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1.
Int J Cancer ; 155(3): 582-594, 2024 Aug 01.
Article in English | MEDLINE | ID: mdl-38380807

ABSTRACT

The proto-oncogene MYCN expression marked a cancer stem-like cell population in hepatocellular carcinoma (HCC) and served as a therapeutic target of acyclic retinoid (ACR), an orally administered vitamin A derivative that has demonstrated promising efficacy and safety in reducing HCC recurrence. This study investigated the role of MYCN as a predictive biomarker for therapeutic response to ACR and prognosis of HCC. MYCN gene expression in HCC was analyzed in the Cancer Genome Atlas and a Taiwanese cohort (N = 118). Serum MYCN protein levels were assessed in healthy controls (N = 15), patients with HCC (N = 116), pre- and post-surgical patients with HCC (N = 20), and a subset of patients from a phase 3 clinical trial of ACR (N = 68, NCT01640808). The results showed increased MYCN gene expression in HCC tumors, which positively correlated with HCC recurrence in non-cirrhotic or single-tumor patients. Serum MYCN protein levels were higher in patients with HCC, decreased after surgical resection of HCC, and were associated with liver functional reserve and fibrosis markers, as well as long-term HCC prognosis (>4 years). Subgroup analysis of a phase 3 clinical trial of ACR identified serum MYCN as the risk factor most strongly associated with HCC recurrence. Patients with HCC with higher serum MYCN levels after a 4-week treatment of ACR exhibited a significantly higher risk of recurrence (hazard ratio 3.27; p = .022). In conclusion, serum MYCN holds promise for biomarker-based precision medicine for the prevention of HCC, long-term prognosis of early-stage HCC, and identification of high-response subgroups for ACR-based treatment.


Subject(s)
Biomarkers, Tumor , Carcinoma, Hepatocellular , Liver Neoplasms , N-Myc Proto-Oncogene Protein , Neoplasm Recurrence, Local , Proto-Oncogene Mas , Adult , Aged , Female , Humans , Male , Middle Aged , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/blood , Liver Neoplasms/genetics , Liver Neoplasms/drug therapy , Liver Neoplasms/prevention & control , Liver Neoplasms/pathology , N-Myc Proto-Oncogene Protein/genetics , Neoplasm Recurrence, Local/prevention & control , Neoplasm Recurrence, Local/blood , Prognosis
2.
Stem Cells ; 38(4): 504-515, 2020 04.
Article in English | MEDLINE | ID: mdl-31828873

ABSTRACT

The role of microRNAs (miRNAs) during mouse early development, especially in endoderm germ layer formation, is largely unknown. Here, via miRNA profiling during endoderm differentiation, we discovered that miR-124a negatively regulates endoderm lineage commitment in mouse embryonic stem cells (mESCs). To further investigate the functional role of miR-124a in early stages of differentiation, transfection of embryoid bodies with miR-124a mimic was performed. We showed that overexpression of miR-124a inhibits endoderm differentiation in vitro through targeting the 3'-untranslated region (UTR) of Sox17 and Gata6, revealing the existence of interplay between miR-124a and the Sox17/Gata6 transcription factors in hepato-specific gene regulation. In addition, we presented a feasible in vivo system that utilizes teratoma and gene expression profiling from microarray to quantitatively evaluate the functional role of miRNA in lineage specification. We demonstrated that ectopic expression of miR-124a in teratomas by intratumor delivery of miR-124a mimic and Atelocollagen, significantly suppressed endoderm and mesoderm lineage differentiation while augmenting the differentiation into ectoderm lineage. Collectively, our findings suggest that miR-124a plays a significant role in mESCs lineage commitment.


