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Epigenetic inactivation of the NORE1gene correlates with malignant progression of colorectal tumors
- Chang Kyun Lee†1,
- Jin-Hee Lee†2,
- Min-Goo Lee2,
- Seong-In Jeong2,
- Tae-Kyu Ha2,
- Min-Ju Kang2,
- Byung-Kyu Ryu2,
- Young Hwangbo1,
- Jae-Jun Shim1,
- Jae Young Jang1,
- Kil Yeon Lee3,
- Hyo Jong Kim1 and
- Sung-Gil Chi2Email author
© Lee et al; licensee BioMed Central Ltd. 2010
Received: 6 July 2010
Accepted: 22 October 2010
Published: 22 October 2010
NORE1 (RASSF5) is a newly described member of the RASSF family with Ras effector function. NORE1 expression is frequently inactivated by aberrant promoter hypermethylation in many human cancers, suggesting that NORE1 might be a putative tumor suppressor. However, expression and mutation status of NORE1 and its implication in colorectal tumorigenesis has not been evaluated.
Expression, mutation, and methylation status of NORE1A and NORE1B in 10 cancer cell lines and 80 primary tumors were characterized by quantitative PCR, SSCP, and bisulfite DNA sequencing analyses. Effect of NORE1A and NORE1B expression on tumor cell growth was evaluated using cell number counting, flow cytometry, and colony formation assays.
Expression of NORE1A and NORE1B transcript was easily detectable in all normal colonic epithelial tissues, but substantially decreased in 7 (70%) and 4 (40%) of 10 cancer cell lines and 31 (38.8%) and 25 (31.3%) of 80 primary carcinoma tissues, respectively. Moreover, 46 (57.6%) and 38 (47.5%) of 80 matched tissue sets exhibited tumor-specific reduction of NORE1A and NORE1B, respectively. Abnormal reduction of NORE1 was more commonly observed in advanced stage and high grade tumors compared to early and low grade tumors. While somatic mutations of the gene were not identified, its expression was re-activated in all low expressor cells after treatment with the demethylating agent 5-aza-dC. Bisulfite DNA sequencing analysis of 31 CpG sites within the promoter region demonstrated that abnormal reduction of NORE1A is tightly associated with promoter CpG sites hypermethylation. Moreover, transient expression and siRNA-mediated knockdown assays revealed that both NORE1A and NORE1B decrease cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress.
Our data indicate that epigenetic inactivation of NORE1 due to aberrant promoter hypermethylation is a frequent event in colorectal tumorigenesis and might be implicated in the malignant progression of colorectal tumors.
Mutational activation of Ras plays a critical role in the development and malignant progression of many human tumors [1, 2]. Ras oncoproteins participate in the regulation of a broad range of biological processes, including cell growth and differentiation, membrane trafficking, transcriptional regulation and apoptosis [2, 3]. Ras proteins exist in an inactive GDP-bound and an active GTP-bound conformation, and in the active GTP-bound state, Ras functions by binding and modulating the activity of a diverse array of effector proteins, such as Raf kinases, RalGEFs, and phosphatidylinositol 3-kinases[3, 4]. Recently, new genes encoding the Ras-association (RA) domain have been identified, and termed the Ras-association domain family (RASSF) [5, 6]. RASSF proteins interact either directly or indirectly with activated Ras and may serve as tumor suppressors by modulating signaling pathways associated with tumor cell growth . Among 8 RASSF genes identified, RASSF1 was the most well-characterized tumor suppressor, which exhibits a frequent epigenetic inactivation in various types of human neoplasms, including lung and breast cancers [6–11]. RASSF1 encodes several isoforms (RASSF1A-C), which are derived from alternative mRNA splicing and promoter usage. Transcriptional silencing of RASSF1A was observed in a considerable proportion of lung, breast, ovarian, and nasopharyngeal cancers by de novo methylation at the CpG islands in the promoter [8–11]. In small cell lung cancers, allelic loss of 3p21.3 was associated with RASSF1A methylation, suggesting that both genetic and epigenetic mechanisms are implicated in RASSF1A inactivation in some tumor types . In addition, RASSF1A was identified to suppress the growth of tumor cells in both in vitro and in vivo, and Ras regulates pro-apoptotic pathway through its interaction with RASSF1A [5, 8, 12].
