BACKGROUND AND AIMS:Intrahepatic metastasis negatively impacts the prognosis of several cancers, including hepatocellular and colorectal carcinoma. Zebrafish larval xenografts serve as a robust vertebrate platform that allows direct visualisation of tumour behaviour within a living organism. However, organ-specific metastasis models in zebrafish remain limited, and liver metastasis has not yet been demonstrated. This study aimed to establish a zebrafish larval intrahepatic metastasis model and to determine the role of c-Met activation in mediating liver tropism of cancer cells. METHODS:An intravenous injection-based zebrafish model (zLiverMet) was developed using a liver-specific fluorescent reporter line to visualise tumour colonisation in vivo. Liver cancer cell lines with distinct c-Met expression and activation levels were injected into 2-day post-fertilisation larvae. The effects of c-Met overexpression and pharmacological inhibition on intrahepatic metastasis were analysed through confocal imaging and quantitative image measurements. The model's applicability was further tested using colorectal cancer (CRC) cell lines. RESULTS:Intravenous injection facilitated efficient intrahepatic colonisation, whereas yolk-sac injection failed to reproduce vascular dissemination. Liver cancer cell lines with high c-Met expression, SNU-449, Mahlavu and SK-HEP-1, exhibited strong liver tropism, while cell lines with no/low c-Met expression, HuH-7 and SNU-398, showed minimal hepatic metastasis. Overexpressing c-Met increased liver colonisation of HCC cells, and inhibiting c-Met activation with the c-Met inhibitor SU11274 reduced this effect. CONCLUSIONS:The zLiverMet model successfully mimics intrahepatic metastasis and highlights c-Met as a driver of liver tropism. This zebrafish-based model offers an 'organism-on-a-chip' platform that is rapid, imageable and scalable-bridging in vitro assays and in vivo models for mechanistic and therapeutic studies of liver metastasis.

Abstract Aberrant HGF–c-MET signaling is a major driver of hepatocellular carcinoma (HCC) progression and a clinically validated therapeutic axis, but current inhibitors predominantly target the intracellular kinase domain and remain vulnerable due to limited selectivity and resistance development. We therefore pursued an upstream strategy based on small molecules that target the extracellular HGF–c-MET interaction interface. We combined large-scale virtual screening of more than one million compounds from the ChemDiv and Enamine libraries with molecular dynamics (MD) simulations, steered MD, MM/GBSA profiling, and iterative lead optimization to identify candidate c-MET inhibitors targeting its extracellular (EC) domain. In HGF-stimulated HuH7 cells, selected compounds suppressed c-MET autophosphorylation, reduced cell viability, and inhibited long-term colony formation. Surface plasmon resonance (SPR) further confirmed direct binding of L083-1287 and 8008-3424 to the recombinant c-MET ectodomain. Mechanistic analyses identified previously unrecognized hotspot residues on the c-MET EC domain and a novel inhibitory network spanning multiple c-MET ectodomain interfaces. L083-0077 displayed the most consistent interaction pattern within this framework, including stabilization of key hotspot residues and preserved binding under acidic conditions relevant to the tumor microenvironment. Zebrafish xenograft assays with selected early hit compounds revealed compound-dependent developmental liabilities supporting the use of this model as an early in vivo prioritization step during lead optimization. These findings establish EC interface-directed c-MET inhibition as a promising therapeutic strategy in HCC and provide a mechanism-guided platform for the development of selective, upstream c-MET inhibitors with the potential to complement or overcome limitations of kinase-directed therapies.

In this chapter, we present a detailed protocol for establishing a three-dimensional (3D) multicellular tumor spheroids (MCTSs) model to simulate the tumor microenvironment (ME) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) for the study of hepatocellular carcinoma (HCC) and colorectal cancer (CRC) cell aggressiveness, growth, and metastasis potential. The MASLD microenvironment (MASLD-ME) is recreated by embedding hepatic stellate cells in a collagen I matrix within a Boyden chamber system. The metabolic medium mimics MASLD conditions, enriched with high glucose, fructose, insulin, and fatty acids, to simulate metabolic stresses associated with the disease.In the protocol, cancer cells are loaded in the upper compartment to analyze their migration toward the MASLD-ME, thereby facilitating studies on cancer cell invasiveness and metastatic capacity. This method offers an adaptable, reproducible model to research disease progression and investigate therapeutic interventions, contributing to preclinical research on MASLD-related liver cancer pathophysiology and potential drug responses.

