Methylation of CpG islands located within the promoter region of tumor-related genes is an epigenetic modification frequently associated with the development of human cancers.1 Aberrant methylation of CpG-rich promoter regions results in inactivation of genes in cancer cells through transcriptional silencing.2 In colorectal carcinomas, epigenetic silencing has been reported for a number of genes; aberrant CpG methylation was found for hMLH1 in 10–20% of carcinomas, for p16 in 20–30%, and for APC in 10–20%. VHL, MGMT and COX2 are also frequently silenced in colorectal cancer.3 Identification of selectively hypermethylated genes in colon cancers may improve our understanding of the role of epigenetic inactivation in colorectal tumorigenesis and may be valuable as a specific diagnostic marker. The combination of high throughput microarray analysis and the induction of methylated genes by demethylating agents can be used to identify genes that are silenced by aberrant hypermethylation and establish a panel of epigenetic markers for specific cancers.4, 5 In this study, we used oligonucleotide microarrays to identify genes that are differentially expressed in colon cancer cells after treatment with a demethylating agent. We identified 70 genes with functions potentially related to tumorigenesis that contain CpG islands in their promoter region and are expressed after treatment with a demethylating agent. We report the identification of secreted protein, acidic and rich in cysteine (SPARC), as a putative colon cancer related gene that is silenced by aberrant hypermethylation in colon cancer cells.
Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2′-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers. © 2007 Wiley-Liss, Inc.
Material and methods
Cell lines and tissue samples
Cell lines were obtained from either the American Type Culture Collection (ATCC; http://www.atcc.org) or the Korean Cell Line Bank (KCLB; http://cellbank.snu.ac.kr). DLD-1, HCT8, HCT116, LOVO and HT29 were grown in RPMI medium supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in the presence of 5% CO2. CCD18-Co, RKO and WiDr were cultured in minimum essential medium supplemented with 10% fetal bovine serum (Life Technologies) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). Fresh tissue samples were collected from 20 patients with colorectal cancer as a test set. In each case, grossly normal mucosa remote from the tumor was included as a control. A total of another 292 colon cancer cases were selected as a validation set, and used for tissue microarray construction. For tissue microarray construction, colorectal cancer specimens were obtained from the archives of the Department of Pathology at Yonsei University, Seoul, Korea. Samples were selected from consecutively identified cases at the Gastrointestinal Tumor Working Group Tissue Bank at Yonsei University Medical Center (Seoul, Korea) between December 1996 and November 1999, and were subjected to immunohistochemical analysis for SPARC using tissue microarray. All cases were assessed independent of any knowledge of molecular status. Histological factors were recorded as follows: (i) histological grade (well/moderately/poorly differentiated), (ii) growth pattern (pushing/mixed/irregular) and (iii) peritumoral lymphoid reaction (absent/present). Tumors with less than 10% SPARC expression were regarded as negative, 10–30% as weak positive and greater than 30% as strong positive. The Kaplan–Meier survival curve of 292 stage I, II, III and IV colorectal cancer patients according to SPARC expression was calculated using SPSS version 11 (Chicago, IL).
Authorization for the use of these tissues for research purposes was obtained from the Institutional Review Board of Yonsei University of College of Medicine. Some of the fresh specimens were supported by the Liver Cancer Specimen Bank from the National Research Resource Bank Program of the Korea Science and Engineering Foundation in the Ministry of Science and Technology. Fresh tissues were obtained immediately after surgical excision and stored at −70°C prior to DNA and RNA extraction. Tumor specimens were microdissected on a cryostat and fractionated to enrich the tumor cell populations more than 70%. Genomic DNA was prepared using the sodium dodecyl sulfate-proteinase K and phenol–chloroform extraction method. For the RT-PCR analysis, 6 out of 20 cases of colon cancer test samples were selectively microdissected with the laser capture microdissection technique. Tumor cells were selectively procured from H&E-stained slides without any contamination from normal cells, using a 30G1/2 hypodermic needle (Becton Dickinson, Franklin Lakes, NJ) affixed to a micromanipulator, as described previously.6
5-Aza-2′-deoxycytidine (5Aza-dC) treatment
Seven colon cancer cell lines (DLD-1, HCT8, HCT116, LOVO, RKO, HT29 and WiDr) were treated with a demethylating agent, 5Aza-dC (Sigma Chemical, St Louis, MO). Exponentially growing cells were seeded in T-75 culture flasks. After overnight incubation cells were exposed continuously to 5 μm 5Aza-dC for 6 days, with a change of drug and culture medium every 48 hr.
