Barrier to autointegration factor 1, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3, and splicing factor 3b subunit 4 as early-stage cancer decision markers and drivers of hepatocellular carcinoma.

An accurate tool enabling early diagnosis of hepatocellular carcinoma (HCC) is clinically important, given that early detection of HCC markedly improves survival. We aimed to investigate the molecular markers underlying early progression of HCC that can be detected in precancerous lesions. We designed a gene selection strategy to identify potential driver genes by integrative analysis of transcriptome and clinicopathological data of human multistage HCC tissues, including precancerous lesions, low- and high-grade dysplastic nodules. The gene selection process was guided by detecting the selected molecules in both HCC and precancerous lesion. Using various computational approaches, we selected 10 gene elements as a candidate and, through immunohistochemical staining, showed that barrier to autointegration factor 1 (BANF1), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (PLOD3), and splicing factor 3b subunit 4 (SF3B4) are HCC decision markers with superior capability to diagnose early-stage HCC in a large cohort of HCC patients, as compared to the currently popular trio of HCC diagnostic markers: glypican 3, glutamine synthetase, and heat-shock protein 70. Targeted inactivation of BANF1, PLOD3, and SF3B4 inhibits in vitro and in vivo liver tumorigenesis by selectively modulating epithelial-mesenchymal transition and cell-cycle proteins. Treatment of nanoparticles containing small-interfering RNAs of the three genes suppressed liver tumor incidence as well as tumor growth rates in a spontaneous mouse HCC model. We also demonstrated that SF3B4 overexpression triggers SF3b complex to splice tumor suppressor KLF4 transcript to nonfunctional skipped exon transcripts. This contributes to malignant transformation and growth of hepatocyte through transcriptional inactivation of p27Kip1 and simultaneously activation of Slug genes. CONCLUSION: The findings suggest molecular markers of BANF1, PLOD3, and SF3B4 indicating early-stage HCC in precancerous lesion, and also suggest drivers for understanding the development of hepatocarcinogenesis. (Hepatology 2018;67:1360-1377).

MicroRNA-495-3p functions as a tumor suppressor by regulating multiple epigenetic modifiers in gastric carcinogenesis.

MicroRNAs (miRNAs) engage in complex interactions with the machinery that controls the transcriptome and concurrently target multiple mRNAs. Here, we demonstrate that microRNA-495-3p (miR-495-3p) functions as a potent tumor suppressor by governing ten oncogenic epigenetic modifiers (EMs) in gastric carcinogenesis. From the large cohort transcriptome datasets of gastric cancer (GC) patients available from The Cancer Genome Atlas (TCGA) and the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), we were able to recapitulate 15 EMs as significantly overexpressed in GC among the 51 EMs that were previously reported to be involved in cancer progression. Computational target prediction yielded miR-495-3p, which targets as many as ten of the 15 candidate oncogenic EMs. Ectopic expression of miRNA mimics in GC cells caused miR-495-3p to suppress ten EMs, and inhibited tumor cell growth and proliferation via caspase-dependent and caspase-independent cell death processing. In addition, in vitro metastasis assays showed that miR-495-3p plays a role in the metastatic behavior of GC cells by regulating SLUG, vimentin, and N-cadherin. Furthermore, treatment of GC cells with 5-aza-2'-deoxcytidine restored miR-495-3p expression; sequence analysis revealed hypermethylation of the miR-495-3p promoter region in GC cells. A negative regulatory loop is proposed, whereby DNMT1, among ten oncogenic EMs, regulates miR-495-3p expression via hypermethylation of the miR-495-3p promoter. Our findings suggest that the functional loss or suppression of miR-495-3p triggers overexpression of multiple oncogenic EMs, and thereby contributes to malignant transformation and growth of gastric epithelial cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gastrokine 1 protein is a potential theragnostic target for gastric cancer.

BACKGROUND: Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer. METHODS: We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay. RESULTS: In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas. CONCLUSIONS: The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.

An exploratory analysis for Lean and Six Sigma implementation in hospitals: Together is better?