Fusion transcripts (FTs) are RNA molecules, also known as chimeric transcripts, formed through chromosomal rearrangements or transcriptional processes, contributing to tumorigenesis. This study systematically examined tumour-specific FTs in hepatocellular carcinoma (HCC) using high-throughput RNA sequencing data from independent datasets and The Cancer Genome Atlas (TCGA). Our meta cohort analysis included 328 HCC samples. Using STAR-Fusion, we identified 15 novel tumour-specific FTs, with SERPINA1-H19 as the most recurrent fusion event. Comparative expression analysis of fusion partner genes revealed significant downregulation in HCC tumours relative to normal adjacent liver tissues (NAT). We validated the expression levels of the key partner genes with 436 TCGA samples serving as an in silico validation cohort and in wet lab validation cohorts with 42 samples. ALB, APOA2, IGF2, MT2A, SERPINA1, and H19, which are key liver-associated genes, were frequently involved in tumour-specific fusion events suggesting either a loss of tumour suppressor property or gaining a novel function playing a role in hepatocarcinogenesis. Detailed characterization of SERPINA1-H19 identified 16 transcript variants with distinct structural modifications that may impact its functional output. Furthermore, low expression of SERPINA1 and H19 was associated with more aggressive HCC phenotypes. Overall, this study established a comprehensive repository of FTs for the first time, offering valuable insights into their role in HCC and their potential to serve as diagnostic and prognostic biomarkers for HCC.

Research biobanks have become crucial collaborators in a variety of basic and clinical research projects with comprehensive biological sample collection and associated data storage. Medical students, who are the most important stakeholders of biobanks as future physicians, need to be trained in biobanking; however, there is no consensus on how to include it in formal education. This study aimed to determine and increase awareness among medical students regarding biobanks through peer training organized online by medical student research association networks. Volunteer medical or graduate students were trained by biobank professionals at the Izmir Biomedicine and Genome Center (IBG) biobank for 6-9 months. Then, a biobank event was planned by these trainees, the Ege Scientific Research Team (ESRT), and IBG-Biobank with the support of The Biobanking and BioMolecular Resources Research Infrastructure (BBMRI) Turkey. The study reached students of 46 different medical faculties. Before the event, students' level of knowledge about biobanks was identified using a pre-event questionnaire (n = 239). Following 2 days (4 main sessions) of online events, a post-event questionnaire was administered to event participants (n = 110) and 80.9% of them answered (n = 89). The pre-event survey revealed that only 34.3% of the medical students had heard of the term "Biobank" in Turkey. After the event, medical students were significantly more enthusiastic about putting effort into biobanking and using and sharing stored biobank samples of their patients compared with the pre-event (p < 0.0001). Moreover, 92% of the students stated that they would consider attending an advanced course in biobanking. In conclusion, the current study demonstrates that extracurricular courses with peer learning methods coordinated with medical student associations can be valuable in increasing future physicians' awareness and knowledge of biobanking.