High-density spotted-oligonucleotide microarrays were manufactured at the array core facility in the Department of Pathology, College of Medicine of the Catholic University of Korea. The human Oligolibrary™ was purchased from Compugen/Sigma-Genosys and consisted of 18,861 oligonucleotides, representing 18,664 LEADS™ clusters plus 197 controls of glyceraldehyde-3-phosphate dehydrogenase. Thus, a total of 18,861 synthesized 60-mer oligos were robotically printed and processed.
RNA preparation and hybridization
Total mRNA was extracted from cells grown to 80–90% confluence using TRIZOL reagent (Invitrogen, San Diego, CA). Twenty microgram RNA was used for synthesis of cDNA target molecules.7 Target cDNA and Universal Human Reference RNA (Stratagene, La Jolla, CA) were hybridized to an oligonucleotide microarray containing 18,664 probe sets representing 18,664 unique (LEADS™) genes, and the array was scanned using GenePix scanners. Expression values for each gene were calculated using GenePix Pro 4.0 analysis software. Some hybridization were carried out in duplicate with fluororeversal to compensate for the different chemical properties of the fluorescent dye molecules and for potential biases associated with normalization.
Classification of colorectal cancer cell lines by molecular pattern analysis
Unsupervised hierarchical clustering analysis was used to analyze gene expression in 7 colorectal cancer cell lines (with or without 5Aza-dC treatment) and 2 normal colonic mucosae. We selected a data set of genes that satisfied the filtering criteria: genes having more than 60% of log-transformed ratio values presenting across all arrays were chosen, and genes with less than 0.3 standard deviations of log-transformed ratio were discarded. The selected gene data set was then applied to average linkage hierarchical clustering analysis using the uncentered correlation similarity metric method in Cluster version 2.20, and the resulting expression map was visualized with Treeview version 1.60 (http://rana.lbl.gov/EisenSoftware.htm).
First-strand cDNA was synthesized from 1 mg total RNA using random hexamer primers (Qiagen, Valencia, CA) and M-MLV reverse transcriptase (Invitrogen) according to the manufacturers' instructions. Twenty nanogram cDNA was used in each RT-PCR reaction. β-Actin was used as a quantitative internal control. Primer sets were designed to contain an exon–exon junction, and were as follows: SPARC: 5′-TGA TGA GAC AGA GGT GGT GG-3′ (sense), 5′-AAG TGG CAG GAA GAG TCG AAG-3′ (antisense) and β-actin, 5′-ACA GAG TAC TTG CGC TCA GGA G-3′ (sense) and 5′-TGT ATG CCT CTG GTC GTA CCA C-3′ (antisense). Five microliters aliquots of the RT-PCR product were subjected to 2% agarose gel electrophoresis and stained with ethidium bromide.
