Angiogenesis In Hepatocellular Carcinoma Research Articles

TCBIR/CD320: a potential therapeutic target upregulated in endothelial cells and associated with immune cell infiltration in liver hepatocellular carcinoma

CD320, which is a transmembrane protein responsible for facilitating the absorption of vitamin B12, plays a key role in this process. However, the relationships between CD320 and immune cell infiltration levels remain unclear, with limited studies investigating the diagnostic and prognostic significance of CD320 in hepatocellular carcinoma. We used various databases, including the TIMER, GEPIA, UALCAN and TCGA databases to investigate the expression levels of CD320 in hepatocellular carcinoma. Subsequently, we analyzed the prognosis of hepatocellular carcinoma patients with different expression levels of CD320. Furthermore, we also performed Western blot, immunohistochemistry, and immunofluorescence analyses to validate the results of the database analysis. Finally, the functions of CD320 in hepatocellular carcinoma were also confirmed via relevant cell experiments and angiogenesis assays. We found that CD320 expression was significantly upregulated in tumor vascular endothelial cells. Moreover, the knockdown of CD320 led to a reduction in angiogenesis in endothelial cells. Increased expression of CD320 was also correlated with a poor prognosis in patients with hepatocellular carcinoma, which suggested that CD320 may be a potential prognostic marker. Finally, TIMER analysis demonstrated that the infiltration of six immune cell types was significantly associated with high expression levels of CD320 in hepatocellular carcinoma. Herein, we demonstrated that CD320 may play an important role in angiogenesis in hepatocellular carcinoma. These findings suggested that CD320 may be a potential clinical prognostic marker and immunotherapy target for hepatocellular carcinoma.

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.

CircDCAF8 promotes the progression of hepatocellular carcinoma through miR-217/NAP1L1 Axis, and induces angiogenesis and regorafenib resistance via exosome-mediated transfer

BackgroundCircular RNAs (circRNAs), which are a new type of single-stranded circular RNA, have significant involvement in progression of many diseases, including tumors. Currently, multiple circRNAs have been identified in hepatocellular carcinoma (HCC). Our study aims to investigate the function and mechanism of circDCAF8 in HCC.MethodsThe expression of circDCAF8 (hsa_circ_0014879) in HCC and para-carcinoma tissue samples was determined using quantitative real-time polymerase chain reaction (qRT-PCR). The biological function of circDCAF8 in HCC was confirmed by experiments conducted both in vitro and in vivo. And the relationship between circDCAF8, miR-217 and NAP1L1 was predicted by database and verified using qRT-PCR, RNA-binding protein immunoprecipitation (RIP) and dual-luciferase reporter assays. Exosomes isolated from HCC cells were utilized to assess the connection of exosomal circDCAF8 with HCC angiogenesis and regorafenib resistance.ResultsCircDCAF8 is upregulated in HCC tissues and cell lines, and is linked to an unfavourable prognosis for HCC patients. Functionally, circDCAF8 was proved to facilitate proliferation, migration, invasion and Epithelial-Mesenchymal Transformation (EMT) in HCC cells. Animal examinations also validated the tumor-promoting characteristics of circDCAF8 on HCC. Besides, exosomal circDCAF8 promoted angiogenesis in HUVECs. Mechanistically, circDCAF8 interacted with miR-217 and NAP1L1 was a downstream protein of miR-217. CircDCAF8 promoted NAP1L1 expression by sponging miR-217. In addition, exosomes may transfer circDCAF8 from regorafenib-resistant HCC cells to sensitive cells, where it would confer a resistant phenotype.ConclusionCircDCAF8 facilitates HCC proliferation and metastasis via the miR-217/NAP1L1 axis. Meanwhile, circDCAF8 can promote angiogenesis and drive resistance to regorafenib, making it a viable therapeutic target for HCC patients.

Mitochondria-Derived Vesicles, Sterile Inflammation, and Pyroptosis in Liver Cancer: Partners in Crime or Innocent Bystanders?

