Hepatocellular Carcinoma Biology Research Articles

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

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

Celestial Insights: Unraveling the Role of miR-3682-3p in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains a formidable oncological challenge, calling for innovative therapeutic strategies to improve patient outcomes. MicroRNAs have emerged as key regulators in cancer, and miR-3682-3p shows potential as a diagnostic and prognostic biomarker in HCC. We conducted a comprehensive study to uncover its role in HCC biology, revealing dysregulation and clinical associations. Target gene analysis provided insights into potential molecular mechanisms. Moreover, we explored its impact on the tumor microenvironment, immune cell infiltration, and therapy responses. Our findings highlight miR-3682-3p as a promising candidate for further investigations and potential therapeutic strategies in HCC management.

A transcriptome-wide association study identified susceptibility genes for hepatocellular carcinoma in East Asia

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is prevalent in East Asia. Although genome-wide association studies (GWASs) of HCC have identified 23 risk regions, the susceptibility genes underlying these associations largely remain unclear. To identify novel candidate genes for HCC, we conducted liver single-tissue and cross-tissue transcriptome-wide association studies (TWASs) in two populations of East Asia. Methods GWAS summary statistics of 2,514 subjects (1,161 HCC cases and 1,353 controls) from the Chinese Qidong cohort and 161,323 subjects (2,122 HCC cases and 159,201 controls) from the BioBank Japan project were used to conduct TWAS analysis. The single-tissue and cross-tissue TWAS approaches were both used to detect the association between susceptible genes and the risk of HCC. TWAS identified genes were further annotated by Metascape, UALCAN, GEPIA2, and DepMap. Results We identified 22 novel genes at 16 independent loci significantly associated with HCC risk after Bonferroni correction. Of these, 13 genes were located in novel regions. Besides, we found 83 genes overlapped in the Chinese and Japanese cohorts with P < 0.05, of which, three genes (NUAK2, HLA-DQA1, and ATP6V1G2) were discerned by both single-tissue and cross-tissue TWAS approaches. Among the genes identified through TWAS, a significant proportion of them exhibit a credible role in HCC biology, such as FAM96B, HSPA5, POLRMT, MPHOSPH10, and RABL2A. HLA-DQA1, NUAK2, and HSPA5 associated with the process of carcinogenesis in HCC as previously reported. Conclusions Our findings highlight the value of leveraging the gene expression data to identify new candidate genes beyond the GWAS associations and could further provide a genetic insight for the biology of HCC.

Identification and validation of microtubule depolymerizing agent, CYT997, as a potential drug candidate for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a typically fatal malignancy with limited treatment options and poor survival rates, despite recent FDA approvals of newer treatment options. We aim to address this unmet need by using a proprietary computational drug discovery platform that identifies drug candidates with the potential to advance rapidly and successfully through preclinical studies. We generated an in silico model of HCC biology to identify the top 10 small molecules with predicted efficacy. The most promising candidate, CYT997, was tested for its invitro effects on cell viability and cell death, colony formation, cell cycle changes, and cell migration/invasion in HCC cells. We used an HCC patient-derived xenograft (PDX) mouse model to assess its invivo efficacy. CYT997 was significantly more cytotoxic against HCC cells than against primary human hepatocytes, and sensitized HCC cells to sorafenib. It arrested cell cycle at the G2/M phase with associated up-regulations of p21, p-MEK1/2, p-ERK, and down-regulation of cyclin B1. Cell apoptosis and senescence-like morphology were also observed. CYT997 inhibited HCC cell migration and invasion, and down-regulated the expressions of acetylated tubulins, β-tubulin, glypican-3 (GPC3), β-catenin, and c-Myc. Invivo, CYT997 (20 mg/kg, three times weekly by oral gavage) significantly inhibited PDX growth, while being non-toxic to mice. Immunohistochemistry confirmed the down-regulation of GPC3, c-Myc, and Ki-67, supporting its anti-proliferative effect. CYT997 is a potentially efficacious and non-toxic drug candidate for HCC therapy. Its ability to down-regulate GPC3, β-catenin, and c-Myc highlights a novel mechanism of action.

