Hepatocyte Tumor Research Articles

Pharmacological Effects and Related Mechanisms of Ganoderma Lucidum Polysaccharides at the Cellular Level

Ganoderma lucidum polysaccharides are the primary active constituents of Ganoderma lucidum. Existing researches indicate that they possess multiple pharmacological effects and benefits, including anti-tumor, antioxidant, anti-aging, hypoglycemic, and immune-modulating properties. Studies on the cellular impact of Ganoderma polysaccharides have predominantly focused on various cell types, including malignant tumor cells, immune cells, cardiac myocytes, neurons, hepatocytes, and fibroblasts. In this paper, we review and summarize recent literature primarily related to these pharmacological effects at the cellular level and their underlying mechanisms. Our aim is to provide insights and references for the deep clinical development and utilization of Ganoderma lucidum polysaccharides.

Abstract 1100: Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1

Abstract Introduction: Hepatoblastoma (HB) is a liver tumor that arises in children. It's a sporadic malignancy that is often overly aggressive. The current treatment consists of chemotherapy. However, chemotherapy in young patients has disastrous and long-term side effects such as ototoxicity, cardiomyopathy, and infertility. Thus, alternative strategies are needed. One hint is to target the most common mutations in HB. It has been demonstrated that 90% of HB tumors are mutated for the Wnt pathway effector ß-catenin. This mutation leads to an aberrant constitutive activation of Wnt/ß-catenin signaling. Here, we investigate one of ß-catenin transcriptional targets, Fascin-1 that is found up-regulated in many tumors. Fascin1 affects actin organization into bundles, and this leads to cell migration and invasion. Whereas Fascin-1 is absent from normal hepatocytes, we found its expression associated to the poor prognosis C2 subtype of HB. In both human and murine HB samples, Fascin-1 is associated to undifferentiated tumor cells. We further demonstrated that Fascin-1 expression modulates tumor hepatocyte differentiation status through gene expression. In this study, we investigate how Fascin-1 can regulate tumor cell plasticity and whether Fascin-1 is a druggable target in HB tumors. Methods: We use two classical HB model cells Huh6 and HepG2 and 3 Patient-Derived-Xenograft cell lines. We explore the effect of Fascin-1 actin-bundling activity impairment by using inhibitors, on invasion and migration using Trans-well and wound-healing assays. We follow proliferation and cell death by Flow cytometry and investigate gene expression by PCR and reporter assay. We investigate Fascin modulation of the kinome with PamGene technology. Results: We show that the inhibition of Fascin actin-binding activity decreases cell invasion and migration as well as proliferation. We show an increase of cell death in Huh6/HepG2 cells but not in the PDX models. A cell cycle arrest is induced, and the cells become senescent. Differentiation genes are overexpressed and EMT genes are repressed. Yap expression is downregulated; Yap promoter activity is downregulated, and Yap is found translocated into the cytoplasm upon Fascin-1 inhibition. These data suggest that Fascin inhibition decreases tumors cell invasion, induces cell senescence, and favors a switch towards a differentiated and these effects on the cells are mediated via the Hippo pathway. The kinome is also modulated. Conclusion: Fascin-1 is an interesting target in hepatoblastoma, commercialized phase-2 drugs are available, and this study will confirm the potential use of those drugs in HB treatment and elucidate by which mechanism Fascin-1 inhibition impacts tumors. Citation Format: Lydia DIF, Amandine Martin, Sara Basbous, Gregoire Manaud, Violaine Moreau. Fascin-1 inhibitor decreases hepatoblastoma cells tumorigenicity via YAP 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1100.

