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Cyclooxygenase-2 (COX-2), aromatase and breast cancer:a possible role for COX-2 inhibitors in breast cancer chemoprevention
Annals of Oncology 13: 669–678, 2002 DOI: 10.1093/annonc/mdf125
Review
Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention G. Davies1,2*, L.-A. Martin1, N. Sacks2 & M. Dowsett1 Academic Departments of 1Biochemistry and 2Surgery, Royal Marsden Hospital, London, UK Received 14 August 2001; revised 6 November 2001; accepted 23 November 2001
Non-steroidal anti-inflammatory drugs, cyclooxygenase-2 and breast cancer Non-steroidal anti-inflammatory drugs and cancer There have been a number of studies over the past 25 years linking non-steroidal anti-inflammatory drugs (NSAIDs) and cancer incidence [1]. Much of the work relating to NSAIDs and cancer has focused on colorectal cancer, including familial adenomatous polyposis (FAP). Several studies have reported an inverse relationship between colon cancer incidence and regular NSAID use including aspirin [2–6]. Studies using animal models of intestinal tumorigenesis have shown cyclooxygenase-2 (COX-2) expression in intestinal adenomas [7–10]. Further human studies of colon carcinomas have shown marked upregulation of COX-2 in carcinomas compared with normal mucosa [11–14]. A number of retrospective and prospective studies have shown a reduced incidence of colorectal cancer with NSAID use [15, 16]. In addition a number of randomised placebo-controlled double-blind trials
*Correspondence to: Dr G. Davies, Academic Departments of 1 Biochemistry and 2Surgery, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK. Tel: +44-207-808-2883; Fax: +44-207-376-3918; E-mail: [email protected] © 2002 European Society for Medical Oncology
have produced data showing a regression in polyp size [17, 18]. This process was reversible with tumours resuming growth after removal of the NSAID. There are fewer human data in relation to NSAID use and breast cancer incidence. A large cohort study of 89 528 registered nurses in the USA found no association between regular aspirin use and the incidence of breast cancer [19]. The analyses were based on 2414 cases identified over 12 years of follow-up [relative risk (RR) 1.0]. By contrast, in their casecontrol study of 511 women with newly diagnosed breast cancer and 1534 women who had undergone screening mammography, Harris et al. [20] found a reduced risk of breast cancer associated with the use of any NSAID three or more times a week for at least a year (RR 0.66). The protection was similar for users of aspirin alone, ibuprofen alone and all NSAIDs combined. The most heavily exposed women had the lowest risk, although the study was limited by incomplete descriptions of the study population and the participation rates. The epidemiological data relating NSAID use and the incidence of breast cancer are summarised in Table 1.
NSAIDs: mechanisms of action Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is the precursor for prostaglandin (PG) synthesis. The first step is
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Interest in chemoprevention in oncology using suppressants of prostaglandin (PG) synthesis has been stimulated by epidemiological observations that the use of aspirin and other non-steroidal inflammatory drugs (NSAIDs) is associated with reduced incidence of some cancers, including cancer of the breast. The main target of NSAID activity is the cyclooxygenase (COX) enzyme. Two isoforms of COX have been identified: COX-1, the constitutive isoform; and COX-2, the inducible form of the enzyme. COX-2 can undergo rapid induction in response to many factors such as bacterial lipopolysaccharides, growth factors, cytokines and phorbol esters. COX-2 is overexpressed in some malignancies including carcinoma of the breast. It has been suggested that such enhanced expression may lead to increased angiogenesis such that the inhibition of COX-2 might have a general anticancer effect via decreased blood vessel formation. In addition, an association between COX-2, its main product PGE2 and aromatase activity in human breast cancer suggests that such inhibitors might have an additional, specific prophylactic mechanism for this tumour. New COX-2 inhibitors are already licensed for use in the treatment of arthritis and are well tolerated. Their potential role in chemoprevention of mammary carcinogenesis in rats has already been investigated. What remains to be seen is if these findings can be extrapolated to human studies. Key words: aromatase, breast cancer, cyclooxygenase-2, oestrogens, prevention, prostaglandins
670 Table 1. Use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of human breast cancer Authors
Year of study
Study size
NSAID used
Relative risk (95% confidence interval)
Prospective studies Paganini-Hill et al. [98]
1989
13 987
Aspirin
0.95–1.67
Thun et al. [2]
1993
635 031
Aspirin
0.98 (0.76–1.26)
Schreinemachers and Everson [16]
1994
12 668
Aspirin
0.70 (0.50–0.96)
Egan et al. [19]
1996
89 528
Aspirin
1.01 (0.80–1.27)
Harris et al. [97]
1999
32 505
Aspirin, ibuprofen
0.6
Case control studies Harris et al. [20]
1996
511
Aspirin, ibuprofen
0.66 (0.52–0.83)
Coogan et al. [99]
1999
6558
Aspirin
0.8 (0.7–1.0)
Sharpe et al. [100]
2000
5882
Aspirin, ibuprofen
0.76 (0.63–0.92)
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Figure 1. Prostaglandin synthesis via arachidonic acid. COX, cyclooxygenase; PG, prostaglandin; TXA2, thromboxane A2.
the hydrolysis of phospholipids to produce free arachidonic acid catalysed by phospholipase A2 (Figure 1). The next step is catalysed by COX, which inserts molecular oxygen into arachidonic acid [21]. This reaction produces an unstable product, PGG2. PGG2 is then converted by the peroxidase activity of COX to PGH2. PGH2 is the common precursor for all other prostanoids. The production of individual prostanoids
is catalysed by different, specific synthases, which may vary in their expression between different types of cells. Each of the products derived from PGH2 has a distinct biological function. The main target of NSAID action is COX. Two isoenzymes exist in the human form. The enzyme which is now called COX-1 was first purified and characterised from bull vesicular
671 role of PPAR-γ in carcinogenesis, and in particular in relation to mammary carcinogenesis.
Transcriptional regulation of COX-2 Increased expression of COX-2 in malignancy is likely to occur via multiple routes. COX-2 induction by lipopolysaccharide (LPS) has been shown to occur through both the mitogen-activated protein kinase (MAPK) and protein kinase C-ζ (PKC-ζ) pathways [30]. It has also been shown that ceramide-stimulated activation of MAPK can activate c-Jun N-terminal kinase (JNK), which in turn can lead to increased COX-2 gene expression in human mammary epithelial cells [31]. This occurs via a cAMP response element (CRE) in the COX-2 promoter. Further evidence for the role of MAPK and c-Jun pathways in tumour necrosis factor-α (TNF-α)-stimulated COX-2 expression in human epithelial cells was provided recently by Chen et al. [32]. These pathways are illustrated in Figure 2. Transient transfection experiments have demonstrated that nuclear factor κB (NFκB), nuclear factor IL-6 (NF-IL6) and CRE promoter sites mediate gene transcription independently in response to LPS treatment [30]. LPS can activate different pathways to induce COX-2 gene transcription: through NFκB
Figure 2. Signal transduction pathways influencing cyclooxygenase-2 (COX-2) expression. TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide; PKC-ζ, protein kinase C-ζ; MAPK, mitogen-activated protein kinase; CRE, cAMP response element.
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glands by Miyamoto in 1976 [22]. In 1991 cDNAs for COX-2, a novel isoenzyme of COX, were isolated and sequenced by two independent groups [23, 24]. Before the discovery of COX-2 it was known that PG synthesis could be stimulated by a variety of substances including cytokines, growth factors and tumour promoters. These effects were due to activation of phospholipases, which supply arachidonic acid to COX [25]. The two isoenzymes are regulated independently: COX-1 is constitutively expressed, whereas the inducible isoenzyme COX-2 is expressed only in response to certain stimuli such as tumour promoters, endotoxin, cytokines and hormones [26, 27]. There are also data suggesting that COX-independent mechanisms may be responsible for some of the effects seen with NSAIDs in cancer models. It has been reported that NSAIDs can disrupt the binding of a nuclear transcription factor (PPAR-γ) to its cis recognition sequence [28]: PPAR-γ expression is increased after disruption of the tumour suppressor gene Apc and could be an important downstream effector. This NSAID effect is not likely to be related to inhibition of COX. In contrast, another report demonstrated that PPAR-γ can be activated by binding PGI2 derived from COX-2 [29]. More work therefore needs to be done to elucidate the exact
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COX-2 and carcinogenesis A number of studies have shown overexpression of COX-2 in solid malignancies including breast [37], pancreas, prostate [38] and colon [39]. The most compelling data to support a causal relationship between overexpression of COX-2 and carcinogenesis have come from studies of the Apc∆ 716 mouse. This is an animal model for human FAP, a condition caused by a germline mutation of the Apc gene in which individuals develop numerous adenomatous colorectal polyps, which predispose to colorectal carcinomas. Several strains of mice have been developed that carry mutations in one Apc allele, including the Min mouse [40] and Apc∆ 716 [41]. Analysis of adenomatous polyps from Min mice show increased expression of COX-2 relative to normal mucosa [9]. The effect of the absence of COX-2 in the Apc∆ 716 mouse has been studied by introduction of a knockout mutation of the COX-2 gene. Removal of the COX-2 gene in the mouse reduces the number and size of intestinal polyps [42]. Genetic and pharmacological evidence has been gathered to show that specific COX-2 inhibition is more effective than traditional NSAIDs in suppressing polyposis in mouse models of FAP, where COX-2 is induced [42].