Alterations in cellular signaling, chronic inflammation, and tissue remodeling contribute to hepatocellular carcinoma (HCC) development. The release of damage-associated molecular patterns (DAMPs) upon tissue injury and the ensuing sterile inflammation have also been attributed a role in HCC pathogenesis. Cargoes of extracellular vesicles (EVs) and/or EVs themselves have been listed among circulating DAMPs but only partially investigated in HCC. Mitochondria-derived vesicles (MDVs), a subpopulation of EVs, are another missing link in the comprehension of the molecular mechanisms underlying the onset and progression of HCC biology. EVs have been involved in HCC growth, dissemination, angiogenesis, and immunosurveillance escape. The contribution of MDVs to these processes is presently unclear. Pyroptosis triggers systemic inflammation through caspase-dependent apoptotic cell death and is implicated in tumor immunity. The analysis of this process, together with MDV characterization, may help capture the relationship among HCC development, mitochondrial quality control, and inflammation. The combination of immune checkpoint inhibitors (i.e., atezolizumab and bevacizumab) has been approved as a synergistic first-line systemic treatment for unresectable or advanced HCC. The lack of biomarkers that may allow prediction of treatment response and, therefore, patient selection, is a major unmet need. Herein, we overview the molecular mechanisms linking mitochondrial dysfunction, inflammation, and pyroptosis, and discuss how immunotherapy targets, at least partly, these routes.

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.

Extracellular Vesicle-Related Non-Coding RNAs in Hepatocellular Carcinoma: An Overview.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. It is a major public health problem worldwide, and it is often diagnosed at advanced stages, when no effective treatment options are available. Extracellular vesicles (EVs) are nanosized double-layer lipid vesicles containing various biomolecule cargoes, such as lipids, proteins, and nucleic acids. EVs are released from nearly all types of cells and have been shown to play an important role in cell-to-cell communication. In recent years, many studies have investigated the role of EVs in cancer, including HCC. Emerging studies have shown that EVs play primary roles in the development and progression of cancer, modulating tumor growth and metastasis formation. Moreover, it has been observed that non-coding RNAs (ncRNAs) carried by tumor cell-derived EVs promote tumorigenesis, regulating the tumor microenvironment (TME) and playing critical roles in the progression, angiogenesis, metastasis, immune escape, and drug resistance of HCC. EV-related ncRNAs can provide information regarding disease status, thus encompassing a role as biomarkers. In this review, we discuss the main roles of ncRNAs present in HCC-derived EVs, including micro(mi) RNAs, long non-coding (lnc) RNAs, and circular (circ) RNAs, and their potential clinical value as biomarkers and therapeutic targets.

HOXD3 promotes the migration and angiogenesis of hepatocellular carcinoma via modifying hepatocellular carcinoma cells exosome-delivered CCR6 and regulating chromatin conformation of CCL20

Angiogenesis plays an essential role in the microenvironment of hepatocellular carcinoma (HCC). HOXD3 is involved in the metastasis and invasion of HCC cells; Whereas the underlying molecular mechanisms in the microenvironment of HCC remain unknown. Wound healing, transwell invasion, tube formation and spheroid sprouting assays were carried out to identify the effects of HCC-HOXD3-exosomes and genes on the migration of HCC cells. ChIP–PCR was applied to test the binding region of HOXD3 on CCR6, Med15, and CREBBP promoter. Exosome isolation and mRNA-seq were applied to examine the morphological characteristics of exosomes and the contained mRNA in exosomes. Co-IP and Immunofluorescence assays were used to demonstrate the role of CREBBP in the chromatin conformation of CCL20. The nude mice were used to identify the function of genes in regulating migration of HCC in vivo. In this study, integrated cellular and bioinformatic analyses revealed that HOXD3 targeted the promoter region of CCR6 and induced its transcription. CCR6 was delivered by exosomes to endothelial cells and promoted tumour migration. Overexpression of CCR6 promoted metastasis, invasion in HCCs and angiogenesis in endothelial cells (ECs), whereas its downregulation suppressed these functions. The role of HOXD3 in the metastasis and invasion of HCC cells was reversed after the suppression of CCR6. Furthermore, CCL20 was demonstrated as the ligand of CCR6, and its high expression was found in HCC tissues and cells, which was clinically associated with the poor prognosis of HCC. Mechanistically, HOXD3 targets the promoter regions of CREBBP and Med15, which affect CCL20 chromatin conformation by regulating histone acetylation and expression of Pol II to enhance the migration of HCCs. This study demonstrated the function of the HOXD3–CREBBP/Med15–CCL20–CCR6 axis in regulating invasion and migration in HCC, thus providing new therapeutic targets for HCC.

