Hepatocellular Carcinoma Cell Tumor Research Articles

Research on the Construction of Bispecific-Targeted Sustained-Release Drug-Delivery Microspheres and Their Function in Treatment of Hepatocellular Carcinoma.

Lenvatinib (LEN) is approved as one of the commonly used drugs in the treatment of hepatocellular carcinoma (HCC). It is recognized to be a novel therapeutic choice for the direct and targeted delivery of effective drugs to HCC tumor sites. The key to the proposed method lies in the requirement for efficient targeted drug delivery carriers with targeting performance to deliver effective drugs directly and safely to tumor lesions. Methods: Here, magnetic liposomes (MLs) were modified by phosphatidylinositol proteoglycan 3 (GPC3) and epithelial cell adhesion molecules (EpCAMs). Subsequently, bispecific-targeted sustained-release drug-loaded microspheres containing LEN (GPC3/EpCAM-LEN-MLs) were constructed. In addition, both cytotoxicity and magnetic resonance imaging (MRI) analyses were performed to establish a mouse model and further perform corresponding performance assessments. Results: The corresponding results showed that GPC3/EpCAM-LEN-MLs were spherical-shaped and evenly dispersed. The encapsulation and drug-loading efficiencies were 91.08% ± 1.83% and 8.22% ± 1.24%, respectively. Meanwhile, GPC3/EpCAM-LEN-MLs showed a high inhibition rate on the proliferation of HCC cells and significantly increased their apoptosis. Furthermore, MRI revealed that the system possessed the function of tracking and localizing tumor cells, and animal experiments verified that it could exert the function of disease diagnosis. Conclusions: Our experiments successfully constructed a safe and efficient bispecific-targeted sustained-release drug delivery system for HCC tumor cells. It provides a useful diagnostic and therapeutic scheme for the clinical diagnosis and targeted therapy of HCC. Moreover, it can be used as a potential tumor-specific MRI contrast agent for the localization and diagnosis of malignant tumors.

Cytological Comparison between Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma by Image Analysis Software Using Touch Smear Samples of Surgically Resected Specimens.

Simple SummaryThis study cytologically compared hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) using image analyzing software. The results indicated that the major/minor axis ratio of ICC was significantly larger than in HCC in Papanicolaou staining. This difference was consistently observed in clinical samples of cytology such as fine-needle aspiration, brushing and ascites. This study indicated a significant difference in the nuclear morphology of HCC (round shape) and ICC (oval shape) in Papanicolaou-stained cytology specimens. This simple and objective finding is considered to be useful for differential cytodiagnosis of HCC and ICC.To investigate useful cytological features for differential diagnosis of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), this study cytologically compared HCC to ICC using image analysis software. Touch smear specimens of surgically resected specimens were obtained from a total of 61 nodules of HCC and 16 of ICC. The results indicated that the major/minor axis ratio of ICC is significantly larger than that of HCC (1.67 ± 0.27 vs. 1.32 ± 0.11, p < 0.0001) in Papanicolaou staining. This result means that the nucleus of HCC is close to round and the nucleus of ICC is close to an oval. This significant difference in the major/minor axis ratio between ICC and HCC was consistently observed by the same analyses using clinical samples of cytology (4 cases of HCC and 13 cases of ICC) such a fine-needle aspiration, brushing and ascites (ICC: 1.45 ± 0.13 vs. HCC: 1.18 ± 0.056, p = 0.004). We also confirmed that nuclear position center-positioned nucleus (p < 0.0001) and granular cytoplasm (p < 0.0001) are typical features of HCC tumor cells compared to ICC tumor cells. The research study found a significant difference in the nuclear morphology of HCC (round shape) and ICC (oval shape) in Papanicolaou-stained cytology specimens. This simple and objective finding will be very useful for the differential cytodiagnosis of HCC and ICC.