BACKGROUND: Despite the increasing interest for Lean and Six Sigma implementations in hospitals, there has been little empirical evidence that goes beyond descriptive case studies to address the current status and the effectiveness of the implementations. PURPOSE: The aim of this study was to explore existing patterns of Lean and Six Sigma implementation in U.S. hospitals and compare the performance of the different patterns. METHODOLOGY/APPROACH: We collected data from 215 U.S. hospitals via a survey that includes measurement items developed from related literature. Using the cross-sectional data, we conducted a cluster analysis, followed by t tests, chi-square tests, and regression analyses for cluster verification. RESULTS: The cluster analysis identifies two clusters, a Moderate Six Sigma group and a Lean Six Sigma group. Results show that the Lean Six Sigma group outperforms the Moderate Six Sigma group across many performance dimensions: responsiveness capability, patient safety, and possibly cost saving. In addition, the Lean Six Sigma group tends to be composed of larger, private teaching hospitals located in more urban areas, and they employ more resources for quality improvement. CONCLUSION: Our research contributes to the quality management literature by supporting the possible complementary relationship between Lean and Six Sigma in hospitals. PRACTICE IMPLICATIONS: Our study encourages practitioners and managers to pay more attention to Lean implementation. Although Lean seems to be conducted in a limited fashion in many hospitals, it should be expanded and combined with Six Sigma for better results.

Gastrokine 1 inhibits gastric cancer cell migration and invasion by downregulating RhoA expression.

BACKGROUND: We investigated whether GKN1, a gastric tumor suppressor, contributes to the progression of gastric cancer by regulating RhoA expression. METHODS: We analyzed the expression of GKN1, RhoA, miR-185, and miR-34a in 35 gastric cancer tissues, and compared their expression with T category and TNM stage. Cell migration and invasion, as well as the expression of epithelial-to-mesenchymal transition (EMT)-related proteins, were assessed in GKN1- and RhoA small interfering RNA (siRhoA)-transfected and recombinant-GKN1-treated AGS and MKN1 gastric cancer cells. RESULTS: Expression of RhoA protein and messenger RNA (mRNA) was increased in 15 (42.9 %) and 17 (48.6 %) of 35 gastric cancer tissues respectively, and was associated with higher T category and TNM stage. GKN1 expression was significantly decreased in 27 gastric cancers (77.1 %) with a higher T category, and was inversely correlated with RhoA mRNA expression. In AGS and MKN1 cells, GKN1 expression increased miR-185 and miR-34a expression and reduced RhoA mRNA and protein expression. A positive relationship between GKN1 and miR-34a and miR-185 expression and an inverse relationship between miR-34a and RhoA expression were observed in gastric cancer tissues. Cell migration and invasiveness were markedly decreased in GKN1- and siRhoA-transfected cells. GKN1 expression and silencing of RhoA decreased the expression of the proteins Snail, Slug, and vimentin. Furthermore, miR-185 and miR-34a silencing in MKN1 cells transfected with GKN1 stimulated cell migration and invasion, and increased the expression of EMT-related proteins. CONCLUSION: Our data suggest that GKN1 may inhibit gastric cancer cell migration and invasion by downregulating RhoA expression in a miR-185- and miR-34a-dependent manner.

Heterodimeric interaction between GKN2 and TFF1 entails synergistic antiproliferative and pro-apoptotic effects on gastric cancer cells.

BACKGROUND: GKN2 and TFF1 form a heterodimer that is only generated in the mucus-secreting cells of the normal stomach. The formation of this heterodimer is frequently disrupted in gastric cancer. However, the precise roles of GKN2 alone and in the heterodimer with TFF1 as well as the contributions of GKN2 and the heterodimer to gastric carcinogenesis are poorly understood. METHODS: Cell viability, proliferation, and apoptosis were analyzed in AGS, MKN1, MKN28, and MKN45 gastric cancer cells transfected with GKN2 and/or TFF1 using MTT, BrdU incorporation, and apoptosis assays, respectively. In addition, cell viability was examined in HFE-145 non-neoplastic gastric epithelial cells after GKN2 and/or TFF1 silencing. Furthermore, the cell cycle and the expression of cell cycle and apoptosis related proteins were assessed. The interaction between GKN2 and TFF1 was confirmed by co-immunoprecipitation. Immunohistochemistry was employed to explore TFF1 expression in 169 gastric cancer tissues. RESULTS: Co-transfection with GKN2 and TFF1 significantly inhibited cell viability and proliferation by inducing G1/S cell cycle arrest and suppressing positive cell cycle regulators. Simultaneous knockdown of GKN2 and TFF1 in HFE-145 cells resulted in markedly increased cell viability. Moreover, the interaction of GKN2 and TFF1 promoted cell death by enhancing caspase-3/7 activity and upregulating pro-apoptotic proteins. At the mRNA level, GKN2 and TFF1 were found to be positively correlated in non-tumor and tumor samples. Immunohistochemistry revealed loss of TFF1 expression in 128 (75.73%) of 169 gastric cancers. There was a borderline-significant association between GKN2 and TFF1 protein expression in gastric cancers (P = 0.0598). CONCLUSION: Collectively, our data demonstrated that the interaction between GKN2 and TFF1 can have synergistic antiproliferative and pro-apoptotic effects on gastric cancer.