Multi-b-value DWI to evaluate the synergistic antiproliferation and anti-heterogeneity effects of bufalin plus sorafenib in an orthotopic HCC model

BackgroundMulti-b-value diffusion-weighted imaging (DWI) with different postprocessing models allows for evaluating hepatocellular carcinoma (HCC) proliferation, spatial heterogeneity, and feasibility of treatment strategies. We assessed synergistic effects of bufalin+sorafenib in orthotopic HCC-LM3 xenograft nude mice by using intravoxel incoherent motion (IVIM), diffusion kurtosis imaging (DKI), a stretched exponential model (SEM), and a fractional-order calculus (FROC) model.MethodsTwenty-four orthotopic HCC-LM3 xenograft mice were divided into bufalin+sorafenib, bufalin, sorafenib treatment groups, and a control group. Multi-b-value DWI was performed using a 3-T scanner after 3 weeks’ treatment to obtain true diffusion coefficient Dt, pseudo-diffusion coefficient Dp, perfusion fraction f, mean diffusivity (MD), mean kurtosis (MK), distributed diffusion coefficient (DDC), heterogeneity index α, diffusion coefficient D, fractional order parameter β, and microstructural quantity μ. Necrotic fraction (NF), standard deviation (SD) of hematoxylin-eosin staining, and microvessel density (MVD) of anti-CD31 staining were evaluated. Correlations of DWI parameters with histopathological results were analyzed, and measurements were compared among four groups.ResultsIn the final 22 mice, f positively correlated with MVD (r = 0.679, p = 0.001). Significantly good correlations of MK (r = 0.677), α (r = -0.696), and β (r= -0.639) with SD were observed (all p < 0.010). f, MK, MVD, and SD were much lower, while MD, α, β, and NF were higher in bufalin plus sorafenib group than control group (all p < 0.050).ConclusionEvaluated by IVIM, DKI, SEM, and FROC, bufalin+sorafenib was found to inhibit tumor proliferation and angiogenesis and reduce spatial heterogeneity in HCC-LM3 models.Relevance statementMulti-b-value DWI provides potential metrics for evaluating the efficacy of treatment in HCC.Key points• Bufalin plus sorafenib combination may increase the effectiveness of HCC therapy.• Multi-b-value DWI depicted HCC proliferation, angiogenesis, and spatial heterogeneity.• Multi-b-value DWI may be a noninvasive method to assess HCC therapeutic efficacy.Graphical

