Human Hepatocellular Carcinoma Tumor Research Articles

Mechanisms of Sorafenib Resistance in HCC Culture Relate to the Impaired Membrane Expression of Organic Cation Transporter 1 (OCT1).

Sorafenib, an FDA-approved drug for advanced hepatocellular carcinoma (HCC) treatment, encounters resistance in many patients. Deciphering the mechanisms underlying sorafenib resistance is crucial for devising alternative strategies to overcome it. This study aimed to investigate sorafenib resistance mechanisms using a diverse panel of HCC cell lines. HCC cell lines were subjected to continuous sorafenib treatment, and stable cell lines (Huh 7.5 and Huh 7PX) exhibiting sustained growth in its presence were isolated. The investigation of drug resistance mechanisms involved a comparative analysis of drug-targeted signal transduction pathways (EGFR/RAF/MEK/ERK/Cyclin D), sorafenib uptake, and membrane expression of the drug uptake transporter. HCC cell lines (Huh 7.5 and Huh 7PX) with a higher IC50 (10μM) displayed a more frequent development of sorafenib resistance compared to those with a lower IC50 (2-4.8μM), indicating a potential impact of IC50 variation on initial treatment response. Our findings reveal that activated overexpression of Raf1 kinases and impaired sorafenib uptake, mediated by reduced membrane expression of organic cation transporter-1 (OCT1), contribute to sorafenib resistance in HCC cultures. Stable expression of the drug transporter OCT1 through cDNA transfection or adenoviral delivery of OCT1 mRNA increased sorafenib uptake and successfully overcame sorafenib resistance. Additionally, consistent with sorafenib resistance in HCC cultures, cirrhotic liver-associated human HCC tumors often exhibited impaired membrane expression of OCT1 and OCT3. Intrinsic differences among HCC cell clones, affecting sorafenib sensitivity at the expression level of Raf kinases, drug uptake, and OCT1 transporters, were identified. This study underscores the potential of HCC tumor targeted OCT1 expression to enhance sorafenib treatment response.

Abstract 5004: Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models

Abstract The members of the vesiculovirus genus have recently become attractive immunotherapeutic agents due to their natural selectivity for tumors and lack of toxicity in humans. Here, the research shows Intratumoral (IT) or Intraperitoneal (IP) injection of JURV was found to induce dynamic tumor regression in human hepatocellular carcinoma (HCC) xenografts and syngeneic models. Furthermore, IT injections of JURV were also shown to recruit and activate cytotoxic T lymphocytes and decrease tumor-associated macrophage infiltration, resulting in the delay of tumor growth in both local and distant murine HCC tumors established in a syngeneic model. Additionally, the concurrent use of JURV and anti-PD-1 antibodies was found to synergistically modulate the tumor microenvironment (TME) via increased tumor-infiltrating CD4+ T cells and depleted CD8+ PD-1+ and NK cells. Furthermore, using RIL-175, an aggressive metastatic HCC tumor mouse model, we show that intraperitoneal (IP) (systemic) administration of JURV and anti-PD1 significantly improved long-term survival (90%) compared to the (20%) JURV or anti-PD1 alone. Through proteogenomic analysis, the activation of different effectors of the immune system was observed to cooperate to alter the TME to a favorable anti-tumor immune state. These findings further support the development of JURV as an immunovirotherapy platform for HCC, with the potential to induce abscopal effects via the activation of several tumor suppressor genes and the mechanism regulating the T helper cell responses. Citation Format: Mulu Z. Tesfay, Khandoker U. Ferdous, Aleksandra Cios, Randal S. Shelton, Camila C. Simoes, Steven R. Post, Thomas J. Kelly, Martin J. Cannon, Alexei Basnakian, Bolni M. Nagalo. Oncolytic Jurona-driven systemic and intratumoral immunotherapy combined with immune checkpoint blockade boost immune response and survival in hepatocellular carcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5004.

