NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing PINK1/Parkin-Mediated Mitophagy.

A long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) variant 1 (NEAT1v1), confers radioresistance to hepatocellular carcinoma (HCC) cells by inducing autophagy via γ-aminobutyric acid A receptor-associated protein (GABARAP). Radiation induces oxidative stress to damage cellular components and organelles, but it remains unclear how NEAT1v1 protects HCC cells from radiation-induced oxidative stress via autophagy. To address this, we precisely investigated NEAT1v1-induced autophagy in irradiated HCC cell lines. X-ray irradiation significantly increased cellular and mitochondrial oxidative stress and mitochondrial DNA content in HCC cells while NEAT1v1 suppressed them. NEAT1v1 concomitantly induced the phosphatase and tensin homolog-induced kinase 1 (PINK1)/parkin-mediated mitophagy. Interestingly, parkin expression was constitutively upregulated in NEAT1v1-overexpressing HCC cells, leading to increased mitochondrial parkin levels. Superoxide dismutase 2 (SOD2) was also upregulated by NEAT1v1, and GABARAP or SOD2 knockdown in NEAT1v1-overexpressing cells increased mitochondrial oxidative stress and mitochondrial DNA content after irradiation. Moreover, it was suggested that SOD2 was involved in NEAT1v1-induced parkin expression, and that GABARAP promoted parkin degradation via mitophagy. This study highlights the unprecedented roles of NEAT1v1 in connecting radioresistance and mitophagy in HCC.

NEAT1 Confers Radioresistance to Hepatocellular Carcinoma Cells by Inducing Autophagy through GABARAP.

A long noncoding RNA (lncRNA), nuclear enriched abundant transcript 1 (NEAT1) variant 1 (NEAT1v1), is involved in the maintenance of cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). CSCs are suggested to play important roles in therapeutic resistance. Therefore, we investigated whether NEAT1v1 is involved in the sensitivity to radiation therapy in HCC. Gene knockdown was performed using short hairpin RNAs, and NEAT1v1-overexpressing HCC cell lines were generated by stable transfection with a NEAT1v1-expressing plasmid DNA. Cells were irradiated using an X-ray generator. We found that NEAT1 knockdown enhanced the radiosensitivity of HCC cell lines and concomitantly inhibited autophagy. NEAT1v1 overexpression enhanced autophagy in the irradiated cells and conferred radioresistance. Gamma-aminobutyric acid receptor-associated protein (GABARAP) expression was downregulated by NEAT1 knockdown, whereas it was upregulated in NEAT1v1-overexpressing cells. Moreover, GABARAP was required for NEAT1v1-induced autophagy and radioresistance as its knockdown significantly inhibited autophagy and sensitized the cells to radiation. Since GABARAP is a crucial protein for the autophagosome-lysosome fusion, our results suggest that NEAT1v1 confers radioresistance to HCC by promoting autophagy through GABARAP.

Secreted matrix metalloproteinase-14 is a predictor for antifibrotic effect of IC-2-engineered mesenchymal stem cell sheets on liver fibrosis in mice.

INTRODUCTION: Transplantation of IC-2-engineered bone marrow-derived mesenchymal stem cell (BM-MSC) sheets (IC-2 sheets) was previously reported to potentially reduce liver fibrosis. METHODS: This study prepared IC-2-engineered cell sheets from multiple lots of BM-MSCs and examined the therapeutic effects of these cell sheets on liver fibrosis induced by carbon tetrachloride in mice. The predictive factors for antifibrotic effect on liver fibrosis were tried to identify in advance. RESULTS: Secreted matrix metalloproteinase (MMP)-14 was found to be a useful predictive factor to reduce liver fibrosis. Moreover, the cutoff index of MMP-14 for 30% reduction of liver fibrosis was 0.918 fg/cell, judging from univariate analysis and receiver operating curve analysis. In addition, MMP-13 activity and thioredoxin contents in IC-2 sheets were also inversely correlated with hepatic hydroxyproline contents. Finally, IC-2 was also found to promote MMP-14 secretion from BM-MSCs of elderly patients. Surprisingly, the values of secreted MMP-14 from BM-MSCs of elderly patients were much higher than those of young persons. CONCLUSION: The results of this study suggest that the IC-2 sheets would be applicable to clinical use in autologous transplantation for patients with cirrhosis regardless of the patient's age.