NORE1 (RASSF5), a human homologue of the mouse Ras effector Nore1, is newly identified member of the RASSF family [13, 14]. The NORE1 gene is located on chromosome 1q32.1, and two major transcripts (NORE1A and NORE1B) are derived from different promoter usage . NORE1A is epigenetically inactivated by promoter hypermethylation in various cancer cell lines and primary tumors, including lung, breast, and kidney tumors [15, 16]. It was also found that NORE1A mediates Ras-dependent apoptosis, and its reintroduction in defective cell lines impairs tumor cell growth in soft agar and suppresses colony formation [12, 17–19]. Interestingly, it was reported that the proapoptotic effect of RASSF1 may require heterodimerization with NORE1A . Very recently, it was identified that NORE1A activates the cyclin-dependent kinase inhibitor p21WAF1 via promoting p53 nuclear localization, and loss of NORE1A expression correlates with loss of p21WAF1 in human hepatocellular carcinoma . All of these studies suggest that NORE1A might be a putative tumor suppressor in human cancers.
In the present study, we investigated the expression and mutation status of two NORE1 isoforms, NORE1A and NORE1B in a series of primary colorectal carcinoma tissues and cancer cell lines to explore its candidacy as a tumor suppressor in colorectal tumorigenesis. Our data demonstrate that expression of NORE1A and NORE1B is abnormally down-regulated in a substantial fraction of colorectal cancer cell lines and primary carcinomas by aberrant promoter hypermethylation. Furthermore, altered expression of NORE1 correlated with tumor stage and grade. We also found that both NORE1A and NORE1B exert growth suppression effect via inhibition of cellular growth and colony formation of tumor cells and enhancement of cellular response to apoptotic stress. Together, our data suggest that inactivation of NORE1 may play a critical role in the malignant progression of colorectal tumors.
Human colorectal cancer cell lines and tissue specimens
Ten human colon cancer cell lines (Caco2, Colo320, HCT116, SW403, SW620, DLD-1, WiDr, SNU-C1, SNU-C4, and SNU-C5) were obtained from American Type Culture Collection (Rockville, MD, USA) and Korea Cell Line Bank (Seoul National University, Seoul, Korea). Cell lines were maintained at 37°C in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Carlsbad, CA, USA). A total of 80 primary colorectal tumor specimens and their adjacent normal tissues were obtained by surgical resection in the Kyung Hee University Medical Center (Seoul, Korea). Tissue specimens were snap-frozen immediately in liquid N2 and stored at -80°C until used. Signed informed consent was obtained from each patient. Bits of primary tumors and adjacent portions of each tumor were fixed and used for hematoxylin and eosin staining for histopathological evaluation. Tumor specimens composed of at least 70% carcinoma cells and adjacent tissues found not to contain tumor cells were chosen for molecular analysis.
Semiquantitative reverse transcription (RT)- and DNA-polymerase chain reaction (PCR) analysis
Total cellular RNA was extracted from tissues and cell lines by standard method. One μg of DNase1-treated RNA was converted to cDNA by reverse transcription using random hexamer primers and MoMuLV reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD, USA). PCR was initially performed over a range of cycles (24, 26, 28, 30, 32, 34, 36, and 38 cycles) and 2 μl of 1:4 diluted cDNA (12.5 ng/50 μl PCR reaction) undergoing 28-36 cycles was observed to be within the logarithmic phase of amplification with primers NORE1-10 (sense: 5'-CAGTTGGACTGCAGTCAGCA-3') and NORE1-9 (antisense: 5'-GGAGAGTTTCTGGAAGAGCA-3') for NORE1A, NORE1-5 (sense: 5-CATGAGCAGTGGGTACTGCA-3') and NORE1-9 for NORE1B, and G2 (sense; 5'- CATGTGGGCCATGAGGTCCACCAC-3') and G3 (antisense; 5'-AACCATGAGAAGTATGACAACAGC-3') for an endogenous expression standard gene GAPDH. PCR primers for NORE1A and NORE1B were designed to amplify the transcript regions of exons 2α-5 and 2β-5, respectively. PCR was done for 34 cycles at 95°C (1 min), 58-62°C (0.5 min), and 72°C (1 min) in 1.5 mM MgCl2-containing reaction buffer (PCR buffer II, Perkin Elmer). For genomic PCR analysis, 200 ng of genomic DNA, which was extracted from the same cells by dialysis of the DNA phase after RNA was extracted, were used for amplification of the exon 2 region of NORE1 with intron-specific primers G-RSF5-S (sense; 5'-GCGTGCGGGGAGACCGCAGTCT-3') and G-RSF5-AS (antisense; 5'-CTCCCAAGAACTCACAACAAA-3'). Ten μl of PCR products were resolved on 2% agarose gels. Quantitation was achieved by densitometric scanning of the ethidium bromide-stained gels. Absolute area integrations of the curves representing each specimen were then compared after adjustment for GAPDH level. Integration and analysis was performed using Molecular Analyst software program (Bio-Rad, Hercules, CA, USA). Semi-quantitative PCR was repeated at least three times for each specimen and mean level was calculated.
