Assessing the Utility of Acoustic Radiation Force Impulse in the Evaluation of Non-Alcoholic Fatty Liver Disease with Severe Obesity or Steatosis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) encompasses a heterogeneous spectrum ranging from simple steatosis to fibrosis and cirrhosis. Fibrosis, associated with long-term overall mortality and liver-related events, requires evaluation. Traditionally, liver biopsy has been the gold standard for diagnosing fibrosis. However, its invasive nature, potential complications, and sampling variability limit widespread use. Consequently, various non-invasive tests have been developed as alternatives for diagnosing fibrosis in NAFLD patients. AIM: This study aimed to compare the accuracy of non-invasive tests (NITs) and evaluate the diagnostic accuracy of acoustic radiation force impulse (ARFI), one of the point shear wave techniques, compared to conventional methods, assessing its effective role in diagnosis. METHODS: This is a retrospective study; a total of 136 patients diagnosed with fatty liver disease through ultrasonography were enrolled. The anthropometric data of the patients were collected on the day of admission and blood tests, measurements of ARFI, and a point shear test were conducted using abdominal ultrasound; a biopsy was performed the following day. In addition, we calculated the aspartate aminotransferase-to-platelet ratio index (APRI) index based on four factors (FIB-4) and the NAFLD fibrosis score (NFS). Subsequently, we assessed the diagnostic accuracy of NITs within various subgroups based on the extent of obesity, steatosis, or NAFLD activity score. RESULTS: ARFI has been shown to have the highest diagnostic value among various NITs, with AUROC values of 0.832, 0.794, 0.767, and 0.696 for ARFI, APRI, FIB-4, and NFS, respectively. In the morbidly obese subgroup, the AUROC values of ARFI, APRI, FIB-4, and NFS were 0.805, 0.769, 0.736, and 0.674. In the group with severe steatosis or non-alcoholic steatohepatitis (NASH), the AUROC values were 0.679, 0.596, 0.661, and 0.612, respectively, for severe steatosis and 0.789, 0.696, 0.751, and 0.691, respectively, for NASH. CONCLUSIONS: In conclusion, ARFI is not affected by various factors and maintains diagnostic accuracy compared to serum NITs. Therefore, we can recommend ARFI as a valuable diagnostic test to screen for advanced fibrosis in patients with NAFLD.

Identification of signature gene set as highly accurate determination of metabolic dysfunction-associated steatotic liver disease progression.

BACKGROUND/AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by fat accumulation in the liver. MASLD encompasses both steatosis and MASH. Since MASH can lead to cirrhosis and liver cancer, steatosis and MASH must be distinguished during patient treatment. Here, we investigate the genomes, epigenomes, and transcriptomes of MASLD patients to identify signature gene set for more accurate tracking of MASLD progression. METHODS: Biopsy-tissue and blood samples from patients with 134 MASLD, comprising 60 steatosis and 74 MASH patients were performed omics analysis. SVM learning algorithm were used to calculate most predictive features. Linear regression was applied to find signature gene set that distinguish the stage of MASLD and to validate their application into independent cohort of MASLD. RESULTS: After performing WGS, WES, WGBS, and total RNA-seq on 134 biopsy samples from confirmed MASLD patients, we provided 1,955 MASLD-associated features, out of 3,176 somatic variant callings, 58 DMRs, and 1,393 DEGs that track MASLD progression. Then, we used a SVM learning algorithm to analyze the data and select the most predictive features. Using linear regression, we identified a signature gene set capable of differentiating the various stages of MASLD and verified it in different independent cohorts of MASLD and a liver cancer cohort. CONCLUSION: We identified a signature gene set (i.e., CAPG, HYAL3, WIPI1, TREM2, SPP1, and RNASE6) with strong potential as a panel of diagnostic genes of MASLD-associated disease.

Six-Transmembrane Epithelial Antigen of Prostate 4: An Indicator of Prognosis and Tumor Immunity in Hepatocellular Carcinoma.

