Promotes Hepatocellular Carcinoma Growth Research Articles

LYRM2 Promotes the Growth and Metastasis of Hepatocellular Carcinoma via Enhancing HIF-1α-Dependent Glucose Metabolic Reprogramming.

Hepatocellular carcinoma (HCC) is a foetal malignancy with dismal overall survival. The molecular mechanism underlying the progression of HCC remain largely unknown. LYR motif containing 2 (LYRM2) has been identified as an oncogene in colorectal cancer; however, its expression, functions and molecular mechanism in the context of HCC has not been investigated. Data derived from The Cancer Gemome Atlas, along with findings from our patients' cohort, indicate that LYRM2 expression is elevated in HCC tissues and correlates with adverse clinicopathological features and prognosis in HCC patients. Subsequent research into the biological functions of LYRM2 has revealed that it promotes the proliferation, migration, invasion and epithelial-mesenchymal transition of HCC cells, both invitro and invivo. Mechanistic insights have shown that LYRM2 interacts with HIF-1α, enhancing the protein stability of HIF-1α, which in turn increases cellular glycolysis and inhibits mitochondrial respiration. Moreover, the glucose metabolic reprogramming mediated by LYRM2 is implicated in its role in promoting HCC growth and metastasis. Collectively, this study identifies that LYRM2 as a novel oncogenic protein in HCC and elucidates its contribution to HCC progression through enhancing HIF-1α-dependent glucose metabolic reprogramming.

Tumor-derived CCL15 regulates RNA m6A methylation in cancer-associated fibroblasts to promote hepatocellular carcinoma growth.

Hepatocellular carcinoma (HCC) is a lethal malignancy characterized by rapid growth. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) significantly influences HCC progression. CCL15, a CC chemokine family member, is predominantly expressed in HCC and strongly correlates with tumor size, indicating its critical role in HCC growth. However, previous studies suggest that CCL15 does not directly stimulate cancer cell proliferation. The specific role and mechanism of CCL15 in HCC proliferation remain unknown. Here, we identified that CCL15 was predominantly overexpressed by HCC cells through single-cell RNA sequencing data and immunofluorescence. We discovered that CCL15 promotes HCC growth by stimulating the crosstalk between HCC cells and CAFs via CCR1 signaling, as evidenced by co-culture assays, organoid models, and allograft models. Mechanistically, CCL15 induced the expression of FTO in CAFs through the STAT3 pathway. By m6A sequencing and RNA sequencing, we found that CEBPA mRNA, a transcription factor regulating CXCL5 expression, was a target of FTO. CXCL5, secreted by CAFs, activated the CXCR2 receptor on HCC cells and enhanced their proliferation. Notably, we found that interfering with CCL15 signaling using a neutralizing antibody attenuated HCC growth in heterotypic co-injection and patient-derived xenograft murine models. Finally, CXCL5 also upregulated CCL15 expression in HCC cells by modulating P53 expression through MDM2, forming a positive feedback loop. Our study unveiled CCL15 as a key mediator in HCC progression, facilitating communication between HCC cells and CAFs. This highlights a novel regulatory axis in HCC and suggests that targeting CCL15 could be a potential therapeutic strategy.

PRMT1-mediated arginine methylation promotes YAP activation and hepatocellular carcinoma proliferation.

The activity of Hippo signaling is commonly dysregulated in various human malignancies, including hepatocellular carcinoma (HCC). YAP, the key effector of Hippo pathway, is regulated through several posttranslational modifications. However, the mechanism by which YAP is regulated by arginine methylation remains unknown. In this study, immunoprecipitation and mass spectrometry were used to identify the arginine methylation site of YAP in HCC cells. The transcriptional activity of YAP and TEAD were further characterized by real-time qPCR and immunofluorescence assay, and a subcutaneous and orthotopic tumor mouse model was used to assess the effect of PRMT1-knockdown on HCC tumor growth. The result of mass spectrometry analysis identified that YAP was methylated at arginine 124. Moreover, we found that arginine methyltransferase PRMT1 interacted with YAP to mediate its arginine methylation, thus inhibited YAP phosphorylation and promoted YAP activity in the nucleus. PRMT1 was up-regulated in HCC tissues and positively associated with the expressions of YAP target genes. Silencing PRMT1 in HCC cells inhibited cell proliferation and tumor growth, while PRMT1-overexpression promoted HCC growth through YAP methylation. Our study reveals that PRMT1-mediated arginine methylation at R124 is mutually exclusive with YAP S127 phosphorylation, thereby facilitating YAP activity in the nucleus and promoting tumorigenesis in HCC.