Subject(s)
GATA6 Transcription Factor/metabolism , MicroRNAs/metabolism , Mouse Embryonic Stem Cells/metabolism , SOXF Transcription Factors/metabolism , Animals , Cell Lineage , Endoderm , Mice , Transfection
3.
Int J Mol Sci ; 22(21)2021 Oct 28.
Article in English | MEDLINE | ID: mdl-34769072

ABSTRACT

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Interferon-alpha/pharmacology , RNA, Viral/metabolism , SARS-CoV-2/genetics , Cell Line, Tumor , Dose-Response Relationship, Drug , Exoribonucleases/genetics , Genetic Vectors , HeLa Cells , Humans , Interferon-alpha/administration & dosage , Luciferases/genetics , Luciferases/metabolism , Naphthyridines/administration & dosage , Naphthyridines/pharmacology , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , RNA, Viral/drug effects , Replicon
4.
Cancer Sci ; 111(2): 395-405, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31854049

ABSTRACT

The onset of breast cancer among young patients is a major issue in cancer etiology. Our previous study has shown that poor prognosis in young women with breast cancer is associated with lower expression of the microRNA miR-1285-5p. In this study, we showed that the expression of miR-1285-5p is lower in tumor tissues than in normal tissues. Accumulating evidence suggests that miR-1285-5p plays critical roles in various types of cancers. However, the functional role of miR-1285-5p in breast cancer remains to be elucidated. Here, we showed the tumor-suppressive role of miR-1285-5p and detailed its mechanism of action in breast cancer. Overexpression of miR-1285-5p significantly inhibited cell proliferation in breast cancer cells regardless of the tumor subtype. Among the target genes of miR-1285-5p, we found that transmembrane protein 194A (TMEM194A) was directly regulated by miR-1285-5p. Notably, separation of centrosomes from the nuclear envelope was observed upon knockdown of TMEM194A or overexpression of miR-1285-5p. In conclusion, our findings show that miR-1285-5p is a tumor suppressor via TMEM194A inhibition in breast cancer.


Subject(s)
Breast Neoplasms/genetics , MicroRNAs/genetics , Nuclear Proteins/genetics , ran GTP-Binding Protein/genetics , 3' Untranslated Regions , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Centrosome/metabolism , Down-Regulation , Female , Gene Expression Regulation, Neoplastic , Humans , MCF-7 Cells , Nuclear Proteins/metabolism , ran GTP-Binding Protein/metabolism
5.
Cancer Sci ; 111(3): 869-880, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31883160

ABSTRACT

Primary hepatic tumors mainly include hepatocellular carcinoma (HCC), which is one of the most frequent causes of cancer-related deaths worldwide. Thus far, HCC prognosis has remained extremely poor given the lack of effective treatments. Numerous studies have described the roles played by microRNAs (miRNAs) in cancer progression and the potential of these small noncoding RNAs for diagnostic or therapeutic applications. The current consensus supports the idea that direct repression of a wide range of oncogenes by a single key miRNA could critically affect the malignant properties of cancer cells in a synergistic manner. In this study, we aimed to investigate the oncogenes controlled by miR-493-5p, a major tumor suppressor miRNA that inactivates miR-483-3p oncomir in hepatic cancer cells. Using global gene expression analysis, we highlighted a set of candidate genes potentially regulated by miR-493-5p. In particular, the canonical MYCN protooncogene (MYCN) appeared to be an attractive target of miR-493-5p given its significant inhibition through 3'-UTR targeting in miR-493-5p-rescued HCC cells. We showed that MYCN was overexpressed in liver cancer cell lines and clinical samples from HCC patients. Notably, MYCN expression levels were inversely correlated with miR-493-5p in tumor tissues. We confirmed that MYCN knockdown mimicked the anticancer effect of miR-493-5p by inhibiting HCC cell growth and invasion, whereas MYCN rescue hindered miR-493-5p activity. In summary, miR-493-5p is a pivotal miRNA that modulates various oncogenes after its reexpression in liver cancer cells, suggesting that tumor suppressor miRNAs with a large spectrum of action could provide valuable tools for miRNA replacement therapies.


