Hepatocellular Carcinoma Growth Research Articles

Hepatitis B virus-induced hepatocellular carcinoma: a persistent global problem.

Hepatitis B virus (HBV) infections are highly prevalent globally, representing a serious public health problem. The diverse modes of transmission and the burden of the chronic carrier population pose challenges to the effective management of HBV. Vaccination is the most effective preventive measure available in the current scenario. Still, HBV is one of the significant health issues in various parts of the globe due to non-response to vaccines, the high number of concealed carriers, and the lack of access and awareness. Universal vaccination programs must be scaled up in neonates, especially in the developing parts of the world, to prevent new HBV infections. Novel treatments like combinational therapy, gene silencing, and new antivirals must be available for effective management. The prolonged infection of HBV, direct and indirect, can promote the growth of hepatocellular carcinoma (HCC). The present review emphasizes the problems and probable solutions for better managing HBV infections, causal risk factors of HCC, and mechanisms of HCC.

HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia

The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

HIGD2A silencing impairs hepatocellular carcinoma growth via inhibiting mitochondrial function and the MAPK/ERK pathway

BackgroundThe Hypoxia inducible gene domain family member 2A (HIGD2A) protein is indispensable for the assembly of the mitochondrial respiratory supercomplex, which has been implicated in cell proliferation and cell survival under hypoxic conditions. Because the liver has a naturally low oxygen microenvironment, the role of HIGD2A in the development of hepatocellular carcinoma (HCC) remains largely unknown.MethodsGene expression data and clinical information were obtained from multiple public databases. A lentivirus-mediated gene knockdown approach was conducted to explore the function and mechanism of HIGD2A activity in HCC cells. In vivo and in vitro assays were performed to investigate the biological roles of HIGD2A.ResultsHIGD2A was overexpressed in HCC tissues and cell lines and was associated with a worse prognosis. Silencing HIGD2A expression significantly attenuated cell proliferation and migration, caused S-phase cell cycle arrest, and decreased tumor formation in nude mice. Mechanistically, HIGD2A depletion greatly decreased cellular ATP levels by disrupting mitochondrial ATP production. Moreover, HIGD2A knockdown cells displayed impaired mitochondrial function, such as mitochondrial fusion, increased expression of the mitochondrial stress response protein, and decreased oxygen consumption. Furthermore, knockdown of HIGD2A markedly attenuated the activation of the MAPK/ERK pathway.ConclusionsHIGD2A promoted liver cancer cell growth by fueling mitochondrial ATP synthesis and activating the MAPK/ERK pathway, suggested that targeting HIGD2A may represent a new strategy for HCC therapy.

MiR-3180 inhibits hepatocellular carcinoma growth and metastasis by targeting lipid synthesis and uptake

PurposeReprogrammed lipid metabolism is a hallmark of cancer that provides energy, materials, and signaling molecules for rapid cancer cell growth. Cancer cells acquire fatty acids primarily through de novo synthesis and uptake. Targeting altered lipid metabolic pathways is a promising anticancer strategy. However, their regulators have not been fully investigated, especially those targeting both synthesis and uptake.MethodsImmunohistochemistry was performed on samples from patients with hepatocellular carcinoma (HCC) to establish the correlation between miR-3180, stearoyl-CoA desaturase-1 (SCD1), and CD36 expression, quantified via qRT-PCR and western blotting. The correlation was analyzed using a luciferase reporter assay. Cell proliferation, migration, and invasion were analyzed using CCK-8, wound healing, and transwell assays, respectively. Oil Red O staining and flow cytometry were used to detect lipids. Triglycerides and cholesterol levels were analyzed using a reagent test kit. CY3-labeled oleic acid transport was analyzed using an oleic acid transport assay. Tumor growth and metastasis were detected in vivo in a xenograft mouse model.ResultsMiR-3180 suppressed de novo fatty acid synthesis and uptake by targeting the key lipid synthesis enzyme SCD1 and key lipid transporter CD36. MiR-3180 suppressed HCC cell proliferation, migration, and invasion in an SCD1- and CD36-dependent manner in vitro. The mouse model demonstrated that miR-3180 inhibits HCC tumor growth and metastasis by inhibiting SCD1- and CD36-mediated de novo fatty acid synthesis and uptake. MiR-3180 expression was downregulated in HCC tissues and negatively correlated with SCD1 and CD36 levels. Patients with high miR-3180 levels showed better prognosis than those with low levels.ConclusionsOur investigation indicates that miR-3180 is a critical regulator involved in de novo fatty acid synthesis and uptake, which inhibits HCC tumor growth and metastasis by suppressing SCD1 and CD36. Therefore, miR-3180 is a novel therapeutic target and prognostic indicator for patients with HCC.

