The EP1 receptor for prostaglandin E2 promotes the development and progression of malignant murine skin tumors.

High levels of prostaglandin E2 (PGE2) synthesis resulting from the up-regulation of cyclooxygenase (COX)-2 has been shown to be critical for the development of non-melanoma skin tumors. This effect of PGE2 is likely mediated by one or more of its 4 G-protein coupled membrane receptors, EP1-4. A previous study showed that BK5.EP1 transgenic mice produced more carcinomas than wild type (WT) mice using initiation/promotion protocols, although the tumor response was dependent on the type of tumor promoter used. In this study, a single topical application of either 7,12-dimethylbenz[a]anthracene (DMBA) or benzo[a]pyrene (B[a]P), alone, was found to elicit squamous cell carcinomas (SCCs) in the BK5.EP1 transgenic mice, but not in WT mice. While the epidermis of both WT and transgenic mice was hyperplastic several days after DMBA, this effect regressed in the WT mice while proliferation continued in the transgenic mice. Several parameters associated with carcinogen initiation were measured and were found to be similar between genotypes, including CYP1B1 and aromatase expression, B[a]P adduct formation, Ras activity, and keratinocyte stem cell numbers. However, EP1 transgene expression elevated COX-2 levels in the epidermis and SCC could be completely prevented in DMBA-treated BK5.EP1 mice either by feeding the selective COX-2 inhibitor celecoxib in their diet or by crossing them onto a COX-2 null background. These data suggest that the tumor promoting/progressing effects of EP1 require the PGE2 synthesized by COX-2.

Genetic reduction of insulin-like growth factor-1 mimics the anticancer effects of calorie restriction on cyclooxygenase-2-driven pancreatic neoplasia.

Risk of pancreatic cancer, the fourth deadliest cancer in the United States, is increased by obesity. Calorie restriction (CR) prevents obesity, suppresses carcinogenesis in many models, and reduces serum levels of IGF-1. In the present study, we examined the impact of CR on a model of inflammation-associated pancreatitis and pancreatic dysplasia, with a focus on the mechanistic contribution of systemic IGF-1. Administration of a 30% CR diet for 14 weeks decreased serum IGF-1 levels and hindered pancreatic ductal lesion formation and dysplastic severity, relative to a higher calorie control diet, in transgenic mice overexpressing COX-2 [bovine keratin-5 promoter (BK5.COX-2)]. These findings in CR mice correlated with reductions in Ki-67-positive cells, vascular luminal size, VEGF expression, and phosphorylation and total expression of downstream mediators of the IGF-1 pathway. Cell lines derived from BK5.COX-2 ductal lesions (JC101 cells) formed pancreatic tumors in wild-type FVB mice that were significantly reduced in size by a 14-week CR regimen, relative to the control diet. To further understand the impact of circulating levels of IGF-1 on tumor growth in this model, we orthotopically injected JC101 cells into liver-specific IGF-1-deficient (LID) mice. The approximate 65% reduction of serum IGF-1 levels in LID mice resulted in significantly decreased burden of JC101 tumors, despite modestly elevated levels of circulating insulin and leptin. These data show that CR prevents development of dysplasia and growth of pancreatic cancer through alterations in IGF-1, suggesting that modulation of this pathway with dietary and/or pharmacologic interventions is a promising pancreatic cancer prevention strategy.

Upregulation of the EP1 receptor for prostaglandin E2 promotes skin tumor progression.

Prostaglandin E(2) (PGE(2) ) has been shown to promote the development of murine skin tumors. EP1 is 1 of the 4 PGE(2) G-protein-coupled membrane receptors expressed by murine keratinocytes. EP1 mRNA levels were increased ∼2-fold after topical treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or exposure to ultraviolet (UV) light, as well as increased ∼3- to 12-fold in tumors induced by 7,12-dimethyl-benz[a]anthracene (DMBA) initiation/TPA promotion or by UV exposure. To determine the effect of EP1 levels on tumor development, we generated BK5.EP1 transgenic mice that overexpress EP1 in the basal layer of the epidermis. Skins of these mice were histologically indistinguishable from wild type (WT) mice and had similar levels of proliferation after TPA treatment. Using a DMBA/TPA carcinogenesis protocol, BK5.EP1 mice had a reduced tumor multiplicity compared to WT mice, likely due to the observed down-regulation of protein kinase C (PKC). However, the BK5.EP1 mice had an ∼8-fold higher papilloma to carcinoma conversion rate. When DMBA/anthralin was used, BK5.EP1 mice produced more tumors than WT mice, as well as a ninefold increase in carcinomas, indicating that the tumor response is dependent on the type of tumor promoter agent used. Additionally, although almost undetectable in WT mice, cyclooxygenase-2 (COX-2) was expressed in the untreated epidermis of BK5.EP1 mice. While TPA highly induced COX-2 in WT mice, COX-2 expression in the BK5.EP1 mice did not change after TPA treatment; PGE(2) levels were likewise affected. These data indicate that EP1 is more important in tumor progression than in tumor promotion and that it indirectly regulates COX-2 expression.

Progressive metaplastic and dysplastic changes in mouse pancreas induced by cyclooxygenase-2 overexpression.

