Poly‐S‐nitrosated human albumin enhances the antitumor and antimetastasis effect of bevacizumab,partly by inhibiting autophagy through the generation of nitric oxide

Autophagy is one of the major causes of drug resistance. For example, the angiogenesis inhibitor bevacizumab shows only transient and short‐term therapeutic effects, whereas long‐term therapeutic benefits are rarely observed, probably due to hypoxia‐induced autophagy. Nitric oxide (NO) is an important molecule with multiple functions, and it has recently been reported to function as a regulator of autophagy. Therefore, a reasonable therapeutic strategy for overcoming drug resistance by NO would involve it being directly delivered to the tumor. Here, we investigated the inhibitory effect of NO on autophagy by using a macromolecular NO donor S‐nitrosated human serum albumin (SNO‐HSA) with a high degree of NO loading and tumor targeting potential. In colon 26 (C26) cells, SNO‐HSA significantly suppressed hypoxia‐induced autophagy by inhibiting the phosphorylation of JNK1 and the expression of its downstream molecule Beclin1. The effect of SNO‐HSA was also confirmed in vivo by combining it with Bev. In C26‐bearing mice, significant suppression of tumor growth as well as lung metastasis was achieved in the combination group compared to the SNO‐HSA or bevacizumab alone group. Similar to the in vitro experiments, the immunostaining of tumor tissues clearly showed that SNO‐HSA inhibited the autophagy of tumor cells induced by bevacizumab treatment. In addition to other known antitumor effects of SNO‐HSA, that is, the induction of apoptosis and the inhibition of multidrug efflux pumps, these data may open alternate strategies for cancer chemotherapy by taking advantage of the ability of SNO‐HSA to suppress autophagy‐mediated drug resistance and enhance the efficacy of chemotherapy.     

Pro-survival and pro-growth effects of stress-induced nitric oxide in a prostate cancer photodynamic therapy model

We discovered recently that human breast cancer cells subjected to photodynamic therapy (PDT)-like oxidative stress localized in mitochondria rapidly upregulated nitric oxide synthase-2 (NOS2) and nitric oxide (NO), which increased resistance to apoptotic photokilling. In this study, we asked whether human prostate cancer PC-3 cells would exploit NOS2/NO similarly and, if so, how proliferation of surviving cells might be affected. Irradiation of photosensitized PC-3 cells resulted in a rapid (<1 h), robust (∼12-fold), and prolonged (∼20 h) post-irradiation upregulation of NOS2. Caspase-3/7 activation and apoptosis were stimulated by NOS2 inhibitors and a NO scavenger, implying that induced NO was acting cytoprotectively. Cyclic GMP involvement was ruled out, whereas suppression of pro-apoptotic JNK and p38 MAPK activation was clearly implicated. Cells surviving photostress grew back ∼2-times faster than controls. NOS2 inhibition prevented this and the large increase in cell cycle S-phase occupancy observed after irradiation. Thus, photostress upregulation of NOS/NO elicited both a pro-survival and pro-growth response, both of which could compromise clinical PDT efficacy unless suppressed, e.g. by pharmacological intervention with a NOS2 inhibitor.     

Cyclooxygenase 2 rescues LNCaP prostate cancer cells from sanguinarine-induced apoptosis by a mechanism involving inhibition of nitric oxide synthase activity

