Human Hepatocellular Carcinoma Tissues Research Articles

Sn‐1 Specificity of Lysophosphatidylcholine Acyltransferase‐1 Revealed by a Mass Spectrometry‐Based Assay

AbstractLysophosphatidylcholine acyltransferase‐1 (LPCAT1) plays a critical role in the remodeling of phosphatidylcholines (PCs) in cellular lipidome. However, evidence is scarce regarding its sn‐selectivity, viz. the preference of assembling acyl‐Coenzyme A (CoA) at the C1 or C2‐hydroxyl on a glycerol backbone because of difficulty to quantify the thus‐formed PC sn‐isomers. We have established a multiplexed assay to measure both sn‐ and acyl‐chain selectivity of LPCAT1 toward a mixture of acyl‐CoAs by integrating isomer‐resolving tandem mass spectrometry. Our findings reveal that LPCAT1 shows exclusive sn‐1 specificity regardless of the identity of acyl‐CoAs. We further confirm that elevated PC 18 : 1/16:0 relative to its sn‐isomer results from an increased expression of LPCAT1 in human hepatocellular carcinoma (HCC) tissue as compared to normal liver tissue. MS imaging via desorption electrospray ionization of PC 18 : 1/16:0 thus enables visualization of HCC margins in human liver tissue at a molecular level.

Reduced Klotho expression and its prognostic significance in canine hepatocellular carcinoma.

Klotho is an anti-ageing gene and is known to act as a tumour suppressor in human hepatocellular carcinoma (HCC). According to a previous study, Klotho is present in normal canine mammary glands, and down-expression in tumours is positively associated with negative prognosis. However, the presence and significance of Klotho in canine HCC has not yet been reported. This study aimed to confirm Klotho expression in normal canine liver tissues using western blotting and immunohistochemistry, and whether the expression differed in non-neoplastic liver disease and HCC. Furthermore, correlation between clinicopathologic features and expression of Klotho was evaluated. All of the normal liver tissues showed the presence of Klotho, and Klotho expression was significantly decreased in the HCC tissue as compared to the non-neoplastic hepatic tissue. Additionally, Klotho expression was significantly associated with tumour size (P=.045), liver enzyme (alanine aminotransferase (ALT)) (P=.018), and metastasis (P=.024). Analysis of the survival curve revealed that reduced Klotho expression was significantly associated with poor disease-free survival (P=.041) in HCC. These results show that Klotho expression is present in normal canine liver tissue and that reduced Klotho expression is associated with poor prognosis in canine HCC. Thus, Klotho was presumed to be a potential clinical prognostic marker for canine HCC.

DN200434, an orally available inverse agonist of estrogen-related receptor γ, induces ferroptosis in sorafenib-resistant hepatocellular carcinoma

Sorafenib, originally identified as an inhibitor of multiple oncogenic kinases, induces ferroptosis in hepatocellular carcinoma (HCC) cells. Several pathways that mitigate sorafenib-induced ferroptosis confer drug resistance; thus strategies that enhance ferroptosis increase sorafenib efficacy. Orphan nuclear receptor estrogen-related receptor γ (ERRγ) is upregulated in human HCC tissues and plays a role in cancer cell proliferation. The aim of this study was to determine whether inhibition of ERRγ with DN200434, an orally available inverse agonist, can overcome resistance to sorafenib through induction of ferroptosis. Sorafenib-resistant HCC cells were less sensitive to sorafenibinduced ferroptosis and showed significantly higher ERRγ levels than sorafenib-sensitive HCC cells. DN200434 induced lipid peroxidation and ferroptosis in sorafenib-resistant HCC cells. Mechanistically, DN200434 increased mitochondrial ROS generation by reducing glutathione/glutathione disulfide levels, which subsequently reduced mTOR activity and GPX4 levels. DN200434 induced amplification of the antitumor effects of sorafenib was confirmed in a tumor xenograft model. The present results indicate that DN200434 may be a novel therapeutic strategy to re-sensitize HCC cells to sorafenib. [BMB Reports 2022; 55(11): 547-552].

