COX-2 gene expression in colon cancer tissue related to regulating factors and promoter methylation status

Tumor and colon tissue samples were collected from 48 unselected patients at primary operation for colorectal carcinoma between 2001 to 2004 in Uddevalla county of Sweden. All patients underwent surgery as the only curative treatment and none received neoadjuvant radio-chemotherapy, according to individual decisions and institutional indications. Normal colon tissue was collected as a minimum of 10 cm away from the macroscopically seen tumor tissue. The group of patients consisted of 54% males and 46% females with a mean age of 72.5 years (range 40 to 91 years) at surgery. Mean survival time was 27.3 ± 4.2 months (range 0 to 73 months) following surgery according to a recent update of survival (Dec 2008), where 21 patients were still alive. Tumors were histologically classified as Dukes A (8), Dukes B (19), Dukes C (11) and Dukes D (10). PCR analysis was performed to quantify COX-2 mRNA expression [2]. Mean COX-2 expression in tumor tissue for the entire group was 6.27 ± 0.60 mol/mol GAPDH (range 0.11 to 24.67) (n = 48) and 14.6 ± 1.7 (range 1.7 to 38.08) (n = 29) in normal colon tissue. Patients used in subsequent experiments were selected according to COX-2 expression in tumors; one group of 10 patients with highest and one group of 10 patients with lowest COX-2 expression (Figure 1, Table 1).

Tumor and normal colon tissue samples were collected down to the serosa layer during surgery and kept fresh frozen in liquid nitrogen and stored in -80°C until analysis. Certified pathologists staged all tumors. Tumor samples were secured by the surgeon in charge and contained around 55-75% tumor cells according to random visual inspection. RNA extraction and cDNA synthesis were performed as described [2]. A quality check of RNA in all samples was done in Bioanalyzer 2100 with limit RIN 6.0 for further analysis (Agilent). DNA was extracted with QIAamp^® DNA Mini Kit (Qiagen) according to instructions. RNase A solution (Promega) was used as extra step in DNA purification. Concentration of RNA and DNA was determined in NanoDrop^® ND-1000 Spectrophotometer (NanoDrop Technologies).

Microarray analysis was performed on pooled tumor mRNA from 9 out of 10 patients with high COX-2 mRNA expression versus 9 out of 10 patients with low COX-2 mRNA expression. The two patients were excluded due to low RIN indicating poor RNA quality. 500 ng of mRNA from tumors with high COX-2 patients was labeled with Cy-3-dCTP (Amersham BioSciences) in a cDNA synthesis reaction with Agilent Flourescent Direct Label. Tumor samples with low COX-2 expression (500 ng) were labeled with Cy-5-dCTP. Pooled tumor cDNA from patients with high COX-2 versus pooled DNA from patients with low COX-2 expression were then hybridized on whole human oligo genome microarrays (4 × 44K expression arrays, Agilent) during 18 hrs followed by post-hybridization washes according to in situ instructions (Hybridization Kit Plus, Agilent). Microarrays were quantified on Agilent G2565 AA microarray scanner and data were pre-processed in Feature Extraction 9.1 software program (Agilent). The same procedures were performed with normal colon mucosa tissue from all patients with only 4 patients per pool due to poor RNA quality. One hybridization with high COX-2 tumor expression versus corresponding normal colon mucosa tissue was also performed. Four technical replicates of all tumor tissue analyses and two technical replicates of normal colon tissue and tumor versus normal colon tissue analyses were performed. Dye-normalized, outlier- and background subtracted values were analyzed in GeneSpring GX 10 software program (Agilent) according to standard procedures. A quality control was performed with QC metrics. Analysis used were filter on flags (present or marginal) were 27194 probes (out of 41078) passed in 2 out of 4 technical replicates with tumor tissue. In normal tissue 25735 probes passed and in tumor vs normal tissue 27919 probes passed filter on flags for further analysis with t-test (p < 0.05). Fold change 1.5 of log2 transformed ratios was considered a statistically significant change in gene expression. Significant pathway analysis with entity list FC 1.5 was used to define significantly altered pathways. PCR analyses were performed at Tataa Biocenter (Gothenburg, Sweden) in a LightCycler 480 Probe Master (Roche). PCR assays (AKT1 HS00178289_m1, IL1B HS01555410_m1, IL6 HS00985639_m1, TRA@ HS 00612292_m1, CARD11 HS01060626_m1, Applied Biosystems) were tested and validated for efficiency and specificity. All samples were run in duplicate and negative controls were negative. Results were related to assay efficiency and GAPDH (GAPDH assay from Tataa Biocenter's Reference Gene Panel Human) and calculated according to comparative Ct method.

