Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

Expression of the tumor suppressor gene ARHI in epithelial ovarian cancer is associated with increased expression of p21WAF1/CIP1 and prolonged progression-free survival.

PURPOSE: ARHI, an imprinted putative tumor suppressor gene, is expressed in normal ovarian epithelial cells, but its expression is down-regulated or lost in most ovarian cancer cell lines. Reexpression of ARHI in cancer cells induces p21(WAF1/CIP1), down-regulates cyclin D1 promoter activity and inhibits growth in cell culture and in heterografts. To determine the relevance of these observations to clinical cancer, we have now measured ARHI expression in normal, benign and malignant ovarian tissues using immunohistochemistry and in situ hybridization. EXPERIMENTAL DESIGN: Paraffin embedded tissues from 7 normal ovaries, 22 cystadenomas and 42 borderline lesions were analyzed using standard immunoperoxidase and in situ hybridization techniques to assess ARHI expression. In addition, immunohistochemistry against ARHI was performed on a tissue microarray containing 441 consecutive cases of ovarian carcinoma. RESULTS: Strong ARHI expression was found in normal ovarian surface epithelial cells, cysts and follicles using immunohistochemistry and in situ hybridization. Reduced ARHI expression was observed in tumors of low malignant potential as well as in invasive cancers. ARHI expression was down-regulated in 63% of invasive ovarian cancer specimens and could not be detected in 47%. When immunohistochemistry and in situ hybridization were compared, ARHI protein expression could be down-regulated in the presence of ARHI mRNA. ARHI expression was correlated with expression of p21(WAF1/CIP1) (P = 0.0074) but not with cyclin D1 and associated with prolonged disease free survival (P = 0.001). On multivariate analysis, ARHI expression, grade and stage were independent prognostic factors. ARHI expression did not correlate with overall survival. CONCLUSIONS: Persistence of ARHI expression in epithelial ovarian cancers correlated with prolonged disease free survival and expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).     

Silencing of bidirectional promoters by DNA methylation in tumorigenesis

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.     

Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers.

A cDNA clone encoding human SRBC [serum deprivation response factor (sdr)-related gene product that binds to c-kinase] was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe. The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors. hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines. More significantly, the expression of hSRBC protein was down-regulated in a large fraction [30 (70%) of 43] of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells. The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated. Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation. Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene.     

Reexpression of the tumor suppressor gene ARHI induces apoptosis in ovarian and breast cancer cells through a caspase-independent calpain-dependent pathway

ARHI, an imprinted putative tumor suppressor gene, encodes a M(r) 26,000 GTP-binding protein that is 60% homologous to ras and rap but has a dramatically different function. ARHI expression is down-regulated in a majority of breast and ovarian cancers. Using a dual adenovirus system, we have reexpressed ARHI in ovarian cancer and breast cancer cells that have lost ARHI expression. Reexpression of ARHI inhibited growth, decreased invasiveness, and induced apoptosis. At 5 days after infection with ARHI adenovirus, 30-45% of MDA-MB-231 breast cancer cells and 5-11% of SKOv3 ovarian cancer cells were apoptotic as judged by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay and by Annexin V staining with flow cytometric analysis. Although poly(ADP-ribose) polymerase could be detected immunohistochemically in the nuclei of apoptotic cells, no activation of the effector caspases (caspase 3, 6, 7, or 12) or the initiator caspases (caspase 8 or 9) could be detected in cell lysates using Western blotting. When gene expression was analyzed on a custom cDNA array that contained 2304 known genes, infection with ARHI adenovirus up-regulated 15 genes relative to control cells infected with LacZ adenovirus. The greatest degree of mRNA up-regulation was observed in a Homo sapiens calpain-like protease. On Western blot analysis, calpain protein was increased 2-3-fold at 3-5 days after infection with ARHI adenovirus. No increase in calpain protein was observed after LacZ adenovirus infection. Calpain cleavage could be detected after ARHI reexpression, and inhibitors of calpain, but not inhibitors of caspase, partially prevented ARHI-induced apoptosis. Consequently, reexpression of ARHI in breast and ovarian cancer cells appears to induce apoptosis through a caspase-independent, calpain-dependent mechanism.     