Expression of cyclooxygenase-2 (Cox-2), an inducible enzyme responsible for the production of prostaglandins from arachidonic acid, is elevated in human prostate tumor samples. The aim of this study was to investigate whether expression of Cox-2 is effective against prostate cancer cell apoptosis triggered by sanguinarine, the quaternary benzophenanthridine alkaloid with antineoplastic properties. Sanguinarine effectively induced apoptosis in LNCaP human prostate cancer epithelial cells as assessed by caspase-3 activation assay, Annexin V staining assay, or by visual analysis for the apoptotic morphology changes. Sanguinarine-mediated apoptosis was associated with the increase of nitric oxide (NO) formation in prostate cancer cells as assessed by measurements of nitrites with Sievers nitric oxide analyzer as well as flow cytometry analysis using NO fluorescent sensor. Activation of NO synthase (NOS) activity was crucial for sanguinarine-induced cell death because NOS inhibitor L-NMMA efficiently protected cells from apoptosis. Adenovirus-mediated transfer of Cox-2 into LNCaP cells inhibited sanguinarine-induced apoptosis and prevented an increase in NO production. Surprisingly, NO donors failed to induce apoptosis in LNCaP cells, suggesting that constitutive NO generation is not sufficient for triggering apoptosis in these cells. Besides NO generation, NOS is also capable of producing superoxide radicals. Sanguinarine-induced production of superoxide radicals, and the addition of MnTBAP, a scavenger of superoxide radicals, efficiently inhibited sanguinarine-mediated apoptosis. These results suggest that Cox-2 expression rescues prostate cancer cells from sanguinarine-induced apoptosis by a mechanism involving inhibition of NOS activity, and that coadministration of Cox-2 inhibitors with sanguinarine may be developed as a strategy for the management of prostate cancer.     

N-(4-Hydroxyphenyl)retinamide is more potent than other phenylretinamides in inhibiting the growth of BRCA1-mutated breast cancer cells

Women with germline mutations in the breast cancer susceptibility gene BRCA1 are at an increased risk of developing breast cancer. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been shown to have a clinical chemopreventive activity in patients with premenopausal breast cancer. Since BRCA1 mutations are associated with an early-onset breast cancer, usually before menopause, we hypothesized that 4-HPR may be an effective chemopreventive agent against breast tumors exhibiting BRCA1 mutations. The objective of this study was to determine the effectiveness and mechanisms of action of 4-HPR and its phenylretinamide analogues in BRCA1-mutated breast cancer cells. At clinically relevant doses, 4-HPR induced apoptosis in human (HCC1937) and murine (W0069, W525) BRCA1-mutated breast cancer cells. Among the various phenylretinamides tested, N-(2-carboxyphenyl)retinamide (2-CPR) and 3-CPR significantly inhibited the growth of HCC1937 cells; however, they were not as potent as 4-HPR in this respect. We also determined the mechanisms by which 4-HPR induces apoptosis in BRCA1-mutated breast cancer cells. The extent to which 4-HPR induced apoptosis in BRCA1-mutated cells correlated with the increases in nitric oxide (NO) production and nitric oxide synthase (NOS) II and NOSIII expression. Use of a NOS inhibitor to block NO production suppressed the inhibitory effects of 4-HPR in all cell lines. These in vitro results suggest that 4-HPR may be an effective chemopreventive agent against breast tumors that exhibit BRCA1 mutations because of its ability to induce NO-mediated apoptosis in such tumors.     

Hepatitis C virus targets over‐expression of arginase I in hepatocarcinogenesis

Hepatitis C virus (HCV) infection is often associated with chronic liver disease, which is a major risk factor for the development of hepatocellular carcinoma (HCC). To study the HCV host‐cell relationship on the molecular level, HepG2 and Huh7 cells were stably transfected with an infectious cDNA clone of HCV or with empty vector. Evidence for HCV replication was obtained in both culture systems. HCV also stimulated growth in vitro. To identify genes whose altered expression by HCV are important to the pathogenesis of infection, RNAs were isolated from HepG2‐HCV and HepG2‐vector cells and subjected to microarray analysis. The results showed that arginase 1 mRNA and protein were elevated about threefold in HCV positive compared with negative cells (p < 0.01). Arginase 1 expression was elevated in more than 75% of HCV infected liver samples compared with paired HCC from the same patients (>33% positive) and to uninfected liver tissues (0% positive). Arginase 1 specific siRNA inhibited the ability of HCV to stimulate hepatocellular growth in culture by >70%, suggesting that the metabolism of arginine to ornithine may contribute to HCV mediated stimulation of hepatocellular growth. Introduction of arginase specific siRNA also resulted in increased nitric oxide synthase (iNOS) (>1.2‐fold), nitric oxide (NO) production (>3‐fold) and increased cell death (>2.5‐fold) in HCV positive compared with negative cells, suggesting that these molecules potentially contribute to hepatocellular damage. Hence, an important part of the mechanism whereby HCV regulates hepatocellular growth and survival may be through altering arginine metabolism. © 2009 UICC     