Cells were lysed in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM sodium fluoride, 2 mM sodium pyrophosphate, 1 mM sodium orthovanadate, protease inhibitor cocktail, and 1 mM PMSF. The cell lysate was clarified by centrifugation and 20 to 50 μg of total protein was supplemented with Laemmli buffer and loaded on an 8% SDS-polyacrylamide gel for electrophoresis. Immunoblotting analyses were performed using antibodies specific for NORE1A (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and Actin (Santa Cruz Biotechnology). Antibody binding was detected by enhanced chemiluminescence (Santa Cruz Biotechnology) using a secondary antibody conjugated to horseradish peroxidase. For reprobing with other antibodies, the membranes were incubated in a stripping buffer (0.2M glycine pH 2.2, 0.1% SDS, 1% Tween-20) at 50°C for 60 min.
Nonisotopic PCR-single strand conformation polymorphism (SSCP) analysis
To detect possible sequence alterations in NORE1, we performed nonisotopic PCR-SSCP analysis. The NORE1 gene was amplified with 7 sets of primers that were designed to cover the entire coding region of the gene. The PCR products of over 300 bp in lengths were digested with endonuclease(s) to increase the sensitivity of SSCP analysis. Twenty μl of PCR products were mixed with 10 μl of 0.5 N NaOH, 10 mM EDTA, and 15 μl of denaturing loading buffer (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). After heating at 95°C for 5 min, samples were loaded in wells pre-cooled to 4°C and run using 9% nondenaturating acrylamide gels containing 10% glycerol at 4-8°C and 18-22°C.
5-Aza-2'-deoxycytidine (5-Aza-dC) and TSA (Trichostantin A) treatment
To assess reactivation of NORE1 expression, cell lines showing no or low levels of NORE1A and/or NORE1B transcripts were plated in six-well tissue plates 48 h before treatment. 5-Aza-dC (Sigma, St. Louis, MO, USA) was added to the fresh medium at concentration of 1, 5, and 10 μM in duplicate and cells were harvested after 5 days. To evaluate effect of TSA on NORE1 expression, cells were incubated with 5-Aza-dC for 48 h and then treated with TSA (Sigma) at concentration of 250 ng/ml for 48 h.
Bisulfite DNA sequencing analysis
Fifty ng of bisulfite-modified DNA was subjected to PCR amplification of the NORE1A promoter region using primers MS-NORE3 (5'-AAAGAGGCAGGGCTGAAGGACCTAGG-3') and MS-NORE6 (antisense; 5'- CCGATGGCCGGGGACGCCATGGCC-3'). The PCR products were cloned into pCRII vectors (Invitrogen, Carlsbad, CA, USA) and 5 clones of each specimen were sequenced by automated fluorescence-based DNA sequencing to determine the methylation status.
Construction of expression plasmids, siRNA and transfection
Expression vectors encoding wild-type NORE1A and NORE1B were constructed by a PCR based approach using primers NORE1A-S (sense; 5'-ATGGCCATGGCGTCCCCGGC-3') and NORE1-AS (antisense; 5'-CCCAGGTTTGCCCTGGGATT-3') for NORE1A and NORE1B-S (sense; 5'-ATGACCGTGGACAGCAGCAC-3') and NORE1-AS for NORE1B. The PCR products were cloned into pEF6/V5-His-TOPO expression vector (Invitrogen, Carlsbad, CA, USA). Transfection of expression plasmids was performed using Effectene (QIAGEN, Hilden, Germany) according to the instruction of manufacturer. Briefly, 1 × 105 cells were transfected with plasmids-Effectene mixture for 4 h. The transfected cells were incubated with RPMI1640 medium with 10% serum for indicated duration. Detectable toxicity and apoptosis by reagent or vectors was rare. Each transfection experiment was carried out in triplicate. The transfection efficiency was monitored using a CAT assay (Roche, Mannheim, Germany) according to the instruction of manufacturer. Small interfering RNA (siRNA) duplex against NORE1A (5'-GCGCUGCACUAACUGUAAA-3'), NORE1B (5'-GCGCAGAGCAAACAUCUUU-3') and control siRNA duplex which served as negative control were synthesized by Invitrogen (Carlsbad, CA, USA). For transfection, 1 × 105 cells were plated on 60-mm-diameter dishes 24 h and incubated with a siRNA-Oligofectamine mixture at 37°C for 4 h. Fresh medium containing 1% fetal bovine serum was added and incubated for 20 h.
