Skin Papillomas Research Articles

Photodynamic therapy using a novel irradiation source, LED lamp, is similarly effective to photodynamic therapy using diode laser or metal-halide lamp on DMBA- and TPA-induced mouse skin papillomas.

Photodynamic therapy (PDT) is useful for superficial skin tumors such as actinic keratosis and Bowen disease. Although PDT is non-surgical and easily-performed treatment modality, irradiation apparatus is large and expensive. Using 7, 12-dimethylbenz[a]anthracene (DMBA) and 12-ο-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin papilloma model, we compared the efficacy of TONS501- and ALA-PDT with a LED lamp, a diode laser lamp or a metal-halide lamp on the skin tumor regression. TONS501-PDT using 660nm LED lamp showed anti-tumor effect at 1day following the irradiation and the maximal anti-tumor effect was observed at 3days following the irradiation. There was no significant difference in the anti-tumor effects among TONS501-PDT using LED, TONS501-PDT using diode laser, and 5-aminolevulinic acid hydrochloride (ALA)-PDT using metal-halide lamp. Potent anti-tumor effect on DMBA- and TPA-induced mouse skin papilloma was observed by TONS501-PDT using 660nm LED, which might be more useful for clinical applications.

SOX2 controls tumour initiation and cancer stem-cell functions in squamous-cell carcinoma.

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here we find that Sox2, a transcription factor expressed in various types of embryonic and adult stem cells, was the most upregulated transcription factor in the CSCs of squamous skin tumours in mice. SOX2 is absent in normal epidermis but begins to be expressed in the vast majority of mouse and human pre-neoplastic skin tumours, and continues to be expressed in a heterogeneous manner in invasive mouse and human SCCs. In contrast to other SCCs, in which SOX2 is frequently genetically amplified, the expression of SOX2 in mouse and human skin SCCs is transcriptionally regulated. Conditional deletion of Sox2 in the mouse epidermis markedly decreases skin tumour formation after chemical-induced carcinogenesis. Using green fluorescent protein (GFP) as a reporter of Sox2 transcriptional expression (SOX2-GFP knock-in mice), we showed that SOX2-expressing cells in invasive SCC are greatly enriched in tumour-propagating cells, which further increase upon serial transplantations. Lineage ablation of SOX2-expressing cells within primary benign and malignant SCCs leads to tumour regression, consistent with the critical role of SOX2-expressing cells in tumour maintenance. Conditional Sox2 deletion in pre-existing skin papilloma and SCC leads to tumour regression and decreases the ability of cancer cells to be propagated upon transplantation into immunodeficient mice, supporting the essential role of SOX2 in regulating CSC functions. Transcriptional profiling of SOX2-GFP-expressing CSCs and of tumour epithelial cells upon Sox2 deletion uncovered a gene network regulated by SOX2 in primary tumour cells in vivo. Chromatin immunoprecipitation identified several direct SOX2 target genes controlling tumour stemness, survival, proliferation, adhesion, invasion and paraneoplastic syndrome. We demonstrate that SOX2, by marking and regulating the functions of skin tumour-initiating cells and CSCs, establishes a continuum between tumour initiation and progression in primary skin tumours.

Peroxiredoxin 6 in skin carcinogenesis.