COX-2 and mammary carcinogenesis COX-2 has been implicated in mammary carcinogenesis in several ways. COX-2 expression was detected using reverse transcription polymerase chain reaction (RT-PCR) in 13 of 13 human breast tumours, with no detectable expression in normal breast tissue [37]. A correlation was also observed between COX-2 expression and increasing tumour cell density. The COX-2 expression was localised to the epithelial compartment with no expression within the stromal component. A further study of 21 human breast tumour specimens found no detectable COX-2 expression in normal breast tissue specimens, but detectable and heterogeneous COX-2 gene
expression in each of the 21 tumour specimens [43]. In addition, a significant linear association between the tumour cell density and COX-2 gene expression was found (P <0.0001). In contrast Hwang et al. [44] analysed 44 samples by western blotting and only detected COX-2 protein in two of the 44 samples. A possible explanation for this is the differential sensitivity of the approaches to measuring the expression of COX-2. A study by Subbaramaiah et al. [45] looked at 29 microdissected breast cancers and found high levels of COX-2 protein in 14 of 15 HER-2/neu-positive samples. This was in contrast to the 14 HER-2/neu-negative tumours, where COX-2 was only detected in four of the cases, and at significantly lower levels than in the positive cases. The presence and levels of tissue PGs have been studied extensively in breast cancer over the past 30 years. Early studies demonstrated a relationship between tissue PG levels in human breast tumours, poor postoperative survival and development of metastases [46–48]. The main product of COX-2, namely PGE2, is found in high levels in tumour cells [49], and is synthesised by several human breast cancer cell lines. In clinical studies, high PGE2 concentrations have been associated with both high metastatic potential and a lack of oestrogen and progesterone receptors [48, 50]. The inhibition of COX-1 and COX-2 has been investigated in rat models. A 35-day course of ibuprofen in female Sprague– Dawley rats with 7,12-dimethylbenzathracene (DMBA)induced mammary carcinomas resulted in significant reduction of tumour volume (P <0.05) and a significant reduction in gene expression of both COX-1 and COX-2 [51]. Other studies including studies with indomethacin have yielded similar results [52, 53]. More recently the chemopreventive effect of a specific COX-2 inhibitor, celecoxib, against DMBA-induced mammary carcinogenesis in female Sprague–Dawley rats has been investigated [54]. Dietary administration of celecoxib produced striking reductions in the incidence, multiplicity and volume of mammary tumours relative to the control group. The chemopreventive properties of another COX-2 inhibitor, nimesulide, have been assessed in a study in which COX-2 expression and mammary tumours were induced by the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, together with a 24% corn oil diet [55]. Tumour incidence was significantly reduced from 71% in the control group to 51% in the treatment group, as well as a significant reduction in the size and multiplicity of tumours. More definitive evidence has now been provided by the recent demonstration that COX-2 overexpression is sufficient to induce mammary tumorigenesis in transgenic mice [56]. Overexpression of COX-2 in the mammary glands of transgenic mice was achieved by the use of the mouse mammary tumour virus promoter. Multiparous but not virgin females showed a much higher incidence of mammary gland hyperplasia, dysplasia and transformation to metastatic tumours. Studies of breast cancer cell lines have also provided data supporting the overexpression of COX-2 in breast cancer. A study of the differential expression of COX-1 and COX-2 in
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via extracellular signal related kinase (ERK-2), p38 and JNK pathways, through NF-IL6 via a p38 pathway, and through CRE via ERK-2 and JNK pathways. Moreover, PKC-ζ signalling seems to mediate transcription after LPS treatment through all three promoter sites. Therefore, individual signalling pathways, such as ERK-2, p38, JNK or PKC-ζ, appear to be sufficient to mediate COX-2 gene transcription by virtue of their ability to recruit transcription factors to at least two promoter sites. This may indicate redundancy in the signalling pathways and promoter elements regulating COX-2 transcription, at least in endotoxin-treated cells of macrophage/ monocyte lineage. Associations have also been made between mutated ras gene and COX-2 expression in human breast cancer cell lines [33], and c-myb expression is upregulated in colon tumours and breast cancers [34, 35]; c-myb overexpression causes a modest induction of COX-2 promoter activity [36].
673 human breast cancer cell lines found that in the MCF7 oestrogen receptor-positive cell line, COX-2 was barely detectable, whereas in the highly invasive, metastatic cell line MDA-MB231 there was a low level of COX-1 but a high level of COX-2 expression [57]. The concentration of PGE2, the major COX-2 product, correlated well with the level of COX-2 protein.
COX-2 overexpression and mammary carcinogenesis: potential mechanisms
Intratumoural aromatase, COX-2 and breast cancer Intratumoural aromatase and breast cancer Several areas of research have implicated sex hormones in the aetiology of breast cancer. Laboratory studies have shown that hormones, and in particular oestrogens control the growth of breast epithelial cells and affect the course of established disease. In premenopausal women, studies of serum oestradiol
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The expression of COX-2 in human solid cancers is not confined to the epithelial component of the tumour. The neovasculature associated with colonic adenomas, carcinomas and metastatic liver lesions also demonstrates COX-2 staining by immunohistochemical methods [58]. This expression seems to be a general characteristic of epithelial tumours including head and neck [59] and pancreas [60]. Significant correlations between COX-2 and tumour vascularisation (P <0.0001), microvessel density (P <0.007) and vascular endothelial growth factor (VEGF) in 35 head and neck cancers have been reported [61]. Uefuji et al. [62] found that microvessel density positively correlated with COX-2 expression in 42 cases of primary human gastric adenocarcinoma. The COX-2-overexpressing cases showed significantly elevated levels of PGE2 compared with normal gastric mucosa. The effects of specific COX-2 inhibitors have been tested in animal models of angiogenesis and celecoxib, a specific COX-2 inhibitor, has been shown to cause inhibition of the angiogenic response in fibroblast growth factor (FGF)induced corneal angiogenesis in rats [58]. Celecoxib reduced both the number and length of sprouting capillaries in a dosedependent fashion. These data suggest COX-2 overexpression is associated with increased PGE2 biosynthesis and angiogenesis in gastric cancer, and provide supportive data for COX-2 overexpression being functionally significant in tumour angiogenesis. The mechanism by which COX-2 expression produces these effects is unclear, however. Activated human microvascular endothelial cells produce a number of eicosanoid products including thromboxane A2 (TXA2) [63]. Selective COX-2 antagonists have been shown to inhibit TXA2 production and endothelial migration as well as corneal angiogenesis, an effect that is reversed with the use of a TXA2 agonist U46619 under COX-2-inhibited conditions [63]. TXA2 may represent an important intermediary of the angiogenic process. It has also been suggested that combined expression of inducible nitric oxide synthase and COX -2 may contribute to tumour angiogenesis. The exact mechanism by which this may occur is far from understood. Inducible nitric oxide synthase and COX-2 have been positively correlated with VEGF in non-small cell lung cancers [64]. In addition a study of 100 patients with resectable hepatocellular carcinoma showed that tumours that were negative for inducible nitric oxide synthase and COX-2 by immunhistochemical assess-
ment had a significantly better overall (P = 0.041) and recurrence-free survival (P = 0.018) [65]. A recent study by Dormond et al. [66] investigated the potential links between αVβ3 integrin, an adhesion receptor critically involved in mediating tumour angiogenesis and COX-2. They demonstrated that inhibition of endothelial-cell COX-2 by NSAIDs suppresses αVβ3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of FGF-2-induced angiogenesis in vivo. These results provide a functional link between COX-2, integrin αVβ3 and Cdc42-/Rac-dependent endothelial-cell migration. Thus, COX-2 appears to be related to induction of angiogenesis in a variety of tumours, which provides a general target for the chemopreventive use of COX-2 inhibitors for a variety of malignancies. In addition to angiogenic effects, COX-2 expression may have effects on apoptosis. The specific COX-2 inhibitor celecoxib induces apoptosis in human prostate cancer cells, whereas the COX-1 inhibitor piroxicam has no appreciable effect [67]. The forced expression of COX-2 inhibits programmed cell death, and the growth of COX-2-positive human colonic cancer xenografts has been shown to be inhibited by a COX-2 inhibitor (SC-58125) [68]. PGE2 inhibited the SC-58125-stimulated apoptosis and also induced expression of the anti-apoptotic protein Bcl-2 but did not affect expression of BAX, the death-promoting member of the Bcl-2 family, in human colonic cancer (HCA-7) cells. In the human colorectal cancer cell line HCT116, which contains normal p53 and BAX genes, deletion of the BAX gene provides profound protection against the apoptosis induced by the NSAIDs, sulindac and indomethacin [69]. This mechanism may also explain resistance to NSAID-induced apoptosis in conditions with a hereditary predisposition such as hereditary non-polyposis colorectal cancer, where inherited defects of BAX may be present. The enhanced tumorigenesis seen in transgenic mice carrying COX-2 under the control of the mouse mammary tumour virus promoter is preceded by reduced levels of BAX and Bcl-x(L) and increased levels of Bcl-2 in the mammary epithelial cells [56]. Thus the interactions between COX-2, Bcl-2 and other members of the Bcl-2 family may represent a further mechanism by which COX-2 expression influences tumour growth, including that of the mammary gland.
674
and its correlation with breast cancer risk have been inconsistent [70]. Variations in concentrations throughout the menstrual cycle may account for this inconsistency. A number of studies [71–75], however, have reported an association between high serum concentrations of oestradiol and increased risk of breast cancer in postmenopausal women. Some studies, e.g. Thomas et al. [71], report a steep gradient of association. If the association is causative, this gradient would result in a small decrease in serum oestradiol concentrations leading to substantial reduction in breast cancer risk. Oestrogens are produced from androgens by the action of the enzyme aromatase. In postmenopausal women, plasma oestrogens result from peripheral aromatisation, particularly in adipose tissue. Many breast cancers, however, also contain aromatase, and studies using radioactive isotopes indicate that both of these sources contribute to the oestrogen detectable in breast cancers [76]. The proportional significance varies markedly from patient to patient but the mean contribution appears to be approximately equal from the local and distal sources. This capacity of certain breast cancers to synthesise oestrogens by intratumoural aromatase activity has been known for many years [77], but the exact cell types or breast
tumours which contain functionally active aromatase remain an area of debate. In different studies, aromatase has been detected in both epithelial and stromal cells [78], in just the stromal elements [79] and only in breast cancer epithelial cells [80]. Aromatase is also detectable in normal breast tissue. The association between high levels of aromatase in the quadrant of normal tissue containing a breast carcinoma has been suggested as causative [81]. A small number of studies have suggested that breast carcinomas with aromatase activity show an increased response to aromatase inhibitors [82, 83] but larger studies are needed to confirm this. Aromatasetransfected MCF7 breast cancer cells can be mitogenically stimulated with androgen and their proliferation can be suppressed with aromatase inhibitors [84]. All of these findings provide support for the role of intratumoural aromatase being of importance in breast cancer incidence and progression.
Regulation of breast aromatase gene expression and transcription Transcriptional regulation of the aromatase gene is complex and differs between tissues, providing tissue-specific control
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Figure 3. Transcriptional regulation of aromatase via cyclooxygenase-2 (COX-2). CRE, cAMP response element; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; GRE, glucocorticoid response element.
675
COX-2 and aromatase in breast cancer COX-2 expression has been found to correlate with aromatase expression within human breast cancer tissue. A study of 23 human breast tumours found that COX-2 and aromatase expression, as measured by semi-quantitative RT-PCR showed a significant positive correlation [43]. The highest levels of COX-2 expression were in tumours with high cellularity and those that showed evidence of invasion. A strong linear association between aromatase expression and the sum of COX-1 and COX-2 expression was also found. The COX-2 product PGE2 and cytokines such as interleukin-6 (IL-6) or TNF-α can regulate aromatase activity [90]. Studies of aromatase activity in fibroblasts from breast tissue proximal to a carcinoma have shown a higher basal aromatase activity than those derived from the tumour itself, and the ability of PGE2, TNF-α and IL-6 to stimulate aromatase activity was greater in the tissue adjacent to the tumour [92, 93].
