Rna profiling of cyclooxygenases 1 and 2 in colorectal cancer

ABSTRACT Cyclooxygenases (particularily Cox-2) are involved in carcinogenesis and metastatic cancer progression. The expression profiles of the cyclooxygenases and the roles they play in

established tumours of similar stage remains unclear. We report that Cox-1 and Cox-2 expression is highly variable in Dukes' C tumours, and changes in Cox-1 expression may be of

importance. MAIN Colorectal cancer is the third most common cancer in the Western world and despite advances in surgery, adjuvant therapies and screening, little impact on the mortality

rates has been seen (Jemal et al, 2003). A greater understanding of the molecular mechanisms underlying carcinogenesis and progression is leading to novel treatment strategies.

Cyclooxygenases (Cox) are responsible for the metabolism of arachidonic acid into prostaglandins (Church et al, 2003). Two isoforms exist, termed Cox-1 and Cox-2 (Vane, 1971; Xie et al,

1991). Increased expression of Cox-2 has been implicated in carcinogenesis and metastatic progression in many forms of human cancer (Church et al, 2003). For example, increased expression of

Cox-2 protein has been shown to correlate with tumour invasiveness and metastasis (Chen et al, 2001). In addition, 100% of metastatic lesions had positive immunohistochemical staining for

Cox-2 _vs_ 72% of primary tumours (Zhang and Sun, 2002). Cox-2 inhibitors are now being evaluated as adjuncts to chemotherapy for colon cancer (Blanke, 2002; Blanke and Masferrer, 2003).

Initial evidence with regard to the expression of Cox-1 suggested a minimal role in colonic neoplasia, with several studies demonstrating minimal expression of Cox-1 with little variability

in polyps and established tumours (Eberhart et al, 1994; Sano et al, 1995). More recent evidence suggests that Cox-1 expression and activity may have a role to play in the carcinogenic

process (Takeda et al, 2003). For example, reduced polyp formation was seen in MIN mice lacking a functional Cox-1 gene (Chulada et al, 2000) and Cox-1 expression may promote carcinogenesis

in lung and gynaecological tissues, both synergistically with and independently of Cox-2 (Hasturk et al, 2002; Sales et al, 2002; Gupta et al, 2003). We used real time PCR to investigate

Cyclooxygenase 1 and 2 expression profiles in invasive colonic tumours. Our aims were to define cyclooxygenase expression patterns in established colorectal tumours compared to adjacent

normal mucosa and correlate this with clinicopathological variables and patient outcome. MATERIALS AND METHODS PATIENTS In total, 51 stage III (Dukes' C) colorectal cancer patients had

tumour and adjacent normal bowel mucosa samples collected at the time of surgical resection by The Siteman Cancer Center Tissue Procurement Core. The median age of these patients was 68

(range 39–96 years). All samples were snap frozen and stored at −80°C until used for RNA extraction. In total, 29 patients (56.9%) were male. Approval for this study, including the genomic

analysis of the tissue samples, was obtained from the Washington University in St Louis Human Studies Committee. All patients gave informed consent. Clinical data were collected

prospectively and used to compare expression with tumour differentiation, anatomic location (either left or right colon), survival, recurrence (both metastatic and local recurrence), patient

gender and age. RNA EXTRACTION AND REAL TIME PCR FOR CYCLOOXYGENASES The TRIzol RNA isolation kit (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction from the paired tumour and

normal mucosa. Areas of high cellularity on light microscopy (median 86%, range 65–95%) were chosen from each tissue sample. RNA was quantified and assessed for purity by measurement of

OD260 and OD280 using a UV fiberoptic spectrophotometer (Nanodrop Technologies, Rockland, DE, USA) and was qualitatively assessed by measurement of relative 28S and 18S ribosomal band

intensities using a Bioanalyzer and RNA NanoChip capillary gel electrophoresis assay (Agilent Technologies, Palo Alto, CA, USA). RNAs were reverse-transcribed into cDNA samples using

Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Primers and probes for the Real Time PCR for Cox-1 and Cox-2 RNA were designed using the Primer Express Software (ABI,

Foster City, CA, USA) (Table 1). The probe and primer sets were synthesised by Integrated DNA Technologies (Coralvile, IA, USA). The relative RNA quantitation was assessed by Taqman real

time PCR using an ABI PRISM 7700 analyser (Applied Biosystems, Foster City, CA, USA). All real time PCR assays were performed in triplicate. MEASUREMENT OF RELATIVE RNA EXPRESSION LEVELS The

relative expression levels were calculated using the modified comparative CT method (Pfaffl, 2001). The PCR efficiencies were calculated from standard curves using the formula