Less frequent promoter hypermethylation of DLC-1 gene in primary breast cancers

Absence or low expression of DLC-1, a tumor suppressor gene, in breast cancers has been shown recently. LOH of 8p12-p22, on which DLC-1 is located, is frequent in breast cancers, but the correlation between low expression of DLC-1 and LOH has not been confirmed. To determine the implication of aberrant methylation, one of the most frequent mechanisms of silencing the tumor suppressor or cancer-related genes, we examined the methylation status of DLC-1 promoter region in breast cancer cell lines and primary breast tumors. The hypermethylation status was examined by MSP and 25% of cell lines harbored a methylated allele. The gene silencing by methylation was also confirmed by the re-expression of DLC-1 by the 5-aza-2'-deoxycytidine treatment in DLC-1 hypermethylated cell line. But the methylation of DLC-1 gene was less frequently shown in primary breast cancers (10%). These data suggest that hypermethylation is responsible for silencing of DLC-1 gene in a limited portion of breast cancers.     

Epigenetic silencing of PEG3 gene expression in human glioma cell lines

Genomic imprinting, the phenomenon in which alleles of genes are expressed differentially depending on their parental origins, has important consequences for mammalian development, and disturbance of normal imprinting leads to abnormal embryogenesis and some inherited diseases and is also associated with various cancers. In the context of screening for novel imprinted genes on human chromosome 19q13.4 with mouse A9 hybrids, we identified a maternal allele-specific methylated CpG island in exon 1 of paternally expressed imprinted gene 3 (PEG3), a gene that exhibits paternal allele-specific expression. Because PEG3 expression is downregulated in some gliomas and glioma cell lines, despite high-level expression in normal brain tissues, we investigated whether the loss of PEG3 expression is related to epigenetic modifications involving DNA methylation. We found monoallelic expression of PEG3 in all normal brain tissues examined and five of nine glioma cell lines that had both unmethylated and methylated alleles; the remaining four glioma cell lines exhibited gain of imprinting with hypermethylated alleles. In addition, treatment of glioma cell lines with the DNA demethylating agent 5-aza-2'-deoxycytidine reversed the silencing of PEG3 biallelically. In this article, we report that the epigenetic silencing of PEG3 expression in glioma cell lines depends on aberrant DNA methylation of an exonic CpG island, suggesting that PEG3 contributes to glioma carcinogenesis in certain cases.     

Hypermethylation of CpG islands in primary and metastatic human prostate cancer

Aberrant DNA methylation patterns may be the earliest somatic genome changes in prostate cancer. Using real-time methylation-specific PCR, we assessed the extent of hypermethylation at 16 CpG islands in DNA from seven prostate cancer cell lines (LNCaP, PC-3, DU-145, LAPC-4, CWR22Rv1, VCaP, and C42B), normal prostate epithelial cells, normal prostate stromal cells, 73 primary prostate cancers, 91 metastatic prostate cancers, and 25 noncancerous prostate tissues. We found that CpG islands at GSTP1, APC, RASSF1a, PTGS2, and MDR1 were hypermethylated in >85% of prostate cancers and cancer cell lines but not in normal prostate cells and tissues; CpG islands at EDNRB, ESR1, CDKN2a, and hMLH1 exhibited low to moderate rates of hypermethylation in prostate cancer tissues and cancer cell lines but were entirely unmethylated in normal tissues; and CpG islands at DAPK1, TIMP3, MGMT, CDKN2b, p14/ARF, and CDH1 were not abnormally hypermethylated in prostate cancers. Receiver operator characteristic curve analyses suggested that CpG island hypermethylation changes at GSTP1, APC, RASSF1a, PTGS2, and MDR1 in various combinations can distinguish primary prostate cancer from benign prostate tissues with sensitivities of 97.3-100% and specificities of 92-100%. Hypermethylation of the CpG island at EDNRB was correlated with the grade and stage of the primary prostate cancers. PTGS2 CpG island hypermethylation portended an increased risk of recurrence. Furthermore, CpG island hypermethylation patterns in prostate cancer metastases were very similar to the primary prostate cancers and tended to show greater differences between cases than between anatomical sites of metastasis.     

Hypermethylation and silencing of the putative tumor suppressor Tazarotene-induced gene 1 in human cancers

A variety of tumor suppressor genes are down-regulated by hypermethylation during carcinogenesis. Using methylated CpG amplification-representation difference analysis, we identified a DNA fragment corresponding to the Tazarotene-induced gene 1 (TIG1) promoter-associated CpG island as one of the genes hypermethylated in the leukemia cell line K562. Because TIG1 has been proposed to act as a tumor suppressor, we tested the hypothesis that cytosine methylation of the TIG1 promoter suppresses its expression and causes a loss of responsiveness to retinoic acid in some neoplastic cells. We examined TIG1 methylation and expression status in 53 human cancer cell lines and 74 primary tumors, including leukemia and head and neck, breast, colon, skin, brain, lung, and prostate cancer. Loss of TIG1 expression was strongly associated with TIG1 promoter hypermethylation (P < 0.001). There was no correlation between TIG1 promoter methylation and that of retinoid acid receptor beta2 (RARbeta2), another retinoic-induced putative tumor suppressor gene (P = 0.78). Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine for 5 days restored TIG1 expression in all eight silenced cell lines tested. TIG1 expression was also inducible by treatment with 1 micro M all-trans-retinoic acid for 3 days except in densely methylated cell lines. Treatment of the K562 leukemia cells with demethylating agent combined with all-trans-retinoic acid induced apoptosis. These findings indicate that silencing of TIG1 promoter by hypermethylation is common in human cancers and may contribute to the loss of retinoic acid responsiveness in some neoplastic cells.     

