Susan M. Fischer

CHEMOPREVENTIVE ACTIVITY OF CELECOXIB, A SPECIFIC CYCLOOXYGENASE-2 INHIBITOR, AND INDOMETHACIN AGAINST ULTRAVIOLET LIGHT-INDUCED SKIN CARCINOGENESIS

Epidemiological and dietary studies suggest that nonsteroidal anti‐inflammatory drugs (NSAIDs) reduce the risk of colon cancer, possibly through a mechanism involving inhibition of cyclooxygenase (COX)‐2, which is overexpressed in premalignant adenomatous polyps and colon cancer. Because ultraviolet light (UV) can induce COX‐2 and nonspecific NSAIDs can decrease UV‐induced skin cancer, we evaluated the ability of two compounds, celecoxib (a specific COX‐2 inhibitor) and indomethacin (a nonspecific NSAID), to block UV‐induced skin tumor development in SKH:HR‐1‐hrBr hairless mice. Mice fed 150 or 500 ppm celecoxib showed a dose‐dependent reduction (60% and 89%, respectively) in tumor yield. Indomethacin (4 ppm) reduced tumor yield by 78%. Although both acute and chronic UV exposure increased cell proliferation and edema, neither compound reduced these parameters. In contrast, UV‐induced prostaglandin synthesis in the epidermis was effectively blocked by both compounds. UV‐induced increases in COX‐2 expression in skin were also not altered in any of the treatment groups. Similarly, tumors that constitutively express high levels of COX‐2 displayed no reduction by treatment with celecoxib or indomethacin. The dramatic protective effects of celecoxib suggests that specific COX‐2 inhibitors may offer a way to safely reduce the risk of skin cancer in humans. Mol. Carcinog. 25:231–240, 1999.

Transcriptional Regulation of Cyclooxygenase-2 in Mouse Skin Carcinoma Cells REGULATORY ROLE OF CCAAT/ENHANCER-BINDING PROTEINS IN THE DIFFERENTIAL EXPRESSION OF CYCLOOXYGENASE-2 IN NORMAL AND NEOPLASTIC TISSUES

Many studies have suggested that overexpression of cyclooxygenase-2 (COX-2) contributes to the development of tumors in several tissues. COX-2 expression tends to be up-regulated in various types of tumors and transformed cell lines, and the overexpression of COX-2 is caused by enhanced transcription of the gene. In an attempt to characterize the signaling pathway leading to the overexpression of COX-2 in the mouse skin carcinoma cell line JWF2, we investigatedcis- and trans-acting factors required for COX-2 expression and demonstrated a molecular mechanism by which COX-2 is expressed differentially in normal and neoplastic tissues. Two regions of the COX-2 promoter containing an E-box and nuclear factor IL6 site were identified as the positive regulatory elements through transient transfections with luciferase reporter vectors containing the various 5′-flanking regions of the promoter. Moreover, electrophoretic mobility shift assays and cotransfection experiments showed that upstream stimulatory factors and CCAAT/enhancer-binding proteins (C/EBPs) bind to the E-box and nuclear factor IL6 site, respectively, and functionally transactivate the COX-2 promoter. We also found that C/EBP isoforms are expressed differentially during mouse skin carcinogenesis, suggesting that overexpression of COX-2 in tumors may be caused by a change in C/EBP expression levels.

The 15-lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-δ to induce apoptosis in colorectal cancer cells

Diminished apoptosis, a critical event in tumorigenesis, is linked to down-regulated 15-lipoxygenase-1 (15-LOX-1) expression in colorectal cancer cells. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which is the primary product of 15-LOX-1 metabolism of linoleic acid, restores apoptosis. Nonsteroidal antiinflammatory drugs (NSAIDs) transcriptionally up-regulate 15-LOX-1 expression to induce apoptosis. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for linoleic and arachidonic acid metabolites. PPAR-δ promotes colonic tumorigenesis. NSAIDs suppress PPAR-δ activity in colon cancer cells. The mechanistic relationship between 15-LOX-1 and PPAR-δ was previously unknown. Our current study shows that (i) 13-S-HODE binds to PPAR-δ, decreases PPAR-δ activation, and down-regulates PPAR-δ expression in colorectal cancer cells; (ii) the induction of 15-LOX-1 expression is a critical step in NSAID down-regulation of PPAR-δ and the resultant induction of apoptosis; and (iii) PPAR-δ is an important signaling receptor for 13-S-HODE-induced apoptosis. The in vivo relevance of these mechanistic findings was demonstrated in our tumorigenesis studies in nude mouse xenograft models. Our findings indicate that the down-regulation of PPAR-δ by 15-LOX-1 through 13-S-HODE is an apoptotic signaling pathway that is activated by NSAIDs.