CAF-Released Exosomal miR-20a-5p Facilitates HCC Progression via the LIMA1-Mediated β-Catenin Pathway

Currently, exosomes derived from Cancer-associated fibroblast (CAF) have reportedly been involved in regulating hepatocellular carcinoma (HCC) tumour microenvironment (TME). LIM domain and actin binding 1 (LIMA1) is an actin-binding protein that is involved in controlling the biological behaviour and progression of specific solid tumours. We aimed to determine the effect of LIMA1 and exosome-associated miR-20a-5p in HCC development. LIMA1 and miR-20a-5p expression levels were examined by real-time quantitative PCR (qRT-PCR), western blotting or immunohistochemistry (IHC). Functional experiments, including Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) assays, colony formation assays, wound healing assays, and Transwell invasion assays, were performed to investigate the effect of LIMA1 and miR-20a-5p. A dual-luciferase reporter gene assay was performed to confirm the interaction of miR-20a-5p and LIMA1. Exosomes were characterised by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We noted that LIMA1 was downregulated in human HCC tissues and cells and remarkably correlated with overall survival (OS) and recurrence-free survival (RFS). LIMA1 overexpression suppressed HCC cell proliferation and metastasis in vitro and in vivo, while LIMA1 knockdown had the opposite effects. A mechanistic investigation showed that LIMA1 inhibited the Wnt/β-catenin signalling pathway by binding to BMI1 and inducing its destabilisation. Additionally, we found that LIMA1 expression in HCC cells could be suppressed by transferring CAF-derived exosomes harbouring oncogenic miR-20a-5p. In summary, LIMA1 is a tumour suppressor that inhibits the Wnt/β-catenin signalling pathway and is downregulated by CAF-derived exosomes carrying oncogenic miR-20a-5p in HCC.

MicroRNA 424-5p promotes the sensitivity of hepatocellular carcinoma cells to sorafenib by targeting Kinesin family member 23

Objective: To explore the effect of MicroRNA 424-5p/Kinesin family member 23(miR-424-5p/KIF23)axis on the malignant phenotype of hepatoma cells and its sensitivity of sorafenib. Methods: Real-time quantitative reverse PCR(qRT-PCR) and/or Western blot were used to detect the expression of miR-424-5p and KIF23 in liver cancer tissues and paracancerous tissues, human hepatocellular carcinoma(HCC) cells HepG2 and normal hepatocyte LO2. HepG2 cells transfected with miR-424-5p mimic and miR-424-5p mimic NC were respectively defined as miR-424-5p mimic group and mimic NC group, HepG2 cells transfected with KIF23 overexpression vector pcDNA3.1-KIF23 or empty vector pcDNA3.1 respectively were defined as OE-KIF23 group and Vector group, and HepG2 cells co-transfected with miR-424-5p mimic and overexpression vector pcDNA3.1-KIF23 were defined as mimic+OE-KIF23 group: The KIF23-3'UTR wild-type vector (KIF23-WT) and the mutant vector (KIF23-MT) were co-transfected with miR-424-5p micic and mimic NC, respectively, and the targeting relationship between miR-424-5p and KIF23 was verified by dual-luciferase reporting experiments. The cell counting Kit-8 (CCK-8) was used to detect HepG2 cell proliferation and sensitivity to sorafenib. Flow cytometry was used to assess apoptosis in HepG2 cells. Transwell and scratch experiments were used to detect HepG2 cell migration and invasion capabilities. Intergroup data were compared using t-tests or analysis of variance. Results: Compared with the paracancerous tissue and normal hepatocytes, miR-424-5p in the HCC tissue and hepatocellular cells was significantly down-regulated (the relative expression was 0.604±0.121, 0.585±0.064), and KIF23 was significantly up-regulated (the relative expression was 5.451±1.834, 2.482±0.545), P<0.05. miR-424-5p mimic can inhibit the proliferation, migration and invasion of HCC cells and promote apoptosis of HCC cells (P<0.05). Overexpression of KIF23 can promote the proliferation, migration and invasion of HCC cells and inhibit apoptosis of HCC cells (P<0.05). The luciferase activity of HepG2 cells in the mimic and KIF23-WT co-transfection groups was significantly reduced compared with HepG2 cells in the mimic NC and KIF23-WT co-transfection groups (the relative fluorescence intensities were 3.668±0.091 and 2.629±0.056, respectively, P<0.05),however, there was no significant comparison between the luciferase activity of cells in the mimic and KIF23-MT co-transfection groups compared with those in the mimic NC and KIF23-MT co-transfection groups. miR-424-5p mimic can reverse the role of overexpression of KIF23 in promoting the ability of HCC cells to proliferate, migrate and invade (P<0.05). The inhibition rates of sorafenib on HepG2 cells in the mimic+OE-KIF23 group and the OE-KIF23 group were 47.491%±3.863% and 36.246%±6.063% (t=3.027, P<0.05). Conclusion: miR-424-5p can inhibit the proliferation, migration and invasion of HCC cells and can increase the sensitivity of HCC cells to sorafenib by targeting the expression level of KIF23.