Non-melanoma skin cancer represents a serious and globally increasing health problem. Therefore, a detailed knowledge on the mechanisms of skin cancer formation is required to develop innovative, efficient and cost-effective strategies for skin cancer prevention and treatment. A major risk factor for cancer formation is oxidative stress, which results from excessive levels of reactive oxygen species (ROS). This can cause oxidative damage of lipids, proteins and DNA, which enhances the risk of mutations [1]. A major enzyme involved in the detoxification of ROS is peroxiredoxin 6 (Prdx6). As a “moonlighting” enzyme it bears two distinct catalytic activities: a cytosolic, selenium-independent peroxidase activity and a lysosomal, calcium-independent phospholipase A2 (PLA2) activity [2]. Acting as PLA2 in lipid catabolism at low pH, Prdx6 hydrolyses the sn-2 ester linkage of phospholipids to release a fatty acid and a lysophospholipid. As a peroxidase, Prdx6 can reduce H2O2, short chain organic hydroperoxides and phospholipid hydroperoxides at cytosolic, neutral pH [2]. Due to these functions it was of particular interest to determine the role of Prdx6 in skin carcinogenesis. For this purpose we made use of transgenic mice overexpressing Prdx6 in the epidermis as well as of Prdx6 knockout mice. Interestingly, overexpression of Prdx6 protected keratinocytes of the skin from oxidative damage in vitro and in vivo [3]. Vice versa, mice lacking Prdx6 were more susceptible to oxidative damage in the skin [4]. Based on these data, we analyzed the function of Prdx6 in skin tumorigenesis using genetic and chemically-induced skin cancer models [5]. In the genetic model we used transgenic mice, which express the oncogenes of the human papilloma virus type 8 (HPV8) in keratinocytes and which develop spontaneous skin papillomas within a few weeks. These mice were mated with either Prdx6 transgenic mice or Prdx6 knockout mice. Interestingly, loss of Prdx6 enhanced tumor multiplicity in the HPV8 model, while overexpression of Prdx6 significantly reduced the tumor number. Results obtained with Prdx6 transgenic mice subjected to a model where tumors are induced with a mutagen (DMBA) and a tumor promoter (TPA) confirmed the tumor-preventive effect of this enzyme. In both tumor models, Prdx6 activation had no influence on keratinocyte proliferation, apoptosis or the inflammatory response of the mice. However, following TPA treatment of wild-type mice, we detected an increase in the levels of several epidermal phospholipid hydroperoxides. This increase was much less pronounced in Prdx6 transgenic mice, but more pronounced in Prdx6 knockout mice. This finding suggests that Prdx6 reduces oxidative stress and thus protects from lipid peroxidation and most likely also from oxidative DNA damage. Figure 1 The dual role of Prdx6 in skin carcinogenesis On the other hand, we found an increase in tumor progression in Prdx6 overexpressing mice, most likely due to protection of tumor cells from inflammation-induced ROS damage and subsequent cell death. Therefore, our data revealed a surprising dual function of Prdx6 in skin tumorigenesis depending on the stage of tumor development: While it protects normal cells from oxidative damage and thus reduces mutagenesis and tumorigenesis, it is also beneficial for cancer cells, thus promoting their survival under stress conditions and subsequent tumor progression. These results are likely to be of human relevance, since we detected strong PRDX6 expression in normal skin and also in cutaneous SCCs [5]. Surprisingly, however, the expression levels were highly variable between different patients. Therefore, it will be important to determine if individuals with strong PRDX6 expression in normal skin are less susceptible to tumor development and vice versa, and if PRDX6 overexpression in skin tumors correlates with tumor aggressiveness. These results will provide the basis for the potential use of PRDX6 activators for skin tumor prevention, whereas PRDX6 inhibitors might have therapeutic potential for the treatment of malignant epithelial skin cancers. Consistent with this assumption, thiacremonone, a compound isolated from garlic, was shown to bind to the catalytic site Cys47 of Prdx6 and it reduced the tumor volume of lung carcinoma cells allografted to wild-type mice and in particular of cells allografted to Prdx6 transgenic mice [6]. This promising result points to the usefulness of PRDX6 inhibitors in cancer therapy. These data combined with our results obtained in the skin also demonstrate that Prdx6 activation/ overexpression and ROS-detoxifying strategies in general are not always beneficial. This is also reflected by the recently described acceleration of lung cancer progression in mice by antioxidants [7]. Therefore, activation of ROS-detoxifying enzymes and reduction of ROS levels should be carefully evaluated, in particular in patients with diagnosed cancer.

Congenic mapping and allele-specific alteration analysis of Stmm1 locus conferring resistance to early-stage chemically induced skin papillomas.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