Purpose: The six-transmembrane epithelial antigen of prostate 4 (STEAP4) has been linked to tumor progression via its involvement in inflammatory responses, oxidative stress, and metabolism. However, STEAP4 has rarely been studied in hepatocellular carcinoma (HCC). We explored STEAP4 expression associated with tumor prognosis to understand its role in tumor biology in HCC. Patients and Methods: STEAP4 mRNA and protein expressions were primarily analyzed using bioinformatics tools based on The Cancer Genome Atlas database to understand the expression pattern, molecular mechanism, prognostic impact, and association with immune cell infiltration. We further investigated the association between STEAP4 protein expression and clinicopathological parameters and their predictive value in HCC patients using immunohistochemical staining of tissue microarrays. Results: The expression of STEAP4 mRNA and protein in HCC tissues was significantly lower than in normal liver tissues. Reduced expression of STEAP4 was linked to advanced HCC stages, poor recurrence-free survival (RFS), and overall survival. Furthermore, reduced STEAP4 expression was a significant predictor of worse RFS in univariate and multivariate analyses in the immunohistochemical cohort. GO, KEGG, and GSEA analyses revealed that STEAP4 is related to numerous biological processes and pathways, including drug metabolism, DNA replication, RNA metabolism, and immune response. In terms of the immune system, the decreased level of STEAP4 was correlated with the immunosuppressive microenvironment. Conclusion: Our data indicated that reduced STEAP4 expression was significantly associated with tumor aggressiveness and poor prognosis, possibly because of its link to various biological processes and induction of HCC immune evasion. Therefore, STEAP4 expression may serve as a potential prognostic biomarker for cancer progression and immunity, as well as a therapeutic target in HCC.

Targeted suicide gene therapy for liver cancer based on ribozyme-mediated RNA replacement through post-transcriptional regulation.

Hepatocellular carcinoma (HCC) has high fatality rate and limited therapeutic options. Here, we propose a new anti-HCC approach with high cancer-selectivity and efficient anticancer effects, based on adenovirus-mediated Tetrahymena group I trans-splicing ribozymes specifically inducing targeted suicide gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To confer potent anti-HCC effects and minimize hepatotoxicity, we constructed post-transcriptionally enhanced ribozyme constructs coupled with splicing donor and acceptor site and woodchuck hepatitis virus post-transcriptional regulatory element under the control of microRNA-122a (miR-122a). Adenovirus encoding post-transcriptionally enhanced ribozyme improved trans-splicing reaction and decreased human TERT (hTERT) RNA level, efficiently and selectively retarding hTERT-positive liver cancers. Adenovirus encoding miR-122a-regulated ribozyme caused selective liver cancer cytotoxicity, the efficiency of which depended on ribozyme expression level relative to miR-122a level. Systemic administration of adenovirus encoding the post-transcriptionally enhanced and miR-regulated ribozyme caused efficient anti-cancer effects at a single dose of low titers and least hepatotoxicity in intrahepatic multifocal HCC mouse xenografts. Minimal liver toxicity, tissue distribution, and clearance pattern of the recombinant adenovirus were observed in normal animals administered either systemically or via the hepatic artery. Post-transcriptionally regulated RNA replacement strategy mediated by a cancer-specific ribozyme provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Hepatic STAMP2 mediates recombinant FGF21-induced improvement of hepatic iron overload in nonalcoholic fatty liver disease.

Although previous studies have shown that the administration of fibroblast growth factor 21 (FGF21) reverses hepatic steatosis, the mechanism by which FGF21 exerts a therapeutic effect on nonalcoholic fatty liver disease (NAFLD) is not yet entirely understood. We previously demonstrated that hepatic six transmembrane protein of prostate 2 (STAMP2) may represent a suitable target for NAFLD. We investigated the mechanism underlying the therapeutic effect of recombinant FGF21 on NAFLD, focusing on the involvement of hepatic STAMP2. In this study, we used human nonalcoholic steatosis patient pathology samples, C57BL/6 mice for a high-fat diet (HFD)-induced in vivo NAFLD model, and used human primary hepatocytes and HepG2 cells for oleic acid (OA)-induced in vitro NAFLD model. We observed that recombinant FGF21 treatment ameliorated hepatic steatosis and insulin resistance through the upregulation of STAMP2 expression. We further observed hepatic iron overload (HIO) and reduced iron exporter, ferroportin expression in the liver samples obtained from human NAFLD patients, and HFD-induced NAFLD mice and in OA-treated HepG2 cells. Importantly, recombinant FGF21 improved HIO through the hepatic STAMP2-mediated upregulation of ferroportin expression. Our data suggest that hepatic STAMP2 may represent a suitable therapeutic intervention target for FGF21-induced improvement of NAFLD accompanying HIO.

A scoring system for the diagnosis of non-alcoholic steatohepatitis from liver biopsy.

