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1.  Characteristic promoter hypermethylation signatures in male germ cell tumors 
Molecular Cancer  2002;1:8.
Human male germ cell tumors (GCTs) arise from undifferentiated primordial germ cells (PGCs), a stage in which extensive methylation reprogramming occurs. GCTs exhibit pluripotentality and are highly sensitive to cisplatin therapy. The molecular basis of germ cell (GC) transformation, differentiation, and exquisite treatment response is poorly understood.
To assess the role and mechanism of promoter hypermethylation, we analyzed CpG islands of 21 gene promoters by methylation-specific PCR in seminomatous (SGCT) and nonseminomatous (NSGCT) GCTs. We found 60% of the NSGCTs demonstrating methylation in one or more gene promoters whereas SGCTs showed a near-absence of methylation, therefore identifying distinct methylation patterns in the two major histologies of GCT. DNA repair genes MGMT, RASSF1A, and BRCA1, and a transcriptional repressor gene HIC1, were frequently methylated in the NSGCTs. The promoter hypermethylation was associated with gene silencing in most methylated genes, and reactivation of gene expression occured upon treatment with 5-Aza-2' deoxycytidine in GCT cell lines.
Our results, therefore, suggest a potential role for epigenetic modification of critical tumor suppressor genes in pathways relevant to GC transformation, differentiation, and treatment response.
PMCID: PMC149411  PMID: 12495446
Germ cell tumor; promoter hypermethylation; MGMT; RASSF1A; BRCA1; gene expression
2.  Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1 
Neoplasia (New York, N.Y.)  2005;7(3):200-206.
We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma.
PMCID: PMC1501141  PMID: 15799820
Retinoblastoma; methylation; RASSF1A; MGMT; RB
Neuro-Oncology  2014;16(Suppl 3):iii23.
BACKGROUND: Intracranial germ cell tumors (iGCTs) are rare in the Western countries, however they are the second most common brain tumors in patients under 14 in Japan. Unlike other common pediatric brain tumors, the biology of iGCTs is largely unknown. METHODS: We performed a whole exome sequencing in a large series of iGCTs to elucidate their molecular pathogenesis. A total of 198 germ cell tumors (GCTs) including 133 iGCTs (69 pure germinomas, 56 NGGCTs and 8 metastatic tumors) as well as 65 testicular germ cell tumors (tGCTs) (39 seminomas and 26 non-seminoma GCTs) were collected from 13 centers participating in the Intracranial Germ Cell Tumor Consortium in Japan. Somatic mutations in all coding exons were investigated by whole exome sequencing (WES) in 41 tumors and the matched normal DNAs. Based on the WES data, 41 candidate genes were selected according to the frequency and/or significance of the mutations found. All coding exons of these 41 genes spanning over 160kb were PCR-amplified in a further 157 GCTs and sequenced using the IonTorrent system. The results were integrated with the patients' clinical information that was available for 124 iGCT patients. RESULTS: On average, 15.4 non-synonymous somatic mutations were observed in each tumor, ranging from 1 to 140 by WES in 41 iGCTs. The combined WES and IonTorrent screenings showed that KIT was the most frequently mutated gene in both iGCTs (27%) and tGCTs (18%). MTOR was the second most frequently mutated also in both iGCTs (7%) and tGCTs (6%). RAS mutations (KRAS, HRAS, NRAS) were altogether found in 13% of iGCTs and 12% of tGCTs. These mutations were mutually exclusive to each other and also to KIT mutations. Collectively, the genes involved in the MAPK pathway (e.g., KIT, RAS, NF1) and the PI3K/MTOR pathway (e.g., MTOR, PTEN) were mutated in 44% and 13% of all GCTs. Among the iGCTs, these alterations were significantly more common among pure germinomas than NGGCTs. The mutated MTOR protein was shown to have increased kinase activity, which was suppressed by specific MTOR inhibitors. CONCLUSIONS: Our comprehensive mutational genomic analysis of GCTs revealed that alterations of the MAPK and/or PI3K/MTOR pathways play a critical role in the pathogenesis of both iGCTs and tGCTs, although the extent of their involvement depends on the histopathological subtypes. Our findings will hopefully lead to the development of a targeted therapy for treatment-resistant iGCTs. SECONDARY CATEGORY: Tumor Biology.