DEK promotes HPV-positive and -negative head and neck cancer cell proliferation.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, and patient outcomes using current treatments remains poor. Tumor development is etiologically associated with tobacco or alcohol use and/or HPV infection. HPV positive HNSCCs, which frequently harbor wild-type p53, carry a more favorable prognosis and are a biologically distinct subgroup when compared to their HPV negative counterparts. HPV E7 induces expression of the human DEK gene, both in vitro and in vivo. In keratinocytes, DEK overexpression is sufficient for causing oncogenic phenotypes in the absence of E7. Conversely, DEK loss results in cell death in HPV positive cervical cancer cells at least in part through p53 activation, and Dek knockout mice are relatively resistant to the development of chemically induced skin papillomas. Despite the established oncogenic role of DEK in HPV associated cervical cancer cell lines and keratinocytes, a functional role of DEK has not yet been explored in HNSCC. Using an established transgenic mouse model of HPV16 E7 induced HNSCC, we demonstrate that Dek is required for optimal proliferation of E7-transgenic epidermal cells and for the growth of HNSCC tumors. Importantly, these studies also demonstrate that DEK protein is universally up-regulated in both HPV positive and negative human HNSCC tumors relative to adjacent normal tissue. Furthermore, DEK knockdown inhibited the proliferation of HPV positive and negative HNSCC cells, establishing a functional role for DEK in human disease. Mechanistic studies reveal that attenuated HNSCC cell growth in response to DEK loss was associated with reduced expression of the oncogenic p53 family member, ΔNp63. Exogenous ΔNp63 expression rescued the proliferative defect in the absence of DEK, thereby establishing a functional DEK-ΔNp63 oncogenic pathway that promotes HNSCC. Taken together, our data demonstrate that DEK stimulates HNSCC cellular growth and identify ΔNp63 as a novel DEK effector.

Pten Loss Induces Autocrine FGF Signaling to Promote Skin Tumorigenesis

Inactivation of the Pten tumor suppressor negatively regulates the PI3K-mTOR pathway. In a model of cutaneous squamous cell carcinoma (SCC), we demonstrate that deletion of Pten strongly elevates Fgf10 protein levels without increasing Fgf10 transcription in vitro and in vivo. The translational activation of Fgf10 by Pten deletion is reversed by genetic disruption of the mTORC1 complex, which also prevents skin tumorigenesis in Pten mutants. We further show that ectopic expression of Fgf10 causes skin papillomas, whereas Pten deletion-induced skin tumors are inhibited by epidermal deletion of Fgfr2. Collectively, our data identify autocrine activation of FGF signaling as an essential mechanism in promoting Pten-deficient skin tumors.

S100A9 has a protective role in inflammation‐induced skin carcinogenesis

The S100A8/A9 heterodimer is expressed by myeloid cells where its function has been extensively investigated. Immune cell S100A8/A9 promotes proinflammatory effects, and its absence is often associated with lack of leukocyte recruitment resulting in protection in terms of disease progression. S100A8/A9 is also expressed by certain epithelia, either constitutively as in mucosal epithelia or following stimulation as in skin keratinocytes. The role of the heterodimer in this context has not been as frequently explored. In this study, the incidence of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) in S100a9(-/-) mice has been investigated. Unlike the immune disorders and certain models of cancer, absence of S100A8/A9 caused an increased incidence in skin of papillomas and, subsequently, squamous cell carcinomas. Although associated in S100a9(-/-) mice with increased recruitment of neutrophils and T cells, a bone marrow chimera experiment revealed the major defect to be primarily due to the absence of S100A8/A9 in the skin keratinocytes. S100a9(-/-) skin displayed enhanced Ki-67 expression over the time period of appearance of the papillomas suggesting an effect of S100A8/A9 in regulating proliferation in the epidermal layer. Thus, despite immune cell recruitment in S100a9(-/-) mouse skin that might have been predicted to promote tumor growth, it was the absence of S100A8/A9 in skin keratinocytes that dominated in terms of papilloma formation. The study highlights the importance of the S100A8/A9-expressing skin epidermal layer in controlling skin tumor formation and suggests that the influence of the heterodimer is dependent on the tissue context in which it is expressed.

Chemopreventive potential of Apium leptophyllum (Pers.) against DMBA induced skin carcinogenesis model by modulatory influence on biochemical and antioxidant biomarkers in Swiss mice.

Aim:The study was designed to investigate the chemopreventive potential of flavonoidal fractions of Apium leptophyllum fruits (FFALF) on Swiss mice.Materials and Methods:Skin tumor or papilloma was developed by topical application of DMBA (25 μg in 0.1 ml acetone) on intrascapular region of mice, twice weekly for 8 weeks. The animals were divided into six groups: Group I (vehicle control); group II (FFALF control, 5 mg/kg); group III (carcinogenic control, DMBA treated initially for 8 weeks); and group IV, V and VI as pre-treated group (FFALF 5, 10 and 20 mg/kg respectively for 16 weeks along with DMBA treatment). After the 16th week of treatment; the tumor morphology, skin histopathology, and biochemical and antioxidant biomarkers were measured and compared with carcinogenic control as well as vehicle control.Results:The co-administration of FFALF with DMBA-treated groups showed significant (P ≤ 0.001) prevention against skin papilloma and normalized the status of lipid peroxidation with antioxidant biomarkers in a dose-dependent manner as compared to carcinogenic control.Conclusions:Thus, the present study suggests that the FFALF is non-carcinogenic and has chemopreventive potential on DMBA-induced carcinogenesis in mouse, which may be due to the modulation of cutaneous lipid peroxidation or enhancement of total antioxidant capacity.