Subject(s)
Carcinoma, Hepatocellular/genetics , Cell Proliferation/genetics , Liver Neoplasms/genetics , N-Myc Proto-Oncogene Protein/genetics , Oncogenes/genetics , 3' Untranslated Regions/genetics , Aged , Aged, 80 and over , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Genes, Tumor Suppressor/physiology , Hep G2 Cells , Humans , Liver Neoplasms/pathology , Male , MicroRNAs , Middle Aged , Prognosis , Proto-Oncogenes/genetics
6.
Mol Ther ; 26(7): 1840-1854, 2018 07 05.
Article in English | MEDLINE | ID: mdl-29759938

ABSTRACT

Primary liver tumors are mainly represented by hepatocellular carcinoma (HCC), one of the most aggressive and resistant forms of cancer. Liver tumorigenesis is characterized by an accumulation of epigenetic abnormalities, leading to gene extinction and loss of hepatocyte differentiation. The aim of this work was to investigate the feasibility of converting liver cancer cells toward a less aggressive and differentiated phenotype using a process called epigenetic reconditioning. Here, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine (5-AZA) promoted an anti-cancer response by inhibiting HCC cell tumorigenicity. Furthermore, epigenetic reconditioning improved sorafenib response. Remarkably, epigenetic treatment was associated with a significant restoration of differentiation, as attested by the increased expression of characteristic hepatocyte markers in reconditioned cells. In particular, we showed that reexpression of these epigenetically silenced liver genes following 5-AZA treatment or after knockdown of DNA methyltransferase 1 (DNMT1) was the result of regional CpG demethylation. Lastly, we confirmed the efficacy of HCC differentiation therapy by epigenetic reconditioning using an in vivo tumor growth model. In summary, this work demonstrates that epigenetic reconditioning using the demethylating compound 5-AZA shows therapeutic significance for liver cancer and is potentially attractive for the treatment of solid tumors.


Subject(s)
Carcinogenesis/genetics , Cell Differentiation/genetics , Epigenesis, Genetic/genetics , Liver Neoplasms/genetics , Animals , Azacitidine/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/pathology , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Differentiation/drug effects , Cell Line, Tumor , DNA Methylation/drug effects , DNA Methylation/genetics , Epigenesis, Genetic/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/pathology , Humans , Liver/drug effects , Liver/pathology , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Mice , Mice, Nude
7.
Int Immunol ; 29(1): 11-19, 2017 01 01.
Article in English | MEDLINE | ID: mdl-28184439

ABSTRACT

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular plaques resulting from the accumulation of beta-amyloid peptide (Aß). To date, a definitive cure for this disease is still lacking as the currently approved drugs used are mainly symptomatic treatments. The revolutionary discovery of extracellular vesicles (EVs) has shed new light on the development of disease-modifying treatments for AD, owing to their potential in delivering the therapeutic agents to the brain. The feasibility of harnessing EVs for clinical applications is highly dependent on the donor cell, which determines the intrinsic properties of EVs. The merit of mesenchymal stem cells (MSCs) as therapeutic delivery vehicles, and the proven therapeutic effects of the EVs derived from these cells, make researchers esteem MSCs as ideal producers of EVs. Therefore, MSC-derived EVs (MSC-EVs) emerge to be an appealing therapeutic delivery approach for the treatment of AD. Here, we discuss perspectives on the therapeutic strategies using MSC-EVs to treat AD and the associated challenges in clinical application.


Subject(s)
Alzheimer Disease/therapy , Cell-Derived Microparticles/transplantation , Mesenchymal Stem Cells , Alzheimer Disease/immunology , Animals , Humans
8.
J Cell Physiol ; 229(7): 903-15, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24501087