PMCID: PMC4144564
4.  Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: Pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells 
Oncology Reports  2012;27(4):1135-1141.
Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature of primary cancer cells. However, to date the somatic epigenetic events that occur in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined. In the present study, we analyzed the promoter methylation status of the genes mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of the three proteins was quantified by semi-quantitative immunohistochemical analysis. The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing. Proliferation was determined by Alamar blue staining. We found that the MGMT promoter in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated. The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples while in the examined cell line the MLH promoter was unmethylated. The RASSF1A promoter showed methylation in 13% of the tumor samples and in the cell line. MGMT expression in the group of tumor samples with a hypermethylated promoter was statistically significantly lower compared to the group of tumors with no measured hypermethylation of the MGMT promoter. After treatment of the cell line with the demethylating agent 5-Aza no demethylation of the methylated MGMT and RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results, however, increased numbers of unmethylated CpG islands in the promoter region of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05) reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza application resulted in partial demethylation of the MGMT and RASSF1A TSGs and reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza for HNSCC treatment.
PMCID: PMC3583513  PMID: 22246327
O-6-methylguanine-DNA methyltransferase; mutL homolog 1; Ras association domain family member 1; tumor suppressor gene; head and neck squamous cell carcinoma; 5-azacytidine
5.  Resistance to Platinum-Containing Chemotherapy in Testicular Germ Cell Tumors Is Associated with Downregulation of the Protein Kinase SRPK11 
Neoplasia (New York, N.Y.)  2004;6(4):297-301.
Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPK1 levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPK1 staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.
PMCID: PMC1502111  PMID: 15256051
Chemotherapy resistance; germ cell tumors; chemotherapy sensitivity; protein kinase SRPK1; immunohistochemistry
6.  Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients 
Oncogene  2004;23(22):4014-4022.
Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hyper-methylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methy-latransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n = 20) compared to metastatic melanomas. However, hypermethylation of RAR-β2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had ≥1 gene(s) and 59% had ≥2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis of melanoma patients’ plasma (preoperative blood; n = 31) demonstrated circulating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.
PMCID: PMC2856469  PMID: 15064737
MGMT; RAR-β2; RASSF1A; methylation; melanoma
7.  Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma 
BMC Cancer  2004;4:65.
Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma.
We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading:. I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control.
Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6,, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma.
Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.
PMCID: PMC520749  PMID: 15367334
8.  Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer 
BMC Medical Genomics  2009;2:34.
Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s) underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI) methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition.
To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis.
Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99) between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance) following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells.
Selective epigenetic disruption of distinct biological pathways was observed during development of platinum resistance in ovarian cancer. Integrated analysis of DNA methylation and gene expression may allow for the identification of new therapeutic targets and/or biomarkers prognostic of disease response. Finally, our results suggest that epigenetic therapies may facilitate the prevention or reversal of transcriptional repression responsible for chemoresistance and the restoration of sensitivity to platinum-based chemotherapeutics.
PMCID: PMC2712480  PMID: 19505326
9.  CpG methylation of the FHIT, FANCF, cyclin-D2, BRCA2 and RUNX3 genes in Granulosa cell tumors (GCTs) of ovarian origin 
Molecular Cancer  2004;3:33.
Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.
We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were also included as the control.
Frequencies of methylation in GCTs were 7/25 (28 % for FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.
These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment.
PMCID: PMC538268  PMID: 15574200
10.  High DNA Methyltransferase 3B Expression Mediates 5-aza-deoxycytidine Hypersensitivity in Testicular Germ Cell Tumors 
Cancer research  2009;69(24):9360-9366.
