A lactate-induced response to hypoxia.

Organisms must be able to respond to low oxygen in a number of homeostatic and pathological contexts. Regulation of hypoxic responses via the hypoxia-inducible factor (HIF) is well established, but evidence indicates that other, HIF-independent mechanisms are also involved. Here, we report a hypoxic response that depends on the accumulation of lactate, a metabolite whose production increases in hypoxic conditions. We find that the NDRG3 protein is degraded in a PHD2/VHL-dependent manner in normoxia but is protected from destruction by binding to lactate that accumulates under hypoxia. The stabilized NDRG3 protein binds c-Raf to mediate hypoxia-induced activation of Raf-ERK pathway, promoting angiogenesis and cell growth. Inhibiting cellular lactate production abolishes the NDRG3-mediated hypoxia responses. Our study, therefore, elucidates the molecular basis for lactate-induced hypoxia signaling, which can be exploited for the development of therapies targeting hypoxia-induced diseases.

SH3RF2 functions as an oncogene by mediating PAK4 protein stability.

SH3RF (SH3-domain-containing RING finger protein) family members, SH3RF1-3, are multidomain scaffold proteins involved in promoting cell survival and apoptosis. In this report, we show that SH3RF2 is an oncogene product that is overexpressed in human cancers and regulates p21-activated kinase 4 (PAK4) protein stability. Immunohistochemical analysis of 159 colon cancer tissues showed that SH3RF2 expression levels are frequently elevated in cancer tissues and significantly correlate with poor prognostic indicators, including increased invasion, early recurrence and poor survival rates. We also demonstrated that PAK4 protein is degraded by the ubiquitin-proteasome system and that SH3RF2 inhibits PAK4 ubiquitination via physical interaction-mediated steric hindrance, which results in the upregulation of PAK4 protein. Moreover, ablation of SH3RF2 expression attenuates TRADD (TNFR-associated death domain) recruitment to tumor necrosis factor-α (TNF-α) receptor 1 and hinders downstream signals, thereby inhibiting NF-κB (nuclear factor-kappaB) activity and enhancing caspase-8 activity, in the context of TNF-α treatment. Notably, ectopic expression of SH3RF2 effectively prevents apoptosis in cancer cells and enhances cell migration, colony formation and tumor growth in vivo. Taken together, our results suggest that SH3RF2 is an oncogene that may be a definitive regulator of PAK4. Therefore, SH3RF2 may represent an effective therapeutic target for cancer treatment.

Phosphorylations of Sds23/Psp1/Moc1 by stress-activated kinase and cAMP-dependent kinase are essential for regulating cell viability in prolonged stationary phase.

Under nutritional deprivation caused by prolonged culture, actively growing cells prepare to enter stationary phase. We showed here that Sds23/Psp1/Moc1 was phosphorylated by both cAMP-dependent kinase and stress-activated MAP kinase Sty1 upon entry into stationary phase. Overexpression of the phosphorylation-defective mutant Sds23/Psp1/Moc1 induced cell death in prolonged culture and blocked re-entry into the cell division cycle. These phosphorylations are likely to be required for cell survival during stationary phase and for binding of Ufd2, a Schizosaccharomyces pombe homologue of multi-ubiquitin chain assembly factor E4. Deletion of the Ufd2 gene and overexpression of Sds23/Psp1/Moc1 increased cell viability in prolonged stationary phase. These results suggested that Ufd2 induces cell death in prolonged nutrient deprivation, that Sds23/Psp1/Moc1 may be a target protein of the ubiquitin-fusion degradation pathway for regulation of cell viability under this stress condition, and that Sty1 and PKA activity in stationary phase is essential for interaction between Sds23/Psp1/Moc1 and Ufd2.

Pyruvate kinase M2 promotes the growth of gastric cancer cells via regulation of Bcl-xL expression at transcriptional level.