MiR-17-5p slows progression of hepatocellular carcinoma by downregulating TGFβR2.

Downregulation of miR-17-5p has been reported in several cancers, but whether and how miR-17-5p is downregulated in hepatocellular carcinoma (HCC) is unknown. Here, we examined whether miR-17-5p is downregulated in HCC and whether that affects expression of its target gene encoding transforming growth factor β receptor 2 (TGFβR). We screened for potential microRNAs (miRNAs) involved in HCC by analyzing published transcriptomes from HCC patients. Expression of miR-17-5p was measured in HCC cell lines and in tissues from HCC patients using quantitative real-time PCR. The in vitro effects of miR-17-5p on HCC cells were assessed by EdU proliferation assay, CCK-8 cell proliferation assay, colony-formation assay, transwell migration/invasion assay, wound healing assay, and flow cytometry. Effects of miR-17-5p were evaluated in vivo using mice with subcutaneous tumors. Effects of the miRNA on the epithelial-mesenchymal transition (EMT) were assessed, while its effects on TGFβR2 expression were analyzed using bioinformatics and a dual luciferase reporter assay. Patients with low miR-17-5p expression showed lower rates of overall and recurrence-free survival than patients with high miR-17-5p expression, and multivariate Cox regression identified low miR-17-5p expression as an independent predictor of poor overall survival in HCC patients. In vitro, miR-17-5p significantly inhibited HCC cell proliferation, migration, invasion, and the EMT, while promoting apoptosis. In vivo, it slowed the development of tumors. These protective effects of miR-17-5p were associated with downregulation of TGFβR2. The miRNA miR-17-5p can negatively regulate the expression of TGFβR2 and inhibit the EMT, thereby slowing tumor growth in HCC, suggesting a potential therapeutic approach against HCC.

Dyrk2 gene transfer suppresses hepatocarcinogenesis by promoting the degradation of Myc and Hras

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and has a poor prognosis. However, the molecular mechanisms underlying hepatocarcinogenesis and progression remain unknown. Invitro gain- and loss-of-function analyses in cell lines and xenografts revealed that dual-specificity tyrosine-regulated kinase 2 (DYRK2) influences tumour growth in HCC. To investigate the role of Dyrk2 during hepatocarcinogenesis, we developed liver-specific Dyrk2 conditional knockout mice and an invivo gene delivery system with a hydrodynamic tail vein injection and the Sleeping Beauty transposon. The antitumour effects of Dyrk2 gene transfer were investigated in a murine autologous carcinogenesis model. Dyrk2 expression was reduced in tumours, and that its downregulation was induced before hepatocarcinogenesis. Dyrk2 gene transfer significantly suppressed carcinogenesis. It also suppresses Myc-induced de-differentiation and metabolic reprogramming, which favours proliferative, and malignant potential by altering gene profiles. Dyrk2 overexpression caused Myc and Hras degradation at the protein level rather than at the mRNA level, and this degradation mechanism was regulated by the proteasome. Immunohistochemical analyses revealed a negative correlation between DYRK2 expression and MYC and longer survival in patients with HCC with high-DYRK2 and low-MYC expressions. Dyrk2 protects the liver from carcinogenesis by promoting Myc and Hras degradation. Our findings would pave the way for a novel therapeutic approach using DYRK2 gene transfer. Hepatocellular carcinoma (HCC) is one of the most common cancers, with a poor prognosis. Hence, identifying molecules that can become promising targets for therapies is essential to improve mortality. No studies have clarified the association between DYRK2 and carcinogenesis, although DYRK2 is involved in tumour growth in various cancer cells. This is the first study to show that Dyrk2 expression decreases during hepatocarcinogenesis and that Dyrk2 gene transfer is an attractive approach with tumour suppressive activity against HCC by suppressing Myc-mediated de-differentiation and metabolic reprogramming that favours proliferative and malignant potential via Myc and Hras degradation.