ABSTRACT

The extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway has been involved in the positive and negative regulation of cell proliferation. Upon mitogen stimulation, ERK1/ERK2 activation is necessary for G1- to S-phase progression whereas when hyperactived, this pathway could elicit cell cycle arrest. The mechanisms involved are not fully elucidated but a kinase-independent function of ERK1/2 has been evidenced in the MAPK-induced growth arrest. Here, we show that p70S6K, a central regulator of protein biosynthesis, is essential for the cell cycle arrest induced by overactivation of ERK1/2. Indeed, whereas MEK1 silencing inhibits cell cycle progression, we demonstrate that active mutant form of MEK1 or MEK2 triggers a G1 phase arrest by stimulating an activation of p70S6K by ERK1/2 kinases. Silencing of ERK1/2 activity by shRNA efficiently suppresses p70S6K phosphorylation on Thr421/Ser424 and S6 phosphorylation on Ser240/244 as well as p21 expression, but these effects can be partially reversed by the expression of kinase-dead mutant form of ERK1 or ERK2. In addition, we demonstrate that the kinase p70S6K modulates neither the p21 gene transcription nor the stability of the protein but enhances the translation of the p21 mRNA. In conclusion, our data emphasizes the importance of the translational regulation of p21 by the MEK1/2-ERK1/2-p70S6K pathway to negatively control the cell cycle progression.


Subject(s)
MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 2/genetics , MAP Kinase Signaling System/genetics , Ribosomal Protein S6 Kinases, 70-kDa/genetics , Cell Cycle Checkpoints/genetics , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Cyclin-Dependent Kinase Inhibitor p21/genetics , G1 Phase Cell Cycle Checkpoints/genetics , Hep G2 Cells , Humans , MAP Kinase Kinase 1/biosynthesis , MAP Kinase Kinase 2/biosynthesis , Phosphorylation , Protein Biosynthesis , RNA, Small Interfering , Ribosomal Protein S6 Kinases, 70-kDa/biosynthesis , Signal Transduction
9.
Hepatology ; 58(3): 1153-65, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23532995

ABSTRACT

UNLABELLED: MicroRNAs (miRNAs) are evolutionary conserved small RNAs that post-transcriptionally regulate the expression of target genes. To date, the role of miRNAs in liver development is not fully understood. By using an experimental model that allows the induced and controlled differentiation of mouse fetal hepatoblasts (MFHs) into mature hepatocytes, we identified miR-148a as a hepatospecific miRNA highly expressed in adult liver. The main finding of this study revealed that miR-148a was critical for hepatic differentiation through the direct targeting of DNA methyltransferase (DNMT) 1, a major enzyme responsible for epigenetic silencing, thereby allowing the promotion of the "adult liver" phenotype. It was also confirmed that the reduction of DNMT1 by RNA interference significantly promoted the expression of the major hepatic biomarkers. In addition to the essential role of miR-148a in hepatocyte maturation, we identified its beneficial effect through the repression of hepatocellular carcinoma (HCC) cell malignancy. miR-148a expression was frequently down-regulated in biopsies of HCC patients as well as in mouse and human HCC cell lines. Overexpressing miR-148a led to an enhancement of albumin production and a drastic inhibition of the invasive properties of HCC cells, whereas miR-148a silencing had the opposite consequences. Finally, we showed that miR-148a exerted its tumor-suppressive effect by regulating the c-Met oncogene, regardless of the DNMT1 expression level. CONCLUSION: miR-148a is essential for the physiology of the liver because it promotes the hepatospecific phenotype and acts as a tumor suppressor. Most important, this report is the first to demonstrate a functional role for a specific miRNA in liver development through regulation of the DNMT1 enzyme.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cell Transformation, Neoplastic/pathology , Hepatocytes/pathology , Liver Neoplasms/pathology , MicroRNAs/physiology , Phenotype , Albumins/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Cell Differentiation , Cell Line, Tumor , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/metabolism , Disease Models, Animal , Down-Regulation/physiology , Humans , Liver Neoplasms/metabolism , Mice , Neoplasm Invasiveness/pathology
10.
Cell Death Dis ; 14(6): 358, 2023 06 13.
Article in English | MEDLINE | ID: mdl-37308486