PYCR2 promotes growth and aerobic glycolysis in human liver cancer by regulating the AKT signaling pathway

Hepatocellular carcinoma (HCC) is the world's third most fatal cancer. Because metabolic rewiring is a hallmark of HCC, studies into the causes of aberrant glycolysis could provide insight into novel HCC therapeutic strategies. Pyrroline-5-carboxylate reductase 2 (PYCR2), a key enzyme of proline synthesis, has previously been found to play vital roles in various malignancies regarding amino acid metabolism and oxidative stress response. Our study investigated the mechanistic function of PYCR2 in HCC. We used Gene Expression Profiling Interactive Analysis to perform bioinformatics analysis of PYCR2 expression and survival in human HCC patients based on the Cancer Genome Atlas database. The function of PYCR2 in cell viability and glycolysis was assessed using CCK-8 and ECAR assays. Transducing shRNA or overexpression vectors into the HCC cell line altered the expression status of PYCR2. PYCR2 expression was validated using quantitative real-time PCR and Western blot. In mouse xenograft models, the role of PYCR2 in HCC tumor formation was confirmed. PYCR2 was overexpressed in human HCC tumor tissue and was associated with a poor prognosis. The functional assay revealed that silencing PYCR2 inhibited cell viability, glycolysis, and AKT activation. Furthermore, the xenograft experiment demonstrated that silencing PYCR2 significantly inhibited tumor growth and Ki67 expression. On the other hand, PYCR2 overexpression significantly promoted cell viability and glycolysis, which could be inhibited by either a glycolysis inhibitor or an AKT inhibitor, indicating that PYCR2 may function via glycolysis and the AKT pathway. Moreover, despite the overexpression of PYCR2 in vivo, treatment with a glycolysis inhibitor may considerably suppress tumor growth. Our findings suggest that PYCR2 may play an oncogenic role in HCC growth by promoting glycolysis and activating AKT, emphasizing PYCR2's clinical relevance in HCC management as a novel potential therapeutic target.

Cholesterol Efflux Drives the Generation of Immunosuppressive Macrophages to Promote the Progression of Human Hepatocellular Carcinoma.

Cholesterol is often enriched in tumor microenvironment (TME); however, its impact on disease progression varies in different tissues and cells. Monocytes/macrophages (Mφ) are major components and regulators of the TME and play pivotal roles in tumor progression and therapeutic responses. We aimed to investigate the profile, effects, and regulatory mechanisms of Mφ cholesterol metabolism in the context of human hepatocellular carcinoma (HCC). Here, we found that patients with high serum levels of cholesterol had shorter survival times and lower response rates to anti-PD-1 treatment. However, the cholesterol content in tumor-infiltrating monocytes/Mφ was significantly lower than that in their counterparts in paired nontumor tissues. The expression of the cholesterol efflux transporter, ABCA1, was upregulated in tumor monocytes/Mφ, and ABCA1 upregulation positively associated with decreased cellular cholesterol content and increased serum cholesterol levels. Mechanistically, autocrine cytokines from tumor-treated monocytes increased LXRα and ABCA1 expression, which led to the generation of immature and immunosuppressive Mφ. Although exogenous cholesterol alone had little direct effect on Mφ, it did act synergistically with tumor-derived factors to promote ABCA1 expression in Mφ with more immunosuppressive features. Moreover, high numbers of ABCA1+ Mφ in HCC tumors associated with reduced CD8+ T-cell infiltration and predicted poor clinical outcome for patients. Our results revealed that dysregulated cholesterol homeostasis, due to the collaborative effects of tumors and exogenous cholesterol, drives the generation of immunosuppressive Mφ. The selective modulation of cholesterol metabolism in Mφ may represent a novel strategy for cancer treatment.

