Systematic evaluation of RNA-Seq preparation protocol performance.

BACKGROUND: RNA-Seq is currently the most widely used tool to analyze whole-transcriptome profiles. There are numerous commercial kits available to facilitate preparing RNA-Seq libraries; however, it is still not clear how some of these kits perform in terms of: 1) ribosomal RNA removal; 2) read coverage or recovery of exonic vs. intronic sequences; 3) identification of differentially expressed genes (DEGs); and 4) detection of long non-coding RNA (lncRNA). In RNA-Seq analysis, understanding the strengths and limitations of commonly used RNA-Seq library preparation protocols is important, as this technology remains costly and time-consuming. RESULTS: In this study, we present a comprehensive evaluation of four RNA-Seq kits. We used three standard input protocols: Illumina TruSeq Stranded Total RNA and mRNA kits, a modified NuGEN Ovation v2 kit, and the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5' and 3' end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes. CONCLUSIONS: At the manufacturers' recommended input RNA levels, all the RNA-Seq library preparation protocols evaluated were suitable for distinguishing between experimental groups, and the TruSeq Stranded mRNA kit was universally applicable to studies focusing on protein-coding gene profiles. The TruSeq protocols tended to capture genes with higher expression and GC content, whereas the modified NuGEN protocol tended to capture longer genes. The SMARTer Ultra Low RNA Kit may be a good choice at the low RNA input level, although it was inferior to the TruSeq mRNA kit at standard input level in terms of rRNA removal, exonic mapping rates and recovered DEGs. Therefore, the choice of RNA-Seq library preparation kit can profoundly affect data outcomes. Consequently, it is a pivotal parameter to consider when designing an RNA-Seq experiment.

Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer.

The Slug transcription factor plays an important role in ultraviolet radiation (UVR)-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT) occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2) pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis.

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.

The tumor promoting activity of the EP4 receptor for prostaglandin E2 in murine skin.

To determine whether the EP4 receptor for prostaglandin E2 (PGE2) contributes to the tumor promoting activity of PGs in murine skin, EP4 over-expressing mice (BK5.EP4) were generated and subjected carcinogenesis protocols. An initiation/promotion protocol resulted in 25-fold more squamous cell carcinomas (SCCs) in the BK5.EP4 mice than wild type (WT) mice. An increase in SCCs also occurred following treatment with initiator alone or UV irradiation. The initiator dimethylbenz[a]anthracene caused cytotoxicity in BK5.EP4, but not WT mice, characterized by sloughing of the interfollicular epidermis, regeneration and subsequent SCC development. A comparison of transcriptomes between BK5.EP4 and WT mice treated with PGE2 showed a significant upregulation of a number of genes known to be associated with tumor development, supporting a pro-tumorigenic role for the EP4 receptor.

In vivo functional studies of tumor-specific retrogene NanogP8 in transgenic animals.

The current study was undertaken to investigate potential oncogenic functions of NanogP8, a tumor-specific retrogene homolog of Nanog (expressed in pluripotent cells), in transgenic animal models. To this end, human primary prostate tumor-derived NanogP8 was targeted to the cytokeratin 14 (K14) cellular compartment, and two lines of K14-NanogP8 mice were derived. The line 1 animals, expressing high levels of NanogP8, experienced perinatal lethality and developmental abnormalities in multiple organs, including the skin, tongue, eye, and thymus in surviving animals. On postnatal day 5 transgenic skin, for example, there was increased c-Myc expression and Ki-67(+) cells accompanied by profound abnormalities in skin development such as thickened interfollicular epidermis and dermis and lack of hypodermis and sebaceous glands. The line 3 mice, expressing low levels of NanogP8, were grossly normal except cataract development by 4-6 mo of age. Surprisingly, both lines of mice do not develop spontaneous tumors related to transgene expression. Even more unexpectedly, high levels of NanogP8 expression in L1 mice actually inhibited tumor development in a two-stage chemical carcinogenesis model. Mechanistic studies revealed that constitutive NanogP8 overexpression in adult L1 mice reduced CD34(+)α6(+) and Lrig-1(+) bulge stem cells, impaired keratinocyte migration, and repressed the expression of many stem cell-associated genes, including Bmp5, Fgfr2, Jmjd1a, and Jun. Our study, for the first time, indicates that transgenically expressed human NanogP8 is biologically functional, but suggests that high levels of NanogP8 may disrupt normal developmental programs and inhibit tumor development by depleting stem cells.