LTR retrotransposon-derived LncRNA LINC01446 promotes hepatocellular carcinoma progression and angiogenesis by regulating the SRPK2/SRSF1/VEGF axis

The causal link between long terminal repeat (LTR) retrotransposon-derived lncRNAs and hepatocellular carcinoma (HCC) remains elusive and whether these cancer-exclusive lncRNAs contribute to the effectiveness of current HCC therapies is yet to explore. Here, we investigated the activation of LTR retrotransposon-derived lncRNAs in a broad range of liver diseases. We found that LTR retrotransposon-derived lncRNAs are mainly activated in HCC and is correlated with the proliferation status of HCC. Furthermore, we discovered that an LTR retrotransposon-derived lncRNA, LINC01446, exhibits specific expression in HCC. HCC patients with higher LINC01446 expression had shorter overall survival times. In vitro and in vivo assays showed that LINC01446 promoted HCC growth and angiogenesis. Mechanistically, LINC01446 bound to serine/arginine protein kinase 2 (SRPK2) and activated its downstream target, serine/arginine splicing factor 1 (SRSF1). Furthermore, activation of the SRPK2-SRSF1 axis increased the splicing and expression of VEGF isoform A165 (VEGFA165). Notably, inhibiting LINC01446 expression dramatically impaired tumor growth in vivo and resulted in better therapeutic outcomes when combined with antiangiogenic agents. In addition, we found that the transcription factor MESI2 bound to the cryptic MLT2B3 LTR promoter and drove LINC01446 transcription in HCC cells. Taken together, our findings demonstrate that LTR retrotransposon-derived LINC01446 promotes the progression of HCC by activating the SRPK2/SRSF1/VEGFA165 axis and highlight targeting LINC01446 as a potential therapeutic strategy for HCC patients.

Unveiling the role of UPF3B in hepatocellular carcinoma: Potential therapeutic target.

RNA-binding proteins can regulate nucleotide metabolism and gene expression. UPF3B regulator of nonsense mediated mRNA decay (UPF3B) exhibits dysfunction in cancers. However, its role in the progression of hepatocellular carcinoma (HCC) is still insufficiently understood. Here, we found that UPF3B was markedly upregulated in HCC samples and associated with adverse prognosis in patients. UPF3B dramatically promoted HCC growth both invivo and invitro. Mechanistically, UPF3B was found to bind to PPP2R2C, a regulatory subunit of PP2A, boosting its mRNA degradation and activating the PI3K/AKT/mTOR pathway. E2F transcription factor 6 (E2F6) directly binds to the UPF3B promoter to facilitate its transcription. Together, the E2F6/UPF3B/PPP2R2C axis promotes HCC growth through the PI3K/AKT/mTOR pathway. Hence, it could be a promising therapeutic target for treating HCC.

USP40 promotes hepatocellular carcinoma progression through a YAP/USP40 positive feedback loop

Yes-associated protein (YAP) is an essential driver of hepatocellular carcinoma (HCC) progression and the ubiquitin-proteasome system controls its abundance. However, the role of ubiquitin-specific protease 40 (USP40) in YAP stability remains unclear. Here, USP40 was first identified as a novel regulator of YAP abundance and its target genes in HCC cells. USP40 interacted with YAP to remove the lysine 48 (K48)-linked polyubiquitination of YAP at K252 and K315 sites, thereby maintaining YAP stability. USP40 facilitated the proliferation, colony formation, migration and spheroid formation of HCC cells in vitro and promoted HCC growth in vivo in a YAP-dependent manner. In turn, YAP transcriptionally activated USP40 expression in HCC cells. RNA sequencing analysis showed that about 37% of USP40-regulated genes overlapped with YAP-regulated genes. Interestingly, stiffness-induced USP40 upregulation was abolished by YAP knockdown, and USP40 knockdown attenuated stiffness-induced YAP accumulation in HCC cells. Clinical data demonstrated that USP40 was positively associated with YAP expression in HCC tissues and its high expression indicated a poor prognosis. In conclusion, the USP40/YAP positive feedback loop contributes to HCC progression, suggesting that USP40 may be a promising drug target for anti-HCC.