Dissecting the role of the NADPH oxidase NOX4 in TGF-beta signaling in hepatocellular carcinoma

The NADPH oxidase NOX4 has been proposed as necessary for the apoptosis induced by the Transforming Growth Factor-beta (TGF-β) in hepatocytes and hepatocellular carcinoma (HCC) cells. However, whether NOX4 is required for TGF-β-induced canonical (SMADs) or non-canonical signals is not fully understood yet, neither its potential involvement in other parallel actions induced by TGF-β. In this work we have used CRISPR Cas9 technology to stable attenuate NOX4 expression in HCC cells. Results have indicated that NOX4 is required for an efficient SMAD2/3 phosphorylation in response to TGF-β, whereas non-canonical signals, such as the phosphorylation of the Epidermal Growth Receptor or AKT, are higher in NOX4 silenced cells. TGF-β-mediated inhibition of cell proliferation and viability is attenuated in NOX4 silenced cells, correlating with decreased response in terms of apoptosis, and maintenance of high expression of MYC and CYCLIN D1. These results would indicate that NOX4 is required for all the tumor suppressor actions of TGF-β in HCC. However, analysis in human HCC tumors has revealed a worse prognosis for patients showing high expression of TGF-β1-related genes concomitant with high expression of NOX4. Deepening into other tumorigenic actions of TGF-β that may contribute to tumor progression, we found that NOX4 is also required for TGF-β-induced migratory effects. The Epithelial-Mesenchymal transition (EMT) program does not appear to be affected by attenuation of NOX4 levels. However, TGF-β-mediated regulation of cytoskeleton dynamics and focal adhesions require NOX4, which is necessary for TGF-β-induced increase in the chaperone Hsp27 and correct subcellular localization of Hic-5 within focal adhesions, as well for upregulation of the metalloprotease MMP9. All these results together point to NOX4 as a key element in the whole TGF-β signaling in HCC cells, revealing an unknown role for NOX4 as tumor promoter in HCC patients presenting activation of the TGF-β pathway.

LncRNA HClnc1 facilitates hepatocellular carcinoma progression by regulating PKM2 signaling and indicates poor survival outcome after hepatectomy.

Long noncoding RNAs (lncRNAs) are key mediators with a wide range of pathophysiological functions, but their role in human hepatocellular carcinoma (HCC) is still unclear. An unbiased microarray study evaluated a novel lncRNA, HClnc1, that is linked to the development of HCC. In vitro cell proliferation assays and an in vivo xenotransplanted HCC tumor model were performed to determine its functions, followed by antisense oligo-coupled mass spectrometry to identify HClnc1-interacting proteins. To study relevant signaling pathways, in vitro experiments were performed, including chromatin isolation by RNA purification, RNA immunoprecipitation, luciferase, and RNA pull-down assay. HClnc1 levels were considerably greater in patients with advanced tumor-node-metastatic stages, and it was found to be inversely connected to survival rates. Moreover, the proliferative and invasive potential of the HCC cells was attenuated by HClnc1 RNA knockdown in vitro, while HCC tumor growth and metastasis were found to be reduced in vivo. HClnc1 interacted with pyruvate kinase M2 (PKM2) to prevent its degradation and thus facilitated aerobic glycolysis and PKM2-STAT3 signaling. HClnc1 is involved in a novel epigenetic mechanism of HCC tumorigenesis and PKM2 regulation. HClnc1 is not only a more accurate prognostic indicator of HCC but also a potential therapeutic target for HCC treatment.

MALT1 paracaspase is overexpressed in hepatocellular carcinoma and promotes cancer cell survival and growth

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, therapeutic management of HCC remains a challenge, emphasizing the importance of exploring novel targets. MALT1 paracaspase is a druggable signaling molecule whose dysregulation has been linked to hematological and solid tumors. However, the role of MALT1 in HCC remains poorly understood, leaving its molecular functions and oncogenic implications unclear. Here we provide evidence that MALT1 expression is elevated in human HCC tumors and cell lines, and that correlates with tumor grade and differentiation state, respectively. Our results indicate that ectopic expression of MALT1 confers increased cell proliferation, 2D clonogenic growth, and 3D spheroid formation in well differentiated HCC cell lines with relatively low MALT1 levels. In contrast, stable silencing of endogenous MALT1 through RNA interference attenuates these aggressive cancer cell phenotypes, as well as migration, invasion, and tumor-forming ability, in poorly differentiated HCC cell lines with higher paracaspase expression. Consistently, we find that pharmacological inhibition of MALT1 proteolytic activity with MI-2 recapitulates MALT1 depletion phenotypes. Finally, we show that MALT1 expression is positively correlated with NF-kB activation in human HCC tissues and cell lines, suggesting that its tumor promoting functions may involve functional interaction with the NF-kB signaling pathway. This work unveils new insights into the molecular implications of MALT1 in hepatocarcinogenesis and places this paracaspase as a potential marker and druggable liability in HCC.