Transcription Factor MAZ Potentiates the Upregulated NEIL3-mediated Aerobic Glycolysis, Thereby Promoting Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is characterized by high vascularity and notable abnormality of blood vessels, where angiogenesis is a key process in tumorigenesis and metastasis. The main functions of Nei Like DNA Glycosylase 3 (NEIL3) include DNA alcoholization repair, immune response regulation, nervous system development and function, and DNA damage signal transduction. However, the underlying mechanism of high expression NEIL3 in the development and progression of HCC and whether the absence or silencing of NEIL3 inhibits the development of cancer remain unclear. Therefore, a deeper understanding of the mechanisms by which increased NEIL3 expression promotes cancer development is needed. Expression of NEIL3 and its upstream transcription factor MAZ in HCC tumor tissues was analyzed in bioinformatics efforts, while validation was done by qRT-PCR and western blot in HCC cell lines. The migration and tube formation capacity of HUVEC cells were analyzed by Transwell and tube formation assays. Glycolytic capacity was analyzed by extracellular acidification rate, glucose uptake, and lactate production levels. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter gene assays were utilized to investigate specific interactions between MAZ and NEIL3. NEIL3 and MAZ were substantially upregulated in HCC tissues and cells. NEIL3 was involved in modulating the glycolysis pathway, suppression of which reversed the stimulative impact of NEIL3 overexpression on migration and angiogenesis in HUVEC cells. MAZ bound to the promoter of NEIL3 to facilitate NEIL3 transcription. Silencing MAZ reduced NEIL3 expression and suppressed the glycolysis pathway, HUVEC cell migration, and angiogenesis. MAZ potentiated the upregulated NEIL3-mediated glycolysis pathway and HCC angiogenesis. This study provided a rationale for the MAZ/NEIL3/glycolysis pathway as a possible option for anti-angiogenesis therapy in HCC.

Eriodictyol inhibits the motility, angiogenesis and tumor growth of hepatocellular carcinoma via NLRP3 inflammasome inactivation

NLRP3 involves in the development of hepatocellular carcinoma (HCC). Eriodictyol has shown its inhibitory effect on HCC cell proliferation. However, the underlying mechanism of eriodictyol in HCC is still unclear. This study aimed to explore the effect of and mechanism of eriodictyol on HCC. In this study, compared with eriodictyol (0 μM) group, eriodictyol significantly suppressed HepG2 cells (eriodictyol of 25, 50 and 100 μM) and Huh-7 cells (eriodictyol of 50 and 100 μM) viability, invasion, tube formation, metastasis-related genes MMP3, MMP16 and angiogenesis regulator VEGFA expressions with IC50 of 45.63 μM and 78.26 μM in vitro, respectively. Besides, eriodictyol significantly repressed NLRP3 expression, and reduced the protein levels of NLRP3 inflammasome-related proteins, adapter protein ASC, caspase-1, interleukin (IL)-18, and IL-1β in HepG2 (eriodictyol of 25, 50 and 100 μM) and Huh-7 cells (eriodictyol of 50 and 100 μM), respectively. Meanwhile, compared with control group, NLRP3 overexpression reversed the anti-metastatic effects of 100 μM eriodictyol on HCC cells invasion, tube formation, and metastasis-related genes MMP3, MMP16 and angiogenesis regulator VEGFA expressions, whereas NLRP3 knockdown enhanced the anti-metastatic effects of 100 μM eriodictyol on HCC cells. In vivo, compared with control group, eriodictyol significantly reduced the tumor growth, liver damage, inhibited the activation of NLRP3 inflammasome, and improved liver function, whereas NLRP3 overexpressing neutralized the anti-tumor effects of eriodictyol and degraded liver function. Hence, eriodictyol inhibited HCC cell viability, motility, angiogenesis and tumor growth via NLRP3 inflammasome inactivation both in vitro and in vivo.

Exosome-derived miR-23a-5p inhibits HCC proliferation and angiogenesis by regulating PRDX2 expression: MiR-23a-5p/PRDX2 axis in HCC progression

microRNAs (miRNAs) are closely related to the progression of hepatocellular carcinoma (HCC). Cancer-derived exosomes play an essential role in the establishment of the HCC microenvironment. However, the possible effects and underlying mechanisms of exosome (exo) microRNA-23a-5p (miR-23a-5p) in the progression of HCC remain unknown. In this study, we aimed to determine the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression and to investigate whether exo miR-23a-5p levels can serve as an indicator of the prognosis of transarterial chemoembolization in patients with HCC. Our findings illustrated that miR-23a-5p was downregulated in exosomes separated from the serum of HCC patients and that miR-23a-5p carried by exosomes inhibited HCC cell proliferation and angiogenesis. Mechanistically, miR-23a-5p negatively targeted peroxiredoxin-2 (PRDX2). Functionally, PRDX2 overexpression relieved exosome-induced inhibition of HCC cell proliferation and angiogenesis by promoting vascular endothelial growth factor (VEGF) expression. In conclusion, Exo miR-23a-5p inhibited HCC proliferation and angiogenesis by regulating PRDX2 expression. Our results revealed the role and specific molecular mechanism of exo miR-23a-5p in regulating HCC progression.