Identification of <i>Stmm3</i> locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.

Anti-carcinogenic effects of non-polar components containing licochalcone A in roasted licorice root.

BACKGROUND/OBJECTIVELicorice has been shown to possess cancer chemopreventive effects. However, glycyrrhizin, a major component in licorice, was found to interfere with steroid metabolism and cause edema and hypertension. The roasting process of licorice modifies the chemical composition and converts glycyrrhizin to glycyrrhetinic acid. The purpose of this study was to examine the anti-carcinogenic effects of the ethanol extract of roasted licorice (EERL) and to identify the active compound in EERL.MATERIALS/METHODSEthanol and aqueous extracts of roasted and un-roasted licorice were prepared. The active fraction was separated from the methylene chloride (MC)-soluble fraction of EERL and the structure of the purified compound was determined by nuclear magnetic resonance spectroscopy. The anti-carcinogenic effects of licorice extracts and licochalcone A was evaluated using a MTT assay, Western blot, flow cytometry, and two-stage skin carcinogenesis model.RESULTSEERL was determined to be more potent and efficacious than the ethanol extract of un-roasted licorice in inhibiting the growth of DU145 and MLL prostate cancer cells, as well as HT-29 colon cancer cells. The aqueous extracts of un-roasted and roasted licorice showed minimal effects on cell growth. EERL potently inhibited growth of MCF-7 and MDA-MB-231 breast, B16-F10 melanoma, and A375 and A2058 skin cancer cells, whereas EERL slightly stimulated the growth of normal IEC-6 intestinal epithelial cells and CCD118SK fibroblasts. The MC-soluble fraction was more efficacious than EERL in inhibiting DU145 cell growth. Licochalcone A was isolated from the MC fraction and identified as the active compound of EERL. Both EERL and licochalcone A induced apoptosis of DU145 cells. EERL potently inhibited chemically-induced skin papilloma formation in mice.CONCLUSIONSNon-polar compounds in EERL exert potent anti-carcinogenic effects, and that roasted rather than un-roasted licorice should be favored as a cancer preventive agent, whether being used as an additive to food or medicine preparations.

Bovine papillomavirus in beef cattle: first description of BPV-12 and putative type BAPV8 in Brazil.

Bovine papillomavirus (BPV) is an oncogenic virus associated with benign and malignant lesions, which result in notable economic losses. Peripheral blood samples and cutaneous papillomas were obtained from four adult beef cattle. Viral molecular identification was performed using specific primers for BPV-1, -2 and -4 in blood diagnosis and FAP59/FAP64 for skin papillomas. Histopathologic examination was done as a complementary and differential diagnosis. The fragments were purified, sequenced, and compared using BLASTn. The blood diagnosis showed the presence of BPV-2 and the analysis of cutaneous papillomas showed the presence of BPV-4, a new putative virus type BAPV8, and BPV-12, revealing for the first time the presence of BPV-12 and the putative type BAPV8 in beef cattle in Brazil. The sequences were deposited in the GenBank. Histopathology revealed acanthosis, hyperkeratosis, and koilocytosis in all samples analyzed. The presence of BAPV8 and BPV-12 in Brazil emphasizes the ubiquitous dissemination of BPVs in the herds of Brazil.

Human Papillomavirus Deoxyribonucleic Acid may not be Detected in Non-genital Benign Papillomatous Skin Lesions by Polymerase Chain Reaction.