PKM2 is an isoenzyme of the glycolytic enzyme pyruvate kinase that promotes aerobic glycolysis. Here, we describe an important role for PKM2 in regulating the survival of gastric cancer (GC) cells. We showed that PKM2 was overexpressed in gastric tumor tissues compared to normal tissues and its expression level was associated with poor survival of gastric cancer patients. We also showed that PKM2 affected cell survival by regulating Bcl-xL at the transcriptional level. PKM2 knockdown partially affected the stability of NF-kB subunit p65, suggesting that post-translational regulation of p65 by PKM2 is one of plausible mechanisms for the increased cell growth. Therefore, PKM2 may function as an upstream molecule that regulates p65 function and thus enhances the growth of tumor cells.

Liverome: a curated database of liver cancer-related gene signatures with self-contained context information.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. A number of molecular profiling studies have investigated the changes in gene and protein expression that are associated with various clinicopathological characteristics of HCC and generated a wealth of scattered information, usually in the form of gene signature tables. A database of the published HCC gene signatures would be useful to liver cancer researchers seeking to retrieve existing differential expression information on a candidate gene and to make comparisons between signatures for prioritization of common genes. A challenge in constructing such database is that a direct import of the signatures as appeared in articles would lead to a loss or ambiguity of their context information that is essential for a correct biological interpretation of a gene's expression change. This challenge arises because designation of compared sample groups is most often abbreviated, ad hoc, or even missing from published signature tables. Without manual curation, the context information becomes lost, leading to uninformative database contents. Although several databases of gene signatures are available, none of them contains informative form of signatures nor shows comprehensive coverage on liver cancer. Thus we constructed Liverome, a curated database of liver cancer-related gene signatures with self-contained context information. DESCRIPTION: Liverome's data coverage is more than three times larger than any other signature database, consisting of 143 signatures taken from 98 HCC studies, mostly microarray and proteome, and involving 6,927 genes. The signatures were post-processed into an informative and uniform representation and annotated with an itemized summary so that all context information is unambiguously self-contained within the database. The signatures were further informatively named and meaningfully organized according to ten functional categories for guided browsing. Its web interface enables a straightforward retrieval of known differential expression information on a query gene and a comparison of signatures to prioritize common genes. The utility of Liverome-collected data is shown by case studies in which useful biological insights on HCC are produced. CONCLUSION: Liverome database provides a comprehensive collection of well-curated HCC gene signatures and straightforward interfaces for gene search and signature comparison as well. Liverome is available at http://liverome.kobic.re.kr.

Functional switching of TGF-beta1 signaling in liver cancer via epigenetic modulation of a single CpG site in TTP promoter.

BACKGROUND & AIMS: Acquisition of resistance to the antiproliferative effect of transforming growth factor (TGF)-beta1 is crucial for the malignant progression of cancers. In this study, we sought to determine whether deregulated expression of tristetrapolin (TTP), a negative posttranscriptional regulator of c-Myc, confers resistance to the antiproliferative effects of TGF-beta1 on liver cancer cells. METHODS: The epigenetics of TTP promoter regulation and its effects on TGF-beta1 signaling were examined in hepatocellular carcinoma (HCC) cell lines and patient tissues. RESULTS: TTP was down-regulated in HCC cell lines (10/11), compared with normal liver, as well as in tumor tissues (19/24) from paired HCC specimens. Methylation of a specific single CpG site located within the TGF-beta1-responsive region (TRR) of the TTP promoter was significantly associated with TTP down-regulation in both HCC cell lines and tumor tissues (r = -0.606383, P < .001). The singly methylated CpG site was specifically bound by a transcriptional repressor complex consisting of MECP2/c-Ski/DNMT3A and abolished the TGF-beta1-induced as well as basal-level expression of TTP. The epigenetic inactivation of TTP led to an increased half-life of c-Myc mRNA and blocked the cytostatic effect of TGF-beta1. Statistically significant correlations were observed between the single CpG site methylation and expression levels of TTP or c-Myc in clinical samples of HCC. CONCLUSIONS: Abrogation of the post-transcriptional regulation of c-Myc via methylation of a specific single CpG site in the TTP promoter presents a novel mechanism for the gain of selective resistance to the antiproliferative signaling of TGF-beta1 in HCC.