Testicular germ cell tumors (TGCTs) are the most common solid tumors of 15–35 year old men. TGCT patients are frequently cured with cytotoxic cisplatin-based therapy. However, TGCT patients refractory to cisplatin-based chemotherapy have a poor prognosis, as do those having a late relapse. Pluripotent embryonal carcinomas (ECs) are the malignant counterparts to embryonic stem (ES) cells and are considered the stem cells of TGCTs. Here we demonstrate that human EC cells are highly sensitive to 5-aza-deoxycytidine (5-aza-CdR) as compared to somatic solid tumor cells. Decreased proliferation and survival with low nanomolar concentrations of 5-aza-CdR is associated with ATM activation, H2AX phosphorylation, increased expression of p21, and the induction of genes known to be methylated in TGCTs (MGMT, RASSF1A and HOXA9). Notably, 5-aza-CdR hypersensitivity is associated with markedly abundant expression of the pluripotency-associated DNA methyltransferase 3B (DNMT3B) as compared to somatic tumor cells. Knockdown of DNMT3B in EC cells results in substantial resistance to 5-aza-CdR, strongly indicating that 5-aza-CdR sensitivity is mechanistically linked to high levels of DNMT3B. Intriguingly, cisplatin-resistant EC cells retain an exquisite sensitivity to low dose 5-aza-CdR treatment and pretreatment of 5-aza-CdR re-sensitizes these cells to cisplatin-mediated toxicity. This re-sensitization is also partially dependent on high DNMT3B levels. These novel findings indicate that high expression of DNMT3B, a likely byproduct of their pluripotency and germ cell origin, sensitizes TGCT-derived EC cells to low dose 5-aza-CdR treatment.
PMCID: PMC2795063  PMID: 19951990
DNMT3B; 5-aza-deoxycytidine; embryonal carcinoma; cisplatin; resistance; testicular cancer
11.  Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas 
ISRN Neurology  2012;2012:576578.
While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14ARF, and p16INK4A), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14ARF and p16INK4A did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14ARF and p16INK4A, in which other alterations (mutations, homozygous deletions) are prevalent.
PMCID: PMC3263565  PMID: 22389839
12.  Hypermethylation of CpG island in O6-methylguanine-DNA methyltransferase gene was associated with K-ras G to A mutation in colorectal tumor 
AIM: To investigate the functions of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) gene in colorectal tumorigenesis and progression.
METHODS: The promoter hypermethylation of MGMT gene was detected in 27 sporadic colorectal adenomas, 62 sporadic colorectal carcinomas and 20 normal colorectal mucosa tissues by methylation-specific PCR. At the same time, the expression of MGMT protein was carried out in the same samples using immunohistochemistry. Mutant-allele-specific amplification was used to detect K-ras G to A point mutation in codon 12.
RESULTS: None of the normal colorectal mucosa tissues showed methylated bands. Promoter hypermethylation was detected in 40.7% (11 of 27) of adenomas and 43.5% (27 of 62) of carcinomas. MGMT proteins were expressed in nucleus and cytoplasm of normal colorectal mucosa tissues. Loss of MGMT expression was found in 22.2% (6 of 27) of adenomas and 45.2% (28 of 62) of carcinomas. The difference between them was significant (P = 0.041). In the 6 adenomas and 28 carcinomas losing MGMT expression, 5 and 24 cases presented methylation, respectively (P = 0.027, P<0.001). Thirteen of the 19 colorectal tumors with K-ras G to A point mutation in codon 12 had methylated MGMT (P = 0.011). The frequencies of K-ras G to A point mutation were 35.3% (12 of 34) and 12.7% (7 of 55) in tumors losing MGMT expression and with normal expression, respectively.
CONCLUSION: Promoter hypermethylation and loss of expression of MGMT gene were common events in colorectal tumorigenesis, and loss of expression of MGMT occurs more frequently in carcinomas than in adenomas in sporadic patients. Hypermethylation of the CpG island of MGMT gene was associated with loss of MGMT expression and K-ras G to A point mutation in colorectal tumor. The frequency of K-ras G to A point mutation was increased in tumors losing MGMT expression. It suggests that epigenetic inactivation of MGMT plays an important role in colorectal neoplasia.
PMCID: PMC4305730  PMID: 15800999
O6-methylguanine-DNA methyltransferase; CpG island; DNA methylation; Epigenetic change; K-ras mutation
13.  Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma 
Molecular Cancer  2013;12:87.
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with high mutational heterogeneity and a generally poor clinical outcome. Despite implicated roles of deregulated TP53, IGF-2 and Wnt signaling pathways, a clear genetic association or unique mutational link to the disease is still missing. Recent studies suggest a crucial role for epigenetic modifications in the genesis and/or progression of ACC. This study specifically evaluates the potential role of epigenetic silencing of RASSF1A, the most commonly silenced tumor suppressor gene, in adrenocortical malignancy.