Role of cytoplasmic localization of maspin in promoting cell invasion in breast cancer with aggressive phenotype.

Mammary serine protease inhibitor (maspin) is a tumor suppressor gene that is downregulated during carcinogenesis and breast cancer progression. While the nuclear localization of maspin is essential for tumor suppression, we previously reported that the cytoplasmic localization of maspin was significantly correlated with poor prognosis in breast cancer patients. To understand the mechanisms that underlie oncogenic role of cytoplasmic maspin, we studied its biological function in breast cancer cell lines. Subcellular localization of maspin in MDA-MB-231 breast cancer cells was mainly detected in the cytoplasm, whereas in MCF10A mammary epithelial cells, maspin was present in both cytoplasm and nucleus. In MDA-MB-231 cells, maspin overexpression promoted cell proliferation and cell invasion, whereas maspin downregulation resulted in the opposite effect. Further, we observed that SRGN protein levels were increased in MDA-MB-231 cells stably overexpressing maspin. Finally, maspin overexpression in MDA-MB-231 cells resulted in the N-cadherin and epithelial mesenchymal transition (EMT)-related transcription factors upregulation, and TGFß signaling pathway activation. These results suggested that cytoplasmic maspin enhances the invasive and metastatic potential in breast cancer cells with aggressive phenotype by inducing EMT via SRGN/TGFß axis. This study demonstrated a novel biological function of cytoplasmic maspin in progression of breast cancer cells with an aggressive phenotype.

Clinical and Biological Implications of Cancer Stem Cells in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a malignant tumor with poor prognosis, and is one of the leading causes of cancer-related deaths worldwide. Recently, the development of therapeutic drugs via novel mechanisms of action, involving molecular-targeted drugs and immune checkpoint inhibitors, has progressed in the field of HCC. However, the recurrence rate remains high, and further improvement of the prognosis of patients with HCC is urgently needed. Cancer stem cells (CSCs) are a promising target for further development of novel anti-cancer drugs because they are reportedly involved in tumor initiation, maintenance, recurrence, and resistance to conventional therapies. Although several studies have already been conducted, the functions and roles of CSCs in the development and progression of tumors remain to be elucidated. In this review article, we will clarify the fundamental knowledge of CSCs necessary for the understanding of CSCs and will outline so-far identified markers specific to liver CSCs and the pathological and therapeutic implications of CSCs in HCC.

Immune evasion by cancer stem cells.

Tumor immunity represents a new avenue for cancer therapy. Immune checkpoint inhibitors have successfully improved outcomes in several tumor types. In addition, currently, immune cell-based therapy is also attracting significant attention. However, the clinical efficacy of these treatments requires further improvement. The mechanisms through which cancer cells escape the immune response must be identified and clarified. Cancer stem cells (CSCs) play a central role in multiple aspects of malignant tumors. CSCs can initiate tumors in partially immunocompromised mice, whereas non-CSCs fail to form tumors, suggesting that tumor initiation is a definitive function of CSCs. However, the fact that non-CSCs also initiate tumors in more highly immunocompromised mice suggests that the immune evasion property may be a more fundamental feature of CSCs rather than a tumor-initiating property. In this review, we summarize studies that have elucidated how CSCs evade tumor immunity and create an immunosuppressive milieu with a focus on CSC-specific characteristics and functions. These profound mechanisms provide important clues for the development of novel tumor immunotherapies.

Hepatocellular carcinoma development in diabetic patients: a nationwide survey in Japan.