Transmembrane protein 88 suppresses hepatocellular carcinoma progression and serves as a novel prognostic factor.

Transmembrane protein 88 (TMEM88) is known to be involved in the canonical Wnt signaling pathway and is implicated in several malignancies. However, the expression, function, and prognostic significance of TMEM88 in hepatocellular carcinoma (HCC) remain unclear. In this study, we analyzed mRNA levels of TMEM88 in HCC specimens from the TCGA dataset (n=374) to explore the correlation between TMEM88 and HCC. We also overexpressed TMEM88 in the Huh7 human HCC cell line to investigate its tumor-related role in HCC. Additionally, we conducted in vivo experiments using a mouse model to further validate the critical function of TMEM88 in modulating HCC growth. Our results showed that TMEM88 is negatively correlated with the T stage, TNM stage, and pathological grade of HCC. Higher levels of TMEM88 can help predict better overall survival of HCC in both univariate and multivariate analyses. Similarly, higher TMEM88 is a novel prognostic factor for better disease-specific survival of HCC. Overexpression of TMEM88 in Huh7 cells led to a decreased cell proliferation capacity. Xenograft experiments in a mouse model showed that TMEM88 overexpression can remarkably suppress HCC progression. Transmembrane protein 88 suppresses HCC growth both in vitro and in vivo, which can act as a potential prognostic factor with clinical application potential.

Abstract 4456: Inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX2) inhibition reprogram the tumor microenvironment and suppress tumor growth in hepatocellular carcinoma

Abstract Hepatocellular carcinoma is a common cancer worldwide and a leading cause of cancer-related death. Chronic inflammation, accumulation of genetic changes and alternations of the liver microenvironment are critical during the process of carcinogenesis and development of HCC. Inducible nitric oxide synthase (iNOS or NOS2)-derived NO and cyclooxygenase-2 (COX2) are important parts of the neoplastic inflammatory environment. Continuous exposure to moderate to high concentrations of NO, produced by inducible NO synthase (iNOS) and COX2, promotes neoplastic transformation, chemotherapeutic resistance, enhanced cell proliferation, increased inflammation and immune resistance. Our previous study reported that iNOS promoted stem-like characteristics and activation of liver cancer stem cells through Notch signaling pathway. iNOS activity in both the liver cancer stem cells and the microenvironment contributes to tumor progression in mice, suggesting that targeting iNOS in the entire tumor could have therapeutic benefit in HCC. iNOS-derived NO and COX2-derived prostaglandin E2 (PGE2) were shown to promote feed-forward iNOS/COX2 crosstalk because NO induced COX2 and PGE2 induced NOS2. Our research showed that iNOS inhibition with the iNOS inhibitor (1400W) and COX2 inhibitor (Celebrex) diminished HCC tumor growth. The cytokine analysis revealed considerably lower levels of inflammatory cytokines including, IL-6, tissue inhibitor of matrix metalloproteinase-1 (TIMP1), macrophage inflammatory protein-3alpha (MIP-3alpha), IL-1B, CC chemokine subfamily of eosinophil chemotactic proteins eotaxins (CCL11), M-CSF, thymus and activation-regulated chemokine (TARC, CCL17) and leukemia inhibitory factor (LIF) in HCC with iNOS/COX2 inhibition. iNOS/COX2 levels influence the polarization and spatial location of lymphoid cells including CD8+ T cells. Targeting iNOS/COX2 blockade improved CD8+ T cell penetration into the tumor core. We also found that iNOS/COX2 blockade result in more CD4+ T helper cells and CD8+ tumor infiltrating lymphocyte (TIL), but reduce the number of exhausted CD4+ T cells and CD8+ T cells (PD1-high, Lag3+, CD39+ Tex). The results suggesting that iNOS/COX2 inhibitor therapy may alleviate HCC growth by promoting a anti-tumorigenic TME, modifying lymphoid spatial localization and gene expression phenotypes and decreasing T-cell exhaustion. Citation Format: Ronghua Wang, Christof Kaltenmeier, Ruiqi Yang, Tony Haykal, Hamza Yazdani, Celine Tohme, David Geller, Samer Tohme, Timothy Billiar. Inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX2) inhibition reprogram the tumor microenvironment and suppress tumor growth in hepatocellular carcinoma. [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 4456.

