Epigenetic programing of cancer stemness by transcription factors-non-coding RNAs interactions.

Cancer 'stemness' is fundamental to cancer existence. It defines the ability of cancer cells to indefinitely perpetuate as well as differentiate. Cancer stem cell populations within a growing tumor also help evade the inhibitory effects of chemo- as well as radiation-therapies, in addition to playing an important role in cancer metastases. NF-κB and STAT-3 are representative transcription factors (TFs) that have long been associated with cancer stemness, thus presenting as attractive targets for cancer therapy. The growing interest in non-coding RNAs (ncRNAs) in the recent years has provided further insight into the mechanisms by which TFs influence cancer stem cell characteristics. There is evidence for a direct regulation of TFs by ncRNAs, such as, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) as well as circular RNAs (circRNAs), and vice versa. Additionally, the TF-ncRNAs regulations are often indirect, involving ncRNA-target genes or the sponging of other ncRNA species by individual ncRNAs. The information is rapidly evolving and this review provides a comprehensive review of TF-ncRNAs interactions with implications on cancer stemness and in response to therapies. Such knowledge will help uncover the many levels of tight regulations that control cancer stemness, providing novel opportunities and targets for therapy in the process.

Epigenetic regulation of CXCR4 signaling in cancer pathogenesis and progression.

Signaling involving chemokine receptor CXCR4 and its ligand SDF-1/CXL12 has been investigated for many years for its possible role in cancer progression and pathogenesis. Evidence emerging from clinical studies in recent years has further established diagnostic as well as prognostic importance of CXCR4 signaling. CXCR4 and SDF-1 are routinely reported to be elevated in tumors, distant metastases, which correlates with poor survival of patients. These findings have kindled interest in the mechanisms that regulate CXCR4/SDF-1 expression. Of note, there is a particular interest in the epigenetic regulation of CXCR4 signaling that may be responsible for upregulated CXCR4 in primary as well as metastatic cancers. This review first lists the clinical evidence supporting CXCR4 signaling as putative cancer diagnostic and/or prognostic biomarker, followed by a discussion on reported epigenetic mechanisms that affect CXCR4 expression. These mechanisms include regulation by non-coding RNAs, such as, microRNAs, long non-coding RNAs and circular RNAs. Additionally, we also discuss the regulation of CXCR4 expression through methylation and acetylation. Better understanding and appreciation of epigenetic regulation of CXCR4 signaling can invariably lead to identification of novel therapeutic targets as well as therapies to regulate this oncogenic signaling.

Molecular and Morphological Characterization of Inflammatory Infiltrate in Rosacea Reveals Activation of Th1/Th17 Pathways.

Rosacea is a common chronic inflammatory skin disease of unknown etiology. Our knowledge about an involvement of the adaptive immune system is very limited. We performed detailed transcriptome analysis, quantitative real-time reverse-transcriptase-PCR, and quantitative immunohistochemistry on facial biopsies of rosacea patients, classified according to their clinical subtype. As controls, we used samples from patients with facial lupus erythematosus and healthy controls. Our study shows significant activation of the immune system in all subtypes of rosacea, characterizing erythematotelangiectatic rosacea (ETR) already as a disease with significant influx of proinflammatory cells. The T-cell response is dominated by Th1/Th17-polarized immune cells, as demonstrated by significant upregulation of IFN-γ or IL-17, for example. Chemokine expression patterns support a Th1/Th17 polarization profile of the T-cell response. Macrophages and mast cells are increased in all three subtypes of rosacea, whereas neutrophils reach a maximum in papulopustular rosacea. Our studies also provide evidence for the activation of plasma cells with significant antibody production already in ETR, followed by a crescendo pattern toward phymatous rosacea. In sum, Th1/Th17 polarized inflammation and macrophage infiltration are an underestimated hallmark in all subtypes of rosacea. Therapies directly targeting the Th1/Th17 pathway are promising candidates in the future treatment of this skin disease.

Proteinase-activated receptors 1 and 2 regulate invasive behavior of human melanoma cells via activation of protein kinase D1.

