Hepatocellular Carcinoma Tumor Biology Research Articles

Establishment and characterization of six canine hepatocellular carcinoma cell lines.

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor in dogs. Although surgical resection is a major treatment option for canine HCC, there are no distinct strategies for unresectable tumor subtypes or adjuvant chemotherapy for tumors with positive margins. We aimed to establish and characterize novel HCC cell lines from canine patients. The cellular morphology, general growth features and tumorigenicity of the established cell lines were evaluated. We also examined the sensitivity of the cell lines to multi-target tyrosine kinase inhibitors (TKIs). We established novel canine HCC cell lines from hepatic tumors and an additional kidney tumor of six canine patients. All cell lines showed colony forming and migratory ability. KU-cHCC-001 and KU-cHCC-001-Kidney, two cell lines exhibiting high epithelial-mesenchymal transition characteristics, showed tumorigenicity in xenografted mice. Toceranib, a veterinary TKI that targets vascular endothelial growth factor (VEGFR)/platelet-derived growth factor receptor (PDGFR)/c-kit, effectively inhibited the mitogen-activated protein kinase pathway and induced apoptosis. The established canine HCC cell lines showed greater sensitivity to toceranib than to sorafenib, a first-line treatment for human HCC targeting RAF/VEGFR/PDGFR. Sorafenib showed improved anti-tumor effects when co-treated with SCH772984, an extracellular signal-regulated kinase inhibitor. Our study suggests new therapeutic strategies for canine HCC, and these cell lines are valuable research materials for understanding HCC tumor biology in both humans and dogs.

AHSA1 Regulates Hepatocellular Carcinoma Progression via the TGF-β/Akt-Cyclin D1/CDK6 Pathway.

Activator of heat shock protein 90 (HSP90) ATPase Activity 1 (AHSA1) regulates proliferation, apoptosis, migration, and invasion of osteosarcoma and hepatocellular carcinoma (HCC). However, the novel mechanism of AHSA1 in the tumor biology of hepatocellular carcinoma (HCC) remains unclear. We analyzed AHSA1 expression in 85 pairs of clinical samples of HCC and the Cancer Genome Atlas database. The role of AHSA1 in HCC was proved by cell proliferation, colony formation, migration, cell cycle analysis in vitro, xenograft models and tumor metastasis assay in vivo, and bioinformatics. High AHSA1 expression was demonstrated in HCC and associated with invasive depth, clinical stage, and poor overall survival of patients. UnivariateCox analysis confirmed that AHSA1 was an independent prognostic factor for patients with HCC. Meanwhile, AHSA1 upregulation promoted cell proliferation, colony formation, and cell migration in vitro and tumor cell proliferation and metastasis of HCC cells in vivo. AHSA1 upregulation increased the cell cycle transition from G1 to S phase by increasing the expression of cyclinD1, cyclinD3, and cyclin-dependent kinase 6(CD). Transforming growth factor beta 1 (TGF-β1)-induced protein kinase B (Akt) signaling regulated the expression of downstream targets, including cyclinD1. AHSA1 expression was closely correlated with the expression of TGF-β, Akt, cyclinD1, cyclinD3, and CDK6 using the Gene Expression Profiling Interactive Analysis database. AHSA1 upregulation participated in HCC progression by regulating TGF-β/Akt-cyclinD1/CDK6 signaling. AHSA1 might serve as a biomarker for predicting the clinical outcome of patients with HCC. It is vital in tumor metastasis and disease progression of HCC and may facilitate the development of clinical intervention strategies against HCC.