Abstract 3808: MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs

Abstract Kupffer cells (KCs) account for 15% of the total liver cells. However, their roles in hepatocellular carcinoma (HCC) is poorly described. Clec4fCre-tdTomato knock-in mice expressing Cre recombinase and tdTomato-NLS under the KC-specific Clec4f promoter were hydrodynamically injected with oncogenic AKT and Nras (AKT/Ras) to induce HCC. A LoxP-stop-LoxP (LSL) system was used to express miR-206 in KCs of mice. AKT activation was observed in hepatocytes and KCs of HCC patients. Hydrodynamic injection (HDI) of AKT/Ras led to activation of AKT signaling in KCs and hepatocytes. AKT/Ras mice developed lethal HCC within 6-8 weeks post injection. Activation of AKT signaling led to M2 polarization of KCs, increased hepatic Tregs and exhaustion of CTLs. MicroRNA (miR) profiling revealed that AKT/Ras impaired miR-206 biogenesis in KCs by driving translation of Yin Yang 1 (YY1), a transcription repressor of miR-206. HDI of miR-206 into AKT/Ras mice fully prevented HCC, while all control mice died of HCC 6-8 weeks post injection. However, HDI of miR-206 only transfected ~ 20% of hepatocytes, indicating that it is unreasonable to conclude that the full prevention of HCC in AKT/Ras/miR-206 mice is caused by inhibited proliferation of HCC cells. We, therefore, posited that miR-206-mediated recovery of immune surveillance, at least in part, accounted for the full prevention of HCC in AKT/Ras/miR-206 mice. We next used the LSL system to overexpress miR-206 in KCs of Clec4fCretdTomato mice injected with AKT/Ras. KC-specific expression of miR-206 fully prevented HCC in AKT/Ras mice, while 100% control mice died of HCC. Mechanistically, AKT signaling in KCs drove CCL22 (C-C motif chemokine ligand 22) production by phosphorylating β-catenin that activates transcription of Ccl22. In contrast, miR-206 disrupted β-catenin signaling by targeting LEF1 (lymphoid enhancer-binding factor 1), an “architectural” transcription factor of β-catenin. CCL22 recruits Tregs. Indeed, KC-specific expression of miR-206 led to elevated CCL22, M2 to M1 transition of KCs, reduced hepatic Tregs, and elevated CTLs. Depletion of CTLs in AKT/Ras mice fully offset the ability of miR-206 to prevent HCC. Conclusions: AKT-educated KCs initiate Treg recruitment via a regulatory loop consisting of YY1, β-catenin/LEF1, miR-206, and CCL22; once this loop is activated, AKT activates β-catenin axis, which drives CCL22 production, Treg recruitment and HCC growth; while KC-specific expression of miR-206 reversed this process by blocking β-catenin axis in KCs. miR-206 represents a novel immunotherapy against HCC. Citation Format: Guisheng Song, Ningning Liu. MicroRNA-206 drives antitumor immunity by disrupting the communication between Kupffer cells and Tregs. [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 3808.

Abstract 3959: Novel spliced isoform of the proteasome subunit ADRM1/Rpn13 promotes hepatocellular carcinoma (HCC) development through selective degradation of tumor suppressor p53