Prospects for COX-2 inhibitors in breast cancer prophylaxis The body of evidence cited above supports a link between the overexpression of COX-2 and mammary carcinogenesis which may at least in part be dependent on the induction of aromatase by PGE2. The selective inhibition of COX-2 may modulate a critical step in the initiation and promotion, as well as progression of breast cancer. COX-2 inhibitors are well tolerated and have minimal side-effects, and as such may be well suited to prophylactic use. The long-term use of COX-2 inhibitors has been evaluated in several large randomised trials including the Vioxx Gastrointestinal Outcomes Research Study (VIGOR) [94] and the Celecoxib Long-term Safety Study (CLASS) [95]. These
Figure 4. Possible effects of cyclooxygenase-2 (COX-2) inhibitors as chemopreventives of breast cancer. PGE2, prostaglandin E2.
studies confirm the improved gastrointestinal safety profile compared with conventional NSAIDs. Recent concerns have been raised, however, about possible cardiovascular risks associated with COX-2 inhibitor usage [96]. An increased RR of thrombotic cardiovascular events (2.38; P = 0.002) was seen with the use of rofecoxib compared with naproxen in the VIGOR study. This difference was not seen between celecoxib and other NSAIDs in the CLASS study. Whether these findings reflect a prothrombotic effect of rofecoxib or a greater antithrombotic effect of naproxen is uncertain. Further prospective evaluation is needed to fully characterise any increased risk and these data may affect the prospects for COX-2 inhibition in breast cancer prophylaxis. Aromatase inhibitors are in pilot trials to evaluate their possible utility in chemoprevention, but their profound suppression of oestrogen throughout the body may lead to unacceptable side-effects. In contrast, the possible link between the COX-2 products and breast aromatase in and around malignant breast tissue suggests a possible means for selective suppression of oestrogenic stimulation of the breast. It is likely that such targeted oestrogen suppression will lead to only partial withdrawal of oestrogen, but the epidemiological data indicate that there may be a steep dose relationship between oestrogen exposure and breast cancer incidence, suggesting that partial oestrogen suppression may have profound effects on breast cancer incidence. Thus the current understanding of the role of COX-2 in breast cancer suggests that COX-2 inhibitors may have a role in chemoprevention which is based in part on the generic issues of anti-angiogenesis and pro-apoptotic processes, and in part on a tissue-specific inhibition of oestrogen synthesis (Figure 4). It might be considered
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of the enzyme. At least eight exons I have been reported. In breast carcinomas exons 1.3 and II are the most frequent exons I [85], suggesting that promoters 1.3 and II are the major promoters directing aromatase expression in the malignant and surrounding tissue. This contrasts with the adipose stromal cells and fibroblasts of normal breast tissue, in which exon 1.4 is the dominant exon I [86, 87]. Exon 1.4 is preceded by a TATA-less promoter and upstream CRE and Sp1 sequences, which are required for upregulation of transcription by serum glucocorticoids [88]. A series of in vitro experiments point to exons 1.3 and II being under the control of cAMP [89, 90]. Thus the concept has been developed of promoter switching on the aromatase gene between normal and malignant tissues (Figure 3). PGE2, which is synthesised by many breast carcinomas, as noted above, is a known stimulant of cAMP in breast cancer cells [91]; its production by breast cancer may be instrumental in the switching of aromatase promoters. If these transcriptional relationships are important in breast cancer, a relationship between COX-2 and aromatase activity might be expected.
676 that the effects on angiogenesis and apoptosis may be more important since these would provide a means of non-oestrogendependent prevention, while current strategies for prevention in breast cancer are targeted at oestrogen-dependent effects. Pilot studies of the chemopreventive use of COX-2 inhibitors are merited to test these concepts. The presurgical setting offers an opportunity to test the effects of a short course of these compounds on biological markers of angiogenesis, apoptosis, proliferation as well as conventional markers such as oestrogen, progesterone and HER-2 receptor status in human breast cancer tissue. This would provide direct data to indicate whether these compounds have biological effects of potential clinical significance within human breast cancer tissue, and would provide the platform for larger chemopreventive studies.
1. Mehta RG, Moon RC. Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol. Anticancer Res 1991; 11: 593–596. 2. Thun MJ, Namboodiri MM, Heath CW Jr. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 1991; 325: 1593–1596. 3. Greenberg ER, Baron JA, Freeman DH et al. Reduced risk of largebowel adenomas among aspirin users. The Polyp Prevention Study Group. J Natl Cancer Inst 1993; 85: 912–916. 4. Logan RF, Little J, Hawtin PG, Hardcastle JD. Effect of aspirin and non-steroidal anti-inflammatory drugs on colorectal adenomas: casecontrol study of subjects participating in the Nottingham faecal occult blood screening programme. BMJ 1993; 307: 285–289. 5. Suh O, Mettlin C, Petrelli NJ. Aspirin use, cancer, and polyps of the large bowel. Cancer 1993; 72: 1171–1177. 6. Reeves MJ, Newcomb PA, Trentham-Dietz A et al. Nonsteroidal anti-inflammatory drug use and protection against colorectal cancer in women. Cancer Epidemiol Biomarkers Prev 1996; 5: 955–960. 7. Boolbol SK, Dannenberg AJ, Chadburn A et al. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 1996; 56: 2556–2560. 8. DuBois RN, Radhika A, Reddy BS, Entingh AJ. Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 1996; 110: 1259–1262. 9. Williams CS, Luongo C, Radhika A et al. Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology 1996; 111: 1134– 1140. 10. Singh J, Hamid R, Reddy BS. Dietary fat and colon cancer: modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res 1997; 57: 3465–3470. 11. Eberhart CE, Coffey RJ, Radhika A et al. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994; 107: 1183–1188. 12. Kargman SL, O’Neill GP, Vickers PJ et al. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res 1995; 55: 2556–2559. 13. Sano H, Kawahito Y, Wilder RL et al. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 1995; 55: 3785– 3789.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
References
14. Kutchera W, Jones DA, Matsunami N et al. Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc Natl Acad Sci USA 1996; 93: 4816–4820. 15. Rosenberg L, Palmer JR, Zauber AG et al. A hypothesis: nonsteroidal anti-inflammatory drugs reduce the incidence of large-bowel cancer. J Natl Cancer Inst 1991; 83: 355–358. 16. Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994; 5: 138–146. 17. Nugent KP, Farmer KC, Spigelman AD et al. Randomized controlled trial of the effect of sulindac on duodenal and rectal polyposis and cell proliferation in patients with familial adenomatous polyposis. Br J Surg 1993; 80: 1618–1619. 18. Giardiello FM, Hamilton SR, Krush AJ et al. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N Engl J Med 1993; 328: 1313–1316. 19. Egan KM, Stampfer MJ, Giovannucci E et al. Prospective study of regular aspirin use and the risk of breast cancer. J Natl Cancer Inst 1996; 88: 988–993. 20. Harris RE, Namboodiri KK, Farrar WB. Nonsteroidal antiinflammatory drugs and breast cancer. Epidemiology 1996; 7: 203–205. 21. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971; 231: 232–235. 22. Miyamoto T, Ogino N, Yamamoto S, Hayaishi O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 1976; 251: 2629–2636. 23. Xie WL, Chipman JG, Robertson DL et al. Expression of a mitogenresponsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 1991; 88: 2692–2696. 24. Kujubu DA, Fletcher BS, Varnum BC et al. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 1991; 266: 12866–12872. 25. Marnett LJ. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res 1992; 52: 5575–5589. 26. Herschman HR. Prostaglandin synthase 2. Biochim Biophys Acta 1996; 1299: 125–140. 27. Wu KK. Cyclooxygenase-2 induction: molecular mechanism and pathophysiologic roles. J Lab Clin Med 1996; 128: 242–245. 28. He TC, Chan TA, Vogelstein B, Kinzler KW. PPAR∆ is an APCregulated target of nonsteroidal anti-inflammatory drugs. Cell 1999; 99: 335–345. 29. Lim H, Gupta RA, Ma WG et al. Cyclooxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPAR∆. Genes Dev 1999; 13: 1561–1574. 30. Mestre JR, Mackrell PJ, Rivadeneira DE et al. Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells. J Biol Chem 2001; 276: 3977–3982. 31. Subbaramaiah K, Chung WJ, Dannenberg AJ. Ceramide regulates the transcription of cyclooxygenase-2. Evidence for involvement of extracellular signal-regulated kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways. J Biol Chem 1998; 273: 32943–32949. 32. Chen CC, Sun YT, Chen JJ, Chang YJ. Tumor necrosis factor-αinduced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IκB kinase 1/2 in human alveolar epithelial cells. Mol Pharmacol 2001; 59: 493–500.