BACKGROUND: Although type 2 diabetes mellitus (T2DM) is a known risk factor for hepatocellular carcinoma (HCC) development, the annual incidence in diabetes patients is far below the threshold of efficient surveillance. This study aimed to elucidate the risk factors for HCC in diabetic patients and to determine the best criteria to identify surveillance candidates. METHODS: The study included 239 patients with T2DM who were diagnosed with non-viral HCC between 2010 and 2015, with ≥ 5 years of follow-up at diabetes clinics of 81 teaching hospitals in Japan before HCC diagnosis, and 3277 non-HCC T2DM patients from a prospective cohort study, as controls. Clinical data at the time of and 5 years before HCC diagnosis were collected. RESULTS: The mean patient age at HCC diagnosis was approximately 73 years, and 80% of the patients were male. The proportion of patients with insulin use increased, whereas the body mass index (BMI), proportion of patients with fatty liver, fasting glucose levels, and hemoglobin A1c (HbA1c) levels decreased significantly in 5 years. In the cohort study, 18 patients developed HCC during the mean follow-up period of 4.7 years with an annual incidence of 0.11%. Multivariate logistic regression analyses showed that the FIB-4 index was an outstanding predictor of HCC development along with male sex, presence of hypertension, lower HbA1c and albumin levels, and higher BMI and gamma-glutamyl transpeptidase levels. Receiver-operating characteristic analyses showed that a FIB-4 cut-off value of 3.61 could help identify high-risk patients, with a corresponding annual HCC incidence rate of 1.1%. CONCLUSION: A simple calculation of the FIB-4 index in diabetes clinics can be the first step toward surveillance of HCC with a non-viral etiology.

Liver-specific dysregulation of clock-controlled output signal impairs energy metabolism in liver and muscle.

The liver is the major organ maintaining metabolic homeostasis in animals during shifts between fed and fasted states. Circadian oscillations in peripheral tissues including the liver are connected with feeding-fasting cycles. We generated transgenic mice with hepatocyte specific E4BP4, D-box negative regulator, overexpression. Liver-specific E4BP4 overexpression was also achieved by adenoviral gene transfer. Interestingly, hepatic E4BP4 overexpression induced marked insulin resistance, that was rescued by DBP, a competing D-box positive regulator, overexpression. At basal conditions hepatocyte E4BP4 transgenic mice exhibited increased gluconeogenesis with reduced AKT phosphorylation in liver. In muscle, AKT phosphorylation was impaired after insulin stimulation. Such muscle insulin resistance was associated with elevated free fatty acid flux from the liver and reduced fatty acid utilization as an energy source during the inactive phase. E4BP4, one of the clock-controlled output genes, are key metabolic regulators in liver adjusting liver and muscle metabolism and insulin sensitivity in the feeding-fasting cycles. Its tuning is critical for preventing metabolic disorders.

Identification of a Novel Deactivating Small-Molecule Compound for Fibrogenic Hepatic Stellate Cells.

BACKGROUND: Liver fibrosis progresses to decompensated liver cirrhosis, for which medical needs remain unmet. We recently developed IC-2, a small-molecule compound that suppresses Wnt/ß-catenin signaling, and found that IC-2 also suppresses liver fibrosis. In this study, we performed three-step screening of newly synthesized IC-2 derivatives to identify other small-molecule compounds that suppress liver fibrosis. METHODS: The screening system consisted of three steps: a cell viability assay, a transcription factor 4 (TCF4) reporter assay, and induction of α-smooth muscle actin (α-SMA) and collagen 1α1 (Col1A1) expression in response to each compound. Screening using human LX-2 hepatic stellate cells (HSCs) was performed to target HSCs, which are the driver cells of liver fibrosis. RESULTS: In the first step, since 9b and 9b-CONH2 at 100 µM did not have any effects on cell viability, they were omitted in the next screening. Additionally, the conditions that led to > 40% inhibition of the controls were also excluded in subsequent screening. The second step was performed under 31 conditions for 19 small-molecule compounds. Sixteen small-molecule compounds caused significant reduction of TCF4 activity relative to that of 0.1% DMSO. Of the 16 compounds, the 10 showing the greatest suppression of TCF4 activity were selected for the third step. Expressions of mRNA for α-SMA and Col1A1 were significantly reduced by seven and three small-molecule compounds, respectively. The greatest reductions in the α-SMA and Col1A1 mRNA expressions were observed in the cells treated with IC-2-F. Protein expressions of α-SMA and Col1A1 caused by IC-2-F were also comparable to those caused by IC-2. CONCLUSION: IC-2-F was identified as a novel deactivating small-molecule compound for HSCs in vitro. These data suggest that IC-2-F is a promising medicine for liver fibrosis.