Racial and Ethnic Disparities in Liver Transplantation in Patients with Hepatocellular Carcinoma

Introduction: Racial and ethnic disparities in outcomes of hepatocellular carcinoma (HCC) have been inadequately studied, especially in the context of liver transplantation (LT). We sought to evaluate disparities among racial/ethnic groups in this patient population to identify ways to mitigate them. Methods: Patient and tumor characteristics were obtained from UNOS STAR files (April 12, 2012 to June 6, 2021). One-way ANOVA and Chi-square tests were conducted for differences among racial/ethnic groups, using non-Hispanic White (NHW) patients as the comparator and Kaplan-Meier plots for survival. Statistical analyses were performed using SAS 9.4. Results: Ninety percent of patients listed for liver transplantation were transplanted or removed from the waitlist after 654 days; ninety-five percent were transplanted or removed after 3 years. Mean graft and patient survival times were the lowest in Hispanics. Hispanics also had the highest physiologic MELD at listing in both recipient and waitlist groups. Among LT recipients, Non-Hispanic Blacks (NHBs) had significantly lower physiologic MELDs at listing and transplantation compared with NHWs. However, NHBs had lower survival at 3 and 5 years after transplant. Hispanics had the highest percentage of poorly differentiated tumors in the explants compared with NHWs and NHBs. Hispanics also had the highest waitlist dropout at 654 days and 3 years. Conclusion: There are significant disparities in HCC outcomes for both the Hispanic and NHB populations compared with NHWs. Whether this is a result of a more advanced cancer stage at listing, differences in HCC tumor biology, or social determinants of health will need further study.

Sex-based Disparities in Hepatocellular Carcinoma Recurrence After Liver Transplantation.

Women with chronic liver disease have lower rates of hepatocellular carcinoma (HCC) as compared to men; it is unknown if there are sex-based differences in HCC recurrence postliver transplant. We conducted an analysis of patients who underwent liver transplant for HCC in the United Network for Organ Sharing/Organ Procurement and Transplantation Network from January 1, 2012 through December 31, 2017. A total of 12 711 patients underwent liver transplant for HCC: 2909 (23%) women and 9802 (73%) men. Women had significantly lower rates of postliver transplant HCC recurrence than men (4.0% versus 5.4%, P = 0.002). A cox-regression analysis for postliver transplant HCC recurrence highlighted that even after accounting for etiology of cirrhosis, alpha-fetoprotein at liver transplant, tumor diameter, tumor pathology, and vascular invasion, female sex was associated with a 25% lower risk of postliver transplant HCC recurrence (95% confidence interval: 0.57-0.99). There were no interactions between female sex and the following variables: age, type of locoregional therapy, alpha-fetoprotein, donor sex, body mass index, or nonalcoholic steatohepatitis etiology (P > 0.05 for each). This study demonstrates an independent effect of sex on risk for HCC recurrence postliver transplant. Our data highlight an opportunity to better understand HCC tumor biology by investigating the drivers of this sex-based difference in HCC recurrence.

Absence of association between pretransplant serum soluble PD-1 level and prognosis following living donor liver transplantation in patients with hepatocellular carcinoma

Programmed death protein 1 (PD-1) pathway is one of the most critical mechanisms in tumor biology of hepatocellular carcinoma (HCC). The study aimed to assess the prognostic influence of pretransplant serum soluble PD-1 (sPD-1) in patients undergoing liver transplantation for treatment of HCC. Data from 229 patients with HCC who underwent living donor liver transplantation between January 2010 and December 2015 were retrospectively evaluated. Stored serum samples were used to measure sPD-1 concentrations. Overall survival (OS) and disease-free survival (DFS) rates were 94.3% and 74.5% at 1 year; 78.2% and 59.2% at 3 years; and 75.4% and 55.5% at 5 years, respectively. Prognostic analysis using pretransplant serum sPD-1 with a cutoff of 93.6 µg/mL (median value of the study cohort) did not have significant prognostic influence on OS (p = 0.69) and DFS (p = 0.26). Prognostic analysis using sPD-1 with a cutoff of 300 µg/mL showed similar OS (p = 0.46) and marginally lower DFS (p = 0.070). Combination of Milan criteria and sPD-1 with a cutoff of 300 µg/mL showed similar outcomes of OS and DFS in patients within and beyond Milan criteria. Multivariate analysis revealed that only Milan criteria was an independent prognostic for OS and DFS, but pretransplant sPD1 with a cutoff of 300 µg/mL did not become a prognostic factor. The results of this study demonstrate that pretransplant serum sPD-1 did not show significant influences on post-transplant outcomes in patients with HCC. Further large-scale, multicenter studies are necessary to clarify the role of serum sPD-1 in liver transplantation recipients.

