PGs have been implicated in the etiology of epithelial cancers, primarily because of their role in promoting proliferation, angiogenesis, immune suppression, and cell survival [reviewed in 24
]. PGs can be synthesized by both COX-1 and COX-2 but the contribution of each depends on their level of expression and affinity for the substrate, arachidonic acid [26
]. COX-1 expression is considered to be constitutive under a range of physiological conditions, while COX-2 is an immediate early response gene that is normally not expressed in most epithelial cells under non-stimulated conditions. It is, however, highly induced in response to growth factors, cytokines, irritants including phorbol ester tumor promoters, and UV light [reviewed in 27
]. COX-2 is also constitutively upregulated in most epithelial tumors, including murine squamous cell tumors [7
As has been carried out in colon cancer studies, two approaches, pharmacological and genetic, have been taken to determine whether upregulated COX-2 contributes to skin tumor development. Müller-Decker et al. [14
] showed that a selective COX-2 inhibitor, SC-58125, applied topically at doses that inhibited TPA-induced PGE2
synthesis, substantially reduced skin tumor development. We and others have shown that celecoxib, another selective COX-2 inhibitor, have dramatic chemopreventive activity against UV-induced skin cancer [9
]. However, because these pharmacological agents have additional targets, including Akt [reviewed in 29
], it is difficult to interpret this data in terms of the contribution of PGs to tumorigenesis. To overcome this obstacle, mice deficient in either COX-1 or COX-2 were developed [21
]. Homozygous deficiency of either COX isoform reduced DMBA/TPA skin tumorigenesis by 75% [18
]. Because exposure to UV light is the major cause of human skin cancer [1
], there has been interest in demonstrating a role for COX-2 in UV-induced skin cancer as well.
Pentland et al. [15
] recently reported crossing the null alleles for COX-1 onto the SKH-1 mouse strain and with these animals in a UV carcinogenesis protocol. They reported that tumor number, tumor size, and time of tumor onset in COX-1 −/− mice were identical to wild-type SKH-1 mice, even though there was a fourfold increase in apoptosis in the null mice [15
]. As would be expected, we show here that heterozygous COX-1 mice also have the same tumor response as wild-type control. We attempted to take a similar approach with COX-2 knockout mice, however, the homozygous nulls on a SKH-1 background have a significantly reduced viability, negating the possibility that they could be used in long-term experiments. The heterozygous COX-2 mice, however, are viable and were used in a UV carcinogenesis protocol to determine if the loss of just one allele would affect tumor outcome. As shown in both experiments reported here, we observed a 50–75% reduction in tumor multiplicity, as well as a reduction in tumor size and an increased latency. In addition, tumor development was enhanced in transgenic mice overexpressing COX-2. These findings support the pharmacological studies on COX-2 inhibition and strengthen the conclusion that COX-2 is required for the development of murine skin tumors.
As previously reported [15
], COX-1 does not appear to be required for UV-induced skin tumors but is for DMBA/TPA-induced tumors [18
]. This is strongly suggested in the accompanying article by Akunda et al. [20
], in which it was shown that COX-2 is protective against acute UV sunburn effects, while, as shown here, it has tumor-promoting activity when chronically upregulated. This may reflect the significant differences in the carcinogenic and promoting agents. UV imparts both mutagenic and tumor-promoting activity with each exposure and frequent, prolonged exposure is required for tumor development. In chemical initiation-promotion protocols, a single sub-threshold dose of carcinogen is used, resulting in a one-time, low level of DNA damage [31
] and is followed by repeated promotion without further DNA damage. Another consideration is that the DMBA/TPA experiments were performed on a B6/129 strain of mouse while the UV studies were carried out with the SKH-1 mouse [21
]. Different strain backgrounds are well known to influence tumor outcome and are likely to be due to a number of modifier genes [32
With regard to PGE2
synthesis, we observed a >three fold increase in wild-type SKH-1 mice 6 h after UV. The ability of UV exposure to induce PGE2
synthesis was reduced in both the COX-1 and COX-2 heterozygous mice, which likely reflects the contribution of each of the isoforms to the total PGE2
level. However, the similar reduction in both genotypes does not correlate with their respective tumor response. Although different time points were used by Pentland et al. [15
], they also found a reduction in PGE2
in UV-treated COX-1 hetero- and homozygous mice, which did not correlate with the unaltered tumor response.
It is possible that that the single UV treatment protocols used for measurement of biomarkers do not reflect what occurs during chronic treatment. UV activates cytoplasmic phospholipase A2
) that translocates to membranes and hydrolyzes arachidonic acid from phospholipids, thus making it available for metabolism. With a single UV irradiation the only COX isoform that is initially present in the epidermis is COX-1; as COX-2 is induced [9
], more of the metabolism may be taken over by COX-2. However, this concept is clouded by evidence that at least in some cell types COX-1 can only metabolize exogenous arachidonic acid. This may be due to differences in subcellular localization of the isoforms, with COX-2 being more highly concentrated in the nuclear envelope, a site of translocated cPLA2
]. Additionally, it has been reported that low levels of arachidonic acid (<2.5 µM) are not metabolized by COX-1 but are by COX-2; high levels of arachidonate (>10 µM), however, are preferentially metabolized by COX-1 [33
]. Our data suggest that under basal conditions (no COX-2 present), COX-1 can metabolize low levels of arachidonic acid. This is based on the observed reduction in basal PGE2
levels with the loss of one COX-1 allele. Under stimulated conditions, it appears that both isoforms contribute to the total PG pool. However, it is also clear that PGE2
levels measured at an early time (6 h) after UV do not correlate with long-term tumor outcome. Whether this is due to changes in the expression or activity of degradative enzymes such as 15-hydroxyprostaglandin dehydrogenase, and/or the expression of one or more of the four membrane receptors that mediate the action of PGE2
is not known at this time [34
Pentland et al. [15
] also reported that although loss of both COX-1 alleles did not alter tumor outcome, it did result in an increase in apoptosis. We did not observe an increase in our heterozygous COX-1 mice but did observe a small increase in the heterozygous COX-2 mice. PG-induced increased resistance to apoptosis has been proposed as a major mechanism by which COX-2 contributes to tumor development in a number of epithelial cells and tumors [26
]. An anti-apoptotic activity could affect tumorigenesis by allowing cells that have acquired a genetic alteration to survive.
In conclusion, the present study as well as reports by others, indicate that COX-1 and COX-2 can play very different roles in skin tumor development, depending on the nature of the agents used to induce tumors. While COX-1 is important in phorbol ester promotion [18
], it does not contribute to UV carcinogenesis [15
]. COX-2, on the other hand, is a prerequisite for skin tumor development with both protocols. COX-2 thus remains a viable target for topical skin cancer prevention agents.