Activation of Piezo1 contributes to matrix stiffness-induced angiogenesis in hepatocellular carcinoma.

Despite integrin being highlighted as a stiffness-sensor molecule in matrix stiffness-driven angiogenesis, other stiffness-sensor molecules and their mechanosensory pathways related to angiogenesis in hepatocellular carcinoma (HCC) remain obscure. Here, we explored the interplay between Piezo1 and integrin β1 in the mechanosensory pathway and their effects on HCC angiogenesis to better understand matrix stiffness-induced angiogenesis. The role of Piezo1 in matrix stiffness-induced angiogenesis was investigated using orthotopic liver cancer SD rat models with high liver stiffness background, and its clinical significance was evaluated in human HCC tissues. Matrix stiffness-mediated Piezo1 upregulation and activation were assayed using an in vitro fibronectin (FN)-coated cell culture system with different stiffness, Western blotting and Ca2+ probe. The effects of shPiezo1-conditioned medium (CM) on angiogenesis were examined by tube formation assay, wound healing assay and angiogenesis array. The underlying mechanism by which Piezo1 participated in matrix stiffness-induced angiogenesis was analyzed by microRNA quantitative real-time polymerase chain reaction (qRT-PCR), matrix stiffness measurement, dual-luciferase reporter assay, ubiquitination assay and co-immunoprecipitation. Increased matrix stiffness significantly upregulated Piezo1 expression at both cellular and tissue levels, and high expression of Piezo1 indicated an unfavorable prognosis. High matrix stiffness also noticeably enhanced the activation level of Piezo1, similar to its expression level. Piezo1 knockdown significantly suppressed tumor growth, angiogenesis, and lung metastasis of HCC rat models with high liver stiffness background. shPiezo1-CM from HCC cells attenuated tube formation and migration abilities of vascular endothelial cells remarkably, and analysis of differentially expressed pro-angiogenic factors revealed that Piezo1 promoted the expression and secretion of vascular endothelial growth factor (VEGF), CXC chemokine ligand 16 (CXCL16) and insulin-like growth factor binding protein 2 (IGFBP2). Matrix stiffness-caused Piezo1 upregulation/activation restrained hypoxia inducible factor-1α (HIF-1α) ubiquitination, subsequently enhanced the expression of downstream pro-angiogenic factors to accelerate HCC angiogenesis. Besides, collagen 1 (COL1)-reinforced tissue stiffening resulted in more expression of Piezo1 via miR-625-5p. This study unravels a new mechanism by which the integrin β1/Piezo1 activation/Ca2+ influx/HIF-1α ubiquitination/VEGF, CXCL16 and IGFBP2 pathway participates in matrix stiffness-driven HCC angiogenesis. Simultaneously, a positive feedback regulation loop as stiff matrix/integrin β1/miR-625-5p/Piezo1 and COL1/stiffer matrix mediates matrix stiffness-caused Piezo1 upregulation.