Unveiling the Role of Concanavalin A in a Rodent Model of Chemical-Induced Hepatocellular Carcinoma: A Promising Guide in Understanding Liver Cancer Development

Hepatocellular carcinoma is a pressing global health issue, ranking as the third leading cause of cancer-related mortality in humans. Chronic liver diseases, such as hepatitis B and C infections and cirrhosis, are often associated with hepatocellular carcinoma, necessitating ongoing research for improved diagnostic and therapeutic strategies. Animal models, including both spontaneous and chemically induced models like diethylnitrosamine, play a pivotal role in understanding hepatocellular carcinoma mechanisms. Metabolic alterations in tumoral hepatocytes contribute significantly to cancer initiation and progression, impacting energy metabolism and cell survival. Lectins, specifically Concanavalin A, provide valuable insights into altered glycosylation patterns in cancer cells. This study employs lectin histochemistry to assess hepatic alterations in Concanavalin A expression in a murine model of diethylnitrosamine-induced hepatocellular carcinoma. Utilizing confocal laser scanning microscopy, our study unveils notable changes in Concanavalin A subcellular localization and intensity distribution in hepatocellular carcinoma compared with healthy liver tissue. A significant increase in the Concanavalin A labeling within the tumoral cells and a shifting of the expression within the perinuclear space is observed. These findings offer valuable insights into molecular changes in hepatocellular carcinoma, providing potential avenues for diagnostic and therapeutic advancements.

Cancer Development in Hepatocytes by Long-Term Induction of Hypoxic Hepatocellular Carcinoma Cell (HCC)-Derived Exosomes In Vivo and In Vitro.

Hypoxic tumor cell-derived exosomes play a key role in the occurrence, development, and metastasis of tumors. However, the mechanism of hypoxia-mediated metastasis remains unclear. In this study, hypoxic hepatocellular carcinoma cell (HCC-LM3)-derived exosomes (H-LM3-exos) were used to induce hepatocytes (HL-7702) over a long term (40 passages in 120 days). A nude mouse experiment further verified the effect of H-LM3-exos on tumor growth and metastasis. The process of cancer development in hepatocytes induced by H-LM3-exos was analyzed using both biological and physical techniques, and the results showed that the proliferation and soft agar growth abilities of the transformed cells were enhanced. The concentration of tumor markers secreted by transformed cells was increased, the cytoskeleton was disordered, and the migration ability was enhanced and was accompanied by epithelial-mesenchymal transition (EMT). Transcriptome results showed that differentially expressed genes between transformed cells and hepatocytes were enriched in cancer-related signaling pathways. The degree of cancer development in transformed cells was enhanced by an increase in H-LM3-exos-induced passages. Nude mice treated with different concentrations of H-LM3-exos showed different degrees of tumor growth and liver lesions. The physical properties of the cells were characterized by atomic force microscopy. Compared with the hepatocytes, the height and roughness of the transformed cells were increased, while the adhesion and elastic modulus were decreased. The changes in physical properties of primary tumor cells and hepatocytes in nude mice were consistent with this trend. Our study linking omics with the physical properties of cells provides a new direction for studying the mechanisms of cancer development and metastasis.

An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma.

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response.

Abstract IA004: Targeting STAT3 with TTI-101, an oral small molecule, to prevent colorectal and hepatocellular cancer

