Fig 2: 1-MNA prevents ubiquitin-mediated CD44 degradation. (A-B) Effect of 1-MNA on CD44 protein and mRNA levels in QGY-7701 and LM3 cells. (C) CD44 protein levels in control, NNMT-overexpressing and 1-MNA-treated SMMC-7721 cells after treatment with CHX (20 µg·mL-1 for 20 h). (D) CD44 protein degradation curve in the above three groups. (E) Co-IP assay showing NNMT-CD44 binding. (F-G) Co-IP and immunoblots showing CD44 ubiquitination levels in control, NNMT-overexpressing and 1-MNA-treated SMMC-7721 cells. The t-test was used in B, G. The error bars represent SD. Each experiment was performed in triplicate. ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 3: Activated HSCs increase HCC migration and invasion by upregulating NNMT. (A–B) TGF-ß upregulated ACTA2, Colla1, and a-SMA and downregulated NPC1 in the LX2 cells. (C) Number of BODIPY493/503-stained lipid droplets decreased in LX2 cells upon activation. (D) The in vitro migration and invasion of SMMC-7721 cells cocultured with HSCs were assessed by the Transwell assay. (E) Western blot images showing expression of NNMT in SMMC-7721 and Huh7 cell lines cocultured with HSCs. (F–G) Efficiency of NNMT KD in Huh7 cells cocultured with HSCs and its effect on migration and invasion. (H) In situ NNMT protein expression in 92 HCC and corresponding adjacent tissues, and the IOD values (original magnification ×200). (I) Immunoblot showing NNMT protein expression in paired HCC tumor and adjacent samples and the IOD values. (J) Relative expression of NNMT mRNA in the paired HCC tumor and adjacent tissues. (K-L) Kaplan–Meier curves showing OS and relapse-free survival in NNMT-high (n = 46) and NNMT-low (n = 46) patients. The t-test was used in A, D, G. The two-related samples Wilcoxon nonparametric test was used in H, I, J. The error bars represent SD. Scale bars = 200 µm. Each experiment was performed in triplicates. ***P < 0.001, **P < 0.01, and *P < 0.05.

Fig 4: NNMT activation and DNMT1 inhibition render OXPHOS inhibition-sensitive cancer cells resistant. A–C) NNMT decreases intracellular SAM levels in OXPHOS inhibition-sensitive and resistant cancer cells. OXPHOS inhibition sensitive cancer cells have higher intracellular SAM levels than that in resistant ones (A); NNMT overexpression decreases intracellular SAM levels in OXPHOS inhibition sensitive cancer cells (NCI-H82 and G-401) (B). NNMT knockdown increases intracellular SAM level in OXPHOS inhibition-resistant cancer cells (CFPAC-1 and 786-O); the intracellular SAM level was measured by Elisa analysis and normalized by total protein in indicated cell lines (C). Mean ± SD. n = 3. Paired t-test. D) Boxplot shows DNMT1 transcription positively correlated with cancer cell sensitivity to Gboxin treatment. 57 cancer cell lines in Gboxin sensitivity screen were divided into five groups according to their Gboxin sensitivity as indicated. DNMT1 gene transcription data was extracted from DepMap and CCLE, and the expression value of DNMT1 was rescaled to a range from 0 to 10. The p value is calculated using the Mann–Whitney test. E–G) NNMT overexpression and DNMT1 knockdown have a synergistic effect on reducing NCI-H82 sensitivity to treatment of Gboxin (E), Oligomycin A (F), and Berberine (G). NCI-H82 cells with NNMT overexpression, DNMT1 knockdown, or both were treated with a series dilution of Gboxin, Oligomycin A, and Berberine for 3 days. Cell viability was measured by CellTilter Glo reagent. Two-way ANOVA test shows the lowest p value. n = 2. H–J) Western blotting showed attenuated ATF4 upregulation and p-S6 inhibition in NCI-H82 cells with NNMT overexpression and DNMT1 knocked down. NCI-H82 transfected with empty vector or overexpression of NNMT and knockdown of DNMT1 were treated with different concentrations of Gboxin (H), Oligomycin A (I), and Berberine (J) as indicated for 30 h, and ATF4 and p-S6 expression were measured by Western blot. n = 2 at least. K–M) NCI-H82 cells with NNMT overexpression, Decitabine treatment or both were incubated with a series dilution of Gboxin (K), Oligomycin A (L), and Berberine (M) for 3 days. Cell viability was then measured by CellTilter Glo reagent. Paired t-test shows the lowest p value compared to the vector. Mean ± SD. n = 2.

Fig 5: OXPHOS inhibitors, S-Gboxin and Berberine, inhibit growth of xenograft tumors by OXPHOS sensitive cells but not resistant ones. A–D) OXPHOS inhibition-sensitive and resistant cancer cells maintain their NNMT and DNMT1 status in vivo. 5 × 106 NCI-H82 (sensitive to OXPHOS inhibition), MDA-MB-453 (sensitive to OXPHOS inhibition), NCI-H82-OE-NNMT/sh-DNMT1 (resistant to OXPHOS inhibition), and CFPAC-1 (resistant to OXPHOS inhibition) cancer cells were subcutaneously injected into flanks of nude mice. Mice were sacrificed when tumor volume (1/2 × length × width2) was ˜1000 mm3. Representative immunohistochemistry staining images show expression of NNMT and DNMT1 in tumor samples by cancer cells as indicated. Scale bar: 100 um (A). Quantification of NNMT expression (B), DNMT1 expression (C), and DNMT1/NNMT ratio (D) of (A). Mean± SD. n = 3. One-way ANOVA test. E–G) NCI-H82 (E), NCI-H82-OE-NNMT/sh-DNMT1 (F), and CFPAC-1 (G) cancer cells were subcutaneously injected into flanks of nude mice as in (A). 5 days after transplant, mice were administrated intraperitoneally with S-Gboxin (10 mg kg-1 per day) or Berberine (10 mg kg-1 per day). Up panel, growth for NCI-H82 (E), NCI-H82-OE-NNMT/sh-DNMT1 (F), and CFPAC-1 (G) tumors were assessed every 2 to 3 days and calculated with the formula (1/2 × length × width2). Tumor n = 7 for NCI-H82; n = 9 for NCI-H82-OE-NNMT/sh-DNMT1; and n = 9 for CFPAC-1 xenografts. Paired t-test shows the lowest p value compared to vehicle. Bottom panel, images show five or six NCI-H82 (E), NCI-H82-OE-NNMT/sh-DNMT1 (F), and CFPAC-1 (G) tumors each on the day all mice were sacrificed after the treatments as indicated.