Isolation and bisulfite treatment of genomic DNA
Genomic DNA was isolated from fresh frozen tissue by overnight incubation in 100 μg/ml proteinase K (Sigma) and 0.1% SDS (Sigma) at 55°C with subsequent phenol/chloroform extraction and isopropanol precipitation. Herring sperm DNA (1 μg; Promega, Madison, WI) was added as a carrier. Two micrograms of DNA was denatured in 0.2 mol/l NaOH for 10 min at 37°C in 50 μl total volume. Thirty microlitres of 10 mmol/l hydroquinone (Sigma) and 520 μl 3.5 mol/l sodium bisulfite (pH 5.0; Sigma) was added. After 16 hr at 50°C, the DNA was purified and incubated in 0.3 mol/l NaOH for 5 min at room temperature. After ethanol precipitation, DNA was dissolved in 40 μl Tris-EDTA.8
Sequencing analysis of bisulfite treated genomic DNA
A 221-bp fragment of the 5′ region of the SPARC gene was amplified using the primers ATT TAG TTT AGA GTT TTG AGT GG (sense) and ACA AAA CTT CCC TCC CTT AC (antisense). PCR conditions were as follows: 95°C for 5 min; 40 cycles of 95°C for 20 sec, 60°C for 20 sec and 72°C for 30 sec; and a final extension of 4 min at 72°C. After incubation with exonuclease I and shrimp alkaline phosphatase (USB, Cleveland, OH), PCR products were sequenced using the SequiTherm Excel II cycle sequencing kit as recommended by the manufacturer (Epicentre Technologies, Madison, WI). Products of the sequencing reactions were resolved by electrophoresis and visualized by autoradiography. The methylation status of each sample was evaluated by determining the percentage of the intensity of the cytosine band versus the thymine band for each CpG site.9
Methylation-specific polymerase chain reaction
Methylation status of the SPARC gene was also determined by MSP as described previously.10 Bisulfite treated DNA (1 μg) was amplified using primers specific for either methylated or unmethylated DNA under the following conditions: 95°C for 5 min; 40 cycles of 95°C for 20 sec, 62°C for 20 sec and 72°C for 30 sec; and a final extension of 4 min at 72°C. Primer sequences were TTT TTT AGA TTG TTT GGA GAG TG (sense) and AAC TAA CAA CAT AAA CAA AAA TAT C (antisense) for unmethylated reactions (132 bp product), and GAG AGC GCG TTT TGT TTG TC (sense) and AAC GAC GTA AAC GAA AAT ATC G (antisense) for methylated reactions (112 bp product). Five microliters PCR product was loaded onto 3% agarose gels and visualized by ethidium bromide staining.9
Western blot analysis
Whole lysates were prepared from cell lines using lysis buffer [50 mM Tris (pH 7.4), 1% Triton X-100, 5 mM EDTA, 1 mM KCl, 140 mM NaCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium fluoride, 1% aprotinin, 1 μM leupeptin and 1 mM sodium ortho-vanadate]. Twenty microgram of total protein lysate was loaded into each lane, size-fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane that was blocked with tris-buffered saline-Tween 20 containing 5% skim milk. Membranes were incubated with primary antibodies, SPARC (AON-5031; Haematologic Technologies, Essex Junction, VT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Trevigen, Gaitherburg, MD) for 2 hr at room temperature. After washing, membranes were incubated with HRP-conjugated secondary antibody (Santacruz Biotech, Santa Cruz, CA), washed and developed with ECL-Plus (Amersham Pharmacia Biotech, UK).
Colorectal carcinoma tissue arrays containing 292 carcinomas and matched normal mucosae were constructed from formalin-fixed and paraffin-embedded tissues (IsuAbxis, Seoul, Korea). Sections (5 μm) were cut onto coated slides and deparaffinized using routine techniques. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) heated at 95°C in a steamer for 20 min. After blocking endogenous peroxidase activity with a 3% aqueous H2O2 solution for 5 min, sections were incubated with anti-SPARC monoclonal antibody (Haematologic Technologies) at a final concentration of 4 μg/ml for 60 min. Labeling was detected with the Envision Plus Detection Kit (DAKO, Carpinteria, CA), as recommended by the manufacturer, and all sections were counterstained with hematoxylin. SPARC expression was detected in the mesenchymal and stromal cells, normal colonic epithelical cells and infrequently in colon cancer cells.10 The extent of immunostaining was categorized based on the proportion of cancer cells with SPARC expression: <10%, negative; 10–30%, weak positive; and >30%, strong positive.11 Comparisons according to SPARC expression treated these as 2 groups: without SPARC expression (negative) versus with SPARC expression (weak positive and strong positive).