Abstract Persistent STAT3 activation contributes to 10 of 14 hallmarks of cancer, including inflammation; successful targeting of STAT3 has the potential to prevent and/or treat cancer. However, no small molecule that directly targets STAT3 has been FDA approved. The Tweardy lab used computer-based docking of drug-like compounds into the SH2 domain of STAT3, along with hit-to-lead optimization and medicinal chemistry, to identify TTI-101; TTI-101 treatment was safe, hit its target in tumor cells, and resulted in clinical benefit in a Phase I trial of patients with advanced solid tumors. The incidence of colorectal cancer (CRC) is increased 20-30 fold in patients with inflammatory bowel disease (IBD), while 90% of hepatocellular carcinomas (HCC) arise in the setting of chronic inflammation. To assess the contribution of STAT3 to CRC secondary to IBD and to HCC, we performed immunohistochemistry (IHC) staining and computer-based scoring for activated STAT3 (phosphorylated on Y705, pY-STAT3) of epithelial and stromal cells within colonic endoscopic biopsies and surgically resected CRC from IBD patients, as well as of tumor cells and hepatocytes within surgically resected HCC tumors. Compared to epithelium of normal tissue, levels of pY-STAT3 were increased 1.9-fold in dysplastic epithelium (p=0.05) and 1.8-fold in the stroma of normal tissue (p<0.0001). In surgically resected HCC tumors, lower pY-STAT3 scores in tumor cells, but not hepatocytes, correlated with longer recurrence free survival (RFS; p=0.003). TTI-101 administration in three AOM-DSS mouse models of IBD resulted in a dose-dependent reduction in polyps, adenomas, and/or adenocarcinomas. TTI-101 administration to the HepPten- mouse model of NASH-induced HCC resulted in a dose-dependent reduction in liver pY-STAT3 levels. Thus, STAT3 may be a valid target for chemoprevention using TTI-101 in CRC arising from IBD and in HCC. We thank Tvardi Therapeutics for providing TTI-101 for these studies. Citation Format: Prema Robinson, Leticia Hamana, Laura Beretta, Moses Kasembeli, Uddalak Bharadwaj, Yun Shin Chun, Yinghong Wang, Xeumei Wang, Laura Reyes, Luisa Solis, Asif Rashid, Dipen Maru, Eduardo Vila, Apostolia Tsimberidou, Ahmed Kaseb, Scott Kopetz, David Tweardy. Targeting STAT3 with TTI-101, an oral small molecule, to prevent colorectal and hepatocellular cancer [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA004.

Cytotoxic activity of Solanum tuberosum polyphenolic extracts in human hepatocarcinoma cells is mediated by apoptosis and autophagy.

Hepatocellular carcinoma (HCC) is one of the most frequent tumors worldwide, associated with a high mortality rate. HCC patient's prognosis remains unfavorable, mainly due to late diagnosis and treatment instauration delay. Thus, there is an urgent requirement for the development of new therapeutical options, particularly for advanced HCC patients. Polyphenols are specialized plant metabolites, which have been broadly characterized for their beneficial effects on human health. Potato is one of the main sources of these compounds in human diet, due to its high consumption. The objectives of this study were to: characterize the polyphenolic composition, analyze the antioxidant properties, evaluate the cytotoxic activity in human tumoral hepatocytes, and describe the molecular mechanisms involved in cell death, of potato polyphenolic extracts (PPEs) from the pigmented Andigena cultivar CL658. The results showed that phenolic acids were the main group of polyphenols in the extracts, chlorogenic acid being the major compound, followed by caffeic acid. HPLC-DAD profile showed pelargonidin as the principal anthocyanin. The treatment of HCC cell lines with the cytotoxic concentration 50 (CC50 ) of the PPEs resulted in cytotoxicity, evidenced by increases in the percentage of dead cells (5-10%) after treatments. This cytotoxic effect was mainly due to apoptosis activation, evidenced by modulation of the Bcl-2 family proteins expression, in favor of pro-apoptotic members over anti-apoptotic in the final balance. PPEs also induced autophagy, indicated by increased LC3 expression (122-274%) and autophagosomes, and reduced p62 expression (32-48%), in variable magnitudes according to the treated cell line. Overall, these results support the potential use of PPEs from the cultivar CL658 as a source of bioactive compounds for novel treatments against HCC.

The immune landscape of hepatocellular carcinoma-where we are?