Imaging prognostication and tumor biology in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, and represents a significant global health burden with rising incidence rates, despite a more thorough understanding of the etiology and biology of HCC, as well as advancements in diagnosis and treatment modalities. According to emerging evidence, imaging features related to tumor aggressiveness can offer relevant prognostic information, hence validation of imaging prognostic features may allow for better noninvasive outcomes prediction and inform the selection of tailored therapies, ultimately improving survival outcomes for patients with HCC.

Tumor steatosis and glutamine synthetase expression in patients with advanced hepatocellular carcinoma receiving atezolizumab plus bevacizumab therapy.

The anti-programmed death-ligand 1 antibody atezolizumab and vascular endothelial growth factor-neutralizing antibody bevacizumab in combination (Atezo+Bev) have become the first-line therapy in advanced hepatocellular carcinoma (HCC). Distinct types of tumor immune microenvironment (TIME) and their associations with specific molecular subclasses and driver gene mutations have been identified in HCC; however, these insights are mainly based on surgically resected early-stage tumors. The current study aimed to reveal the biology and TIME of advanced HCC and their significance in predicting clinical outcomes of Atezo+Bev therapy. Thirty-three patients with advanced HCC who were scheduled for treatment with Atezo+Bev therapy were included in this study. Pretreatment tumor biopsy, pre- and posttreatment diffusion-weighted magnetic resonance imaging (MRI) with nine b values (0-1500s/mm2 ), and other clinicopathologic factors were analyzed. Compared with resectable HCC, advanced HCC was characterized by higher proliferative activity, a higher frequency of Wnt/β-catenin-activated HCC, and lower lymphocytic infiltration. Prognostically, two metabolism-related factors, histopathologically determined tumor steatosis and/or glutamine synthetase (GS) expression, and MRI-determined tumor steatosis, were the most significant prognostic indicators for progression-free survival (PFS) and overall survival after Atezo + Bev therapy. Furthermore, changes in the pre- and posttreatment true diffusion coefficients on MRI, which might reflect changes in TIME after treatment, were significantly associated with better PFS. The biology and TIME of HCC were strikingly different in advanced HCC compared with those of surgically resected HCC. Two metabolism-related factors, pathologically determined tumor steatosis and/or GS expression, and MRI-determined tumor steatosis, were found to be the most significant prognostic indicators for Atezo+Bev therapy in advanced HCC.

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 4783: ADO is essential for redox homeostasis in liver cancer

Abstract 2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that plays a direct role in both metabolism and protein stability. Oxidation of the metabolite cysteamine produces hypotaurine, while oxidation of N-terminal cysteines targets protein substrates for N-degron pathway-mediated degradation. Despite these known functions, the (patho)physiological roles of ADO remain obscure. By analyzing TCGA datasets we discovered that high ADO expression is associated with poor outcome for patients with hepatocellular carcinoma (HCC) (HR 1.2, p<0.001). HCC is linked to viral hepatitis, alcohol, metabolic syndrome, non-alcoholic fatty liver disease and chronic inflammation. It is the third leading cause of cancer-related deaths worldwide and is significantly more common in males than females. With poor outcomes, there is a need to better understand HCC biology and vulnerabilities. To assess the functional roles of ADO, we created ADO knockout mice as well as two liver cancer cell line models where ADO was depleted by siRNA, doxycycline-inducible shRNA, or CRISPR/Cas9. While male ADO knockout mice were viable, fertile and healthy, depletion of ADO in liver cancer models significantly reduced cancer cell proliferation and colony formation. ADO was also essential for supporting xenograft growth when implanted subcutaneously in immune-compromised mice. Taken together, this suggested that ADO depletion represents a cancer-specific vulnerability. Comprehensive metabolomic, transcriptomic and proteomic characterization of HCC cells isogenic for ADO demonstrated that loss of ADO resulted in dysregulation of glutathione, ascorbate, polyamine and proline metabolism. Consistent with this, ADO depleted cells had high levels of reactive oxygen species (ROS) measured by CellROX flowcytometry, and an increased ratio of oxidized to reduced glutathione, indicative of oxidative stress. Since mitochondria represent a substantial source of ROS, we assessed mitochondrial mass and function using MitotrackerTM flow cytometry and Seahorse stress tests, respectively. ADO depleted cancer cells had higher mitochondrial mass and higher maximal respiration rates compared to control cells. Finally, exogenously supplied antioxidants could rescue the survival of HCC cells with ADO depletion, demonstrating that the observed loss of viability is due to oxidative stress. This work shows that ADO is essential for redox homeostasis in liver cancer models and suggests that interfering with ADO function may represent a novel targeting strategy for HCC. Citation Format: Sandy C-E Lee, Andrea H. Pyo, Helia Mohammadi, Ji Zhang, Anna Dvorkin-Gheva, Lucie Malbeteau, Stephen Chung, Shahbaz Khan, Thomas Kislinger, Julie A. Reisz, Courtney Jones, Marianne Koritzinsky. ADO is essential for redox homeostasis in liver cancer. [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 4783.