BACKGROUND: Liver biopsy is the essential method to diagnose non-alcoholic steatohepatitis (NASH), but histological features of NASH are too subjective to achieve reproducible diagnoses in early stages of disease. We aimed to identify the key histological features of NASH and devise a scoring model for diagnosis. METHODS: Thirteen pathologists blindly assessed 12 histological factors and final histological diagnoses ('not-NASH,' 'borderline,' and 'NASH') of 31 liver biopsies that were diagnosed as non-alcoholic fatty liver disease (NAFLD) or NASH before and after consensus. The main histological parameters to diagnose NASH were selected based on histological diagnoses and the diagnostic accuracy and agreement of 12 scoring models were compared for final diagnosis and the NAFLD Activity Score (NAS) system. RESULTS: Inter-observer agreement of final diagnosis was fair (κ = 0.25) before consensus and slightly improved after consensus (κ = 0.33). Steatosis at more than 5% was the essential parameter for diagnosis. Major diagnostic factors for diagnosis were fibrosis except 1C grade and presence of ballooned cells. Minor diagnostic factors were lobular inflammation ( ≥ 2 foci/ × 200 field), microgranuloma, and glycogenated nuclei. All 12 models showed higher inter-observer agreement rates than NAS and post-consensus diagnosis (κ = 0.52-0.69 vs. 0.33). Considering the reproducibility of factors and practicability of the model, summation of the scores of major (× 2) and minor factors may be used for the practical diagnosis of NASH. CONCLUSIONS: A scoring system for the diagnosis of NAFLD would be helpful as guidelines for pathologists and clinicians by improving the reproducibility of histological diagnosis of NAFLD.

Clinicopathologic analysis of intraductal papillary neoplasm of bile duct: Korean multicenter cohort study.

BACKGROUND: IPNB is very rare disease and most previous studies on IPNB were case series with a small number due to low incidence. The aim of this study is to validate previously known clinicopathologic features of intraductal papillary neoplasm of bile duct (IPNB) based on the first largest multicenter cohort. METHODS: Among 587 patients previously diagnosed with IPNB and similar diseases from each center in Korea, 387 were included in this study after central pathologic review. We also reviewed all preoperative image data. RESULTS: Of 387 patients, 176 (45.5%) had invasive carcinoma and 21 (6.0%) lymph node metastasis. The 5-year overall survival was 80.9% for all patients, 88.8% for IPNB with mucosal dysplasia, and 70.5% for IPNB with invasive carcinoma. According to the "Jang & Kim's modified anatomical classification," 265 (68.5%) were intrahepatic, 103 (26.6%) extrahepatic, and 16 (4.1%) diffuse type. Multivariate analysis revealed that tumor invasiveness was a unique predictor for survival analysis. (p = 0.047 [hazard ratio = 2.116, 95% confidence interval 1.010-4.433]). CONCLUSIONS: This is the first Korean multicenter study on IPNB through central pathologic and radiologic review process. Although IPNB showed good long-term prognosis, relatively aggressive features were also found in invasive carcinoma and extrahepatic/diffuse type.

Analysis of intrahepatic sarcomatoid cholangiocarcinoma: Experience from 11 cases within 17 years.

BACKGROUND: Intrahepatic sarcomatoid chonalgiocarcinoma (s-CCC) is an extremely rare disease, accounting for less than 1% of hepatobiliary system malignancies, and its pathophysiology is not well known. On the hypothesis that its clinical, serologic, or radiologic diagnosis are not fully understood and its prognosis is poor, we investigated the distinguishing features of s-CCC compared with those of intrahepatic bile duct adenocarcinoma [cholangiocellular carcinoma (CCC)] in patients from a single center. AIM: To analyze the clinical, serologic, imaging, and histopathologic characteristics of intrahepatic s-CCC patients diagnosed in a single center. METHODS: The clinical, serologic, imaging, and histopathologic features of 227 patients diagnosed with intrahepatic cholangiocarcinoma (IHCC) in a single medical center during the last 17 years were analyzed. The characteristics of 11 patients with s-CCC were compared with those of 216 patients with CCC. RESULTS: The number of patients with s-CCC who presented fever and abdominal pain and past history of chronic viral hepatitis or liver cirrhosis (LC) was higher than that of patients with CCC. In imaging studies, patients with s-CCC showed relatively aggressive features. However, no clear distinction was observed between s-CCC and CCC based on other clinical, serologic or radiologic examination results. An accurate diagnosis could be made only via a histopathologic examination through immunohistochemical staining. The clinical course of s-CCC was generally aggressive, and patients had a relatively poor prognosis. CONCLUSION: In patients with s-CCC, early diagnosis through biopsy and aggressive treatment, including surgical resection, are important.