Hepatocellular carcinoma (HCC) is one of the most common types of cancer diagnosed worldwide. After a decade of stagnation, several novel compounds have recently been shown to be effective in the treatment of HCC. Since immunotherapy is associated with important clinical benefits in some, but not all patients, it is essential to identify reliable predictive biomarkers. As the complex interplay between hepatocytes and immune cells is highly dependent on the tumor microenvironment, the tumor microenviroment has been suggested to be an important factor associated with the response to therapy and is currently being extensively investigated. Within this network, several important factors should be highlighted. Most of the cells are hepatocytes, but fibroblasts, endothelial cells, and immune cells are also present. Tumor-infiltrating leukocytes include several populations of cells and each of them plays a role in forming the tumor environment. Some of these cells may have antitumor effects, whereas others may be associated with the progression of the disease. The most important subsets include tumor-associated macrophages, tumor-associated neutrophils, and lymphocytes. These groups are described in the present review. The immune response is controlled by immune checkpoint molecules. One of the most important molecules involved in this checkpoint process seems to be the programmed death-1 (PD-1) receptor, which typically is induced on activated T cells, natural killer (NK) cells, B cells, and antigen-presenting cells. On the other hand, programmed death ligand 1 (PD-L1) is expressed by tumor cells, hepatocytes and hepatic stellate cells, and Kupffer cells or liver sinusoidal cells. Complex interactions between ligands and receptors are dependent on the signals from the microenvironment leading to either cancer development or apoptosis. Evidence from several studies indicates that patients with higher expression levels of PD-L1 on tumor cells or immune cells are more likely to achieve beneficial results from treatment with checkpoint blockers. This review focuses on the basic information regarding the microenvironment and its components, particularly on immune system involvement.

A modified kidney-sparing portal vein arterialization model of heterotopic auxiliary liver transplantation increases liver IL-6, TNF-α, and HGF levels and enhances liver regeneration: an animal model

Background and AimThe success of partial donor liver transplantation is affected by the implantation site of the donor liver and the vascular reconstruction approach. We investigated the effects of different donor liver implantation sites and vascular reconstruction approaches on liver regeneration using a rat kidney-sparing heterotopic auxiliary liver transplantation model, with portal vein arterialization (PVA).MethodsSixty male Sprague–Dawley rats underwent end-to-end anastomosis of the donor liver portal vein and the right renal artery stent (control group), or end-to-side anastomosis of the donor liver portal vein and the left common iliac artery (experimental group).ResultsThe experimental group had significantly lower plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, and cholinesterase than the control group (all, P < 0.05). The levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and hepatocyte growth factor (HGF) in the liver were significantly higher in the experimental group than that in the control group (all, P < 0.05). Hematoxylin and eosin (HE) staining of the liver tissue specimens indicated that the experimental group had greater hepatocyte regeneration compared to the control group.ConclusionsThe modified kidney-sparing PVA model of heterotopic auxiliary liver transplantation is more conducive to liver regeneration with quicker return of liver function.

Monitoring One-carbon metabolism by mass spectrometry for early diagnosis of cirrhosis and HCC