677 52. Alshafie GA, Harris RE, Robertson FM et al. Comparative chemopreventive activity of ibuprofen and N-(4-hydroxyphenyl) retinamide against the development and growth of rat mammary adenocarcinomas. Anticancer Res 1999; 19: 3031–3036. 53. McCormick DL, Madigan MJ, Moon RC. Modulation of rat mammary carcinogenesis by indomethacin. Cancer Res 1985; 45: 1803–1808. 54. Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase-2 inhibitor. Cancer Res 2000; 60: 2101–2103. 55. Nakatsugi S, Ohta T, Kawamori T et al. Chemoprevention by nimesulide, a selective cyclooxygenase-2 inhibitor, of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats. Jpn J Cancer Res 2000; 91: 886–892. 56. Liu CH, Chang SH, Narko K et al. overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276: 18563–18569. 57. Liu XH, Rose DP. Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res 1996; 56: 5125–5127. 58. Masferrer JL, Leahy KM, Koki AT et al. Antiangiogenic and antitumour activities of cyclooxygenase-2 inhibitors. Cancer Res 2000; 60: 1306–1311. 59. Chan G, Boyle JO, Yang EK et al. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck. Cancer Res 1999; 59: 991–994. 60. Tucker ON, Dannenberg AJ, Yang EK et al. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res 1999; 59: 987–990. 61. Gallo O, Franchi A, Magnelli L et al. Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis. Neoplasia 2001; 3: 53– 61. 62. Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer. Clin Cancer Res 2000; 6: 135–138. 63. Daniel TO, Liu H, Morrow JD et al. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis. Cancer Res 1999; 59: 4574–4577. 64. Marrogi AJ, Travis WD, Welsh JA et al. Nitric oxide synthase, cyclooxygenase 2, and vascular endothelial growth factor in the angiogenesis of non-small-cell lung carcinoma. Clin Cancer Res 2000; 6: 4739–4744. 65. Rahman MA, Dhar DK, Yamaguchi E et al. Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possible involvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res 2001; 7: 1325–1332. 66. Dormond O, Foletti A, Paroz C et al. NSAIDs inhibit αVβ3 integrinmediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nature Med 2001; 7: 1041–1047. 67. Hsu AL, Ching TT, Wang DS et al. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000; 275: 11397–11403. 68. Sheng H, Shao J, Morrow JD et al. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998; 58: 362–366. 69. Zhang L, Yu J, Park BH et al. Role of BAX in the apoptotic response to anticancer agents. Science 2000; 290: 989–992.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
33. Gilhooly EM, Rose DP. The association between a mutated ras gene and cyclooxygenase-2 expression in human breast cancer cell lines. Int J Oncol 1999; 15: 267–270. 34. Guerin M, Sheng ZM, Andrieu N, Riou G. Strong association between c-myb and oestrogen-receptor expression in human breast cancer. Oncogene 1990; 5: 131–135. 35. Ramsay RG, Thompson MA, Hayman JA et al. Myb expression is higher in malignant human colonic carcinoma and premalignant adenomatous polyps than in normal mucosa. Cell Growth Differ 1992; 3: 723–730. 36. Ramsay RG, Friend A, Vizantios Y et al. Cyclooxygenase-2, a colorectal cancer nonsteroidal anti-inflammatory drug target, is regulated by c-MYB. Cancer Res 2000; 60: 1805–1809. 37. Parrett ML, Harris RE, Joarder FS et al. Cyclooxygenase-2 gene expression in human breast cancer. Int J Oncol 1997; 10: 503–507. 38. Gupta S, Srivastava M, Ahmad N et al. Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate 2000; 42: 73–78. 39. Maekawa M, Sugano K, Sano H et al. Increased expression of cyclooxygenase-2 to -1 in human colorectal cancers and adenomas, but not in hyperplastic polyps. Jpn J Clin Oncol 1998; 28: 421–426. 40. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990; 247: 322–324. 41. Oshima M, Oshima H, Kitagawa K et al. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci USA 1995; 92: 4482–4486. 42. Oshima M, Dinchuk JE, Kargman SL et al. Suppression of intestinal polyposis in Apc∆ 716 knockout mice by inhibition of cyclooxygenase-2 (COX-2). Cell 1996; 87: 803–809. 43. Brueggemeier RW, Quinn AL, Parrett ML et al. Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens. Cancer Lett 1999; 140: 27–35. 44. Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 1998; 90: 455–460. 45. Subbaramaiah K, Norton L, Gerald W et al. Increased expression of cyclooxygenase-2 in HER-2-overexpressing human breast cancer cells. NCI 7th SPORE Investigators Workshop 1999. 46. Bennett A, Berstock DA, Raja B, Stamford IF. Survival time after surgery is inversely related to the amounts of prostaglandins extracted from human breast cancers. Br J Pharmacol 1979; 66: 451P. 47. Bennett A. The production of prostanoids in human cancers, and their implications for tumor progression. Prog Lipid Res 1986; 25: 539– 542. 48. Rolland PH, Martin PM, Jacquemier J et al. Prostaglandin in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. J Natl Cancer Inst 1980; 64: 1061–1070. 49. Schrey MP, Patel KV. Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators. Br J Cancer 1995; 72: 1412–1419. 50. Fulton AM, Heppner GH. Relationships of prostaglandin E and natural killer sensitivity to metastatic potential in murine mammary adenocarcinomas. Cancer Res 1985; 45: 4779–4784. 51. Robertson FM, Parrett ML, Joarder FS et al. Ibuprofen-induced inhibition of cyclooxygenase isoform gene expression and regression of rat mammary carcinomas. Cancer Lett 1998; 122: 165–175.
678 86. Harada N. A unique aromatase (P-450arom) mRNA formed by alternative use of tissue-specific exons I in human skin fibroblasts. Biochem Biophys Res Commun 1993; 189: 1001–1007. 87. Mahendroo MS, Mendleson CR, Simpson ER. Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. J Biol Chem 1993; 268: 19463–19470. 88. Zhao Y, Mendleson CR, Simpson ER. Characterization of the sequences of the human CYP19(aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Mol Endocrinol 1995; 9: 340–349. 89. Michael MD, Kilgore MW, Morohashi K, Simpson ER. Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 1995; 270: 13561–13566. 90. Michael MD, Michael LF, Simpson ER. A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 1997; 134: 147–156. 91. Zhao Y, Agarwal VR, Mendelson CR, Simpson ER. Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 1996; 137: 5739–5742. 92. Reed MJ, Coldham NG, Patel SR et al. Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells. J Endocrinol 1992; 132: 5–8. 93. Singh A, Purohit A, Ghilchik MW, Reed MJ. The regulation of aromatase activity in breast fibroblasts: the role of interleukin-6 and prostaglandin E2. Endocr Relat Cancer 1999; 6: 139–147. 94. Bombardier C, Laine L, Reicin A et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med 2000; 343: 1520–1528. 95. Silverstein FE, Faich G, Goldstein JL et al. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 2000; 284: 1247–1255. 96. Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 2001; 286: 954– 959. 97. Harris RE, Prospective study of nonsteroidal anti-inflammatory drugs and breast cancer. Oncol Rep 1999; 6: 71–73. 98. Paganini-Hill A, Chao A, Ross RK et al. Aspirin use and chronic diseases: a cohort study of the elderly. BMJ 1989; 299: 1247–1250. 99. Coogan PF, Rao SR, Rosenberg L et al. The relationship of nonsteroidal anti-inflammatory drugs use to the risk of breast cancer. Prev Med 1999; 29: 72–76. 100.Sharpe CR, Collet JP, McNutt M et al. Nested case–control study of the effects of non-steroidal anti-inflammatory drugs on breast cancer risk and stage. Br J Cancer 2000; 83: 112–120.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
70. Key TJ, Pike MC. The role of oestrogens and progestagens in the epidemiology and prevention of breast cancer. Eur J Cancer Clin Oncol 1988; 24: 29–43. 71. Thomas HV, Key TJ, Allen DS et al. A prospective study of endogenous serum hormone concentrations and breast cancer risk in postmenopausal women on the island of Guernsey. Br J Cancer 1997; 76: 401–405. 72. Helzlsouer KJ, Alberg AJ, Bush TL et al. A prospective study of endogenous hormones and breast cancer. Cancer Detect Prev 1994; 18: 79–85. 73. Toniolo PG, Levitz M, Zeleniuch-Jacquotte A et al. A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J Natl Cancer Inst 1995; 87: 190–197. 74. Berrino F, Muti P, Micheli A et al. Serum sex hormone levels after menopause and subsequent breast cancer. J Natl Cancer Inst 1996; 88: 291–296. 75. Dorgan JF, Longcope C, Stephenson HE et al. Relation of prediagnostic serum estrogen and androgen levels to breast cancer risk. Cancer Epidemiol Biomarkers Prev 1996; 5: 533–539. 76. Reed MJ, Owen AM, Lai LC et al. In situ oestrone synthesis in normal breast and breast tumour tissues: effect of treatment with 4-hydroxyandrostenedione. Int J Cancer 1989; 44: 233–237. 77. Miller WR, Forrest AP. Oestradiol synthesis from C19 steroids by human breast cancers. Br J Cancer 1976; 33: 116–118. 78. Esteban JM, Warsi Z, Haniu M et al. Detection of intratumoral aromatase in breast carcinomas. An immunohistochemical study with clinicopathologic correlation. Am J Pathol 1992; 140: 337–343. 79. Sasano H, Nagura H, Harada N et al. Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders. Hum Pathol 1994; 25: 530–535. 80. Lu Q, Nakmura J, Savinov A et al. Expression of aromatase protein and messenger ribonucleic acid in tumor epithelial cells and evidence of functional significance of locally produced estrogen in human breast cancers. Endocrinology 1996; 137: 3061–3068. 81. O’Neill JS, Elton RA, Miller WR. Aromatase activity in adipose tissue from breast quadrants: a link with tumour site. BMJ 1988; 296: 741–743. 82. Miller WR, O’Neill J. The importance of local synthesis of estrogen within the breast. Steroids 1987; 50: 537–548. 83. Bulun SE, Price TM, Aitken J et al. link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 1993; 77: 1622–1628. 84. Dowsett M, Lee K, Macaulay VM et al. The control and biological importance of intratumoural aromatase in breast cancer. J Steroid Biochem Mol Biol 1996; 56: 145–150. 85. Zhou C, Zhou D, Esteban J et al. Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription–polymerase chain reaction. J Steroid Biochem Mol Biol 1996; 59: 163–171.
Review
Cyclooxygenase-2 (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention G. Davies1,2*, L.-A. Martin1, N. Sacks2 & M. Dowsett1 Academic Departments of 1Biochemistry and 2Surgery, Royal Marsden Hospital, London, UK Received 14 August 2001; revised 6 November 2001; accepted 23 November 2001
Non-steroidal anti-inflammatory drugs, cyclooxygenase-2 and breast cancer Non-steroidal anti-inflammatory drugs and cancer There have been a number of studies over the past 25 years linking non-steroidal anti-inflammatory drugs (NSAIDs) and cancer incidence [1]. Much of the work relating to NSAIDs and cancer has focused on colorectal cancer, including familial adenomatous polyposis (FAP). Several studies have reported an inverse relationship between colon cancer incidence and regular NSAID use including aspirin [2–6]. Studies using animal models of intestinal tumorigenesis have shown cyclooxygenase-2 (COX-2) expression in intestinal adenomas [7–10]. Further human studies of colon carcinomas have shown marked upregulation of COX-2 in carcinomas compared with normal mucosa [11–14]. A number of retrospective and prospective studies have shown a reduced incidence of colorectal cancer with NSAID use [15, 16]. In addition a number of randomised placebo-controlled double-blind trials
*Correspondence to: Dr G. Davies, Academic Departments of 1 Biochemistry and 2Surgery, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK. Tel: +44-207-808-2883; Fax: +44-207-376-3918; E-mail: [email protected] © 2002 European Society for Medical Oncology
have produced data showing a regression in polyp size [17, 18]. This process was reversible with tumours resuming growth after removal of the NSAID. There are fewer human data in relation to NSAID use and breast cancer incidence. A large cohort study of 89 528 registered nurses in the USA found no association between regular aspirin use and the incidence of breast cancer [19]. The analyses were based on 2414 cases identified over 12 years of follow-up [relative risk (RR) 1.0]. By contrast, in their casecontrol study of 511 women with newly diagnosed breast cancer and 1534 women who had undergone screening mammography, Harris et al. [20] found a reduced risk of breast cancer associated with the use of any NSAID three or more times a week for at least a year (RR 0.66). The protection was similar for users of aspirin alone, ibuprofen alone and all NSAIDs combined. The most heavily exposed women had the lowest risk, although the study was limited by incomplete descriptions of the study population and the participation rates. The epidemiological data relating NSAID use and the incidence of breast cancer are summarised in Table 1.
