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Role of epidermal lipoxygenases in differentiation and carcinogenesis

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From left to right: Silvia deJuanes, Peter Krieg, Mareen Neumann, Timo Kehl, Lena Ehret, Susanne Latzko.

Introduction

Lipoxygenases (LOX) comprise a widespread family of non-heme iron dioxygenases, representing key enzymes in the biosynthesis of a variety of eicosanoids, highly active lipid mediators that play an important role in the regulation of proliferation, differentiation, inflammation, and carcinogenesis (Fig. 1).
We were the first group to characterize a previously undescribed subclass of mammalian LOX isoforms characterized by unique structural and enzymatic properties. They comprise epidermis-type 12-LOX, 15-LOX-2 and its mouse ortholog 8-LOX, 12R-LOX, and eLOX-3. These epidermis-type LOX were shown to be expressed in a differentiation dependent manner in the epidermis and few other epithelial tissues and their expression was shown to be deregulated in the course of epithelial carcinogenesis.
Our current research aims at the functional analysis of these enzymes in differentiation and in cancer development focussing on three topics:

1. Antitumorigenic effects of 15-LOX metabolism in pancreatic cancer evelopment
2. The role of a unique 12R-LOX / eLOX-3 pathway in skin barrier function and in the pathogenesis of inherited skin disorders
3. The role of eLOX-3 in adipocyte differentiation

Our experimental strategy is to establish expression and activity profiles of individual LOX in mouse and human biopsies in order to bridge animal data and clinical investigations. The experimental models used for functional studies are transgenic mouse lines with targeted overexpression or deletion of LOX genes in vivo and inducible LOX expression systems in vitro.




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Fig. 1 LOX as key enzymes in the Eicosanoid biosynthesis.
Eicosanoids, the oxidative metabolites of arachidonic acid are highly potent lipid mediators involved in diverse biological processes including proliferation, differentiation, inflammation and carcinogenesis. Upon internal or external stimuli arachidonic acid (AA) is released by the activity of phospholipases from the membrane bound phospholipids and then metabolized along one of three pathways. The cyclooxygenases (COX) metabolize AA to prostanoids, the P450 monooxygenases (CYP) convert AA to midchain EETs and HETEs, and the lipoxygenase (LOX) catalysed pathway produces HETEs, leukotrienes, hepoxilins and lipoxins.

1. Antitumorigenic effects of 15-LOX metabolism in pancreatic cancer development

Pancreatic cancer is the fourth leading cause of cancer-related death. Because of late diagnosis and lack of therapeutic options it has an abysmal prognosis, with less than 20% of patients surviving one year after diagnosis and less than 5% surviving five years. Obesity and high intake of ω-6 polyunsaturated fatty acids are identified as risk factors. Oxidative lipid metabolism, particularly the eicosanoid metabolism, has been shown to play an important role in pancreatic tumor progression. LOX representing key enzymes in the eicosanoid pathway, therefore, might be valuable as potential targets for early diagnosis, prevention and treatment of pancreatic cancer.
Clinical data on LOX expression in human tumours and in animal models indicate differing roles of distinct LOX in carcinogenesis in a tissue-specific manner: in general, 5- and 12-LOX are thought to exhibit pro-tumorigenic effects, while 15-LOX isoforms rather show anti-tumorigenic activities (Fig. 2).
Our studies on LOX expression in pancreatic carcinogenesis revealed overexpression of 5-LOX but loss of expression of 15-LOX-1 and 15-LOX-2 in pancreatic tumours and preneoplastic lesions. Forced expression of 15-LOX-1 and 15-LOX-2 or treatment with the LOX product 15-HETE strongly decreased cell growth in pancreatic tumour cells. The results suggest that loss of both 15-LOX isoforms may be implicated in pancreatic carcinogenesis and induction of 15-LOX may be a potential therapeutic option.
Ongoing studies are aimed to analyse the signaling pathways involved in the growth inhibitory effects of 15-LOX on pancreatic cell growth and to evaluate 15-LOX metabolism as potential targets for early diagnosis, prevention and treatment of pancreatic cancer.
Functional analyses include pharmacologic and genetic intervention studies in normal pancreatic duct and pancreatic cancer cells by using tetracycline-inducible LOX expression systems in vitro and an orthotopic GFP nude mouse model in vivo.