Identification of genes induced by 5Aza-dC in colon cancer celllines
The gene expression profiles in 7 colon cancer cell lines before and after induction by 5Aza-dC were determined using oligonucleotide microarrays. The expression profiles of 2 normal colonic mucosae were used as controls. The relative expression of each gene was measured by comparing its expression ratio to that of Universal Human Reference RNA. We initially performed molecular pattern analysis to determine whether our spotted-oligoarray system was able to identify colorectal carcinoma cell lines, colon cancer cell lines treated with 5Aza-dC and normal mucosa by molecular profiling. Unsupervised hierarchical clustering analysis of 7 colorectal cancer cell lines treated with and without 5Aza-dC, and 2 normal colonic mucosae was performed according to gene expression profile. Genes that passed the filtering criteria were selected (genes expressed in more than 60% of all arrays were selected, and genes with standard deviation of less than 0.3 were discarded). A total of 6,490 genes were selected and applied to average linkage hierarchical clustering analysis using the uncentered correlation similarity metric method. When we compared the gene expression in 7 colon cancer cell lines before and after 5Aza-dC treatment, there was a substantial increase (>2-fold) in signal intensities of 83 transcripts in DLD-1, 99 transcripts in HCT116, 57 transcripts in HCT8, 30 transcripts in HT29, 62 transcripts in LOVO 17 transcripts in RKO and 35 transcripts in WiDr after 5Aza-dC treatment. Overall, 226 transcripts were upregulated >2-fold in at least 1 of the 7 cancer cell lines after drug treatment. Notably, this large panel of genes, including several testis cancer antigens such as G antigens, is consistent with previous reports of genes inducible by 5Aza-dC.12 Although several genes have been reported to be induced by 5Aza-dC treatment independent of the methylation status of their 5′ CpG islands, many of the genes identified here represent potential targets for aberrant methylation in colorectal cancer. Among the genes that were upregulated after 5Aza-dC treatment, genes known to be related to tumorigenesis (such as those involved in cell cycle regulation, induction of apoptosis, differentiation or transcriptional regulation) were examined for the presence of CpG islands in their promoters. In total, 70 genes met these criteria and are listed in Table I according to the expression fold change after 5Aza-dC treatment (Table I).
|SPARC||5q31.3-q32||Cell adhesion growth|
|FSTL1||3q13.33||Function as a negative regulator of cell growth|
|CKB||14q32||Amino acid and derivative metabolism|
|HMGB1||13q12||Bind DNA with low specificity|
|GAGE1||Xp11.4-p11.2||Cellular defense response|
|ID1||20q11||Transcription regulation from Pol II promoter|
|APOH||17q23-qter||Complement activation, classical pathway|
|EMP1||12p12.3||Ionic insulation of neurons by glial cells|
|SPRY2||13q22.1||Histogenesis and organogenesis|
|ELF3||1q32.2||Transcription from Pol II promoter|
|BTG1||12q22||Negative control of cell proliferation|
|GMNN||6p22.1||Cell cycle arrest|
|CDC45L||22q11.21||DNA replication initiation|
|TYR||11q14-q21||Malanin biosynthesis from tyrosine|
|ST5||11p15||RNA dependent DNA replication|
|RAC3||17q25.3||Cell growth and/or maintenance|
|PMP22||17p12-p11.2||Ionic insulation of neurons by glial cells|
|EPLIN||12q13||Transcription regulation from Pol II promoter|
|INSIG1||7q36||Metabolism; cell proliferation;|
|GPC3||Xq26.1||Cell growth and/or maintenance|
|SIAT1||3q27-q28||Humoral immune response; protein modification|
|MDK||11p11.2||Cell cycle control|
|KLF3||4p14||Repression of transcription from Pol II promoter|
|CCND3||6p21||Cell cycle control|
Identification of SPARC as a gene that is inactivated in colon cancer cell lines
Among the 70 genes identified by oligonucleotide microarray analysis, expression of SPARC (secreted protein acidic and rich in cystein) was suppressed 10.3 to 30.8 fold in all 7 colon cancer cell lines compared with normal mucosa. A 3.1 to 8.7 fold induction of SPARC after 5Aza-dC treatment was observed in DLD-1, HCT8, HCT116 and HT29 cell lines, and LOVO and WiDr cells also showed a slight increase in SPARC expression (Table II). RKO cells did not show an increased level of SPARC expression after treatment with 5Aza-dC. These results indicate that expression of SPARC is suppressed through epigenetic inactivation in colon cancers.