HCC: role of pre- and post-treatment tumor biology in driving adverse outcomes and rare responses to therapy.

Liver cancer is the fastest-growing cause of cancer deaths in the United States and is a complex disease. The response of hepatocellular carcinoma (HCC) to treatment can be variable. Predicting response to determine the most effective therapy is an active area of research. Our understanding of underlying factors which drive response to therapy is continually increasing. As more therapies for the treatment of this disease evolve, it is crucial to identify and match the ideal therapy for a particular tumor and patient. The potential predicative imaging features of tumor behavior, while of research interest, have not been validated for clinical use and do not currently inform treatment planning. If further validated though, prognostic features may be used in the future to personalize treatment plans according to individual patients and tumors. Unexpected post-treatment responses such as potential tumor biology changes and abscopal effect which are important to be aware of. This review is intended for radiologists who routinely interpret post treatment HCC imaging and is designed to increase their cognizance about how HCC tumor biology drives response to therapy and explore rare responses to therapy.

Transcriptional Profiling of Porcine HCC Xenografts Provides Insights Into Tumor Cell Microenvironment Signaling.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, representing the most common form of liver cancer. As HCC incidence and mortality continue to increase, there is a growing need for improved translational animal models to bridge the gap between basic HCC research and clinical practice to improve early detection and treatment strategies for this deadly disease. Recently the Oncopig cancer model—a novel transgenic swine model that recapitulates human cancer through Cre recombinase induced expression of KRASG12D and TP53R167H driver mutations—has been validated as a large animal translational model for human HCC. Due to the similar size, anatomy, physiology, immunology, genetics, and epigenetics between pigs and humans, the Oncopig has the potential to improve translation of novel diagnostic and therapeutic modalities into clinical practice. Recent studies have demonstrated the importance of tumor cells in shaping its surrounding microenvironment into one that is more proliferative, invasive, and metastatic; however, little is known about the impact of microenvironment signaling on HCC tumor biology and differential gene expression between HCC tumors and its tumor microenvironment (TME). In this study, transcriptional profiling was performed on Oncopig HCC xenograft tumors (n = 3) produced via subcutaneous injection of Oncopig HCC cells into severe combined immunodeficiency (SCID) mice. To differentiate between gene expression in the tumor and surrounding tumor microenvironment, RNA-seq reads originating from porcine (HCC tumor) and murine (microenvironment) cells were bioinformatically separated using Xenome. Principle component analysis (PCA) demonstrated clustering by group based on the expression of orthologous genes. Genes contributing to each principal component were extracted and subjected to functional analysis to identify alterations in pathway signaling between HCC cells and the microenvironment. Altered expression of genes associated with hepatic fibrosis deposition, immune response, and neo angiogenesis were observed. The results of this study provide insights into the interplay between HCC and microenvironment signaling in vivo, improving our understanding of the interplay between HCC tumor cells, the surrounding tumor microenvironment, and the impact on HCC development and progression.