Disease Etiology and Outcomes After Atezolizumab Plus Bevacizumab in Hepatocellular Carcinoma: Post-Hoc Analysis of IMbrave150

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of hepatocellular carcinoma (HCC) by inducing meaningful rates of antitumor responses and having favorable toxicity profiles. Based on large, randomized phase III trials, including the IMbrave150 study testing atezolizumab plus bevacizumab vs sorafenib, combination ICI regimens have emerged as the preferred frontline treatments for HCC based on their overall survival (OS) benefit. 1 Finn R.S. et al. N Engl J Med. 2020; 382: 1894-1905 Crossref PubMed Scopus (2345) Google Scholar ,2 Abou-Alfa G.K. et al. N Engl J Med. 2022; 1 Google Scholar HCC biology may be influenced by distinct etiologies underlying tumorigenesis including viral infections, alcohol-related injury, and metabolic causes. Based on in vivo models and retrospective studies, nonalcoholic fatty liver disease (NAFLD)-related HCC is purported to be resistant to ICIs. 3 Pfister D. et al. Nature. 2021; 592: 450-456 Crossref PubMed Scopus (345) Google Scholar , 4 Heinrich B. et al. Gastroenterology. 2021; 160: 331-345 Abstract Full Text Full Text PDF PubMed Google Scholar , 5 Foerster F. et al. Hepatology. 2022; 75: 1604-1626 Crossref Scopus (21) Google Scholar , 6 Kelley R.K. et al. N Engl J Med. 2021; 385: 280-282 Crossref PubMed Scopus (0) Google Scholar , 7 Haber P.K. et al. Gastroenterology. 2021; 161: 879-898 Abstract Full Text Full Text PDF PubMed Google Scholar Despite their limitations, these lines of evidence have led to the prevailing notion that the underlying etiology of liver disease influences ICI outcomes in HCC, with NASH being a barrier to ICI benefits, although this finding remains controversial. 5 Foerster F. et al. Hepatology. 2022; 75: 1604-1626 Crossref Scopus (21) Google Scholar , 6 Kelley R.K. et al. N Engl J Med. 2021; 385: 280-282 Crossref PubMed Scopus (0) Google Scholar , 7 Haber P.K. et al. Gastroenterology. 2021; 161: 879-898 Abstract Full Text Full Text PDF PubMed Google Scholar , 8 Ho W.J. et al. J Immunother Cancer. 2020; 8 Google Scholar Herein, we performed a post-hoc patient-level analysis of the IMbrave150 trial to evaluate if cirrhosis etiology affects survival or antitumor responses after atezolizumab plus bevacizumab.

Long non-coding RNA H19 promotes proliferation in hepatocellular carcinoma cells via H19/miR-107/CDK6 axis.