NSAIDs: mechanisms of action Arachidonic acid, a 20-carbon polyunsaturated fatty acid, is the precursor for prostaglandin (PG) synthesis. The first step is
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Interest in chemoprevention in oncology using suppressants of prostaglandin (PG) synthesis has been stimulated by epidemiological observations that the use of aspirin and other non-steroidal inflammatory drugs (NSAIDs) is associated with reduced incidence of some cancers, including cancer of the breast. The main target of NSAID activity is the cyclooxygenase (COX) enzyme. Two isoforms of COX have been identified: COX-1, the constitutive isoform; and COX-2, the inducible form of the enzyme. COX-2 can undergo rapid induction in response to many factors such as bacterial lipopolysaccharides, growth factors, cytokines and phorbol esters. COX-2 is overexpressed in some malignancies including carcinoma of the breast. It has been suggested that such enhanced expression may lead to increased angiogenesis such that the inhibition of COX-2 might have a general anticancer effect via decreased blood vessel formation. In addition, an association between COX-2, its main product PGE2 and aromatase activity in human breast cancer suggests that such inhibitors might have an additional, specific prophylactic mechanism for this tumour. New COX-2 inhibitors are already licensed for use in the treatment of arthritis and are well tolerated. Their potential role in chemoprevention of mammary carcinogenesis in rats has already been investigated. What remains to be seen is if these findings can be extrapolated to human studies. Key words: aromatase, breast cancer, cyclooxygenase-2, oestrogens, prevention, prostaglandins
670 Table 1. Use of non-steroidal anti-inflammatory drugs (NSAIDs) and the incidence of human breast cancer Authors
Year of study
Study size
NSAID used
Relative risk (95% confidence interval)
Prospective studies Paganini-Hill et al. [98]
1989
13 987
Aspirin
0.95–1.67
Thun et al. [2]
1993
635 031
Aspirin
0.98 (0.76–1.26)
Schreinemachers and Everson [16]
1994
12 668
Aspirin
0.70 (0.50–0.96)
Egan et al. [19]
1996
89 528
Aspirin
1.01 (0.80–1.27)
Harris et al. [97]
1999
32 505
Aspirin, ibuprofen
0.6
Case control studies Harris et al. [20]
1996
511
Aspirin, ibuprofen
0.66 (0.52–0.83)
Coogan et al. [99]
1999
6558
Aspirin
0.8 (0.7–1.0)
Sharpe et al. [100]
2000
5882
Aspirin, ibuprofen
0.76 (0.63–0.92)
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Figure 1. Prostaglandin synthesis via arachidonic acid. COX, cyclooxygenase; PG, prostaglandin; TXA2, thromboxane A2.
the hydrolysis of phospholipids to produce free arachidonic acid catalysed by phospholipase A2 (Figure 1). The next step is catalysed by COX, which inserts molecular oxygen into arachidonic acid [21]. This reaction produces an unstable product, PGG2. PGG2 is then converted by the peroxidase activity of COX to PGH2. PGH2 is the common precursor for all other prostanoids. The production of individual prostanoids
is catalysed by different, specific synthases, which may vary in their expression between different types of cells. Each of the products derived from PGH2 has a distinct biological function. The main target of NSAID action is COX. Two isoenzymes exist in the human form. The enzyme which is now called COX-1 was first purified and characterised from bull vesicular
671 role of PPAR-γ in carcinogenesis, and in particular in relation to mammary carcinogenesis.
Transcriptional regulation of COX-2 Increased expression of COX-2 in malignancy is likely to occur via multiple routes. COX-2 induction by lipopolysaccharide (LPS) has been shown to occur through both the mitogen-activated protein kinase (MAPK) and protein kinase C-ζ (PKC-ζ) pathways [30]. It has also been shown that ceramide-stimulated activation of MAPK can activate c-Jun N-terminal kinase (JNK), which in turn can lead to increased COX-2 gene expression in human mammary epithelial cells [31]. This occurs via a cAMP response element (CRE) in the COX-2 promoter. Further evidence for the role of MAPK and c-Jun pathways in tumour necrosis factor-α (TNF-α)-stimulated COX-2 expression in human epithelial cells was provided recently by Chen et al. [32]. These pathways are illustrated in Figure 2. Transient transfection experiments have demonstrated that nuclear factor κB (NFκB), nuclear factor IL-6 (NF-IL6) and CRE promoter sites mediate gene transcription independently in response to LPS treatment [30]. LPS can activate different pathways to induce COX-2 gene transcription: through NFκB
Figure 2. Signal transduction pathways influencing cyclooxygenase-2 (COX-2) expression. TNF-α, tumour necrosis factor-α; LPS, lipopolysaccharide; PKC-ζ, protein kinase C-ζ; MAPK, mitogen-activated protein kinase; CRE, cAMP response element.
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glands by Miyamoto in 1976 [22]. In 1991 cDNAs for COX-2, a novel isoenzyme of COX, were isolated and sequenced by two independent groups [23, 24]. Before the discovery of COX-2 it was known that PG synthesis could be stimulated by a variety of substances including cytokines, growth factors and tumour promoters. These effects were due to activation of phospholipases, which supply arachidonic acid to COX [25]. The two isoenzymes are regulated independently: COX-1 is constitutively expressed, whereas the inducible isoenzyme COX-2 is expressed only in response to certain stimuli such as tumour promoters, endotoxin, cytokines and hormones [26, 27]. There are also data suggesting that COX-independent mechanisms may be responsible for some of the effects seen with NSAIDs in cancer models. It has been reported that NSAIDs can disrupt the binding of a nuclear transcription factor (PPAR-γ) to its cis recognition sequence [28]: PPAR-γ expression is increased after disruption of the tumour suppressor gene Apc and could be an important downstream effector. This NSAID effect is not likely to be related to inhibition of COX. In contrast, another report demonstrated that PPAR-γ can be activated by binding PGI2 derived from COX-2 [29]. More work therefore needs to be done to elucidate the exact
672
COX-2 and carcinogenesis A number of studies have shown overexpression of COX-2 in solid malignancies including breast [37], pancreas, prostate [38] and colon [39]. The most compelling data to support a causal relationship between overexpression of COX-2 and carcinogenesis have come from studies of the Apc∆ 716 mouse. This is an animal model for human FAP, a condition caused by a germline mutation of the Apc gene in which individuals develop numerous adenomatous colorectal polyps, which predispose to colorectal carcinomas. Several strains of mice have been developed that carry mutations in one Apc allele, including the Min mouse [40] and Apc∆ 716 [41]. Analysis of adenomatous polyps from Min mice show increased expression of COX-2 relative to normal mucosa [9]. The effect of the absence of COX-2 in the Apc∆ 716 mouse has been studied by introduction of a knockout mutation of the COX-2 gene. Removal of the COX-2 gene in the mouse reduces the number and size of intestinal polyps [42]. Genetic and pharmacological evidence has been gathered to show that specific COX-2 inhibition is more effective than traditional NSAIDs in suppressing polyposis in mouse models of FAP, where COX-2 is induced [42].
COX-2 and mammary carcinogenesis COX-2 has been implicated in mammary carcinogenesis in several ways. COX-2 expression was detected using reverse transcription polymerase chain reaction (RT-PCR) in 13 of 13 human breast tumours, with no detectable expression in normal breast tissue [37]. A correlation was also observed between COX-2 expression and increasing tumour cell density. The COX-2 expression was localised to the epithelial compartment with no expression within the stromal component. A further study of 21 human breast tumour specimens found no detectable COX-2 expression in normal breast tissue specimens, but detectable and heterogeneous COX-2 gene
expression in each of the 21 tumour specimens [43]. In addition, a significant linear association between the tumour cell density and COX-2 gene expression was found (P <0.0001). In contrast Hwang et al. [44] analysed 44 samples by western blotting and only detected COX-2 protein in two of the 44 samples. A possible explanation for this is the differential sensitivity of the approaches to measuring the expression of COX-2. A study by Subbaramaiah et al. [45] looked at 29 microdissected breast cancers and found high levels of COX-2 protein in 14 of 15 HER-2/neu-positive samples. This was in contrast to the 14 HER-2/neu-negative tumours, where COX-2 was only detected in four of the cases, and at significantly lower levels than in the positive cases. The presence and levels of tissue PGs have been studied extensively in breast cancer over the past 30 years. Early studies demonstrated a relationship between tissue PG levels in human breast tumours, poor postoperative survival and development of metastases [46–48]. The main product of COX-2, namely PGE2, is found in high levels in tumour cells [49], and is synthesised by several human breast cancer cell lines. In clinical studies, high PGE2 concentrations have been associated with both high metastatic potential and a lack of oestrogen and progesterone receptors [48, 50]. The inhibition of COX-1 and COX-2 has been investigated in rat models. A 35-day course of ibuprofen in female Sprague– Dawley rats with 7,12-dimethylbenzathracene (DMBA)induced mammary carcinomas resulted in significant reduction of tumour volume (P <0.05) and a significant reduction in gene expression of both COX-1 and COX-2 [51]. Other studies including studies with indomethacin have yielded similar results [52, 53]. More recently the chemopreventive effect of a specific COX-2 inhibitor, celecoxib, against DMBA-induced mammary carcinogenesis in female Sprague–Dawley rats has been investigated [54]. Dietary administration of celecoxib produced striking reductions in the incidence, multiplicity and volume of mammary tumours relative to the control group. The chemopreventive properties of another COX-2 inhibitor, nimesulide, have been assessed in a study in which COX-2 expression and mammary tumours were induced by the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, together with a 24% corn oil diet [55]. Tumour incidence was significantly reduced from 71% in the control group to 51% in the treatment group, as well as a significant reduction in the size and multiplicity of tumours. More definitive evidence has now been provided by the recent demonstration that COX-2 overexpression is sufficient to induce mammary tumorigenesis in transgenic mice [56]. Overexpression of COX-2 in the mammary glands of transgenic mice was achieved by the use of the mouse mammary tumour virus promoter. Multiparous but not virgin females showed a much higher incidence of mammary gland hyperplasia, dysplasia and transformation to metastatic tumours. Studies of breast cancer cell lines have also provided data supporting the overexpression of COX-2 in breast cancer. A study of the differential expression of COX-1 and COX-2 in
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via extracellular signal related kinase (ERK-2), p38 and JNK pathways, through NF-IL6 via a p38 pathway, and through CRE via ERK-2 and JNK pathways. Moreover, PKC-ζ signalling seems to mediate transcription after LPS treatment through all three promoter sites. Therefore, individual signalling pathways, such as ERK-2, p38, JNK or PKC-ζ, appear to be sufficient to mediate COX-2 gene transcription by virtue of their ability to recruit transcription factors to at least two promoter sites. This may indicate redundancy in the signalling pathways and promoter elements regulating COX-2 transcription, at least in endotoxin-treated cells of macrophage/ monocyte lineage. Associations have also been made between mutated ras gene and COX-2 expression in human breast cancer cell lines [33], and c-myb expression is upregulated in colon tumours and breast cancers [34, 35]; c-myb overexpression causes a modest induction of COX-2 promoter activity [36].