Using adrenocortical tumor and normal tissue specimens, we show a significant reduction in expression of RASSF1A mRNA and protein in ACC. Methylation-sensitive and -dependent restriction enzyme based PCR assays revealed significant DNA hypermethylation of the RASSF1A promoter, suggesting an epigenetic mechanism for RASSF1A silencing in ACC. Conversely, the RASSF1A promoter methylation profile in benign adrenocortical adenomas (ACAs) was found to be very similar to that found in normal adrenal cortex. Enforced expression of ectopic RASSF1A in the SW-13 ACC cell line reduced the overall malignant behavior of the cells, which included impairment of invasion through the basement membrane, cell motility, and solitary cell survival and growth. On the other hand, expression of RASSF1A/A133S, a loss-of-function mutant form of RASSF1A, failed to elicit similar malignancy-suppressing responses in ACC cells. Moreover, association of RASSF1A with the cytoskeleton in RASSF1A-expressing ACC cells and normal adrenal cortex suggests a role for RASSF1A in modulating microtubule dynamics in the adrenal cortex, and thereby potentially blocking malignant progression.
Downregulation of RASSF1A via promoter hypermethylation may play a role in the malignant progression of adrenocortical carcinoma possibly by abrogating differentiation-promoting RASSF1A- microtubule interactions.
PMCID: PMC3750604  PMID: 23915220
Adrenal cortex; Carcinoma; Adenoma; RASSF1A; Hypermethylation; Epigenetic silencing; Cytoskeleton
14.  The role of gene body cytosine modifications in MGMT expression and sensitivity to temozolomide 
Molecular cancer therapeutics  2014;13(5):1334-1344.
The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is known to play a role in sensitivity to temozolomide. Promoter hypermethylation of MGMT is commonly used to predict low expression levels of MGMT in gliomas, despite observed discordance between promoter methylation and protein levels. Here, we investigated the functional role of gene body cytosine modification in regulating levels of MGMT gene expression and sensitivity to temozolomide. In 91 human glioblastoma samples, we observed significant variation in MGMT expression levels in patients with an unmethylated promoter, with higher levels of gene body cytosine modification correlating with higher gene expression levels. Furthermore, inducing hypomethylation across the MGMT gene body with decitabine corresponded with decreased levels of MGMT gene expression in lymphoblastoid and glioblastoma cell lines, indicating an important functional role for gene body cytosine modifications in maintaining gene expression. We reasoned that the decrease in MGMT expression induced by decitabine may render resistant glioblastoma cell lines more sensitive to temozolomide. Consistent with this reasoning, we found that the MGMT-expressing glioblastoma cell lines exhibiting an unmethylated MGMT promoter that were pre-treated with decitabine became significantly more sensitive to temozolomide. Overall, our results suggest a functional role for gene body cytosine modification in regulating gene expression of MGMT and indicate that pre-treating patients whose tumors have an unmethylated MGMT promoter with decitabine prior to temozolomide treatment may increase their response to therapy.
PMCID: PMC4013207  PMID: 24568970
MGMT; temozolomide; glioblastoma; decitabine; DNA methylation
15.  Signification of Hypermethylated in Cancer 1 (HIC1) as Tumor Suppressor Gene in Tumor Progression 
Cancer Microenvironment  2012;5(3):285-293.
Hypermethylated in cancer 1(HIC1) was identified as a strong suppressor gene in chromosome region 17p13.3 telomeric to TP53. This gene encodes a transcriptional repressor and is ubiquitously expressed in normal tissues but downexpressed in different tumor tissues where it is hypermethylated. The hypermethylation of this chromosomal region leads to epigenetic inactivation of HIC1, which would prompt cancer cells to alter survival and signaling pathways or specific transcription factors during the period of tumorigenesis. In vitro, HIC1 function is mainly a sequence-specific transcriptional repressor interacting with a still growing range of histone deacetylase(HDAC)-dependent and HDAC-independent corepressor complexes. Furthermore, a role for HIC1 in tumor development is firmly supported by Hic1 deficient mouse model and two double heterozygote models cooperate with p53 and Ptch1. Notably, our findings suggest that potential factors derived from tumor microenviroment may play a role in modulating HIC1 expression in tumor cells by epigenetic modification, which is responsible for tumor progression. In this review, we will describe genomic and proteinic structure of HIC1, and summary the potential role of HIC1 in human various solid tumors and leukemia, and explore the influence of tumor microenviroment on inducing HIC1 expression in tumor cells.