Hepatocellular carcinoma (HCC) is the leading cause of cancer death worldwide; nevertheless, current therapeutic options are limited or ineffective for many patients. Therefore, elucidation of molecular mechanisms in HCC biology could yield important insights for the intervention of novel therapies. Recently, various studies have reported dysregulation of long non-coding RNAs (lncRNAs) in the initiation and progression of HCC, including H19; however, the biological function of H19 in HCC remains unclear. Here, we show that knockdown of H19 disrupted HCC cell growth, impaired the G1-to-S phase transition, and promoted apoptosis, while overexpression of H19 yielded the opposite results. Screening for expression of cell cycle-related genes revealed a significant downregulation of CDK6 at both RNA and protein levels upon H19 suppression. Bioinformatic analysis of the H19 sequence and the 3' untranslated region (3' UTR) of CDK6 transcripts showed several binding sites for microRNA-107 (miR-107), and the dual luciferase reporter assay confirmed their direct interaction with miR-107. Consistently, blockage of miR-107 activity alleviated the growth suppression phenotypes induced by H19 downregulation, suggesting that H19 serves as a molecular sponge for miR-107 to promote CDK6 expression and cell cycle progression. Together, this study demonstrates a mechanistic function of H19 in driving the proliferation of HCC cells and suggests H19 suppression as a novel approach for HCC treatment.

MYC and MET cooperatively drive hepatocellular carcinoma with distinct molecular traits and vulnerabilities

Enhanced activation of the transcription factor MYC and of the receptor tyrosine kinase MET are among the events frequently occurring in hepatocellular carcinoma (HCC). Both genes individually act as drivers of liver cancer initiation and progression. However, their concomitant alteration in HCC has not been explored, nor functionally documented. Here, we analysed databases of five independent human HCC cohorts and found a subset of patients with high levels of MYC and MET (MYChigh/METhigh) characterised by poor prognosis. This clinical observation drove us to explore the functionality of MYC and MET co-occurrence in vivo, combining hydrodynamic tail vein injection for MYC expression in the R26stopMet genetic setting, in which wild-type MET levels are enhanced following the genetic deletion of a stop cassette. Results showed that increased MYC and MET expression in hepatocytes is sufficient to induce liver tumorigenesis even in the absence of pre-existing injuries associated with a chronic disease state. Intriguingly, ectopic MYC in MET tumours increases expression of the Mki67 proliferation marker, and switches them into loss of Afp, Spp1, Gpc3, Epcam accompanied by an increase in Hgma1, Vim, and Hep-Par1 levels. We additionally found a switch in the expression of specific immune checkpoints, with an increase in the Ctla-4 and Lag3 lymphocyte co-inhibitory responses, and in the Icosl co-stimulatory responses of tumour cells. We provide in vitro evidence on the vulnerability of some human HCC cell lines to combined MYC and MET targeting, which are otherwise resistant to single inhibition. Mechanistically, combined blockage of MYC and MET converts a partial cytostatic effect, triggered by individual blockage of MYC or MET, into a cytotoxic effect. Together, these findings highlight a subgroup of HCC characterised by MYChigh/METhigh, and document functional cooperativity between MYC and MET in liver tumorigenesis. Thus, the MYC-R26Met model is a relevant setting for HCC biology, patient classification and treatment.

Abstract PO010: Molecular characterization of pediatric hepatocellular carcinoma: genomic, methylomic and transcriptomic analysis