_E_=10[−1/slope] where _E_ is the efficiency and slope is the slope of the standard curve. Standard curves for the reference and cyclooxygenase genes constituted separate experiments using

pooled colorectal cancer RNA samples (data not shown). The APP gene was used as the internal reference. The relative expression level of the RNA for each Cox gene was normalised to the APP

gene and to one of all of the 102 tissue samples. The calibrator sample chosen was that which had the maximum _C_T value, that is, the lowest expression level. The normalised relative

expression levels for each gene was calculated using the following formula (Pfaffl, 2001): where _E_target is the real-time PCR frequency of the target gene transcript and _E_reference is

the real time PCR efficiency of the reference gene transcript. STATISTICS Statistical analyses were performed using GraphPad InStat version 3.05 for GraphPad Software (San Diego, CA USA).

Wilcoxon matched pairs test and Spearman's Rank Correlation coefficient were used to evaluate the differences seen in expression levels of the Cox enzymes between the samples.

Kruskal–Wallis Test (Nonparametric ANOVA) and Mann–Whitney _U_ test was used to compare cyclooxygenase expression and clinical and pathological variables. Kaplan–Meier analyses were carried

out when comparing survival times. The _P_-values of <0.05 were considered to be significant. RESULTS Substantial variation in the expression of Cyclooxygenase 2 mRNA was observed in

normal mucosa (33-fold) and tumour tissues (51-fold). Variable Cox-1 expression was also seen in normal mucosa (68-fold) and tumour (40-fold). Cox-2 was significantly upregulated in the

tumour samples compared to paired mucosal tissues (median tumour : normal ratio=1.54, range 0.20–8.96, _P_=0.012, Figure 1B). In contrast, tumour Cox-1 expression was significantly lower

than normal mucosal samples (median tumour : normal ratio=0.48, range 0.01–2.85, _P_<0.0001, Figure 1A). The expression levels of each enzyme in normal mucosa also correlated to the

expression seen in paired malignant mucosa (Cox-1, _r_s=0.63, _P_<0.0001; Cox-2, _r_s=0.33, _P_=0.008). Cyclooxygenase-2 expression in tumour tissues did not correlate with disease

recurrence (_P_=0.16), tumour differentiation (_P_=0.26), gender (_P_=0.2), age >70 (_P_=0.06), or site of tumour (_P_=0.84). Cox-1 expression similarly did not show any significantly

different expression in tumour or normal mucosa in relation to these clinicopathological variables. The relationships between Cox-1 and Cox-2 were also examined (Figure 3A and B). In normal

tissue a linear relationship could be seen between Cox-1 and Cox-2 expression (Figure 3A, _r_2=0.32). However this relationship was clearly not maintained in the tumour tissues (Figure 3B,

_r_2=0.003) with an increased expression of Cox-2 protein relative to Cox-1 expression. Patients were divided into high or low expression groups using the median expression values for each

cyclooxygenase gene. No significant differences in cancer-specific survival were seen using Cox-1 expression in normal (_P_=0.26) or malignant tissues (_P_=0.36). Cox-2 expression in the

tumour did not correlate with survival (_P_=0.85, Figure 2A) but patients expressing high levels of Cox-2 in the normal mucosa appeared to have a survival advantage (_P_=0.02; Figure 2B).

DISCUSSION Cox-1 and Cox-2 expression seen in normal and malignant mucosa showed wide variation, even in the context of patients with the same clinical disease stage. The validity of such

variation could be confirmed with immunohistochemistry but the recovery of tissue slides for inclusion in this pilot study was not possible. The previously reported immunohistochemical

studies have also shown large differences in the staining intensity, and the numbers of cells expressing the Cox-2 protein (Hao et al, 1999; Masunaga et al, 2000; Cianchi et al, 2001). Our

RNA expression data highlight such previously observed variability. These and other studies have been able to show that such elevated expression of Cox-2 correlated with clinicopathological

variables. However, thresholds for positivity in these studies were low, including cells weakly stained, and sections with less than 10% of epithelial cell population deemed to be positive