XPA serves as an autophagy and apoptosis inducer by suppressing hepatocellular carcinoma in a PI3K/Akt/mTOR dependent manner

BackgroundTo explore the potential biological function of XPA (Xeroderma pigmentosum group A) in hepatic neoplasms and the underlying molecular mechanisms.MethodsLiver cells were used as experimental models to establish HCC (hepatocellular carcinoma) in vitro. Protein extractions were subjected to Western blotting to detect the proteins expression. The lentivirus transfection efficiency was confirmed by Western blot and RT-qPCR, Tunnel staining was used to detect apoptosis, and Transwell assays were used to observe cell migration and invasion. Cell proliferation was detected with colony formation and CCK-8 (cell counting kit-8) assays.ResultsXPA expression was obviously lower in HCC tissue and liver cancer cell lines. XPA overexpression induced autophagy and apoptosis by increasing LC3B II/I, Beclin1, cleaved-caspase-3, and Bax expression and decreasing p62 and Bcl2 protein levels. XPA also suppressed HCC EMT (Epithelial-Mesenchymal Transition) by increasing E-cadherin and decreasing N-cadherin and vimentin protein expression. Cell proliferation, migration and invasion in vivo were significantly inhibited by the overexpression of XPA, and p-PI3K, p-Akt, and p-mTOR expression were decreased in LV-XPA cells. In general, XPA inhibited HCC by inducing autophagy and apoptosis and by modulating the expression of PI3K/Akt/mTOR proteins.ConclusionsXPA overexpression was found to suppress HCC by inducing autophagy and apoptosis and repressing EMT and proliferation. Each of these effects may be involved in modulating the PI3K/Akt/mTOR signaling pathway.     

The mechanism of fucoidan‐induced apoptosis in leukemic cells: Involvement of ERK1/2, JNK,glutathione, and nitric oxide

Fucoidan, a sulfated polysaccharide in brown seaweed, has various biological activities including anti‐tumor activity. We investigated the effects of fucoidan on the apoptosis of human promyeloid leukemic cells and fucoidan‐mediated signaling pathways. Fucoidan induced apoptosis of HL‐60, NB4, and THP‐1 cells, but not K562 cells. Fucoidan treatment of HL‐60 cells induced activation of caspases‐8, ‐9, and ‐3, the cleavage of Bid, and changed mitochondrial membrane permeability. Fucoidan‐induced apoptosis, cleavage of procaspases, and changes in the mitochondrial membrane permeability were efficiently blocked by depletion of mitogen‐activated protein kinase (MAPK) kinase kinase 1 (MEKK1), and inhibitors of MAPK kinase 1 (MEK1) and c Jun NH₂‐terminal kinase (JNK). The phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and JNK was increased in fucoidan‐treated HL‐60, NB4, and THP‐1 cells, but not K562 cells. ERK1/2 activation occurred at earlier times than JNK activation and JNK activation was blocked by MEK1 inhibitor. In addition, fucoidan‐induced apoptosis was inhibited by addition of glutathione and/or L ‐NAME, and fucoidan decreased intracellular glutathione concentrations and stimulated nitric oxide (NO) production. Buthionine‐[R,S]‐sulfoximine rendered HL‐60 cells more sensitive to fucoidan. Depletion of MEKK1 and inhibition of MEK1 restored the intracellular glutathione content and abrogated NO production, whereas inhibition of JNK activation by SP600125 restored intracellular glutathione content but failed to inhibit NO production in fucoidan‐treated HL‐60 cells. These results suggest that activation of MEKK1, MEK1, ERK1/2, and JNK, depletion of glutathione, and production of NO are important mediators in fucoidan‐induced apoptosis of human leukemic cells. © 2010 Wiley‐Liss, Inc.     