Circ_MAPK9 promotes STAT3 and LDHA expression by silencing miR-642b-3p and affects the progression of hepatocellular carcinoma

BackgroundAberrant expression and activation of circular RNAs (circRNAs) are closely associated with various cancers. The role of circ_MAPK9 (hsa_circ_0001566) in cancer progression remains unknown. This study aims to investigate the function, mechanism and clinical significance of circ_MAPK9 in hepatocellular carcinoma (HCC).MethodsCirc_MAPK9 expression on the microarray of tumor from clinical HCC patients was detected by in situ hybridization (ISH). Circ_MAPK9 knockdown was achieved with siRNAs in SMMC-7721 and SK-Hep1 HCC cell lines. The biological function of circ_MAPK9 was verified in vitro by CCK8 test, colony formation assay, transwell assay, PI-Annexin V staining, and in vivo by xenograft tumor in nude mice. Fluorescent in situ hybridization (FISH), subcellular fractionation assay, a dual-luciferase reporter assay and rescue experiments were employed for further mechanistic investigation.ResultsThe expression of circ_MAPK9 was significantly up-regulated in HCC tissues and cells, which was found to be associated with poor prognosis. Patients with high expression of circ_MAPK9 had a shorter overall survival and disease-free survival in comparison to those with low circ_MAPK9 expression. Functional assays showed that circ_MAPK9 knockdown suppressed cellular proliferation, migration, invasion and tumor growth in vivo, and promoted apoptosis in HCC cells. Moreover, we found that circ_MAPK9 knockdown could inhibit aerobic glycolysis by decreasing the production of adenosine triphosphate (ATP) and lactic acid, which was mediated by lactate dehydrogenase (LDHA). Mechanistically, circ_MAPK9 functioned as ceRNA via sponging miR-642b-3p and alleviated the inhibitory effect of miR-642b-3p on its target signal transducer and activator of transcription 3 (STAT3) and LDHA, thereby leading to STAT3 activation and LDHA expression.ConclusionsCirc_MAPK9, as an oncogene, promotes HCC growth and metastasis through miR-642b-3p/STAT3-LDHA axis. Circ_MAPK9 could serve as a potential biomarker for HCC poor prognosis and diagnosis.

Aldo-keto reductase family member C3 (AKR1C3) promotes hepatocellular carcinoma cell growth by producing prostaglandin F2α.

Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Current therapies are effective for HCC patients with early disease, but many patients suffer recurrence after surgery and have a poor response to chemotherapy. Therefore, new therapeutic targets are needed. We analyzed gene expression profiles between HCC tissues and normal adjacent tissues from public databases and found that the expression of genes involved in lipid metabolism was significantly different. The analysis showed that AKR1C3 was upregulated in tumors, and high AKR1C3 expression was associated with a poorer prognosis in HCC patients. In vitro, assays demonstrated that the knockdown of AKR1C3 or the addition of the AKR1C3 inhibitor indomethacin suppressed the growth and colony formation of HCC cell lines. Knockdown of AKR1C3 in Huh7 cells reduced tumor growth in vivo. To explore the mechanism, we performed pathway enrichment analysis, and the results linked the expression of AKR1C3 with prostaglandin F2 alpha (PGF2α) downstream target genes. Suppression of AKR1C3 activity reduced the production of PGF2α, and supplementation with PGF2α restored the growth of indomethacin-treated Huh7 cells. Knockdown of the PGF receptor (PTGFR) and treatment with a PTGFR inhibitor significantly reduced HCC growth. We showed that indomethacin potentiated the sensitivity of Huh7 cells to sorafenib. In summary, our results indicate that AKR1C3 upregulation may promote HCC growth by promoting the production of PGF2α, and suppression of PTGFR limited HCC growth. Therefore, targeting the AKR1C3-PGF2α-PTGFR axis may be a new strategy for the treatment of HCC.