Peroxisome proliferator-activated receptor γ coactivator-1α is a predictor of lymph node metastasis and poor prognosis in human colorectal cancer.

Peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ coactivator-1α (PGC-1α) expression levels are correlated with clinical outcome in breast cancer. However, the potential biological and clinical significance of PPARγ and PGC-1α in colorectal cancer remains unknown. Here we investigated PPARγ and PGC-1α expression in colorectal cancer, and the associations of these expression levels with clinicopathological features. We also evaluated the roles of PPARγ and PGC-1α as prognostic factors in colorectal cancer. We performed immunohistochemical analysis to investigate PPARγ and PGC-1α expression in human colorectal cancer tissues and adjacent normal tissues from 108 primary colorectal cancer patients. We then examined how these expression levels correlated with clinicopathological features. Using the Kaplan-Meier method, we evaluated 3-year disease-free survival (DFS) and overall survival (OS) in patients with tumors expressing different levels of PPARγ and PGC-1α. Our results revealed that PPARγ expression was not significantly correlated with age at surgery, gender, differentiation, depth of infiltration, relapse, or TNM stage. Additionally, PGC-1α expression was not significantly correlated with age at surgery, differentiation, depth of infiltration, relapse, or TNM stage. However, PGC-1α expression was significantly correlated with nodal metastasis (p=0.020). Survival analysis demonstrated reduced OS in the PGC-1α-positive group compared to the PGC-1α-negative group (p=0.03). Our present findings suggest that PGC-1α may be useful for predicting nodal metastasis, and may represent a biomarker for poor prognosis in colorectal cancer.

In vitro and in vivo anti-leukemic effects of cladoloside C2 are mediated by activation of Fas/ceramide synthase 6/p38 kinase/c-Jun NH2-terminal kinase/caspase-8.

We previously demonstrated that the quinovose-containing hexaoside stichoposide C (STC) is a more potent anti-leukemic agent than the glucose-containing stichoposide D (STD), and that these substances have different molecular mechanisms of action. In the present study, we investigated the novel marine triterpene glycoside cladoloside C2 from Cladolabes schmeltzii, which has the same carbohydrate moiety as STC. We assessed whether cladoloside C2 could induce apoptosis in K562 and HL-60 cells. We also evaluated whether it showed antitumor action in mouse leukemia xenograft models, and its molecular mechanisms of action. We investigated the molecular mechanism behind cladoloside C2-induced apoptosis of human leukemia cells, and examined the antitumor effect of cladoloside C2 in a HL-60 and K562 leukemia xenograft model. Cladoloside C2 dose- and time-dependently induced apoptosis in the analyzed cells, and led to the activation of Fas/ceramide synthase 6 (CerS6)/p38 kinase/JNK/caspase-8. This cladoloside C2-induced apoptosis was partially blocked by specific inhibition by Fas, CerS6, and p38 siRNA transfection, and by specific inhibition of JNK by SP600125 or dominant negative-JNK transfection. Cladoloside C2 exerted antitumor activity through the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 without showing any toxicity in xenograft mouse models. The antitumor effect of cladoloside C2 was reversed in CerS6 shRNA-silenced xenograft models. Our results suggest that cladoloside C2 has in vitro and in vivo anti-leukemic effects due to the activation of Fas/CerS6/p38 kinase/JNK/caspase-8 in lipid rafts. These findings support the therapeutic relevance of cladoloside C2 in the treatment of human leukemia.

T-Cell Immunoglobulin Mucin 3 Expression on Tumor Infiltrating Lymphocytes as a Positive Prognosticator in Triple-Negative Breast Cancer.