The chemopreventive efficacies of nonsteroidal anti-inflammatory drugs: the relationship of short-term biomarkers to long-term skin tumor outcome.

The ultraviolet B (UVB) component of sunlight, which causes DNA damage and inflammation, is the major cause of nonmelanoma skin cancer (NMSC), the most prevalent of all cancers. Nonsteroidal anti-inflammatory drugs (NSAID) and coxibs have been shown to be effective chemoprevention agents in multiple preclinical trials, including NMSC, colon, and urinary bladder cancer. NSAIDs, however, cause gastrointestinal irritation, which led to the recent development of nitric oxide (NO) derivatives that may partially ameliorate this toxicity. This study compared the efficacy of several NSAIDs and NO-NSAIDs on UV-induced NMSC in SKH-1 hairless mice and determined whether various short-term biomarkers were predictive of long-term tumor outcome with these agents. Naproxen at 100 (P = 0.05) and 400 ppm (P < 0.01) in the diet reduced tumor multiplicity by 26% and 63%, respectively. The NO-naproxen at slightly lower molar doses shows similar activities. Aspirin at 60 or 750 ppm in the diet reduced tumor multiplicity by 19% and 50%, whereas the equivalent doses (108 and 1,350 ppm) were slightly less effective. Sulindac at 25 and 150 ppm in the diet, doses far below the human equivalent dose was the most potent NSAID with reductions of 50% and 94%, respectively. In testing short-term biomarkers, we found that agents that reduce UV-induced prostaglandin E2 synthesis and/or inhibit UV-induced keratinocyte proliferation yielded long-term tumor efficacy.

Transgenic insulin-like growth factor-1 stimulates activation of COX-2 signaling in mammary glands.

Studies show that elevated insulin-like growth factor-1 (IGF-1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the pro-tumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA-induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE(2) levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE(2) showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE(2) /EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE(2) levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE(2) /EP3 signaling pathway is involved in IGF-1-stimulated mammary tumorigenesis and that COX-2-selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans. © 2011 Wiley Periodicals, Inc.

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.

Hydroxyethyl starch (130 kD) inhibits Toll-like receptor 4 signaling pathways in rat lungs challenged with lipopolysaccharide.

BACKGROUND: A number of studies have shown that hydroxyethyl starch (HES) solutions are able to down-regulate the expression of inflammatory mediators and inhibit neutrophil-mediated tissue injuries when they are used in patients with sepsis or other diseases with severe inflammatory responses. However, our knowledge about the underlying mechanisms is limited. Toll-like receptor 4 (TLR4) signaling has a pivotal role in inflammatory processes. In this study, we examined the possible involvement of TLR4 signaling in the antiinflammatory effects of HES. METHODS: Male Sprague-Dawley rats were exposed to lipopolysaccharide (LPS) (10 mg/kg, IV) and received IV saline (30 mL/kg) or HES 130/0.4 (15 or 30 mL/kg). Six hours after LPS challenge, rats were killed and their lungs harvested. Lung injury was examined by hematoxylin and eosin staining. TLR4 mRNA expression, p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1/2 MAPK activation, and activator protein 1 (AP-1) activity in the lungs were detected with quantitative polymerase chain reaction, Western blotting, and electrophoretic mobility shift assay, respectively. RESULTS: Compared with saline, HES profoundly attenuated the histological changes induced by LPS in the lungs at both dose levels. Molecular analysis showed that both 15 and 30 mL/kg HES significantly decreased TLR4 mRNA levels and inhibited activation of p38 MAPK and AP-1 in rats challenged with LPS, whereas activation of extracellular signal-regulated kinases 1/2 MAPK was not affected by either dose of HES. CONCLUSIONS: These findings indicate that the beneficial effects of HES 130/0.4 on inflammation are mediated at least in part by inhibiting the TLR4/p38 MAPK/AP-1 pathway in lungs from rats challenged with LPS.

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.

Molecular mechanisms of mouse skin tumor promotion.