SRSF10 facilitates HCC growth and metastasis by suppressing CD8+T cell infiltration and targeting SRSF10 enhances anti-PD-L1 therapy

Background and AimsRNA splicing is an essential step in regulating the gene posttranscriptional expression. Serine/arginine-rich splicing factors (SRSFs) are splicing regulators with vital roles in various tumors. Nevertheless, the expression patterns and functions of SRSFs in hepatocellular carcinoma (HCC) are not fully understood. MethodsFlow cytometry and immunofluorescent staining were used to determine the CD8+T cell infiltration. Orthotopic HCC model, lung metastasis model, DEN/CCl4 model, Srsf10△hep model, and Srsf10HepOE model were established to evaluate the role of SRSF10 in HCC and the efficacy of combination treatment. ResultsSRSF10 was one of the most survival-relevant genes among SRSF members and was an independent prognostic factor for HCC. SRSF10 facilitated HCC growth and metastasis by suppressing CD8+T cell infiltration. Mechanistically, SRSF10 down-regulated the p53 protein by preventing the exon 6 skipping (exon 7 in mouse) mediated degradation of MDM4 transcript, thus inhibiting CD8+T cell infiltration. Elimination of CD8+T cells or overexpression of MDM4 removed the inhibitory role of SRSF10 knockdown in HCC growth and metastasis. SRSF10 also inhibited the IFNα/γ signaling pathway and promoted the HIF1α-mediated up-regulation of PD-L1 in HCC. Hepatocyte-specific SRSF10 deficiency alleviated the DEN/CCl4-induced HCC progression and metastasis, whereas hepatocyte-specific SRSF10 overexpression deteriorated these effects. Finally, SRSF10 knockdown enhanced the anti-PD-L1-mediated anti-tumor activity. ConclusionsSRSF10 promoted HCC growth and metastasis by repressing CD8+T cell infiltration mediated by the MDM4-p53 axis. Furthermore, SRSF10 suppressed the IFNα/γ signaling pathway and induced the HIF1α signal mediated PD-L1 up-regulation. Targeting SRSF10 combined with anti-PD-L1 therapy showed promising efficacy.

New and Old Key Players in Liver Cancer.

Liver cancer represents a major health problem worldwide with growing incidence and high mortality, hepatocellular carcinoma (HCC) being the most frequent. Hepatocytes are likely the cellular origin of most HCCs through the accumulation of genetic alterations, although hepatic progenitor cells (HPCs) might also be candidates in specific cases, as discussed here. HCC usually develops in a context of chronic inflammation, fibrosis, and cirrhosis, although the role of fibrosis is controversial. The interplay between hepatocytes, immune cells and hepatic stellate cells is a key issue. This review summarizes critical aspects of the liver tumor microenvironment paying special attention to platelets as new key players, which exert both pro- and anti-tumor effects, determined by specific contexts and a tight regulation of platelet signaling. Additionally, the relevance of specific signaling pathways, mainly HGF/MET, EGFR and TGF-β is discussed. HGF and TGF-β are produced by different liver cells and platelets and regulate not only tumor cell fate but also HPCs, inflammation and fibrosis, these being key players in these processes. The role of C3G/RAPGEF1, required for the proper function of HGF/MET signaling in HCC and HPCs, is highlighted, due to its ability to promote HCC growth and, regulate HPC fate and platelet-mediated actions on liver cancer.