Aim: Hepatocellular carcinoma (HCC) in patients with Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD) is expected to be a significant public health issue in the near future. Therefore, understanding the tumor microenvironment interactions in MASLD-induced HCC is crucial, and the development of relevant preclinical models is needed. Hence, we aimed to determine the effects of a MASLD-mimicking microenvironment (ME) on the aggressiveness of HCC cells and identify target genes that drive HCC by developing a 3D-in vitro co-culture system. Methods: A 3D co-culture system mimicking the MASLD-ME was created with LX-2 liver stellate cells embedded in 3D collagen gel in the lower and SNU-449 HCC cells on the upper parts of Boyden chambers, and cells were grown in an optimized metabolic medium (MM). The effects of MASLD-ME on motility, sphere formation, proliferation, and cell cycle of SNU-449 cells were tested by Boyden chamber, 3D sphere formation, XTT, and Flow cytometry, respectively. The protein expression/activation profiles of motile SNU-449 cells that passed the membrane toward MASLD-ME or control condition were investigated using a multiplex protein profiling system DigiWest and confirmed with RT-PCR, WB, and Flow cytometry. IDH2 levels were examined in primary human HCC and adjacent liver tissues by IHC and in TCGA and CPTAC cohorts by bioinformatics tools. Results: MM treatment increased fat accumulation, motility, and spheroid formation of both SNU-449 and LX-2 cells. MASLD-ME induced activation of LX2 cells, leading to the formation of bigger colonies with many intrusions compared to related controls. DigiWest analysis showed that metabolism-related proteins such as IDH2 were the most affected molecules in SNU-449 cells that migrated toward the MASLD-ME compared to those that migrated toward the control condition. Downregulation of IDH2 expression was confirmed in SNU-449 cells grown in MASLD-ME, in primary HCC tumor samples by IHC, and in HCC patient cohorts by bioinformatics analysis. Conclusion: This study reports the potential involvement of MASLD-ME in the downregulation of IDH2 expression and promoted motility and colonization capacity of HCC cells. The 3D MASLD model presented in this study may be useful in investigating the mechanistic roles of MASLD-ME in HCC.

Purpose The contribution of androgen receptors (AR) on bladder cancer has been demonstrated in pre-clinical studies, however in clinical studies, only the canonical AR (AR-FL) protein was measured by immunohistochemistry and conflicting results were obtained. To get better insight into the alterations of AR signalling, we used western blotting (WB) method and simultaneously measured both mRNA and protein levels of AR-FL and AR-V7. Methods 23 naive non-muscle invasive bladder cancer patients and 12 healthy individuals were included. AR-FL protein, AR-FL mRNA, AR-V7 protein and AR-V7 mRNA levels were quantitatively measured by WB and qRT-PCR. Results While AR-FL protein and AR-V7 mRNA were significantly higher in bladder cancer, AR-FL mRNA and AR-V7 protein were lower. AR-V7 mRNA level was higher in patients with tumour size over 3 cm and AR-FL protein was higher in single tumours (p < 0,005). The small sampling size and the inclusion of only male participants were the main limitations. Conclusions The increase of AR-FL protein in bladder cancer supports the contribution of the AR pathway in bladder cancer. The presence of high AR-FL protein despite low mRNA levels may be due to a disruption in post-transcriptional regulatory mechanisms. AR-V7 was demonstrated for the first time in bladder tissue and found significantly different in bladder cancer tissues. Our study reached new and valuable findings and will shed light on the studies that aim to clarify the role of the AR pathway in bladder cancer.

Activation of c-Met signaling is associated with an aggressive phenotype and poor prognosis in hepatocellular carcinoma (HCC); however, its contribution to organ preference in metastasis remains unclear. In this study, using a Lab on a Chip device, we defined the role of aberrant c-Met activation in regulating the extravasation and homing capacity of HCC cells. Our studies showed that (i) c-Met overexpression and activation direct HCC cells preferentially towards the hepatocytes-enriched microenvironment, and (ii) blockage of c-Met phosphorylation by a small molecule inhibitor attenuated extravasation and homing capacity of HCC cells. These results, thus, demonstrate the role of c-Met signaling in regulating the colonization of HCC cells preferentially in the liver.