NEAT1 is Required for the Expression of the Liver Cancer Stem Cell Marker CD44.

CD44, a cancer stem cell (CSC) marker, is required for maintaining CSC properties in hepatocellular carcinoma (HCC). Nuclear enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), is an oncogenic driver in HCC. In the present study, we investigated the significance of the NEAT1 gene in association with CD44 expression in liver CSCs of human HCC cell lines. The CSC properties were evaluated by spheroid culture, CSC marker expression, and sensitivity to anti-cancer drugs. The expression of both NEAT1 variant 1 (NEAT1v1) and variant 2 (NEAT1v2) as well as CD44 was significantly increased in the spheroid culture, compared with that in monolayer culture. Overexpression of Neat1v1, but not Neat1v2, enhanced the CSC properties, while knockout of the NEAT1 gene suppressed them. CD44 expression was increased by the overexpression of Neat1v1 and abrogated by NEAT1 knockout. The overexpression of NEAT1v1 restored the CSC properties and CD44 expression in NEAT1-knockout cells. NEAT1v1 expression in HCC tissues was correlated with poor prognosis and CD44 expression. These results suggest that NEAT1v1 is required for CD44 expression. To our surprise, NEAT1v1 also restored the CSC properties even in CD44-deficient cells, suggesting that NEAT1v1 maintains the properties of CSCs in a CD44-independent manner.

WNT/ß-catenin signal inhibitor IC-2-derived small-molecule compounds suppress TGF-ß1-induced fibrogenic response of renal epithelial cells by inhibiting SMAD2/3 signalling.

Renal fibrosis compromises kidney function, and it is a risk factor for chronic kidney disease (CKD). CKD ultimately progresses to end-stage kidney disease that can be cured only by kidney transplantation. Owing to the increasing number of CKD patients, effective treatment strategies are urgently required for renal fibrosis. TGF-ß is a well-established fibrogenic factor that signals through SMAD2/3 signaling pathway. It was shown that there is a cross-talk between TGF-ß/SMAD and WNT/ß-catenin signaling pathways in renal tubular epithelial cells, and that a WNT/ß-catenin inhibitor, ICG-001, ameliorates TGF-ß1induced renal fibrosis. IC-2, a derivative of ICG-001, has been shown to potently induce hepatocyte differentiation of human mesenchymal stem cells by inhibiting WNT/ß-catenin signaling. In the present study, we examined the effect of ICG-001, IC-2, and IC-2 derivatives (IC-2-506-1, IC-2-506-2, IC-2-506-3, IC-2-Ar-Cl, IC-2-OH, IC-2-OTBS, and IC-2-F) on TGF-ß1-induced SMAD activation and fibrogenic response in immortalized human renal tubular epithelial HK-2 cells. All these compounds inhibited LiCl-induced WNT/ß-catenin reporter activation to a similar extent, whereas ICG-001, IC-2-OTBS, and IC-2-F almost completely suppressed TGF-ß1-induced SMAD reporter activation without apparent cytotoxicity. Phosphorylation of SMAD2/3 by TGF-ß1 was more potently inhibited by IC-2-OTBS and IC-2-F than by ICG-001 and IC-2. IC-2-F suppressed TGF-ß1-induced COL1A1 protein expression, whereas IC-2-506-1 and IC-2-OTBS suppressed TGF-ß1-induced epithelial-mesenchymal transition. These results demonstrated that IC-2 derivatives suppress the TGF-ß1-induced fibrogenic response of tubular epithelial cells and thus could be promising therapeutic agents for the treatment of renal fibrosis.

A report of the 18th congress of the Japanese Society for Regenerative Medicine.