Cell proliferation and apoptosis analysis
Cells were seeded in 6-well plate at the density of 1 × 105 cells per well in duplicate and were maintained in the presence of 10% FBS. The following day, cells were transfected with expression vector or siRNA as described above. Cell numbers were counted using a hemocytometer for 4 days at 24 h intervals. For flow cytometry analysis, cells were harvested 48 h after transfection and fixed with 70% ethanol and resuspended in 1 ml of PBS containing 50 mg/ml RNase and 50 mg/ml propidium iodide (Sigma, St. Louis, MO, USA). For apoptosis analysis, the cells were treated with 25 μM of etoposide for 24 h and fraction of apoptotic sub G1 phase was determined using a FACScan flow cytometry and Modfit software (Becton Dickinson, San Jose, CA, USA).
Colony formation assay
Caco-2 cells (1 × 105 ) were transfected with 2 μg of expression vectors encoding WT-NORE1A, WT-NORE1B or empty vector (pcDNA3.1). HCT116 cells were transfected with 20 pM of siNORE1A, siNORE1B or siControl. The transfected cells were maintained in the presence of G418 (400 ng) for 7 days. Colonies were fixed with methanol for 15 min and stained with 0.05% crystal violet in 20% ethanol.
A student's t-test was used to determine the statistical significance of the difference. The Chi-square test was used to determine the statistical significance of expression and methylation levels between tumor and normal tissues. A P value of less than 0.05 was considered significant.
Frequent reduction of NORE1 expression in primary carcinoma tissues and cancer cell lines
Association of NORE1 reduction with tumor progression
Re-activation of NORE1 expression by 5-Aza-dC treatment
Aberrant promoter CpG sites hypermethyation of NORE1A in colorectal cancers
Inhibition of tumor cell growth by NORE1A and NORE1B
In the present study, we demonstrated that expression of NORE1A and NORE1B is down-regulated at the transcription level in a substantial fraction of colorectal cancer cell lines and primary carcinomas. Abnormal reduction of NORE1A and NORE1B was identified to be a tumor-specific phenomenon and correlated with advanced stage and high grade of tumors. In both established cell lines and primary tumors, downregulation of NORE1A expression was tightly associated with aberrant promoter CpG sites hypermethylation. Moreover, NORE1A and NORE1B were found to suppress cellular growth and colony forming ability of tumor cells and enhance tumor cell response to apoptotic stress, indicating that both NORE1 isoforms have growth suppression functions. Collectively, these results suggest that epigenetic silencing of NORE1 might contribute to the malignant progression of human colorectal cancers.
Chromosome 1q32 region exhibits loss of heterozygosity in many types of human cancer including colorectal cancer, thus suggested to harbor tumor suppressor genes [22, 23]. NORE1 was predicted as a target of deletion at 1q32.1, and allelic loss of the gene was suggested as a plausible mechanism underlying its low expression in tumor cells . In this study, however, genomic analysis of NORE1 revealed that deletion or sequence alteration of NORE1 is very rare in cancers, suggesting that genomic deletion of the gene might not be a main cause leading to loss or abnormal reduction of its expression in colorectal cancers.