Background:Papillomatosis is a known histopathologic pattern usually seen in human papillomavirus (HPV) infection and verruca vulgaris is the typical example. This pattern is also detected in some other benign cutaneous lesions such as nevus sebaceous (NS), seborrheic keratosis (SK), trichilemmoma (TL) and inverted follicular keratosis (IFK). The association between papillomatous lesions and HPV infection is questionable.Objective:The objective of this study was to investigate the presence of HPV deoxyribonucleic acid (DNA) in non-genital benign papillomatous skin lesions (NS, SK, TL and IFK) by polymerase chain reaction (PCR).Materials and Methods:A total of 100 specimens of non-genital NS, SK, TL and IFK were retrieved from archives of Dermatopathology Department of Razi Hospital, between 2003 and 2010. The conventional PCR using consensus GP5+/GP6+ primer and hydroxymethylbilane synthase gene as inner control was performed.Results:PCR for HPV DNA revealed no positive results in any of 28 seborrheic keratosis (SK), 28 nevus sebaceous (NS), 28 inverted follicular keratosis (IFK) and 13 trichilemmoma (TL) studied specimens.Conclusion:Papillomatosis is usually a characteristic pattern of HPV infection. However, we found no association between HPV infection and non-genital benign papillomatous lesions.

Resveratrol and P-glycoprotein Inhibitors Enhance the Anti-Skin Cancer Effects of Ursolic Acid

Ursolic acid, present in apples, rosemary, and other sources, is known to inhibit tumor formation and tumor cell viability in multiple systems, including skin. However, various cancers are resistant to ursolic acid treatment. Herein, skin carcinoma cells (Ca3/7) as compared with skin papilloma cells (MT1/2) displayed more resistance to ursolic acid-induced cytotoxicity. Interestingly, Ca3/7 cells had elevated levels of P-glycoprotein (P-gp), an ATP-dependent efflux pump that mediates resistance to chemotherapy in preclinical and clinical settings, and not only accumulated less but also more rapidly expelled the P-gp substrate rhodamine 123 (Rh123) indicating ursolic acid is transported by P-gp. To determine whether P-gp inhibition can enhance ursolic acid-mediated cytotoxicity, cells were challenged with P-gp inhibitors verapamil or cyclosporin A. Alternatively, cells were pretreated with the natural compound resveratrol, a known chemotherapy sensitizer. Verapamil and resveratrol enhanced the effects of ursolic acid in both cell lines, whereas cyclosporin A only did so in Ca3/7 cells. Similarly, verapamil inhibited Rh123 efflux in both lines, whereas cyclosporin A only inhibited Rh123 efflux in Ca3/7 cells. Resveratrol did not inhibit Rh123 efflux in either line, indicating the synergistic effects of resveratrol and ursolic acid are not manifest by inhibition of P-gp-mediated efflux of ursolic acid. These results indicate that the anti-skin cancer effects of ursolic acid are enhanced with P-gp inhibitors. In addition, resveratrol and ursolic acid interact synergistically, but not through inhibition of P-gp. Resveratrol and/or p-glycoprotein inhibitors in combination with ursolic acid are an effective anti-skin cancer regimen.

Prevalence of skin neoplastic lesions in bream (Abramis brama L.) from the Curonian Lagoon of the Baltic Sea

Skin neoplastic lesions have been studied in bream inhabiting the Curonian Lagoon of the Baltic Sea in 1999–2010. A total of 12163 fish specimens have been examined. Two types of lesions have been revealed: epidermal hyperplasia and skin papilloma, considered to be the successive stages of the epizootic cutaneous papillomatosis in fish. This disease has been registered in 18.5% of fish older than 5 years. The share of diseased specimens increases with age and reaches 62.5% in 15-year-old fish. The prevalence of lesions in males is significantly higher (12%) than in females (5.3%). The number of diseased fish in winter (30.2%) is higher than in summer (15.6%). The maximum share of fish with neoplasms has been recorded in 2006 (40%). Since 2007, there has been a trend towards a decrease in the prevalence of skin neoplastic lesions, and the average occurrence of diseased fish has reached 7.3%. The high prevalence of skin neoplasms (SNs) in bream is evidence of the unfavorable ecological situation in the Curonian Lagoon.

Study on extraction process of tannins from Semen Cuscutae and their anti-papilloma activity.

The objective of this paper was to study the extraction methods of tannin constituents from Semen Cuscutae and their anti-papilloma effects. Single factor test and orthogonal design methods were used to determine the optimal extraction method; the mouse skin papilloma model induced by DMBA/croton oil was established, which was a classic two-stage carcinogenesis model being used to observe and evaluate the anti-carcinogenic effects of tannins extracted from Semen Cuscutae in different stages. The optimal extraction method of Semen Cuscutae was a 20-fold volume of solvent, a temperature of 50 °C, three times of extraction, with 20 min each, skin papilloma experiment revealed that the number of bearing tumors gradually reduced, and the inhibition rate gradually increased with the increase of dose, in the high-dose group, its inhibition rate reached 70.2%. Tannin extract from Semen Cuscutae has an obvious inhibitory effect on skin papilloma development.