Transcriptional regulation of alcohol induced liver fibrosis in a translational porcine hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. HCC risk factors include alcohol induced liver cirrhosis, which prompts hepatic inflammation, cell necrosis, and fibrosis deposition. As 25% of HCC cases are associated with alcohol induced liver disease, understanding the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic responses are critical. This study utilized the Oncopig Cancer Model—a transgenic pig model that recapitulates human HCC through induced expression of KRASG12D and TP53R167H driver mutations—to investigate the molecular mechanisms underlying alcohol induced liver disease. Oncopigs (n = 5) underwent fibrosis induction via infusion of ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Eight-weeks post induction, liver tissue samples from fibrotic and age-matched control (n = 5) Oncopigs were collected for histological evaluation and transcriptional profiling. Increased hepatic inflammation and fibrosis was observed in fibrotic Oncopigs via pathological assessment. Transcriptional profiling (RNA-seq) resulted in the identification of 4387 differentially expressed genes between Oncopig fibrotic and control livers. GO term enrichment analysis identified pathway alterations associated with cirrhosis progression in humans, including cell proliferation, angiogenesis, extracellular matrix deposition, and oxidation-reduction. Key alterations include activation of hepatic stellate cells, increased matrix metalloproteinase production, and altered expression of ABC and SLC transporter genes involved in transport of anticancer drugs.These results demonstrate Oncopig liver fibrosis recapitulates transcriptional hallmarks of human cirrhosis, making the Oncopig an ideal model for studying the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic response.

Alpha-fetoprotein: A molecular bootstrap for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is well known for its aggressive nature and high recurrence rates. Alpha- fetoprotein (AFP) secreting tumors are more common in HCC. However, a few proportion of HCC do not produce AFP more than the basal level. AFP secreting tumors are more aggressive in nature since the ability of AFP to promote effective progression, growth, and metastasis of tumor. AFP also intervenes the immune system to evade the immune responses against cancer cells. AFP-producing tumors contain poorly differentiated cells similar to embryonic stem cells of liver mimicking rapid replication, proliferation, and AFP secretion by fetal liver. In this review, we highlight the crucial roles of AFP in immune evasion, aggressiveness, progression, and tumor biology of HCC.

MicroRNA-126-5p suppresses cell proliferation, invasion and migration by targeting EGFR in liver cancer

The expression status and potential mechanistic involvement of microRNA (miR)-126-5p in hepatocellular carcinoma (HCC) are still elusive currently. Here we set out to address this issue both in cell lines and in patients' tissues. The relative expression levels of endogenous miR-126-5p and epidermal growth factor receptor (EGFR) were quantified by real-time polymerase chain reaction. Cell viability and proliferation were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays, respectively. Cell invasive and migrative capacities were determined by transwell and wound healing assays, respectively. The regulatory action of miR-126-5p on EGFR was interrogated by luciferase reporter assay. Translational level of EGFR was analyzed by Western blotting. MiR-126-5p was significantly down-regulated in both HCC patients' tissues and cell lines. Forced expression of miR-126-5p greatly compromised cell viability, proliferation, invasion and migration, while miR-126-5p-specific inhibitor promoted these oncogenic phenotypes. MiR-126-5p mimics inhibited endogenous expression of EGFR and suppressed EGFR 3'-untranslated region-fused luciferase activity. Co-expression of EGFR in miR-126-5p-proficient cells completely restored cell migrative and invasive capacities, while co-transfection with EGFR siRNA significantly inhibited miR-126-5p inhibitor-induced cell invasion and migration. MiR-126-5p was aberrantly decreased in HCC and subsequently relieved the suppression on EGFR expression, which consequently contributed to the tumor biology of HCC.

The long noncoding RNA AK002107 negatively modulates miR-140-5p and targets TGFBR1 to induce epithelial-mesenchymal transition in hepatocellular carcinoma.