|Log ratio value||Fold change (Cell line + 5-aza: Cell line)||Log ratio value||Fold change (Normal tissue/Cell line)|
|Cell line||Cell line + 5-aza||Normal tissue|
We next performed RT-PCR and Western blot analysis to examine the expression of SPARC mRNA and protein in 7 colon cancer cell lines and a normal colonic fibroblast cell line, CCD-18Co. The SPARC transcript was detectable in CCD-18Co, but undetectable in 5 (71%) of the 7 colon cancer cell lines, and protein expression was evident only in CCD-18Co (Figs. 1a and 1b). These results confirm the difference in SPARC expression between most colon cancer cell lines and fibroblasts. PCR-LOH analysis at the SPARC (5q31.3-q32) gene locus was performed on 20 colorectal carcinoma test samples (Fig. 1c), demonstrating that inactivation was not due to allelic deletion, since only 2 (Cases 2 and 9) out of 20 cases showed allelic loss at SPARC.
SPARC mRNA expression was measured before and after 5Aza-dC treatment using RT-PCR analysis. Before treatment the cell lines DLD-1, HCT116, LOVO, RKO and WiDr did not express SPARC mRNA, while HCT8 and HT29 expressed low levels of SPARC mRNA (Fig. 1d). Following treatment with 5Aza-dC, SPARC mRNA was induced in DLD-1, HCT8, HCT116, LOVO and HT29, but not in RKO or WiDr cells.
Methylation analysis of the SPARC gene in colon cancer cell lines and primary colorectal cancer samples
The 5′ region of the SPARC gene contains a CpG-rich sequence fulfilling the criteria of CpG islands (%GC = 61.9, ObsCpG/ExpCpG = 0.602, length = 291)13 (Figs. 2a and 2b). The methylation status of the CpG sites in the SPARC promoter region was determined in 7 colon cancer cell lines and 20 colon cancer test samples using MSP.
We initially performed MSP using primers specific for unmethylated and methylated DNA. Among the 16 CpG sites, the 3rd and 4th sites were included in the sense primer and the 9–11th sites were included in the antisense primer (Fig. 2b). MSP for methylated DNA gave no product in the normal human colon fibroblast cell line, CCD18-Co. Six colon cancer cell lines, DLD-1, HCT116, LOVO, RKO, HT29 and WiDr, gave PCR products only with primers for methylated DNA, indicating fully methylated CpG islands. The HCT8 cell line gave products from both primers specific for methylated and unmethylated DNA, indicating a hemimethylated status of CpG sites (Fig. 2c). Direct sequencing analysis of bisulfite modified PCR products was used to determine whether any CpG sites were preferentially methylated in the 7 cell lines that were found to contain methylated DNA (Fig. 2b). In the 5 cell lines (DLD-1, HCT116, LOVO, RKO and WiDr) with no SPARC expression, complete methylation was found in all 16 CpG sites in HCT116, RKO and WiDr, while full methylation at CpG 1-15 and hemimethylation at the 16th CpG sites were found in DLD-1 and LOVO (Fig. 2d). In the 2 cell lines with low level of SPARC expression (HCT8 and HT29), full methylation was present at the 1–6th and 9–16th CpG sites and hemimethylation was found at the 7th and 8th CpG sites in HCT8, while full methylation at 1–6th and 8–16th CpG sites and unmethylation at the 7th CpG site was found in HT29 (Fig. 2d).