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Fig. 2. Antagonistic role of LOX in carcinogenesis.
The inverse expression pattern of individual LOX isoenzymes in normal versus malignant tissues and the biological effects of the corresponding LOX products indicate a dynamic balance among LOX shifting toward the pro-carcinogenic 5- and p12-LOX and away from anti-carcinogenic such as 15-LOX-1 and -2 in the course of cancer development.

2. The role of a unique 12R-LOX/eLOX-3 pathway in skin barrier function and in the pathogenesis of inherited skin disorders

The epidermis is a self-renewing stratified epithelium that serves as a protective barrier against mechanical, chemical, and biological insults; it is also a water-impermeable barrier that prevents excessive loss of body fluids. This function is critical for the survival of all terrestrial vertebrates and is established during late embryonic development. Defective barrier function has been shown to be the underlying defect of various inherited skin disorders referred to as ichthyoses and a major contributing factor to common inflammatory skin disorders such as atopic dermatitis and psoriasis. Furthermore, barrier function also plays an important role in bacterial and viral infections including cutaneous HPV transmission suspected to be involved in the development of non melanoma skin cancer.
Recent studies by our group and others have identified 12R-lipoxygenase (12R-LOX) and epidermal LOX-3 (eLOX-3) as key enzymes of a novel lipid signalling pathway that plays a pivotal role in the establishment and maintenance of the epidermal barrier function. This view is supported by genetic studies showing that inactivating mutations in 12R-LOX and eLOX-3 are linked to the development of an inherited skin disorder called autosomal recessive congenital ichthyosis (ARCI) in humans. By using the Cre/LoxP system we have established mouse models with targeted inactivation of either LOX gene. Constitutive 12R-LOX or eLOX-3 gene inactivation leads to a post-natal lethal phenotype which is due to a severely impaired permeability barrier function. The mature 12R-LOX deficient mouse skin, as analysed in skin grafts and in a conditional knockout model with skin–specific gene inactivation, develops an ichthyosiform phenotype that closely resembles the phenotype observed in ichthyosis patients indicating that LOX knockout mice represent a suitable animal model for ARCI.
The main goals of the ongoing project are to use these mouse models and in vitro keratinocyte systems to decipher the mechanisms of action of the LOX pathway related to the diseased phenotype (Fig. 3) and to develop molecular approaches towards a therapy of ichthyosis.


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Fig. 3. Putative role of the 12R-LOX/eLOX-3 pathway in epidermal barrier function.
Specific protein („cornified cell envelope“) and lipid structures („cornified lipid envelope“ and intercellular lipid lamellae) within the stratum corneum are the major structural components for the development and maintenance of the epidermal barrier function. These structures, often referred to as „bricks and mortar”, are formed via a complex series of processing steps during terminal differentiation. The interruption of the 12R-LOX/eLOX-3 pathway in 12R-LOX-deficient mice leads to impaired processing of the CE-Protein filaggrin as well as to a disturbed metabolism of protein bound ceramides. Metabolites of the 12R-LOX/e-LOX-3 pathway possibly act as endogenous ligands of nuclear receptors of the PPAR-family thereby influencing the transcriptional regulation of key enzymes of the protein and lipid metabolism.

3. The role of eLOX-3 in adipogenesis

Adipocytes play a central role in whole body energy homeostasis. Adipocyte differentiation is a complex process that is accompanied by changes in cell morphology, hormone sensitivity and gene expression. These alterations are regulated by a network of several transcription factors that coordinate these processes. The nuclear hormone receptor PPARγ is known to play a central role in this network, the nature of its activating ligand, however, is still unknown.
In cooperation with the group of K. Kristiansen (University of Southern Denmark, Odense, Denmark) a LOX activity has been assigned to the initial stages of adipocyte differentiation that proved to be sensitive to LOX inhibitors. Our data indicate that eLOX-3 is critically involved in the production of the endogenous ligand for PPARγ (Fig. 4).
Ongoing work is focussed on the identification of the LOX-derived PPARγ activator and the characterization of the eLOX-3 metabolic pathway involved in the synthesis of this ligand by using LOX over-expressing and LOX-knocked down adipocytes and conditional knock-out mice for adipocyte-specific ablation of eLOX-3.


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Fig. 4. The role of eLOX-3 in the transcriptional control of adipogenesis. Potential LOX pathways or LOX-derived endogenous ligands critically involved in transactivation of PPARγ are essential in early adipocyte differentiation.