Cisplatin alters nitric oxide synthase levels in human ovarian cancer cells: involvement in p53 regulation and cisplatin resistance

The present study determines if (1) basal protein levels of nitric oxide (NO) synthases (eNOS, iNOS, and nNOS) are different in cisplatin-sensitive (OV2008) and counterpart cisplatin-resistant (C13(*)) human ovarian cancer cells, (2) cisplatin alters NOS levels, (3) NO donor causes apoptosis and p53 upregulation, (4) NO donor sensitizes C13(*) cells to cisplatin via p53 upregulation (determined by p53 siRNA gene-knockdown), and (5) inhibition of endogenous NOS alters cisplatin-induced apoptosis. Basal iNOS levels were higher in OV2008 cells than in C13(*) cells. Cisplatin upregulated iNOS, but dramatically reduced eNOS and nNOS, in OV2008 cells only. Failure of cisplatin to upregulate iNOS and downregulate eNOS/nNOS in cisplatin-resistant C13(*) cells may be an aetiological factor in the development of resistance. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased p53 protein levels and induced apoptosis in both cell types, and enhanced cisplatin-induced apoptosis in C13(*) cells in a p53-dependent manner (i.e., enhancement blocked by p53 siRNA). Specific iNOS inhibitor 1400W partially blocked cisplatin-induced apoptosis in OV2008 cells. In cisplatin-resistant C13(*) cells, blocking all NOSs with N(G)-amino-L-arginine dramatically changed these cells from cisplatin-resistant to cisplatin-sensitive, greatly potentiating cisplatin-induced apoptosis. The data suggest important roles for the three NOSs in regulating chemoresistance to cisplatin in ovarian cancer cells.     

Role of nitric oxide in tumour progression: Lessons from human tumours

Varied cellular expression and localisation of nitric oxide synthase (NOS) isoforms has been shown in human cancers, including tumours of the breast, ovary, stomach, cervix and central nervous system. Mapping of NOS expression within tumour tissue from breast and gastric cancers shows inducible NOS (iNOS) is expressed predominantly in stromal (macrophage and endothelial) cells, although the level of NOS activity is at least 1–2 orders of magnitude lower than the enzyme activity associated with cytotoxicity and apoptosis. There is evidence that the intratumoural environment may provide chemoattractant signals for monocyte-macrophage recruitment and their subsequent activation via expression of interleukin-4, IgE, and CD23. Such signals lead to induction of iNOS in human macrophages in vitro. The correlation between NOS activity and grade for breast cancer suggests that NO may provide a positive growth signal within the tumour microenvironment. In vivo studies showing increased growth rate, vascular density and invasiveness of a human tumour cell line transfected to constitutively express iNOS support this. Furthermore, in vivo administration of a highly selective inhibitor of iNOS limited invasion and growth rate of iNOS transfected tumours and other murine tumours expressing this isoform. Inhibition of NO generation in the intratumoural microenvironment may prove a useful cancer therapy by preventing angiogenesis, invasion and metastasis.     

Association of beclin 1 expression with response to neoadjuvant chemoradiation therapy in patients with locally advanced rectal carcinoma

Beclin 1 is an essential regulator of autophagy that is induced in response to cellular stress and serves to maintain cell survival in established tumors. We recently demonstrated that Beclin 1 suppression can sensitize colorectal cancer cells to radiation‐induced DNA damage and apoptosis. Therefore, we hypothesized that the level of Beclin 1 expression may be associated with radiation sensitivity in vivo. We determined the association of Beclin 1 expression in pretreatment rectal cancer tissues with response to neoadjuvant chemoradiation in surgical resection specimens. Stages II and III (n = 96) rectal adenocarcinoma patients were treated with neoadjuvant chemoradiation followed by surgical resection with curative intent. Beclin 1 was analyzed by immunohistochemistry and the expression level was dichotomized at the median value with categorization into low and high groups. We identified 56 (58.3%) and 40 (41.7%) patients whose tumors had high‐ versus low‐level Beclin 1 expression, respectively. Rectal cancers with high versus low Beclin 1 expression were significantly less likely to be downstaged after chemoradiation treatment (45% [25/55] vs. 58% [22/38]; p = 0.02). In a multivariable analysis adjusted for age, sex, histological grade and baseline tumor node metastasis (TNM) stage, the impact of Beclin 1 expression on tumor downstaging remained statistically significant (p = 0.03). The association of the level of Beclin 1 expression with the rate of tumor downstaging after chemoradiation is consistent with in vitro data, and suggests that Beclin 1 may be a predictive biomarker for the efficacy of chemoradiation in patients with rectal cancer.     