PMCID: PMC3460058  PMID: 22528874
HIC1; Epigenetic modification; Tumor microenviroment; Tumor progression
16.  Epigenetic-Genetic Interactions in the APC/WNT, RAS/RAF, and P53 Pathways in Colorectal Carcinoma 
Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O6-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C→A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist.
Experimental Design
We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas.
We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non – MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P <0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002).
Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.
PMCID: PMC3544184  PMID: 18451217
17.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
PMCID: PMC1716188  PMID: 17194187
18.  TLR4 activates NFkB in human ovarian granulosa tumor cells 
Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-κB) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFαplus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-κB does not sensitize GCTs to TRAIL or cisplatin.
PMCID: PMC3118942  PMID: 21616060
Granulosa cell tumor; Toll-like receptor; TLR; ovary; LPS; lipopolysaccharide
19.  The Intronic Long Noncoding RNA ANRASSF1 Recruits PRC2 to the RASSF1A Promoter, Reducing the Expression of RASSF1A and Increasing Cell Proliferation 
PLoS Genetics  2013;9(8):e1003705.
The down-regulation of the tumor-suppressor gene RASSF1A has been shown to increase cell proliferation in several tumors. RASSF1A expression is regulated through epigenetic events involving the polycomb repressive complex 2 (PRC2); however, the molecular mechanisms modulating the recruitment of this epigenetic modifier to the RASSF1 locus remain largely unknown. Here, we identify and characterize ANRASSF1, an endogenous unspliced long noncoding RNA (lncRNA) that is transcribed from the opposite strand on the RASSF1 gene locus in several cell lines and tissues and binds PRC2. ANRASSF1 is transcribed through RNA polymerase II and is 5′-capped and polyadenylated; it exhibits nuclear localization and has a shorter half-life compared with other lncRNAs that bind PRC2. ANRASSF1 endogenous expression is higher in breast and prostate tumor cell lines compared with non-tumor, and an opposite pattern is observed for RASSF1A. ANRASSF1 ectopic overexpression reduces RASSF1A abundance and increases the proliferation of HeLa cells, whereas ANRASSF1 silencing causes the opposite effects. These changes in ANRASSF1 levels do not affect the RASSF1C isoform abundance. ANRASSF1 overexpression causes a marked increase in both PRC2 occupancy and histone H3K27me3 repressive marks, specifically at the RASSF1A promoter region. No effect of ANRASSF1 overexpression was detected on PRC2 occupancy and histone H3K27me3 at the promoter regions of RASSF1C and the four other neighboring genes, including two well-characterized tumor suppressor genes. Additionally, we demonstrated that ANRASSF1 forms an RNA/DNA hybrid and recruits PRC2 to the RASSF1A promoter. Together, these results demonstrate a novel mechanism of epigenetic repression of the RASSF1A tumor suppressor gene involving antisense unspliced lncRNA, in which ANRASSF1 selectively represses the expression of the RASSF1 isoform overlapping the antisense transcript in a location-specific manner. In a broader perspective, our findings suggest that other non-characterized unspliced intronic lncRNAs transcribed in the human genome might contribute to a location-specific epigenetic modulation of genes.
Author Summary
RASSF1A is a tumor suppressor gene whose expression is repressed through epigenetic events in a wide range of different cancers. Repression is effected by DNA hypermethylation of the RASSF1A promoter, which in turn is triggered through histone H3K9/H3K27 trimethylation repressive marks. The addition of the H3K27me3 mark at the RASSF1A promoter locus involves the polycomb repressive complex 2 (PRC2). The molecular mechanisms that control the recruitment of PRC2 to the promoter to initiate H3K27 trimethylation and repress RASSF1A expression have not been described. Here, we identified a long noncoding RNA (lncRNA), termed ANRASSF1 for antisense noncoding RASSF1, that is transcribed from the opposite strand of the RASSF1A gene and is responsible for recruiting PRC2 to the RASSF1A promoter region in a highly location-specific manner. No effect of ANRASSF1 was detected on the promoter of the RASSF1C isoform or the promoters of the four other genes within the vicinity of RASSF1, including two other well-characterized tumor suppressor genes. This work provides evidence that the epigenetic modulation of the tumor suppressor gene RASSF1A is dependent on the lncRNA ANRASSF1 and highlights the importance of further studies on the involvement of ANRASSF1 in tumorigenesis.
