Gerhard Fürstenberger

RAGE signaling sustains inflammation and promotes tumor development

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis

Genetic and pharmacological evidence suggests that overexpression of cyclooxygenase-2 (COX-2) is critical for epithelial carcinogenesis and provides a major target for cancer chemoprevention by nonsteroidal antiinflammatory drugs. Transgenic mouse lines with keratin 5 promoter-driven COX-2 overexpression in basal epidermal cells exhibit a preneoplastic skin phenotype. As shown here, this phenotype depends on the level of COX-2 expression and COX-2-mediated prostaglandin accumulation. The transgenics did not develop skin tumors spontaneously but did so after a single application of an initiating dose of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Long-term treatment with the tumor promoter phorbol 12-myristate 13-acetate, as required for tumorigenesis in wild-type mice, was not necessary for transgenics. The ratios of squamous cell carcinomas to papillomas and of sebaceous gland adenomas to papillomas plus squamous cell carcinomas were increased markedly in transgenic mice treated with DMBA alone compared with DMBA/phorbol 12-myristate 13-acetate-treated transgenic and wild-type mice. Thus, COX-2 overexpression, which leads to high levels of epidermal prostaglandin E2, prostaglandin F2α, and 15-deoxyΔ12,14-PGJ2, is insufficient for tumor induction but transforms epidermis into an “autopromoted” state, i.e., dramatically sensitizes the tissue for genotoxic carcinogens.

Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin

In prostanoid biosynthesis, the first two steps are catalyzed by cyclooxygenases (COX). In mice and humans, deregulated expression of COX-2, but not of COX-1, is characteristic of epithelial tumors, including squamous cell carcinomas of skin. To explore the function of COX-2 in epidermis, a keratin 5 promoter was used to direct COX-2 expression to the basal cells of interfollicular epidermis and the pilosebaceous appendage of transgenic mouse skin. COX-2 overexpression in the expected locations, resulting in increased prostaglandin levels in epidermis and plasma, correlated with a pronounced skin phenotype. Heterozygous transgenic mice exhibited a reduced hair follicle density. Moreover, postnatally hair follicle morphogenesis and thinning of interfollicular dorsal epidermis were delayed. Adult transgenics showed a body-site-dependent sparse coat of greasy hair, the latter caused by sebaceous gland hyperplasia and increased epicutaneous sebum levels. In tail skin, hyperplasia of scale epidermis reflecting an increased number of viable and cornified cell layers was observed. Hyperplasia was a result of a disturbed program of epidermal differentiation rather than an increased proliferation rate, as reflected by the strong suppression of keratin 10, involucrin, and loricrin expression in suprabasal cells. Further pathological signs were loss of cell polarity, mainly of basal keratinocytes, epidermal invaginations into the dermis, and formation of horn perls. Invaginating hyperplastic lobes were surrounded by CD31-positive vessels. These results demonstrate a causal relationship between transgenic COX-2 expression in basal keratinocytes and epidermal hyperplasia as well as dysplastic features at discrete body sites.

Sequential expression of mRNA-encoded keratin sets in neonatal mouse epidermis: Basal cells with properties of terminally differentiating cells

The keratin pattern of newborn mouse epidermis was investigated during terminal differentiation. In highly pure fractions of basal and suprabasal cells, obtained by Percoll density gradient centrifugation, we identified two sets of mRNA-encoded proteins: a basal set of 58.5, 52, and 47 kd subunits and a suprabasal set of 67 and 60 kd subunits. The large subunits of each set were alkaline to neutral, while the small subunits were acidic. Polyclonal antibodies against the suprabasal, acidic 60 kd protein and the basal, alkaline 58.5 kd protein selectively recognized their antigens in immunoblots of NEPHGE -resolved keratins and decorated the corresponding epidermal compartments in frozen sections. The antibody to the suprabasal 60 kd protein also recognized distinct cells in the basal cell layer. Quantification of this cell population revealed a 10% cell fraction, morphologically indistinguishable from the total cell population, that, in addition to expressing basal keratin proteins, was already synthesizing suprabasal keratin subunits.

