression of RACGAP1 and NE markers (CHGA, and SYP) in prostate cancer cells following transient transfection with control (shNC) or RACGAP1 shRNA (sh1, sh2) and FLAG-RACGAP1 or an empty vector were determined by Western blot analysis. (J, K, L) Western blot and qRT-PCR were performed to detect protein and relative mRNA expression of RACGAP1, NE markers, and AR in NE-like cells (C4-2B-N). (M) Morphological changes of C4-2B-N compared with C4-2B cells under a microscope. Bar graphs show the statistical analysis of three independent experiments. ***, p < 0.001; **, p < 0.01; *, p < 0.05, p = ns (no significance); t test for two groups or ANOVA for more than two groups.
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