Lipidomic study and diagnosis of hepatocellular carcinoma tumor with rapid evaporative ionization mass spectrometry.

Liver cancer is generally considered the leading cause of cancer deaths worldwide, and hepatocellular carcinoma (HCC) contributes to more than 90% of liver cancers. The altered lipid metabolism for rapid cancer cell growth and tumor formation has been frequently proven. In this study, an ambient ionization mass spectrometry technique, rapid evaporative ionization mass spectrometry (REIMS) using a monopolar electric knife, called iKnife, was systematically optimized and employed for ex vivo analysis of 12 human HCC tumor tissue specimens together with the paired paracancerous tissue (PT) and noncancerous liver tissue (NCT) specimens. Nine free fatty acids and 34 phospholipids were tentatively identified according to their extract masses and/or tandem mass spectra. With the help of statistical methods, 7 free fatty acids and 10 phospholipids were distributed differently in 3 types of liver tissue specimens (95% confidence interval). The box plots showed these characterized lipid metabolites varied in PT, HCC, and NCT. Compared with PT and NCT, the upregulations of four common fatty acids FA 18:0, FA 20:4, FA 16:0, and FA 18:1, together with phospholipids PC 36:1, PE 38:3, PE (18:0/20:4), PA (O-36:1), PC (32:1), PC 32:0, PE 34:0, and PC (16:0/18:1), were found in HCC specimens. The sensitivity and specificity of the established statistic model for real-time HCC tumor diagnosis were 100% and 90.5%, respectively. This study demonstrated that the described REIMS technique is a potential method for rapid lipidomic analysis and characterization of HCC tumor tissue.

Abstract 296: Clinical significance of glycolytic metabolic activity in liver cancer and its implication to immunotherapy

Abstract Background & Aims: High metabolic activity is a hallmark of cancers including hepatocellular carcinoma (HCC), a major form of liver cancer. However, the molecular features of HCC with high metabolic activity contributing to the clinical outcomes and their therapeutic implications are poorly understood. We aimed to uncover the metabolic characteristics of HCC that are associated with clinical relevance such as overall survival and response to immunotherapy. Methods: By applying cross-species comparison of genomic data, we integrated gene expression profile data from well-defined mouse models and human HCC tumor tissues to stratify HCC tumors according to their intrinsic metabolic activity. For stratification of HCC tumors, we developed a robust genomic predictor and validated it in 5 HCC cohorts (n=1038). Statistics and informatics approaches were performed to assess clinical significance such as overall survival and response to immunotherapy in metabolic subtypes. Genomic data from patient derived xenograft (PDX) models were integrated to the analysis. Results: Analysis of systematically integrated genomic data revealed three distinct metabolic subtypes of HCC (high, moderate and low metabolic subtypes). The high metabolic subtype is characterized by poor survival, the strongest stem cell features, high genomic instability, high expression of alpha-fetoprotein and KRT19, and activation of EPCAM and SALL4, and poor response to immunotherapy. Importantly, analysis of immune cell population in HCC tumors showed that immune-suppressive regulatory T-cells (Treg) are highly enriched in high metabolic HCC tumors, suggesting that high metabolic activity of HCC cells may trigger activation or infiltration of Treg cells, leading to evasion of HCC cells from anti-cancer immune cells. Interestingly, recent study showed that Treg cells can effectively utilize lactic acid as metabolic fuel for its proliferation. Because high metabolic activity of HCC tumors eventually leads to accumulation of glycolysis by-product lactic acid in tumor microenvironment, we postulate that accumulated lactic acid in tumor microenvironment might account for aggregation of Treg cells in high metabolic HCC tumors, leading to resistance to immunotherapy. In agreement with this, 18F-FDG PET/CT imaging data showed that high metabolic activity of tumors reflected in SUVmax is significantly associated with poorer survival of cancer patients after immunotherapy. Conclusions: We identified clinically and metabolically distinct subtypes of HCC tumors, potential biomarkers associated with these subtypes, and potential mechanism of metabolism-mediated evasion of HCC cells to anti-cancer immunotherapy. Moreover, the metabolic subtypes are well conserved in PDX models, offering a tool for selecting the best preclinical models for future study. Citation Format: Sowon Grace Park, Joann Jung, Yeonwoo Grace Jang, Sung Hwan Lee, Yun-Seong Jeong, Sun Young Yim, Ju-Seog Lee. Clinical significance of glycolytic metabolic activity in liver cancer and its implication to immunotherapy [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 296.