Genomic DNA was isolated from primary colorectal samples and subjected to methylation-specific PCR analysis (Figs. 3a and 3b). CpG methylation in SPARC was detected in all of the 20 colorectal cancer samples. In contrast, methylation was detected in only 3 of the corresponding 20 normal colonic mucosa samples, suggesting that CpG methylation of the SPARC gene is a frequent event in colorectal cancer. We measured expression of SPARC in 6 of the 20 samples by semiquantitive RT-PCR of the extraction of RNA with the laser capture microdissction microscope and found that SPARC is suppressed in 5 out of 6 colon cancers (Fig. 3c). We could not perform the RT-PCR in remaining 14 cases because adequate amount of samples were not available in these 14 cases. We could not perform Western blotting analysis, because adequate amount of protein could not be obtained by our laser capture dissection method. Subsequent immunohistochemical analysis demonstrated that SPARC is expressed in normal colonic epithelial cells, stromal cells and some of the tumors (Figs. 4a, 4b and 4c). SPARC was diffusely expressed in the cytoplasm in both colonic epithelial and tumor cells (Fig. 4d). Immunohistochemistry without primary antibody application did not give any positive signal (data not shown). When we evaluated SPARC expression of colon cancer cells in our 20 colon tumors, SPARC expression was absent in 5 cases, suppressed in 12 cases and intense in 3 cases.
Inactivation of SPARC expression is related to poor patient outcome
The expression of SPARC protein was examined in the validation set of 292 primary colorectal cancers by immunostaining (Figs. 4c–4h and Table III). Similar to the result of test samples, SPARC expression was not detected in 81 cases (27.7%), weak SPARC expression was found in 165 cases (56.5%) and strong SPARC expression was found in 46 cases (15.8%). In most cases the stromal cells near to or surrounding the cancer cells showed strong expression. Suppression of SPARC is not related to any clinicopathological factors except mucin production, which is more frequent in cases without SPARC expression (20% vs. 9% p = 0.011). Comparing the 5-year survival rate of patients according to SPARC expression levels reveals significant differences; 5-year survival was 56.79% in patients with negative expression, 70.91% in those with weak positive expression and 93.48% in patients with strongly positive expression (p = 0.0001, Fig. 5a). Overall, patients with SPARC expression (weak positive and strong positive) showed a better survival rate than patients lacking SPARC expression (75.83% vs. 56.79% 5-year survival rate, p = 0.0014, Fig. 5b).
|Features||Number of carcinomas without SPARC expression (%)||Number of carcinomas with SPARC expression (%)||p value|
|<60 years||40 (49.4)||112 (53.1)||0.851|
|≥60 years||41 (50.6)||99 (46.9)|
|Male||47 (58.0)||115 (54.5)||0.480|
|Female||34 (42.0)||96 (45.5)|
|I||6 (7.4)||26 (12.3)||0.398|
|II||37 (45.7)||80 (37.9)|
|III||26 (32.1)||80 (37.9)|
|IV||12 (14.8)||25 (11.9)|
|≥6.0 cm||38 (46.9)||96 (45.5)||0.336|
|<6.0 cm||43 (53.1)||115 (54.5)|
|Pushing||10 (12.3)||35 (16.6)||0.955|
|Mixed||36 (44.4)||98 (46.4)|
|Irregular||35 (43.2)||78 (37.0)|
|Well||13 (16.0)||41 (19.4)||0.983|
|Moderate||37 (45.7)||101 (47.9)|
|Poor||31 (38.3)||69 (32.7)|
|Right colon||18 (22.2)||51 (24.2)||0.725|
|Left colon||63 (77.8)||160 (75.8)|
|Absent||65 (80.2)||192 (91.0)||0.011|
|Present||16 (19.8)||19 (9)|
|Absent||33 (40.7)||87 (41.2)||0.247|
|Present||48 (59.3)||124 (58.8)|
|High||10 (12.3)||26 (12.3)||0.578|
|Low or stable||71 (87.7)||185 (87.7)|
Hypermethylation of DNA promoter CpG islands results in transcriptional silencing of genes on the inactivated X chromosome, imprinted genes and exogenous integrated genes. Aberrant DNA methylation is now recognized as a common and important event in cancer development. In colorectal carcinomas, silencing of the expression of certain genes by CpG island methylation is associated with carcinogenesis.14 To identify genes that are targets for inactivation by CpG methylation in colon carcinomas, colon cancer cell lines were treated with a methylation inhibitor. Using DNA microarray analysis, we identified 70 genes with functions putatively related to tumorigenesis that contain CpG islands and were induced following 5Aza-dC treatment. From these 70 genes, we selected SPARC for further analysis because: (i) SPARC was suppressed in 5 of the 7 colon cancer cell lines, (ii) induction of SPARC was observed in 5 of the 7 cell lines after 5Aza-dC treatment and (iii) SPARC is known to be involved in tumor cell growth, differentiation and metastasis.