GSDME has prognostic and immunotherapeutic significance in residual hepatocellular carcinoma after insufficient radiofrequency ablation

BackgroundHeat stress can induce programmed cell death (PCD). Pyroptosis is a gasdermin-mediated PCD. This study hypothesized that insufficient radiofrequency ablation (IRFA) induced pyroptosis in hepatocellular carcinoma (HCC) and investigated its underlying mechanism and clinical significance. MethodsThermostatic water bath was used to stimulate IRFA in vitro. Cell viability was assessed by MTT assay. IL-1β and HMGB1 were measured by ELISA assay. LDH level was measured by LDH cytotoxicity detection kit. Permeability of cell membrane was assessed by Hoechst33342/PI fluorescence staining. RNA expression was evaluated by qRT-PCR, and protein was assessed by Western Blotting or immunofluorescence or immunohistochemistry. Gene expression with clinicopathological characteristics from HCC patients treated by RFA were analyzed for associations between GSDME expression and prognosis. ResultsOur study revealed that IRFA induced pyroptosis in HCCLM3 and HepG2 cells. GSDME, rather than GSDMD, was cleaved in heat stress-induced pyroptosis in HCCLM3 and HepG2 cells due to caspase-3 activation. However, GSDME overexpression promoted HCC growth in vivo and predicted poor PFS and OS in HCC patients treated by RFA. Heat stress modulated gene expression related to PD-L1 signaling and caspase inhibitors inhibited heat-induced PD-L1 expression in residual HCC after IRFA. Gsdme overexpression caused resistance to PD-L1 inhibitor in residual HCC after IRFA by increasing infiltrating of CD3+PD-1+ or CD3+CTLA-4+ exhausted T cells. ConclusionsThis study indicated that GSDME could serve as a potential prognostic biomarker and help to prescribe personalized sequential immunotherapy for HCC patients receiving RFA.

Low glucose metabolite 3-phosphoglycerate switches PHGDH from serine synthesis to p53 activation to control cell fate.

Glycolytic intermediary metabolites such as fructose-1,6-bisphosphate can serve as signals, controlling metabolic states beyond energy metabolism. However, whether glycolytic metabolites also play a role in controlling cell fate remains unexplored. Here, we find that low levels of glycolytic metabolite 3-phosphoglycerate (3-PGA) can switch phosphoglycerate dehydrogenase (PHGDH) from cataplerosis serine synthesis to pro-apoptotic activation of p53. PHGDH is a p53-binding protein, and when unoccupied by 3-PGA interacts with the scaffold protein AXIN in complex with the kinase HIPK2, both of which are also p53-binding proteins. This leads to the formation of a multivalent p53-binding complex that allows HIPK2 to specifically phosphorylate p53-Ser46 and thereby promote apoptosis. Furthermore, we show that PHGDH mutants (R135W and V261M) that are constitutively bound to 3-PGA abolish p53 activation even under low glucose conditions, while the mutants (T57A and T78A) unable to bind 3-PGA cause constitutive p53 activation and apoptosis in hepatocellular carcinoma (HCC)cells, even in the presence of high glucose. In vivo, PHGDH-T57A induces apoptosis and inhibits the growth of diethylnitrosamine-induced mouse HCC, whereas PHGDH-R135W prevents apoptosis and promotes HCC growth, and knockout of Trp53 abolishes these effects above. Importantly, caloric restriction that lowers whole-body glucose levels can impede HCC growth dependent on PHGDH. Together, these results unveil a mechanism by which glucose availability autonomously controls p53 activity, providing a new paradigm of cell fate control by metabolic substrate availability.

N6 -methyladenosine-modified FAM111A-DT promotes hepatocellular carcinoma growth via epigenetically activating FAM111A.

As an epitranscriptomic modulation manner, N6 -methyladenosine (m6 A) modification plays important roles in various diseases, including hepatocellular carcinoma (HCC). m6 A modification affects the fate of RNAs. The potential contributions of m6 A to the functions of RNA still need further investigation. In this study, we identified long noncoding RNA FAM111A-DT as an m6 A-modified RNA and confirmed three m6 A sites on FAM111A-DT. The m6 A modification level of FAM111A-DT was increased in HCC tissues and cell lines, and increased m6 A level was correlated with poor survival of HCC patients. m6 A modification increased the stability of FAM111A-DT transcript, whose expression level showed similar clinical relevance to that of the m6 A level of FAM111A-DT. Functional assays found that only m6 A-modified FAM111A-DT promoted HCC cellular proliferation, DNA replication, and HCC tumor growth. Mutation of m6 A sites on FAM111A-DT abolished the roles of FAM111A-DT. Mechanistic investigations found that m6 A-modified FAM111A-DT bound to FAM111A promoter and also interacted with m6 A reader YTHDC1, which further bound and recruited histone demethylase KDM3B to FAM111A promoter, leading to the reduction of the repressive histone mark H3K9me2 and transcriptional activation of FAM111A. The expression of FAM111A was positively correlated with the m6 A level of FAM111A-DT, and the expression of methyltransferase complex, YTHDC1, and KDM3B in HCC tissues. Depletion of FAM111A largely attenuated the roles of m6 A-modified FAM111A-DT in HCC. In summary, the m6 A-modified FAM111A-DT/YTHDC1/KDM3B/FAM111A regulatory axis promoted HCC growth and represented a candidate therapeutic target for HCC.