Aberrant up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer.

The LAMB3 and LAMC2 genes encode the laminin-5 ß3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis.

Galectin-3 binding protein promotes cell motility in colon cancer by stimulating the shedding of protein tyrosine phosphatase kappa by proprotein convertase 5.

It has previously been reported that shedding of the PTPκ ectodomain drives enhanced motility of colon cancer cells. Herein, we provide mechanism underlying the regulation of PTPκ shedding by galectin-3 binding protein. PTPκ was inarguably scissored by the processed form of proprotein convertase 5 (subtilisin/kexin type 5), and galectin-3 binding protein which is over-produced in colon cancer cells and tissues contributed to increased cancer cell motility by acting as a negative regulator of galectin-3 at the cell surface. The high expression ratio of galectin-3 binding protein to galectin-3 was clinically correlated to lymphatic invasion. These results suggest that galectin-3 binding protein may be a potential therapeutic target for treatment of, at least, colon cancer patients with high expression of galectin-3 binding protein.

Optimization of phage-immobilized ELISA for autoantibody profiling in human sera.

Phage libraries displaying cDNA or random peptides have been used for profiling autoantibodies in cancer. The detection of autoantibodies in human sera using phages displaying specific epitopes is usually performed by phage-immobilized ELISAs which can detect specific antibodies without identification of whole antigens. However, these ELISAs can give feeble detection signals that are indistinguishable from background signals which are caused by human sera. To improve the usefulness of phage ELISA for human sera, the conditions for each step in phage ELISA were optimized. The antigenicity of phage antigens was maximal when using coating buffer of neutral pH. By using protein-free blocking buffer and pre-adsorbing human sera with phage host cell ER2738 extracts significantly decreased non-specific signals. Finally, when these conditions were applied to phage ELISA using K10P1, the values of the negative controls were concentrated near cutoff values, which made the assay more reliable. The optimized phage ELISA conditions described here would increase the efficacy of detection specific autoantibodies in human sera.

Frequent silencing of popeye domain-containing genes, BVES and POPDC3, is associated with promoter hypermethylation in gastric cancer.

The Popeye domain-containing (POPDC) genes BVES, POPDC2 and POPDC3 encode proteins that regulate cell-cell adhesion and cell migration during development. Herein, we report the frequent downregulation of BVES and POPDC3 by promoter hypermethylation in gastric cancer. POPDC expression in 11 gastric cancer cell lines and 96 paired gastric tumor and normal adjacent tissues was analyzed with quantitative reverse transcription-polymerase chain reaction. The methylation status of BVES and POPDC3 was analyzed with methylated DNA immunoprecipitation sequencing, bisulfite sequencing and pyrosequencing. Expression of BVES and POPDC3 was downregulated in 73% of the gastric cancer cell lines and in 69% (BVES) and 87% (POPDC3) of the gastric cancer tissues. The BVES and POPDC3 promoter regions were hypermethylated in the gastric cancer cell lines in which they were silenced. Combined treatment with a DNA methylation inhibitor and a histone deacetylase inhibitor strongly induced BVES and POPDC3 expression. BVES and POPDC3 were hypermethylated in 69% (BVES) and 64% (POPDC3) of the gastric cancer tissues. We knocked down POPDC3 expression with short hairpin RNAs and examined the consequences on cell migration and invasion. Knockdown of POPDC3 in SNU-216 cells caused increased cell migration and invasion. Thus, epigenetic inactivation of BVES and POPDC3 occurs frequently in gastric tumors and may promote gastric cancer cell migration and invasion.