Abstract The Ubiquitin-Proteasome System (UPS) is the major pathway in eukaryotic cells that regulates intracellular protein degradation and turnover. Cumulative evidence has suggested that defects in the UPS machinery are involved in the pathogenesis of human diseases, including cancer. The proteasome-associated polyubiquitin receptor ADRM1 (also known as Rpn13) orchestrates the process of substrate recruitment with deubiquitination in the proteasome through its N-terminal ubiquitin-binding domain and C-terminal activation of deubiquitinating enzyme. Using long-read SMRT-seq to profile the RNA splicing landscape in hepatocellular carcinoma (HCC), we previously reported on an unannotated alternative spliced variant of ADRM1 in human hepatocellular carcinoma (HCC) (Hepatology 2019). This novel isoform exhibits an exon 9 skipping (ADRM1-ΔEx9) that resulted in an altered C-terminus of the protein. In this study, we undertook further investigations to characterize the detailed functional effects of ADRM1-ΔEx9 in HCC biology and the underlying mechanism. Initial study by junction-specific Taqman PCR assay showed that ADRM1-ΔEx9 is more frequently upregulated in HCC tumors than the canonical full-length counterpart (ADRM1-FL). There is an interchange between the two isoforms as tumor develops which underscores an isoform-switch in favor of HCC development. Indeed, our result showed that ADRM1-ΔEx9, but not ADRM1-FL, correlated significantly with inferior patient survival. Functionally, ADRM1-ΔEx9 knockdown profoundly suppressed proliferation by inducing spontaneous apoptosis in HCC cell lines and patient-derived HCC organoids. The corresponding effects were less apparent from ADRM1-FL knockdown. Consistently, overexpression of ADRM1-ΔEx9 in normal human liver organoids promoted pre-malignant features alongside increased in-vitro propagation time. Ubiquitin Proteome profiling revealed that ADRM1-ΔEx9 led to a reduced expression of the pivotal tumor suppressor p53. A parallel transcriptome also substantiated that ADRM1-ΔEx9 modulated genes involved in the p53 and apoptosis pathways. Collectively, our study demonstrated that the non-canonical spliced isoform ADRM1-ΔEx9 confers growth and pro-survival advantages in HCC through mediating selective degradation of tumor suppressor p53 protein. Citation Format: Yanmei Sun, Mingjing Xu, Ho Lee Wan, Alissa M. Wong, Kelvin K. Ng, Nathalie Wong. Novel spliced isoform of the proteasome subunit ADRM1/Rpn13 promotes hepatocellular carcinoma (HCC) development through selective degradation of tumor suppressor p53. [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 3959.

Abstract 3683: Activation of purine anabolism creates a therapeutic vulnerability in hepatocellular carcinoma via m6A-mediated epitranscriptomic regulation

Abstract Introduction Purines are building blocks for genomics and the abundance of purine nucleotides is controlled by purine synthesis and purine degradation. Imbalance of purine nucleotide pool in tumors has been shown, but how synthesis and degradation of purine integrates in tumors has not been well characterized. Hepatocellular carcinoma (HCC) is the most common liver cancer with a high mortality rate and limited treatments. Aberrant purine metabolism was observed in HCC, but the functional status of global purine metabolism in HCC and how that drive HCC fitness and therapeutic response remain unclear. Method HCC-specific purine metabolic changes were identified from the transcriptomic and metabolic data of tumor and paired non-tumor liver tissues obtained from 62 HCC patients from Thailand (discovery cohort) and validated in an additional 672 HCC with different race/ethnicities and etiologies. Correlation analysis of purine metabolic alteration and tumor-associated genomic, transcriptome and metabolome were conducted. The impacts of purine metabolism on HCC survival and therapeutic vulnerability were investigated. Biological functions of purine metabolic alterations were assessed in vitro and in vivo on multiple HCC cell lines. Results Using multi-omics integrative analyses, we found a tumor-specific activation of purine anabolism, induced by upregulation of purine de novo biosynthesis and inhibition of purine degradation, in HCC. A high purine anabolic status was associated with dysregulation of the DNA damage repairing (DDR) machinery in HCC, accompanied by unique somatic mutational signatures linked to patient prognosis. By examining drug responses to 180 molecular targeted agents in HCC cells, we found that increasing purine anabolism induced a therapeutic vulnerability of HCC to DDR targeting agents. Mechanistically, we found that excessive purine metabolites induced N6-methyladenosine (m6A) modification on DTL, a DDB1 cullin4 associated factor, thereby delaying the degradation of DTL mRNA transcriptomic remodeling of the DDR machinery and tumor fitness. Experimentally, we demonstrated that suppressing purine anabolism or exposing to DDR targeting agent, such as berzosertib, inhibited the growth of HCCs with high purine anabolism in vitro and in vivo. Conclusion Our study suggests that targeting purine anabolism or DDR may represent as attractive strategies in patients with high purine anabolic HCCs, and purine anabolic status could be a valuable biomarker to allocate systemic treatments for patients with advanced HCC. Citation Format: Man-Hsin Hung, Ching Wen Chang, Kathy Cheng Wang, Jittiporn Chaisaingmongkol, Mathuros Ruchirawat, Tim F. Greten, Xin Wei Wang. Activation of purine anabolism creates a therapeutic vulnerability in hepatocellular carcinoma via m6A-mediated epitranscriptomic regulation. [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 3683.