The abnormal expression of long noncoding RNAs (lncRNAs) is associated with human carcinoma. The present study aimed to investigate the mechanisms underlying the function of lncRNA AK002107 in the progression of hepatocellular carcinoma (HCC). The differential expression of lncRNAs between HCC and paired nontumor tissues was identified using microarrays, and the correlation between the expression of lncRNA AK002107 and the clinical prognosis of HCC was analyzed. We investigated the role of lncRNA AK002107 in HCC tumor biology in vitro using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT), colony formation, and Matrigel invasion assays and in vivo by assessing the growth of xenografted HCC tumors. The potential microRNAs that interact with lncRNA AK002107 were identified using online tools and were verified using PCR and luciferase reporter assay. The levels of TGFBR1, E‐cadherin, and vimentin were determined using western blot assays. We then further investigated the correlation between expression of lncRNA AK002107 with miR‐140‐5p and TGFBR1 expression in HCC tissues. The expression of lncRNA AK002107 is frequently upregulated in HCC samples and cell lines. Patients with HCC who have elevated lncRNA AK002107 expression exhibit poorer overall survival and disease‐free survival. Silencing lncRNA AK002107 expression significantly inhibited HCC cell proliferation, colony formation, and invasion both in vitro and in vivo. Furthermore, lncRNA AK002107 directly binds to miR‐140‐5p and significantly inhibits miR‐140‐5p expression. The functions of lncRNA AK002107 in cell growth and tumor invasion are mediated via miR‐140‐5p. lncRNA AK002107 upregulated TGFBR1 expression and then induced epithelial–mesenchymal transition (EMT) by inhibiting miR‐140‐5p expression. The expression of lncRNA AK002107 inversely correlated with miR‐140‐5p expression and positively correlated with TGFBR1 expression in HCC tissues. In summary, lncRNA AK002107 functions as an oncogene in tumors by inhibiting miR‐140‐5p, targeting TGFBR1, and then inducing EMT. The lncRNA AK002107/miR‐140‐5p/TGFBR1/EMT regulatory network may be a valuable target for the development of novel diagnostic and treatment methods for HCC.

Phosphoprotein enriched in diabetes (PED/PEA15) promotes migration in hepatocellular carcinoma and confers resistance to sorafenib

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death with limited treatment options and frequent resistance to sorafenib, the only drug currently approved for first-line therapy. Therefore, better understanding of HCC tumor biology and its resistance to treatment is urgently needed. Here, we analyzed the role of phosphoprotein enriched in diabetes (PED) in HCC. PED has been shown to regulate cell proliferation, apoptosis and migration in several types of cancer. However, its function in HCC has not been addressed yet. Our study revealed that both transcript and protein levels of PED were significantly high in HCC compared with non-tumoral tissue. Clinico-pathological correlation revealed that PEDhigh HCCs showed an enrichment of gene signatures associated with metastasis and poor prognosis. Further, we observed that PED overexpression elevated the migration potential and PED silencing the decreased migration potential in liver cancer cell lines without effecting cell proliferation. Interestingly, we found that PED expression was regulated by a hepatocyte specific nuclear factor, HNF4α. A reduction of HNF4α induced an increase in PED expression and consequently, promoted cell migration in vitro. Finally, PED reduced the antitumoral effect of sorafenib by inhibiting caspase-3/7 activity. In conclusion, our data suggest that PED has a prominent role in HCC biology. It acts particularly on promoting cell migration and confers resistance to sorafenib treatment. PED may be a novel target for HCC therapy and serve as a predictive marker for treatment response against sorafenib.

Advances in How We Prioritize Liver Allocation for Hepatocellular Carcinoma in the USA

Treatment options for hepatocellular carcinoma (HCC) have changed significantly, beginning in 1994 with the establishment of successful long-term outcomes for HCC patients within Milan criteria who went on to receive liver transplantation (LT). Improved outcomes for patients with HCC undergoing LT led to challenges in determining the optimal liver allocation policy, which could provide relatively equal access for patients with and without HCC in the face of a long-standing critical shortage of available donor organs. United Network for Organ Sharing/Organ Procurement Transplantation Network (UNOS/OPTN) policy changes have aimed to optimize data collection for patients transplanted with HCC, improve the diagnostic criteria required for HCC, and reduce the disparity in access to transplantation for HCC and non-HCC patients observed between high- and low-model for end-stage liver disease (MELD) regions. Although current understanding of HCC tumor biology remains suboptimal, continued careful analysis of waitlist and post-transplant outcomes for both HCC and non-HCC patients combined with refinements to the allocation policy will hopefully result in improved survival both for waitlisted and post-transplant patients.