Abstract Background: The main pediatric liver tumors are hepatoblastoma (HB) and, to a lesser extent, pediatric hepatocellular carcinoma (HCC). HB appears at an early age (<3 years) while HCC is diagnosed mainly in older children (>8 years) or adolescents and it is usually associated to underlying metabolic diseases. HEM-NOS (Hepatocellular malignant neoplasm, not otherwise specified) is a recent entity with histopathological features of HB and HCC. Due to the rarity of the disease (<1 case per million children), the biology of HCC and their similarity with HB has been poorly explored. Aims: To molecularly characterize pediatric HCC at the genomic, transcriptomic and epigenomic level and to perform a comparative study with HB and HEM-NOS. Methodology: We performed a genomic (Cytoscan, ThermoFisher), epigenomic (EPIC/850k, Illumina) and transcriptomic (HTA 2.0 array, ThermoFisher) studies on 8 HCC and compared them with already published omics data of 31 HB, 2 HEM-NOS and 19 control non-tumor liver (NT) samples (Carrillo-Reixach et al, J Hepatol, 2020). The results obtained were validated in an independent series of 19 HCC, including 10 fibrolamellar HCC (FL-HCC), 56 HB, 7 HEM-NOS and 5 NTs using QUAlu and Nanostring techniques for assessing DNA methylation and gene expression, respectively. Results: We identified CTNNB1 gene mutations in 73%, 50% and 14% of HB, HEM-NOS and HCC, respectively. Contrarily, TERT mutations were only found in HEM-NOS and HCC tumors. FL-HCC had the DNAJB1:PRKACA fusion transcript and in one case without this fusion, a deletion in the PRKCA gene was identified. The genome of HCC and HEM-NOS had higher number of chromosomal losses than HB. Moreover, HBs and HEM-NOS showed a higher DNA hypomethylation than HCC whereas HCC was characterized by CpG island hypermethylation. Two imprinting regions were differently expressed in 14q32 (DLK1/DIO3) and 15q11.2 (SNORD115/116) in HB and HCC, respectively. Finally, we identified the SPINK1 gene (Serine peptidase inhibitor, kazal type 1) as an overexpressed gene in HCC that was strongly associated with prognosis of patients with liver cancer in both training (Log-Rank=0.008) and validation sets (Log-rank=0.028). Using XENA database, we found also that different SPINK1 expression was also associated with adult HCC prognosis (n=357, Log-rank=0.017). Conclusions: The characterization of HCC and its comparison with HB allowed us to identify potential diagnostic and prognostic markers that could help to improve the clinical management of childhood liver cancer. Citation Format: Juan Carrillo-Reixach, Álvaro del Río-Álvarez, Laura Royo, Montserrat Domingo-Sàbat, Rita Alaggio, Ronald de Krijger, Christian Vokuhl, Marta Garrido, Laura Guerra, Constantino Sábado, Francisco Hernández, Manuel López-Santamaría, Viera Bajčiová, Rudolf Maibach, Margaret Childs, Piotr Czauderna, Keith Wheatley, Roland Kappler, Stefano Cairo, Bruce Morland, Margarita Sala, Maria Rosa Sarrias, Carolina Armengol. Molecular characterization of pediatric hepatocellular carcinoma: genomic, methylomic and transcriptomic analysis [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO010.

Abstract PO023: Uncovering hepatitis B virus-induced signaling changes in hepatocellular carcinoma

Abstract Although precision medicine has revolutionized the treatment for other solid tumors, comprehensive genome profiling of hepatocellular carcinoma (HCC) has demonstrated that there are few oncogenic mutational drivers. HCC arises nearly universally in the context of co-morbid hepatitis, driven by hepatitis C virus or hepatitis B virus (HBV). Previous work has highlighted the effects of HBV protein X (HBx) on signaling and proliferation. However, it remains unclear how these signaling changes interact with the stress imposed by HBV replication. We hypothesize that HBV replication and maintenance of HBV proteins like HBx creates specific vulnerabilities that can be uncovered and exploited. We performed whole-genome clustered regularly interspaced short palindromic repeat interference (CRISPRi) screening in two engineered, patient-derived HBV+ cell models, Hep3B and SNU-368, with cellular proliferation as the readout. A conditional induction of HBx was used to overexpress HBx in each cell model for the whole genome CRISPRi screen. Genes where HBx expression was significantly associated with a more negative dependency score were classified as HBV differential dependencies. 37 genes were identified as shared HBV-induced dependencies across both cell lines. Of these, 16 were also differentially expressed in HCC specimens with ongoing HBV replication vs. non-HBV expressing tumors collected in The Cancer Genome Atlas (TCGA), suggesting in vivo selective pressure to alter the expression of these genes. We focused on Zinc Fingers and Homeoboxes 2 (ZHX2) and methionine adenosyltransferase 2A (MAT2A). ZHX2 was identified as an HCC tumor suppressor, and data from TCGA showed differential gene expression of ZHX2 in HBV+HCC. Validation of ZHX2 sgRNA knockdown confirmed that ZHX2 knockdown preferentially reduced cell proliferation in the presence of HBx, consistent with an HBV-induced dependency. ZHX2 also can activate the HIF pathway; we identified alterations of HIF targets with ZHX2 knockdown and HBx expression. Similarly, MAT2A knockdown in HBV+HCC cell lines showed decreased cell proliferation with HBx expression. MAT2A is an emerging therapeutic target, with sensitivity to MAT2A inhibitors conferred by deletions in methylthioadenosine phosphorylase (MTAP), seen in 3% of HCC. Ongoing investigations include alteration of splicing and cell cycle changes with depletion of MAT2A in the presence of HBx. With small molecule inhibitors of MAT2A in clinical development, this finding may be therapeutically actionable. Thus, using functional genomics to interrogate HBV-associated gene dependencies has illuminated the biology of HCC and identified candidate therapeutic targets for pre-clinical validation. Citation Format: Rigney E Turnham, Huat Chye Lim, Lucille Ferret, Katherine Lo, LeeAnn Wang, Alex Choi, John D Gordan. Uncovering hepatitis B virus-induced signaling changes in hepatocellular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO023.