Abstract 2679: Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models

Abstract Introduction: The cell cycle inhibitor palbociclib has been shown to have a cytostatic effect on hepatocellular carcinoma (HCC) cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested HCC cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining palbociclib with the mTOR inhibitor rapamycin on preclinical models of HCC. Methods: We identified commonly altered molecular markers in HCC, including cell cycle related genes, using single sample gene set enrichment analysis (ssGSEA) of 13 paired (tumor and adjacent non-tumor) RNA-seq and 15 paired proteomic data sets as well as 50 paired RNA-seq data sets from TCGA. Subsequently, we studied the in-vitro effects of palbociclib and rapamycin by evaluating induction of senescence, inhibition of cell growth with fixed ratio combination treatment as well as Western Blotting to establish mechanistic effects. We tested the in-vivo anti-tumor efficacy of palbociclib plus rapamycin in athymic nude mice with growing xenograft tumors. We then generated immunocompetent murine HCC models using hydrodynamic transfection of oncogenes ΔN90-β-catenin and c-Met, and measured plasma AFP levels as a marker of HCC to assess the efficacy of treatment with palbociclib and/or rapamycin. Results: ssGSEA revealed significant positive enrichment of several cell cycle related gene sets in all our HCC cases. Palbociclib treatment in vitro showed an increase in senescence associated β-galactosidase staining as well as increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27, indicating senescence. Fixed ratio treatment with palbociclib and rapamycin established a synergistic interaction between the drugs, as indicated by a combination index of less than 1. We also observed an increase in levels of proteins of the mTOR pathway (p-mTOR, p-4eBP1, p-p70 and p-s6) after palbociclib treatment, followed by suppression of the levels on addition of rapamycin. In the xenograft HCC model, combination treatment with palbociclib and rapamycin resulted in slower tumor growth and significantly increased time to critical tumor volume (2000 mm3) (log rank, p <0.001). In the hydrodynamic transfection HCC model, we observed a significantly lower mean AFP level in the combination treatment group, along with lower liver weights which were closer to the liver weights of normal mice. Conclusion: HCC cells overexpress genes and proteins that drive cell cycle progression. Palbociclib induces senescence in HCC cells along with an associated upregulation in mTOR signaling, and combination with rapamycin has a synergistic effect on inhibition of tumor growth in vitro as well as in vivo, possibly by abrogating both actively growing and quiescent/senescent HCC cells. The combination of the two drugs may have potential as a novel therapy for HCC. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models [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 2679.

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

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

Development of a risk score to predict portal vein tumor thrombosis in patients with hepatocellular carcinoma.

Portal vein tumor thrombosis (PVTT) is a common complication of hepatocellular carcinoma and is one of the most negative prognostic factors. The management of patients with PVTT is challenging. The aim of the study was to develop a score predictive of tumor thrombosis. Data from a large cohort of 2243 hepatocellular carcinoma patients (all stages) recorded in the Progetto Epatocarcinoma Campania (January 2013-April 2021) database were analyzed. To construct the score, univariate generalized estimated equation models, the bootstrap approach for internal validation, and a regression coefficient-based scoring system were used. PVTT (any location) was found in 14.4% of cases and was related to shorter survival. Males, younger patients, and symptomatic cases were more prevalent among the PVTT group. At multivariate analysis, size ≥5 cm, massive or infiltrative hepatocellular carcinoma growth, and alpha-fetoprotein ≥400 ng/mL were significantly associated with PVTT. A risk prediction score of PVTT based on eight variables was developed. Using a continuous score, the risk was associated with an odds ratio (OR) of 1.30 (1.27-1.34; P < 0.001). Considering a dichotomous score >8 versus a score ≤8 the OR for PVTT was 11.33 (8.55-15.00; P < 0.001). The risk score for PVTT might be useful for clinicians to optimize hepatocellular carcinoma management by picking out patients with more aggressive cancers and higher mortality rates. Prospective validation of the score is needed before its application in daily clinical practice.