Background Liver cancer is the third leading cause of cancer-related deaths worldwide, and hepatocellular carcinoma (HCC) is the most common type of liver cancer. Transarterial interventions are among the chemotherapeutic approaches used in hardly operable regions prior to transplantation, and in electrochemotherapy, where doxorubicin is used. However, the efficacy of treatment is affected by resistance mechanisms. Previously, we showed that overexpression of the CUE5 gene results in doxorubicin resistance in Saccharomyces cerevisiae (S. cerevisiae). In this study, the effect of Toll-interacting protein ( TOLLIP ), the human ortholog of CUE5 , on doxorubicin resistance was evaluated in HCC cells to identify its possible role in increasing the efficacy of transarterial interventions. Methods and results The NIH Gene Expression Omnibus (GEO) and Oncomine datasets were analyzed for HCC cell lines with relatively low and high TOLLIP expression, and SNU449 and Hep3B cell lines were chosen, respectively. TOLLIP expression was increased by plasmid transfection and decreased by TOLLIP- siRNA in both cell lines and evaluated by RT-PCR and ELISA. Cell proliferation and viability were examined using xCELLigence and MTT assays after doxorubicin treatment, and growth inhibitory 50 (GI 50) concentrations were evaluated. Doxorubicin GI 50 concentrations decreased approximately 2-folds in both cell lines upon silencing TOLLIP after 48 h of drug treatment. Conclusions Our results showed for the first time that silencing TOLLIP in hepatocellular carcinoma cells may help sensitize these cells to doxorubicin and increase the efficacy of chemotherapeutic regimens where doxorubicin is used.

INTRODUCTION:Hepatocellular carcinoma (HCC) is a highly complex and deadly cancer. There is an urgent need for new and effective treatment modalities. Since the primary goal in the management of cancer is to cure and improve survival, personalized therapy can increase survival, reduce mortality rates, and improve quality of life. Biobanks hold potential in leading to breakthroughs in biomedical research and precision medicine (PM). They serve as a biorepository, collecting, processing, storing, and supplying specimens and relevant data for basic, translational, and clinical research.OBJECTIVE:We aimed to highlight the fundamental role of biobanks, harboring high quality, sustainable collections of patient samples in adequate size and variability, for developing diagnostic, prognostic, and predictive biomarkers to develop and PM approaches in the management of HCC.METHOD:We obtained information from previously published articles and BBMRI directory.RESULTS AND CONCLUSION:Biobanking of high-quality biospecimens along with patient clinical information provides a fundamental scientific infrastructure for basic, translational, and clinical research. Biobanks that control and eliminate pre-analytical variability of biospecimens, provide a platform to identify reliable biomarkers for the application of PM. We believe, establishing HCC biobanks will empower to underpin molecular mechanisms of HCC and generate strategies for PM. Thus, first, we will review current therapy approaches in HCC care. Then, we will summarize challenges in HCC management. Lastly, we will focus on the best practices for establishing HCC biobanking to support research, translational medicine in the light of new experimental research conducted with the aim of delivering PM for HCC patients.

PURPOSE:Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide with lack of effective systemic chemotherapy. In this study, we aimed to evaluate the value of ATPase family AAA domain-containing protein 2 (ATAD2) as a biomarker and potential therapeutic target for HCC.METHODS:The expression of ATAD2 was tested in different HCC patient cohorts by immunohistochemistry and comparative transcriptional analysis. The co-expression of ATAD2 and proliferation markers was compared during liver regeneration and malignancy with different bioinformatics tools. The cellular effects of ATAD2 inactivation in liver malignancy was tested on cell cycle, apoptosis, and colony formation ability as well as tumor formation using RNA interference. The genes affected by ATAD2 inactivation in three different HCC cell lines were identified by global gene expression profiling and bioinformatics tools.RESULTS:ATAD2 overexpression is closely correlated with HCC tumor stage. There was gradual increase from dysplasia, well-differentiated and poorly-differentiated HCC, respectively. We also observed transient upregulation of ATAD2 expression during rat liver regeneration in parallel to changes in Ki-67 expression. ATAD2 knockdown resulted in apoptosis and decreased cell survival in vitro and decreased tumor formation in some HCC cell lines. However, three other HCC cell lines tested were not affected. Similarly, gene expression response to ATAD2 inactivation in different HCC cell lines was highly heterogeneous.CONCLUSIONS:ATAD2 is a potential proliferation marker for liver regeneration and HCC. It may also serve as a therapeutic target despite heterogeneous response of malignant cells.