The impact of tumor burden at the initial hepatectomy on the recurrence-to-death survival after repeat surgical resection/radiofrequency ablation: a retrospective study

BackgroundPrevious studies have reported the surgical resection (SR) and radiofrequency ablation (RFA) could achieve comparable recurrence-to-death survival (RTDS). However, the impact of primary tumor burden on RTDS of patients with recurrent hepatocellular carcinoma (HCC) following SR or RFA has not been clarified.MethodsFrom January 2009 to March 2015, 171 patients who underwent initial hepatectomy and second curative treatments in West China Hospital were retrospectively analyzed. Survival analysis was performed by the Kaplan–Meier method. Risk factors were identified using the Cox proportional hazard model.ResultsAt initial hepatectomy, 96 patients (56.1%) were diagnosed with HCC within the Milan criteria (MC), and 75 patients (43.9%) were HCC beyond the MC. The clinicopathological features and re-treatment methods of recurrent HCC were similar between patients with primary HCC within or beyond the MC. Patients with primary HCC within the MC had longer recurrence time (31.4 ± 24.2 months vs. 20.2 ± 16 months, P < 0.001). The 1- and 3- year RTDS within and beyond the MC group were 88.8%, 57.6% and 79.0%, 46.3%, respectively (P = 0.093). In multivariate analysis, the recurrence time, tumor size and AFP > 400 ng/mL at the time of recurrence were associated with RTDS.ConclusionsThe primary tumor burden had no impact on RTDS, but had an impact on recurrence time. The recurrence time had an impact on RTDS and might be a good index to reflect the biology of recurrent HCC.

Understanding Molecular Heterogeneity in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the 5th most common cause of cancer‐related death in the United States with an estimated 32,000 annual deaths. Roughly 30% of HCC cases demonstrate mutations in the CTNNB1 oncogene that encodes a form of the ꞵ‐catenin protein that cannot be degraded. Recently, we developed a mouse model of HCC that genetically represents ~9‐12% of all human HCCs. Here, we combine hydrodynamic tail vein injections with the Sleeping Beauty Transposase – a non‐viral method of expressing genes of interest. Our model uses a clinically observed mutant of ꞵ‐catenin‐(T41A) that is co‐expressed with a mutant form of the nuclear factor erythroid 2–related factor 2 (Nrf2‐G31A) in the livers of wild‐type mice (B+N). This system results in microscopic HCC within 3 weeks and evident macroscopic disease by 7 weeks after induction.ꞵ‐catenin activation can be identified by the expression of its downstream target gene Glutamine Synthetase (GS) in the liver. Patient HCCs with ꞵ‐catenin mutations strongly correlate with GS expression. The evolution and function of GS in b‐catenin‐mutated HCC, remains unclear. Also, there is a gap in the knowledge whether elevated GS expression correlates with HCC biology, which were all subjects of the current study. B+N mouse model of HCC was examined temporally for development of HCC along with GS expression. B+N mice develop HCCs with heterogeneous GS expression in the form of GS‐high and GS‐low to GS‐negative foci. We next asked whether GS expression is associated with cell proliferation or death in the B+N model. We stained tissues for TUNEL and PCNA to mark cell apoptosis and proliferation, respectively in 7‐ and 9.5 weeks post‐injection livers, which were then correlated to GS staining of HCC nodules. While we found no correlation between apoptosis and GS, we found nodules with lower GS expression to have higher proliferation and vice versa. Finally, we asked if eliminating GS expression from B+N HCCs will impact tumor burden. Five weeks after B+N tumor induction in GS‐floxed mice, these animals were injected with AAV8‐TBG‐Cre virus to delete Glul,and mice were harvested after 2‐ or 4.5‐weeks. We found B+N mice with Glul deletion show increased liver weight to body weight ratio, a measurement of HCC burden, compared to controls. The livers of these mice were enriched for GS‐negative tumors. Together, our data suggests that HCC nodules with lower GS expression may outperform GS‐positive foci, and thus are more aggressive compared to the nodules with higher GS expression, which may be more differentiated. The mechanisms of GS heterogeneity and elucidation of its functional significance may help select the most effective targeted therapies for HCC patients with ꞵ‐catenin mutations.

