Xiaolu Zhang

Nephrolithiasis is a common urinary disorder characterized by high prevalence and recurrence, but the molecular mechanisms underlying calcium oxalate (CaOx)-crystal-induced renal injury remain incompletely understood. We applied integrated proteomic, phosphoproteomic, acetylomic, and lactylomic analyses to kidney tissues from a mouse model of CaOx nephrolithiasis followed by bioinformatic analysis and experimental validation. We identified 658 differentially expressed proteins, 735 differential phosphorylation sites, 335 differential acetylation sites, and 113 differential lactylation sites. Functional enrichment indicated immune activation, fibrotic remodeling, and alterations in PI3K-Akt, NOD-like receptor, p53, and Toll-like receptor signaling together with changes in fatty acid degradation, the tricarboxylic acid cycle, and glycolysis. Kinase activity prediction suggested the relative activation of multiple cyclin-dependent kinases. Proteins associated with ferroptosis, autophagy, necroptosis, and pyroptosis, including ACSL4, BNIP3, RIPK3, and GSDMD, showed coordinated abundance and modification changes. Several candidate sites, including MTOR_S1849, GCLM_K94, GCLM_S59, and GSS_K172, were also dysregulated. These data provide a multiomics resource for CaOx nephrolithiasis and identify candidate PTM events and regulatory pathways for future mechanistic validation.

Histone lactylation acts as a master regulator in tumor development, but its role in a noncoding RNA (ncRNA) network remains unclear. This study aims to reveal the interaction between H3K18la and the lncRNA-miRNA-mRNA regulatory network in hepatocellular carcinoma (HCC). Transcriptome sequencing and ChIP sequencing were performed in HCC and adjacent normal tissues. Cut&Run and qPCR were used to validate the H3K18la enrichment on LINC02732 and CD44 promoter. Dual luciferase reporter assay, qPCR and Western blotting were used to verify the LINC02732-miR-1291-ASF1B axis. Co-Immunoprecipitation was performed to validate ASF1B recruiting p300. CCK8 and mouse subcutaneous tumor formation were performed to demonstrate this axis promoting HCC. H3K18la enrichment on LINC02732 promoter elevates its expression in both HCC samples and cell lines, therefore enhancing ASF1B expression via sponging miR-1291. Moreover, ASF1B, a histone chaperone, promotes H3K18la by recruiting lactyltransferase p300, forming an ASF1B-H3K18la positive feedback loop. The axis upregulates CD44 expression and promotes HCC in vitro and in vivo. These findings demonstrated the influence of H3K18la on the LINC02732-miR-1291-ASF1B axis and the novel role of ASF1B in histone lactylation by recruiting p300, which together promoted HCC proliferation.

Supplementary Figure 6. The USP10-PKLR axis serves as the key effector of creatine/SERPINE1-mediated Warburg effect and parthanatos antagonism. (A, B) Confocal microscopy (A) and cell fractionation (B) assays determining the subcellular localization of SERPINE1 in HCC cells. Scale bars, 10 μm. (C) Confocal microscopy analysis showing the colocalization of SERPINE1 and PKLR in HCC cells. Scale bars, 10 μm. (D) Endogenous co-IP assays confirmed the interaction between SERPINE1 and PKLR in HCC cells. (E, F) qRT-PCR was used to analyze the mRNA level of PKLR in SERPINE1-knockdown (E) and overexpression (F) cells. (G) Relative PKLR protein levels after CHX treatment. (H) Western blot analysis of the ubiquitination of PKLR upon SERPINE1 knockdown in HCC cells. (I, J) Endogenous co-IP assays confirmed the presence of the SERPINE1-PKLR-USP10 complex based on the LC-MS/MS data. (K) Confocal microscopy analysis showing the colocalization of PKLR and USP10 in HCC cells. Scale bars, 10 μm. (L, M) Endogenous and exogenous co-IP assays confirmed the interaction of PKLR and USP10 in HCC cells. (N, O) Western blotting analysis assessed the PARyaltion of USP10 in HCC cells upon MNNG or Olaparib administration. (P) Western blot analysis showing the degradation rate of USP10 in control and MNNG-treated groups. n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.