ABSTRACT

Transglutaminase 2 (TG2) is a multifunctional protein that promotes or suppresses tumorigenesis, depending on intracellular location and conformational structure. Acyclic retinoid (ACR) is an orally administered vitamin A derivative that prevents hepatocellular carcinoma (HCC) recurrence by targeting liver cancer stem cells (CSCs). In this study, we examined the subcellular location-dependent effects of ACR on TG2 activity at a structural level and characterized the functional role of TG2 and its downstream molecular mechanism in the selective depletion of liver CSCs. A binding assay with high-performance magnetic nanobeads and structural dynamic analysis with native gel electrophoresis and size-exclusion chromatography-coupled multi-angle light scattering or small-angle X-ray scattering showed that ACR binds directly to TG2, induces oligomer formation of TG2, and inhibits the transamidase activity of cytoplasmic TG2 in HCC cells. The loss-of-function of TG2 suppressed the expression of stemness-related genes, spheroid proliferation and selectively induced cell death in an EpCAM+ liver CSC subpopulation in HCC cells. Proteome analysis revealed that TG2 inhibition suppressed the gene and protein expression of exostosin glycosyltransferase 1 (EXT1) and heparan sulfate biosynthesis in HCC cells. In contrast, high levels of ACR increased intracellular Ca2+ concentrations along with an increase in apoptotic cells, which probably contributed to the enhanced transamidase activity of nuclear TG2. This study demonstrates that ACR could act as a novel TG2 inhibitor; TG2-mediated EXT1 signaling is a promising therapeutic target in the prevention of HCC by disrupting liver CSCs.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Protein Glutamine gamma Glutamyltransferase 2 , Neoplastic Stem Cells , Glycosyltransferases
11.
J Cell Physiol ; 227(1): 59-69, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21437905

ABSTRACT

Recent reports suggest that extracellular signal-regulated kinase (ERK1) and ERK2 mitogen-activated protein kinases (MAPK) may direct specific biological functions under certain contexts. In this study, we investigated the role of early and sustained epidermal growth factor (EGF) stimulation on long-term hepatocyte differentiation and the possible role of ERK1 and ERK2 in this process. We demonstrate a long-term survival and an elevated level of differentiation up to 3 weeks. The differentiation state of hepatocytes is supported by sustained expression of aldolase B, albumin, and the detoxifying enzymes CYP1A2, 2B2, and 3A23. Similarly to freshly isolated cells, cultured hepatocytes also retain the ability to respond to 3-methylcholanthrene (3MC) and phenobarbital (PB), two known CYP inducers. In addition, we show evidence that continuous MAPK/ERK kinase (MEK) inhibition enhances the level of differentiation. Using RNA interference approaches against ERK1 and ERK2, we demonstrate that this effect requires both ERK1 and ERK2 activity, whereas the specific ERK1 knockdown promotes cell survival and the specific ERK2 knockdown regulates cell proliferation. In conclusion, we demonstrate that early and sustained EGF stimulation greatly extends long-term hepatocyte survival and differentiation, and that inhibition of the ERK1/2 MAPK pathway potentiates these pro-survival/pro-differentiation phenotypes. We clearly attest that specific ERK1 and ERK2 MAPKs determine hepatocyte survival and proliferation, respectively, whereas dual inhibition is required to stabilize a highly differentiated state.


Subject(s)
Cell Differentiation/physiology , Hepatocytes/cytology , MAP Kinase Signaling System/physiology , Animals , Cell Survival/physiology , Cells, Cultured , Epidermal Growth Factor/metabolism , Hepatocytes/physiology , Immunoblotting , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , RNA, Small Interfering , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Transfection
12.
Cell Death Discov ; 7(1): 130, 2021 Jun 02.
Article in English | MEDLINE | ID: mdl-34078875