PMCID: PMC3749938  PMID: 23990798
20.  Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin 
Molecular and Clinical Oncology  2014;3(2):408-414.
The development of cisplatin resistance in human cancers is controlled by multiple genes and leads to therapeutic failure. Hypermethylation of specific gene promoters is a key event in clinical resistance to cisplatin. Although the usage of multiple promoters is frequent in the transcription of human genes, the role of alternative promoters and their regulatory sequences have not yet been investigated in cisplatin resistance genes. In a new approach, we hypothesized that human cancers exploit the specific transcription factor-binding sites (TFBS) and CpG islands (CGIs) located in the alternative promoters of certain genes to acquire platinum drug resistance. To provide a useful resource of regulatory elements associated with cisplatin resistance, we investigated the TFBS and CGIs in 48 alternative promoters of 14 hypermethylated cisplatin resistance genes previously reported. CGIs prone to methylation were identified in 28 alternative promoters of 11 hypermethylated genes. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, whereas the ones lacking CGIs were distributed in two unrelated subclasses. Regulatory sequences, initiator and TATA-532 prevailed over TATA-8 and were found in all the promoters. B recognition element (BRE) sequences were present only in alternative promoters harboring CGIs, but CCAAT and TAACC were found in both types of alternative promoters, whereas downstream promoter element sequences were significantly less frequent. Therefore, it was hypothesized that BRE and CGI sequences co-localized in alternative promoters of cisplatin resistance genes may be used to design molecular markers for drug resistance. A more extensive knowledge of alternative promoters and their regulatory elements in clinical resistance to cisplatin is likely to usher novel avenues for sensitizing human cancers to treatment.
PMCID: PMC4360857  PMID: 25798277
alternative promoters; cisplatin resistance; CpG islands; DNA methylation; transcription factor-binding sites; bioinformatics
21.  Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences 
Molecular Cancer  2010;9:264.
The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.
Frequent methylation of the RASSF2 and RASSF5A CpG island promoters in thyroid tumors was observed. RASSF2 was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. RASSF2 methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for RASSF2 in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). RASSF2 promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated RASSF2 transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05).
These results suggest that RASSF2 encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.
PMCID: PMC2956732  PMID: 20920251
22.  Identification of Hypermethylated Genes Associated with Cisplatin Resistance in Human Cancers 
Cancer research  2010;70(7):2870-2879.
Cisplatin is among the most widely used cytotoxic anti-cancer agents in solid tumors, however, the development of secondary resistance remains a major obstacle to clinical efficacy. Treatment-related DNA hypermethylation may play a role in creating drug resistant phenotypes by inactivating genes that are required for cytotoxicity. We applied a pharmacologic unmasking approach to detect hypermethylated genes whose inactivation contributes to cisplatin resistance. Utilizing three pairs of isogeneic, cisplatin-sensitive and -resistant cell lines derived from two parental cell lines (KB-3-1 and SCC25), we identified several hundred genes that were down-regulated in each resistant cell line and re-activated by the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC). Among them, 30 genes were common to ≥ 2 cell lines, and/or reported to be down-regulated in previous studies. Bisulfite sequencing confirmed that 14 genes were hypermethylated in resistant cell lines, but not in the sensitive parental cell lines. Six of 14 genes (SAT, C8orf4, LAMB3, TUBB, G0S2, MCAM) were cisplatin-inducible in sensitive, but not in resistant cell lines. siRNA knockdown of two genes, SAT and S100P, increased cell viability with cisplatin treatment in sensitive parental cell lines. S100P knockdown significantly decreased the S-phase fraction (SPF) of parental sensitive cell lines and slowed cell proliferation, which was associated with decreased sensitivity to cisplatin. Based on these findings, we conclude that DNA methylation is a frequent event in cells that are chronically exposed to cisplatin, and that methylation-induced gene silencing may play a role in the development of resistance to cytotoxic chemotherapeutic agents.
PMCID: PMC2849007  PMID: 20215521
cisplatin; drug resistance; S100P; methylation; epigenetics
23.  Epigenetic silencing of microRNA-199b-5p is associated with acquired chemoresistance via activation of JAG1-Notch1 signaling in ovarian cancer 
Oncotarget  2013;5(4):944-958.