Novel low-avidity glypican-3 specific CARTs resist exhaustion and mediate durable antitumor effects against hepatocellular carcinoma

Abstract Background &amp; Aims Chimeric antigen receptor engineered T cells (CARTs) for hepatocellular carcinoma (HCC) and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAb) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects. Methods and results New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained 3 hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with ~17 fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects. In vitro, low-avidity 8F8 CARTs killed both hGPC3high and hGPC3low HCC tumor cells to the same extent as high-avidity GC33 CARTs. 8F8 CARTs expanded and persisted to a greater extent than GC33 CARTs, resulting in durable responses against HCC xenografts. Importantly, compared to GC33 CARTs, there were 5 folds more of 8F8-BBz CARTs in the tumor mass for a longer period of time. Remarkably, the tumor infiltrating 8F8 CARTs were less exhausted and apoptotic, and more functional than GC33 CARTs. Conclusion The novel low-avidity 8F8-BBz CART resists exhaustion and apoptosis inside tumor lesions, demonstrating a greater therapeutic potential than high-avidity CARTs.

An Alternatively Spliced Variant of METTL3 Mediates Tumor Suppression in Hepatocellular Carcinoma.

Many post-transcriptional mRNA processing steps play crucial roles in tumorigenesis and the progression of cancers, such as N6-methyladenosine (m6A) modification and alternative splicing. Upregulation of methyltransferase-like 3 (METTL3), the catalytic core of the m6A methyltransferase complex, increases m6A levels and results in significant effects on the progression of hepatocellular carcinoma (HCC). However, alternative splicing of METTL3 has not been fully investigated, and the functions of its splice variants remain unclear. Here, we analyzed both our and online transcriptomic data, obtaining 13 splice variants of METTL3 in addition to canonical full-length METTL3-A in HCC cell lines and tissues. Validated by RT–qPCR and Western blotting, we found that METTL3-D, one of the splice variants expressing a truncated METTL3 protein, exhibits higher levels than METTL3-A in normal human livers but lower levels than METTL3-A in HCC tumor tissues and cell lines. Further functional assays demonstrated that METTL3-D expression decreased cellular m6A modification, inhibited the proliferation, migration, and invasion of HCC cells, and was negatively associated with the malignancy of patient tumors, exhibiting functions opposite to those of full-length METTL3-A. This study demonstrates that the METTL3-D splice variant is a tumor suppressor that could potentially be used as a target for HCC therapy.

Role of DTL in Hepatocellular Carcinoma and Its Impact on the Tumor Microenvironment.

BackgroundThe crucial role of DTL has been previously implicated in genomic stability; however, its prognostic value and its relation with tumor immunity in hepatocellular carcinoma (HCC) remain to be further explored.MethodsTranscriptional and mutational datasets as well as clinical information were retrieved from the GEO, ICGC, and TCGA databases. Differentially expressed genes (DEGs) were obtained from the comparison of DTLhigh and DTLlow expression groups of the TCGA-HCC cohort. Those genes were under KEGG and gene ontology (GO) analyses to decipher the influence of the DTL gene on the biological behavior of HCC tumor cells. The survival status and mutational characteristics of patients according to DTL levels were depicted and analyzed. The DTL overexpression in HCC and its impact on prognosis were further confirmed by a cohort of 114 HCC patients (validation cohort). The TIMER, GEPIA, and TISIDB databases were adopted to investigate the potential relations between DTL levels and the status of immune cells, as well as immune cell infiltrations.ResultsThe DTL gene is overexpressed in tumor tissues compared with distant non-malignant liver tissues, and DTL overexpression in HCC would enhance the HCC cells in the activities of cell cycle and division. HCC patients with high DTL expression have unfavorable clinical outcomes and harbor more somatic mutations than those with low DTL expression, and multivariate analysis also revealed that DTL overexpression could act as an independent biomarker for prognosis. Moreover, the DTL gene was positively linked to marker sets of infiltrating activated CD8+ and CD4+ T cells; however, these cells demonstrated to be functionally exhausted.ConclusionsPatients with a DTL overexpression phenotype in HCC have poorer prognosis than those in the DTLlow group due to the role of the DTL gene in the process of pro-cell proliferation, accompanied by the immunosuppressive microenvironment and T cell exhaustion.