LncRNA miR4458HG modulates hepatocellular carcinoma progression by activating m6A-dependent glycolysis and promoting the polarization of tumor-associated macrophages

Long non-coding RNAs (lncRNAs) play significant roles in different biological functions of cancers. However, their function in the metabolism of glucose in patients with human hepatocellular carcinoma (HCC) remains largely unknown. In this study, HCC and paired intact liver tissues were utilized to examine the miR4458HG expression using qRT-PCR and human HCC cell lines to examine cell proliferation, colony formation, and glycolysis after transfection of siRNAs targeting miR4458HG or miR4458HG vectors. The molecular mechanism of miR4458HG was clarified through in situ hybridization, Western blotting, qRT-PCR, RNA pull-down, and RNA immunoprecipitation analysis. The results showed that the miR4458HG affected HCC cell proliferation, activated the glycolysis pathway, and promoted the polarization of tumor-associated macrophage in vitro and in vivo models. Mechanistically, miR4458HG bound IGF2BP2 (a key RNA m6A reader) and facilitated IGF2BP2-mediated target mRNA stability, including HK2 and SLC2A1 (GLUT1), and consequently altered HCC glycolysis and tumor cell physiology. At the same time, HCC-derived miR4458HG could be wrapped in the exosomes and promoted the polarization of tumor-associated macrophage by increasing ARG1 expression. Hence, miR4458HG is oncogenic in nature among patients with HCC. To develop an effective treatment strategy of HCC patients presenting with high glucose metabolism, physicians should focus on miR4458HG and its pathway.

CD36+ cancer-associated fibroblasts provide immunosuppressive microenvironment for hepatocellular carcinoma via secretion of macrophage migration inhibitory factor

Hepatocellular carcinoma (HCC) is an immunotherapy-resistant malignancy characterized by high cellular heterogeneity. The diversity of cell types and the interplay between tumor and non-tumor cells remain to be clarified. Single cell RNA sequencing of human and mouse HCC tumors revealed heterogeneity of cancer-associated fibroblast (CAF). Cross-species analysis determined the prominent CD36+ CAFs exhibited high-level lipid metabolism and expression of macrophage migration inhibitory factor (MIF). Lineage-tracing assays showed CD36+CAFs were derived from hepatic stellate cells. Furthermore, CD36 mediated oxidized LDL uptake-dependent MIF expression via lipid peroxidation/p38/CEBPs axis in CD36+ CAFs, which recruited CD33+myeloid-derived suppressor cells (MDSCs) in MIF- and CD74-dependent manner. Co-implantation of CD36+ CAFs with HCC cells promotes HCC progression in vivo. Finally, CD36 inhibitor synergizes with anti-PD-1 immunotherapy by restoring antitumor T-cell responses in HCC. Our work underscores the importance of elucidating the function of specific CAF subset in understanding the interplay between the tumor microenvironment and immune system.

Targeting Glutamine Metabolism with a Novel Na+/K+-ATPase Inhibitor RX108 in Hepatocellular Carcinoma.