Investigating the role of tumour cell derived iNOS on tumour growth and vasculature in vivo using a tetracycline regulated expression system

Nitric oxide (NO) is a free radical signalling molecule involved in various physiological and pathological processes, including cancer. Both tumouricidal and tumour promoting effects have been attributed to NO, making its role in cancer biology controversial and unclear. To investigate the specific role of tumour‐derived NO in vascular development, C6 glioma cells were genetically modified to include a doxycycline regulated gene expression system that controls the expression of an antisense RNA to inducible nitric oxide synthase (iNOS) to manipulate endogenous iNOS expression. Xenografts of these cells were propagated in the presence or absence of doxycycline. Susceptibility magnetic resonance imaging (MRI), initially with a carbogen (95% O₂/5% CO₂) breathing challenge and subsequently an intravascular blood pool contrast agent, was used to assess haemodynamic vasculature (ΔR₂*) and fractional blood volume (fBV), and correlated with histopathological assessment of tumour vascular density, maturation and function. Inhibition of NO production in C6 gliomas led to significant growth delay and inhibition of vessel maturation. Parametric fBV maps were used to identify vascularised regions from which the carbogen‐induced ΔR₂* was measured and found to be positively correlated with vessel maturation, quantified ex vivo using fluorescence microscopy for endothelial and perivascular cell staining. These data suggest that tumour‐derived iNOS is an important mediator of tumour growth and vessel maturation, hence a promising target for anti‐vascular cancer therapies. The combination of ΔR₂* response to carbogen and fBV MRI can provide a marker of tumour vessel maturation that could be applied to non‐invasively monitor treatment response to iNOS inhibitors.     

Nitric Oxide and Angiogenesis

The steps required for new vessel growth are biologically complex and require coordinate regulation of contributing components, including modifications of cell–cell interactions, proliferation and migration of endothelial cells and matrix degradation. The observation that in vivo angiogenesis is accompanied by vasodilation, that many angiogenesis effectors possess vasodilating properties and that tumor vasculature is in a persistent state of vasodilation, support the existence of a molecular/biochemical link between vasodilation and angiogenesis. Several pieces of evidence converge in the indication of a role for nitric oxide (NO), the factor responsible for vasodilation, in physiological and pathological angiogenesis. Data originated in different labs indicate that NO can act both as an 'actor' of angiogenesis and as a 'director of angiogenesis', both functions being equally expressed during physiological and pathological processes. NO significantly contributes to the prosurvival/proangiogenic program of capillary endothelium by triggering and transducing cell growth and differentiation via endothelial-constitutive NO synthase (ec-NOS) activation, cyclic GMP (cGMP) elevation, mitogen activated kinase (MAPK) activation and fibroblast growth factor-2 (FGF-2) expression. Re-establishment of a balanced NO production in the central nervous system results in a reduction of cell damage during inflammatory and vascular diseases. Elevation of NOS activity in correlation with angiogenesis and tumor progression has been extensively reported in experimental and human tumors. In the brain, tumor expansion and edema formation are sensitive to NOS inhibition. On this basis, the nitric oxide pathway appears to be a promising target for consideration in pro- and anti-angiogenic therapeutic strategies. The use of NOS inhibitors seems appropriate to reduce edema, block angiogenesis and facilitate antitumor drug delivery.     