Introduction: Liver cancer represents one of the most frequent causes of death by cancer, ranked as the sixth most prevalent and the second in lethality. Hepatocellular carcinoma (HCC) is the predominant type of liver cancer and presents an increasing incidence during the last years. Late diagnosis is one of the reasons explaining the low survival rate of HCC patients (5 years survival after diagnosis below 20%). Remarkably, 80% of HCC cases develop in cirrhotic tissue (1). Main risk factors for HCC are well known and include hepatitis B and C viral infections or abusive alcohol consumption. However, the underlying molecular mechanisms remain unknown and its research will lead to the characterization of new biomolecular markers for the early diagnosis, prognosis and therapy of HCC. Metabolic remodeling is a common feature among several hepatic disorders, from steatosis to HCC (2). Tumoral hepatocytes modify their metabolism to satisfy cancer’s proliferative requirements. One-carbon metabolism (OCM) plays a fundamental role maintaining the differentiation and quiescent state of hepatocytes, and is recognized as the link between intermediate metabolism and epigenetic regulation. Owing to these reasons, it might be a potential source of biomarkers for early diagnosis and prognosis of HCC. Accordingly, it has been demonstrated that some OCM enzymes are differentially expressed in murine HCC models (3). Materials and methods: We have developed a robust targeted mass spectrometry-based method, using SRM mode (Selected Reaction Monitoring), for the systematic quantification of 13 enzymes that participate in OCM. For this purpose, purified synthetic heavy standard peptides, as well as OCM recombinant proteins have been used. Sixty-four human liver samples, including 28 control samples, 21 tumoral samples and15 cirrhotic samples have been used. Results and conclusions: We have demonstrated that there is a profound remodeling of the OCM cycle in HCC versus control samples, while cirrhotic samples tend to show intermediate expression levels between both physiological situations. Machine learning- based analysis of our results suggests that monitoring a panel of functionally related proteins might be useful for future clinical developments and improve the management of HCC patients. However, further experiments with larger cohorts are required to confirm these findings. References 1) Ryerson, A. B., et al (2016). Annual Report to the Nation on the Status of Cancer, 1975-2012, featuring the increasing incidence of liver cancer. Cancer, 122(9), 1312-1337. 2) Vander Heiden, et al (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science, 324(5930), 1029-1033 3) Mora, M. I., Corrales, F. J., et al (2017). Prioritizing popular proteins in liver cancer: remodelling one-carbon metabolism. Journal of proteome research, 16(12), 4506-4514.

Silencing of RND3/RHOE inhibits the growth of human hepatocellular carcinoma and is associated with reversible senescence.

Rnd3/RhoE is an atypical Rho GTPase family member, known to be deregulated in many types of cancer. Previously, we showed that RND3 expression is downregulated in hepatocellular carcinoma (HCC) cell lines and tissues. In cancer cells, Rnd3 is involved in the regulation of cell proliferation and cell invasion. The implication of Rnd3 in HCC invasion was importantly studied whereas its role in cell growth needs further investigation. Thus, in this work, we aimed to better understand the impact of Rnd3 on tumor hepatocyte proliferation. Our results indicate that the silencing of RND3 induces a cell growth arrest both in vitro in 2D and 3D culture conditions and in vivo in tumor xenografts. The growth alteration after RND3 silencing in HCC cells is not due to an increase of cell death but to the induction of senescence. This RND3 knockdown-mediated phenomenon is dependent on the decrease of hTERT expression. Interestingly, after re-expression of RND3, these cells are able to bypass senescence and regain the ability to proliferate, with a re-expression of hTERT. Given that a low expression of Rnd3 is linked to the presence of satellite nodules in HCC, the transient senescence state observed might play a role in cancer progression.

Well-differentiated liver cancers reveal the potential link between ACE2 dysfunction and metabolic breakdown

Angiotensin-converting enzyme 2 (ACE2) is the receptor of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causing Coronavirus disease 2019 (COVID-19). Transmembrane serine protease 2 (TMPRSS2) is a coreceptor. Abnormal hepatic function in COVID-19 suggests specific or bystander liver disease. Because liver cancer cells express the ACE2 viral receptor, they are widely used as models of SARS-CoV-2 infection in vitro. Therefore, the purpose of this study was to analyze ACE2 and TMPRSS2 expression and localization in human liver cancers and in non-tumor livers. We studied ACE2 and TMPRSS2 in transcriptomic datasets totaling 1503 liver cancers, followed by high-resolution confocal multiplex immunohistochemistry and quantitative image analysis of a 41-HCC tissue microarray. In cancers, we detected ACE2 and TMPRSS2 at the biliary pole of tumor hepatocytes. In whole mount sections of five normal liver samples, we identified ACE2 in hepatocyte’s bile canaliculi, biliary epithelium, sinusoidal and capillary endothelial cells. Tumors carrying mutated β-catenin showed ACE2 DNA hypomethylation and higher mRNA and protein expression, consistently with predicted β-catenin response sites in the ACE2 promoter. Finally, ACE2 and TMPRSS2 co-expression networks highlighted hepatocyte-specific functions, oxidative stress and inflammation, suggesting a link between inflammation, ACE2 dysfunction and metabolic breakdown.