Cyclooxygenase-2 (COX-2) overexpression is an established factor linking chronic inflammation with metaplastic and neoplastic change in various tissues. We generated transgenic mice (BK5.COX-2) in which elevation of COX-2 and its effectors trigger a metaplasia-dysplasia sequence in exocrine pancreas. Histologic evaluation revealed a chronic pancreatitis-like state characterized by acinar-to-ductal metaplasia and a well-vascularized fibroinflammatory stroma that develops by 3 months. By 6 to 8 months, strongly dysplastic features suggestive of pancreatic ductal adenocarcinoma emerge in the metaplastic ducts. Increased proliferation, cellular atypia, and loss of normal cell/tissue organization are typical features in transgenic pancreata. Alterations in biomarkers associated with human inflammatory and neoplastic pancreatic disease were detected using immunohistochemistry. The abnormal pancreatic phenotype can be completely prevented by maintaining mice on a diet containing celecoxib, a well-characterized COX-2 inhibitor. Despite the high degree of atypia, only limited evidence of invasion to adjacent tissues was observed, with no evidence of distant metastases. However, cell lines derived from spontaneous lesions are aggressively tumorigenic when injected into syngeneic or nude mice. The progressive nature of the metaplastic/dysplastic changes observed in this model make it a valuable tool for examining the transition from chronic inflammation to neoplasia.

The effect of cyclooxygenase-2 overexpression on skin carcinogenesis is context dependent.

The up-regulation of the inducible form of cyclooxygenase (COX-2), a central enzyme in the prostaglandin (PG) biosynthetic pathway, occurs in many epithelial tumors and has been associated with tumor cell proliferation and angiogenesis. To better understand the role of COX-2 in skin tumor development, we generated transgenic mice that overexpress COX-2 under the control of the keratin 14 promoter. We previously reported (Cancer Res. 62: 2516, 2002) that these mice, referred to as keratin 14 (K14).COX2 mice, were unexpectedly very resistant to 12-O-tetradecanoylphorbol 13-acetate (TPA) tumor promotion. The current studies were undertaken to determine the mechanism of this resistance and determine if it was restricted to TPA promotion. Transgenic and wild-type mice were subjected to a complete carcinogenesis protocol using 7,12-dimethylbenz[a]anthracene (DMBA) only, as well as a two-stage protocol using DMBA plus an unrelated tumor promoter, anthralin. In addition, the responses of transgenic and wild-type mice to TPA in terms of induction of proliferation and various down-stream mediators were examined. The TPA resistance phenotype correlated with a reduced ability to induce ornithine decarboxylase, interleukin-1alpha, and tumor necrosis factor-alpha and a reduced proliferation response. This resistance phenotype appears to be restricted to phorbol ester promotion because K14.COX2 mice developed six times more tumors than wild-type mice when anthralin was used as the tumor promoter. Additionally, K14.COX2 mice treated only with DMBA developed approximately 3.5 times more tumors than wild-type mice, suggesting that PGs have intrinsic tumor promoting activity. We conclude that the role of PGs in skin tumorigenesis is context dependent.

A role for cyclooxygenase-2 in ultraviolet light-induced skin carcinogenesis.

Nonmelanoma skin cancer is the most prevalent cancer in the United States and its incidence is on the rise. These cancers generally arise on sun-exposed areas of the body and the ultraviolet (UV) B spectrum of sunlight has been clearly identified as the major carcinogen responsible for skin cancer development. Besides inducing DNA damage directly, UV exposure of the skin induces the expression of the enzyme cyclooxygenase-2 (COX-2), which catalyzes the first step in the conversion of arachidonic acid to prostaglandins, the primary product in skin being prostaglandin E(2) (PGE(2)). COX-2 has been shown to be overexpressed in premalignant lesions as well as in nonmelanoma skin cancers in both humans and mice chronically exposed to UV. Through the use of COX-2-selective inhibitors and COX-2 knockout mice, it has been shown that UV-induced COX-2 expression plays a major role in UV-induced PGE(2) production, inflammation, edema, keratinocyte proliferation, epidermal hyperplasia, and generation of a pro-oxidant state leading to oxidative DNA damage. Chronic exposure to UV leads to chronic up-regulation of COX-2 expression and chronic inflammation along with the accumulation of DNA damage and mutations, all of which combine to induce malignant changes in epidermal keratinocytes and skin cancers. Both inhibition of COX-2 activity and reduction in COX-2 expression by genetic manipulations significantly reduce, while overexpression of COX-2 in transgenic mice significantly increases UV-induced skin carcinogenesis. Together these studies demonstrate that COX-2 expression/activity is critical to the development of UV-related nonmelanoma skin cancers.

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry.

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.

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.

SAGE profiling of UV-induced mouse skin squamous cell carcinomas, comparison with acute UV irradiation effects.

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

Regulation of transcription of the intracellular interleukin-1 receptor antagonist gene by AP-1 in mouse carcinoma cells.

Interleukin-1 receptor antagonist (IL-1Ra) is involved in many processes, including epidermal inflammation and hyperplasia after irritation or injury. However, the mechanism by which intracellular IL-1Ra (icIL-1Ra) expression is regulated in mouse keratinocytes has not been reported. We found that the CH72 mouse carcinoma cell line constitutively expresses the icIL-1Ra mRNA. To study the transcriptional factors responsible for the constitutive expression of icIL-1Ra, we functionally characterized 4.5 kb of the 5' flanking region of the human icIL-1Ra gene in these cells. We first demonstrated that icIL-1Ra expression in these cells was regulated at the level of transcription. Deletion analysis of the promoter showed that regulatory elements for constitutive expression were located -158 to -49 bp upstream of the transcription start site for icIL-1Ra. We investigated the cis- and trans-acting factors required for icIL-1Ra expression. An activating protein-1 (AP-1) site was identified as the positive regulatory element necessary for the constitutive expression of the icIL-1Ra promoter in CH72 cells. Moreover, electrophoretic mobility shift assay and cotransfection experiments showed that c-jun and c-fos proteins bound to the AP-1 site and functionally transactivated the icIL-1Ra promoter in mouse carcinoma CH72 cells.