Genetic and epigenetic analysis of the TIMP-3 gene in ovarian cancer

Chromosome 22q shows a high frequency of loss of heterozygosity (LOH) in ovarian cancers suggesting the existence of one or more important tumor suppressor genes (TSGs). The tissue inhibitor of metalloproteinase-3 (TIMP-3) is a plausible TSG candidate since it is often encompassed within these regions of LOH. TIMP-3 has not previously been investigated for somatic mutations or promoter hypermethylation in ovarian cancer. We analyzed 65 ovarian cancers for both somatic genetic mutations and TIMP-3 promoter hypermethylation. Screening of all coding exons of TIMP-3 did not reveal any somatic genetic mutations and only 1/65 showed TIMP-3 methylation. Our data indicate that inactivation of TIMP-3 by somatic mutation or promoter hypermethylation is rare in ovarian cancer.     

Identification of genes uniquely involved in frequent microsatellite instability colon carcinogenesis by expression profiling combined with epigenetic scanning

Gene silencing through CpG island hypermethylation has been associated with genesis or progression of frequent microsatellite instability (MSI-H) cancers. To identify novel methylation sites unique to MSI-H colon cancers in an unbiased fashion, we conducted a global expression profiling-based methylation target search. We identified 81 genes selectively down-regulated in MSI-H cancers using cDNA microarray analysis of 41 primary colon cancers. Forty six of these 81 genes contained CpG islands overlapping their 5'untranslated regions. Initial screening of six genes in 57 primary colon cancers detected the following gene with MSI-H cancer-specific hypermethylation: RAB32, a ras family member and A-kinase-anchoring protein, was methylated in 14 of 25 (56%) MSI-H cancers but in none of 32 non-MSI-H cancers or 23 normal colonic specimens. RAB32 hypermethylation correlated with RAB32 mRNA down-regulation and with hMLH1 hypermethylation. In addition, the protein-tyrosine phosphatase receptor type O gene, PTPRO, was frequently methylated in right-sided tumors. This methylation screening strategy should identify additional genes inactivated by epigenetic silencing in colorectal and other cancers.     

Genetic and epigenetic analysis of CHEK2 in sporadic breast, colon, and ovarian cancers.

PURPOSE: Germ-line variants in CHEK2 have been associated with increased breast, thyroid, prostate, kidney, and colorectal cancer risk; however, the prevalence of somatic inactivation of CHEK2 in common cancer types is less clear. The aim of this study was to determine if somatic mutation and/or epigenetic modification play a role in development of sporadic breast, colon, or ovarian cancers. EXPERIMENTAL DESIGN: We undertook combined genetic and epigenetic analysis of CHEK2 in sporadic primary breast, ovarian, and colon tumors [all exhibiting chromosome 22q loss of heterozygosity (LOH)] and cancer cell lines. Expression of Chk2 was assessed by immunohistochemistry in 119 ovarian tumors. RESULTS: Two novel germ-line variants were identified; however, none of the primary tumors harbored somatic mutations. Two CpG clusters previously implicated in CHEK2 silencing were investigated for evidence of hypermethylation. No methylation was detected at the distal CpG island. The proximal CpG cluster was methylated in all tumor and normal DNA, suggesting that this might not represent a true CpG island and is not relevant in the control of CHEK2 expression. Twenty-three percent of ovarian tumors were negative for Chk2 protein by immunohistochemistry, but there was no significant correlation between LOH across the CHEK2 locus and intensity of Chk2 staining (P = 0.12). CONCLUSIONS: LOH across the CHEK2 locus is common in sporadic breast, ovarian, and colorectal cancers, but point mutation or epigenetic inactivation of the retained allele is uncommon. Loss of Chk2 protein in ovarian cancer was not associated with allelic status, suggesting that inactivation does not occur as a consequence of haploinsufficiency.     