A comprehensive analysis of FOX family in HCC and experimental evidence to support the oncogenic role of FOXH1.

Hepatocellular carcinoma (HCC) remains the second leading cause of cancer related deaths worldwide. Understanding about the molecular biology of HCC and development of targeted therapies are still the main focuses of this type of disease. Here, by connecting the expression levels of FOX proteins with their associated clinical characteristics using TCGA LIHC dataset, we found that 27/40 FOX proteins were highly expressed in HCC tumors compared to normal liver tissues and their expression levels were tightly associated with HCC tumor stage, tumor grade and overall survival. Our experimental results also confirmed that FOXH1 indeed played an oncogenic role in HCC development by promoting cell growth and cell migration/invasion. Mechanistic dissection demonstrated that FOXH1-induced cell growth and cell migration/invasion relied on mTOR signaling because inhibition of mTOR signaling by rapamycin could attenuate FOXH1-mediated phenotypic alterations of HCC cells. The results from orthotopic mouse model also validated that FOXH1 promoted HA22T tumor growth via triggering mTOR activation. Overall, this study not only comprehensively examines the clinical values of FOX proteins in HCC but also provides experimental evidence to support the role of FOXH1 in HCC development, building rationale to develop more effective therapies to treat HCC patients.

Asiatic Acid Encapsulated Exosomes of Hepatocellular Carcinoma Inhibit Epithelial-Mesenchymal Transition Through Transforming Growth Factor Beta/Smad Signaling Pathway.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death in many countries, which accounts for more than 80% of primary liver cancers. Better understanding of the biology of HCC and more therapeutic strategies are urgently needed to improve the current situation. Exosomes, lipid-bound particles derived from cells, have been revealed to play versatile roles in mediating communication between tumor and its microenvironment. Thus, exosomes could act as potential drug delivery systems in cancer treatment. This study aimed to investigate the effect of asiatic acid (AA)-loaded exosomes on the proliferation and migration of HCC cells and clarify the underlying mechanisms. HCC cells were treated with AA-loaded exosomes and cell vitality, migration and invasion were examined. Compared with free AA, AA-loaded exosomes significantly reduced cell vitality, migration, invasion and epithelial mesenchymal transition (EMT). And the inhibition was enhanced as AA concentration went up. Moreover, the expression of proteins involved in EMT and TGF-β/Smad pathway such as TGF-β1, Smad4 and Vimentin were decreased while E-cadherin was up-regulated. Collectively, these findings demonstrate that HCC derived exosomes display as potential drug delivery vehicles in HCC treatment. And AA-loaded exosomes might work by inhibiting EMT through inactivating TGF-β/Smad pathway.