Abstract LBA012: Engineered mini-livers for high-throughput tumor organoid screening of prodrugs

Abstract Several anti-cancer compounds, including chemotherapy and targeted agents such as ifosfamide, cyclophosphamide, and imatinib, are administered as prodrugs which are metabolized to become pharmacologically active. Prodrugs are particularly difficult to study in vitro or ex vivo as many are activated in the liver by Cytochrome P450 enzymes. Preclinical studies of these compounds are restricted to in vivo models, as cancer cells typically do not express the enzymes required to transform prodrugs into their active form. While animal models effectively recapitulate the complexity of systemic drug metabolism and distribution, in vivo studies are time-consuming, expensive, and unsuitable for high-throughput drug screening studies. In previous work, we have developed a platform for screening patient-derived tumor organoids that yields results within a week of surgery (Phan et al, Commun Biol 2, 78, 2019; Nguyen and Soragni, STAR Protoc 1, 2, 2020; Al Shihabi et al, bioRxiv, 2021). Tumor organoids are promising pre-clinical models as they closely recapitulate features of the parent tumor, including drug responses. Here, we expand the physiological relevance of tumor organoid screenings by including mini-livers to metabolize prodrugs of interest. To retain the ability to perform automated, high-throughput screenings, we engineered a miniature liver insert (MLI). The MLI is a removable system that facilitates the addition of liver organoids to specific wells of 96-well plates in which prodrugs are tested. The MLI is designed to integrate with our existing tumor organoid screening platform (Phan et al, Commun Biol 2, 78, 2019; Nguyen and Soragni, STAR Protoc 1, 2, 2020; Al Shihabi et al, bioRxiv, 2021), and allows for simultaneous monitoring of tumor organoids and hepatocytes with brightfield and fluorescent imaging. Hepatocytes seeded in the MLI are viable and maintain their expression of the key Cytochrome P450 enzymes responsible for the metabolism of many clinically used prodrugs. Finally, we demonstrate that incorporating the MLI into prodrug screening experiments sensitizes tumor organoids to treatment with chemotherapeutic and targeted agents. Due to the facile integration with existing screening protocols, the MLI is a simple and effective system for studying anti-cancer prodrugs ex vivo. The MLI is a novel tool that facilitates the co-culture of functional liver organoids with clinically relevant tumor organoid models for drug discovery studies and precision medicine applications. Citation Format: Peyton J Tebon, Alice Soragni. Engineered mini-livers for high-throughput tumor organoid screening of prodrugs [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA012.

Isolation of Primary Hepatocytes for Testing Tumor Penetrating Peptides.

Methods for isolation of human primary cells is an important tool to be able to test the effect of several drugs for the treatment of diseases. Protocols have been described for the isolation of healthy and tumoral hepatocytes; however, the quality and the amount of the isolated cells is not satisfactory. We describe here protocols for the isolation of healthy and tumoral hepatocytes as well as the use of tumor penetrating and interfering peptides as new therapeutics. Peptides are showed to be safe drugs and taking into account recent improvements in peptide administration, biodelivery, and bioavailability. Tumor penetrating peptides fused to an interfering peptide have a great potential to become therapeutic agents.

A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma.

Simple SummaryLiver cancer is one of the most commonly diagnosed cancers worldwide and the fourth leading cause of cancer-related deaths. Hepatocellular carcinoma (HCC) accounts for at least 80% of all malignant liver primary tumors. A better characterization of molecular mechanisms underlying HCC onset and progression may lead to discover new therapeutic targets and biomarkers. In this study, we performed an integrative transcriptomics analysis to evaluate the clinical relevance of genes associated with hepatocyte differentiation in human HCC. The HepaRG cell line model was used to define a gene expression signature reflecting the status of tumor hepatocyte differentiation. This signature was able to stratify HCC patients into clinically relevant molecular subtypes. Then, a minimal subset of seven differentiation-associated genes was identified to predict a poor prognosis in several cancer datasets.Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.