PURPOSE: T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) is an emerging immune response molecule related to T-cell anergy. There has been tremendous interest in breast cancer targeting immune checkpoint molecules, especially in the triple-negative breast cancer (TNBC). This study was designed to investigate TIM-3 expression on tumor infiltrating lymphocytes (TILs), its relationships with clinicopathological para-meters and expression of programmed death receptor 1 (PD-1)/programmed death receptor ligand 1 (PD-L1), and its prognostic role. METHODS: Immunohistochemistry on tissue microarray blocks produced from 109 samples of invasive ductal carcinoma type TNBC was performed with antibodies toward TIM-3, PD-1, PD-L1 and breast cancer-related molecular markers. Associations between their expression and clinicopathological parameters as well as survival analyses were performed. RESULTS: TIM-3 was expressed in TILs from all 109 TNBCs, consisting of 17 cases (<5%), 31 cases (6%-25%), 48 cases (26%-50%), and 13 cases (>51%). High TIM-3 was significantly correlated with younger patients (p=0.0101), high TILs (p=0.0029), high tumor stage (p=0.0018), high PD-1 (p=0.0001) and high PD-L1 (p=0.0019), and tended to be associated with higher histologic grade, absence of extensive in situ components and microcalcification. High TIM-3 expression was significantly associated with a combinational immunophenotype group of high PD-L1 and high PD-1 (p<0.0001). High TIM-3 demonstrated a significantly better disease-free survival (DFS) (p<0.0001) and longer overall survival (OS) (p=0.0001), together with high TILs and high PD-1. In univariate survival analysis, high TIM-3 showed reduced relapse risk (p<0.0001) and longer OS (p=0.0003), together with high PD-1 expression. In multivariate analysis, high TIM-3 was statistically significant in predicting prognosis, showing better DFS (hazard ratio [HR], 0.0994; 95% confidence interval [CI], 0.0296-0.3337; p=0.0002) and longer OS (HR, 0.1109; 95% CI, 0.0314-0.3912; p=0.0006). CONCLUSION: In this study, we demonstrate that TIM-3 expression is an independent positive prognostic factor in TNBC, despite its association with poor clinical and pathologic features.

Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement.

Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation.

Clinicopathologic significance of tumor microenvironment CD11c, and FOXP3 expression in diffuse large B-cell lymphoma patients receiving rituximab, cyclophosphamide, anthracycline, vincristine, and prednisone (R-CHOP) combination chemotherapy.

BACKGROUND/AIMS: CD11c is a dendritic cell marker in humans, which potentially induces a cytotoxic effect on lymphoma cells. Forkhead boxP3 (FOXP3) is a regulator of T lymphocyte in the microenvironment of the lymphoma. The principal objective of this study was to determine whether the tumors' microenvironment expressions of CD11c and FOXP3 are predictive of clinical outcomes in diffuse large B-cell lymphoma (DLBCL) patients receiving treatment with rituximab, cyclophosphamide, anthracycline, vincristine, and prednisone (R-CHOP) combination chemotherapy. METHODS: The study population consisted of 100 patients with DLBCL. The CD11c and FOXP3 expression in primary tumors' microenvironment were evaluated using an immunohistochemistry (IHC). RESULTS: CD11c and FOXP3 expression positivity in microenvironment were 25% and 35%, respectively. Each one counted for 1 point. In CD11c and FOXP3 stain, positive was counted as 0 and negative was 1. The points were separated into low risk (0 to 1) and high risk (2) groups. Only the extranodal DLBCL patient group analysis conveyed significant differences of progression-free survival (p = 0.019) and overall survival (p = 0.039) between the two groups. CONCLUSIONS: We can achieve possible clinical significance of lymphoma tumor microenvironments through CD11c and FOXP3 IHC stains in extranodal DLBCL patients receiving R-CHOP therapy.

Interobserver Agreement on Pathologic Features of Liver Biopsy Tissue in Patients with Nonalcoholic Fatty Liver Disease.

BACKGROUND: The histomorphologic criteria for the pathological features of liver tissue from patients with non-alcoholic fatty liver disease (NAFLD) remain subjective, causing confusion among pathologists and clinicians. In this report, we studied interobserver agreement of NAFLD pathologic features and analyzed causes of disagreement. METHODS: Thirty-one cases of clinicopathologically diagnosed NAFLD from 10 hospitals were selected. One hematoxylin and eosin and one Masson's trichrome-stained virtual slide from each case were blindly reviewed with regard to 12 histological parameters by 13 pathologists in a gastrointestinal study group of the Korean Society of Pathologists. After the first review, we analyzed the causes of disagreement and defined detailed morphological criteria. The glass slides from each case were reviewed a second time after a consensus meeting. The degree of interobserver agreement was determined by multi-rater kappa statistics. RESULTS: Kappa values of the first review ranged from 0.0091-0.7618. Acidophilic bodies (k = 0.7618) and portal inflammation (k = 0.5914) showed high levels of agreement, whereas microgranuloma (k = 0.0984) and microvesicular fatty change (k = 0.0091) showed low levels of agreement. After the second review, the kappa values of the four major pathological features increased from 0.3830 to 0.5638 for steatosis grade, from 0.1398 to 0.2815 for lobular inflammation, from 0.1923 to 0.3362 for ballooning degeneration, and from 0.3303 to 0.4664 for fibrosis. CONCLUSIONS: More detailed histomorphological criteria must be defined for correct diagnosis and high interobserver agreement of NAFLD.

