Kyoko Fujiwara

Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Me thylated D NA I mmunoP recipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify T issue and D evelopmental S tage specific D ifferentially M ethylated DNA R egions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

Characterization of Dysplastic Aberrant Crypt Foci in the Rat Colon Induced by 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]Pyridine

The multistage model of colon carcinogenesis is well established in both humans and experimental animals, and aberrant crypt foci (ACF) are generally assumed to be putative preneoplastic lesions of the colon. However, morphological analyses of ACF have suggested that they are highly heterogeneous in nature and their role in tumorigenesis is still controversial. To better understand the biological significance of ACF in carcinogenesis, morphological and genetic analyses were performed using a rat colon cancer model induced by a food-borne colon carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). ACF of different sizes were collected at weeks 6, 18, 25, and 32 after three cycles of 2-week PhIP feeding (400 ppm in diet) with 4-week intervals on a high-fat diet, and a total of 110 ACF, representing approximately three-quarters of the total ACF, were subjected to histological evaluation. Thirty (27%) were diagnosed as dysplastic ACF, based on cytological and structural abnormalities of crypts. Dysplastic ACF were detected even at week 6 (0.4 per rat), and the numbers increased slightly at later time points, being 0.8, 1.4, and 0.8 per rat at weeks 18, 25, and 32, respectively. The sizes of these dysplastic ACF varied widely from 1 to 16 crypts and 50% (15 of 30) were composed of less than 4 crypts. Immunohistochemical analysis revealed that 83% (25 of 30) of dysplastic ACF demonstrated beta-catenin accumulation; 22 only in the cytoplasm and 3 in both the cytoplasm and nucleus, the latter manifesting a higher grade of dysplasia as compared with the former. Seven dysplastic ACF harbored beta-catenin mutations at codon 32, 34, or 36 in exon 2, and one had an Apc mutation at the boundary of intron 10 and exon 11. Mutations at these sites were also commonly found in colon tumors induced by PhIP. The results of our present study indicate that dysplastic ACF, which accounted for approximately one-fourth of the total ACF, are preneoplastic lesions of colon cancers induced by PhIP in rats.

A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines contained in cooked meat and fish, and induces aberrant crypt foci (ACF), putative preneoplastic lesions of the colon, and colon cancers in male rats when administered orally. As has been reported previously, F344 rats are susceptible to induction of ACF by PhIP, while ACI rats being relatively resistant. Approximately one-fourth of ACF induced by PhIP in F344 rats are dysplastic; exhibiting lesions with structural distortion of the crypt, decrease of goblet cells, nuclear stratification and enlargement of nuclei. Dysplastic ACF demonstrate beta-catenin accumulation, mainly in the cytoplasm, and increased cell proliferation in crypts. These dysplastic ACF are, therefore, strongly considered to be putative preneoplastic lesions of the colon.A genetic trait affecting the susceptibility to colon carcinogenesis in F344 rats was mapped to chromosome 16, between D16Rat17 and D16Wox3, using the number of ACF as a surrogate biomarker for colon carcinogenesis. Since the number of dysplastic lesions is well correlated with the total number of ACF, being approximately one-fourth of the total ACF as described above in F344 rats and will be described elsewhere in ACI rats, the gene involved in the susceptibility to ACF induction may possibly be partly responsible for the susceptibility to colon carcinogenesis by PhIP. We, thus, tentatively referred the name of the candidate susceptibility gene on rat chromosome 16 as susceptibility to colon tumor (Sct). In the present study, the colonic lesions induced by PhIP were well refined histologically and genetically, and the multi-step profiles of colon cancer development by PhIP were well characterized and revealed to be similar to the multi-step model of colon carcinogenesis in humans. The PhIP-induced colon cancer model in rats, thus contributes as a relevant tool to elucidate genetic factors responsible for susceptibility to colon carcinogenesis in human. Other unknown genetic or epigenetic alterations, which are essential for the development of early lesions of colon carcinogenesis, could also be clarified using this system.