Isoginkgetin, a potential CDK6 inhibitor, suppresses SLC2A1/GLUT1 enhancer activity to induce AMPK-ULK1-mediated cytotoxic autophagy in hepatocellular carcinoma

ABSTRACT Isoginkgetin (ISO), a natural biflavonoid, exhibited cytotoxic activity against several types of cancer cells. However, its effects on hepatocellular carcinoma (HCC) cells and mechanism remain unclear. Here, we revealed that ISO effectively inhibited HCC cell proliferation and migration in vitro. LC3-II expression and autophagosomes were increased under ISO treatment. In addition, ISO-induced cell death was attenuated by treatment with chloroquine or knockdown of autophagy-related genes (ATG5 or ULK1). ISO significantly suppressed SLC2A1/GLUT1 (solute carrier family 2 member 1) expression and glucose uptake, leading to activation of the AMPK-ULK1 axis in HepG2 cells. Overexpression of SLC2A1/GLUT1 abrogated ISO-induced autophagy. Combining molecular docking with thermal shift analysis, we confirmed that ISO directly bound to the N terminus of CDK6 (cyclin-dependent kinase 6) and promoted its degradation. Overexpression of CDK6 abrogated ISO-induced inhibition of SLC2A1/GLUT1 transcription and induction of autophagy. Furthermore, ISO treatment significantly decreased the H3K27ac, H4K8ac and H3K4me1 levels on the SLC2A1/GLUT1 enhancer in HepG2 cells. Finally, ISO suppressed the hepatocarcinogenesis in the HepG2 xenograft mice and the diethylnitrosamine+carbon tetrachloride (DEN+CCl4)-induced primary HCC mice and we confirmed SLC2A1/GLUT1 and CDK6 as promising oncogenes in HCC by analysis of TCGA data and human HCC tissues. Our results provide a new molecular mechanism by which ISO treatment or CDK6 deletion promotes autophagy; that is, ISO targeting the N terminus of CDK6 for degradation inhibits the expression of SLC2A1/GLUT1 by decreasing the enhancer activity of SLC2A1/GLUT1, resulting in decreased glucose levels and inducing the AMPK-ULK1 pathway.

Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Given that HCC is an extraordinarily heterogeneous malignant disease, finding an effective therapeutic strategy for treating it has been difficult. Because of the importance of angiogenesis in tumorigenesis, targeting the more homogenous HCC endothelial cells may be a better therapeutic strategy. In a unpublished manuscript, we found that the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase 2/9 (MMP2/9) were reduced in human HCC tissues that overexpressed DNA damage repair gene general transcription factor II subunit H2 (GTF2H2). This suggested that GTF2H2 may have an inhibitory effect on angiogenesis. Therefore, we hypothesized that GTF2H2 acts as an anti-angiogenesis gene. However, our results showed that GTF2H2 overexpression had no effect on endothelial cell viability, migration, or permeability. To our surprise, treating human umbilical vein endothelial cells (HUVECs) with the culture medium of Huh 7 cells overexpressing GTF2H2 could inhibit their viability, migration, and permeability. We then isolated the culture medium into exosomes and other components from the culture medium. Only GTF2H2-enriched exosomes could inhibit the viability, migration, tube formation, and permeability of HUVECs. Our results suggest that overexpressing GTF2H2 had no effect on HUVECs, while GTF2H2 enriched exosomes from Huh7 cells could inhibit HUVEC phenotypes such as proliferation and migration. Therefore, GTF2H2-enriched exosomes can possibly be utilized as a novel drug for treating HCC and also serve as a potential molecular target for inhibiting tumor angiogenesis.

Tumor-Promoting Actions of HNRNP A1 in HCC Are Associated with Cell Cycle, Mitochondrial Dynamics, and Necroptosis.

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in the world. Although increasing evidence supports the role of heterogeneous ribonucleoprotein particle A1 (HNRNP A1) in tumor progression, the function of HNRNP A1 in HCC remains unclear. Here, we focused on the role of HNRNP A1 in the development of HCC. In this study, we found HNRNP A1 participates in many aspects of HCC, such as progression and prognosis. Our results showed that HNRNP A1 is upregulated in human HCC tissues and cell lines. High expression of HNRNP A1 can promote the proliferation, migration, and invasion in HCC cells and accelerate tumor progression in mice. Moreover, we found that HNRNP A1 prevents the senescence process of HCC cells. Knocking down of HNRNP A1 promotes the expression of P16INK4, which arrests the cell cycle and then induces the senescence phenotype in HCC cells. Furthermore, we found that HNRNP A1 regulated necroptosis and mitochondrial dynamics. In summary, our study indicates that HNRNP A1 promotes the development of HCC, which suggests a potential therapeutic target for HCC.