ABSTRACT

Chronic hepatitis B virus (HBV) infections remain a health burden affecting ~250 million people worldwide. Thus far, available interferon-alpha (IFNα)-based therapies have shown unsatisfactory cure rates, and alternative therapeutic molecules are still required. However, their development has been hampered because accessible cell models supporting relevant HBV replication and appropriate antiviral activity are lacking. Strategies that reverse epigenetic alterations offer a unique opportunity for cell reprogramming, which is valuable for restoring altered cellular functions in human cell lines. This work aimed to investigate the feasibility of converting HepG2 cells that stably overexpress the HBV entry receptor (sodium/taurocholate cotransporting polypeptide, NTCP) toward IFNα-responsive cells using epigenetic reprogramming. Herein, we showed that an epigenetic regimen with non-cytotoxic doses of the demethylating compound 5-azacytidine restored the anti-HBV action of IFNα in epigenetically reprogrammed HepG2-NTCP-C4 cells, named REP-HepG2-NTCP cells. Thus, a significant inhibition in HBV DNA levels was measured in REP-HepG2-NTCP cells after IFNα treatment. This inhibitory effect was associated with the enhancement of IFNα-mediated induction of critical interferon-stimulated genes (ISGs), which was limited in non-reprogrammed cells. In particular, our data indicated that re-expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) and interferon regulatory factor 9 (IRF9) was the result of an epigenetically driven unmasking of these genes in reprogrammed cells. At last, we evaluated the therapeutic potential of the IFN analog CDM-3008 in REP-HepG2-NTCP cells and demonstrated the efficiency of this chemical compound in triggering ISG induction and HBV inhibition. In summary, this study shows that epigenetic reprogramming promotes the IFNα response in HBV-infected cells and is potentially attractive for cell-based experimental screening of IFN-like compounds.

13.
Int J Cancer ; 126(6): 1367-77, 2010 Mar 15.
Article in English | MEDLINE | ID: mdl-19816936

ABSTRACT

The mitogen-activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock-down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.


Subject(s)
Carcinoma, Hepatocellular/prevention & control , Liver Neoplasms, Experimental/prevention & control , MAP Kinase Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , RNA Interference , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation , Enzyme Activation/genetics , Female , Humans , Immunoblotting , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/pathology , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 2/genetics , MAP Kinase Kinase 2/metabolism , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Phosphorylation , Signal Transduction , Xenograft Model Antitumor Assays
14.
J Hepatol ; 52(3): 398-406, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20149472

ABSTRACT

BACKGROUND & AIMS: Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. METHODS: We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index). RESULTS: The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination. CONCLUSIONS: Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.


Subject(s)
Collagen/metabolism , Liver Cirrhosis/metabolism , Liver Cirrhosis/pathology , Microscopy, Fluorescence, Multiphoton/methods , Severity of Illness Index , Biopsy , Cohort Studies , Extracellular Matrix Proteins/metabolism , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/diagnosis , Reproducibility of Results , Sensitivity and Specificity
15.
Hepatology ; 49(3): 930-9, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19177593

ABSTRACT

UNLABELLED: We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. CONCLUSION: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.


Subject(s)
Cell Cycle/physiology , Cell Proliferation , Hepatocytes/cytology , Hepatocytes/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Animals , Cell Survival/physiology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Male , Mice , Mice, Knockout , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , RNA Interference , Rats , Rats, Sprague-Dawley
16.
Opt Express ; 18(25): 25794-807, 2010 Dec 06.
Article in English | MEDLINE | ID: mdl-21164924

ABSTRACT

Second Harmonic Generation (SHG) microscopy offers the opportunity to image collagen of type I without staining. We recently showed that a simple scoring method, based on SHG images of histological human liver biopsies, correlates well with the Metavir assessment of fibrosis level (Gailhouste et al., J. Hepatol., 2010). In this article, we present a detailed study of this new scoring method with two different objective lenses. By using measurements of the objectives point spread functions and of the photomultiplier gain, and a simple model of the SHG intensity, we show that our scoring method, applied to human liver biopsies, is robust to the objective's numerical aperture (NA) for low NA, the choice of the reference sample and laser power, and the spatial sampling rate. The simplicity and robustness of our collagen scoring method may open new opportunities in the quantification of collagen content in different organs, which is of main importance in providing diagnostic information and evaluation of therapeutic efficiency.