Gastrokine 1 inhibits gastrin-induced cell proliferation.

BACKGROUND: Gastrokine 1 (GKN1) acts as a gastric tumor suppressor. Here, we investigated whether GKN1 contributes to the maintenance of gastric mucosal homeostasis by regulating gastrin-induced gastric epithelial cell growth. METHODS: We assessed the effects of gastrin and GKN1 on cell proliferation in stable AGS(GKN1) and MKN1(GKN1) gastric cancer cell lines and HFE-145 nonneoplastic epithelial cells. Cell viability and proliferation were analyzed by MTT and BrdU incorporation assays, respectively. Cell cycle and expression of growth factor receptors were examined by flow cytometry and Western blot analyses. RESULTS: Gastrin treatment stimulated a significant time-dependent increase in cell viability and proliferation in AGS(mock) and MKN1(mock), but not in HFE-145, AGS(GKN1), and MKN1(GKN1), cells, which stably expressed GKN1. Additionally, gastrin markedly increased the S-phase cell population, whereas GKN1 significantly inhibited the effect of gastrin by regulating the expression of G1/S cell-cycle regulators. Furthermore, gastrin induced activation of the NF-kB and ß-catenin signaling pathways and increased the expression of CCKBR, EGFR, and c-Met in AGS and MKN1 cells. However, GKN1 completely suppressed these effects of gastrin via downregulation of gastrin/CCKBR/growth factor receptor expression. Moreover, GKN1 reduced gastrin and CCKBR mRNA expression in AGS and MKN1 cells, and there was an inverse correlation between GKN1 and gastrin, as well as between GKN1 and CCKBR mRNA expression in noncancerous gastric mucosae. CONCLUSION: These data suggest that GKN1 may contribute to the maintenance of gastric epithelial homeostasis and inhibit gastric carcinogenesis by downregulating the gastrin-CCKBR signaling pathway.

MicroRNA-221 governs tumor suppressor HDAC6 to potentiate malignant progression of liver cancer.

BACKGROUND & AIMS: Most common reason behind changes in histone deacetylase (HDAC) function is its overexpression in cancer. However, among HDACs in liver cancer, HDAC6 is uniquely endowed with a tumor suppressor, but the mechanism underlying HDAC6 inactivation has yet to be uncovered. METHODS: Microarray profiling and target prediction programs were used to identify miRNAs targeting HDAC6. A series of inhibitors, activators and siRNAs was introduced to validate regulatory mechanisms for microRNA-221-3p (miR-221) governing HDAC6 in hepatocarcinogenesis. RESULTS: Comprehensive miRNA profiling analysis identified seven putative endogenous miRNAs that are significantly upregulated in hepatocellular carcinoma (HCC). While miR-221 was identified as a suppressor of HDAC6 by ectopic expression of miRNA mimics in Dicer knockdown cells, targeted-disruption of miR-221 repressed cancer cell growth through derepressing HDAC6 expression. Suppression of HDAC6 via miR-221 was induced by JNK/c-Jun signaling in liver cancer cells but not in normal hepatic cells. Additionally, cytokine-induced NF-κBp65 independently regulated miR-221, thereby suppressing HDAC6 expression in HCC cells. HCC tissues derived from chemical-induced rat and H-ras12V transgenic mice liver cancer models validated that JNK/c-Jun activation and NF-κBp65 nuclear translocation are essential for the transcription of miR-221 leading to repression of HDAC6 in HCC. CONCLUSIONS: Our findings suggest that the functional loss or suppression of the tumor suppressor HDAC6 is caused by induction of miR-221 through coordinated JNK/c-Jun- and NF-κB-signaling pathways during liver tumorigenesis, providing a novel target for the molecular treatment of liver malignancies.