Cyclo‐oxygenase‐2 Plays a Critical Role in UV‐induced Skin Carcinogenesis

Besides induction of DNA damage and p53 mutations, chronic exposure to UV irradiation leads to the constitutive up‐regulation of cyclo‐oxygenase‐2 (COX‐2) expression and to increased production of its primary product in skin, prostaglandin E2 (PGE2). COX‐2 has also been shown to be constitutively overexpressed in mouse, as well as human, UV‐induced skin cancers and premalignant lesions. UV exposure results in ligand‐independent activation of the epidermal growth factor receptor and subsequent activation of mitogen‐activated protein kinase and phosphatidylinositol 3‐kinase/Akt pathways leading to transcriptional activation of the COX‐2 gene. Use of COX‐2‐specific inhibitors and genetic manipulation of COX‐2 expression have demonstrated that UV induction of COX‐2 in the skin contributes to the induction of epidermal hyperplasia, edema, inflammation, and counters the induction of apoptosis after UV exposure. Likewise, inhibition of COX‐2 activity or reduced expression in COX‐2 knockout mice resulted in significantly reduced UV‐induced tumorigenesis, while overexpression of COX‐2 in transgenic mice enhanced UV‐induced tumor development. A combination of signaling from the PGE2 EP1, EP2 and/or EP4 receptors mediates the effects of COX‐2 overexpression. These studies demonstrate the crucial role of COX‐2 in the development of UV‐related nonmelanoma skin cancers.

Prostaglandin D2 Inhibits Hair Growth and Is Elevated in Bald Scalp of Men with Androgenetic Alopecia

Prostaglandin D2 inhibits hair growth through its receptor, GPR44, and this pathway could serve as a new target for developing treatments for male pattern baldness. A Not-So-Hairy Situation Everybody wishes his or her hair was different; curly hair wants straight locks, straight hair desires some curl. Patients with androgenetic alopecia (AGA), however, would take either one, as long as it meant having hair. AGA is a disorder that affects both men and women, leading to hair thinning and loss. Here, Garza and colleagues provide new insight into the pathogenesis of AGA, in hopes of developing new therapeutics that target specific pathways responsible for baldness. The authors first examined bald and haired scalp from five men with AGA and showed that the enzyme prostaglandin D2 (PGD2) synthase was elevated at the mRNA and protein levels in bald scalp only. In a larger group of 17 men, they confirmed that the synthase product PGD2 was also elevated in bald versus haired scalp. In mice with synchronized hair follicle cycling, Garza et al. uncovered a temporal relationship between PGD2 gene expression and hair follicle regression. The authors further found that PGD2 and a related metabolite, 15-dPGJ2, inhibited hair growth in both mice and human hair follicles, providing a crucial functional link between the prostaglandin pathway and AGA. Garza and coauthors identified the receptor GPR44 to be responsible for mediating the negative effects of PGD2. By discovering such therapeutic targets for downstream drug development, such as a topical treatment, Garza et al. may have given patients with AGA a long-awaited choice: curly or straight? Testosterone is necessary for the development of male pattern baldness, known as androgenetic alopecia (AGA); yet, the mechanisms for decreased hair growth in this disorder are unclear. We show that prostaglandin D2 synthase (PTGDS) is elevated at the mRNA and protein levels in bald scalp compared to haired scalp of men with AGA. The product of PTGDS enzyme activity, prostaglandin D2 (PGD2), is similarly elevated in bald scalp. During normal follicle cycling in mice, Ptgds and PGD2 levels increase immediately preceding the regression phase, suggesting an inhibitory effect on hair growth. We show that PGD2 inhibits hair growth in explanted human hair follicles and when applied topically to mice. Hair growth inhibition requires the PGD2 receptor G protein (heterotrimeric guanine nucleotide)–coupled receptor 44 (GPR44), but not the PGD2 receptor 1 (PTGDR). Furthermore, we find that a transgenic mouse, K14-Ptgs2, which targets prostaglandin-endoperoxide synthase 2 expression to the skin, demonstrates elevated levels of PGD2 in the skin and develops alopecia, follicular miniaturization, and sebaceous gland hyperplasia, which are all hallmarks of human AGA. These results define PGD2 as an inhibitor of hair growth in AGA and suggest the PGD2-GPR44 pathway as a potential target for treatment.

Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E3 (PGE3) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE3. The extracellular ratio of PGE3 to PGE2 increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE3, respectively, than that produced by COX-1. Exposure of A549 cells to 1 μM PGE3 inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE3, however, had no effect. When A549 cells were exposed to EPA (25 μM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 μM). This effect appears to be associated with a 50% reduction of PGE3 formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE2, an increase in the level of PGE3, and PGE3-mediated inhibition of tumor cell proliferation.