Tumor associated macrophages-derived exosomes facilitate hepatocellular carcinoma malignance by transferring lncMMPA to tumor cells and activating glycolysis pathway

BackgroundTumor-associated macrophages (TAMs), which form a large part of the tumor microenvironment, are normally regulated by metabolic reprogramming. However, the potential mechanisms of the immune-metabolism interaction between hepatocellular carcinoma (HCC) cells and TAMs remain unclear.MethodsThe candidate long non-coding RNAs (lncRNAs) were screened by Smart-seq based scRNA-seq method and then validated by qPCR. Immunostaining analysis was done to examine the levels of markers for TAMs and glycolysis. Exosomes from primary TAMs of human HCC tissues were isolated by centrifugation, and their internalization with lncRNAs was confirmed by immunofluorescence. The underlying mechanism of TAMs-derived exosomal lncRNA to HCC was confirmed by luciferase reporter assay and RNA immunoprecipitation. Metabolism regulation was evaluated through glucose consumption, lactate productions and extracellular acidification rates (ECARs). Mouse xenograft models were used to elucidate the in vivo effect of candidate lncRNAs on tumor growth.ResultsTAMs augment the aerobic glycolysis in HCC cells and their proliferation by the extracellular exosome transmission of a myeloid-derived lncRNA, M2 macrophage polarization associated lncRNA (lncMMPA). Mechanistically, lncMMPA not only could polarize M2 macrophage, but also could act as an microRNA sponge to interact with miR-548 s and increase the mRNA level of ALDH1A3, then further promote glucose metabolism and cell proliferation in HCC. Moreover, lncMMPA increased HCC cell multiplication through interacting with miR-548 s in vivo. Clinically, lncMMPA expression associates with glycolysis in TAMs and reduced survival of HCC patients.ConclusionLncMMPA plays an important role in regulating HCC malignancy and metabolic reprogramming of miR-548 s/ALDH1A3 pathway.

Loss of SLC46A1 decreases tumor iron content in hepatocellular carcinoma.

It is interesting that high iron is an independent inducer or cofactor of hepatocellular carcinoma (HCC) while the amount of iron is decreased in the liver tumor tissues. Due to the previous findings that iron deficiency promoted HCC metastasis, it is of significance to identify the underlying mechanism of iron deficiency in HCC. The tumor iron content and expressions of iron‐metabolic molecules were observed in the primary liver cancers of rats and mice. The molecules that changed independently of iron were identified by comparing the expression profiles in the human HCC tissues and iron‐deprived HCC cells. The downstream effects of these molecules on regulating intracellular iron content were investigated in vitro and further validated in vivo. Both in primary liver cancers of rats and mice, we confirmed the decreased iron content in tumor tissues and the altered expressions of iron‐metabolic molecules, including transferrin receptor 1 (TfR1), six‐transmembrane epithelial antigen of prostate 3 (STEAP3), divalent metal transporter 1 (DMT1), SLC46A1, ferroportin, hepcidin, and ferritin. Among these, STEAP3, DMT1, and SLC46A1 were altered free of iron deficiency. However, only silence or overexpression of SLC46A1 controlled the intracellular iron content of HCC cells. The interventions of STEAP3 or DMT1 could not change the intracellular iron content. Lentivirus‐mediated regain of SLC46A1 expression restored the iron content in orthotopically implanted tumors, with correspondingly changes in the iron‐metabolic molecules as iron increasing. Conclusion: Taken together, these results suggest that the loss of SLC46A1 expression leads to iron deficiency in liver tumor tissues, which would be an effective target to manage iron homeostasis in HCC.