Hypermethylation in CpG-rich region is strongly associated with transcriptional silencing, and hypermethylation at CpG sites in transcription regulatory region is a critical event leading to the epigenetic inactivation of tumor suppressor genes [24, 25]. Hypermethylation of the NORE1 gene has been reported in several cancers, including breast, lung, kidney, and colorectal cancers [15, 26]. It was also reported that epigenetic alteration of NORE1A is confined to lung tumors with a wild-type K-Ras, suggesting that epigenetic inactivation of NORE1A and mutational activation of K-Ras might play a cooperative role in lung tumorigenesis . In the present study, we found that expression of both NORE1A and NORE1B is frequently decreased in primary colorectal carcinoma tissues as well as cancer cell lines. However, no association was identified between expression status of NORE1A or NORE1B and mutational status of K-Ras in 80 primary carcinomas specimens we examined. Interestingly, abnormal reduction of NORE1A was more frequent compared to that of NORE1B and none of cell lines and primary tumors showed NORE1B-specific reduction, suggesting that NORE1A might be a more critical target of inactivation in colorectal tumorigenesis. Expression of both NORE1 isoforms was re-activated in cell lines by treatment with 5-Aza-dC and the NORE1A promoter was identified to be aberrantly hypermethylated in cancer cell lines and primary tumors expressing low levels of NORE1A transcript. Methylation frequency of 31 CpG sites in the proximal region of NORE1A promoter was much higher in tumors showing low mRNA expression compared to adjacent noncancerous tissues with normal mRNA level. Moreover, the methylation levels in both cancer cell lines and primary tumors showed a tight correlation with mRNA expression status. In particular, aberrant methylation of 21 CpG sites (numbered 11-31, Figure 5B) was most tightly associated with gene silencing, indicating that CpG dinucleotides in this region may play an important role for the regulation of NORE1A transcription. Therefore, our data support that NORE1 is epigenetically inactivated in a substantial fraction of colorectal cancers, and CpG sites hypermethylation of the 5' upstream region of the gene is crucial for its transcriptional silencing. It could be assumed that the hypermethylation of CpG sites in this region might block the access of cis-acting transcription factors to their binding sequences presumably by direct inhibition of binding or through the establishment of a repressed chromatin structure at the methylated CpG island [28, 29].
Although NORE1 has been identified as a potential tumor suppressor, the biological function of NORE1 and the significance of its inactivation during tumorigenesis have not been well understood. Previous studies showed that NORE1A mediates Ras-dependent apoptosis and suppresses tumor cell growth and colony formation, and the proapoptotic effect of RASSF1 requires the heterodimerization with NORE1A [12, 17–20]. In the present study we demonstrated that cellular growth and colony forming capability of colorectal cancer cells are significantly suppressed and their responses to apoptotic stress are markedly increased by expression of either NORE1A or NORE1B, indicating that both NORE1A and NORE1B have growth suppression abilities. Transient expression and siRNA-mediated knockdown assays revealed that compared to NORE1B, NORE1A evokes more potent growth inhibition effects in two different cells. Although further molecular analyses are required, this finding might correlate with our expression data supporting that NORE1A is a more critical target of inactivation in colorectal tumorigenesis. Interestingly, a recent study demonstrated that NORE1A activates the cyclin-dependent kinase inhibitor p21WAF1 via promoting p53 nuclear localization, and loss of NORE1A expression correlates with tightly with loss of p21WAF1 in human hepatocellular carcinoma . Considering that p21WAF1 is a crucial target of p53-mediated cell cycle arrest, this finding raises the interesting possibility that NORE1A could be a potential component, which plays an important role in functional connection between Ras and p53 signaling . Although we did not evaluate the possible role of NORE1 in p53 signaling, our finding of the significant growth suppression of mutant p53-carrying Caco-2 cells by NORE1A or NORE1B indicates that both NORE1A and NORE1B have p53-independent growth arrest and apoptosis-enhancing functions. It has been well known that oncogenic activation of Ras and mutational inactivation of p53 play a critical role in colorectal tumorigenesis. In this context, expression status of NORE1 and its association with activation status of Ras and p53 could provide valuable information for the molecular mechanisms underlying colorectal tumor progression. In the present study, we observed that reduced expression of NORE1 is associated with histopathological characteristics of tumors, such as tumor stage and grade. However, no association was recognized between expression of NORE1A or NORE1B and mutation of K-Ras. In contrast to cancerous tissues, abnormal reduction or promoter hypermethylation of NORE1 was not found in corresponding nonneoplastic mucosa. Based on previous reports that DNA methylation occurs early in the multistep process of carcinogenesis [30–32], our data raises the possibility that epigenetic inactivation of NORE1 might be implicated in the development and/or early progression of colorectal tumors.
Both NORE1 and NORE1B is significantly down-regulated in a considerable fraction of colorectal cell lines and primary tumors by aberrant DNA hypermethylation, and its abnormal reduction correlates with advanced stage and higher grade of tumor. Restored expression of NORE1A or NORE1B decreases cellular growth and colony forming ability of colorectal tumor cells and enhances cellular response to apoptotic stress. These findings thus suggest that epigenetic inactivation of NORE1 might play a crucial role in the malignant progression of colorectal tumors, possibly by providing selective growth advantage for colorectal epithelial tumor cells.
This work was supported in part by grants from the Korea Research Foundation (2009-0000794 and 2009-0001197) and the National Cancer Center (0820070), Republic of Korea.
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