The ubiquitin-like protein FAT10 in hepatocellular carcinoma cells limits the efficacy of anti-VEGF therapy

IntroductionThe efficacy of anti-vascular endothelial growth factor (VEGF) therapy is limited. However, the key factors involved in limiting the efficacy of anti-VEGF therapy and the underlying mechanisms remain unclear. ObjectivesTo investigate the effects and mechanisms of human leukocyte antigen F locus-adjacent transcript 10 (FAT10), a ubiquitin-like protein, in limiting the efficacy of anti-VEGF therapy in hepatocellular carcinoma (HCC) cells. MethodsFAT10 was knocked out in HCC cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 technology. Bevacizumab (BV), an anti-VEGF monoclonal antibody, was used to evaluate the efficacy of anti-VEGF therapy in vivo. Mechanisms of FAT10 action were assessed by RNA sequencing, glutathione S-transferase pulldown assays and in vivo ubiquitination assays. ResultsFAT10 accelerated VEGF-independent angiogenesis in HCC cells which limited BV efficacy and BV-aggravated hypoxia and inflammation promoted FAT10 expression. FAT10 overexpression increased levels of proteins involved in several signaling pathways in HCC cells, resulting in upregulation of VEGF and multiple non-VEGF proangiogenic factors. Upregulation of multiple FAT10-mediated non-VEGF signals compensated for the inhibition of VEGF signaling by BV, enhancing VEGF-independent angiogenesis and promoting HCC growth. ConclusionsOur preclinical findings identify FAT10 in HCC cells as a key factor limiting the efficacy of anti-VEGF therapy and elucidate its underlying mechanisms. This study provides new mechanistic insights into the development of antiangiogenic therapies.

CENPA-driven STMN1 Transcription Inhibits Ferroptosis in Hepatocellular Carcinoma.

The growing knowledge of ferroptosis has suggested the regulatory role of ferroptosis in hepatocellular carcinoma (HCC), but the pertinent molecular mechanisms remain unclear. Herein, this study investigated the mechanistic basis of ferroptosis-related genes (ferrGenes) in the growth of HCC. Differentially expressed human ferrGenes and tumor-related transcription factors (TFs) were obtained from the The Cancer Genome Atlas (TCGA) dataset and the GTEx dataset. Spearman method-based correlation analysis were conducted to construct TF-ferrGene coexpression regulatory network. Key genes associated with prognosis were singled out with Lasso regression and multivariate Cox analysis to construct the prognostic risk model. Then the accuracy and independent prognostic ability of the model were evaluated. Expression of CENPA and STMN1 was determined in clinical HCC tissues and HCC cells, and their binding was analyzed with dual-luciferase and chromatin immunoprecipitation (ChIP) assays. Furthermore, ectopic expression and knockdown assays were performed in HCC cells to assess the effect of CENPA and STMN1 on ferroptosis and malignant phenotypes. The prognostic risk model constructed based on the eight TF-ferrGene regulatory network-related genes accurately predicted the prognosis of HCC patients. It was strongly related to the clinical characteristics of HCC patients. Moreover, CENPA/STMN1 might be a key TF-ferrGene regulatory network in ferroptosis of HCC. CENPA and STMN1 were overexpressed in HCC tissues and cells. Additionally, CENPA facilitated STMN1 transcription by binding to STMN1 promoter, thus facilitating the malignant phenotypes and suppressing the ferroptosis of HCC cells. Taken together, CENPA curbs the ferroptosis of HCC cells by upregulating STMN1 transcription, thereby promoting HCC growth.