Subject(s)
Algorithms , Collagen Type I/ultrastructure , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Liver Cirrhosis/pathology , Microscopy, Fluorescence/methods , Pattern Recognition, Automated/methods , Biomarkers, Tumor/analysis , Humans , Reproducibility of Results , Sensitivity and Specificity
17.
Front Oncol ; 10: 618515, 2020.
Article in English | MEDLINE | ID: mdl-33937011

ABSTRACT

Upregulated MYCN gene expression is restricted to specialized cell populations such as EpCAM+ cancer stem cells in liver cancer, regardless of DNA amplification and mutation. Here, we reviewed the role of MYCN gene expression in liver homeostasis, regeneration, and tumorigenesis, and discussed the potential non-genomic mechanisms involved in controlling MYCN gene expression in liver cancer, with a focus on inflammation-mediated signal transduction and microRNA-associated post-transcriptional regulation. We concluded that dynamic MYCN gene expression is an integrated consequence of multiple signals in the tumor microenvironment, including tumor growth-promoting signals, lipid desaturation-mediated endoplasmic reticulum stress adaptation signals, and tumor suppressive miRNAs, making it a potential predictive biomarker of tumor stemness and plasticity. Therefore, understanding and tracing the dynamic changes and functions of MYCN gene expression will shed light on the origin of liver tumorigenesis at the cellular level and the development of novel therapeutic and diagnostic strategies for liver cancer treatment.

18.
Endocrinology ; 150(1): 200-11, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18772239

ABSTRACT

A precise description of the mechanisms by which estrogen receptor-alpha (ERalpha) exerts its influences on cellular growth and differentiation is still pending. Here, we report that the differentiation of PC12 cells is profoundly affected by ERalpha. Importantly, depending upon its binding to 17beta-estradiol (17betaE2), ERalpha is found to exert different effects on pathways involved in nerve growth factor (NGF) signaling. Indeed, upon its stable expression in PC12 cells, unliganded ERalpha is able to partially inhibit the neurite outgrowth induced by NGF. This process involves a repression of MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways, which leads to a negative regulation of markers of neuronal differentiation such as VGF and NFLc. This repressive action of unliganded ERalpha is mediated by its D domain and does not involve its transactivation and DNA-binding domains, thereby suggesting that direct transcriptional activity of ERalpha is not required. In contrast with this repressive action occurring in the absence of 17betaE2, the expression of ERalpha in PC12 cells allows 17betaE2 to potentiate the NGF-induced neurite outgrowth. Importantly, 17betaE2 has no impact on NGF-induced activity of MAPK and Akt signaling pathways. The mechanisms engaged by liganded ERalpha are thus unlikely to rely on an antagonism of the inhibition mediated by the unliganded ERalpha. Furthermore, 17betaE2 enhances NGF-induced response of VGF and NFLc neuronal markers in PC12 clones expressing ERalpha. This stimulatory effect of 17betaE2 requires the transactivation functions of ERalpha and its D domain, suggesting that an estrogen-responsive element-independent transcriptional mechanism is potentially relevant for the neuritogenic properties of 17betaE2 in ERalpha-expressing PC12 cells.


Subject(s)
Cell Division/drug effects , Estrogen Receptor alpha/genetics , PC12 Cells/cytology , Animals , Clone Cells/cytology , Clone Cells/drug effects , Diethylstilbestrol/pharmacology , Estrogen Receptor alpha/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Amplification , Genetic Variation , Ligands , Nerve Growth Factors/pharmacology , Neurites/drug effects , Neurites/physiology , PC12 Cells/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Rats , Transcription, Genetic/drug effects
19.
Cell Death Dis ; 10(8): 553, 2019 07 18.
Article in English | MEDLINE | ID: mdl-31320614