Image-aided Suicide Gene Therapy Utilizing Multifunctional hTERT-targeting Adenovirus for Clinical Translation in Hepatocellular Carcinoma.

Trans-splicing ribozyme enables to sense and reprogram target RNA into therapeutic transgene and thereby becomes a good sensing device for detection of cancer cells, judging from transgene expression. Previously we proposed PEPCK-Rz-HSVtk (PRT), hTERT targeting trans-splicing ribozyme (Rz) driven by liver-specific promoter phosphoenolpyruvate carboxykinase (PEPCK) with downstream suicide gene, herpes simplex virus thymidine kinase (HSVtk) for hepatocellular carcinoma (HCC) gene therapy. Here, we describe success of a re-engineered adenoviral vector harboring PRT in obtaining greater antitumor activity with less off-target effect for clinical application as a theranostics. We introduced liver-selective apolipoprotein E (ApoE) enhancer to the distal region of PRT unit to augment activity and liver selectivity of PEPCK promoter, and achieved better transduction into liver cancer cells by replacement of serotype 35 fiber knob on additional E4orf1-4 deletion of E1&E3-deleted serotype 5 back bone. We demonstrated that our refined adenovirus harboring PEPCK/ApoE-Rz-HSVtk (Ad-PRT-E) achieved great anti-tumor efficacy and improved ability to specifically target HCC without damaging normal hepatocytes. We also showed noninvasive imaging modalities were successfully employed to monitor both how well a therapeutic gene (HSVtk) was expressed inside tumor and how effectively a gene therapy took an action in terms of tumor growth. Collectively, this study suggests that the advanced therapeutic adenoviruses Ad-PRT-E and its image-aided evaluation system may lead to the powerful strategy for successful clinical translation and the development of clinical protocols for HCC therapy.

By activating Fas/ceramide synthase 6/p38 kinase in lipid rafts, stichoposide D inhibits growth of leukemia xenografts.

Stichoposide D (STD) is a marine triterpene glycoside isolated from sea cucumbers. We examined the molecular mechanisms underlying the antitumor activity of STD in human leukemia cells. The role of Fas (CD95), ceramide synthase 6 (CerS6) and p38 kinase during STD-induced apoptosis was examined in human leukemia cells. In addition, the antitumor effects of STD in K562 and HL-60 leukemia xenograft models were investigated. We found that STD induces Fas translocation to lipid rafts, and thus mediates cell apoptosis. We also observed the activation of CerS6 and p38 kinase during STD-induced apoptosis. The use of methyl-ß-cyclodextrin and nystatin to disrupt lipid rafts prevents the clustering of Fas and the activation of CerS6 and p38 kinase, and also inhibits STD-induced apoptosis. Specific inhibition by Fas, CerS6, and p38 kinase siRNA transfection partially blocked STD-induced apoptosis. In addition, STD has antitumor activity through the activation of CerS6 and p38 kinase without displaying any toxicity in HL-60 and K562 xenograft models. We observed that the anti-tumor effect of STD is partially prevented in CerS6 shRNA-silenced xenograft models. We first report that Fas/CerS6/p38 kinase activation in lipid rafts by STD is involved in its anti-leukemic activity. We also established that STD is able to enhance the chemosensitivity of K562 cells to etoposide or Ara-C. These data suggest that STD may be used alone or in combination with other chemotherapeutic agents to treat leukemia.

Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Tumor-to-muscle ratio of 8F-FDG PET for predicting histologic features and recurrence of HCC.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) recurrence is observed in up to 70-80% of patients despite a curative treatment. Microvascular invasion (MVI) and poor differentiation are strong risk factors for recurrence, but these cannot be known preoperatively. The aim of this study was to investigate the correlation of 18F-FDG PET with MVI and differentiation, and predictive role of tumor-to-background ratio of PET for recurrence in HCC. METHODOLOGY: Fifty-four patients had 18F-FDG PET/CT study before surgical resection as a first treatment of HCC between December 2008 and December 2012. We analyzed the predictive role of metabolic parameters of PET for recurrence of HCC. Maximal standardized uptake value, tumor-to-nontumor ratio, tumor-to-muscle ratio (TMR) and tumor-to-blood ratio were tested as metabolic index of 18F-FDG PET. RESULTS: Twenty-seven patients had increased uptake in preoperative PET and 14 (51.9%) of them experienced the recurrence. Increased uptake in PET and TMR were associated with MVI (p = 0.04, p = 0.005) and histologic differentiation (p = 0.018, p = 0.002). MVI was the only predictive factor for re- currence in multivariate analysis although TMR ≥ 6.36 showed a favorable result despite no statistical significance (p = 0.061). CONCLUSIONS: Increased 18F-FDG uptake of HCC, especially high TMR might be correlated with MVI and poor differentiation, and tends to have a risk for recurrence in HCC.

Overexpression of PGC­1α enhances cell proliferation and tumorigenesis of HEK293 cells through the upregulation of Sp1 and Acyl-CoA binding protein.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC­1α), a coactivator interacting with multiple transcription factors, regulates several metabolic processes. Although recent studies have focused on the role of PGC­1α in cancer, the underlying molecular mechanism has not been clarified. Therefore, we evaluated the role of PGC­1α in cell proliferation and tumorigenesis using human embryonic kidney (HEK)293 cells and colorectal cancer cells. We established stable HEK293 cell lines expressing PGC­1α and examined cell proliferation, anchorage-independent growth, and oncogenic potential compared to parental HEK293 cells. To identify the molecular PGC­1α targets for increased cell proliferation and tumorigenesis, the GeneFishing™ DEG (differentially expressed genes) screening system was used. Western blot analysis and immunofluorescence staining were performed for a regulated gene product to confirm the results. Forced expression of PGC­1α in HEK293 cells promoted cell proliferation and anchorage-independent growth in soft agar. In addition, HEK293 cells that highly expressed PGC­1α showed enhanced tumor formation when subcutaneously injected into the bilateral flanks of immunodeficient mice. The results of the GeneFishing DEG screening system identified one upregulated gene (Acyl-CoA binding protein; ACBP). Real-time RT-PCR, western blot analysis, and immunofluorescence staining showed that ACBP was markedly increased in HEK293 cells stably overexpressing PGC­1α (PGC­1α-HEK293 cells) compared to those expressing an empty vector. In PGC­1α, ACBP, and specificity protein 1 (Sp1) siRNA knockdown experiments in PGC­1α-HEK293 and SNU-C4 cells, we also observed inhibition of cell proliferation, reduced expression of antioxidant enzymes, and increased H2O2-induced reactive oxygen species production and apoptosis. These findings suggest that PGC­1α may promote cell proliferation and tumorigenesis through upregulation of ACBP. We provide evidence that increased Sp1 expression might contribute to enhanced ACBP expression by PGC­1α. The current results also suggest that PGC­1α, whose expression is related to enhanced cell proliferation and tumorigenesis, may be a good candidate molecular target for cancer therapy.

Targeted anticancer effect through microRNA-181a regulated tumor-specific hTERT replacement.

We previously generated a group I intron-based ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to stimulate transgene activity in cancer cells expressing the target RNA via an accurate and specific trans-splicing reaction. One of the major concerns of the hTERT RNA targeting anti-cancer approach is the potential side effects to hTERT(+) hematopoietic stem cell-derived blood cells. Thus, here we modified the ribozyme by inserting target sites against microRNA-181a, which is a blood cell-specific microRNA, downstream of its 3' exon. The specificity of transgene induction and anticancer activity in hTERT(+) cancer cells improved significantly with the modified ribozyme, resulting in selective targeting of hTERT(+) cancer cells, but not hematopoietic cells even if they are hTERT-positive. Importantly, the trans-splicing reaction of the microRNA-regulated ribozyme worked equally well in a nude mouse model of hepatocarcinoma-derived intrasplenic carcinomatosis, inducing highly specific expression of a therapeutic transgene and efficiently regressing hTERT-positive liver tumors with minimal liver toxicity when systemically delivered with an adenoviral vector encoding the ribozyme. These results suggest that a combined approach of microRNA regulation with targeted RNA replacement is more useful for effective anti-cancer treatment.