The 18th Congress of the Japanese Society for Regenerative Medicine was held from March 21-23, 2019, at Kobe International Conference Center (Hyogo Prefecture) with 3,576 participants. The theme of this congress was "Message from the Birthplace of Regenerative Medicine" with expectation of disseminating the message of 'saving patients with regenerative medicine' for the future. With this theme, this congress aimed to provide opportunity for accelerating the development of this field through exchanging information among people from all participants (including individuals from academia, various industries, and regulatory authorities). Numerous topics were covered in the one presidential lecture, one congress president's lecture, one keynote lecture, one invited lecture, three special lectures, three educational lectures, four special programs including two joint symposia with oversea society (13 topics), one award lecture program (four lectures), symposia (46 sessions, 460 topics), oral presentations (38 sessions, 219 topics), poster presentations (77 sessions, 417 topics), 32 co-organized seminars (39 talks), and the state-of-the-art technology showcase (178 organizations). Additionally, two sessions for junior high school and high school students (basic course and advanced course) were held during the conference.

HBx and c-MYC Cooperate to Induce URI1 Expression in HBV-Related Hepatocellular Carcinoma.

Unconventional prefoldin RNA polymerase II subunit 5 interactor (URI1) has emerged as an oncogenic driver in hepatocellular carcinoma (HCC). Although the hepatitis B virus (HBV) represents the most common etiology of HCC worldwide, it is unknown whether URI1 plays a role in HBV-related HCC (HCC-B). In the present study, we investigated URI1 expression and its underlying mechanism in HCC-B tissues and cell lines. URI1 gene-promoter activity was determined by a luciferase assay. Human HCC-B samples were used for a chromatin immunoprecipitation assay. We found that c-MYC induced URI1 expression and activated the URI1 promoter through the E-box in the promoter region while the HBx protein significantly enhanced it. The positivity of URI1 expression was significantly higher in HCC-B tumor tissues than in non-HBV-related HCC tumor tissues, suggesting that a specific mechanism underlies URI1 expression in HCC-B. In tumor tissues from HCC-B patients, a significantly higher level of c-MYC was recruited to the E-box than in non-tumor tissues. These results suggest that HBx and c-MYC are involved in URI1 expression in HCC-B. URI1 expression may play important roles in the development and progression of HCC-B because HBx and c-MYC are well-known oncogenic factors in the virus and host, respectively.

Reversal of established liver fibrosis by IC-2-engineered mesenchymal stem cell sheets.

Chronic hepatitis viral infection, alcoholic intoxication, and obesity cause liver fibrosis, which progresses to decompensated liver cirrhosis, a disease for which medical demands cannot be met. Since there are currently no approved anti-fibrotic therapies for established liver fibrosis, the development of novel modalities is required to improve patient prognosis. In this study, we clarified the anti-fibrotic effects of cell sheets produced from human bone marrow-derived mesenchymal stem cells (MSCs) incubated on a temperature-sensitive culture dish with the chemical compound IC-2. Orthotopic transplantation of IC-2-engineered MSC sheets (IC-2 sheets) remarkably reduced liver fibrosis induced by chronic CCl4 administration. Further, the marked production of fibrolytic enzymes such as matrix metalloproteinase (MMP)-1 and MMP-14, as well as thioredoxin, which suppresses hepatic stellate cell activation, was observed in IC-2 sheets. Moreover, the anti-fibrotic effect of IC-2 sheets was much better than that of MSC sheets. Finally, knockdown experiments revealed that MMP-14 was primarily responsible for the reduction of liver fibrosis. Here, we show that IC-2 sheets could be a promising therapeutic option for established liver fibrosis.