Hepatitis B Virus-Encoded MicroRNA (HBV-miR-3) Inhibits FIH-1 Expression to Promote Tumor Angiogenesis in HBV-Related Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a solid tumor with a rich blood supply, and anti-angiogenesis has important clinical significance. Hepatitis B Virus-Encoded MicroRNA 3 (HBV-miR-3) has recently been reported to be involved in HCC development. In this study, we aim to elucidate the role of HBV-miR-3 in promoting HBV-related HCC angiogenesis through Factor Inhibiting Hypoxia-inducible factor 1 (FIH-1). By analyzing HBV-related HCC tissue samples, we found that high expression of HBV-miR-3 was associated with poor overall survival and HBV-miR-3 expression was significantly correlated with VEGFR2 and FIH-1 expressions. In vitro, HBV-miR-3 agomir repressed FIH-1 expression and promoted HIF-1α/VEGFA signaling activation in HepG2 cells, resulting in increased HUVEC lumen formation in HepG2-HUVEC co-culture model. Conversely, HBV-miR-3 antagomir induced FIH-1 expression and inhibited HIF-1α/VEGFA signaling activation in HepG2.2.15 cells, resulting in decreased HUVEC lumen formation in HepG2.2.15-HUVEC co-culture model. The effect of HBV-miR-3 to HCC angiogenesis was also confirmed by a mouse tumor bearing model. We also confirmed that HBV-miR-3 repressed FIH-1 expression via targeting the 3'-UTR of FIH-1 mRNA by luciferase activity assay. HBV-miR-3 was related to HCC patients' overall survival and it promoted angiogenesis by repressing FIH-1 expression. HBV-miR-3 may be a new marker for predicting prognosis and a novel target for anti-angiogenic treatment of HBV-related HCC.

Tripartite motif protein 6 promotes hepatocellular carcinoma progression via multiple pathways.

Hepatocellular carcinoma (HCC) ranks among the most prevalent malignancies worldwide and the third leading cause of cancer-related death. The TRIM (tripartite motif-containing) protein family members had been reported to be involved in carcinogenesis and tumor progression. Here we aimed to explore the expression profile of TRIM6 in HCC and investigate its clinical significance as well as underlying mechanisms. We retrospectively enrolled 138 HCC patients that underwent surgical resection in our hospital and tested protein expression level of TRIM6 through immunohistochemical staining. The correlation between TRIM6 and patients' characteristics was assessed by Chi-square test. Log-rank test and Cox hazard regression test were conducted for univariate and multivariate survival analyses, respectively. Two human HCC cell lines, Huh7 and Hep3B, were subjected for knockdown and overexpression assays, followed by phonotype tests including proliferation and invasion. Nude mice were used to generate xenograft model to validate our findings in vivo. TRIM6 was highly expressed in HCC specimen compared to nontumorous liver tissues. Higher TRIM6 expression was correlated with larger tumor size, later tumor stage, and worse prognosis. According to the cellular experiments, TRIM6-knockdown resulted in decreased expression of cyclin B1, c-Myc, Snail, MMP2, and VEGF-A. Consistently, TRIM6-knockdown led to impaired HCC proliferation, invasion, and angiogenesis. In contrast, TRIM6 overexpression showed opposite effects. Finally, the oncogenic role of TRIM6 in HCC was validated by in vivo mice experiments. TRIM6 can serve as a novel prognostic factor for HCC, which functions by multiple signaling pathways.