(Cianchi et al, 2001; Zhang and Sun, 2002). In addition, these studies utilised samples obtained across various disease stages. The increased expression of cyclooxygenase-2 mRNA in tumour in

this study is consistent with these previous studies (Church et al, 2003). A direct molecular basis for the upregulation of Cox-2 in polyps and cancer is still poorly defined. However, one

mechanism may be the clonal expansion of tumour cells that express Cox-2. Such increased expression seems to increase tumour angiogenesis and decrease cellular apoptosis, leading to improved

overall cellular viability compared to tumours not aberrantly expressing this protein (Church et al, 2003). We were not able to show differences in cancer-specific survival or disease

recurrence in patients expressing high levels of Cox-2 in tumour. This may reflect the fact that our samples are from a well-defined stage of disease progression, that is, Dukes' C

tumours. Previously, it has been suggested that Cox-2 expression is associated with poorer outcomes; however, these studies compared expression across clinical disease stages and were not

able to demonstrate any predictive potential independent of Dukes' stage (Sheehan et al, 1999; Masunaga et al, 2000). Patients with a high level of Cox-2 expression in the normal mucosa

did seem to have survival advantage. The reasons for this observation are not easily explained and conflicts with some previous studies that examined the expression of the cyclooxygenases

in the malignant tumour (Church et al, 2003). The expression levels of Cox-1 also demonstrated considerable variation in RNA expression in normal and malignant tissues. This contrasts with

the previously accepted opinion that Cox-1 exists as a house keeping gene, which is not subject to variable expression (Sano et al, 1995). More recent evidence suggests that Cox-1 is

inducible and can be upregulated in malignant tissues (Sales et al, 2002; Gupta et al, 2003). We have shown that Cox-1 seemed to be downregulated in colorectal tumour specimens. Indeed, a

synergistic relationship of the cyclooxygenases in the early stages of carcinogenesis has been suggested, with Cox-1 having a role initially followed by a rise in Cox-2 expression as the

malignant process continues (Takeda et al, 2003). Our data confirm an altered regulation of Cox-1 expression between normal and malignant tissues, consistent with such suggestions. It has

also been suggested that the increases of Cox-2 expression and the tissue-specific prostaglandin E Synthetase often seen in malignant tissue may be dependent on the expression of Cox-1, at

least initially (Takeda et al, 2003). There is emerging evidence that Cox-1 may have a role to play in carcinogenesis in other solid tumours such as ovarian (Gupta et al, 2003) and skin

cancer (Tiano et al, 2002). This may mean that the nonspecific cyclooxygenase inhibitors, such as sulindac and aspirin, may be more important agents in the prevention of colonic polyps, if

compared to the Cox-2 specific inhibitors, such as celecoxib and rofecoxib, which are currently being studied in this context. However, the reduction in Cox-1 expression in more advanced

disease supports the view that as additions to adjuvant therapy regimes specific Cox-2 inhibitors should be more effective. CHANGE HISTORY * _ 16 NOVEMBER 2011 This paper was modified 12

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97: 1037–1041 Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported in part by the Siteman Cancer Center (P30 CA 091842), The BJH Foundation,

the Pharmacogenetics Research Network (GM 63340; http://pharmacogenetics.wustl.edu/), and the Section of Colorectal Surgery. The efforts of the members of the Siteman Cancer Center Tissue

Procurement Core (Mark Watson MD, PhD, Director) were greatly appreciated. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Surgery, Washington University School of Medicine, St

Louis, MO 63110-1093, USA R D Church & J W Fleshman * Department of Medicine, Washington University School of Medicine, Campus Box 8069, 660 South Euclid Ave, St Louis, 63110-1093, MO,

USA J Yu, W D Shannon, R Govindan & H L McLeod * The Siteman Cancer Center, Washington University School of Medicine, St Louis, 63110-1093, MO, USA J W Fleshman, W D Shannon, R Govindan 

& H L McLeod * Division of Biostatistics, Washington University School of Medicine, St Louis, 63110-1093, MO, USA W D Shannon * Department of Genetics, Washington University School of

Medicine, St Louis, 63110-1093, MO, USA H L McLeod * Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, 63110-1093, MO, USA H L McLeod

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ABOUT THIS ARTICLE CITE THIS ARTICLE Church, R., Yu, J., Fleshman, J. _et al._ RNA profiling of cyclooxygenases 1 and 2 in colorectal cancer. _Br J Cancer_ 91, 1015–1018 (2004).

https://doi.org/10.1038/sj.bjc.6602119 Download citation * Received: 08 April 2004 * Revised: 01 July 2004 * Accepted: 05 July 2004 * Published: 24 August 2004 * Issue Date: 13 September

2004 * DOI: https://doi.org/10.1038/sj.bjc.6602119 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link

is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * cyclooxygenase * colorectal cancer * gene

expression