Abstract 349: LncRNA Myca controls hepatocellular carcinoma growth and progression

Abstract Long non-coding RNAs (lncRNAs) have been shown to play critical roles in the tumorigenesis and progression of cancer. From a clinic and genomic perspective of evidence, the MYC signaling plays a crucial role in triggering hepatocellular carcinoma (HCC) and therefore represents an attractive target for HCC intervention. However, no specific drug is currently available to target MYC directly due to the lack of an enzymatic pocket. Therefore, deciphering the critical lncRNAs that functionally crosstalk with MYC protein levels may offer novel therapeutic strategies in HCC. Here we identify the involvement of a lncRNA Myca (Myc Activator) in HCC through controlling MYC protein levels. Overexpression of Myca triggers tumor growth in a MYC-dependent manner, while inactivation of Myca by antisense oligonucleotides (ASOs) profoundly reduces MYC protein levels, thus suppressing tumor progression in vitro. Mechanistically, we show that Myca attenuates MYC ubiquitination and leads to MYC accumulation. Clinically, overexpression of Myca is positively correlated with Hi-MYC transcriptional profile and HCC progression from the TCGA dataset. Together, our findings not only identify Myca in controlling MYC protein levels but also provide Myca as a new therapeutic lncRNA target for the HCC intervention. Citation Format: Hsin-Yi Chen, Yu-Yun Weng, Wei-Chien Yuan, Yu-Ru Lee. LncRNA Myca controls hepatocellular carcinoma growth and progression [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 349.

Nanodrug removes physical barrier to promote T-cell infiltration for enhanced cancer immunotherapy.

The dense extracellular matrix (ECM) is a key barrier to tumor infiltration of cytotoxic T lymphocytes (CTLs), which greatly compromises T cell-dependent immunotherapy of hepatocellular carcinoma (HCC). Herein, hyaluronidase (HAase), IL-12, and anti-PD-L1 antibody (αPD-L1) were co-delivered using a pH and MMP-2 dual-sensitive polymer/calcium phosphate (CaP) hybrid nanocarrier. The dissolution of CaP triggered by tumor acidity facilitated the release of IL-12 and HAase responsible for ECM digestion, enhancing the tumor infiltration and proliferation of CTLs. Furthermore, the in situ-released αPD-L1 inside tumor, as triggered by an overexpressed MMP-2, prevented the tumor cell from escaping the killing effects of CTLs. Such combination strategy induced a robust antitumor immunity for efficiently suppressing HCC growth in mice. Additionally, tumor acidity-sheddable polyethylene glycol (PEG) coating enhanced the tumor accumulation of nanocarrier and reduced the immune-related adverse events (irAEs) induced by on-target off-tumor αPD-L1. This dual-sensitive nanodrug demonstrates an effective immunotherapy paradigm for other dense ECM-characterized solid tumors.

High Expression of Heterogeneous Nuclear Ribonucleoprotein A1 Facilitates Hepatocellular Carcinoma Growth.

Hepatocellular carcinoma (HCC) represents one of the most common tumors in the world. Our study aims to explore new markers and therapeutic targets for HCC. Heterogeneous Nuclear ribonucleoprotein A1 (hnRNPA1) has recently been found to be involved in the progression of several types of cancer, but its role in HCC remains uncovered. We performed bioinformatic analysis to preliminarily show the relationship between hnRNPA1 and liver cancer. Then the correlation of the hnRNPA1 gene expression with clinicopathological characteristics of HCC patients was verified by human liver cancer tissue microarrays. The functional role of this gene was evaluated by in vivo and vitro experiments. Results showed that the expression of hnRNPA1 was upregulated in HCC tissues and was associated with pathological stage of HCC patients. Knockdown of hnRNPA1 gene markedly inhibited tumor growth in vivo, and reversed the effects on proliferation, migration and invasion and promoted apoptosis in vitro. Furthermore, down-regulation of hnRNPA1 gene expression can inhibit the activity of the MEK/ERK pathway. In our work, we combined bioinformatic analysis with in vivo and in vitro experiments to initially elucidate the function of hnRNPA1 in liver cancer, which may help to explore biomarkers and therapeutic targets for HCC patients.

Histone-lysine N-methyltransferase EHMT2 (G9a) inhibition mitigates tumorigenicity in Myc-driven liver cancer.