Anticancer effects of imidazole nucleus in hepatocellular carcinoma cell lines via the inhibition of AKT and ERK1/2 signaling pathways.

Imidazole nucleus has been used efficiently in the development of many drug molecules due to its therapeutic effects. Many derivatives of it have been produced particularly for use in cancer treatment. However, the anti-cancer effects of imidazole nucleus in liver cancer cells are as yet unclear. In this study, we aimed to investigate the anti-cancer effects of imidazole nucleus in hepatocellular carcinoma (HCC) cell lines. Anti-cancer effect of imidazole nucleus was investigated using cell viability assay, apoptosis analysis, cell migration analysis, cell morphology analysis, colony formation assay and 3D cell culture techniques in HuH-7 and Mahlavu cell lines. Also, effect of imidazole on AKT and ERK1/2 pathways were determined using by western blot analysis. Imidazole decreased cell viability in both HCC cell lines in a dose and time-dependent manner and also suppressed the colony forming ability of the cells (p < 0.05). Imidazole increased the cleaved caspase 3 protein levels and thus induced apoptosis (p < 0.05). Imidazole induced morphological alterations and autophagy by increasing intracellular vacuolization. Also, imidazole decreased the viability and dimensions of HCC cell tumor spheroids produced in 3D cell cultures (p < 0.05). Moreover, it was observed that all of these effects, are defined above, appeared in parallel with suppression of AKT and ERK1/2 signaling pathways by imidazole nucleus. The findings of this present study established the anti-cancer effects of imidazole nucleus in HCC cell lines and showed that it could be a potential molecule in the treatment of HCC via inhibition of AKT and ERK1/2 signaling pathways.

STAT1 and STAT3 Exhibit a Crosstalk and Are Associated with Increased Inflammation in Hepatocellular Carcinoma.

Simple SummaryLiver cancer is the fourth-leading cause of cancer-related mortality worldwide and lacks effective therapies. Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common types of liver cancer and both are associated with underlying inflammatory diseases. Thereby, interleukin-6 (IL-6)-mediated STAT3 signaling is critically involved in early carcinogenesis and disease progression. Here, we assessed the interplay between STAT1 and STAT3 in IL-6 signaling in vitro and studied the activation of STAT1 and STAT3 in a cohort of 124 HCC and a cohort of 138 CCA patients by immunohistochemistry. We found that IL-6 induced STAT1 transcriptional activity upon STAT3 depletion, suggesting that HCC tumor cells may activate both STAT1 and STAT3 signaling under pro-inflammatory conditions. Furthermore, HCC patient tissues showed a strong positive correlation of STAT1 and STAT3 activation in distinct patient groups. These patients also exhibited a high degree of immune cell infiltration, suggesting that these tumors are immune “hot”.Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Circulating Tumor Cells in Hepatocellular Carcinoma (HCC) and their Association with Early Recurrence after Surgery: A Preliminary Study