An Overview of Bacterial and Viral Inhibitors Involved in Gene Therapy of Hepatocellular Carcinoma (Hcc)

Hepatocellular carcinoma (HCC) is a form of cancer that is very widespread around the world and has a high mortality rate. Extensive evidence suggests that, HCC is a multifactorial disease. Hepatic cirrhosis is present, along with systemic inflammation and viral infections such as hepatitis B or C. Thus, giving rise to genetically and phenotypically heterogeneous hepatocellular carcinoma tumors. Researchers have found that bacterial and viral inhibitors can be used to silence targeted genes in hepatocellular carcinoma. Many bacterial species such as; Salmonella, Listeria, and Escherichia, proved to have anti-tumor properties. Up till now, adenoviral, retroviral, herpes-simplex viral and adeno-associated viral vectors have been modified and are being used for HCC gene therapy. In patients, up regulation of TLR signaling have also been observed showing an interesting influence on HCC’s microenvironment. TLR 4 and TLR 9 have positive relationship with tumor whereas, TLR3 is associated with anti-tumor influence. TLRs can cause an inflammatory response in the presence of foreign pathogens including bacteria and fungi. This review reflects an insight into the biology of HCC suggesting that certain signaling pathways and molecular alterations plays a very significant role in HCC development. As well as new experimental approaches, including; anti-angiogenesis, cancer therapy, oncolytic virotherapy, and suppressing the function of oncogenes, leading to apoptosis are successively being applied. The current challenge for the researchers is to identify a medicament which is selective for tumors specific cells only, having minimal noxiousness and harmless to normal tissue. We have scrutinized research articles based on how to merge viral and bacterial anticancer therapies into a single treatment for HCC.

Epigenetic Regulation of Hepatocellular Carcinoma Progression through the mTOR Signaling Pathway.

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is an aggressive tumor with a high mortality rate because of the limited systemic and locoregional treatment modalities. The development and progression of HCC depend on epigenetic changes that result in the activation or inhibition of some signaling pathways. The mTOR signaling pathway is essential for many pathophysiological processes and is considered a major regulator of cancer. Increasing evidence has shown that epigenetics plays a key role in HCC biology by regulating the mTOR signaling pathway. Therefore, epigenetic regulation through the mTOR signaling pathway to diagnose and treat HCC will become a very promising strategy.

Peri-operative outcomes of liver resection for hepatocellular carcinoma in 1,936 pediatric patients: a retrospective population based study (NIS 1998-2014)

Presenter: Sushruta S Nagarkatti MD | Ascension Providence Hospital Background: Hepatocellular carcinoma (HCC) accounts for less than 0.5% of all pediatric malignancies. The etiology and biology of pediatric HCC differs from that seen in adults. However, as in the adult population, surgical resection offers the highest chance of survival. The available literature on the outcomes of hepatic resection in this age group is scarce and is limited by small sample sizes. This is the first population based study analyzing demographic and clinical characteristics as well as peri-operative outcomes of liver resection in pediatric HCC patients Methods: The National Inpatient Database (NIS 1998-2014) was used to identify pediatric patients (age ≤19 years) diagnosed with hepatocellular carcinoma (ICD-9 code 155.0), who had either partial hepatectomy (ICD 9 code 50.22) or liver lobectomy (ICD 9 code 50.3). Discharge weights were applied to obtain national estimates. Demographic, clinical and outcomes data was compared using standard statistical methodology. Results: A total of 1,936 pediatric patients who underwent surgical resection for HCC were identified. The mean age was 4.9 years, with a male to female ratio 1.18:1. Formal anatomic lobectomy was performed in 56.4% with 43.6% of patients undergoing a non-anatomic partial hepatectomy. The all-cause mortality rate was 1.8%, with an overall complication rate of 15.2%. Post-operative hemorrhage occurred in 4.6% patients, with the most common complications being respiratory (7.7%) and GI (7.6%). Overall, the post-operative infection rate was low at 2.4% with < 1.0% of patients developing sepsis. Most patients were discharged home (83.1%) with a mean LOS 11.6 (±12.9) days. Home health services were required for 10.2% of patients with 4.6% requiring subacute rehab services or a skilled nursing facility ( <1.0%). Conclusion: The majority of pediatric HCC patients received a formal anatomic liver lobectomy with reported morbidity and mortality far lower than the published literature on similar procedures in adult HCC patients. Further studies are needed to validate the findings of this study and establish long term survival outcomes in this patient population.