ETV4 facilitates angiogenesis in hepatocellular carcinoma by upregulating MMP14 expression

Abnormal vascularization plays a crucial role in cell proliferation, tumor invasion and metastasis of hepatocellular carcinoma (HCC). It has been reported that ETV4 functions as an oncogenic gene in driving the carcinogenesis and progression, and promoting invasion and metastasis of HCC. However, the function of ETV4 on angiogenesis in HCC remains unclear. In the current study, immunohistochemistry showed that knockdown of ETV4 reduced angiogenesis in HCC xenograft tumor tissues. In vitro, tube formation assay verified that ETV4 expression promoted angiogenesis through simulating the angiogenic environment in HCC cells. Transcriptome sequencing indicated that MMP14 was one of the differentially expressed genes enriched in angiogenesis process. Subsequently, it was confirmed that MMP14 was regulated by ETV4 at the transcription level in HCC cells, clinical tissue samples and online databases. Further, we demonstrated that MMP14 induced angiogenesis in ETV4-mediated HCC microenvironment. Collectively, this research further reveals the biological mechanism of ETV4 in promoting the migration and invasion of HCC, and provides novel mechanistic insights and strategic guidance for anti-angiogenic therapy in HCC.

Non-metabolic enzyme function of PKM2 in hepatocellular carcinoma: A review.

Hepatocellular carcinoma (HCC) is one of the most malignant tumors with the highest incidence and mortality in the world, causing a serious burden on society. Pyruvate kinase M2 (PKM2) is one of the principal metabolic enzymes involved in glycolysis. Studies have shown that PKM2 is highly expressed in HCC and can be translocated to the nucleus, where it interacts with various transcription factors and proteins such as hypoxia-inducible factor-1α, sterol regulatory element-binding protein 1a, signal transducer and activator of transcription 3, nuclear factor erythroid 2-like 2 and histone H3, exerting non-metabolic enzyme functions to regulate the cell cycle, proliferation, apoptosis, immune escape, migration, and invasion, as well as HCC angiogenesis and tumor microenvironment. This review is focused on the recent progress of PKM2 interacting with various transcription factors and proteins affecting the onset and development of HCC, as well as natural drugs and noncoding RNA impacting diverse biological functions of liver cancer cells by regulating PKM2 non-metabolic enzyme functions, thereby providing valuable directions for the prognosis improvement and molecular targeted therapy of HCC in the future.

Endothelial DGKG promotes tumor angiogenesis and immune evasion in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is among the most prevalent and lethal cancers worldwide. The tumor microenvironment (TME) contributes to the poor response of patients with HCC to current therapies, while tumor vascular endothelial cells (ECs) are fundamental TME components that significantly contribute to tumor progression. However, the specific functions and mechanisms of tumor vascular ECs in HCC remain unclear. We screened and validated diacylglycerol kinase gamma (DGKG) hyper-expression specifically in HCC tumor vascular ECs. Single-cell RNA-sequencing, cytometry by time-of-flight, and invitro and invivo studies were performed to investigate the functions of endothelial DGKG. Multiplexed immunohistochemistry staining and flow cytometry were used to evaluate changes in the TME. Functionally, endothelial DGKG promotes tumor angiogenesis and immunosuppressive regulatory T-cell differentiation in HCC. Of significance, we found that HIF-1α activates DGKG transcription by directly binding to its promoter region under hypoxia. Upregulated DGKG promotes HCC progression by recruiting ubiquitin specific peptidase 16 to facilitate ZEB2 deubiquitination, which increases TGF-β1 secretion, thus inducing tumor angiogenesis and regulatory T-cell differentiation. Importantly, targeting endothelial DGKG potentiated the efficiency of dual blockade of PD-1 and VEGFR-2. Hypoxia-induced EC-specific DGKG hyper-expression promotes tumor angiogenesis and immune evasion via the ZEB2/TGF-β1 axis, suggesting EC-specific DGKG as a potential therapeutic target for HCC. Here, we reported that hypoxia-induced endothelial cell-specific DGKG hyper-expression promotes angiogenesis and immune evasion in HCC by recruiting USP16 for K48-linked deubiquitination and inducing the subsequent stabilization of ZEB2, leading to increased TGF-β1 secretion. Most importantly, endothelial DGKG inhibition greatly improved the efficacy of the dual combination of anti-VEGFR2 and anti-PD-1 treatment in a mouse HCC model, significantly inhibiting the malignant progression of HCC and improving survival. This preclinical study supports the targeting of endothelial DGKG as a potential strategy for precision HCC treatment.