The biological mechanisms responsible for depression symptoms are not yet understood. For this reason, it is important to reveal the etiopathogenetic mechanisms in this disease. This study aims to compare the levels of pro-inflammatory cytokines, Brain-Derived Neurotrophic Factor (BDNF), and telomerase activity in patients with major depressive disorder (MDD) and healthy controls. Plasma BDNF, interleukin-6 (IL-6), IL-1beta, and Tumor Necrosis Factor-alpha (TNF-alpha) levels, and telomerase activity were measured in 39 patients with major depression and 39 healthy controls matched with patients in terms of age, gender, and education year. Plasma concentration of BDNF, IL-6 levels, and telomerase activity was significantly different between patients with MDD and healthy controls. Correlation analysis showed a positive trend between plasma BDNF levels and plasma IL-6 levels in patients with MDD with melancholic features. Furthermore, the path analysis results showed that the telomerase activity was indirectly affected by gender, IL-1β, IL-6, BDNF, and BMI, via the severity of depression and anxiety and MDD status as the mediators. Further studies are needed to examine the molecular mechanism of the telomerase activity and the role of BDNF and pro-inflammatory cytokines in the telomerase activation in MDD.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a newly defined stem cell marker in endoderm-derived organs such as the small intestine, colon and pancreas. Recently, LGR5 was demonstrated to be an important factor in liver regeneration and stem cell maintenance. Moreover, LGR5 expression is highly up-regulated in various cancers including hepatocellular carcinoma. Herein, we demonstrate that LGR5 expression is specifically observed in certain subset of HCC cell lines with “hepatoblast-like” appearance, characterized by the expression of liver fetal/progenitor markers. Notably, the activation of the canonical Wnt pathway significantly increases the expression of LGR5 in this subset of cell lines, whereas it does not cause any induction of LGR5 expression in mesenchymal like cell lines SNU-475 and SNU-449. Furthermore, we showed that treatment of the hepatoblast-like HCC cell lines HuH-7 and Hep3B with LGR5 ligand R-Spo1 significantly amplifies the induction of LGR5 expression, the phosphorylation of LRP6 and β-catenin resulting in enhanced TCF/LEF activity either alone or in combination with Wnt3a. Consistently, the silencing of the LGR5 gene attenuates the co-stimulatory effect of R-Spo1/Wnt3a on TCF/LEF activity while overexpression of LGR5 enhances it. On the contrary, overexpression of LGR5 does not change TCF/LEF activity induced by R-Spo1/Wnt3a in mesenchymal-like HCC line, SNU-449. Importantly, LGR5-overexpressing cells have increased expression of several Wnt target genes and stemness-related genes including EpCAM and CK19 upon R-Spo1/Wnt3a treatment. LGR5-overexpressing cells also have increased spheroid forming, migration and invasion abilities and stimulation with R-Spo1/Wnt3a augments these abilities of LGR5-overexpressing cells. In addition, ectopic overexpression of LGR5 significantly increases cell proliferation rate independent of R-Spo1/Wnt3a stimulation. Moreover, in vitro tubulogenesis assay demonstrates that treatment with R-Spo1/Wnt3a enhances the sprouting of capillary tubules in only LGR5-overexpressing cells. Finally, R-Spo1/Wnt3a significantly promotes dissemination of LGR5-overexpressing cells in vivo in a zebrafish xenograft model. Our study unravels a tumor-promoting role for LGR5 through activation of canonical Wnt/β-catenin signaling in the hepatoblast-like HCCs. In conclusion, our results suggest that LGR5/R-Spo1/Wnt3a generates an axis that mediates the acquisition of aggressive phenotype specifically in hepatoblast-like subset of HCCs and might represent a valuable target for treatment of HCC tumors with aberrant activation of Wnt/β-catenin pathway.

Hepatocellular carcinoma (HCC) is strongly associated with metabolic dysregulations/deregulations and hyperglycemia is a common metabolic disturbance in metabolic diseases. Hyperglycemia is defined to promote epithelial to mesenchymal transition (EMT) of cancer cells in various cancers but its molecular contribution to HCC progression and aggressiveness is relatively unclear. In this study, we analyzed the molecular mechanisms behind the hyperglycemia-induced EMT in HCC cell lines. Here, we report that high glucose promotes EMT through activating c-Met receptor tyrosine kinase via promoting its ligand-independent homodimerization. c-Met activation is critical for high glucose induced acquisition of mesenchymal phenotype, survival under high glucose stress and reprogramming of cellular metabolism by modulating glucose metabolism gene expression to promote aggressiveness in HCC cells. The crucial role of c-Met in high glucose induced EMT and aggressiveness may be the potential link between metabolic syndrome-related hepatocarcinogenesis and/or HCC progression. Considering c-Met inhibition in hyperglycemic patients would be an important complementary strategy for therapy that favors sensitization of HCC cells to therapeutics.