Characteristic molecular and proteomic signatures of drug-induced liver injury in a rat model.

Drug-induced liver injury (DILI) is a major safety concern during drug development and remains one of the main reasons for withdrawal of drugs from the market. Although it is crucial to develop methods that will detect potential hepatotoxicity of drug candidates as early and as quickly as possible, there is still a lack of sensitive and specific biomarkers for DILI that consequently leads to a scarcity of reliable hepatotoxic data. Hence, in this study, we assessed characteristic molecular signatures in rat liver treated with drugs (pyrazinamide, ranitidine, enalapril, carbamazepine and chlorpromazine) that are known to cause DILI in humans. Unsupervised hierarchical clustering analysis of transcriptome changes induced by DILI-causing drugs resulted in three different subclusters on dendrogram, i.e., hepatocellular, cholestatic and mixed type of DILI at early time points (2 days), and multiclassification analysis suggested 31 genes as discernible markers for each DILI pattern. Further analysis for characteristic molecular signature of each DILI pattern provided a molecular basis for different modes of DILI action. A proteomics study of the same rat livers was used to confirm the results, and the two sets of data showed 60 matching classifiers. In conclusion, the data of different DILI-causing drug treatments from genomic analysis in a rat model suggest that DILI-specific molecular signatures can discriminate different patterns of DILI at an early exposure time point, and that they provide useful information for mechanistic studies that may lead to a better understanding of the molecular basis of DILI.

Gastrokine 1 inhibits the carcinogenic potentials of Helicobacter pylori CagA.

Helicobacter pylori CagA directly injected by the bacterium into epithelial cells via a type IV secretion system, leads to cellular changes such as morphology, apoptosis, proliferation and cell motility, and stimulates gastric carcinogenesis. We investigated the effects of cytotoxin-associated gene A (CagA) and gastrokine 1 (GKN1) on cell proliferation, apoptosis, reactive oxygen species (ROS) production, epithelial-mesenchymal transition (EMT) and cell migration in CagA- or GKN1-transfected gastric epithelial cells and mucosal tissues from humans and mice infected with H.pylori. On the molecular level, H.pylori CagA induced increased cell proliferation, ROS production, antiapoptotic activity, cell migration and invasion. Moreover, CagA induced activation of NF-κB and PI3K/Akt signaling pathways and EMT-related proteins. In addition, H.pylori CagA reduced GKN1 gene copy number and expression in gastric cells and mucosal tissues of humans and mice. However, GKN1 overexpression successfully suppressed the carcinogenic effects of CagA through binding to CagA. These results suggest that GKN1 might be a target to inhibit the effects from H.pylori CagA.

GKN2 contributes to the homeostasis of gastric mucosa by inhibiting GKN1 activity.

Gastrokine 1 (GKN1) plays an important role in maintaining gastric mucosa integrity. Here, we investigated whether gastrokine 2 (GKN2) contributes to the homeostasis of gastric epithelial cells by regulating GKN1 activity. We analyzed cell viability, proliferation, and death in AGS cells transfected with GKN1, GKN2, GKN1 plus GKN2 using MTT, BrdU incorporation, and apoptosis assays, respectively. In addition, the expression levels of the cell cycle- and apoptosis-related proteins, miR-185, DNMT1, and EZH2 were determined. We also compared the expression of GKN1, GKN2, and CagA in 50 non-neoplastic gastric mucosae and measured GKN2 expression in 169 gastric cancers by immunohistochemistry. GKN2 inhibited anti-proliferative and pro-apoptotic activities, miR-185 induction, and anti-epigenetic modifications of GKN1. There was a positive correlation between GKN1 and GKN2 expression (P = 0.0074), and the expression of GKN1, but not GKN2, was significantly lower in Helicobacter pylori CagA-positive gastric mucosa (P = 0.0013). Interestingly, ectopic GKN1 expression in AGS cells increased GKN2 mRNA and protein expression in a time-dependent manner (P = 0.01). Loss of GKN2 expression was detected in 126 (74.6%) of 169 gastric cancers by immunohistochemical staining and was closely associated with GKN1 expression and differentiation of gastric cancer cells (P = 0.0002 and P = 0.0114, respectively). Overall, our data demonstrate that in the presence of GKN2, GKN1 loses its ability to decrease cell proliferation, induce apoptosis, and inhibit epigenetic alterations in gastric cancer cells. Thus, we conclude that GKN2 may contribute to the homeostasis of gastric epithelial cells by inhibiting GKN1 activity.