12R-lipoxygenase deficiency disrupts epidermal barrier function

12R-lipoxygenase (12R-LOX) and the epidermal LOX-3 (eLOX-3) constitute a novel LOX pathway involved in terminal differentiation in skin. This view is supported by recent studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of autosomal recessive congenital ichthyosis. We show that 12R-LOX deficiency in mice results in a severe impairment of skin barrier function. Loss of barrier function occurs without alterations in proliferation and stratified organization of the keratinocytes, but is associated with ultrastructural anomalies in the upper granular layer, suggesting perturbance of the assembly/extrusion of lamellar bodies. Cornified envelopes from skin of 12R-LOX–deficient mice show increased fragility. Lipid analysis demonstrates a disordered composition of ceramides, in particular a decrease of ester-bound ceramide species. Moreover, processing of profilaggrin to monomeric filaggrin is impaired. This study indicates that the 12R-LOX–eLOX-3 pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing.

Prostaglandin-H-synthase isozyme expression in normal and neoplastic human skin

Expression of prostaglandin‐H‐synthase (PGHS) isozymes was analyzed in 50 biopsies of normal human skin and of pre‐malignant and malignant skin lesions, by means of quantitative RT‐PCR, immunoprecipitation and Western blotting, as well as immunohistochemistry. Normal skin constitutively expressed PGHS‐1 in all cell layers of the epidermis, in endothelial cells of small blood vessels and in sweat‐gland epithelium. PGHS‐2 expression was very low and restricted to a few keratinocytes of the interfollicular and follicular epidermis. Steady‐state concentrations of PGHS‐1 and PGHS‐2 mRNA were similar in normal skin and in basal‐cell carcinomas, but PGHS‐1 mRNA was reduced and PGHS‐2 mRNA was elevated in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas. PGHS‐1 protein was detected in all tumor biopsies, being occasionally increased in basal‐cell carcinomas. High amounts of PGHS‐2 protein were found in actinic keratoses, squamous‐cell carcinomas and keratoacanthomas, but not in basal‐cell carcinomas. Four malignant melanomas included in this study contained PGHS‐1 but no PGHS‐2 protein. Immunohistochemical analysis of the biopsies identified keratinocytes, in addition to cells of inflammatory infiltrates and of dendritic morphology, as the major PGHS‐expressing cell types. PGHS‐2‐specific signals were spread throughout the epidermal part of actinic keratoses and squamous‐cell carcinomas. These data suggest that constitutive up‐regulation of PGHS‐2 expression is a consistent pre‐malignant event in squamous‐cell cancer development in man, as it is in animal models of skin carcinogenesis. Thus, pre‐cancerous lesions such as actinic keratoses present a likely target for chemoprevention of skin cancer by selective PGHS‐2 inhibitors. Int. J. Cancer 82:648–656, 1999.

Localization of prostaglandin H synthase isoenzymes in murine epidermal tumors: Suppression of skin tumor promotion by inhibition of prostaglandin H synthase-2

The growth factor– and phorbol ester–inducible prostaglandin H synthase (PGHS)‐2 has been found to be constitutively overexpressed in epidermal tumors generated by the initiation‐promotion protocol in murine skin, whereas the expression of PGHS‐1 does not change under these conditions. In this paper we report the intra‐tumor distribution of the aberrantly expressed PGHS‐2 and the cancer chemopreventive activity of a specific PGHS‐2 inhibitor. By immunohistochemical methods using isoenzyme‐specific antibodies, we found that the PGHS‐1 protein was expressed in keratinocytes and Langerhans cells dispersed throughout the epithelial part of papillomas and squamous cell carcinomas and in inflammatory infiltrates occasionally seen in these tumors. A uniform pattern of PGHS‐2 expression was observed in the basal keratinocytes of papillomas and in the follicular keratinocytes of carcinomas. In addition, Langerhans cells as well as tumor‐associated inflammatory infiltrates exhibited PGHS‐2–specific immunoreactivity. PGHS‐2–catalyzed prostaglandin synthesis stimulated by the phorbol ester 12‐O‐tetradecanoylphorbol‐13 acetate (TPA) in mouse epidermis in vivo was dose‐dependently suppressed by topical administration of SC‐58125, a specific PGHS‐2 inhibitor. TPA‐induced edema formation, epidermal DNA synthesis, and mitotic activity were not impaired by SC‐58125 applied at a dose that inhibited TPA‐induced prostaglandin E2 synthesis. However, the repetitive epicutaneous administration of SC‐58125 substantially and significantly suppressed papilloma development. Malignant progression of papillomas was slightly retarded by the drug. These results indicate that aberrant expression of PGHS‐2 in epidermal tumors may be a relevant target for prevention of epidermal cancer development in experimental animals and that the PGHS‐2–specific inhibitor SC‐58125, which is a potent inhibitor of tumor promotion in mouse skin, may be important for cancer chemoprevention in humans as well. Mol. Carcinog. 23:36–44, 1998.

