Dinghua Yang

Background: The recently described lactyltransferase activity of alanyl‐tRNA synthetase 1 (AARS1) raises questions about its metabolic functions in hepatocellular carcinoma. We examined phosphoglycerate dehydrogenase (PHGDH) as a potential lactylation substrate linking AARS1 to serine metabolism and tumour growth. Methods: Public expression data and patient tissues were studied alongside proteomic, lactylomic and targeted metabolomic profiles. Cellular and xenograft experiments used gene depletion, overexpression and reconstitution with wild‐type or mutant proteins. Immunoprecipitation and activity measurements assessed PHGDH regulation. Survival was examined with Kaplan‒Meier estimates and Cox regression, while experimental groups were compared by t ‐tests or analysis of variance. Results: Of 140 paired specimens, 119 tumours (85%) had higher AARS1 mRNA than their non‐tumour counterparts. Elevated AARS1 independently predicted poorer survival, and E2F4 depletion lowered AARS1 expression. AARS1 supported malignant phenotypes, lactate production and PHGDH lactylation. Mass spectrometry detected lactylation at PHGDH‐K394. Relative to wild‐type PHGDH, K394R showed less lactylation and supported lower lysate enzyme activity, serine, glycine and glutathione levels. The mutant also increased reactive oxygen species, provided weaker phenotypic rescue and supported less xenograft growth. Depleting AARS1 reduced enzyme activity and malignant phenotypes in the wild‐type background; no further significant reduction was detected with K394R. β‐Alanine inhibited xenograft growth, but PHGDH‐K394 alone could not account for its effects. Conclusions: AARS1‐associated PHGDH‐K394 lactylation may connect serine metabolism and redox regulation to hepatocellular carcinoma progression. Establishing therapeutic relevance will require site‐specific mechanistic experiments and additional preclinical validation.

Purpose Hepatocellular carcinoma (HCC) ranks among the most prevalent cancers globally. Ferroptosis plays a vital role in the resistance of HCC to various cancer treatments. However, the specific molecular mechanisms that inhibit ferroptosis in HCC remain unknown. Therefore, this study aims to elucidate the role of 5, N6-adenosine (METTL5) in regulating HCC development and ferroptosis. Methods Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining were employed to detect the expression of METTL5 and Microsomal glutathione S-transferase 1 (MGST1) in HCC cell lines and clinical samples. Xenotransplantation experiments were conducted to investigate the effects of METTL5 knockdown on HCC cells in vivo. Tandem mass tagging proteomic quantification was utilized to analyze the downstream targets of METTL5. In vitro functional rescue assays were used to explore the influence of METTL5 and MGST1 on cell functions, including proliferation, migration, invasion, and ferroptosis in HCC cell lines. Results METTL5 and MGST1 were aberrantly and highly expressed in HCC tissues than in adjacent normal liver tissues. High expression levels of METTL5 or MGST1 were a prognostic risk factor for patients with HCC. METTL5 regulated the protein expression of MGST1 through its N6-methyladenosine catalytic function. The METTL5-MGST1 axis regulated proliferation, migration, invasion, and suppression of ferroptosis in HCC cells. Conclusion Our study explored and confirmed the oncogenic roles of METTL5 and MGST1 in HCC, uncovering a novel mechanism by which the METTL5-MGST1 axis suppresses ferroptosis in HCC. Our findings establish a molecular foundation for developing therapeutic strategies for HCC.

To investigate the expression of parathyroid hormone-like hormone (PTHLH) in hepatocellular carcinoma (HCC) and analyze its correlation with clinical prognosis, its regulatory effects on HCC cell behaviors, and the signaling pathways mediating its effects. We analyzed the differential expression of PTHLH in HCC and adjacent tissues and its association with patient prognosis based on data from TCGA and GEO databases and from 70 HCC patients treated in our hospital. The effects of PTHLH knockdown and overexpression on proliferation, migration, and invasion of cultured HCC cells were investigated using CCK-8 assay, colony formation assay, Transwell migration and invasion assays, and the signaling pathways activated by PTHLH were detected using Western blotting. TCGA and GEO database analysis showed significant overexpression of PTHLH mRNA in HCC tissues, which was associated with poor prognosis of the patients (PPPP<0.05). The prognostic prediction model based on PTHLH mRNA expression showed an improved accuracy for predicting the risk of postoperative recurrence in HCC patients. In cultured HCC cells, PTHLH overexpression significantly promoted cell proliferation, colony formation, migration and invasion, and caused activation of the ERK/JNK signaling pathway in Huh7 and Hep3B cells. High PTHLH expression promotes HCC progression and is associated with poor patient prognosis. Its pro-tumor effects may be mediated by activation of the ERK/JNK signaling pathway.