Tristetraprolin regulates the stability of HIF-1alpha mRNA during prolonged hypoxia.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor involved in the cancer cell adaptation to hypoxia, a leading cause of tumor malignancy. Thus, control of HIF-1alpha expression may assist in treatment of cancer. The expression of HIF-1alpha is finely regulated via alterations in not only HIF-1alpha protein stability but also mRNA stability. However, the molecular mechanisms of regulation of HIF-1alpha mRNA stability have not yet been fully elucidated. Here, we show that tristetraprolin (TTP) protein, of which the mRNA expression level is downregulated in most of hepatocellular carcinoma tissues, bound directly to the 3'-UTR of HIF-1alpha mRNA containing eight putative TTP-binding motifs, AU-rich elements (AUUUA), to downregulate stability. Furthermore, TTP expression was induced in hypoxic cells, and overexpression of TTP repressed the hypoxic induction of HIF-1alpha protein. Taken together, these data suggest that TTP is a modulator of HIF-1alpha expression during hypoxia and may play a physiological role in regulation between cellular adaptation and apoptosis in prolonged hypoxia. In addition, cancer cells may benefit from the downregulation of TTP, which subsequently increases HIF-1alpha expression and assists with the adaptation of cancer cells to hypoxia.

Epigenetic down-regulation and suppressive role of DCBLD2 in gastric cancer cell proliferation and invasion.

The promoter region of Discoidin, CUB and LCCL domain containing 2 (DCBLD2) was found to be aberrantly methylated in gastric cancer cell lines and in primary gastric cancers, as determined by restriction landmark genomic scanning. DCBLD2 expression was inversely correlated with DCBLD2 methylation in gastric cancer cell lines. Treatment with 5-aza-2'-deoxycytidine and trichostatin A partially reversed DCBLD2 methylation and restored gene expression in DCBLD2-silenced cell lines. In an independent series of 82 paired gastric cancers and adjacent normal tissues, DCBLD2 expression was down-regulated in 79% of gastric cancers as compared with normal tissues as measured by real-time reverse transcription-PCR. Pyrosequencing analysis of the DCBLD2 promoter region revealed abnormal hypermethylation in gastric cancers, and this hypermethylation was significantly correlated with down-regulation of DCBLD2 expression. Furthermore, ectopic expression of DCBLD2 in gastric cancer cell lines inhibited colony formation in both anchorage-dependent and anchorage-independent cultures and also inhibited invasion through the collagen matrix. These data suggest that down-regulation of DCBLD2, often associated with promoter hypermethylation, is a frequent event that may be related to the development of gastric cancer.

Lectin precipitation using phytohemagglutinin-L(4) coupled to avidin-agarose for serological biomarker discovery in colorectal cancer.

N-acetylglucosaminyltransferase V (GnT-V) has been reported to be upregulated in malignant cancer cells, and its targets have been sought after with regard to biomarker identification. The low capacity and high false positive rates of 2-DE gel-based lectin blots using phytohemagglutinin-L(4) (L-PHA) prompted us to develop a novel protocol for identifying GnT-V targets, in which serum proteins were subjected to immunodepletion, alkylation, and lectin precipitation using L-PHA coupled to avidin-agarose bead complexes, and tryptic digestion. Proteins captured by L-PHA conjugates were analyzed by a nano-LC-FT-ICR/LTQ MS. Here, we report 26 candidate biomarkers for colorectal cancer (CRC) that show 100% specificity and sensitivities of greater than 50%. Not only can these candidate proteins be used as analytes for validation, but the novel protocol described herein can be applied to biomarker discovery in nonCRCs.

Epigenetic inactivation of protein kinase D1 in gastric cancer and its role in gastric cancer cell migration and invasion.