Multiple molecular mechanisms are involved in the promotion of skin carcinogenesis. Induction of sustained proliferation and epidermal hyperplasia by direct activation of mitotic signaling pathways or indirectly in response to chronic wounding and/or inflammation, or due to a block in terminal differentiation or resistance to apoptosis is necessary to allow clonal expansion of initiated cells with DNA mutations to form skin tumors. The mitotic pathways include activation of epidermal growth factor receptor and Ras/Raf/mitogen-activated protein kinase signaling. Chronic inflammation results in inflammatory cell secretion of growth factors and cytokines such as tumor necrosis factor-a and interleukins, as well as production of reactive oxygen species, all of which can stimulate proliferation. Persistent activation of these pathways leads to tumor promotion.

Transcriptional regulation of estrogen receptor-alpha by p53 in human breast cancer cells.

Estrogen receptor alpha (ER) and p53 are critical prognostic indicators in breast cancer. Loss of functional p53 is correlated with poor prognosis, ER negativity, and resistance to antiestrogen treatment. Previously, we found that p53 genotype was correlated with ER expression and response to tamoxifen in mammary tumors arising in mouse mammary tumor virus-Wnt-1 transgenic mice. These results lead us to hypothesize that p53 may regulate ER expression. To test this, MCF-7 cells were treated with doxorubicin or ionizing radiation, both of which stimulated a 5-fold increase in p53 expression. ER expression was also increased 4-fold over a 24-h time frame. In cells treated with small interfering RNA (siRNA) targeting p53, expression of both p53 and ER was significantly reduced (>60%) by 24 h. Induction of ER by DNA-damaging agents was p53 dependent as either ionizing radiation or doxorubicin failed to up-regulate ER after treatment with p53-targeting siRNA. To further investigate whether p53 directly regulates transcription of the ER gene promoter, MCF-7 cells were transiently transfected with a wild-type (WT) p53 expression vector along with a luciferase reporter containing the proximal promoter of ER. In cells transfected with WT p53, transcription from the ER promoter was increased 8-fold. Chromatin immunoprecipitation assays showed that p53 was recruited to the ER promoter along with CARM1, CBP, c-Jun, and Sp1 and that this multifactor complex was formed in a p53-dependent manner. These data show that p53 regulates ER expression through transcriptional control of the ER promoter, accounting for their concordant expression in human breast cancer.

Paracrine overexpression of insulin-like growth factor-1 enhances mammary tumorigenesis in vivo.

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

Multiple signaling pathways are responsible for prostaglandin E2-induced murine keratinocyte proliferation.

Although prostaglandin E2 (PGE2) has been shown by pharmacologic and genetic studies to be important in skin cancer, the molecular mechanism(s) by which it contributes to tumor growth is not well understood. In this study, we investigated the mechanisms by which PGE2 stimulates murine keratinocyte proliferation using in vitro and in vivo models. In primary mouse keratinocyte cultures, PGE2 activated the epidermal growth factor receptor (EGFR) and its downstream signaling pathways as well as increased cyclic AMP (cAMP) production and activated the cAMP response element binding protein (CREB). EGFR activation was not significantly inhibited by pretreatment with a c-src inhibitor (PP2), nor by a protein kinase A inhibitor (H-89). However, PGE2-stimulated extracellularly regulated kinase 1/2 (ERK1/2) activation was completely blocked by EGFR, ERK1/2, and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors. In addition, these inhibitors attenuated the PGE2-induced proliferation, nuclear factor-kappa B, activator protein-1 (AP-1), and CREB binding to the promoter regions of the cyclin D1 and vascular endothelial growth factor (VEGF) genes and expression of cyclin D1 and VEGF in primary mouse keratinocytes. Similarly, in vivo, we found that WT mice treated with PGE2 and untreated cyclooxygenase-2-overexpressing transgenic mice had higher levels of cell proliferation and expression of cyclin D1 and VEGF, as well as higher levels of activated EGFR, nuclear factor-kappa B, AP-1, and CREB, than vehicle-treated WT mice. Our findings provide evidence for a link between cyclooxygenase-2 overexpression and EGFR-, ERK-, PI3K-, cAMP-mediated cell proliferation, and the tumor-promoting activity of PGE2 in mouse skin.

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.

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.

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.