ARHI, as a novel suppressor of cell growth and downregulated in human hepatocellular carcinoma, could contribute to hepatocarcinogenesis

The identification of cancer genes differentially expressed in hepatocellular carcinoma (HCC) plays an important role in understanding the molecular mechanisms of hepatocarcinogenesis. Here, ARHI gene expression was analyzed by real-time RT-PCR and it was significantly downregulated in 33 of the 42 (78.6%, more than two folds) HCC specimens compared with adjacent noncancerous livers (P < 0.01). In addition, ARHI expression was reduced in some HCC samples at protein level confirmed by immunohistochemistry. Furthermore, our data suggested that the overexpression of ARHI can significantly inhibit cell growth and colony formation of Hep3B cells (P < 0.01), whilst silencing endogenous ARHI gene by RNAi could promote cell growth of Huh-7 and Focus. LOH of microsatellite markers D1S2806 and D1S2803 was only found in 2.4% (1 of 42 HCCs) of HCC cases. The expression of ARHI was obviously re-expressed in some HCC cells, Bel-7402, Bel-7405, QGY-7703 and Hep3B, by a demethylation agent, 5-aza-2'-deoxycytidine (DAC). DNA hypermethylation within ARHI promoter was identified in 47.1% of HCC specimens without ARHI expression. Our current observations provide evidences that ARHI downregulated in HCCs could play a role in liver cancer via acting as a tumor suppressor gene, which mainly was triggered by the epigenetic events in HCC specimens.     

WT1 and WT1-AS genes are inactivated by promoter methylation in ovarian clear cell adenocarcinoma

BACKGROUND: Ovarian clear cell adenocarcinoma is associated with one of the poorest prognoses among human epithelial ovarian cancers. The authors hypothesized that Wilms tumor suppressor 1 gene (WT1) sense and antisense (WT1-AS) expression and their promoter methylation status could characterize ovarian clear cell adenocarcinoma from ovarian serous adenocarcinoma. METHODS: To test this hypothesis, ovarian cancer cell lines and 42 cancer tissues (17 clear cell and 25 serous adenocarcinoma) were analyzed for expression and methylation of WT1 and WT1-AS genes. RESULTS: These experiments demonstrated that all serous adenocarcinoma tissues expressed both WT1 and WT1-AS genes, although expression of these genes was lacking in clear cell adenocarcinoma. The WT1 and WT1-AS promoter were significantly methylated in clear cell adenocarcinoma (88.2% and 88.2%, respectively) compared with serous adenocarcinoma (24.0% and 20.0%, respectively). Significant correlation between methylation and mRNA expression status was observed for each gene. Also in agreement with these data, WT1 and WT1-AS negative ovarian cancer cell lines reexpressed these genes after treatment with the demethylating agent, 5-aza-2'-deoxycytidine. CONCLUSIONS: The current study shows that CpG hypermethylation is an important mechanism of WT1 and WT1-AS gene inactivation in ovarian clear cell adenocarcinoma. This is the first report that has demonstrated differential expression and methylation of WT1-AS in ovarian clear cell and serous adenocarcinomas. This study presents new molecular characterizations between these two types of adenocarcinoma and may provide insight as to why clear cell adenocarcinoma has a poorer prognosis than serous adenocarcinoma of the ovary.     

Reduced expression of MYO18B, a candidate tumor-suppressor gene on chromosome arm 22q, in ovarian cancer

Allelic imbalance on chromosome arm 22q has been detected in 50-70% of ovarian cancers, suggesting the presence of a tumor-suppressor gene on this chromosome arm that is involved in ovarian carcinogenesis. Recently, we isolated a candidate tumor-suppressor gene, MYO18B, at 22q12.1, which is deleted, mutated and hypermethylated in approximately 50% of lung cancers. In our study, we analyzed genetic and epigenetic alterations of the MYO18B gene in ovarian cancers. Missense MYO18B mutations were detected in 1 of 4 (25%) ovarian cancer cell lines and in 1 of 17 (5.9%) primary ovarian cancers. MYO18B expression was reduced in all 4 ovarian cancer cell lines and in 12 of 17 (71%) of primary ovarian cancers. MYO18B expression was restored by treatment with 5-aza-2'-deoxycytidine and/or trichostatin A in 3 of 4 cell lines with reduced MYO18B expression, and hypermethylation of the promoter CpG island for MYO18B was observed in 2 of these 3 cell lines. Its hypermethylation was also observed in 2 of 15 (13%) primary ovarian cancers. Thus, it was indicated that MYO18B expression is reduced in a considerable fraction of ovarian cancers by several mechanisms, including hypermethylation, while the MYO18B gene is mutated in a small subset of ovarian cancers. The present results suggest that MYO18B alterations, including both epigenetic and genetic alterations, play an important role in ovarian carcinogenesis.