SPARC is a calcium binding glycoprotein of 35 kDa, and is the most common noncollagenous protein in bone. SPARC is known to be predominantly located in the nonorganic component of bone and adheres collagen and calcium; however, the role of SPARC in these tissues has not been clearly elucidated.15, 16, 17, 18 Originally, SPARC was thought to be a glycoprotein involved in the formation of the intracellular calcified matrix. However, many other diverse functions have since been reported, including: interaction with the extracellular matrix, epithelial cells, and stromal cells18; cell motility; cell adhesion; tumor invasion; and proliferation of normal and tumor cells.19, 20, 21, 22SPARC is also known to inhibit DNA synthesis and stimulate production of TGF-β.23 Based on this findings, it is possible to speculate that downregulation of SPARC may affect the behavior of colon cancer cells, although the specific mechanism remains to be determined.
The expression of SPARC in cancer tissues and functional analysis of the SPARC gene in tumor cell lines has been widely studied. The expression of SPARC in both normal and tumor cells is highly dependent on tumor type. In many cancers, upregulation of SPARC has been reported in the peritumoral stromal cells, and this fibroblast-derived SPARC is believed to have diverse effects on the biology of tumor cells.9SPARC has been reported to enhance the invasive capacity of prostate and breast cancer cells, glioma cells, esophageal, bladder and metastatic hepatocellular cancers, invasive meningiomas and malignant melanomas.24, 25, 26, 27, 28, 29, 30, 31, 32
In this study, we demonstrated that expression of SPARC is downregulated in colorectal carcinomas. Similar suppression of SPARC has been reported in lung cancer and pancreatic cancer.9, 24 This suppression may be related to the rapid growth of tumors, since SPARC has an antiproliferative function through the modulation of cell cycle regulatory proteins or growth factors.33
When we evaluated the relationship between SPARC suppression and clinicopathologic factors in 292 colorectal carcinomas, suppression of SPARC was not associated with age, sex, tumor site, MSI status or tumor stage. However, SPARC suppression was associated with decreased mucin production and with poor prognosis. This result raises the possibility that SPARC expression may be used as a biomarker for selecting chemotherapy in colon cancer patients with modest progression.
We demonstrated that the downregulation of SPARC is related to aberrant methylation of CpG islands in the promoter region. SPARC was shown to be methylated in all of the 7 colon cancer cell lines and in 20 colon cancer tissues but in only 3 of the 20 matched normal colonic muscosal samples. These findings suggest that methylation of SPARC is a common event in colorectal carcinogenesis. Analysis of the methylation sites showed that most of the CpG sites were methylated in the cell lines with SPARC inactivation. However, we found that the 7th CpG site is commonly unmethylated in the 2 cell lines with SPARC expression, suggesting that the 7th CpG site is particularly important in the regulation of SPARC expression.
In conclusion, we showed that SPARC expression is frequently suppressed in colon cancer cell lines in conjunction with aberrant DNA methylation, and that suppression of SPARC expression is related to poor patient outcome. These results suggest that suppression of SPARC may be related to colorectal cancer development and progression.