Introduction: HCC recurrence is observed in up to 60% of patients after curative resection. Circulating tumor cells (CTCs) have been recently advocated as a source of recurrence. The aim of this study is to determine the correlation between CTCs from peripheral blood samples and the risk of recurrence after resection. Methods: From 2019, 24 patients with HCC at first diagnosis, no other oncological history and BCLC stage 0-A-B were prospectively enrolled.They underwent liquid biopsies at day 0-30-90-180-365. Median follow-up was 365 days. EpCAM, N-cadherin and CD90 were selected as biomarkers to isolate CTCs. Epithelial-mesenchymal transition (EMT) was analyzed by an index estimated as ratio between the number of EpCAM+/N-cad- and EpCAM+/N-cad+ cells (EMT Index). Relapse occurred in 5 patients, then they were divided in 2 groups according to recurrence. Results: Expression of EpCAM+/Ncad+ (double+) and EpCAM+/Ncad+/CD90+ (triple+) cells was higher in the no-relapse group. EMT index was lower in the no-relapse group before surgery. Cut-offs were established by ROC for triple+ cells and for EMT Index. Higher number of triple+ cells cases did not reach the median recurrence free survival (RFS), while lower expression cases had median RFS of 185 days (p<0.0001). Patients with higher EMT Index before surgery had 1-year RFS of 50.8% vs 100% for lower index (p=0.0038). Conclusion: EMT transition of CTCs appears to be correlated with recurrence and our preliminary data showed that a lower expression of triple positive cells or an increased EMT index before surgery are associated with a higher rate of recurrence.

ITGB1 Drives Hepatocellular Carcinoma Progression by Modulating Cell Cycle Process Through PXN/YWHAZ/AKT Pathways.

Integrin β1 (ITGB1), which acts as an extracellular matrix (ECM) receptor, has gained increasing attention as a therapeutic target for the treatment of hepatocellular carcinoma (HCC). However, the underpinning mechanism of how ITGB1 drives HCC progression remains elusive. In this study, we first found that ITGB1 expression was significantly higher in HCC tissues than in normal controls by bioinformatics analysis. Furthermore, bioinformatics analysis revealed that paxillin (PXN) and 14-3-3 protein zeta (YWHAZ) are the molecules participating in ITGB1-regulated HCC tumor cell cycle progression. Indeed, immunohistochemistry (IHC) revealed that ITGB1, paxillin, and YWHAZ were strongly upregulated in paired HCC tissue compared with adjacent normal tissues. Notably, the inhibition of ITGB1 expression by small interfering RNA (siRNA) resulted in the downregulated expression of PXN and YWHAZ in primary HCC cells, as assessed by western blot and immunostaining. In addition, ITGB1 knockdown markedly impaired the aggressive behavior of HCC tumor cells and delayed cell cycle progression as determined by cell migration assay, drug-resistance analysis, colony formation assay, quantitative real-time polymerase chain reaction (qRT-PCR), and cell cycle analysis as well as cell viability measurements. More importantly, we proved that xenograft ITGB1high tumors grew more rapidly than ITGB1low tumors. Altogether, our study showed that the ITGB1/PXN/YWHAZ/protein kinase B (AKT) axis enhances HCC progression by accelerating the cell cycle process, which offers a promising approach to halt HCC tumor growth.

Circulating Tumor Cells in Hepatocellular Carcinoma: A Comprehensive Review and Critical Appraisal.

Hepatocellular carcinoma (HCC) is the fifth most common neoplasm and a major cause of cancer-related death worldwide. There is no ideal biomarker allowing early diagnosis of HCC and tumor surveillance in patients receiving therapy. Liquid biopsy, and particularly circulating tumor cells (CTCs), have emerged as a useful tool for diagnosis and monitoring therapeutic responses in different tumors. In the present manuscript, we evaluate the current evidence supporting the quantitative and qualitative assessment of CTCs as potential biomarkers of HCC, as well as technical aspects related to isolation, identification, and classification of CTCs. Although the dynamic assessment of CTCs in patients with HCC may aid the decision-making process, there are still many uncertainties and technical caveats to be solved before this methodology has a true impact on clinical practice guidelines. More studies are needed to identify the optimal combination of surface markers, to increase the efficiency of ex-vivo expansion of CTCs, or even to target CTCs as a potential therapeutic strategy to prevent HCC recurrence after surgery or to hamper tumor progression and extrahepatic spreading.

Inositol 1,4,5-trisphosphate receptor type 3 is involved in resistance to apoptosis and maintenance of human hepatocellular carcinoma.