Sirtuin7 oncogenic potential in human hepatocellular carcinoma and its regulation by the tumor suppressors MiR-125a-5p and MiR-125b.

UNLABELLED: Sirtuins are nicotinamide adenine dinucleotide oxidized form (NAD(+) )-dependent deacetylases and function in cellular metabolism, stress resistance, and aging. For sirtuin7 (SIRT7), a role in ribosomal gene transcription is proposed, but its function in cancer has been unclear. In this study we show that SIRT7 expression was up-regulated in a large cohort of human hepatocellular carcinoma (HCC) patients. SIRT7 knockdown influenced the cell cycle and caused a significant increase of liver cancer cells to remain in the G1 /S phase and to suppress growth. This treatment restored p21(WAF1/Cip1) , induced Beclin-1, and repressed cyclin D1. In addition, sustained suppression of SIRT7 reduced the in vivo tumor growth rate in a mouse xenograft model. To explore mechanisms in SIRT7 regulation, microRNA (miRNA) profiling was carried out. This identified five significantly down-regulated miRNAs in HCC. Bioinformatics analysis of target sites and ectopic expression in HCC cells showed that miR-125a-5p and miR-125b suppressed SIRT7 and cyclin D1 expression and induced p21(WAF1/Cip1) -dependent G1 cell cycle arrest. Furthermore, treatment of HCC cells with 5-aza-2'-deoxycytidine or ectopic expression of wildtype but not mutated p53 restored miR-125a-5p and miR-125b expression and inhibited tumor cell growth, suggesting their regulation by promoter methylation and p53 activity. To show the clinical significance of these findings, mutations in the DNA binding domain of p53 and promoter methylation of miR-125b were investigated. Four out of nine patients with induced SIRT7 carried mutations in the p53 gene and one patient showed hypermethylation of the miR-125b promoter region. CONCLUSION: Our findings suggest the oncogenic potential of SIRT7 in hepatocarcinogenesis. A regulatory loop is proposed whereby SIRT7 inhibits transcriptional activation of p21(WAF1/Cip1) by way of repression of miR-125a-5p and miR-125b. This makes SIRT7 a promising target in cancer therapy. (HEPATOLOGY 2013).

Aberrant expression of SOX9 is associated with gastrokine 1 inactivation in gastric cancers.

BACKGROUND: SOX9 belongs to the SOX [sry-related high-mobility group (HMG) box] family and acts as a transcription factor that plays a central role in the development and differentiation of multiple cell lineages. The aim of this study was to determine whether the GKN1 gene is involved in the development of gastric cancer by regulating SOX9. METHODS: The effect of GKN1 and ß-catenin on SOX9 expression was examined in GKN1 and ß-catenin-transfected AGS and MKN-1 gastric cancer cells. SOX9 expression was also determined in gastric cancer tissues and cell lines by Western blot analysis and immunohistochemistry. RESULTS: Ectopic expression of ß-catenin induced increased expression of SOX9 in AGS cells, whereas GKN1 decreased expression of SOX9 in AGS and MKN-1 cells. In addition, we found an inverse correlation between expression of SOX9 and GKN1 in gastric cancer tissues and cell lines. In immunohistochemistry, nuclear SOX9 expression was detected in 64 (34.6 %) of 185 gastric carcinomas and its expression was closely associated with GKN1 immunonegativity. There was no significant relationship between altered expression of SOX9 protein and clinicopathological parameters including overall survival. CONCLUSION: These data suggest that aberrant SOX9 expression by GKN1 inactivation may be involved in the development of sporadic gastric cancers as an early event.