The effect of vitamin E acetate on ultraviolet‐induced mouse skin carcinogenesis

Despite the benefits of sunscreens, ultraviolet (UV) exposure can still lead to skin cancer. In this study we investigated the effect of topical application of the antioxidant vitamin E acetate (VEA) on the inhibition of UV‐induced carcinogenesis. Hairless SKH‐1 mice received 5.2 mg of VEA 30 min before (VEA/UV) or after (UV/VEA) a single minimal erythemic dose of UV light. Vehicle‐control animals received acetone 30 min before UV exposure (Ace/UV). After 24 h, cyclobutane dimer repair was twofold and 1.5‐fold greater in the UV/VEA and VEA/UV groups, respectively. Expression of p53 protein in the UV/VEA group was maximum at 12 h after UV exposure, whereas in the Ace/UV‐ and VEA/UV‐treated mice, maximum p53 immunostaining was statistically higher at 15 h (P = 0.03). DNA synthesis as determined by 5‐bromo‐2′‐deoxyuridine incorporation was twofold higher after 15 h in all groups but was not statistically different among treatment groups. Protein levels of cyclin D1 and p21 were increased in both VEA groups by 6 h. In addition, VEA treatments delayed tumor formation and yield for the first 20 wk, although this difference was lost by 30 wk. The telomerase activity of carcinomas from the UV/VEA‐treated mice was statistically lower than that of the Ace/UV‐treated mice (P = 0.05). This study showed that although VEA may mitigate some of the initial events associated with UV irradiation such as DNA damage and p53 expression, it has limited potential in preventing UV‐induced proliferation and tumor formation. Mol. Carcinog. 23:175–184, 1998.

An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA

Background: CRISPR/Cas systems allow archaea and bacteria to resist invasion by foreign nucleic acids. Results: The CRISPR/Cas system in Haloferax recognized six different PAM sequences that could trigger a defense response. Conclusion: The PAM sequence specificity of the defense response in type I CRISPR systems is more relaxed than previously thought. Significance: The PAM sequence requirements for interference and adaptation appear to differ markedly. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

Lack of Expression of the EP2 but not EP3 Receptor for Prostaglandin E2 Results in Suppression of Skin Tumor Development

The EP2 receptor for prostaglandin E2 (PGE2) is a membrane receptor that mediates at least part of the action of PGE2. It has been shown that EP2 plays a critical role in tumorigenesis in mouse mammary gland and colon. However, the possibility that the EP2 receptor is involved in the development of skin tumors was unknown. The purpose of this study was to investigate the role of the EP2 receptor in mouse skin carcinogenesis. Unlike EP3 knockout mice, the EP2 knockout mice produced significantly fewer tumors and reduced tumor incidence compared with wild type (WT) mice in a 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage carcinogenesis protocol. EP2 knockout mice had significantly reduced cellular proliferation of mouse skin keratinocytes in vivo and in vitro compared with that in WT mice. In addition, the epidermis of EP2 knockout mice 48 hours after topical TPA treatment was significantly thinner compared with that of WT mice. The inflammatory response to TPA was reduced in EP2 knockout mice, based on a reduced number of macrophages in the dermis and a reduced level of interleukin-1alpha mRNA expression, compared with WT mice. EP2 knockout mice also had significantly reduced epidermal cyclic AMP levels after PGE2 treatment compared with WT mice. Tumors from WT mice produced more blood vessels and fewer apoptotic cells than those of EP2 knockout mice as determined by immunohistochemical staining. Our data suggest that the EP2 receptor plays a significant role in the protumorigenic action of PGE2 in skin tumor development.

Cyclooxygenase-2 expression is critical for chronic UV-induced murine skin carcinogenesis.

While it has been established that both the constitutive and inducible forms of cyclooxygenase (COX‐1 and COX‐2, respectively) play important roles in chemical initiation‐promotion protocols with phorbol ester tumor promoters, the contribution of these two enzymes to ultraviolet (UV) light‐induced skin tumors has not been fully assessed. To better understand the contribution of COX‐1 and COX‐2 to UV carcinogenesis, we transferred the null allele for each isoform onto the SKH‐1 hairless strain of mouse. Due to low viability on this background with complete knockout of COX‐2, heterozygous mice were used in UV carcinogenesis experiments. While the lack of one allele of COX‐1 had no effect on tumor outcome, the lack of one allele of COX‐2 resulted in a 50–65% reduction in tumor multiplicity and a marked decrease in tumor size. Additionally, transgenic SKH‐1 mice that overexpress COX‐2 under the control of a keratin 14 promoter developed 70% more tumors than wild‐type SKH‐1 mice. The lack of one allele of either COX‐1 or COX‐2 reduced prostaglandin (PG) E2 levels in response to a single UV treatment. The proliferative response to UV was significantly reduced in COX‐2, but not COX‐1, heterozygous mice. UV‐induced apoptosis, however, was greater in COX‐2 heterozygous mice. Collectively, these results clearly establish the requirement for COX‐2 in the development of skin tumors.