Protein kinase D (PKD) 1 influences cell migration by mediating both trans-Golgi vesicle fission and integrin recycling to the cell surface. Using restriction landmark genomic scanning methods, we found that the promoter region of PKD1 was aberrantly methylated in gastric cancer cell lines. Silencing of PKD1 expression was detected in 72.7% of gastric cancer cell lines examined, and the silencing was associated with CpG hypermethylation in the promoter region of PKD1. Treatment with 5-aza-2'-deoxycytidine and trichostatin A partially reversed PKD1 methylation and restored gene expression in PKD1-silenced cell lines. Real-time reverse transcription-polymerase chain reaction analysis of 96 paired clinical primary gastric cancer samples revealed that 59% of the analyzed tumors had a >2-fold decrease in PKD1 expression compared with each normal-appearing tissue and that this downregulation of PKD1 expression was significantly correlated with increased methylation. We also observed a gradual increase in the level of promoter methylation of PKD1 in aging, normal-appearing mucosal tissues, suggesting that PKD1 methylation may be one of the earliest events that predispose an individual to gastric cancer. PKD1 expression was required for directional migration of gastric cancer cells. Furthermore, knock down of PKD1 by RNA interference promoted the invasiveness of cell lines that expressed PKD1 at relatively high levels. Based on these results, we propose that PKD1 is frequently silenced by epigenetic regulation, which plays a role in cell migration and metastasis in gastric cancer.

CpG methylation in exon 1 of transcription factor 4 increases with age in normal gastric mucosa and is associated with gene silencing in intestinal-type gastric cancers.

Transcriptional factor 4 (TCF4), encoding a basic helix-loop-helix transcriptional factor, has recently been demonstrated as a causative gene for Pitt-Hopkins syndrome, a neurodevelopmental disease. Examination of gastric cancers using the restriction landmark genomic scanning technique revealed methylation at a NotI enzyme site in TCF4 intron 8 and further identified CpG dinucleotide hypermethylation in TCF4 exon 1, strongly associated with gene silencing in gastric cancer cell lines. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A restored TCF4 expression in TCF4-silenced gastric cancer cell lines. Real-time reverse transcription-polymerase chain reaction analysis of 77 paired primary gastric tumor samples revealed that 38% of analyzed tumors had a >2-fold decrease in TCF4 expression compared with adjacent normal-appearing tissue, and the decrease significantly correlated with increased CpG methylation in TCF4 exon 1. Clinicopathologic data showed that decreased TCF4 expression occurred significantly more frequently in intestinal-type (22/37, 59%) than in diffuse-type (7/37, 19%) gastric cancers (P = 0.0004) and likewise more frequently in early (12/18, 67%) than in advanced (17/59, 29%) gastric cancers (P = 0.004). CpG methylation markedly increased with patient age among normal-appearing tissues, suggesting that CpG methylation in gastric mucosa may be one of the earliest events in carcinogenesis of intestinal-type gastric cancers. Furthermore, ectopic expression of TCF4 decreased cell growth in a gastric cancer cell line, and the knock down of TCF4 using small interfering RNA increased cell migration. Based on these results, we propose that the observed frequent epigenetic-mediated TCF4 silencing plays a role in tumor formation and progression.

Rapid screen of human genes for relevance to cancer using fission yeast.

A total of 437 human full-length cDNAs isolated by microarray analysis of liver and/or gastric cancer tissues were evaluated for their relevance to cancer using the fission yeast Schizosaccharomyces pombe. Overexpression of 161 human cDNAs in S. pombe caused growth inhibition and/or morphological changes, which can be considered as cancer-related phenotypes of S. pombe. Sixteen genes causing growth defects and morphological changes at the same time were chosen to validate their ostensible oncogenic properties. They were highly expressed in liver and/or gastric cancer cell lines. Also, when the mouse embryonic fibroblast cell type NIH3T3 was transfected with these genes, the proliferation rates of cells were increased by 32% to 120%. This study demonstrates that fission yeast can be used as an advantageous and powerful tool for the rapid screening of human genes relevant to cancer. Furthermore, the human genes screened can be tested further as diagnostic markers and potential therapeutic targets for liver and stomach cancers. They also can be studied further for the elucidation of mechanisms involved in carcinogenesis.