Supplementary Figure 5. The oncogenic role of creatine depends on SERPINE1 to form a creatine/SERPINE1/HIF-1α positive feedback loop. (A-C) CCK-8 (A), EdU incorporation (B) and colony formation assays (C) were used to determine the role of SERPINE1 in SLC6A8-knockdown cell proliferation and colony formation under hypoxia. Scale bars, 50 μm. (D) Western blot analysis of PAR polymer accumulation in hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (E) IF analysis of AIF nuclear translocation in hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. Scale bars, 10 μm. (F, G) Intracellular pyruvate content and extracellular lactate content of hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (H, I) The ECAR and OCR were measured in hypoxic SLC6A8 knockdown cells with or without ectopic SERPINE1 expression. (J) Representative confocal microscopy images showed the mitochondrial function in HCC cells treated with MitoTracker Green. Scale bars, 10 μm. (K) qRT-PCR analysis of the mtDNA content of hypoxic SLC6A8-knockdown cells with or without ectopic SERPINE1 expression. (L) Gross image and weight statistics of subcutaneous xenografts of HCC cells. (M) Western blot analysis of HIF-1α expression in hypoxic control and SLC6A8-knockdown cells upon creatine supplementation. (N, O) Relative expression of HIF-1α target genes in hypoxic HCC cells. (P, Q) Western blot analysis of HIF-1α expression in knockdown or ectopic expression of SERPINE1 HCC cells under hypoxia. For cell experiments, n = 3 independent experiments; for animal experiments, n = 6 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.

Supplementary Figure 7. SERPINE1 reverses the attenuated deubiquitination activity of USP10 caused by the direct binding of PAR. (A) Western blot analysis of PKLR levels in USP10-knockdown HCC cells transfected with SERPINE1 plasmids. (B) Western blot analysis of PKLR levels in SERPINE1-knockdown HCC cells transfected with USP10 plasmids. (C, D) Co-IP assays demonstrated the effect of SERPINE1 levels on the interaction between USP10 and PKLR. (E, F) Western blot analysis of the ubiquitination of PKLR upon USP10 overexpression in HCC cells. (G) Western blotting analysis of the ubiquitination of PKLR in hypoxic HCC cells treated with MNNG or Olaparib. (H-M) The ECAR and OCR were measured in hypoxic control and PKLR knockdown cells. n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.

Supplementary Figure 3. Creatine antagonizes parthanatos by enhancing the metabolic reprogramming of hypoxic HCC cells. (A) Heatmap of the DEGs in hypoxic Huh7 SLC6A8-knockdown cells compared with control cells. (B-C) KEGG and GO analyses of Huh7 RNA-seq data. (D) Gene set enrichment analysis of Huh7 cells. The normalized enrichment score (NES) was shown. (E, F) 2-NBDG absorbance (E) and glucose uptake (F) of hypoxic HCC cells were measured upon creatine supplementation. (G) HK and PK activity of hypoxic HCC cells were measured. (H, I) The ECAR (H) and OCR (I) were measured upon creatine supplementation in hypoxic HCC cells using a XF Extracellular Flux Analyzer. (J) ROS of hypoxic HCC cells was measured using flow cytometry. (K, L) The MDA level (K) and GSH/GSSG ration (L) of hypoxic HCC cells were measured. (M, N) The ECAR of HCC cells with or without MNNG under normoxia or hypoxia were measured. (O) Western blot examined the PAR polymer accumulation in hypoxic HCC cells with or without 2-DG (10mM) administration. (P) Western blot showed the phosphorylation levels of Smad2 and Smad3 in hypoxic HCC cells upon creatine supplementation. (Q, R) Western blot analysis was performed to evaluate the effects of SB525334 (Q) and AZ3146 (R) on Smad2/3 phosphorylation. (S) Western blot showed the knockdown efficiency of MPS1. (T) Western blot showed the Smad2/3 phosphorylation of hypoxic HCC cells with MPS1-knockdown rescued by wild-type or mutant MPS1 in the presence or absence of creatine treatment. (U) IF and western blot analyses examined the nuclear translocation of Smad4 in hypoxic HCC cells. n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.