The expression of the inositol 1,4,5-trisphosphate receptor type 3 (ITRP3) in hepatocytes is a common event in the pathogenesis of hepatocellular carcinoma (HCC), regardless of the type of underlying liver disease. However, it is not known whether ITPR3 expression in hepatocytes is involved in tumor maintenance. The aim of the present study was to determine whether there is an association between ITPR3 expression and clinical and morphological parameters using HCC samples obtained from liver explants from patients (n=53) with different etiologies of underlying chronic liver disease (CLD). ITPR3 expression, mitosis and apoptosis were analyzed in human liver samples by immunohistochemistry. Clinical and event-free survival data were combined to assess the relationship between ITPR3 and liver cancer growth in patients. RNA sequencing analysis was performed to identify apoptotic genes altered by ITPR3 expression in a liver tumor cell line. ITPR3 was highly expressed in HCC tumor cells relative to adjacent CLD tissue and healthy livers. There was an inverse correlation between ITPR3 expression and mitotic and apoptotic indices in HCC, suggesting that ITPR3 contributed to the maintenance of HCC by promoting resistance to apoptosis. This was confirmed by the upregulation of CTSB, CHOP and GADD45, genes involved in the apoptotic pathway in HCC. The expression of ITPR3 in the liver may be a promising prognostic marker of HCC

Research progress on circulating tumor cells of hepatocellular carcinoma

Circulating tumor cells (CTCs) are the cells released from the primary tumor and found in the peripheral blood, which can colonize and develop at a distance through blood circulation. At present, the commonly used separation and detection methods of CTCs are mainly divided into physical methods, biological methods, and microfluidic chip-based methods. Monitoring CTC count and cell phenotype is of great significance for early screening and diagnosis of hepatocellular carcinoma (HCC). Moreover, the CTC count and cell phenotype are related to assessing the clinical efficacy of the treatment of HCC and the clinical stage of HCC patients. The CTCs count is also closely related to the overall survival, progression-free survival, and postoperative recurrence of patients with HCC.

The lncRNA VPS9D1-AS1 Promotes Hepatocellular Carcinoma Cell Cycle Progression by Regulating the HuR/CDK4 Axis.

Long noncoding RNAs (lncRNAs) represent promising therapeutic targets associated with hepatocellular carcinoma (HCC). lncRNA VPS9D1 antisense RNA 1 (VPS9D1-AS1) regulates colon and prostate cancer, but its relevance in HCC remains to be clarified. Using microarray data from the NCBI Gene Expression Omnibus (GEO) database (GSE65485) and The Cancer Genome Atlas (TCGA) database, VPS9D1-AS1 expression in HCC and normal liver tissue sample HCC were compared. Relative lncRNA expression was also measured through real-time quantitative PCR (qPCR) in 80 pairs of HCC tumor and paracancerous tissues and in human HCC cell lines. VPS9D1-AS1 knockdown was achieved by transfecting these HCC cells with a specific siRNA construct in vitro, and the proliferation of these cells was quantified through cell proliferation assays and colony formation assays, while flow cytometry was employed to assess their cell cycle progression. The role of the VPS9D1-AS1 lncRNA as a regulator of HCC tumorigenesis was also assessed in vivo by subcutaneously implanting BALB/c nude mice with HepG2 cells stably expressing either sh-VPS9D1-AS1 or a control shRNA construct. Mechanistic analyses were additionally conducted by examining in vitro CDK4 and HuR expression through western blotting and qPCR. VPS9D1-AS1 expression was significantly increased in HCC tissues in the analyzed databases and our independent tissue samples. Elevated VPS9D1-AS1 expression was related to larger tumor size and more advanced tumor, node, metastasis (TNM) stage, and HCC patients expressing higher levels of this lncRNA exhibited poorer survival outcomes. Knocking down VPS9D1-AS1 impaired the proliferative and colony formation activity of HepG2 cells while promoting their apoptotic death. Consistently, VPS9D1-AS1 silencing suppressed HCC tumor growth in vivo. Mechanistically, VPS9D1-AS1 was able to bind to the HuR protein and thereby influence the stability and expression of the CDK4 mRNA, thus impacting HCC cell proliferation. The VPS9D1-AS1/HuR/CDK4 signaling axis regulates HCC tumor cell oncogenic activity, highlighting this pathway as a promising therapeutic target.