The poor prognosis and limited therapeutic options for human hepatocellular carcinoma (HCC), the most common form of liver cancer, highlight the urgent need to identify novel therapeutic modalities. Here, we describe the antitumor activity and underlying molecular mechanisms of a novel Na+/K+-ATPase inhibitor RX108 in human HCC cells and its xenograft model. RX108 dose-dependently inhibited HCC cell proliferation in vitro and tumor growth in a xenograft mouse model, and that the inhibition was associated with induction of apoptosis. Mechanistically, RX108 significantly downregulated alanine serine cysteine transporter 2 (ASCT2) protein expression and reduced glutamine and glutamate concentration in HCC cells and tumors. In addition, RX108 exposure led to a significant decrease in cell energy metabolism in Huh7 and Hep3B cells, including decreased levels of glutathione, NADH, NADPH, and mitochondrial respiration oxygen consumption rate. Furthermore, HCC cells exhibited evidence of glutamine addiction; the antiproliferative effect of RX108 was dependent on glutamine transport. Clinically, elevated ASCT2 mRNA expression in HCCs was associated with unfavorable survival. Taken together, these findings reveal a novel approach to target glutamine metabolism through inhibiting Na+/K+-ATPase and provide a rationale for using RX108 to treat HCC in patients whose tumors express ASCT2 at high levels. RX108 is currently under clinical development.

Inhibition of Checkpoint Kinase 1 (CHK1) Upregulates Interferon Regulatory Factor 1 (IRF1) to Promote Apoptosis and Activate Anti-Tumor Immunity via MICA in Hepatocellular Carcinoma (HCC).

CHK1 is considered a key cell cycle checkpoint kinase in DNA damage response (DDR) pathway to communicate with several signaling pathways involved in the tumor microenvironment (TME) in numerous cancers. However, the mechanism of CHK1 signaling regulating TME in hepatocellular carcinoma (HCC) remains unclear. CHK1 expression in HCC tissue was determined by IHC staining assay. DNA damage and apoptosis in HCC cells induced by cisplatin or CHK1 inhibition were detected by WB and flow cytometry. The interaction of CHK1 and IRF1 was analyzed by single-cell RNA-sequence, WB, and immunoprecipitation assay. The mechanism of IRF1 regulating MICA was investigated by ChIP-qPCR. CHK1 expression is upregulated in human HCC tumors compared to the background liver. High CHK1 mRNA level predicts advanced tumor stage and worse prognosis. Cisplatin and CHK1 inhibition augment cellular DNA damage and apoptosis. Overexpressed CHK1 suppresses IRF1 expression through proteolysis. Furthermore, single-cell RNA-sequence analyses confirmed that MICA expression positively correlated with IRF1 in HCC cells. Immunoprecipitation assay showed the binding between CHK1 and IRF1. Cisplatin and CHK1 inhibition upregulate MICA expression through IRF1-mediated transcriptional effects. A novel specific cis-acting IRF response element was identified at -1756 bp in the MICA promoter region that bound IRF1 to induce MICA gene transcription. MICA may increase NK cell and CD8+T cell infiltration in HCC. DNA damage regulates the interaction of CHK1 and IRF1 to activate anti-tumor immunity via the IRF1-MICA pathway in HCC.

Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway

ObjectivePhysical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. MethodsA physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. ResultsSwimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin+/CD31+ ratio, rather than the number of CD31+ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. ConclusionSwimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor.Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184–193.

Dickkopf-1 drives tumor immune evasion by inducing PD-L1 expression in hepatocellular carcinoma

Understanding the mechanisms regulating PD-L1 expression in hepatocellular carcinoma (HCC) is important to improve the response rate to PD-1/PD-L1 blockade therapy. Here, we show that DKK1 expression is positively associated with PD-L1 expression and inversely correlated with CD8+ T cell infiltration in human HCC tumor specimens. In a subcutaneous xenograft tumor model, overexpression of DKK1 significantly promotes tumor growth, tumoral PD-L1 expression, but reduces tumoral CD8+ T cell infiltration; whereas knockdown of DKK1 has opposite effects. Moreover, enforced expression of DKK1 dramatically promotes PD-L1 expression, Akt activation, β-catenin phosphorylation and total protein expression in HCC cells. By contrast, knockdown of DKK1 inhibits all, relative to controls. In addition, CKAP4 depletion, Akt inhibition, or β-catenin depletion remarkably abrogates DKK1 overexpression-induced transcriptional expression of PD-L1 in HCC cells. Reconstituted expression of the active Akt1 largely increased PD-L1 transcriptional expression in HCC cells. Similarly, expression of WT β-catenin, but not the phosphorylation-defective β-catenin S552A mutant, significantly promotes PD-L1 expression. Correlation analysis of human HCC tumor specimens further revealed that DKK1 and PD-L1 expression were positively correlated with p-β-catenin expression. Together, our findings revealed that DKK1 promotes PD-L1 expression through the activation of Akt/β-catenin signaling, providing a potential strategy to enhance the clinical efficacy of PD-1/PD-L1 blockade therapy in HCC patients.