ABSTRACT

Numerous studies have described the critical role played by microRNAs (miRNAs) in cancer progression and the potential of these small non-coding RNAs for diagnostic or therapeutic applications. However, the mechanisms responsible for the altered expression of miRNAs in malignant cells remain poorly understood. Herein, via epigenetic unmasking, we identified a group of miRNAs located in the imprinted delta like non-canonical Notch ligand 1 (DLK1)-maternally expressed 3 (MEG3) locus that were repressed in hepatic tumor cells. Notably, miR-493-5p epigenetic silencing was correlated with hypermethylation of the MEG3 differentially regulated region (DMR) in liver cancer cell lines and tumor tissues from patients. Experimental rescue of miR-493-5p promoted an anti-cancer response by hindering hepatocellular carcinoma (HCC) cell growth in vitro and tumor progression in vivo. We found that miR-493-5p mediated part of its tumor-suppressor activity by abrogating overexpression of insulin-like growth factor 2 (IGF2) and the IGF2-derived intronic oncomir miR-483-3p in HCC cells characterized by IGF2 loss of imprinting (LOI). In summary, this study describes an unknown miRNA-dependent regulatory mechanism between two distinct imprinted loci and a possible therapeutic window for liver cancer patients exhibiting IGF2-miR-483 LOI and amplification.


Subject(s)
Carcinoma, Hepatocellular/genetics , Genomic Imprinting/genetics , Insulin-Like Growth Factor II/genetics , Liver Neoplasms/genetics , MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , Animals , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Survival/genetics , DNA Methylation , Epigenesis, Genetic , Female , Gene Expression Regulation, Neoplastic/genetics , Genes, Tumor Suppressor , Humans , Insulin-Like Growth Factor II/metabolism , Introns , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Mice , Mice, Nude , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Transplantation, Heterologous
20.
Cell Death Dis ; 9(5): 468, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29700299

ABSTRACT

Curative management of pancreatic adenocarcinoma is limited because this malignancy remains resistant to most chemotherapeutic drugs. Strategies that reverse epigenetic alterations offer a unique opportunity for cancer cell reprogramming, which is valuable for development of new treatments. The aim of this work was to reprogram pancreatic ductal adenocarcinoma (PDAC) cells toward a less aggressive and drug-responsive phenotype. The process applied is called "epigenetic reprogramming". To evaluate the efficiency of PDAC epigenetic reprogramming, we assessed tumor growth and drug response in PANC-1 cells after exposure to non-cytotoxic doses of the demethylating agent 5-azacytidine (5-AZA). Here, we showed that an epigenetic regimen using 5-AZA promoted an anti-cancer response by inhibiting PDAC tumor growth in vivo after the engraftment of treated cells. Remarkably, the subsequent addition of gemcitabine (GEM) to the 5-AZA-mediated reprogramming resulted in a marked growth inhibition effect in GEM-resistant pancreatic cancer cells. We observed that various characteristic peptides expressed in the pancreas, which included the antiproliferative hormone somatostatin (SST) and the SST receptor 2 (SSTR2), were significantly upregulated in the epigenetically reprogrammed PDAC cells. The inhibitory effect of octreotide (OCT), an SST analog, was tested on PDAC cells and found to be improved after cell reprogramming. Furthermore, we found that SST gene expression restoration following 5-AZA treatment or following knockdown of the DNA methyltransferase (DNMT) 1 enzyme was associated with the reversion of SST epigenetic silencing through regional CpG demethylation. Lastly, we confirmed the efficacy of 5-AZA-based epigenetic reprogramming in vivo using a PDAC tumor growth model. In conclusion, this study demonstrates that epigenetic reprogramming using the demethylating compound 5-AZA shows anti-cancer effects in PANC-1 cells and is potentially attractive for the treatment of solid tumors.


Subject(s)
Azacitidine/pharmacology , Carcinoma, Pancreatic Ductal , Epigenesis, Genetic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Pancreatic Neoplasms , Animals , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Xenograft Model Antitumor Assays
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