Selective autophagy of the endoplasmic reticulum (ER), namely ER-phagy, is mediated by ER-localized receptors, which are recognized and sequestered by GABARAP/LC3B-decorated phagophores and transferred to lysosomes for degradation. Being one such receptor, FAM134B plays critical roles in cellular processes such as protein quality control and neuronal survival. FAM134B has also been associated with different cancers, although its exact role remains elusive. We report here that the FAM134B gene encodes not one but at least two different protein isoforms: the full-length and the NH2 terminally truncated forms. Their relative expression shows extreme variation, both within normal tissues and among cancer types. Expression of full-length FAM134B is restricted to the brain, testis, spleen, and prostate. In contrast, NH2 terminally truncated FAM134B is dominant in the heart, skeletal muscle, kidney, pancreas, and liver. We compared wild-type and knockout mice to study the role of the Fam134b gene in starvation. NH2 terminally truncated FAM134B-2 was induced in the liver, skeletal muscle, and heart but not in the pancreas and stomach following starvation. Upon starvation, Fam134b-/- mice differed from wild-type mice by less weight loss and less hyperaminoacidemic and hypocalcemic response but increased levels of serum albumin, total serum proteins, and α-amylase. Interestingly, either NH2 terminally truncated FAM134B or both isoforms were downregulated in liver, lung, and colon cancers. In contrast, upregulation was observed in stomach and chromophobe kidney cancers.NEW & NOTEWORTHY We reported tissues expressing FAM134B-2 such as the kidney, muscle, heart, and pancreas, some of which exhibit stimulated expression upon nutrient starvation. We also demonstrated the effect of Fam134b deletion during ad libitum and starvation conditions. Resistance to weight loss and hypocalcemia, accompanied by an increase in serum albumin and α-amylase levels, indicate critical roles of Fam134b in physiology. Furthermore, the differential expression of FAM134B isoforms was shown to be significantly dysregulated in human cancers.

BACKGROUND:Resistin is known as a potential mediator of obesity-associated insulin resistance. The high resistin level disrupts nitric oxide (NO)-mediated relaxation which is also important in erectile function. An antioxidant alkaloid, Boldine, is known as anti-diabetic and protects endothelial functions.OBJECTIVES:We aimed to investigate resistin expression in penile tissue in the presence of insulin resistance (IR) and the effect of Boldine treatment on erectile functions in the metabolic syndrome (MetS) rat model.MATERIALS AND METHODS:Wistar rats were randomly divided into three groups: Control, MetS, and boldine treated MetS group. MetS parameters were assessed by serum triglycerides (TG), uric acid (UA), glucose, insulin levels, HOMA index, and waist circumference (WC)/tibia length (TL) ratio. To evaluate erectile functions, intracavernous pressure (ICP)/mean arterial pressure (MAP) ratio was performed during cavernous nerve stimulation. Protein expressions of resistin, endothelial nitric oxide synthase (eNOS), p(S1177) eNOS, and insulin receptor-β were evaluated by Western blotting.RESULTS:TG, glucose, insulin levels, weight, WC/TL ratio, HOMA index and resistin expression in penile tissue were significantly increased and ICP/MAP values, and p (S1177) eNOS expression in penile tissue were decreased in MetS group. Boldine treatment enhanced ICP/MAP values, insulin receptor-β and p(S1177) eNOS expressions compared with the MetS group.DISCUSSION AND CONCLUSION:MetS caused a deterioration in erectile function accompanied by an increase in resistin expression and a reduction in eNOS enzyme activation in the rat penile tissues. Boldine treatment resulted in an improvement in erectile function, independent of resistin expression.