673 human breast cancer cell lines found that in the MCF7 oestrogen receptor-positive cell line, COX-2 was barely detectable, whereas in the highly invasive, metastatic cell line MDA-MB231 there was a low level of COX-1 but a high level of COX-2 expression [57]. The concentration of PGE2, the major COX-2 product, correlated well with the level of COX-2 protein.
COX-2 overexpression and mammary carcinogenesis: potential mechanisms
Intratumoural aromatase, COX-2 and breast cancer Intratumoural aromatase and breast cancer Several areas of research have implicated sex hormones in the aetiology of breast cancer. Laboratory studies have shown that hormones, and in particular oestrogens control the growth of breast epithelial cells and affect the course of established disease. In premenopausal women, studies of serum oestradiol
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The expression of COX-2 in human solid cancers is not confined to the epithelial component of the tumour. The neovasculature associated with colonic adenomas, carcinomas and metastatic liver lesions also demonstrates COX-2 staining by immunohistochemical methods [58]. This expression seems to be a general characteristic of epithelial tumours including head and neck [59] and pancreas [60]. Significant correlations between COX-2 and tumour vascularisation (P <0.0001), microvessel density (P <0.007) and vascular endothelial growth factor (VEGF) in 35 head and neck cancers have been reported [61]. Uefuji et al. [62] found that microvessel density positively correlated with COX-2 expression in 42 cases of primary human gastric adenocarcinoma. The COX-2-overexpressing cases showed significantly elevated levels of PGE2 compared with normal gastric mucosa. The effects of specific COX-2 inhibitors have been tested in animal models of angiogenesis and celecoxib, a specific COX-2 inhibitor, has been shown to cause inhibition of the angiogenic response in fibroblast growth factor (FGF)induced corneal angiogenesis in rats [58]. Celecoxib reduced both the number and length of sprouting capillaries in a dosedependent fashion. These data suggest COX-2 overexpression is associated with increased PGE2 biosynthesis and angiogenesis in gastric cancer, and provide supportive data for COX-2 overexpression being functionally significant in tumour angiogenesis. The mechanism by which COX-2 expression produces these effects is unclear, however. Activated human microvascular endothelial cells produce a number of eicosanoid products including thromboxane A2 (TXA2) [63]. Selective COX-2 antagonists have been shown to inhibit TXA2 production and endothelial migration as well as corneal angiogenesis, an effect that is reversed with the use of a TXA2 agonist U46619 under COX-2-inhibited conditions [63]. TXA2 may represent an important intermediary of the angiogenic process. It has also been suggested that combined expression of inducible nitric oxide synthase and COX -2 may contribute to tumour angiogenesis. The exact mechanism by which this may occur is far from understood. Inducible nitric oxide synthase and COX-2 have been positively correlated with VEGF in non-small cell lung cancers [64]. In addition a study of 100 patients with resectable hepatocellular carcinoma showed that tumours that were negative for inducible nitric oxide synthase and COX-2 by immunhistochemical assess-
ment had a significantly better overall (P = 0.041) and recurrence-free survival (P = 0.018) [65]. A recent study by Dormond et al. [66] investigated the potential links between αVβ3 integrin, an adhesion receptor critically involved in mediating tumour angiogenesis and COX-2. They demonstrated that inhibition of endothelial-cell COX-2 by NSAIDs suppresses αVβ3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of FGF-2-induced angiogenesis in vivo. These results provide a functional link between COX-2, integrin αVβ3 and Cdc42-/Rac-dependent endothelial-cell migration. Thus, COX-2 appears to be related to induction of angiogenesis in a variety of tumours, which provides a general target for the chemopreventive use of COX-2 inhibitors for a variety of malignancies. In addition to angiogenic effects, COX-2 expression may have effects on apoptosis. The specific COX-2 inhibitor celecoxib induces apoptosis in human prostate cancer cells, whereas the COX-1 inhibitor piroxicam has no appreciable effect [67]. The forced expression of COX-2 inhibits programmed cell death, and the growth of COX-2-positive human colonic cancer xenografts has been shown to be inhibited by a COX-2 inhibitor (SC-58125) [68]. PGE2 inhibited the SC-58125-stimulated apoptosis and also induced expression of the anti-apoptotic protein Bcl-2 but did not affect expression of BAX, the death-promoting member of the Bcl-2 family, in human colonic cancer (HCA-7) cells. In the human colorectal cancer cell line HCT116, which contains normal p53 and BAX genes, deletion of the BAX gene provides profound protection against the apoptosis induced by the NSAIDs, sulindac and indomethacin [69]. This mechanism may also explain resistance to NSAID-induced apoptosis in conditions with a hereditary predisposition such as hereditary non-polyposis colorectal cancer, where inherited defects of BAX may be present. The enhanced tumorigenesis seen in transgenic mice carrying COX-2 under the control of the mouse mammary tumour virus promoter is preceded by reduced levels of BAX and Bcl-x(L) and increased levels of Bcl-2 in the mammary epithelial cells [56]. Thus the interactions between COX-2, Bcl-2 and other members of the Bcl-2 family may represent a further mechanism by which COX-2 expression influences tumour growth, including that of the mammary gland.
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and its correlation with breast cancer risk have been inconsistent [70]. Variations in concentrations throughout the menstrual cycle may account for this inconsistency. A number of studies [71–75], however, have reported an association between high serum concentrations of oestradiol and increased risk of breast cancer in postmenopausal women. Some studies, e.g. Thomas et al. [71], report a steep gradient of association. If the association is causative, this gradient would result in a small decrease in serum oestradiol concentrations leading to substantial reduction in breast cancer risk. Oestrogens are produced from androgens by the action of the enzyme aromatase. In postmenopausal women, plasma oestrogens result from peripheral aromatisation, particularly in adipose tissue. Many breast cancers, however, also contain aromatase, and studies using radioactive isotopes indicate that both of these sources contribute to the oestrogen detectable in breast cancers [76]. The proportional significance varies markedly from patient to patient but the mean contribution appears to be approximately equal from the local and distal sources. This capacity of certain breast cancers to synthesise oestrogens by intratumoural aromatase activity has been known for many years [77], but the exact cell types or breast
tumours which contain functionally active aromatase remain an area of debate. In different studies, aromatase has been detected in both epithelial and stromal cells [78], in just the stromal elements [79] and only in breast cancer epithelial cells [80]. Aromatase is also detectable in normal breast tissue. The association between high levels of aromatase in the quadrant of normal tissue containing a breast carcinoma has been suggested as causative [81]. A small number of studies have suggested that breast carcinomas with aromatase activity show an increased response to aromatase inhibitors [82, 83] but larger studies are needed to confirm this. Aromatasetransfected MCF7 breast cancer cells can be mitogenically stimulated with androgen and their proliferation can be suppressed with aromatase inhibitors [84]. All of these findings provide support for the role of intratumoural aromatase being of importance in breast cancer incidence and progression.
Regulation of breast aromatase gene expression and transcription Transcriptional regulation of the aromatase gene is complex and differs between tissues, providing tissue-specific control
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Figure 3. Transcriptional regulation of aromatase via cyclooxygenase-2 (COX-2). CRE, cAMP response element; PGE2, prostaglandin E2; PKA, protein kinase A; PKC, protein kinase C; GRE, glucocorticoid response element.
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COX-2 and aromatase in breast cancer COX-2 expression has been found to correlate with aromatase expression within human breast cancer tissue. A study of 23 human breast tumours found that COX-2 and aromatase expression, as measured by semi-quantitative RT-PCR showed a significant positive correlation [43]. The highest levels of COX-2 expression were in tumours with high cellularity and those that showed evidence of invasion. A strong linear association between aromatase expression and the sum of COX-1 and COX-2 expression was also found. The COX-2 product PGE2 and cytokines such as interleukin-6 (IL-6) or TNF-α can regulate aromatase activity [90]. Studies of aromatase activity in fibroblasts from breast tissue proximal to a carcinoma have shown a higher basal aromatase activity than those derived from the tumour itself, and the ability of PGE2, TNF-α and IL-6 to stimulate aromatase activity was greater in the tissue adjacent to the tumour [92, 93].