Lipid response of hepatocellular carcinoma cells to anticancer drug detected on nanostructure-assisted LDI-MS platform

High heterogeneity of hepatocellular carcinoma (HCC) tumor has become an obstacle to select effective therapy for the treatment of HCC patients. Methods that can guide the decision on therapy choice for HCC treatment are highly demanded. Evaluating the drug response of heterogeneous tumor cells at the molecular level can help to reveal the toxicity mechanism of anticancer drugs and provide more information than current cell-based chemosensitivity assays. In the present work, nanostructure-assisted laser desorption/ionization mass spectrometry (NALDI-MS) was used to investigate the lipid response of HCC cells to anticancer drugs. Three types of HCC cells (LM3, Hep G2, Huh7) were treated with sorafenib, doxorubicin hydro-chloride, and cisplatin. We found that the lipid profiles of HCC cells changed a lot after the drug treatment, and the degree of lipid changes was related to the cell viability. Two pairs of fatty acids C16:1/C16:0 and C18:1/C18:0 were found to be strongly related to the viability of HCC cells after drug treatment, and were more sensitive than Methyl-thiazolyl tetrazolium (MTT) assay. Accordingly, they can act as sensitive and comprehensive indexes to evaluate the drug susceptibility of HCC cells. In addition, the peak ratio of several neighboring phospholipids displayed high correlation with drug response of specific cell subtype to specific drug. The ratio of neighboring lipids may be traced back to the activity of enzyme and gene expression which regulate the lipidomic pathway. This method provides drug response of heterogenous tumor cells at molecular level and could be a potential candidate to precise tumor chemosensitivity assay.

GINS1 Induced Sorafenib Resistance by Promoting Cancer Stem Properties in Human Hepatocellular Cancer Cells.

Hepatocellular carcinoma (HCC) is characterized by a high rate of incidence and recurrence, and resistance to chemotherapy may aggravate the poor prognosis of HCC patients. Sorafenib resistance is a conundrum to the treatment of advanced/recurrent HCC. Therefore, studies on the molecular pathogenesis of HCC and the resistance to sorafenib are of great interest. Here, we report that GINS1 was highly expressed in HCC tumors, associated with tumor grades, and predicted poor patient survival using Gene Expression Omnibus (GEO) databases exploration. Cell cycle, cell proliferation assay and in vivo xenograft mouse model indicated that knocking down GINS1 induced in G1/S phase cell cycle arrest and decreased tumor cells proliferation in vitro and in vivo. Spheroid formation assay results showed that GINS1 promoted the stem cell activity of HCC tumor cells. Furthermore, GEO database (GSE17112) analysis showed that HRAS oncogenic gene set was enriched in GINS1 high-expressed cancer cells, and quantitative real-time PCR, and Western blot results proved that GINS1 enhanced HCC progression through regulating HRAS signaling pathway. Moreover, knocking down endogenous GINS1 with shGINS1 increased the sensitivity of HCC cells to sorafenib, and restoring HRAS or stem associated pathway partly recovered the sorafenib resistance. Overall, the collective findings highlight GINS1 functions in hepatocarcinogenesis and sorafenib resistance, and indicate its potential use of GINS1 in drug-resistant HCC.

CK-3, A Novel Methsulfonyl Pyridine Derivative, Suppresses Hepatocellular Carcinoma Proliferation and Invasion by Blocking the PI3K/AKT/mTOR and MAPK/ERK Pathways.