Tumor suppressor LHPP suppresses cell proliferation and epithelial-mesenchymal transition in hepatocellular carcinoma cell lines.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in the world with high mortality due to its high potential of metastasis. Epithelial-mesenchymal transition (EMT) plays a key role in the pathogenesis of HCC occurrence and metastasis. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a novel tumor suppressor. There is little study about LHPP in human HCC development. In the present study, we aimed to investigate the role of LHPP in human HCC cell metastasis. We analyzed the LHPP expression level in human HCC tissues compared with normal tissues in the public database. We detected the mRNA level and protein level of LHPP in transformed liver cell line (LO2) and human HCC cell lines (MHCC-97 H, MHCC-97L, and HepG2). We performed genetic gain and loss of function experiments with LHPP using small interfering RNA (siRNA) and lentivirus infection. Then, we detected that LHPP suppressed proliferation and promoted apoptosis in hepatocellular carcinoma cell lines. Also, we investigated the role of LHPP in the EMT process. Finally, we examined the effect of LHPP on TGF-β-induced EMT. Interestingly, we also found that LHPP expression is positively regulated tumor suppressor p53. Our data showed that LHPP is significantly decreased in the human HCC tissues and human HCC cell lines compared with normal liver tissues and transformed liver cells. Knockdown of LHPP promotes HCC cell proliferation and metastasis, and LHPP expression levels negatively correlate with EMT-related genes. Furthermore, LHPP inhibits TGF-β-induced EMT in HCC cell lines. These studies validate LHPP as a tumor suppressor in liver cancer and provide a new genetic target for HCC diagnosis and treatment.

The correlation of fibrinogen-like protein-1 expression with the progression and prognosis of hepatocellular carcinoma

BackgroundFibrinogen-like-protein 1 (FGL1), a member of the fibrinogen-related protein (FREP) family, is a major ligand of the immune inhibitory receptor lymphocyte-activation gene 3 (LAG-3). While FGL1 is strongly implicated in the development and prognosis of a variety of diseases, its role in hepatocellular carcinoma (HCC) is still disputed. Therefore, the role of FGL1 expression in the progression and prognosis of HCC was investigated.Methods and resultsIn the present study, bioinformatics analysis was first used to probe the expression profile of FGL1 in multiple malignant tumor tissues and paired normal tissues, and to explore the possible relationship between FGL1 and prognosis of HCC patients. Thereafter, the expression levels of FGL1 were determined and compared in human HCC cell lines, HCC tissues, peri-tumor tissues and normal liver tissues by western blot analysis. Furthermore, tissue microarrays were used to detect the expression of FGL1 through immunohistochemical staining and to verify whether the FGL1 expression level was associated with clinicopathological features and the prognosis of HCC patients. The results showed that FGL1 was downregulated significantly in most of the HCC cells lines and HCC tissues, corresponding to the results of the bioinformatics and western blot analyses. FGL1 expression level in HCC was found to be correlated to Edmondson grade and metastasis of the HCC. Additionally, high FGL1 expression was associated with better overall survival in HCC patients, suggesting that FGL1 could function as a tumor suppressor.ConclusionsThe expression level of FGL1 can be correlated with the progression and prognosis of HCC, suggesting its potential as a prognostic biomarker.

Immunotherapeutic Targeting of NG2/CSPG4 in Solid Organ Cancers.

Neuro-glia antigen 2/chondroitin sulfate proteoglycan 4 (NG2/CSPG4, also called MCSP, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a large cell-surface antigen and an unusual cell membrane integral glycoprotein frequently expressed on undifferentiated precursor cells in multiple solid organ cancers, including cancers of the liver, pancreas, lungs, and kidneys. It is a valuable molecule involved in cancer cell adhesion, invasion, spreading, angiogenesis, complement inhibition, and signaling. Although the biological significance underlying NG2/CSPG4 proteoglycan involvement in cancer progression needs to be better defined, based on the current evidence, NG2/CSPG4+ cells, such as pericytes (PCs, NG2+/CD146+/PDGFR-β+) and cancer stem cells (CSCs), are closely associated with the liver malignancy, hepatocellular carcinoma (HCC), pancreatic malignancy, and pancreatic ductal adenocarcinoma (PDAC) as well as poor prognoses. Importantly, with a unique method, we successfully purified NG2/CSPG4-expressing cells from human HCC and PDAC vasculature tissue blocks (by core needle biopsy). The cells appeared to be spheres that stably expanded in cultures. As such, these cells have the potential to be used as sources of target antigens. Herein, we provide new information on the possibilities of frequently selecting NG2/CSPG4 as a solid organ cancer biomarker or exploiting expressing cells such as CSCs, or the PG/chondroitin sulfate chain of NG2/CSPG4 on the cell membrane as specific antigens for the development of antibody- and vaccine-based immunotherapeutic approaches to treat these cancers.