DKK1 promotes NUAK1 transcriptional expression through the activation Akt in hepatocellular carcinoma.

NUAK1 is a serine/threonine kinase that has been shown to be associated with poor prognosis in several cancers. Although NUAK1 is frequently overexpressed at the transcript level in hepatocellular carcinoma (HCC), the actual role of NUAK1 and the mechanism of its overexpression in HCC has yet to be reported. In the present study, we found that NUAK1 expression was significantly increased in human HCC tumor tissues. Overexpression of NUAK1 dramatically enhanced HCC cells proliferation and migration in vitro. Stable induction of NUAK1 expression promoted tumor growth and tumor metastases to the lungs in the subcutaneous xenograft models and intravenous metastasis models. At the cellular level, enforced expression of Dickkopf-1 (DKK1) activated the Akt signaling pathway, thereby promoting the mRNA and protein expression of NUAK1 in HCC cells. By contrast, depletion of DKK1 was found to attenuate the mRNA and protein expression of NUAK1. In the subcutaneous xenograft models, stable induction of DKK1 expression not only accelerated tumor growth but also increased p-Akt and NUAK1 expression; whereas knockdown of DKK1 inhibited tumor growth, p-Akt and NUAK1 expression. Furthermore, immunohistochemical analysis of 20 HCC clinical samples showed that the expression level of NUAK1 was positively correlated with DKK1 and p-Akt. Taken together, we provide the first evidence that DKK1 promotes NUAK1 transcriptional expression via the activation Akt in HCC.

Correlation between the complex of small heat shock proteins (HSPBs) and the progression in patients with hepatocellular carcinoma

Small heat shock proteins (HSPBs) regulate various cell functions. We previously reported that HSPB1, HSPB6, and HSPB8 each suppress the progression of hepatocellular carcinoma (HCC). The heterooligomerization of HSPs is speculated to be crucial for functional activities. Here, we investigated the relationship between the complex of HSPBs and the progression of HCC. HSPB1/HSPB6 complex and HSPB1/HSPB8 complex, but not HSPB6/HSPB8 complex, were observed in both HSPB6-overexpressing human HCC-derived HuH-7 cells and resected human HCC tumor tissue. Differentiation, stage, tumor size, and vein invasion of HCC were inversely related to the presence of HSPB complexes in the HCC tissue. Our results strongly suggest that the HSPB complex formation plays a suppressive role in the HCC progression.

SCYL3, as a novel binding partner and regulator of ROCK2, promotes hepatocellular carcinoma progression