Combination of Phenethyl Isothiocyanate and Dasatinib Inhibits Hepatocellular Carcinoma Metastatic Potential through FAK/STAT3/Cadherin Signalling and Reduction of VEGF Secretion.

Cancerous cells are characterised by their ability to invade, metastasise, and induce angiogenesis. Tumour cells use various molecules that can be targeted to reverse these processes. Dasatinib, a potent Src inhibitor, has shown promising results in treating hepatocellular carcinoma (HCC) in vitro and in vivo. However, its effectiveness is limited by focal adhesion kinase (FAK) activation. Isothiocyanates, on the other hand, are phytochemicals with broad anticancer activity and FAK inhibition capabilities. This study evaluated the synergistic effect of dasatinib and phenethyl isothiocyanate (PEITC) on HCC. The combination was tested using various assays, including MTT, adhesion, scratch, Boyden chamber, chorioallantoic membrane (CAM), and yolk sac membrane (YSM) assays to evaluate the effect of the drug combination on HCC metastatic potential and angiogenesis in vitro and in vivo. The results showed that the combination inhibited the adhesion, migration, and invasion of HepG2 cells and reduced xenograft volume in the CAM assay. Additionally, the combination reduced angiogenesis in vitro, diminishing the growth of vessels in the tube formation assay. The inhibition of FAK/STAT3 signalling led to increased E-cadherin expression and reduced VEGF secretion, reducing HCC metastatic potential. Therefore, a combination of PEITC and dasatinib could be a potential therapeutic strategy for the treatment of HCC.

Astragaloside IV Suppresses the Effects of Hepatocellular Carcinoma Cells on Proliferation, Angiogenesis, and Invasion in Human Umbilical Vein Endothelial Cells by Controlling Exosomes by Inhibiting Rab27a

Hepatocellular carcinoma (HCC) is the third most common cancer worldwide, and invasion and metastasis are the leading causes of death. Astragaloside IV (AS-IV), a kind of traditional Chinese medicine, has been widely used in cancer therapy. Cell Counting Kit-8, tube formation, and Transwell analyses were performed to determine cell proliferation, invasion, and angiogenesis of human umbilical vein endothelial cells (HUVECs) under the treatment of Hep3B or SK-Hep1 cells-derived exosomes or Hep3B or SK-Hep1 cells with or without AS-IV treatment. Then, Rab27a overexpression in Hep3B cells was selected to determine the function of AS-IV on the regulation of cell proliferation, invasion, and angiogenesis of HUVECs. Ultimately, the injection of Hep3B cell-derived exosomes or treatment with AS-IV on nude mice was used to comprehensively investigate AS-IV functions in vivo. Hep3B or SK-Hep1 cells promoted proliferation, invasion, and angiogenesis of HUVECs, which were inhibited by AS-IV therapy. The concentrations of Hep3B and SK-Hep1 cells-derived exosomes were markedly reduced after AS-IV therapy. Meanwhile, Rab27a expression was significantly decreased in SK-Hep1 or Hep3B cells after AS-IV therapy, and it inhibited the effects of AS-IV on Hep3B-induced proliferation, invasion, and angiogenesis of HUVECs. Tumor growth was also suppressed by AS-IV, which was accompanied by the decreased expression levels of Ki67 and CD34. Overall, this study demonstrated that AS-IV inhibits the effects of HCC cell-derived exosomes on proliferation, invasion, and angiogenesis of HUVECs via inhibiting Rab27a expression. Practical Applications. To our knowledge, this is the first study to explore the efficacy of AS-IV against HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs to reveal the underlying mechanism. In this study, CCK-8, Transwell, and tube formation assay detection show that AS-IV has an inhibitory effect on HCC cell-induced proliferation, invasion, and angiogenesis of HUVECs by inhibiting Rab27a, which helps in the development of novel nutraceutical/functional food against HCC progression and thus could improve the quality of life in patients with HCC.