Inflammation regulates microRNA expression in cooperation with p53 and nitric oxide

microRNA (miRNA) are small non‐coding RNA targeting mRNAs leading to their instability and diminished translation. Altered expression of miRNA is associated with cancer. Inflammation and nitric oxide modulates the development of lymphomas in p53 knockout mice and there exists a negative feedback loop between p53 and NOS2. Using a genetic strategy, we tested the hypothesis that inflammation‐induced oxidative and nitrosative stress modulates miRNA expression in mouse model deficient in either p53 or NOS2. Mice treated with Corynebacterium parvum (C. parvum), to induce inflammation, clearly separated from controls by their miRNA profiles in wild‐type, p53‐ and NOS2‐knockout genetic backgrounds. C. parvum‐induced inflammation significantly (p < 0.005) increased miR‐21, miR‐29b and miR‐34a/b/c and decreased (p < 0.005) mir‐29c and mir‐181a/c expression in the spleen of C57BL mice. However, p53‐knockout C57BL mice did not show a significant increase in the mir‐34b/c or a decrease in mir‐29c expression following C. parvum‐induced inflammation. Expression of mir‐21, mir‐29b and mir‐181a was independent of p53‐status. NOS2‐knockout C57BL mice showed a significant increase in miR‐21 and miR‐34a/b/c and decrease in miR‐181a similar to the wild‐type (WT) mice following C. parvum‐induced inflammation. However, in contrast to the WT mice, miR‐29b/c expression was not affected following C. parvum‐induced inflammation in NOS2 knockout mice. N‐acetyl cysteine, an anti‐oxidant, reduced the expression of miR‐21 and miR‐29b in C. parvum‐treated WT mice (p < 0.005) as compared with control C. parvum‐treated mice. These data are consistent with the hypothesis that inflammation modulates miRNA expression in vivo and the alteration in specific miRNA under an inflammatory microenvironment, can be influenced by p53 (miR‐34b/c) and NO^? (29b/c).     

MicroRNA‐155‐5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway

MicroRNA‐155‐5p (miR‐155‐5p) has been reported to play an oncogenic role in different human malignancies; however, its role in hepatocellular carcinoma (HCC) progression is not clearly understood. In this study, we used real‐time PCR in 20 rats with chemically‐induced HCC, 28 human HCC tissues, and the matched paracarcinoma tissues, and HCC cell lines to determine the expression patterns of miR‐155‐5p and PTEN mRNA. Algorithm‐based and experimental strategies, such as dual luciferase gene reporter assays, real‐time PCR and western blots were used to identify PTEN as a candidate miR‐155‐5p target. Gain‐ and loss‐of‐function experiments and administration of a PI3K/Akt pathway inhibitor (wortmannin) were used to identify the effects of miR‐155‐5p and PTEN in MTT assays, flow cytometric analysis, wound healing assays and transwell assays. The results showed that miR‐155‐5p was highly overexpressed; however, PTEN was underexpressed in the HCC rat models, human HCC tissues and cell lines. In addition, miR‐155‐5p upregulation and PTEN downregulation were significantly associated with TNM stage (P < 0.05). Through in vitro experiments, we found that miR‐155‐5p promoted proliferation, invasion and migration, but inhibited apoptosis in HCC by directly targeting the 3′‐UTR of PTEN. Western blots showed that miR‐155‐5p inactivated Bax and caspase‐9, but activated Bcl‐2 to inhibit apoptosis, and it activated MMP to promote migration and invasion via the PI3K/Akt pathway. A xenograft tumor model was used to demonstrate that miR‐155‐5p targets PTEN and activates the PI3K/Akt pathway in vivo as well. Our study highlighted the importance of miR‐155‐5p and PTEN associated with aggressive HCC both in vitro and in vivo.     

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin‐related protein 14‐mediated autophagy

Paclitaxel is not as effective for neuroblastoma as most of the front‐line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel‐associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy‐associated proteins were assessed by western blot. Autophagy was induced and the autophagy‐associated proteins LC3‐I, LC3‐II, Beclin 1, and thioredoxin‐related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1‐mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel‐induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel‐induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14.     

Silencing of microRNA‐122 is an early event during hepatocarcinogenesis from non‐alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus‐independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH‐HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR‐122 in non‐tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR‐122 was further decreased in HCCs relative to non‐tumor LC at the age of 18 weeks. Expression of miR‐122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR‐122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR‐122 is an early event during hepatocarcinogenesis from NASH, and that miR‐122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.