Camellia sinesis leaves extract ameliorates high fat diet-induced nonalcoholic steatohepatitis in rats: analysis of potential mechanisms

Our research aims to address and determine the effect of Camellia sinensis extract in the management of nonalcoholic steatohepatitis (NASH) in rats. Forty adult female albino rats were divided into four groups. Group 1 (G1) served as the control group, while the other three groups received high-fat diet for 32 weeks to induce NASH and then were later assigned to the following groups: (G2) NASH-afflicted group which was left untreated, (G3) NASH-afflicted group treated with Camellia sinensis extract in a dose of 400 mg/kg, and (G4) NASH-afflicted group treated with Camellia sinensis extract in a dose of 200 mg/kg. Significant elevation in serum alanine aminotransferase, albumin, bilirubin (total and direct), cholesterol, low density lipoprotein, triglycerides, leptin, Cox-2, and CD40 values was recorded. Moreover, overexpression of hepatic tumor necrosis factor alpha and hepatocyte growth factor genes were recorded, whereas blood platelet count and serum high density lipoprotein concentration revealed significant depletion, which was paralleled by significant downregulation of hepatic adiponectin gene expression level in NASH group versus the control group. On the opposite side, treatment of NASH groups with two different doses of Camellia sinensis extract reversed the values of the measured biochemical parameters and the targeted gene expression levels when compared with the NASH group. Optical micrograph of liver tissue sections of rats treated with Camellia sinensis extract showed the observed improvement in the studied biochemical and genetic markers. This study provides a clear evidence for the promising therapeutic potential of Camellia sinensis extract against NASH. This could be ascribed to its hepatoprotective activity, hypolipidemic effect, and anti-inflammatory potency.

A Monochromophoric Approach to Succinct Ratiometric Fluorescent Probes without Probe-Product Crosstalk

Ratiometric probes facilitate quantitative studies via self-calibration and are cherished for bioimaging. Often, a small probe-product spectral separation leads to crosstalk, but the rational devel...

Bifunctional Therapeutic Peptides for Targeting Malignant B Cells and Hepatocytes: Proof of Concept in Chronic Lymphocytic Leukemia

AbstractProtein–protein interactions are well recognized as therapeutic targets and therefore interfering peptides (IP) that block these interactions are receiving increasing attention. Four different tumor‐penetrating peptides (TPPs) (iRGD, RPARPAR, Linear TT1 (LinTT1), and cyclic TT1 (TT1)) are associated to an IP that blocks the interaction between the protein phosphatase PP2A and its binding protein SET, generating new bifunctional peptides able to intracellularly target the PP2A/SET interaction in malignant B cells and tumoral hepatocytes. The TPPs are able to penetrate into B cells of patients suffering chronic lymphocytic leukemia (CLL) and into tumoral hepatocytes but not into B cells from healthy donors and healthy hepatocytes. The association of cargo does not affect the penetration of the TPPs in CLL B cells. All the bifunctional peptides induce apoptosis in human CLL B cells and tumoral hepatocytes, and stability tests reveal that iRGD‐IP, RPARPAR‐IP, and TT1‐IP are stable after 24 h incubation in human serum. The iRGD associated with the IP significantly increases the survival of mice bearing xenograft models of CLL without any symptom of toxicity, suggesting that the bifunctional peptides may have a therapeutic application for selective tumoral targeting of PP2A/SET interaction, which is deregulated in several cancers, including CLL.

IGM-8444 as a potent agonistic Death Receptor 5 (DR5) IgM antibody: Induction of tumor cytotoxicity, combination with chemotherapy and in vitro safety profile.