LncRNA MEG3 Inhibits Tumor Progression by Modulating Macrophage Phenotypic Polarization via miR-145-5p/DAB2 Axis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the predominant histological type of primary liver cancer, which ranks sixth among the most common human tumors. Tumor-associated macrophages (TAMs) are an important component of tumor microenvironment (TME) and the M2 macrophage polarization substantially contributes to tumor growth and metastasis. Long non-coding RNA (lncRNA) MEG3 was reported to restrain HCC development. However, whether MEG3 regulates macrophage phenotypic polarization in HCC remains unclear. Bone marrow derived macrophages (BMDMs) were treated with LPS/IFNγ and IL4/IL13 to induce the M1 and M2 macrophage polarization, respectively. M2-polarized BMDMs were simultaneously transfected with adenovirus vector overexpressing MEG3 (Adv-MEG3). Subsequently, M2-polarized BMDMs were cultured for 24 h with serum-free medium, the supernatants of which were harvested as conditioned medium (CM). HCC cell line Huh7 was cultured with CM for 24 h. F4/80+CD68+ and F4/80+CD206+ cell percentages in M1-and M2-polarized BMDMs were calculated using flow cytometry. Huh7 cell migration, invasion and angiogenesis were determined via Transwell assay and tube formation experiment. Nude mice were implanted with Huh7 cells and Adv-MEG3-transfected M2-polarizd BMDMs, and tumor growth and M2 macrophage polarization markers were assessed. The binding between miR-145-5p and MEG3 or disabled-2 (DAB2) was verified by luciferase reporter assay. MEG3 presented lower expression in HCC tissues than in normal controls, and low expression of MEG3 was correlated to poorer prognosis of HCC patients. MEG3 expression was enhanced during LPS/IFNγ-induced M1 polarization, but was reduced during IL4/IL13-induced M2 polarization. MEG3 overexpression inhibited the expression of M2 polarization markers in both M2-polarized BMDMs and mice. Mechanically, MEG3 bound with miR-145-5p to regulate DAB2 expression. Overexpressing MEG3 suppressed M2 polarization-induced HCC cell metastasis and angiogenesis by upregulating DAB2 and inhibited in vivo tumor growth. LncRNA MEG3 curbs HCC development by repressing M2 macrophage polarization via miR-145-5p/DAB2 axis.

Angiogenesis and Hepatocellular Carcinoma: From Molecular Mechanisms to Systemic Therapies.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. The hypervascular nature of the majority of HCCs and the peculiar vascular derangement occurring during liver carcinogenesis underscore the importance of angiogenesis in the development and progression of these tumors. Indeed, several angiogenic molecular pathways have been identified as deregulated in HCC. The hypervascular nature and the peculiar vascularization of HCC, as well as deregulated angiogenic pathways, represent major therapeutic targets. To a large extent, intra-arterial locoregional treatments (transarterial-(chemo)embolization) rely on tumor ischemia caused by embolization of tumor feeding arteries, even though this may represent the "primum movens" of tumor recurrence through the activation of neoangiogenesis. Considering systemic therapies, the currently available tyrosine kinase inhibitors (sorafenib, regorafenib, cabozantinib and lenvatinib) and monoclonal antibodies (ramucirumab and bevacizumab, in combination with the anti-PD-L1, atezolizumab) primarily target, among others, angiogenic pathways. Considering the importance of angiogenesis in the pathogenesis and treatment of liver cancer, in this paper, we aim to review the role of angiogenesis in HCC, addressing the molecular mechanisms, available antiangiogenic therapies and prognostic biomarkers in patients receiving these treatments.