Identification of novel regulators of apoptosis using a high-throughput cell-based screen.

High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity.

Meta- and gene set analysis of stomach cancer gene expression data.

We generated gene expression data from the tissues of 50 gastric cancer patients, and applied meta-analysis and gene set analysis to this data and three other stomach cancer gene expression data sets to define the gene expression changes in gastric tumors. By meta-analysis we identified genes consistently changed in gastric carcinomas, while gene set analysis revealed consistently changed biological themes. Genes and gene sets involved in digestion, fatty acid metabolism, and ion transport were consistently down-regulated in gastric carcinomas, while those involved in cellular proliferation, cell cycle, and DNA replication were consistently up-regulated. We also found significant differences between the genes and gene sets expressed in diffuse and intestinal type gastric carcinoma. By gene set analysis of cytogenetic bands, we identified many chromosomal regions with possible gross chromosomal changes (amplifications or deletions). Similar analysis of transcription factor binding sites (TFBSs), revealed transcription factors that may have caused the observed gene expression changes in gastric carcinomas, and we confirmed the overexpression of one of these, E2F1, in many gastric carcinomas by tissue array and immunohistochemistry. We have incorporated the results of our meta- and gene set analyses into a web accessible database (http://human-genome.kribb.re.kr/stomach/).

Yeast-based screening to identify modulators of G-protein signaling using uncontrolled cell division cycle by overexpression of Stm1.

Stm1, a G-protein coupled receptor, which senses nutritional state drives cells to stop the proliferative cell cycle and enter meiosis under nutritionally deficient conditions in Schizosaccharomyces pombe. It was shown that overexpression of Stm1 led growth inhibition and uncontrolled mitotic haploidization presumably by the premature initiation of mitosis. Sty1 and Gpa2 seem to play important roles for Stm1 to deliver starvation signal to induce downstream function. Based on the observation that conversion of diploid to haploid by overexpression of Stm1 can be easily detected as pink or red colonies in the media containing low adenine, HTS drug screening system to identify modulators of GPCR was established and tested using 413 compounds. Four very potent modulators of GPCR including Biochanin A, which possess strong inhibitory activity against uncontrolled cell division, were identified in this screening. This study provides the yeast-based platform that allows robust cellular assays to identify novel modulators of G-protein signaling and MAP kinase pathway.

Novel candidate targets of Wnt/beta-catenin signaling in hepatoma cells.

The activity of beta-catenin/TCF, the key component of Wnt signaling pathway, is frequently deregulated in HCC, resulting in the activation of genes whose dysregulation has significant consequences on tumor development. Therefore, identifying the target genes of Wnt signaling is important for understanding beta-catenin-mediated carcinogenesis. We analyzed the transcriptome profile of human hepatoma cell lines using cDNA microarrays representing 15,127 unique, liver-enriched gene loci to identify the target genes of beta-catenin-mediated transcription (p<0.005). This analysis yielded 130 potential Wnt-associated classifier genes, and we found 33 of them contain consensus TCF-binding sites in presumptive transcriptional regulatory sequences. These genes were, then, tested for their Wnt-dependence of expression in experimental models of Wnt activation. Genes such as RPL29, NEDD4L, FUT8, LYZ, STMN2, STARD7 and KIAA0998 were proven to be up-regulated upon Wnt/beta-catenin activation. Gene ontology analysis of the 33 candidate genes indicated the presence of functional categories relevant to Wnt pathway such as cell growth, proliferation, adhesion and signal transduction. In conclusion, we identified a number of candidate Wnt/beta-catenin target genes that can be useful for studying the role of altered Wnt signaling in liver cancer development, and showed that some of them might be direct targets of Wnt signaling in hepatoma cells.