Prospects for COX-2 inhibitors in breast cancer prophylaxis The body of evidence cited above supports a link between the overexpression of COX-2 and mammary carcinogenesis which may at least in part be dependent on the induction of aromatase by PGE2. The selective inhibition of COX-2 may modulate a critical step in the initiation and promotion, as well as progression of breast cancer. COX-2 inhibitors are well tolerated and have minimal side-effects, and as such may be well suited to prophylactic use. The long-term use of COX-2 inhibitors has been evaluated in several large randomised trials including the Vioxx Gastrointestinal Outcomes Research Study (VIGOR) [94] and the Celecoxib Long-term Safety Study (CLASS) [95]. These
Figure 4. Possible effects of cyclooxygenase-2 (COX-2) inhibitors as chemopreventives of breast cancer. PGE2, prostaglandin E2.
studies confirm the improved gastrointestinal safety profile compared with conventional NSAIDs. Recent concerns have been raised, however, about possible cardiovascular risks associated with COX-2 inhibitor usage [96]. An increased RR of thrombotic cardiovascular events (2.38; P = 0.002) was seen with the use of rofecoxib compared with naproxen in the VIGOR study. This difference was not seen between celecoxib and other NSAIDs in the CLASS study. Whether these findings reflect a prothrombotic effect of rofecoxib or a greater antithrombotic effect of naproxen is uncertain. Further prospective evaluation is needed to fully characterise any increased risk and these data may affect the prospects for COX-2 inhibition in breast cancer prophylaxis. Aromatase inhibitors are in pilot trials to evaluate their possible utility in chemoprevention, but their profound suppression of oestrogen throughout the body may lead to unacceptable side-effects. In contrast, the possible link between the COX-2 products and breast aromatase in and around malignant breast tissue suggests a possible means for selective suppression of oestrogenic stimulation of the breast. It is likely that such targeted oestrogen suppression will lead to only partial withdrawal of oestrogen, but the epidemiological data indicate that there may be a steep dose relationship between oestrogen exposure and breast cancer incidence, suggesting that partial oestrogen suppression may have profound effects on breast cancer incidence. Thus the current understanding of the role of COX-2 in breast cancer suggests that COX-2 inhibitors may have a role in chemoprevention which is based in part on the generic issues of anti-angiogenesis and pro-apoptotic processes, and in part on a tissue-specific inhibition of oestrogen synthesis (Figure 4). It might be considered
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of the enzyme. At least eight exons I have been reported. In breast carcinomas exons 1.3 and II are the most frequent exons I [85], suggesting that promoters 1.3 and II are the major promoters directing aromatase expression in the malignant and surrounding tissue. This contrasts with the adipose stromal cells and fibroblasts of normal breast tissue, in which exon 1.4 is the dominant exon I [86, 87]. Exon 1.4 is preceded by a TATA-less promoter and upstream CRE and Sp1 sequences, which are required for upregulation of transcription by serum glucocorticoids [88]. A series of in vitro experiments point to exons 1.3 and II being under the control of cAMP [89, 90]. Thus the concept has been developed of promoter switching on the aromatase gene between normal and malignant tissues (Figure 3). PGE2, which is synthesised by many breast carcinomas, as noted above, is a known stimulant of cAMP in breast cancer cells [91]; its production by breast cancer may be instrumental in the switching of aromatase promoters. If these transcriptional relationships are important in breast cancer, a relationship between COX-2 and aromatase activity might be expected.
676 that the effects on angiogenesis and apoptosis may be more important since these would provide a means of non-oestrogendependent prevention, while current strategies for prevention in breast cancer are targeted at oestrogen-dependent effects. Pilot studies of the chemopreventive use of COX-2 inhibitors are merited to test these concepts. The presurgical setting offers an opportunity to test the effects of a short course of these compounds on biological markers of angiogenesis, apoptosis, proliferation as well as conventional markers such as oestrogen, progesterone and HER-2 receptor status in human breast cancer tissue. This would provide direct data to indicate whether these compounds have biological effects of potential clinical significance within human breast cancer tissue, and would provide the platform for larger chemopreventive studies.
1. Mehta RG, Moon RC. Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol. Anticancer Res 1991; 11: 593–596. 2. Thun MJ, Namboodiri MM, Heath CW Jr. Aspirin use and reduced risk of fatal colon cancer. N Engl J Med 1991; 325: 1593–1596. 3. Greenberg ER, Baron JA, Freeman DH et al. Reduced risk of largebowel adenomas among aspirin users. The Polyp Prevention Study Group. J Natl Cancer Inst 1993; 85: 912–916. 4. Logan RF, Little J, Hawtin PG, Hardcastle JD. Effect of aspirin and non-steroidal anti-inflammatory drugs on colorectal adenomas: casecontrol study of subjects participating in the Nottingham faecal occult blood screening programme. BMJ 1993; 307: 285–289. 5. Suh O, Mettlin C, Petrelli NJ. Aspirin use, cancer, and polyps of the large bowel. Cancer 1993; 72: 1171–1177. 6. Reeves MJ, Newcomb PA, Trentham-Dietz A et al. Nonsteroidal anti-inflammatory drug use and protection against colorectal cancer in women. Cancer Epidemiol Biomarkers Prev 1996; 5: 955–960. 7. Boolbol SK, Dannenberg AJ, Chadburn A et al. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res 1996; 56: 2556–2560. 8. DuBois RN, Radhika A, Reddy BS, Entingh AJ. Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 1996; 110: 1259–1262. 9. Williams CS, Luongo C, Radhika A et al. Elevated cyclooxygenase-2 levels in Min mouse adenomas. Gastroenterology 1996; 111: 1134– 1140. 10. Singh J, Hamid R, Reddy BS. Dietary fat and colon cancer: modulation of cyclooxygenase-2 by types and amount of dietary fat during the postinitiation stage of colon carcinogenesis. Cancer Res 1997; 57: 3465–3470. 11. Eberhart CE, Coffey RJ, Radhika A et al. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology 1994; 107: 1183–1188. 12. Kargman SL, O’Neill GP, Vickers PJ et al. Expression of prostaglandin G/H synthase-1 and -2 protein in human colon cancer. Cancer Res 1995; 55: 2556–2559. 13. Sano H, Kawahito Y, Wilder RL et al. Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res 1995; 55: 3785– 3789.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
References
14. Kutchera W, Jones DA, Matsunami N et al. Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect. Proc Natl Acad Sci USA 1996; 93: 4816–4820. 15. Rosenberg L, Palmer JR, Zauber AG et al. A hypothesis: nonsteroidal anti-inflammatory drugs reduce the incidence of large-bowel cancer. J Natl Cancer Inst 1991; 83: 355–358. 16. Schreinemachers DM, Everson RB. Aspirin use and lung, colon, and breast cancer incidence in a prospective study. Epidemiology 1994; 5: 138–146. 17. Nugent KP, Farmer KC, Spigelman AD et al. Randomized controlled trial of the effect of sulindac on duodenal and rectal polyposis and cell proliferation in patients with familial adenomatous polyposis. Br J Surg 1993; 80: 1618–1619. 18. Giardiello FM, Hamilton SR, Krush AJ et al. Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis. N Engl J Med 1993; 328: 1313–1316. 19. Egan KM, Stampfer MJ, Giovannucci E et al. Prospective study of regular aspirin use and the risk of breast cancer. J Natl Cancer Inst 1996; 88: 988–993. 20. Harris RE, Namboodiri KK, Farrar WB. Nonsteroidal antiinflammatory drugs and breast cancer. Epidemiology 1996; 7: 203–205. 21. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nat New Biol 1971; 231: 232–235. 22. Miyamoto T, Ogino N, Yamamoto S, Hayaishi O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J Biol Chem 1976; 251: 2629–2636. 23. Xie WL, Chipman JG, Robertson DL et al. Expression of a mitogenresponsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 1991; 88: 2692–2696. 24. Kujubu DA, Fletcher BS, Varnum BC et al. TIS10, a phorbol ester tumor promoter-inducible mRNA from Swiss 3T3 cells, encodes a novel prostaglandin synthase/cyclooxygenase homologue. J Biol Chem 1991; 266: 12866–12872. 25. Marnett LJ. Aspirin and the potential role of prostaglandins in colon cancer. Cancer Res 1992; 52: 5575–5589. 26. Herschman HR. Prostaglandin synthase 2. Biochim Biophys Acta 1996; 1299: 125–140. 27. Wu KK. Cyclooxygenase-2 induction: molecular mechanism and pathophysiologic roles. J Lab Clin Med 1996; 128: 242–245. 28. He TC, Chan TA, Vogelstein B, Kinzler KW. PPAR∆ is an APCregulated target of nonsteroidal anti-inflammatory drugs. Cell 1999; 99: 335–345. 29. Lim H, Gupta RA, Ma WG et al. Cyclooxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPAR∆. Genes Dev 1999; 13: 1561–1574. 30. Mestre JR, Mackrell PJ, Rivadeneira DE et al. Redundancy in the signaling pathways and promoter elements regulating cyclooxygenase-2 gene expression in endotoxin-treated macrophage/monocytic cells. J Biol Chem 2001; 276: 3977–3982. 31. Subbaramaiah K, Chung WJ, Dannenberg AJ. Ceramide regulates the transcription of cyclooxygenase-2. Evidence for involvement of extracellular signal-regulated kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways. J Biol Chem 1998; 273: 32943–32949. 32. Chen CC, Sun YT, Chen JJ, Chang YJ. Tumor necrosis factor-αinduced cyclooxygenase-2 expression via sequential activation of ceramide-dependent mitogen-activated protein kinases, and IκB kinase 1/2 in human alveolar epithelial cells. Mol Pharmacol 2001; 59: 493–500.