Hepatocellular carcinoma (HCC) is a type of cancer characterized by high recurrence rates. Overcoming chemoresistance can reduce HCC recurrence and improve patients’ prognosis. This work aimed to identify HCC chemoresistance-associated long non-coding RNA (lncRNA) and find an effective drug targeting the identified lncRNA for ameliorating the chemoresistance. In this investigation, bioinformatics analysis based on The Cancer Genome Atlas revealed a new chemoresistance index and suggested LINC02331 as an HCC chemoresistance and patients’ prognosis-associated lncRNA that served as an independent prognostic indicator. Moreover, LINC02331 promoted DNA damage repair, DNA replication, and epithelial-mesenchymal transition as well as attenuated cell cycle arrest and apoptosis through regulating Wnt/β-catenin signaling, thus stimulating HCC resistance to cisplatin cytotoxicity, proliferation, and metastasis. Interestingly, we developed a novel oxidative coupling approach to synthesize a dimeric oxyberberine CT4-1, which exerted superior anti-HCC activities without obvious side effects measured by in vivo mice model and could downregulate LINC02331 mice model and could downregulate LINC02331 to mitigate LINC02331-induced HCC progression by suppressing Wnt/β-catenin signaling. RNA sequencing analyses verified the involvement of CT4-1-affected differential expression genes in dysregulated pathways and processes, including Wnt, DNA damage repair, cell cycle, DNA replication, apoptosis, and cell adhesion molecules. Furthermore, CT4-1 was demonstrated to be an effective cytotoxic drug in ameliorating HCC patients’ prognosis with a prediction model constructed based on RNA-sequencing data from CT4-1-treated cancer cells and public cancer database. In summary, HCC chemoresistance-associated LINC02331 independently predicted poor patients’ prognosis and enhanced HCC progression by promoting resistance to cisplatin cytotoxicity, proliferation, and metastasis. Targeting LINC02331 by the dimeric oxyberberine CT4-1 that exhibited synergistic cytotoxicity with cisplatin could alleviate HCC progression and improve patients’ prognosis. Our study identified LINC02331 as an alternative target and suggested CT4-1 as an effective cytotoxic drug in HCC treatment.

Epigenetic modifications are involved in the remodeling of the tumor microenvironment (TME) and the regulation of immune response. Nonetheless, the role of histone H4 methylation (H4M) modification in the TME and immune regulation of hepatocellular carcinoma (HCC) is unknown. As a result, the purpose of this research is to discover H4M-mediated modification patterns and their effects on TME and immunologic characteristics in HCC. A total of 2305 samples were enrolled from 13 different cohorts. With the help of consensus clustering analysis, three distinct H4M modification patterns were identified. The cell-infiltrating characteristics of TME under these three patterns were highly consistent with their enriched biological processes and clinical outcome. The H4Mscore was then created using principal component analysis algorithm to quantify the H4M modification pattern of each individual tumor and was systematically correlated with representative tumor characteristics. We found that analyzing H4M modification patterns within individual tumors could predict TME infiltration, homologous recombination deficiency (HRD), intratumor heterogeneity, proliferation activity, mRNA stemness index, and prognosis. The group with a low H4Mscore had an inflamed TME phenotype and a better immunotherapy response, as well as a better survival outcome. The prognostic value of H4Mscore was validated in three internal cohorts and five external cohorts, respectively. In external immunotherapy cohorts, the low H4Mscore was also linked to an enhanced response to anti-PD-1/L1 and anti-CTLA4 immunotherapy and a better prognosis. This study revealed that H4M modification played an important role in forming TME diversity and complexity. Evaluating the H4M modification pattern of individual tumors could help us learn more about TME and develop more effective immunotherapy strategies.