The cancer-testis lncRNA LINC01977 promotes HCC progression by interacting with RBM39 to prevent Notch2 ubiquitination

Cancer-testis genes are involved in the occurrence and development of cancer, but the role of cancer-testis-associated lncRNAs (CT-lncRNAs) in hepatocellular carcinoma (HCC) remains to be explored. Here, we discovered a novel CT-lncRNA, LINC01977, based on the Genotype-Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) databases. LINC01977 was exclusively expressed in testes and highly expressed in HCC. High LINC01977 levels correlated with poorer overall survival (OS) in individuals with HCC. Functional assays showed that LINC01977 promoted HCC growth and metastasis in vitro and in vivo. Mechanistically, LINC01977 directly bound to RBM39 to promote the further entry of Notch2 into the nucleus, thereby preventing the ubiquitination and degradation of Notch2. Furthermore, the RNA binding protein IGF2BP2, one of the m6A modification readers, enhanced the stability of LINC01977, resulting in its high level in HCC. Therefore, the data suggest that LINC01977 interacts with RBM39 and promotes the progression of HCC by inhibiting Notch2 ubiquitination and degradation, indicating that LINC01977 may be a potential biomarker and therapeutic target for HCC patients.

Abstract 2869: The role of regulatory T cells in NASH-mediated HCC development

Abstract Objective Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, ranging from simple steatosis to an advanced form of non-alcoholic steatohepatitis (NASH) without effective treatment. NASH can progress to liver cirrhosis and hepatocellular carcinoma (HCC) in association with increased production of liver-resident regulatory T cells (Treg). This study aims to identify the role of Tregs in NASH-driven HCC development. Methods Wild-type C57BL/6 mice were fed with a choline-low high-fat high-sugar diet (CL-HFS), which recapitulates the Western-pattern diet, to induce NASH, then received transplantation of oncogenic hepatocytes by intrasplenic injection to induce an orthotopic HCC. Wild-type mice fed with a normal diet were used for control. Hepatic immune cell profiling was characterized by flow cytometry to identify the cellular basis mediating CL-HFS-induced NASH. The metagenomic assay was applied to identify NASH-relevant gut microbiota in the liver tissue. Results CL-HFS caused successful development of NASH and liver fibrosis in wild-type mice. Fast orthotopic tumor growth was detected in NASH-bearing mice compared to wild-type mice. Strongly positive staining of glypican 3 (GPC3), increased frequency of Tregs, and increased expression of Col1α1, Col4α1, and α-SMA were detected in livers and tumors from NASH-bearing mice compared to wild-type mice by IHC and flow cytometry. Metagenomic assay detected the lipid-producing bacterial genus Bacillus in NASH liver, which was demonstrated to increase the frequency of Tregs in healthy spleen cells. Conclusion CL-HFS-caused NASH promotes HCC growth likely by increasing liver-resident Tregs and liver-infiltrating Bacillus. Keywords: Non-alcoholic fatty liver disease (NAFLD), Non-alcoholic steatohepatitis (NASH), choline-low high-fat high-sugar diet (CL-HFS), regulatory T cells (Tregs), Bacillus, hepatocellular carcinoma (HCC). Citation Format: Ming Yang, Eric T. Kimchi, Kevin F. Staveley-O’Carroll, Guangfu Li. The role of regulatory T cells in NASH-mediated HCC development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2869.

PHGDH arginine methylation by PRMT1 promotes serine synthesis and represents a therapeutic vulnerability in hepatocellular carcinoma