Inhibition of KRAS codon 12 mutants using a novel DNA-alkylating pyrrole–imidazole polyamide conjugate

Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours.

Identification and analysis of an early diagnostic marker for malignant melanoma: ZAR1 intra-genic differential methylation

BACKGROUND Epigenetic changes such as aberrant DNA methylation and histone modification have been shown to play an important role in the tumorigenesis of malignant melanoma. OBJECTIVE To identify novel tumor-specific differentially methylated regions (DMRs) in human malignant melanoma. METHODS The aberrant methylation at 14 candidate human genomic regions identified through a mouse model study with quantitative DNA methylation analysis using the Sequenom MassARRAY system was performed. RESULTS The CpG island Exon 1 region of the Zygote arrest 1 (ZAR1) gene, which is responsible for oocyte-to-embryo transition, showed frequent aberrant methylation of 28 out of 30 (93%) melanoma surgical specimens, 16 of 17 (94%) melanoma cell lines, 0% of 4 normal human epidermal melanocyte (NHEM) cell lines, 0% of 10 melanocytic nevi and 100% of 51 various cancer cell lines. According to the real-time RT-PCR, the ZAR1 gene was overexpressed in part of the hypermethylated cell lines, while its low expression with bivalent histone methylation status was seen in unmethylated cell lines. CONCLUSION Our findings suggest that the ZAR1 intra-genic differentially methylated region would be a useful tumor marker for malignant melanoma and may be other type of cancers. The involvement of ZAR1 in the carcinogenesis of melanoma, still remains unclear, although we have examined tumorigenic capacities by exogenous full-length ZAR1 over-expression and siRNA knock-down experiments.

Mitochondrial division inhibitor-1 induces mitochondrial hyperfusion and sensitizes human cancer cells to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer treatment, but some cancer cell types are resistant to TRAIL cytotoxicity. Therefore, overcoming this resistance is necessary for effective TRAIL therapy. Mitochondrial morphology is important for the maintenance of cell function and survival, and is regulated by the delicate balance between fission and fusion. However, the role of mitochondrial morphology dynamics in TRAIL-induced apoptosis is unknown. Here we show that mitochondrial division inhibitor-1 (mdivi-1), an inhibitor of dynamin-related protein1 (Drp1), modulates mitochondrial morphology and TRAIL-induced apoptosis in human cancer cells. mdivi-1 treatment (≥12.5 µM) caused dose- and time‑dependent cell death in malignant melanoma, lung cancer and osteosarcoma cells, while sparing normal cells. mdivi-1 also sensitized cancer cells to TRAIL-induced apoptosis. This potentiation of apoptosis occurred through a caspase-depependent mechanism including the mitochondrial and endoplasmic reticulum (ER) stress pathways. Mdivi-1 potentiated mitochondrial oxidative stress, a major cause of mitochondrial and ER stresses, as evidenced by increases in mitochondrial reactive oxygen species levels, mitochondrial mass, and cardiolipin oxidation. Live cell fluorescence imaging using MitoTracker Red CMXRos revealed that Mdivi-1 caused substantial mitochondrial hyperfusion. Moreover, silencing of Drp1 expression also caused mitochondrial hyperfusion and sensitized cancer cells to TRAIL-induced apoptosis. Our results suggest that cancer cells are more vulnerable than normal cells to a perturbation in mitochondrial morphology dynamics and that this higher susceptibility can be exploited to selectively kill cancer cells and sensitize to TRAIL.

Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole–imidazole polyamide, which targets an E-box motif

Gene amplification and/or overexpression of the transcription factor c‐MYC, which binds to the E‐box sequence (5′‐CACGTG‐3′), has been observed in many human tumors. In this study, we have designed 5 pyrrole–imidazole (PI) polyamides recognizing E‐box, and found that, among them, Myc‐6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc‐6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc‐6 exerts its tumor‐suppressive ability at least in part through the specific down‐regulation of MALAT1.