Hepatocellular carcinoma (HCC) is an aggressive tumor with a poor prognosis that highly expresses phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (ERK). The PI3K/AKT/mTOR and MAPK/ERK signaling pathways play a crucial role in HCC tumor formation, cell cycle, apoptosis and survival. However, no effective targeted therapies against these pathways is available, mainly due to the extensive and complex negative feedback loops between them. Here we used CK-3, a dual blocker of the PI3K/AKT/mTOR and MAPK/ERK pathways, against HCC cell lines to verify its anti-tumor activity in vitro. CK-3 exhibited cytotoxic activity against HCC, as demonstrated with MTT and colony formation assays. The anti-metastatic potential of CK-3 was demonstrated with wound healing and cell invasion assays. The ability of CK-3 to block both the PI3K/AKT/mTOR and MAPK/ERK pathways was also confirmed. CK-3 induced the apoptosis of Hep3B cells, while Bel7402 cells died via mitotic catastrophe (MC). Oral administration of CK-3 also inhibited the subcutaneous growth of BEL7402 cells in nude mice. Simultaneous PI3K/AKT/mTOR and MAPK/ERK pathway inhibition with CK-3 may be superior to single pathway monotherapies by inhibiting their feedback-regulation, and represents a potential treatment for HCC.

MiRNA and long non-coding RNA transcriptional expression in hepatocellular carcinoma cell line-secreted extracellular vesicles.

Extracellular vesicles (EVs) are membrane-released vesicles acting as transporters of proteins, lipids and short/long non-coding RNA (miRNAs and lncRNAs). They are released by normal and pathological cells, including hepatocellular carcinoma (HCC). To date, studies focused on miRNAs and lncRNAs contained in EVs derived from HCC are limited. Our aim was to analyze the transcriptional profile of potential regulating miRNAs and lncRNAs in EVs secreted by HCC tumor cell line (HepG2, n = 6), and from a non-tumorigenic hepatocyte cell line (WRL68, n = 6), to compare their differential expression profile and to identify novel molecular diagnostic markers of HCC. EVs were isolated from the conditioned medium, through differential centrifugations. The expression profile of miRNAs (miR-23a, miR-16-2, miR-181a, miR-373, miR-205, miR-27a, miR-1323, and miR-532) and lncRNAs (HULC, HOTAIR, XIST, MALAT-1, GAS-5, H19) was performed in Real-time PCR, and their transcript was found both in HepG2 and WRL68 EVs. Lower miR-181a, miR-205 and miR-1323 expression were detected in EVs secreted by HepG2 compared to WRL68, while an opposite trend was observed for miR-23a, miR-16-2, miR-373, miR-27a, and miR-532. Several significant correlations were found between miRNA and lncRNA. An in silico analysis was also performed. The results obtained could identified them as new potential diagnostic and prognostic biomarkers of HCC.

Integrated bioinformatics analysis revealed the regulation of angiogenesis by tumor cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, metastasis accounts for most of the cases. Angiogenesis plays an important role in cancer metastasis, but how tumor cells affect the function of endothelial cells by dictating their microRNA (miRNA) expression remains largely unknown.Differentially expressed miRNAs (DEMs) were identified through dataset downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. We then used online tools to obtain potential targets of candidate miRNAs and functional enrichment analysis, as well as the protein-protein interaction (PPI). Finally, the function of miR-302c-3p was validated through in vitro assay.In the current study, we found that HCC cells altered miRNA expression profiles of human umbilical vein endothelial cells (HUVECs) and miR-302c-3p was the most down-regulated miRNA in HUVECs when they were co-cultured with HCC-LM3 cells. Functional enrichment analysis of the candidate targets revealed that these genes were involved in epigenetic regulation of gene expression, in particular, cytosine methylation. In addition, PPI network demonstrated distinct roles of genes targeted by miR-302c-3p. Importantly, inhibition of angiogenesis, migration and permeability by the most down-regulated miR-302c-3p in HUVECs was confirmed in vitro. These findings brought us novel insight into the regulation of angiogenesis by HCC cells and provided potential targets for the development of therapeutic strategies.