The cyclooxygenase-2-mediated prostaglandin signaling is causally related to epithelial carcinogenesis.

Epidemiologic, pharmacologic, clinical, and experimental studies document the importance of prostaglandin (PG) signaling in cancer development, including non‐melanoma skin cancer lesions in humans and mice. First of all, enzymes involved in PG biosynthesis, such as cyclooxygenase (COX)‐2 and/or membrane prostaglandin E synthase (mPGES)‐1, were found to be overexpressed in a wide range of premalignant and malignant epithelial tumors, including those of the skin, breast, esophagus, stomach, colorectum, pancreas, and bladder. On the other hand, 15‐hydroxy‐prostaglandin dehydrogenase (15‐PGDH), which is involved in the degradation pathway of PG including PGE2, thus counteracting the activities of COX‐2 and PGES, was found to be downregulated in human epithelial tumors, indicating a tumor suppressor activity of this enzyme. Most remarkably, genetic studies showed that mice, which are deficient in COX‐2 and/or PGES are resistant to the development of cancer of skin, colon, and stomach. In contrast, the forced overexpression of COX‐2 in proliferative compartments of simple or stratified epithelia such as skin epidermis, urinary bladder, mammary gland, and pancreas results in spontaneous hyperplasia and dysplasia in transgenic mice. In skin, the pathological changes are found to be due to an abnormal process of terminal differentiation, while in other tissues, hyperproliferation seems to be the main contributer to the pre‐invasive neoplasms. Moreover, the COX‐2 transgenic mouse lines are sensitized for cancer development.

Indomethacin inhibition of cell proliferation induced by the phorbolester TPA is reversed by prostaglandin E2 in mouse epidermis in vivo

Abstract The proliferative response of mouse epidermis to the phorbolester TPA (10 nmoles) in vivo is completely inhibited by a single topical application of indomethacin one hour before TPA. DNA labeling in normal mouse epidermis is not significantly depressed by the drug. The inhibition can be reversed by applying prostaglandin E 2 (>3 nmoles) simultaneously with TPA, whereas prostaglandin F 2α (100 nmoles) is ineffective. The indomethacin-sensitive event is restricted to the first hour after phorbol ester treatment. TPA-induced skin inflammation is not influenced by the drug. It is proposed that prostaglandin E 2 , or a closely related compound, mediates the mitogenic effect of TPA in mouse skin.

Calcium and phospholipid-dependent protein kinase activity in mouse epidermis cytosol. Stimulation by complete and incomplete tumor promoters and inhibition by various compounds.

Calcium- and phospholipid-dependent protein kinase (Ca, PL-PK) activity is detectable in mouse epidermis cytosol. It can be stimulated in vitro by complete and incomplete tumor promoters (12-0-tetradecanoylphorbol-13-acetate (TPA) and 12-0-retinoylphorbol-13-acetate (RPA], respectively. Effective inhibition of the enzyme activity is achieved with quercetin and phloretin, whereas the lipoxygenase and cyclooxygenase inhibitors nordihydroguaiaretic acid (NDGA) and esculetin show just weak or no inhibition. Quercetin inhibits the lipoxygenase and cyclooxygenase equally well as the Ca, PL-PK, whereas the strong Ca, PL-PK inhibitor phloretin is absolutely ineffective in inhibiting the lipoxygenase/cyclooxygenase. The application of these inhibitors in differentiating tumor promoter induced effects in vivo is proposed.