Fibrotic liver extracellular matrix induces cancerous phenotype in biomimetic micro-tissues of hepatocellular carcinoma model.

BACKGROUND: Despite considerable advancements in identifying factors contributing to the development of hepatocellular carcinoma (HCC), the pathogenesis of HCC remains unclear. In many cases, HCC is a consequence of prolonged liver fibrosis, resulting in the formation of an intricate premalignant microenvironment. The accumulation of extracellular matrix (ECM) is a hallmark of premalignant microenvironment. Given the critical role of different matrix components in regulating cell phenotype and function, this study aimed to elucidate the interplay between the fibrotic matrix and malignant features in HCC. METHODS: Liver tissues from both control (normal) and carbon tetrachloride (CCl4)-induced fibrotic rats were decellularized using sodium dodecyl sulfate (SDS) and Triton X-100. The resulting hydrogel from decellularized ECM was processed into micro-particles via the water-in-oil emulsion method. Micro-particles were subsequently incorporated into three-dimensional liver biomimetic micro-tissues (MTs) comprising Huh-7 cells, human umbilical vein endothelial cells (HUVECs), and LX-2 cells. The MTs were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay at day 11, immunofluorescence staining, immunoblotting, and spheroid migration assay at day 14 after co-culture. RESULTS: Fibrotic matrix from CCl4-treated rat livers significantly enhanced the growth rate of the MTs and their expression of CCND1 as compared to the normal one. Fibrotic matrix, also induced the expression of epithelial-to-mesenchymal transition (EMT)-associated genes such as TWIST1, ACTA2, MMP9, CDH2, and VIMENTIN in the MTs as compared to the normal matrix. Conversely, the expression of CDH1 and hepatic maturation genes HNF4A, ALB, CYP3A4 was decreased in the MTs when the fibrotic matrix was used. Furthermore, the fibrotic matrix increased the migration of the MTs and their secretion of alpha-fetoprotein. CONCLUSIONS: Our findings suggest a regulatory role for the fibrotic matrix in promoting cancerous phenotype, which could potentially accelerate the progression of malignancy in the liver.

MiR-29a-laden extracellular vesicles efficiently induced apoptosis through autophagy blockage in HCC cells.

BACKGROUND: In spite of significant advancements in theraputic modalities for hepatocellular carcinoma (HCC), there is still a high annual mortality rate with a rising incidence. Major challenges in the HCC clinical managment are related to the development of therapy resistance, and evasion of tumor cells apoptosis which leading unsatisfactory outcomes in HCC patients. Previous investigations have shown that autophagy plays crucial role in contributing to drug resistance development in HCC. Although, miR-29a is known to counteract authophagy, increasing evidence revealed a down-regulation of miR-29a in HCC patients which correlates with poor prognosis. Beside, evidences showed that miR-29a serves as a negative regulator of autophagy in other cancers. In the current study, we aim to investigate the impact of miR-29a on the autophagy and apoptosis in HCC cells using extracellular vesicles (EVs) as a natural delivery system given their potential in the miRNA delivery both in vitro and in vivo. METHOD: Human Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles were lately isolated through 20,000 or 110,000 × g centrifugation (EV20K or EV110K, respectively), characterized by western blot (WB), scanning electron microscopy (SEM), and dynamic light scattering (DLS). miR-29a was subsequently loaded into these EVs and its loading efficiency was evaluated via RT-qPCR. Comprehensive in vitro and in vivo assessments were then performed on Huh-7 and HepG2 cell lines. RESULTS: EV20K-miR-29a treatment significantly induces cell apoptosis and reduces both cell proliferation and colony formation in Huh-7 and HepG2 cell lines. In addition, LC3-II/LC3-I ratio was increased while the expression of key autophagy regulators TFEB and ATG9A were downregulated by this treatment. These findings suggest an effective blockade of autophagy by EV20K-miR-29a leading to apoptosis in the HCC cell lines through concomitant targeting of critical mediators within each pathway.

Circulating Tumor Cells as a Promising Tool for Early Detection of Hepatocellular Carcinoma.

Liver cancer is a significant contributor to the cancer burden, and its incidence rates have recently increased in almost all countries. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is the second leading cause of cancer-related deaths worldwide. Because of the late diagnosis and lack of efficient therapeutic modality for advanced stages of HCC, the death rate continues to increase by ~2-3% per year. Circulating tumor cells (CTCs) are promising tools for early diagnosis, precise prognosis, and follow-up of therapeutic responses. They can be considered to be an innovative biomarker for the early detection of tumors and targeted molecular therapy. In this review, we briefly discuss the novel materials and technologies applied for the practical isolation and detection of CTCs in HCC. Also, the clinical value of CTC detection in HCC is highlighted.

HNF4α is possibly the missing link between epithelial-mesenchymal transition and Warburg effect during hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a heterogeneous, late-diagnosed, and highly recurrent malignancy that often affects the whole body's metabolism. Finding certain theranostic molecules that can address current concerns simultaneously is one of the priorities in HCC management. In this study, performing protein-protein interaction network analysis proposed hepatocyte nuclear factor 4 alpha (HNF4α) as a hub protein, associating epithelial-mesenchymal transition (EMT) to reprogrammed cancer metabolism, formerly known as the Warburg effect. Both phenomena improved the compensation of cancerous cells in competitive conditions. Mounting evidence has demonstrated that HNF4α is commonly downregulated and serves as a tumor suppressor in the HCC. Enhancing the HNF4α mRNA translation through a specific synthetic antisense long non-coding RNA, profoundly affects both EMT and onco-metabolic modules in HCC cells. HNF4α overexpression decreased featured mesenchymal transcription factors and improved hepatocytic function, decelerated glycolysis, accelerated gluconeogenesis, and improved dysregulated cholesterol metabolism. Moreover, HNF4α overexpression inhibited the migration, invasion, and proliferation of HCC cells and decreased metastasis rate and tumor growth in xenografted nude mice. Our findings suggest a central regulatory role for HNF4α through its broad access to a wide variety of gene promoters involved in EMT and the Warburg effect in human hepatocytes. This essential impact indicates that HNF4α may be a potential target for HCC treatment.

Hepatogenesis and hepatocarcinogenesis: Alignment of the main signaling pathways.

Development is a symphony of cells differentiation in which different signaling pathways are orchestrated at specific times and periods to form mature and functional cells from undifferentiated cells. The similarity of the gene expression profile in malignant and undifferentiated cells is an interesting topic that has been proposed for many years and gave rise to the differentiation-therapy concept, which appears a rational insight and should be reconsidered. Hepatocellular carcinoma (HCC), as the sixth common cancer and the third leading cause of cancer death worldwide, is one of the health-threatening complications in communities where hepatotropic viruses are endemic. Sedentary lifestyle and high intake of calories are other risk factors. HCC is a complex condition in which various dimensions must be addressed, including heterogeneity of cells in the tumor mass, high invasiveness, and underlying diseases that limit the treatment options. Under these restrictions, recognizing, and targeting common signaling pathways during liver development and HCC could expedite to a rational therapeutic approach, reprograming malignant cells to well-differentiated ones in a functional state. Accordingly, in this review, we highlighted the commonalities of signaling pathways in hepatogenesis and hepatocarcinogenesis, and comprised an update on the current status of targeting these pathways in laboratory studies and clinical trials.