Methylation analysis was performed with tag-modified bisulfite genomic sequencing [14]. EpiTect^® Bisulfite kit (Qiagen) was used for bisulphite modification of 1 μg tumor and normal colon tissue DNA from each patient (n = 20) according to instructions. Modified DNA samples were amplified with two different sets of primers (Table 2) directed towards two areas of the COX-2 promoter region using touchdown PCR (1 × Reaction Buffer, 0.5 mM dNTPs, 2.0 mM MgCl₂, 0.4 μM forward and reverse primers resp., and 1 unit HotStart Taq (Qiagen). Primer pair 1 was taken from Hur et al. and primer pair 2 was designed with BiSearch [15, 16]. PCR reactions were denatured at 95°C for 10 min, then 20 cycles of 95°C 45 s, 60°C/65°C annealing temperature with a decrease of half a degree per cycle for 45 s, 68°C 60 s followed by 15-20 cycles of 95°C 45 s, 50°C/55°C 45 s and 68°C 60 s ended with a 7 min extension at 68°C. EpiTect^® PCR Controls (Qiagen) was used to ensure that the reaction worked properly; one methylated control sample, one unmethylated control and a 50/50 mixture of methylated and unmethylated controls were included in each PCR to ensure that methylated and unmethylated template were both equally amplified. The specificity of PCR products was inspected by use of 2100 Bioanalyzer according to protocol for DNA 1000 (Agilent). PCR products were then purified using Agencourt AMPure magnetic beads (Agencourt Bioscience Corporation) using the Biomek NX pipetting robot (Beckman Coulter) and eluted in distilled water. Sequence PCR was performed using forward or reverse primer with the ABI Prism BigDye™ cycle sequencing Ready Reaction Kit v1.1 (Applied Biosystems). Sequence PCR was run in 10 μl reactions under following conditions: 96°C 1 min, followed by 25 cycles of 96°C 10 s and 50°C 4 min. Sequencing products were purified using CleanSeq magnetic beads (Agencourt) using the Biomek NX and re-suspended in 10 μl of High Dye formamide (Applied Biosystems). The sequencing products were separated with gel electrophoresis on a 3730 DNA analyser (Applied Biosystems) and the output data were viewed and analysed using Sequence Analysis v5.2 (Applied Biosystems) and BiqAnalyzer [17].

Results are presented as mean ± SEM. Statistical analyses were performed by ANOVA and p < 0.05 was regarded significant in two-tailed tests. This study was approved by the board of Ethics at University of Gothenburg (NCT00473980). Accordingly, all patients participated with informed consent.

Large difference in gene expression was observed when comparing tumor tissue with high COX-2 expression to normal colon mucosa tissue from the same patients (2557↑, 3182↓) (Table 3). A large number of genes with altered expression appeared also in colon cancer tissue of tumors with high COX-2 expression when compared to tumors with low COX-2 expression (3086↑, 3031↓) (Table 4). Expression of COX-2 in normal colon mucosa tissue was significantly increased in patients with tumors of high COX-2 expression compared with mucosa from patients with low COX-2 expression in tumors (776↑, 804↓). Highly expressed genes in tumor tissue with high COX-2 expression were associated to cell motility, cell structure, muscle proteins, and energy homeostasis while down-regulated genes in such tumors seemed to be related to tumor antigens as Melanoma antigen and Chondrosarcoma ass. Gene, etc (Table 4). Several factors of immune response as serpin peptidase inhibitor and inducible metric oxide synthase 2 with antitumoral activities were either up- or down-regulated in normal colon tissue from patients with tumors of high COX-2 expression (Table 5). Patients with tumors with high COX-2 expression did not in the present material display significantly reduced survival compared to patients with tumors with low COX-2 expression (Table 1). However, COX-2 expression predicted survival at borderline significance in a larger patient material [2].

A large number of transcription factors with reported importance for regulation of the COX-2 gene in human cells were evaluated and are listed in Table 6. Eight of these transcription factors showed increased expression, while 5 transcription factors were down-regulated in tumors with high COX-2 expression.

Genes with reported functions in external cell signaling indicated 6 genes with increased expression, while no gene indicated down-regulation related to high COX-2 expression in tumor tissue (Table 7). Table 8 describes genes with significantly altered expression of transcription factors and external cell signaling factors in normal mucosa from patients with tumors of increased COX-2 expression. IL1-β, IL6, and iNOS were up-regulated, probably inducing a variety of transcription factors.

Significantly altered metabolic and signaling pathways (FC 1.5) in tumors with high COX-2 expression were mainly related to immunity according to algorithm analyses (GeneSpring GX 10, Agilent). Five genes that were significantly changed in arrays (AKT1, CARD11, IL1B, IL6, and TRA@) and involved in significantly changed pathways according to the algorithm analyses (TCR, BCR, IL1, and IL6) were tested individually (Table 9). All results from individual PCR analyses agreed with array results with one exception; in tumor tissue with high compared to low COX-2 expression AKT1 expression was significantly higher in PCR analyses, with opposite direction in array analysis.

A majority of tumor tissue specimens displayed no methylation of the promoter region of the COX-2 gene in tumor tissue; only 2 tumors showed methylation of the promoter region (low COX-2, Dukes B tumors with intermediate differentiation). Methylation of the promoter region of the COX-2 gene was not observed in any of the normal colon mucosa specimens.