Recent studies have indicated an important role of proteinases and proteinase-activated receptors (PARs) in tumorigenesis. Although a role for PARs has been described in various skin tumors including melanoma, the underlying cellular mechanisms have not been understood. Recent studies have suggested PAR(1) as a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. Moreover, changes in the expression patterns of PAR(1) and PAR(2) correlate with skin cancer progression, and PAR(1) is overexpressed in melanoma. Therefore, we sought to elucidate the putative role of PAR(1)- and PAR(2)-mediated signal transduction pathways during melanoma progression. Activation of both PAR(1) and PAR(2) led to rapid phosphorylation of protein kinase D1 (PKD1) in cultured WM9 melanoma cells. PKD1 is known to be involved in cell migration, integrin regulation, and intracellular vesicle transport. Downregulation of PKD1 by siRNA resulted in diminished proliferation, decreased αvß3 integrin regulation, and secretion of pro-angiogenic chemokine IL-8 in WM9 cells. In conclusion, our results show that PAR(1) and PAR(2) are involved in WM9 cell proliferation and secretion of IL-8 by activation of PKD1. Inactivation of the PKD1 pathway may be beneficial for the inhibition of PAR-induced melanoma proliferation and for maintenance of the inflammatory tumor environment.

Distribution and expression of non-neuronal transient receptor potential (TRPV) ion channels in rosacea.

Rosacea is a frequent chronic inflammatory skin disease of unknown etiology. Because early rosacea reveals all characteristics of neurogenic inflammation, a central role of sensory nerves in its pathophysiology has been discussed. Neuroinflammatory mediators and their receptors involved in rosacea are poorly defined. Good candidates may be transient receptor potential (TRP) ion channels of vanilloid type (TRPV), which can be activated by many trigger factors of rosacea. Interestingly, TRPV2, TRPV3, and TRPV4 are expressed by both neuronal and non-neuronal cells. Here, we analyzed the expression and distribution of TRPV receptors in the various subtypes of rosacea on non-neuronal cells using immunohistochemistry, morphometry, double immunoflourescence, and quantitative real-time PCR (qRT-PCR) as compared with healthy skin and lupus erythematosus. Our results show that dermal immunolabeling of TRPV2 and TRPV3 and gene expression of TRPV1 is significantly increased in erythematotelangiectatic rosacea (ETR). Papulopustular rosacea (PPR) displayed an enhanced immunoreactivity for TRPV2, TRPV4, and also of TRPV2 gene expression. In phymatous rosacea (PhR)-affected skin, dermal immunostaining of TRPV3 and TRPV4 and gene expression of TRPV1 and TRPV3 was enhanced, whereas epidermal TRPV2 staining was decreased. Thus, dysregulation of TRPV channels also expressed by non-neuronal cells may be critically involved in the initiation and/or development of rosacea. TRP ion channels may be targets for the treatment of rosacea.

Clinical, cellular, and molecular aspects in the pathophysiology of rosacea.

Rosacea is a chronic inflammatory skin disease of unknown etiology. Although described centuries ago, the pathophysiology of this disease is still poorly understood. Epidemiological studies indicate a genetic component, but a rosacea gene has not been identified yet. Four subtypes and several variants of rosacea have been described. It is still unclear whether these subtypes represent a "developmental march" of different stages or are merely part of a syndrome that develops independently but overlaps clinically. Clinical and histopathological characteristics of rosacea make it a fascinating "human disease model" for learning about the connection between the cutaneous vascular, nervous, and immune systems. Innate immune mechanisms and dysregulation of the neurovascular system are involved in rosacea initiation and perpetuation, although the complex network of primary induction and secondary reaction of neuroimmune communication is still unclear. Later, rosacea may result in fibrotic facial changes, suggesting a strong connection between chronic inflammatory processes and skin fibrosis development. This review highlights recent molecular (gene array) and cellular findings and aims to integrate the different body defense mechanisms into a modern concept of rosacea pathophysiology.

Neurovascular and neuroimmune aspects in the pathophysiology of rosacea.