Dietary folate drives methionine metabolism to promote cancer development by stabilizing MAT IIA

Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.

Integrin alpha 6 is upregulated and drives hepatocellular carcinoma progression through integrin α6β4 complex.

Integrin α6 (ITGA6) forms integrin receptors with either integrin β1 (ITGB1) or integrin β4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6β4 complex. Thus, integrin α6β4 may be a therapeutic target for treating patients with HCC.

27-Hydroxycholesterol promotes metastasis by SULT2A1-dependent alteration in hepatocellular carcinoma.

Oxysterol metabolism plays an important role in the initiation and development of various tumors. However, little is known that the metabolic alternation can promote the metastasis of hepatocellular carcinoma (HCC). In this study, we identify the sulfotransferase family 2A member 1 (SULT2A1) to 27‐hydroxycholesterol (27‐OHC) metabolic axis as playing a critical role in HCC metastasis. The level of 27‐OHC closely corresponded with HCC metastasis instead of proliferation in vitro and in vivo. Also, the expression of SULT2A1 is extremely downregulated in human HCC tissues and is correlated with poor prognosis and tumor metastasis. Gain‐ and loss‐of‐function studies reveal that SULT2A1 suppresses the metastasis of HCC by regulating the level of 27‐OHC. Further mechanistic studies indicated that SULT2A1‐dependent alternation of 27‐OHC activates the nuclear factor‐κB signaling pathway and promotes HCC metastasis by enhancing Twist1 expression and epithelial–mesenchymal transition. In conclusion, our findings indicate the relationship between the metabolism of 27‐OHC and the metastasis of HCC. Moreover, SULT2A1 could act as a potential prognostic biomarker and a therapeutic target for preventing HCC metastasis.

NOL12 as an Oncogenic Biomarker Promotes Hepatocellular Carcinoma Growth and Metastasis.

Hepatocellular carcinoma (HCC) is a common malignancy with a poor prognosis worldwide. However, the pathogenesis of HCC remains poorly understood. In this study, we found that NOL12 was significantly overexpressed in independent HCC datasets from TCGA database. We confirmed that the expression level of NOL12 was upregulated in human HCC tissues and cell lines by RT-qPCR. High expression of NOL12 is associated with worse reduced overall survival (OS), high pathological grade, node metastasis, and advanced clinical stage in patients with HCC. Moreover, knockdown of NOL12 dramatically inhibits the proliferation and metastasis of HCC cells in vitro and in vivo. CIBERSORTx analysis revealed that twelve types of tumor-infiltrating immune cells (TICs) are correlated with NOL12 expression. The risk signature based on 8 NOL12-related genes is an independent prognostic factor for patients with HCC. The OS rate of patients in the low-risk score group was better than that in the high-risk score group. In addition, the total tumor mutation burden (TMB) in the high-risk score group increased significantly, and the risk scores could be used as an alternative indicator of immune checkpoint inhibitor (ICI) response. In conclusion, our findings indicated that NOL12 might be involved in the progression of HCC and can be used as a potential therapeutic target. Moreover, the NOL12-related risk signature may have predictive relevance with regard to ICI therapy.

Lipolysis-Stimulated Lipoprotein Receptor Impairs Hepatocellular Carcinoma and Inhibits the Oncogenic Activity of YAP1 via PPPY Motif.