Epithelial ovarian cancer is a highly lethal and aggressive gynecological malignancy. The high mortality rate is due in part to the fact that many advanced cancer patients become refractory to current chemotherapeutic agents, leading to tumor recurrence and death. However, the underlying mechanisms leading to chemoresistance remain obscure. Here, we report that the loss of miR-199b-5p due to progressive epigenetic silencing leads to the activation of the JAG1-mediated Notch1 signaling cascade, thereby leading to the development of acquired chemoresistance in ovarian cancer. Using miRCURY LNA™ microRNA array and Q-PCR analyses of two pairs of cisplatin-sensitive and –resistant ovarian cancer cell lines, we identified miR-199b-5p as significantly down-regulated in cisplatin-resistant ovarian cancer cells and confirmed that miR-199b-5p is clinically associated with advanced and poor survival ovarian cancers. Interestingly, the loss of miR-199b-5p could be restored by 5-Aza-dC-mediated demethylation, and methylated specific PCR (MS-PCR), bisulfite-sequencing and pyrosequencing revealed that the promoter region of miR-199b-5p was hypermethylated. Computational and mechanistic analyses identified JAG1 as a primary target of miR-199b-5p. Notably, the reduced expression of miR-199b-5p was found to be inversely correlated with the increased expression of JAG1 using an ovarian cancer tissue array. Enforced expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity both in vitro and in vivo. Conversely, re-expression of miR-199b-5p and siRNA-mediated JAG1 knockdown or treatment with Notch specific inhibitor γ-secretase (GSI) attenuated JAG1-Notch1 signaling activity, thereby enhancing cisplatin-mediated cell cytotoxicity. Taken together, our study suggests that the epigenetic silencing of miR-199b-5p during tumor progression is significantly associated with acquired chemoresistance in ovarian cancer through the activation of JAG1-Notch1 signaling.
PMCID: PMC4011596  PMID: 24659709
MiR-199b-5p; JAG1-Notch1; acquired chemoresistance; epithelial ovarian cancer
Neuro-Oncology  2014;16(Suppl 3):iii28.
BACKGROUND: Intracranial germ cell tumors (iGCTs) are the second most common CNS tumors in patients under 14 years old in Japan. But, their molecular genetic profile is largely unknown. METHODS: We have analyzed a total of 198 germ cell tumors (GCTs) including 133 iGCTs (69 pure germinomas, 56 non-germinomatous GCTs and 8 metastatic tumors) as well as 65 testicular germ cell tumors (tGCTs) (39 seminomas and 26 non-seminoma GCTs) were collected from 13 centers participating in the Intracranial Germ Cell Tumor Consortium in Japan. Somatic mutations in all coding exons were investigated by whole exome sequencing (WES) using SureSelectXT Human All Exon v4 and a GAIIx or HiSeq 2000 system in 41 tumors and the matched normal DNAs. Targeted sequencing with a set of custom made PCR primers was performed using either an IonTorrent PGM or Proton System. The results were integrated with the patients' clinical information that was available for 124 iGCT patients. RESULTS: On average, 15.4 non-synonymous somatic mutations were observed in each tumor, ranging from 1 to 140 by WES in 41 iGCTs. MTOR was the second most frequently mutated in both iGCTs (9 cases, 7%) and tGCTs (6%). Collectively, the genes involved in the PI3K/MTOR pathway (e.g., MTOR, PTEN) were mutated in 13% of all GCTs. Clinical parameters of the 9 iGCTs with MTOR mutation were: median age of onset = 15 years old; 7 males and 2 females; 5 germinomas, 3 teratomas and one yolk sac tumor; 5 basal ganglia tumors, 2 pineal tumors, one neurohypophyseal tumor and a medulla oblongata tumor. Age, sex, histology and clinical behavior are within the scope of iGCT except for extraordinary high frequency of basal ganglia GCTs. CONCLUSIONS: iGCTs with MTOR mutation are frequent in iGCTs in basal ganglia. SECONDARY CATEGORY: Pediatrics.
PMCID: PMC4144563
25.  O6-methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies 
Molecular Cancer  2007;6:36.
A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways.
We established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents.
Normal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.
PMCID: PMC1892783  PMID: 17547775

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