CD44 standard isoform is involved in maintenance of cancer stem cells of a hepatocellular carcinoma cell line.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide. Cancer stem cells (CSCs) have attracted attention as a novel therapeutic target for cancer because they play important roles in the development and aggravation of cancer. CD44 is expressed as a standard isoform (CD44s) and several variant isoforms. CD44v is a major isoform expressed on CSCs of a variety of tumors and has been extensively studied. However, HCC tissues dominantly express CD44s, whose function in CSCs remains unclear. In the present study, we investigated the roles of CD44s in CSCs of HCC. Knock-out of the CD44 gene in HuH7 HCC cells on which only CD44s is expressed resulted in decreased spheroid formation and increased drug sensitivity. The expression of CSC marker genes, including CD133 and EpCAM, was significantly downregulated in the spheroids of CD44-deficient cells compared with those in the spheroids of HuH7 cells. In addition, CD44 deficiency impaired antioxidant capacity, concomitant with downregulation of glutathione peroxidase 1 (GPX1) and thioredoxin. Because GPX1 uses the reduced form of glutathione (GSH) to regenerate oxidized cellular components, GSH levels were significantly increased in the CD44-deficient cells. We also found that NOTCH3 and its target genes were downregulated in the spheroids of CD44-deficient cells. NOTCH3 expression in HCC tissues was significantly increased compared with that in adjacent nontumor liver tissues and was correlated with CD44 expression. These results suggest that CD44s is involved in maintenance of CSCs in a HCC cell line, possibly through the NOTCH3 signaling pathway.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome-wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55- and C7orf43-responsive sites exist between base pairs -58 and +36 and -169 and -59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes-associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non-tumor tissues, compared to normal liver tissues from non-HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Hepatic cell sheets engineered from human mesenchymal stem cells with a single small molecule compound IC-2 ameliorate acute liver injury in mice.

INTRODUCTION: We previously reported that transplantation of hepatic cell sheets from human bone marrow-derived mesenchymal stem cells (BM-MSCs) with hexachlorophene, a Wnt/ß-catenin signaling inhibitor, ameliorated acute liver injury. In a further previous report, we identified IC-2, a newly synthesized derivative of the Wnt/ß-catenin signaling inhibitor ICG-001, as a potent inducer of hepatic differentiation of BM-MSCs. METHODS: We manufactured hepatic cell sheets by engineering from human BM-MSCs using the single small molecule IC-2. The therapeutic potential of IC-2-induced hepatic cell sheets was assessed by transplantation of IC-2- and hexachlorophene-treated hepatic cell sheets using a mouse model of acute liver injury. RESULTS: Significant improvement of liver injury was elicited by the IC-2-treated hepatic cell sheets. The expression of complement C3 was enhanced by IC-2, followed by prominent hepatocyte proliferation stimulated through the activation of NF-κB and its downstream molecule STAT-3. Indeed, IC-2 also enhanced the expression of amphiregulin, resulting in the activation of the EGFR pathway and further stimulation of hepatocyte proliferation. As another important therapeutic mechanism, we revealed prominent reduction of oxidative stress mediated through upregulation of the thioredoxin (TRX) system by IC-2-treated hepatic cell sheets. The effects mediated by IC-2-treated sheets were superior compared with those mediated by hexachlorophene-treated sheets. CONCLUSION: The single compound IC-2 induced hepatic cell sheets that possess potent regeneration capacity and ameliorate acute liver injury.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid.

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/ß-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

All-trans retinoic acid ameliorates hepatic stellate cell activation via suppression of thioredoxin interacting protein expression.

Activation of hepatic stellate cells (HSCs) is the effector factor of hepatic fibrosis and hepatocellular carcinoma (HCC) development. Accumulating evidence suggests that retinoic acids (RAs), derivatives of vitamin A, contribute to prevention of liver fibrosis and carcinogenesis, however, regulatory mechanisms of RAs still remain exclusive. To elucidate RA signaling pathway, we previously performed a genome-wide screening of RA-responsive genes by in silico analysis of RA-response elements, and identified 26 RA-responsive genes. We found that thioredoxin interacting protein (TXNIP), which inhibits antioxidant activity of thioredoxin (TRX), was downregulated by all-trans retinoic acid (ATRA). In the present study, we demonstrate that ATRA ameliorates activation of HSCs through TXNIP suppression. HSC activation was attenuated by TXNIP downregulation, whereas potentiated by TXNIP upregulation, indicating that TXNIP plays a crucial role in activation of HSCs. Notably, we showed that TXNIP-mediated HSC activation was suppressed by antioxidant N-acetylcysteine. In addition, ATRA treatment or downregulation of TXNIP clearly declined oxidative stress levels in activated HSCs. These data suggest that ATRA plays a key role in inhibition of HSC activation via suppressing TXNIP expression, which reduces oxidative stress levels.