Rosacea is a common skin disease with a high impact on quality of life. Characterized by erythema, edema, burning pain, immune infiltration, and facial skin fibrosis, rosacea has all the characteristics of neurogenic inflammation, a condition induced by sensory nerves via antidromically released neuromediators. To investigate the hypothesis of a central role of neural interactions in the pathophysiology, we analyzed molecular and morphological characteristics in the different subtypes of rosacea by immunohistochemistry, double immunofluorescence, morphometry, real-time PCR, and gene array analysis, and compared the findings with those for lupus erythematosus or healthy skin. Our results showed significantly dilated blood and lymphatic vessels. Signs of angiogenesis were only evident in phymatous rosacea. The number of mast cells and fibroblasts was increased in rosacea, already in subtypes in which fibrosis is not clinically apparent, indicating early activation. Sensory nerves were closely associated with blood vessels and mast cells, and were increased in erythematous rosacea. Gene array studies and qRT-PCR confirmed upregulation of genes involved in vasoregulation and neurogenic inflammation. Thus, dysregulation of mediators and receptors implicated in neurovascular and neuroimmune communication may be crucial at early stages of rosacea. Drugs that function on neurovascular and/or neuroimmune communication may be beneficial for the treatment of rosacea.

Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo.

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

Histamine and antihistamines in atopic dermatitis.

Itching (pruritus) is perhaps the most common symptom associated with inflammatory skin diseases and can be a lead symptom ofextracutaneous disease (e.g., malignancy, infection, metabolic disorders). In atopic dermatitis itching sensations constitute one of the most prominent and distressing features. The most characteristic response to itching is the scratch reflex: a more or less voluntary, often sub-conscious motor activity, to counteract the itch by slightly painful stimuli. The benefit of a short-termed relieve from itching through this scratch reflex though is counteracted by a simultaneous damage of the epidermal layer of the skin which leads to increased transepidermal water loss and drying, which in turn results in a cycle of more itching and more scratching. A wide range of peripheral itch-inducing stimuli generated within or administered to the skin are able to trigger pruritus, one of them being histamine. Based on early experiments, histamine has been suggested to may play a key role in the pathogenesis ofAD. This is reflected by a history for antihistamines in the therapeutic medication of AD patients. Antihistamines are believed to share a common antipruritic effect and therefore are prescribed to the vast majority of AD patient suffering from itch to act alleviating. The level of evidence in support of the benefits of antihistamine treatment, however, is low. To assess the benefit of antihistamines in the treatment of AD in a better way, their mechanisms and specific effects need to be understood more precisely. In particular their precise indication is crucial for successful use. This book chapter will therefore summarize and assess the role of histamine in AD and the efficacy of antihistamines in its treatment based on results of basic research and clinical studies.

Proteinase-activated receptor-2 (PAR2): a tumor suppressor in skin carcinogenesis.

The proteinase-activated receptor PAR(2) has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. However, the role of PAR(2) in cutaneous cancerogenesis is still unknown. Here we could show a protective role of PAR(2) in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. Tumors started to appear after eight weeks. After 13 weeks, PAR(2)-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild type (eight tumors per mouse). Analysis of possible signal transduction pathways activated upon PAR(2) stimulation in HaCaT keratinocytes showed an involvement of extracellular signal-regulated kinase 1/2 and profound epidermal growth factor receptor transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta1. Thus, our results provide early experimental evidence for a tumor-protective role of PAR(2).

Agonists of proteinase-activated receptor-2 stimulate upregulation of intercellular cell adhesion molecule-1 in primary human keratinocytes via activation of NF-kappa B.