Background: Recently, an in vivo study demonstrated that circulating tumor cell-associated white blood cell (CTC-WBC) cluster possess much greater potential than single CTCs. We aim to explore the correlation between the CTC-WBC cluster and the clinicopathological characteristics of hepatocellular carcinoma (HCC) patients to seek novel biomarkers for HCC metastasis and recurrence.Methods: We retrospectively analyzed 136 HCC patients from October 2014 to March 2020 who received CTC tests using the CanPatrol CTC enrichment technique. The correlation between the clinical features and total CTCs, EMT-CTCs, and CTC-WBC cluster were analyzed by a chi-square test. The ROC curves were simulated for evaluating the diagnostic performance of CTC parameters in HCC metastasis. Patients were followed up from February 2015 to November 2021, and the relapse-free survival (RFS) was analyzed using the Kaplan–Meier curve.Results: A total of 93.4% (127/136) and 31.6% (43/136) of HCC patients had detectable CTCs and CTC-WBC clusters. Baseline CTC-WBC cluster was closely correlated with microvascular invasion, portal vein tumor thrombus, and extrahepatic metastasis in pre-treatment HCC patients (P <0.05). The simulated ROC curves presented an AUC of 0.821 for the CTC-WBC cluster (sensitivity of 90.0% and specificity of 93.7%) in discriminating metastasis from non-metastatic HCC, which was higher than that for total CTCs (0.718) and EMT-CTCs (0.716). Further follow-up analysis showed that compared to the CTC-WBC cluster negative group (<1/5 ml), patients in the CTC-WBC cluster positive group (≥1/5 ml) presented an increased relapse ratio (60.0% versus 17.9%) and shorter RFS (22.9 versus 53.8 months). Dynamic analysis of CTCs parameters showed that total CTC level, EMT-CTCs proportion, and CTC-WBC cluster were decreased after microwave ablation treatment, while CTC-WBC cluster increased on average 10 months in advance of imaging (MRI) diagnosed recurrence.Conclusion: The CTC-WBC cluster is a promising biomarker for the metastasis diagnosis and prognosis of HCC metastasis. Dynamic monitoring of the CTC-WBC cluster is an effective method for early detection and intervention of HCC recurrence and metastasis.

Nuclear receptor coactivator 6 (NCOA6), a coactivator of numerous nuclear receptors and transcription factors, regulates multiple critical cellular functions. Nuclear receptor coactivator 6 is dysregulated in various cancers, including hepatocellular carcinoma (HCC); however, its role remains largely unknown. Here we reported that NCOA6 was highly expressed in HCC compared to the adjacent liver tissue, and NCOA6 overexpression was significantly correlated with poor HCC prognosis. Experiments revealed that the knockdown of NCOA6 damaged the proliferation, migration, and invasion of HCC cells. Multiomics and immune infiltration analyses showed a close relationship between NCOA6 expression, multiple cancer‐related malignant pathways, and the immunosuppressive microenvironment. Finally, we established an effective NCOA6‐related microRNA (miRNA) signature to distinguish HCC from hepatitis\liver cirrhosis patients. To the best of our knowledge, this study is the first to provide a comprehensive analysis of NCOA6 expression in HCC. We found that NCOA6 plays an important role in HCC development and has a potential mechanism of action. Establishing an NCOA6‐related miRNA signature will help develop novel diagnostic strategies for HCC patients.