Supplementary Figure 8. Combining RGX-202 with lenvatinib exhibits potent antitumor effects in PDX models. (A, B) Colony formation assay was used to determine the colony formation ability of hypoxic HCC cells across different treatment groups. (C-F) Cell invasion and migration of hypoxic HCC cells across different treatment groups were evaluated by transwell assay. Scale bars, 50 μm. (G) The ECAR and OCR were measured in hypoxic HCC cells across different treatment groups. (H, I) CCK-8 (H) and EdU incorporation assays (I) were performed to evaluate the effect of SERPINE1 overexpression on cell proliferation under RGX-202 treatment. Scale bars, 50 μm. (J) Colony formation assay was used to determine the effect of SERPINE1 overexpression on colony formation ability under RGX-202 treatment. (K, L) Transwell assays were used to evaluate the effect of SERPINE1 overexpression on cell invasion and migration under RGX-202 treatment. Scale bars, 50 μm. (M, N) The ECAR and OCR were measured in hypoxic HCC cells under RGX-202 treatment with or without SERPINE1-overexpression. (O) Analysis of tumor numbers in the livers of the indicated mice (n = 5). (P) Determination of circulating ALT and AST levels in the indicated mice. (Q) Gross images of PDX model derived from low SLC6A8 expression specimens (n = 6). For cell experiments, n = 3 independent experiments. Means ± SD are shown; *P < 0.05, **P < 0.01, ***P < 0.001, and ns indicates no significant difference.

Recently, a PARP1-dependent cell death process termed “parthanatos” that is driven by DNA damage has emerged as a crucial regulator of tissue homeostasis and tumorigenesis. Hypoxia is a hallmark of solid tumors and profoundly affects the malignant phenotypes of cancer cells. In this study, we investigated the cross-talk between parthanatos and hypoxia. Despite causing DNA damage, hypoxia failed to induce parthanatos in hepatocellular carcinoma (HCC). The creatine transporter SLC6A8 promoted parthanatos antagonism and malignant phenotypes in hypoxic HCC cells. Hypoxia-induced creatine accumulation drove metabolic reprogramming and antagonized parthanatos. Mechanistically, creatine elevated serpin peptidase inhibitor class E member 1 (SERPINE1) expression through MPS1-mediated Smad2/3 phosphorylation and formed a creatine/SERPINE1/hypoxia-inducible factor 1α–positive feedback loop. SERPINE1 facilitated ubiquitin-specific protease 10 (USP10)–mediated deubiquitination and stabilization of pyruvate kinase, liver, and red blood cell by forming a SERPINE1–USP10–pyruvate kinase, liver, and red blood cell complex. USP10 contained a strong poly(ADP-ribose)–binding motif, and SERPINE1 reversed the attenuated deubiquitination activity of USP10 caused by the direct binding of poly(ADP-ribose) under hypoxia. The SLC6A8 inhibitor RGX-202 exerted potent antitumor activity alone and in combination with lenvatinib in patient-derived xenografts and primary HCC mouse models. Overall, this study identified intracellular creatine accumulation as a mechanism that allows hypoxic cancer cells to circumvent parthanatos and as a therapeutic target in HCC.Significance:The creatine transporter SLC6A8 drives creatine uptake in hypoxic cancer cells to induce parthanatos antagonism and metabolic reprogramming, which can be overcome by inhibiting SLC6A8 as a strategy for treating hepatocellular carcinoma.