Gastrokine 1 regulates NF-κB signaling pathway and cytokine expression in gastric cancers.

Gastrokine 1 (GKN1) plays an important role in the gastric mucosal defense mechanism and also acts as a functional gastric tumor suppressor. In this study, we examined the effect of GKN1 on the expression of inflammatory mediators, including NF-κB, COX-2, and cytokines in GKN1-transfected AGS cells and shGKN1-transfected HFE-145 cells. Lymphocyte migration and cell viability were also analyzed after treatment with GKN1 and inflammatory cytokines in AGS cells by transwell chemotaxis and an MTT assay, respectively. In GKN1-transfected AGS cells, we observed inactivation and reduced expression of NF-κB and COX-2, whereas shGKN1-transfected HFE-145 cells showed activation and increased expression of NF-κB and COX-2. GKN1 expression induced production of inflammatory cytokines including IL-8 and -17A, but decreased expression of IL-6 and -10. We also found IL-17A expression in 9 (13.6%) out of 166 gastric cancer tissues and its expression was closely associated with GKN1 expression. GKN1 also acted as a chemoattractant for the migration of Jurkat T cells and peripheral B lymphocytes in the transwell assay. In addition, GKN1 significantly reduced cell viability in both AGS and HFE-145 cells. These data suggest that the GKN1 gene may inhibit progression of gastric epithelial cells to cancer cells by regulating NF-κB signaling pathway and cytokine expression.

Functional analysis of the NH2-terminal hydrophobic region and BRICHOS domain of GKN1.

Gastrokine 1 (GKN1) protects the gastric antral mucosa and promotes healing by facilitating restitution and proliferation after injury. GKN1 is down-regulated in Helicobacter pylori-infected gastric epithelial cells and loss of GKN1 expression is tightly associated with gastric carcinogenesis. However, the underlying mechanisms as a tumor suppressor are largely unknown. Presently, the hydrophobic region and BRICHOS domain of GKN1, pGKN1(D13N), pGKN1(Δ68-199), and pGKN1(Δ1-67,165-199) were shown to suppress gastric cancer cell growth and recapitulate GKN1 functions. As well, the hydrophobic region and BRICHOS domain of GKN1 had a synergistic anti-cancer effect with 5-FU on tumor cell growth, implying that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for tumor suppression, thereby suggesting a therapeutic intervention for gastric cancer. Also, its domain inducing endogenous miR-185 directly targeted the epigenetic effectors DNMT1 and EZH2 in gastric cancer cells. Our results suggest that the NH2-terminal hydrophobic region and BRICHOS domain of GKN1 are sufficient for its tumor suppressor activities.

Histone deacetylase 6 functions as a tumor suppressor by activating c-Jun NH2-terminal kinase-mediated beclin 1-dependent autophagic cell death in liver cancer.

UNLABELLED: Ubiquitin-binding histone deacetylase 6 (HDAC6) is uniquely endowed with tubulin deacetylase activity and plays an important role in the clearance of misfolded protein by autophagy. In cancer, HDAC6 has become a target for drug development due to its major contribution to oncogenic cell transformation. In the present study we show that HDAC6 expression was down-regulated in a large cohort of human hepatocellular carcinoma (HCC) patients, and that low expression of HDAC6 was significantly associated with poor prognosis of HCC patients in 5-year overall, disease-free, and recurrence-free survival. Notably, we observed that ectopic overexpression of HDAC6 suppressed tumor cell growth and proliferation in various liver cancer cells, and elicited increased LC3B-II conversion and autophagic vacuole formation without causing apoptotic cell death or cell cycle inhibition. In addition, the sustained overexpression of HDAC6 reduced the in vivo tumor growth rate in a mouse xenograft model. It was also found that HDAC6 mediated autophagic cell death by way of Beclin 1 and activation of the LC3-II pathway in liver cancer cells, and that HDAC6 overexpression activated c-Jun NH2-terminal kinase (JNK) and increased the phosphorylation of c-Jun. In contrast, the induction of Beclin 1 expression was blocked by SP600125 (a specific inhibitor of JNK) or by small interfering RNA directed against HDAC6. CONCLUSION: Our findings suggest that loss of HDAC6 expression in human HCCs and tumor suppression by HDAC6 occur by way of activation of caspase-independent autophagic cell death through the JNK/Beclin 1 pathway in liver cancer and, thus, that a novel tumor suppressor function mechanism involving HDAC6 may be amenable to nonepigenetic regulation.