Tumor-selective mitochondrial network collapse induced by atmospheric gas plasma-activated medium

Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. However, the mechanisms of its tumor-selective cytotoxicity remain unclear. Here we report that AGP-activated medium increases caspase-independent cell death and mitochondrial network collapse in a panel of human cancer cells, but not in non-transformed cells. AGP irradiation stimulated reactive oxygen species (ROS) generation in AGP-activated medium, and in turn the resulting stable ROS, most likely hydrogen peroxide (H2O2), activated intracellular ROS generation and mitochondrial ROS (mROS) accumulation. Culture in AGP-activated medium resulted in cell death and excessive mitochondrial fragmentation and clustering, and these responses were inhibited by ROS scavengers. AGP-activated medium also increased dynamin-related protein 1-dependent mitochondrial fission in a tumor-specific manner, and H2O2 administration showed similar effects. Moreover, the vulnerability of tumor cells to mitochondrial network collapse appeared to result from their higher sensitivity to mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment.

New chemically induced skin tumour susceptibility loci identified in a mouse backcross between FVB and dominant resistant PWK

BackgroundA variety of skin cancer susceptibility among mouse strains has allowed identification of genes responsible for skin cancer development. Fifteen Skts loci for skin tumour susceptibility have been mapped so far by using the two-stage skin carcinogenesis model [induced by 7.12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)]. A few responsible genes have been identified using wild-derived dominant resistant Mus spretus mice, and one has been confirmed as a low penetrance cancer susceptibility gene in a variety of human cancers.ResultsIn the present study, we found that wild-derived PWK mice developed no tumour by treatment with the two-stage skin carcinogenesis protocol. This phenotype is dominant resistant when crossed with the highly susceptible strain FVB. By analyzing the F1 backcross generation between PWK and FVB, we found empirical evidence of significant linkage at the new loci Skts-fp1 on chromosome 4 and suggestive linkage on chromosomes 1, 3, 11, 12 and 14 for skin tumour susceptibility. Skts-fp1 includes the Skts7 interval, which was previously mapped by a Mus spretus and NIH backcross. We also observed suggestive linkage on chromosomes 1 and 2 in the female population only, while suggestive linkage on chromosomes 14 and 15 only was observed in the male population. A significant genetic interaction was seen between markers of D11Mit339 and D16Mit14.ConclusionAnalysis of this new cross may facilitate the identification of genes responsible for mouse skin cancer susceptibility and may reveal their biological interactions.

Targeting Oct1 genomic function inhibits androgen receptor signaling and castration-resistant prostate cancer growth

Androgen receptor (AR) functions as a ligand-dependent transcription factor to regulate its downstream signaling for prostate cancer progression. AR complex formation by multiple transcription factors is important for enhancer activity and transcriptional regulation. However, the significance of such collaborative transcription factors has not been fully understood. In this study, we show that Oct1, an AR collaborative factor, coordinates genome-wide AR signaling for prostate cancer growth. Using global analysis by chromatin immunoprecipitation sequencing (ChIP-seq), we found that Oct1 is recruited to AR-binding enhancer/promoter regions and facilitates androgen signaling. Moreover, a major target of AR/Oct1 complex, acyl-CoA synthetase 3 (ACSL3), contributes to tumor growth in nude mice, and its high expression is associated with poor prognosis in prostate cancer patients. Next, we examined the therapeutic effects of pyrrole-imidazole polyamides that target the Oct1-binding sequence identified in the center of the ACSL3 AR-binding site. We observed that treatment with Oct1 polyamide severely blocked the Oct1 binding at the ACSL3 enhancer responsible for its transcriptional activity and ACSL3 induction. In addition, Oct1 polyamides suppressed castration-resistant tumor growth and specifically repressed global Oct1 chromatin association and androgen signaling in prostate cancer cells, with few nonspecific effects on basal promoter activity. Thus, targeting Oct1 binding could be a novel therapeutic strategy for AR-activated castration-resistant prostate cancer.