Correlation Between Circulating Tumor Cell DNA Genomic Alterations and Mesenchymal CTCs or CTC-Associated White Blood Cell Clusters in Hepatocellular Carcinoma.

PurposeLiquid biopsy is attracting attention as a method of real-time monitoring of patients with tumors. It can be used to understand the temporal and spatial heterogeneity of tumors and has good clinical application prospects. We explored a new type of circulating tumor cell (CTC) enrichment technology combined with next-generation sequencing (NGS) to analyze the correlation between genomic alterations in circulating tumor cells of hepatocellular carcinoma and the counts of mesenchymal CTCs and CTC-associated white blood cell (CTC-WBC) clusters.MethodsWe collected peripheral blood samples from 29 patients with hepatocellular carcinoma from January 2016 to December 2019. We then used the CanPatrol™ system to capture and analyze mesenchymal CTCs and CTC-WBC clusters for all the patients. A customized Illumina panel was used for DNA sequencing and the Mann–Whitney U test was used to test the correlation between mesenchymal CTCs, CTC-WBC cluster counts, and specific genomic changes.ResultsAt least one somatic hotspot mutation was detected in each of the 29 sequenced patients. A total of 42 somatic hot spot mutations were detected in tumor tissue DNA, and 39 mutations were detected in CTC-DNA, all of which included common changes in PTEN, MET, EGFR, RET, and FGFR3. The number of mesenchymal CTCs was positively correlated with the somatic genomic alterations in the PTEN and MET genes (PTEN, P = 0.021; MET, P = 0.008, Mann–Whitney U test) and negatively correlated with the somatic genomic alterations in the EGFR gene (P = 0.006, Mann–Whitney U test). The number of CTC-WBC clusters was positively correlated with the somatic genomic alterations in RET genes (P = 0.01, Mann–Whitney U test) and negatively correlated with the somatic genomic alterations in FGFR3 (P = 0.039, Mann–Whitney U test).ConclusionsWe report a novel method of a CTC enrichment platform combined with NGS technology to analyze genetic variation, which further demonstrates the potential clinical application of this method for spatiotemporal heterogeneity monitoring of hepatocellular carcinoma. We found that the number of peripheral blood mesenchymal CTCs and CTC-WBC clusters in patients with hepatocellular carcinoma was related to a specific genome profile.

Replication Factor C4 in human hepatocellular carcinoma: A potent prognostic factor associated with cell proliferation.

Replication Factor c4 (RFC4) has been found to play important roles in many carcinomas and is correlated with poor prognosis. The present study was performed to investigate the specific role of RFC4 in hepatocellular carcinoma (HCC) and the underlying molecular mechanism. Public datasets including TCGA and GTEx were applied to explore the expression of RFC4 in HCC and its association with HCC prognosis. The results of bioinformatics analysis showed that RFC4 was overexpressed in HCC tissues compared with noncancerous tissues and significantly correlated with poor prognosis for HCC. Through immunohistochemistry, the association between RFC4 expression and clinical-pathological features of HCC patients was evaluated. Western blots were applied to investigate relative protein expression. Then in vivo and in vitro experiments were performed to explore the function of RFC4 in HCC tumor cells. The present results suggest that high level expression of RFC4 is associated with tumor size. In addition, RFC4 knockdown suppressed the cell proliferation and sphere formation of hepatoma cells in vitro. Moreover, silencing of RFC4 significantly decreased the growth of tumors in a xenograft tumor model. In conclusion, our study indicates that RFC4 is a potential prognostic predictor associated with poor outcomes for HCC patients. Furthermore, knocking down RFC4 could significantly inhibit tumor progression both in vitro and in vivo. Therefore, the present study can shed new light on the understanding of molecular mechanisms of HCC and may provide molecular targets and diagnostic biomarkers for the treatment of HCC.