Interconnections between autophagy and the coagulation cascade in hepatocellular carcinoma.

Autophagy has an important role in tumor biology of hepatocellular carcinoma (HCC). Recent studies demonstrated that tissue factor (TF) combined with coagulation factor VII (FVII) has a pathological role by activating a G-protein-coupled receptor called protease-activated receptor 2 (PAR2) for tumor growth. The present study aimed to investigate the interactions of autophagy and the coagulation cascade in HCC. Seventy HCC patients who underwent curative liver resection were recruited. Immunohistochemical staining and western blotting were performed to determine TF, FVII, PAR2 and light chain 3 (LC3A/B) expressions in tumors and their contiguous normal regions. We found that the levels of autophagic marker LC3A/B-II and coagulation proteins (TF, FVII and PAR2) were inversely correlated in human HCC tissues. Treatments with TF, FVII or PAR2 agonist downregulated LC3A/B-II with an increased level of mTOR in Hep3B cells; in contrast, knockdown of TF, FVII or PAR2 increased LC3A/B. Furthermore, mTOR silencing restored the impaired expression of LC3A/B-II in TF-, FVII- or PAR2-treated Hep3B cells and activated autophagy. Last, as an in vivo correlate, we administered TF, FVII or PAR2 agonist in a NOD/severe combined immunodeficiency xenograft model and showed decreased LC3A/B protein levels in HepG2 tumors with treatments. Overall, our present study demonstrated that TF, FVII and PAR2 regulated autophagy mainly via mTOR signaling. The interaction of coagulation and autophagic pathways may provide potential targets for further therapeutic application in HCC.

Double-Contrast Ultrasound: A Novel Surveillance Tool for Hepatocellular Carcinoma

Kudo, Masatoshi MD1; Hatanaka, Kinuyo MD1; Kumada, Takashi MD2; Toyoda, Hidenori MD2; Tada, Toshifumi MD2 Author Information

A three-dimensional cell biology model of human hepatocellular carcinoma in vitro

We established an in vitro 3-D model of metastatic hepatocellular carcinoma (HCC) by culturing MHCC97H cells on molecular scaffolds within a rotating wall vessel bioreactor. Morphological and biochemical analyses revealed that the 3-D HCC model mirrored many clinical pathological features of HCC in vivo, including cancer cell morphology, tissue ultrastructure, protein production and secretion, glucose metabolism, tissue-specific gene expression, and apoptosis. Xenografts into livers of nude mice resulted in tumorigenesis and distant metastasis. This 3-D HCC spheroid is a promising model for HCC tumor biology, anticancer drug screening, and for the establishment of HCC animal models.

Biology of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common cancer in the world due to high prevalence of hepatitis B or C virus infection. Research in recent years has uncovered important molecular pathways involved in development and progression of HCC. Several genetic aberrations and molecular mechanisms responsible for initiation of hepatocarcinogenesis have been identified. Novel biomarkers for HCC are being developed for better detection and prognostication. Alpha-fetoprotein, the conventional marker of HCC, has limited sensitivity and specificity. Serum levels of isoforms of AFP based on differential lectin binding of the glycan moiety appear to be more sensitive and specific than total AFP level in early detection of HCC. The clinical usefulness of other HCC biomarkers such as des-gamma-carboxy prothrombin and glypican-3 are under investigation. HCC is an aggressive tumor with early vascular invasion and metastasis. Studies over the past two decades have elucidated the clinical predictors of outcome, leading to several staging systems for HCC based on clinical parameters. However, the predictive accuracy of clinical staging systems is limited. Recent studies suggested that biological factors may provide additional prognostic information. In particular, gene expression profiling appears to be a promising approach. Study of tumor angiogenesis in HCC reveals that the expression of angiogenic factors such as vascular endothelial growth factor and angiopoietins may also predict prognosis. The elucidation of tumor biology of HCC is of particular importance in the current era of rapid development of anti-cancer molecular targeting agents, which provide hope for an effective systemic therapy for HCC.