3595 Background: Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF) receptor superfamily that multimerizes when bound to its ligand, TNF-related apoptosis inducing ligand (TRAIL), to activate the extrinsic apoptotic pathway. DR5 is broadly expressed on solid and hematologic cancers and has been targeted with both recombinant TRAIL and agonistic antibodies in the clinic. However, these therapeutics have generally been unsuccessful due to toxicity or lack of efficacy. We have developed a multivalent IgM DR5 agonist, IGM-8444, that multimerizes DR5 to selectively and potently induce tumor cell apoptosis while maintaining tolerability. Methods: IGM-8444 is an engineered, pentameric IgM antibody with 10 binding sites specific for DR5. Human tumor cell lines or hepatocytes were evaluated in vitro for dose dependent IGM-8444 induced cytotoxicity. The efficacy of IGM-8444 was evaluated with or without chemotherapy, in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse tumor models, with IGM-8444 administered at various dose levels and schedules when tumors reached approximately 100 mm3. Sera and tumors were analyzed for biomarkers of tumor apoptosis. Results: In vitro cytotoxicity assays identified IGM-8444 activity across cell lines from 18 solid and hematologic malignancies. In IGM-8444 partially resistant cell lines, combination with chemotherapy or a Bcl2 inhibitor enhanced in vitro cytotoxicity. IGM-8444 was efficacious as a monotherapy in CDX and PDX tumor models including colorectal, lung, and gastric indications. In a gastric PDX model, IGM-8444 induced complete and durable dose-dependent tumor regressions. In vivo, combination of IGM-8444 with standard-of-care chemotherapies, such as irinotecan, led to enhanced efficacy. IGM-8444 administration increased markers of tumor apoptosis, identifying potential clinical pharmacodynamic biomarkers. At doses several log-fold higher than efficacious doses, IGM-8444 demonstrated a favorable single agent in vitro safety profile, with little to no in vitro cytotoxicity observed using primary human hepatocytes from multiple donors. Conclusions: These data support the clinical development of IGM-8444 in both solid and hematological malignancies as a single agent and in combination with standard of care therapy. IGM-8444 is projected for IND filing in 2020.

Opposite responses of normal hepatocytes and hepatocellular carcinoma cells to substrate viscoelasticity.

The cellular microenvironment plays a critical role in cell differentiation, proliferation, migration, and homeostasis. Recent studies have shown the importance of substrate viscosity in determining cellular function. Here, we study the mechanoresponse of normal hepatocytes and hepatocellular carcinoma cells (HCC) to elastic and viscoelastic substrates using the Huh7 cell line derived from a human liver tumor and primary human hepatocytes (PHH). Unlike PHH and fibroblasts, which respond to viscoelastic substrates by reducing spreading area and actin bundle assembly compared to purely elastic substrates of the same stiffness, Huh7 cells spread faster on viscoelastic substrates than on purely elastic substrates. The steady state spreading areas of Huh7 cells are larger on viscoelastic substrates, whereas the opposite effect occurs with PHH cells. The viscoelasticity of the microenvironment also promotes motility and multiple long protrusions in Huh7 cells. Pharmacologic disruption of the actin assembly makes cells unable to spread on either elastic or viscoelastic substrates. In contrast, upon vimentin perturbation, cells still spread to a limited degree on elastic substrates but are unable to spread on viscoelastic substrates. The time evolution of cell traction force shows that the peak occurs at an earlier time point on viscoelastic substrates compared to elastic substrates. However, the total force generation at steady state is the same on both substrates after 4 hours. Our data suggest that stress relaxation time scales of the viscoelastic substrate regulate cell dynamics and traction force generation, indicating different binding-unbinding rates of the proteins that form cell attachment sites in HCC cells and normal hepatocytes. These results suggest that liver cancer cells may have different characteristic lifetimes of binding to the substrate in comparision to normal cells, which might cause differences in cell spreading and motility within the diseased tissue.