PurposeLipolysis-stimulated lipoprotein receptor (LSR) is a type I single-pass transmembrane protein which is mainly expressed in the liver. In this study, we investigated if and how LSR is involved in the carcinogenesis of hepatocellular carcinoma (HCC).Experimental DesignTo evaluate if LSR was abnormally expressed in human HCC tissues, and how its expression was associated with the survival probability of patients, we obtained data from Gene Expression Omnibus and The Cancer Genome Atlas Program. To investigate if and how LSR regulates tumor growth, we knocked down and overexpressed LSR in human HCC cell lines. In addition, to evaluate the interaction between LSR and yes-associated protein1 (YAP1), we mutated LSR at PPPY motif, a binding site of YAP1.ResultsTotally, 454 patients were enrolled in the present study, and high expression of LSR significantly decreased the probability of death. Knockdown of LSR significantly increased the expansion of HCC cells and significantly promoted tumor growth. In addition, downregulation of LSR increased the nuclear accumulation and transcriptional function of YAP1. Conversely, overexpression of LSR impairs this function of YAP1 and phosphorylates YAP1 at serine 127. Of note, mutation of LSR at the PPPY motif could block the interaction between LSR and YAP1, and restore the transcriptional ability of YAP1.ConclusionsThe present study suggests that LSR binds to YAP1 via the PPPY motif. Thus, LSR increases the phosphorylation of YAP1 and impairs the growth of HCC. This highlights that targeting LSR might be a promising therapeutic strategy for HCC.

METTL3 Enhances Hepatocellular Carcinoma Progression by Regulating Polycomb Repressive Complex 1 (PRC1) Components BMI1 and RNF2

N6‐methyladenosine (m6A) modification is the most abundant mRNA modification which plays important roles in the regulation of mRNA stability, splicing, translocation, and translation. Methyltransferase‐like 3 (METTL3), a primary RNA methyltransferase that catalyzes RNA m6A modification, is implicated in several human cancers including hepatocellular carcinoma (HCC), although its mechanisms of actions remain to be further defined. In the current study, we aimed to further determine the effect and mechanism of METTL3‐derived m6A in HCC. We have analyzed the TCGA database and observed that METTL3 expression is significantly upregulated in HCC patients which is associated with lower survival rate (P &lt; 0.001). In a mouse model of HCC induced by hydrodynamic tail vein injection of hyperactive form of YAP and β‐catenin in conjunction with the sleeping beauty (SB) transposon system, we observed that the expression of METTL3 was notably high in YAP/β‐catenin‐induced HCC. In cultured human HCC cell lines (Huh7 and PLC/PRF/5), we observed that stable knockdown of METTL3 by shRNA significantly decreased tumor cell proliferation, colony formation and migration, in vitro. When Huh and PLC/PRF/5 cells with shRNA knockdown of METTL3 were inoculated into the livers of SCID mice, we found that METTL3 knockdown significantly inhibited xenograft tumor growth, in vivo. We next delivered METTL3 and YAP expression plasmids to the livers of wild type mice via hydrodynamic tail vein injection and observed that co‐expression of METTL3 plus YAP induced the development of HCC which involves almost the entire livers (20 weeks after tail vein injection). These findings provide important evidence for a tumor‐promoting role of METTL3 in HCC development. Through N6‐methyladenosine‐sequencing (m6A‐Seq) and RNA sequencing (RNA‐Seq), we identified BMI1 and RNF2, two key components of the polycomb repressive complex 1 (PRC1), as direct downstream targets of METTL3 in HCC. Our further analyses revealed that both BMI1 and RNF2 were significantly elevated in HCC patients and were associated with lower survival rate and that the expression of BMI1 and RNF2 were positively correlated with METTL3 in human HCC tissues. Consistent with the above results, our further data showed that knockdown of METTL3 in Huh7 and PLC/PRF/5 significantly decreased the expression of BMI1 and RNF2. Accordingly, treatment of Huh7 and PLC/PRF/5 cells with the METTL3 inhibitor, STM2457, significantly reduced the expression of BMI1 and RNF2 and decreased tumor cell proliferation and colony formation. Moreover, our data showed that inhibition of the m6A reader YTHDF1 by siRNA significantly decreased the expression of BMI1 and RNF2 in human HCC cells. Collectively, our study provides important evidence that METTL3 promotes HCC development and progression through m6A modification of BMI1 and RNF2 mRNAs which involve a YTHDF1 dependent mechanism. It is conceivable that the METTL3‐m6A‐BMI1/RNF2 axis may represent a promising target for HCC treatment.