Characteristic molecular signatures of early exposure to volatile organic compounds in rat liver.

OBJECTIVE: Investigation on whether the characteristic molecular signatures can discriminate individual volatile organic compounds (VOCs) and provide predictive markers for the detection of VOC exposure. METHODS: Transcriptomic analysis of liver tissues was performed 48 h after the single oral administration of three VOCs doses at LD25 or LD5 values, to Sprague-Dawley. RESULTS: Combination analysis of different multi-classifications suggested that 145 genes predicted VOC exposure. Additionally, Gene Set Enrichment Analysis of genes deregulated by VOCs revealed that T cell prolymphatic leukemia signaling was inactivated in all VOCs. CONCLUSIONS: These molecular markers could be widely implemented to assess and predict environmental exposure to VOCs.

HDAC2 overexpression confers oncogenic potential to human lung cancer cells by deregulating expression of apoptosis and cell cycle proteins.

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses, and is often significantly overexpressed in solid tumors, but little is known of its role in human lung cancer. In this study, we demonstrated the aberrant expression of HDAC2 in lung cancer tissues and investigated oncogenic properties of HDAC2 in human lung cancer cell lines. HDAC2 inactivation resulted in regression of tumor cell growth and activation of cellular apoptosis via p53 and Bax activation and Bcl2 suppression. In cell cycle regulation, HDAC2 inactivation caused induction of p21WAF1/CIP1 expression, and simultaneously suppressed the expressions of cyclin E2, cyclin D1, and CDK2, respectively. Consequently, this led to the hypophosphorylation of pRb protein in G1/S transition and thereby inactivated E2F/DP1 target gene transcriptions of A549 cells. In addition, we demonstrated that HDAC2 directly regulated p21WAF1/CIP1 expression in a p53-independent manner. However, HDAC1 was not related to p21WAF1/CIP1 expression and tumorigenesis of lung cancer. Lastly, we observed that sustained-suppression of HDAC2 in A549 lung cancer cells attenuated in vitro tumorigenic properties and in vivo tumor growth of the mouse xenograft model. Taken together, we suggest that the aberrant regulation of HDAC2 and its epigenetic regulation of gene transcription in apoptosis and cell cycle components play an important role in the development of lung cancer.

Programmed cell death 6 (PDCD6) as a prognostic marker for gastric cancers.

Programmed cell death 6 (PDCD6) plays an important role in apoptotic cell death and tumorigenesis. In this study, we investigated whether PDCD6 contributes to the development and/or progression of gastric cancers. PDCD6 protein expression was examined in 169 advanced gastric cancer specimens by immunohistochemistry and then correlated with clinicopathologic parameters. We also analyzed mutations, methylation status, and alterations in DNA copy number and mRNA transcripts, and protein expression of PDCD6 in gastric cancers. The effect of PDCD6 on cell viability and death was further examined in wild- and mutant-type PDCD6 transfected AGS and HEK293T cell lines. Increased expression of PDCD6 expression was detected in 124 (73.4%) out of 169 gastric cancer specimens. Statistically, altered expression of PDCD6 was closely associated with survival rates (P = 0.0069). One non-sense mutation was found at codon 175 of PDCD6, and no hypermethylation was found in gastric cancers. Decreased copy numbers and mRNA expression of PDCD6 were found in 7 (16.7%) and 10 (23.8%) of 42 gastric cancer specimens, respectively. AGS and HEK293T cells transfected with wild-type PDCD6 showed marked inhibition of cell viability and induction of cell death via activation of mitochondrial cell death pathways, whereas mutant-type PDCD6 showed partial ablation of tumor suppressor activity. In addition, AGS cells transfected with wild-type PDCD6 and treated with 5-FU showed synergistic inhibition of cell viability (P < 0.001). These data provide evidence that the PDCD6 gene is a significant prognostic biomarker for advanced gastric cancer patients.