Serine synthesis is crucial for tumor growth and survival, but its regulatory mechanism in cancer remains elusive. Here, using integrative metabolomics and transcriptomics analyses, we show a heterogeneity between metabolite and transcript profiles. Specifically, the level of serine in hepatocellular carcinoma (HCC) tissues is increased, whereas the expression of phosphoglycerate dehydrogenase (PHGDH), the first rate-limiting enzyme in serine biosynthesis pathway, is markedly downregulated. Interestingly, the increased serine level is obtained by enhanced PHGDH catalytic activity due to protein arginine methyltransferase 1 (PRMT1)-mediated methylation of PHGDH at arginine 236. PRMT1-mediated PHGDH methylation and activation potentiates serine synthesis, ameliorates oxidative stress, and promotes HCC growth in vitro and in vivo. Furthermore, PRMT1-mediated PHGDH methylation correlates with PHGDH hyperactivation and serine accumulation in human HCC tissues, and is predictive of poor prognosis of HCC patients. Notably, blocking PHGDH methylation with a TAT-tagged nonmethylated peptide inhibits serine synthesis and restrains HCC growth in an HCC patient-derived xenograft (PDX) model and subcutaneous HCC cell-derived xenograft model. Overall, our findings reveal a regulatory mechanism of PHGDH activity and serine synthesis, and suggest PHGDH methylation as a potential therapeutic vulnerability in HCC.

Squalene epoxidase promotes hepatocellular carcinoma development by activating STRAP transcription and TGF-β/SMAD signalling.

Squalene epoxidase (SQLE) is a key enzyme involved in cholesterol biosynthesis, but growing evidence also reveals that SQLE is abnormally expressed in some types of malignant tumours, even though the underlying mechanism remains poorly understood. Bioinformatics analysis and RNA sequencing were applied to detect differentially expressed genes in clinical hepatocellular carcinoma (HCC). MTT, colony formation, AnnexinV-FITC/PI, EdU, wound healing, transwell, western blot, qRT-PCR, IHC, F-actin, RNA-sequencing, dual-luciferase reporters, and H&E staining were used to investigate the pharmacological effects and possible mechanisms of SQLE. SQLE expression was specifically elevated in HCC, correlating with poor clinical outcomes. SQLE significantly promoted HCC growth, epithelial-mesenchymal transition, and metastasis both in vitro and in vivo. RNA sequencing and functional experiments revealed that the protumourigenic effect of SQLE on HCC was closely related to the activation of TGF-β/SMAD signalling, but the stimulatory effect of SQLE on TGF-β/SMAD signalling and HCC development is critically dependent on STRAP. SQLE expression is well correlated with STRAP in HCC, and further, to amplify TGF-β/SMAD signalling, SQLE even transcriptionally increased STRAP gene expression mediated by AP-2α. Finally, as a chemical inhibitor of SQLE, NB-598 markedly inhibited HCC cell growth and tumour development. Taken together, SQLE serves as a novel oncogene in HCC development by activating TGF-β/SMAD signalling. Targeting SQLE could be useful in drug development and therapy for HCC.

SLC12A5 promotes hepatocellular carcinoma growth and ferroptosis resistance by inducing ER stress and cystine transport changes.

Hepatocellular carcinoma (HCC) has a poor prognosis and new effective treatments are needed. SLC12A5 plays important roles in multiple complex pathological states and is overexpressed in a variety of malignancies. However, the effects of SLC12A5 in HCC have not been determined. SLC12A5 expression was assessed by immunostaining and western blotting. A cell viability assay was used to detect cell proliferation. Flow cytometry was used to evaluate the intracellular calcium concentration and cell cycle. Ferroptosis was detected by transmission electron microscopy, lipid peroxidation, and glutathione assays. Subcutaneous tumor formation experiments were used to validate the tumorigenic effect of SLC12A5 in vivo. RNA-seq was used to evaluate the molecular mechanisms underlying the effects of SLC12A5. The therapeutic efficacy of targeting SLC12A5 was assessed in a patient-derived xenograft (PDX) model. High SLC12A5 expression was strongly associated with a poor clinical prognosis and promoted HCC growth. Mechanistically, SLC12A5 promoted ER stress to enhance calcium release and upregulated PNCK expression levels. Concomitantly, PNCK was significantly activated by calcium ions released from the ER. PNCK activated and induced the phosphorylation of PI3K/AKT/mTOR pathway components. Furthermore, SLC12A5 inhibited ferroptosis in HCC by upregulating the expression of xCT, a cystine transporter. High SLC12A5 levels were correlated with a poor prognosis, promoted tumorigenesis, and inhibited ferroptosis in HCC. These findings suggested that SLC12A5 is a therapeutic target and provide insight into the link between ER stress and ferroptosis in HCC.