Masahiro Kai

CLONING AND CHARACTERIZATION OF TWO HUMAN ISOZYMES OF MG2+-INDEPENDENT PHOSPHATIDIC ACID PHOSPHATASE

We obtained two human cDNA clones encoding phosphatidic acid phosphatase (PAP) isozymes named PAP-2a (M r = 32,158) and -2b (M r = 35, 119), both of which contained six putative transmembrane domains. Both enzymes were glycosylated and cleaved by N-glycanase and endo-β-galactosidase, thus suggesting their post-Golgi localization. PAP-2a and -2b shared 47% identical sequence and were judged to be the human counterparts of the previously sequenced mouse 35-kDa PAP(83% identity) and rat Dri42 protein (94% identity), respectively. Furthermore, the sequences of both PAPs were 34–39% identical to that of DrosophilaWunen protein. In view of the functions ascribed to Wunen and Dri42 in germ cell migration and epithelial differentiation, respectively, these findings unexpectedly suggest critical roles of PAP isoforms in cell growth and differentiation. Although the two PAPs hydrolyzed lysophosphatidate and ceramide-1-phosphate in addition to phosphatidate, the hydrolysis of sphingosine-1-phosphate was detected only for PAP-2b. PAP-2b was expressed almost ubiquitously in all human tissues examined, whereas the expression of PAP-2a was relatively variable, being extremely low in the placenta and thymus. In HeLa cells, the transcription of PAP-2a was not affected by different stimuli, whereas PAP-2b was induced (up to 3-fold) by epidermal growth factor. These findings indicate that despite structural similarities, the two PAP isozymes may play distinct functions through their different patterns of substrate utilization and transcriptional regulation.

DNA methylation and microRNA dysregulation in cancer

DNA methylation plays a key role in the silencing of numerous cancer‐related genes, thereby affecting a number of vital cellular processes, including the cell cycle checkpoint, apoptosis, signal transduction, cell adhesion and angiogenesis. Also widely altered in human malignancies is the expression of microRNAs (miRNAs), a class of small noncoding RNAs that act as posttranscriptional regulators of gene expression. Furthermore, emerging evidence now supports the idea that DNA methylation is crucially involved in the dysregulation of miRNAs in cancer. This is in part the result of technological advances that enable more comprehensive analysis of miRNA expression profiles and the epigenome in cancer cells, which has led to the identification of a number of epigenetically regulated miRNAs. As with protein‐coding genes, it appears that miRNA genes involved in regulating cancer‐related pathways are silenced in association with CpG island hypermethylation. In addition, methylation in CpG island shore regions and DNA hypomethylation also appear to contribute to miRNA dysregulation in cancer. Aberrant DNA methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Moreover, re‐expression of miRNAs and the replacement of tumor suppressive miRNAs using miRNA mimics or expression vectors could be effective approaches to cancer therapy.

Genome-wide Profiling of Chromatin Signatures Reveals Epigenetic Regulation of MicroRNA Genes in Colorectal Cancer

Altered expression of microRNAs (miRNA) occurs commonly in human cancer, but the mechanisms are generally poorly understood. In this study, we examined the contribution of epigenetic mechanisms to miRNA dysregulation in colorectal cancer by carrying out high-resolution ChIP-seq. Specifically, we conducted genome-wide profiling of trimethylated histone H3 lysine 4 (H3K4me3), trimethylated histone H3 lysine 27 (H3K27me3), and dimethylated histone H3 lysine 79 (H3K79me2) in colorectal cancer cell lines. Combining miRNA expression profiles with chromatin signatures enabled us to predict the active promoters of 233 miRNAs encoded in 174 putative primary transcription units. By then comparing miRNA expression and histone modification before and after DNA demethylation, we identified 47 miRNAs encoded in 37 primary transcription units as potential targets of epigenetic silencing. The promoters of 22 transcription units were associated with CpG islands (CGI), all of which were hypermethylated in colorectal cancer cells. DNA demethylation led to increased H3K4me3 marking at silenced miRNA genes, whereas no restoration of H3K79me2 was detected in CGI-methylated miRNA genes. DNA demethylation also led to upregulation of H3K4me3 and H3K27me3 in a number of CGI-methylated miRNA genes. Among the miRNAs we found to be dysregulated, many of which are implicated in human cancer, miR-1-1 was methylated frequently in early and advanced colorectal cancer in which it may act as a tumor suppressor. Our findings offer insight into the association between chromatin signatures and miRNA dysregulation in cancer, and they also suggest that miRNA reexpression may contribute to the effects of epigenetic therapy.