Background: The JAK/STAT pathway, which transmits signals that are crucial for growth, differentiation, immunity and survival, is altered in pan-cancer. And the downstream transcription factor STAT3 acts on the PD-L1 promoter and induced PD-L1 upregulation of in tumor. The roles of JAK-STAT signaling pathway in hepatocellular carcinoma (HCC), and its correlation with tumor immune microenvironment (TIME) and prognosis remain unclear. Method: 34 patients with HCC who underwent radical surgical resection at Southern Medical University Nanfang hospital were enrolled. Tumor tissue samples were collected for next generation sequencing (NGS) testing (733 genes panel) and multiple immunohistochemical fluorescence staining (mIHC). Clinical information was obtained to evaluate the prognostic performance of biomarkers. Whole exome sequencing data of 366 HCC patients was obtained from the Cancer Genome Altas (TCGA), and the tumor-related immune cells infiltration level difference between mutant and wild-type tumors were inferred using TIMER2.0. Results: Characteristics of 34 HCC patients from Nanfang cohort were as follows: 58.5% HBV positive, 41.2% largest tumor diameter≥5cm, 73.5% liver cirrhosis, 20.6% AFP > 400ng/ml, 67.6% MVI, 11.8% PVTT, 73.5% BCLC A, 85.3% CNLC I/II. 44% (15/34) HCC patients harboring JAK-STAT pathway related gene mutation, which EP300, STAT3 and JAK1 were the top three alternation frequency genes. The JAK-STAT pathway mutant tumors with significantly higher inferred M2 macrophages than wild-type (mean density, mutation vs. wild-type = 2.755 vs. 1.407 cells/mm², p= 0.036). The level of infiltration of PD-L1 expression macrophages was significantly increased in the JAK - STAT pathway mutated tumors compared with wild-type tumors (p =0.048). After median follow-up time was 5.4 months, 3 short-term recurrence events were recorded. The recurrence-free survival (RFS) of EP300 and/or STAT3 mutation group (n=5) were longer than wild-type group (n=29) (median RFS, mutation vs. wild-type = 5.6 vs. NE months; HR 7.267[95% CI 0.46-113.8]; P = 0.057). While in TCGA cohort, the level of infiltration of M2 macrophages was increased in both STAT3 and EP300 mutant tumors compared with wild-type tumors (Wilcoxon test, p < 0.05). The significance of STAT3 and EP300 expression in cancer cells were evaluated on samples stratified by tumor-infiltrating macrophages M2 cells, which were divided into 4 groups respectively. Both RFS of high gene expression and high macrophages M2 groups were significantly different among the four groups (log-rank PCitation Format: Dinghua Yang, Sheng Yu, Baitang Guo, Tingting Chen. The Correlation analysis of driver genes of JAK-STAT signaling pathway with tumor immune microenvironment and prognosis in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6119.

Background: Hepatocellular carcinoma (HCC) is a leading cause of tumor-associated death worldwide, owing to its high 5-year postoperative recurrence rate and heterogeneity. Although Molecular profiling has been used to predict prognosis and guide targeted therapy, obtaining liver tissue biopsies was still a clinical challenge of advanced HCC patients. Circulating tumor DNA (ctDNA) testing as a novel and convenient detecting techniques, which can revealed comprehensive information regarding to tumor genome profiles. The purpose of the study was to analyze and compare the genomic landscape characteristics of tumor tissue and ctDNA in Chinese HCC. Methods: 187 tumor tissue samples and 293 plasma samples from patients with HCC were collected from Nanfang hospital and 3DMed cohort, respectively. Next generation sequencing (NGS) assay (381/733 genes panel) was performed on tumor and plasma samples to capture tumor somatic single-nucleotide variants (SNVs). The gene variants information was summarized, analyzed and visualize using the maftools R package (Mayakonda et al. 2018.). Results: In total, DNA alterations were detected in 93.86% (275/293) plasma samples and 90.91% (170/187) in tumor samples, which variant levels included pathogenic, likely pathogenic and variant of uncertain significance. The most commonly altered genes in ctDNA results were TP53 (52%), TERT (24%), LRP1B (22%), FAT1 (15%), CTNNB1 (14%). While in HCC tumor testing results were TP53 (55%), TERT (27%), CTNNB1 (17%), SPTA1 (14%) and ARID1A (12%). Both the most variant classification was missense, next were frameshift indel. Both the top five affected pathways were TP53, RTK-RAS, NOTCH, WNT and Cell Cycle. In WNT pathway related genes, the highest alteration frequency tumor oncogene was CTNNB1, and suppressor gene was APC. We analyzed mutually exclusive and co-occurring event on top 25 mutated genes. In ctDNA analysis results, the probability of co-mutation between LRP1B and KMT2A gene was the highest (p < 0.001), while mutual exclusion between ARID1A and EGFR gene was the highest. Conclusion: Circulating tumor DNA mutation detection in HCC is feasible in the absence of HCC tissue DNA. The analysis results revealed the genomic landscape characteristics of blood-derived ctDNA in HCC were consistent with tumor tissues, which can provided HCC potential prognosis and treatment related genes information to further study. Citation Format: Dinghua Yang, Sheng Yu, Baitang Guo, Tingting Chen. Genomic landscape analysis in tumor tissue and circulating tumor DNA of Chinese patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5119.