Proteinase-activated receptor-2 (PAR2) belongs to a new G protein-coupled receptor subfamily that is activated by various serine proteases. Recent knowledge indicates that PAR2 is involved in cutaneous inflammation and immune response. PAR2 is highly expressed by human keratinocytes (KTC). The underlying mechanisms of PAR2-mediated KTC function and cutaneous immune response are, however, still incomplete. Therefore, we investigated the activation of important signaling cascades in primary human KTC after PAR2-stimulation using specific agonists. Moreover, we compared PAR2-immunoreactivity in the epidermis of inflammatory dermatoses and normal human skin. Electrophoretic mobility shift assays and morphological transduction studies revealed PAR2-induced activation and translocation of nuclear factor kappa B (NF-kappaB) in primary human KTC with a maximum after 1 h. Supershift analysis demonstrated acivation of the p50/p65 heterodimer complex. PAR2 agonists also induced upregulation of intercellular adhesion molecule-1 (ICAM-1) RNA, as shown by RT-PCR. Use of NF-kappaB inhibitors prevented upregulation of the cell adhesion molecule ICAM-1 in KTC indicating a direct role of NF-kappaB in PAR2-mediated upregulation of ICAM-1. Fluorescence-activated cell sorter analysis confirmed PAR2-induced and NF-kappaB-mediated upregulation of ICAM-1 protein after 13 h. Moreover, increased expression of PAR2 was detected in KTC of patients with atopic dermatitis suggesting a role of PAR2 in human skin inflammation. In conclusion, PAR2 induces upregulation of cell adhesion molecules such as ICAM-1 in primary human KTC via NF-kappaB activation, and is upregulated in KTC during cutaneous inflammation. Thus, PAR2 may play an important regulatory role of human KTC during inflammation and immune response.

Expression of vanilloid receptor subtype 1 in cutaneous sensory nerve fibers, mast cells, and epithelial cells of appendage structures.

The vanilloid receptor subtype 1 (VR1)/(TRPV1), binding capsaicin, is a non-selective cation channel that recently has been shown in human keratinocytes in vitro and in vivo. However, a description of VR1 localization in other cutaneous compartments in particular cutaneous nerve fibers is still lacking. We therefore investigated VR1 immunoreactivity as well as mRNA and protein expression in a series (n = 26) of normal (n = 7), diseased (n = 13) [prurigo nodularis (PN) (n = 10), generalized pruritus (n = 1), and mastocytosis (n = 2)], and capsaicin-treated human skin (n = 6). VR1 immunoreactivity could be observed in cutaneous sensory nerve fibers, mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands. Upon reverse transcriptase-polymerase chain reaction and Western blot analysis, VR1 was detected in mast cells and keratinocytes from human skin. In pruritic skin of PN, VR1 expression was highly increased in epidermal keratinocytes and nerve fibers, which was normalized after capsaicin application. During capsaicin therapy, a reduction of neuropeptides (substance P, calcitonin gene-related peptide) was observed. After cessation of capsaicin therapy, neuropeptides re-accumulated in skin nerves. In conclusion, VR1 is widely distributed in the skin, suggesting a major role for this receptor, e.g. in nociception and neurogenic inflammation.

Agonists of proteinase-activated receptor 2 induce cytokine release and activation of nuclear transcription factor kappaB in human dermal microvascular endothelial cells.

Proteinase-activated receptor 2 belongs to a new G protein-coupled receptor subfamily activated by various serine proteases. It has been demonstrated to play a role during inflammation of many tissues including the skin. Proteinase-activated receptor 2 is expressed by endothelial cells and regulates cutaneous inflammation in vivo. The underlying mechanisms of proteinase-activated receptor 2 activation in the skin and the effects on human dermal microvascular endothelial cells, however, are still unknown. Agonists of proteinase-activated receptor 2 such as mast cell tryptase induce widespread inflammation in many organs including the skin. Trypsinogen is generated by endothelial cells during inflammation or tumor growth. Therefore we tested whether human dermal microvascular endothelial cells express functional proteinase-activated receptor 2 and whether agonists of proteinase-activated receptor 2 regulate inflammatory responses in these cells. Calcium mobilization studies revealed that proteinase-activated receptor 2 is functional in human dermal microvascular endothelial cells. Interleukin-6 and interleukin-8 were upregulated as detected by reverse transcription polymerase chain reaction or enzyme-linked immunosorbent assay indicating a role of proteinase-activated receptor 2 in stimulating human dermal microvascular endothelial cells. Electromobility shift assays revealed proteinase-activated-receptor-2-induced activation of nuclear transcription factor kappaB with a maximum after 1 h. In conclusion, agonists of proteinase-activated receptor 2 upregulate interleukin-6 and interleukin-8 expression and release in human dermal microvascular endothelial cells. Thus, proteinase-activated receptor 2 may play an important role in cutaneous inflammation by mediating inflammatory responses on dermal microvascular endothelial cells and activation of nuclear transcription factor kappaB.