677 52. Alshafie GA, Harris RE, Robertson FM et al. Comparative chemopreventive activity of ibuprofen and N-(4-hydroxyphenyl) retinamide against the development and growth of rat mammary adenocarcinomas. Anticancer Res 1999; 19: 3031–3036. 53. McCormick DL, Madigan MJ, Moon RC. Modulation of rat mammary carcinogenesis by indomethacin. Cancer Res 1985; 45: 1803–1808. 54. Harris RE, Alshafie GA, Abou-Issa H, Seibert K. Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase-2 inhibitor. Cancer Res 2000; 60: 2101–2103. 55. Nakatsugi S, Ohta T, Kawamori T et al. Chemoprevention by nimesulide, a selective cyclooxygenase-2 inhibitor, of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats. Jpn J Cancer Res 2000; 91: 886–892. 56. Liu CH, Chang SH, Narko K et al. overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem 2001; 276: 18563–18569. 57. Liu XH, Rose DP. Differential expression and regulation of cyclooxygenase-1 and -2 in two human breast cancer cell lines. Cancer Res 1996; 56: 5125–5127. 58. Masferrer JL, Leahy KM, Koki AT et al. Antiangiogenic and antitumour activities of cyclooxygenase-2 inhibitors. Cancer Res 2000; 60: 1306–1311. 59. Chan G, Boyle JO, Yang EK et al. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinoma of the head and neck. Cancer Res 1999; 59: 991–994. 60. Tucker ON, Dannenberg AJ, Yang EK et al. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer. Cancer Res 1999; 59: 987–990. 61. Gallo O, Franchi A, Magnelli L et al. Cyclooxygenase-2 pathway correlates with VEGF expression in head and neck cancer. Implications for tumor angiogenesis and metastasis. Neoplasia 2001; 3: 53– 61. 62. Uefuji K, Ichikura T, Mochizuki H. Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer. Clin Cancer Res 2000; 6: 135–138. 63. Daniel TO, Liu H, Morrow JD et al. Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis. Cancer Res 1999; 59: 4574–4577. 64. Marrogi AJ, Travis WD, Welsh JA et al. Nitric oxide synthase, cyclooxygenase 2, and vascular endothelial growth factor in the angiogenesis of non-small-cell lung carcinoma. Clin Cancer Res 2000; 6: 4739–4744. 65. Rahman MA, Dhar DK, Yamaguchi E et al. Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possible involvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res 2001; 7: 1325–1332. 66. Dormond O, Foletti A, Paroz C et al. NSAIDs inhibit αVβ3 integrinmediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nature Med 2001; 7: 1041–1047. 67. Hsu AL, Ching TT, Wang DS et al. The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2. J Biol Chem 2000; 275: 11397–11403. 68. Sheng H, Shao J, Morrow JD et al. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998; 58: 362–366. 69. Zhang L, Yu J, Park BH et al. Role of BAX in the apoptotic response to anticancer agents. Science 2000; 290: 989–992.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
33. Gilhooly EM, Rose DP. The association between a mutated ras gene and cyclooxygenase-2 expression in human breast cancer cell lines. Int J Oncol 1999; 15: 267–270. 34. Guerin M, Sheng ZM, Andrieu N, Riou G. Strong association between c-myb and oestrogen-receptor expression in human breast cancer. Oncogene 1990; 5: 131–135. 35. Ramsay RG, Thompson MA, Hayman JA et al. Myb expression is higher in malignant human colonic carcinoma and premalignant adenomatous polyps than in normal mucosa. Cell Growth Differ 1992; 3: 723–730. 36. Ramsay RG, Friend A, Vizantios Y et al. Cyclooxygenase-2, a colorectal cancer nonsteroidal anti-inflammatory drug target, is regulated by c-MYB. Cancer Res 2000; 60: 1805–1809. 37. Parrett ML, Harris RE, Joarder FS et al. Cyclooxygenase-2 gene expression in human breast cancer. Int J Oncol 1997; 10: 503–507. 38. Gupta S, Srivastava M, Ahmad N et al. Over-expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate 2000; 42: 73–78. 39. Maekawa M, Sugano K, Sano H et al. Increased expression of cyclooxygenase-2 to -1 in human colorectal cancers and adenomas, but not in hyperplastic polyps. Jpn J Clin Oncol 1998; 28: 421–426. 40. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990; 247: 322–324. 41. Oshima M, Oshima H, Kitagawa K et al. Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene. Proc Natl Acad Sci USA 1995; 92: 4482–4486. 42. Oshima M, Dinchuk JE, Kargman SL et al. Suppression of intestinal polyposis in Apc∆ 716 knockout mice by inhibition of cyclooxygenase-2 (COX-2). Cell 1996; 87: 803–809. 43. Brueggemeier RW, Quinn AL, Parrett ML et al. Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens. Cancer Lett 1999; 140: 27–35. 44. Hwang D, Scollard D, Byrne J, Levine E. Expression of cyclooxygenase-1 and cyclooxygenase-2 in human breast cancer. J Natl Cancer Inst 1998; 90: 455–460. 45. Subbaramaiah K, Norton L, Gerald W et al. Increased expression of cyclooxygenase-2 in HER-2-overexpressing human breast cancer cells. NCI 7th SPORE Investigators Workshop 1999. 46. Bennett A, Berstock DA, Raja B, Stamford IF. Survival time after surgery is inversely related to the amounts of prostaglandins extracted from human breast cancers. Br J Pharmacol 1979; 66: 451P. 47. Bennett A. The production of prostanoids in human cancers, and their implications for tumor progression. Prog Lipid Res 1986; 25: 539– 542. 48. Rolland PH, Martin PM, Jacquemier J et al. Prostaglandin in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. J Natl Cancer Inst 1980; 64: 1061–1070. 49. Schrey MP, Patel KV. Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators. Br J Cancer 1995; 72: 1412–1419. 50. Fulton AM, Heppner GH. Relationships of prostaglandin E and natural killer sensitivity to metastatic potential in murine mammary adenocarcinomas. Cancer Res 1985; 45: 4779–4784. 51. Robertson FM, Parrett ML, Joarder FS et al. Ibuprofen-induced inhibition of cyclooxygenase isoform gene expression and regression of rat mammary carcinomas. Cancer Lett 1998; 122: 165–175.
678 86. Harada N. A unique aromatase (P-450arom) mRNA formed by alternative use of tissue-specific exons I in human skin fibroblasts. Biochem Biophys Res Commun 1993; 189: 1001–1007. 87. Mahendroo MS, Mendleson CR, Simpson ER. Tissue-specific and hormonally controlled alternative promoters regulate aromatase cytochrome P450 gene expression in human adipose tissue. J Biol Chem 1993; 268: 19463–19470. 88. Zhao Y, Mendleson CR, Simpson ER. Characterization of the sequences of the human CYP19(aromatase) gene that mediate regulation by glucocorticoids in adipose stromal cells and fetal hepatocytes. Mol Endocrinol 1995; 9: 340–349. 89. Michael MD, Kilgore MW, Morohashi K, Simpson ER. Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450 (CYP19) gene in the ovary. J Biol Chem 1995; 270: 13561–13566. 90. Michael MD, Michael LF, Simpson ER. A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 (CYP19) gene. Mol Cell Endocrinol 1997; 134: 147–156. 91. Zhao Y, Agarwal VR, Mendelson CR, Simpson ER. Estrogen biosynthesis proximal to a breast tumor is stimulated by PGE2 via cyclic AMP, leading to activation of promoter II of the CYP19 (aromatase) gene. Endocrinology 1996; 137: 5739–5742. 92. Reed MJ, Coldham NG, Patel SR et al. Interleukin-1 and interleukin-6 in breast cyst fluid: their role in regulating aromatase activity in breast cancer cells. J Endocrinol 1992; 132: 5–8. 93. Singh A, Purohit A, Ghilchik MW, Reed MJ. The regulation of aromatase activity in breast fibroblasts: the role of interleukin-6 and prostaglandin E2. Endocr Relat Cancer 1999; 6: 139–147. 94. Bombardier C, Laine L, Reicin A et al. Comparison of upper gastrointestinal toxicity of rofecoxib and naproxen in patients with rheumatoid arthritis. N Engl J Med 2000; 343: 1520–1528. 95. Silverstein FE, Faich G, Goldstein JL et al. Gastrointestinal toxicity with celecoxib vs nonsteroidal anti-inflammatory drugs for osteoarthritis and rheumatoid arthritis: the CLASS study: A randomized controlled trial. Celecoxib Long-term Arthritis Safety Study. JAMA 2000; 284: 1247–1255. 96. Mukherjee D, Nissen SE, Topol EJ. Risk of cardiovascular events associated with selective COX-2 inhibitors. JAMA 2001; 286: 954– 959. 97. Harris RE, Prospective study of nonsteroidal anti-inflammatory drugs and breast cancer. Oncol Rep 1999; 6: 71–73. 98. Paganini-Hill A, Chao A, Ross RK et al. Aspirin use and chronic diseases: a cohort study of the elderly. BMJ 1989; 299: 1247–1250. 99. Coogan PF, Rao SR, Rosenberg L et al. The relationship of nonsteroidal anti-inflammatory drugs use to the risk of breast cancer. Prev Med 1999; 29: 72–76. 100.Sharpe CR, Collet JP, McNutt M et al. Nested case–control study of the effects of non-steroidal anti-inflammatory drugs on breast cancer risk and stage. Br J Cancer 2000; 83: 112–120.
Downloaded from http://annonc.oxfordjournals.org/ at Kainan University on March 28, 2015
70. Key TJ, Pike MC. The role of oestrogens and progestagens in the epidemiology and prevention of breast cancer. Eur J Cancer Clin Oncol 1988; 24: 29–43. 71. Thomas HV, Key TJ, Allen DS et al. A prospective study of endogenous serum hormone concentrations and breast cancer risk in postmenopausal women on the island of Guernsey. Br J Cancer 1997; 76: 401–405. 72. Helzlsouer KJ, Alberg AJ, Bush TL et al. A prospective study of endogenous hormones and breast cancer. Cancer Detect Prev 1994; 18: 79–85. 73. Toniolo PG, Levitz M, Zeleniuch-Jacquotte A et al. A prospective study of endogenous estrogens and breast cancer in postmenopausal women. J Natl Cancer Inst 1995; 87: 190–197. 74. Berrino F, Muti P, Micheli A et al. Serum sex hormone levels after menopause and subsequent breast cancer. J Natl Cancer Inst 1996; 88: 291–296. 75. Dorgan JF, Longcope C, Stephenson HE et al. Relation of prediagnostic serum estrogen and androgen levels to breast cancer risk. Cancer Epidemiol Biomarkers Prev 1996; 5: 533–539. 76. Reed MJ, Owen AM, Lai LC et al. In situ oestrone synthesis in normal breast and breast tumour tissues: effect of treatment with 4-hydroxyandrostenedione. Int J Cancer 1989; 44: 233–237. 77. Miller WR, Forrest AP. Oestradiol synthesis from C19 steroids by human breast cancers. Br J Cancer 1976; 33: 116–118. 78. Esteban JM, Warsi Z, Haniu M et al. Detection of intratumoral aromatase in breast carcinomas. An immunohistochemical study with clinicopathologic correlation. Am J Pathol 1992; 140: 337–343. 79. Sasano H, Nagura H, Harada N et al. Immunolocalization of aromatase and other steroidogenic enzymes in human breast disorders. Hum Pathol 1994; 25: 530–535. 80. Lu Q, Nakmura J, Savinov A et al. Expression of aromatase protein and messenger ribonucleic acid in tumor epithelial cells and evidence of functional significance of locally produced estrogen in human breast cancers. Endocrinology 1996; 137: 3061–3068. 81. O’Neill JS, Elton RA, Miller WR. Aromatase activity in adipose tissue from breast quadrants: a link with tumour site. BMJ 1988; 296: 741–743. 82. Miller WR, O’Neill J. The importance of local synthesis of estrogen within the breast. Steroids 1987; 50: 537–548. 83. Bulun SE, Price TM, Aitken J et al. link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription. J Clin Endocrinol Metab 1993; 77: 1622–1628. 84. Dowsett M, Lee K, Macaulay VM et al. The control and biological importance of intratumoural aromatase in breast cancer. J Steroid Biochem Mol Biol 1996; 56: 145–150. 85. Zhou C, Zhou D, Esteban J et al. Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription–polymerase chain reaction. J Steroid Biochem Mol Biol 1996; 59: 163–171.