A novel pit pattern identifies the precursor of colorectal cancer derived from sessile serrated adenoma.

OBJECTIVES:Sessile serrated adenomas (SSAs) are known to be precursors of sporadic colorectal cancers (CRCs) with microsatellite instability (MSI), and to be tightly associated with BRAF mutation and the CpG island methylator phenotype (CIMP). Consequently, colonoscopic identification of SSAs has important implications for preventing CRCs, but accurate endoscopic diagnosis is often difficult. Our aim was to clarify which endoscopic findings are specific to SSAs.METHODS:The morphological, histological and molecular features of 261 specimens from 226 colorectal tumors were analyzed. Surface microstructures were analyzed using magnifying endoscopy. Mutation in BRAF and KRAS was examined by pyrosequencing. Methylation of p16, IGFBP7, MLH1 and MINT1, -2, -12 and -31 was analyzed using bisulfite pyrosequencing.RESULTS:Through retrospective analysis of a training set (n=145), we identified a novel surface microstructure, the Type II open-shape pit pattern (Type II-O), which was specific to SSAs with BRAF mutation and CIMP. Subsequent prospective analysis of an independent validation set (n=116) confirmed that the Type II-O pattern is highly predictive of SSAs (sensitivity, 65.5%; specificity, 97.3%). BRAF mutation and CIMP occurred with significant frequency in Type II-O-positive serrated lesions. Progression of SSAs to more advanced lesions was associated with further accumulation of aberrant DNA methylation and additional morphological changes, including the Type III, IV and V pit patterns.CONCLUSIONS:Our results suggest the Type II-O pit pattern is a useful hallmark of the premalignant stage of CRCs with MSI and CIMP, which could serve to improve the efficacy of colonoscopic surveillance.

Identification and cDNA Cloning of 35-kDa Phosphatidic Acid Phosphatase (Type 2) Bound to Plasma Membranes POLYMERASE CHAIN REACTION AMPLIFICATION OF MOUSE H2O2-INDUCIBLE hic53 CLONE YIELDED THE cDNA ENCODING PHOSPHATIDIC ACID PHOSPHATASE

We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F. (1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35-kDa protein, which was converted to a 29-kDa form when treated with N-glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2-inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K. (1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated Mr of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.

Identification and Characterization of a Novel Human Type II Diacylglycerol Kinase, DGKκ

Diacylglycerol kinase (DGK) plays an important role in signal transduction through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. Here we identified a tenth member of the DGK family designated DGKκ. The κ-isozyme (1271 amino acids, calculated molecular mass, 142 kDa) contains a pleckstrin homology domain, two cysteine-rich zinc finger-like structures, and a separated catalytic region as have been found commonly for the type II isozymes previously cloned (DGKδ and DGKη). The new DGK isozyme has additionally 33 tandem repeats of Glu-Pro-Ala-Pro at the N terminus. Reverse transcriptase-PCR showed that the DGKκ mRNA is most abundant in the testis, and to a lesser extent in the placenta. DGKκ, when expressed in HEK293 cells, was persistently localized at the plasma membrane even in the absence of cell stimuli. Deletion analysis revealed that the short C-terminal sequence (amino acid residues 1199–1268) is necessary and sufficient for the plasma membrane localization. Interestingly, DGKκ, but not other type II DGKs, was specifically tyrosine-phosphorylated at Tyr78 through the Src family kinase pathway in H2O2-treated cells. Moreover, H2O2 selectively inhibited DGKκ activity in a Src family kinase-independent manner, suggesting that the isozyme changes the balance of signaling lipids in the plasma membrane in response to oxidative stress. The expression patterns, subcellular distribution, and regulatory mechanisms of DGKκ are distinct from those of DGKδ and DGKη despite high structural similarity, suggesting unique functions of the individual type II isozymes.

