Fig. 7: G9a inhibitor, UNC0642, suppresses SLC7A2-mediated HCC immune escape, invasion, and metastasis.

A, B MHCC97H and HCCLM3 HCC cells treated with 5-aza (2 μm, 3 days), EPZ-6438 (1 μm, 3 days), UNC0642 (5 μm, 2 days), ITF-2357 (1 μm, 3 days) then qRT-RCR and western blotting was used to test the expression of SLC7A2. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. C ChIP-quantitative real-time PCR analysis to detect H3K9me2 association with SLC7A2 gene after treatment of G9a inhibitor UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. D Representative IHC staining images for SLC7A2 and G9a in human HCC tissues. Scale bars, 200 µm (upper), 50 µm (lower). E Bioinformatics analysis of G9a mRNA expression on the TCGA database in human HCC tissues. F Kaplan–Meier analysis of the associations between G9a expression and overall survival in the HCC cohort. G–I Treatment of UNC0642 promoted HCC cell proliferation and metastasis in vitro. G Usage of UNC0642 on HCC cell colony formation. H The effects of UNC0642 on HCC cell proliferation were measured by a CCK-8 assay. I Transwell assay shown the abilities of migration and invasion. The scale bar represents 100 μm. J Western blotting was used to detect the G9a, SLC7A2, and its downstream target genes expression in MHCC97H and HCCLM3 cells treated with UNC0642. The data are shown as the mean ± SD from the at least three independent experiments; *P < 0.05. K, L Tumor growth in mice subcutaneously injected with H22-control or H22-SLC7A2 cells, treated with UNC0642 daily by intraperitoneal injection at 5 mg/kg. The tumor volume was monitored every 3 days. n = 5. M Flow cytometric images of subcutaneous H22-control or H22-SLC7A2 cells treated with UNC0642 or its control at day 27. CD8⁺ T cells (upper) and MDSCs (lower). Flow cytometric analyses the ratio of CD8⁺ T cells and MDSCs between groups at day 27, respectively, n = 5. The data are shown as the mean ± SD from the at least three independent experiments; **P < 0.01. N Immunofluorescent staining of the CD11b, CD8, and granzyme B protein expression patterns in mice tumor cells. Scale bars, 20 µm. O–R In vivo assays shown that the treatment of UNC0642 can block knock-down SLC7A2-mediated HCC metastasis. O The representative Bioluminescence images were shown in the different groups, treated with UNC0642 or DMSO in the BALB/C injected with the indicated cells in the liver and Incidence of lung metastasis, n = 10. The Bioluminescence intensity in the tumors at the indicated time point was presented as the total photon flux. P The number of metastatic lung nodules in lung in the BALB/C mice. n = 10. Q Overall survival of the mice in each group. R Representative H&E-stained lung metastatic nodules. The scale bars represent 1 mm (upper panel) and 100 μm (lower panel). *P < 0.05, **P < 0.01. S A schematic diagram of the SLC7A2 signaling in HCC immune evasion. SLC7A2 deficiency upregulated CXCL1 expression through PI3K/Akt/NF-κB pathway. CXCL1 promoted HCC growth and metastasis through recruiting MDSCs to the tumors. Neutralizing or suppressing MDSC infiltration abolished deficient SLC7A2-mediated HCC growth and metastasis. Usage of G9a inhibitor (UNC0642) suppressed loss of SLC7A2-meidated HCC metastasis.