SCY1-like pseudokinase 3 (SCYL3) was identified as a binding partner of ezrin, implicating it in metastasis. However, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. In this study, we aimed to elucidate the role of SCYL3 in the progression of hepatocellular carcinoma (HCC). The clinical significance of SCYL3 in HCC was evaluated in publicly available datasets and by qPCR analysis of an in-house HCC cohort. The functional significance and mechanistic consequences of SCYL3 were examined in SCYL3-knockdown/overexpressing HCC cells. Invivo tumor progression was evaluated in Tp53 KO/c-Myc OE mice using the sleeping beauty transposon system. Potential downstream pathways were investigated by co-immunoprecipitation, western blotting analysis and immunofluorescence staining. SCYL3 is often overexpressed in HCC; it is preferentially expressed in metastatic human HCC tumors and is associated with worse patient survival. Suppression of SCYL3 in HCC cells attenuated cell proliferation and migration as well as invivo metastasis. Intriguingly, endogenous SCYL3 overexpression increased tumor development and metastasis in Tp53 KO/c-Myc OE mice. Mechanistic investigations revealed that SCYL3 physically binds and regulates the stability and transactivating activity of ROCK2 (Rho kinase 2) via its C-terminal domain, leading to the increased formation of actin stress fibers and focal adhesions. These findings reveal that SCYL3 plays a critical role in promoting the progression of HCC and have implications for developing new therapeutic strategies to tackle metastatic HCC. SCYL3 was first reported to be a binding partner of a metastasis-related gene, ezrin. To date, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. Herein, we uncover its crucial role in liver cancer progression. We show that it physically binds and regulates the stability and transactivating activity of ROCK2 leading to HCC tumor progression. Our data provide mechanistic insight that SCYL3-mediated ROCK2 protein stability plays a pivotal role in growth and metastasis of HCC cells. Targeting SCYL3/ROCK2 signaling cascade may be a novel therapeutic strategy for treatment of HCC patients.

The Elevated Circ_0067835 Could Accelerate Cell Proliferation and Metastasis via miR-1236-3p/Twist2 Axis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant cancer with leading mortality worldwide. Circ_0067835 is a circRNA which plays an important role in various kinds of tumor, while the potential functions of circ_0067835 in HCC remains unclear. In this study, our results of microarray and real-time PCR (RT-PCR) showed that it was obviously elevated in human HCC tumor tissues and HCC cell lines. Inhibition of circ_0067835 restrained cell proliferation and migration in vitro. Furthermore, miR-1236-3p was decreased in tumor samples, and it was indicated to be a target of circ_0067835. Moreover, Twist2 was established to be elevated in HCC tissues, and we identified it as the direct target of miR-1236-3p. Finally, we found that knockdown of miR-1236-3p could reverse the circ_0067835 inhibition effects in HCC cells. In conclusion, our study demonstrated that circ_0067835 contributed to promoting hepatocellular carcinoma cell proliferation and metastasis through downregulating miR-1236-3p expression and then elevating Twist2 expression, which might provide a new vision for HCC patients.

Isoginkgetin synergizes with doxorubicin for robust co-delivery to induce autophagic cell death in hepatocellular carcinoma

Doxorubicin (DOX) widely used in hepatocellular carcinoma (HCC) can induce serious side effects and drug resistance. Herein, we aimed to seek a strategy to improve the efficacy and reduce the side effects of DOX in HCC based on an autophagy inducer drug called isoginkgetin (ISO). The design of multifunctional nanocarriers based on hyaluronic acid-conjugated and manganese-doped mesoporous silica nanoparticles (HM) for the co-delivery of antitumor drugs against HCC provided an effective and promising antitumor strategy. Our results showed that HM@ISO@DOX could efficiently inhibit HCC cell proliferation through activating autophagy through AMPKa-ULK1 pathway. Moreover, intravenous injection of HM@ISO@DOX significantly suppressed HCC tumor progression in nude mouse HCC model. Collectively, our findings revealed an anti-HCC mechanism of HM@ISO@DOX through autophagy and provide an effective therapeutic strategy for HCC. Statement of significanceIn our study, we constructed a co-delivery system by loading ISO and DOX in the mesoporous channels of manganese-doped mesoporous silica nanoparticles, which could be further conjugated with hyaluronic acid to obtain HM@ISO@DOX. The nanocarriers had been demonstrated to be biodegradable under the acidic and reducing tumor microenvironment, as well as to possess the tumor targeting capability via the conjugated hyaluronic acid. In addition, HM@ISO@DOX enhanced the therapeutic efficacy against human HCC tumor through the combinatorial therapies of chemotherapeutics, Mn2+-mediated chemodynamic therapeutics and autophagic cell death, which might be achieved through AMPK-ULK1 signaling. This work revealed that such a nanomedicine exhibited superior tumor accumulation and antitumor efficiency against HCC with extremely low systemic toxicity in an autophagy-boosted manner.