Hepatocellular carcinoma (HCC) is the third deadliest and sixth most common cancer in the world. Histone-lysine N-methyltransferase EHMT2 (also known as G9a) is a histone methyltransferase frequently overexpressed in many cancer types, including HCC. We showed that Myc-driven liver tumors have a unique H3K9 methylation pattern with corresponding G9a overexpression. This phenomenon of increased G9a was further observed in our c-Myc-positive HCC patient-derived xenografts. More importantly, we showed that HCC patients with higher c-Myc and G9a expression levels portend a poorer survival with lower mean survival months. We demonstrated that c-Myc interacts with G9a in HCC and cooperates to regulate c-Myc-dependent gene repression. In addition, G9a stabilizes c-Myc to promote cancer development, contributing to the growth and invasive capacity in HCC. Furthermore, combination therapy between G9a and synthetic-lethal target of c-Myc, CDK9, demonstrates strong efficacy in patient-derived avatars of Myc-driven HCC. Our work suggests that targeting G9a could prove to be a potential therapeutic avenue for Myc-driven liver cancer. This will increase our understanding of the underlying epigenetic mechanisms of aggressive tumor initiation and lead to improved therapeutic and diagnostic options for Myc-driven hepatic tumors.

Hypoxia-induced lncRNA MRVI1-AS1 accelerates hepatocellular carcinoma progression by recruiting RNA-binding protein CELF2 to stabilize SKA1 mRNA

BackgroundLong non-coding RNAs (lncRNAs) perform a vital role during the progression of hepatocellular carcinoma (HCC). Here, we aimed to identify a novel lncRNA involved in HCC development and elucidate the underlying molecular mechanism.MethodsThe RT-qPCR and TCGA dataset analysis were applied to explore the expressions of MRVI1-AS1 in HCC tissues and cell lines. Statistical analysis was applied to analyze the clinical significance of MRVI1-AS1 in HCC. The functions of MRVI1-AS1 in HCC cells metastasis and growth were explored by transwell assays, wound healing assay, MTT assay, EdU assay, the intravenous transplantation tumor model, and the subcutaneous xenograft tumor model. Microarray mRNA expression analysis, dual luciferase assays, and actinomycin D treatment were used to explore the downstream target of MRVI1-AS1 in HCC cells. RIP assay was applied to assess the direct interactions between CELF2 and MRVI1-AS1 or SKA1 mRNA. Rescue experiments were employed to validate the functional effects of MRVI1-AS1, CELF2, and SKA1 on HCC cells.ResultsMRVI1-AS1 was found to be dramatically upregulated in HCC and the expression was strongly linked to tumor size, venous infiltration, TNM stage, as well as HCC patients’ outcome. Cytological experiments and animal experiments showed that MRVI1-AS1 promoted HCC cells metastasis and growth. Furthermore, SKA1 was identified as the downstream targeted mRNA of MRVI1-AS1 in HCC cells, and MRVI1-AS1 increased SKA1 expression by recruiting CELF2 protein to stabilize SKA1 mRNA. In addition, we found that MRVI1-AS1 expression was stimulated by hypoxia through a HIF-1-dependent manner, which meant that MRVI1-AS was a direct downstream target gene of HIF-1 in HCC.ConclusionIn a word, our findings elucidated that hypoxia-induced MRVI1-AS1 promotes metastasis and growth of HCC cells via recruiting CELF2 protein to stabilize SKA1 mRNA, pointing to MRVI1-AS1 as a promising clinical application target for HCC therapy.

Stratifin promotes the growth and proliferation of hepatocellular carcinoma

ObjectiveHepatocellular carcinoma (HCC) is the second leading cancer cause of death worldwide. SFN plays a vital role in some malignancies. The purpose of this study was to investigate the role of SFN in the development of HCC. MethodsThe bioinformatics database was used to detect the expression of SFN and its prognosis in HCC patients. And the protein-protein interaction network was established. IHC and Elisa were used to analyze the expression level and clinical characteristics of SFN in HCC patients. Subsequently, siRNA knockdown of SFN expression in HCC cell lines was used to explore whether SFN could promote the development of HCC. ResultsSFN was highly expressed in the tissues and serum of hepatocellular carcinoma, and its expression level was correlated with the tumor which was single or not in patients. Bioanalysis and histochemistry results showed that CDC25B was co-expressed with SFN in HCC, which may be the upstream and downstream signaling molecule of SFN. Knockdown of SFN can inhibit cell proliferation, migration, invasion and promote apoptosis. ConclusionsOur results suggest that SFN may play an important role in HCC progression and may interact with CDC25B to promote malignant progression of HCC, providing a molecular target for future HCC therapy.