Epigenetic alteration and microRNA dysregulation in cancer

MicroRNAs (miRNAs) play pivotal roles in numerous biological processes, and their dysregulation is a common feature of human cancer. Thanks to recent advances in the analysis of the cancer epigenome, we now know that epigenetic alterations, including aberrant DNA methylation and histone modifications, are major causes of miRNA dysregulation in cancer. Moreover, the list of miRNA genes silenced in association with CpG island hypermethylation is rapidly growing, and various oncogenic miRNAs are now known to be upregulated via DNA hypomethylation. Histone modifications also play important roles in the dysregulation of miRNAs, and histone deacetylation and gain of repressive histone marks are strongly associated with miRNA gene silencing. Conversely, miRNA dysregulation is causally related to epigenetic alterations in cancer. Thus aberrant methylation of miRNA genes is a potentially useful biomarker for detecting cancer and predicting its outcome. Given that many of the silenced miRNAs appear to act as tumor suppressors through the targeting of oncogenes, re-expression of the miRNAs could be an effective approach to cancer therapy, and unraveling the relationship between epigenetic alteration and miRNA dysregulation may lead to the discovery of new therapeutic targets.

Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N‐linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP‐glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP‐glucose enhanced the amount of folded transferrin and reduced the disulfide‐linked aggregates. Analysis of transferrin folding in briefly heat‐treated microsomes revealed that UDP‐glucose was also effective in elimination of heat‐induced misfolding. Incubation of the microsomes with an α–glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.

Biological significance of the CpG island methylator phenotype

Cancers exhibiting the CpG island methylator phenotype (CIMP) are found among a wide variety of human malignancies and represent a subclass of tumors showing concurrent hypermethylation of multiple CpG islands. These CIMP-positive tumors often exhibit characteristic molecular and clinicopathological features, suggesting CIMP represents a distinct carcinogenic pathway. However, marker genes to define CIMP have been largely inconsistent among studies, which has caused results to vary. Nonetheless, recent advances in genome-wide methylation analysis have enabled the existence of CIMP to be confirmed, and large-scale cancer genome analyses have begun to unravel the previously unknown molecular basis of CIMP tumors. CIMP is strongly associated with clinical outcome, suggesting it may be a predictive biomarker.

Molecular Dissection of Premalignant Colorectal Lesions Reveals Early Onset of the CpG Island Methylator Phenotype

The concept of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC) is widely accepted, although the timing of its occurrence and its interaction with other genetic defects are not fully understood. Our aim in this study was to unravel the molecular development of CIMP cancers by dissecting their genetic and epigenetic signatures in precancerous and malignant colorectal lesions. We characterized the methylation profile and BRAF/KRAS mutation status in 368 colorectal tissue samples, including precancerous and malignant lesions. In addition, genome-wide copy number aberrations, methylation profiles, and mutations of BRAF, KRAS, TP53, and PIK3CA pathway genes were examined in 84 colorectal lesions. Genome-wide methylation analysis of CpG islands and selected marker genes revealed that CRC precursor lesions are in three methylation subgroups: CIMP-high, CIMP-low, and CIMP-negative. Interestingly, a subset of CIMP-positive malignant lesions exhibited frequent copy number gains on chromosomes 7 and 19 and genetic defects in the AKT/PIK3CA pathway genes. Analysis of mixed lesions containing both precancerous and malignant components revealed that most aberrant methylation is acquired at the precursor stage, whereas copy number aberrations are acquired during the progression from precursor to malignant lesion. Our integrative genomic and epigenetic analysis suggests early onset of CIMP during CRC development and indicates a previously unknown CRC development pathway in which epigenetic instability associates with genomic alterations.