Abstract 1418: CLIC4 regulates carcinogenesis in a TGF-β context-dependent manner.

Abstract CLIC4 is a 28kD, ubiquitously expressed, redox-regulated, multifunctional protein. It is dimorphic and can transition between membrane bound or soluble forms in the cytoplasm. Cytoplasmic CLIC4 translocates to the nucleus in multiple cell types under conditions of metabolic stress and nuclear CLIC4 causes growth arrest, terminal differentiation and apoptosis. In vivo, CLIC4 is nuclear in quiescent epithelial cells with little stromal expression. In contrast, nuclear CLIC4 is lost from tumor epithelium and is highly upregulated in tumor stroma. We show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts of oncogenic ras transformed keratinocytes inhibits tumor growth, while elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth. These results identify CLIC4 as a tumor suppressor. We show that CLIC4 is an integral intermediate in TGF-β signaling, that overexpressing CLIC4 in tumor cell lines restores TGF-β mediated growth inhibition, and tumor cells in vivo overexpressing CLIC4 have enhanced TGF-β signaling. We have also analyzed the substantial upregulation of CLIC4 in tumor stroma. Reconstituting orthografts of mammary or squamous tumors with stromal cells overexpressing CLIC4 enhances tumor growth.Correspondingly, tumor growth is significantly inhibited in orthografts of tumor cells to hosts that lack stromal CLIC4. CLIC4 expression is increased in stromal cells by conditioned medium from tumor cells in a TGF-β dependent manner. In stromal cells genetically deleted of CLIC4, the conversion of fibroblasts to cancer associated myofibroblasts by TGF-β through p38 activation is prevented. CLIC4 is essential for preventing the de-activation of p38 by its phosphatase PPM1a. Stromal cells that overexpress CLIC4 enhance tumor cell invasion and EMT in vitro. Thus CLIC4, like TGF-β, has context dependent dual influence on tumor cell growth and progression. CLIC4 is an attractive therapeutic target both in cancer stages where TGF-β signal augmentation or inhibition is required due to responses in separate tissue compartments. Targeting CLIC4 would also be a more specific approach in therapy that would mitigate some of the severe side effects of global targeting of the multifunctional TGF-β pathway. Citation Format: Anjali Shukla, Rebecca Edwards, Yihan Yang, Alexandra Hahn, VC Padmakumar, Andrew Ryscavage, Kwang S. Suh, Stuart H. Yuspa. CLIC4 regulates carcinogenesis in a TGF-β context-dependent manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1418. doi:10.1158/1538-7445.AM2013-1418

Abstract 5371: Epigenetic regulation in skin cancer: global DNA methylation profiling in DMBA/TPA induced mice.

Abstract Hypermethylation of CpG islands in the promoter regions of tumor suppressor genes is postulated to be the most relevant epigenetic changes resulting in gene silencing of tumor suppressor gene in cancer. Transcriptionally repressed genes by epigenetic regulation can be reactivated by epigenetic modifications because these silenced genes are still genetically intact. Thus, it is relevant to profile global DNA methylation changes that occur in early stages of carcinogenesis (Jones & Baylin, 2002). To begin addressing this issue, we conducted global profiling of DNA methylation changes in a well-established multistage skin carcinogenesis model using methylated DNA immunoprecipitation (Medip) coupled with CpG island microarray. Tumorigenesis was induced by sequential topical application of mutagen 7,12-Dimethylbenz(a)anthracene (DMBA), and promoting agent 12-O-tetra-decanoylphorbol-13-acetate (TPA), on the dorsal skin of normal CD-1 mice. After 12 weeks, animals were sacrificed and DNA extracted from papillomas. Methylation enriched DNA fragments were pulled down and hybridized to CpG microarray covering 16,030 CpG islands (Agilent, Santa Clara, CA) The results show that significant changes in DNA methylation profile between normal skin and DMBA/TPA induced papilloma, analyzed by Genespring and Bioconductor. After normalization of probe with the gene, 186 genes from 9,867 of up-regulated methylation genes showed at least average 2-fold changes and these include some DNA damage and repair, oxidative stress and inflammatory genes such as Gadd45b and Arnt2. In summary, DMBA/TPA induced mouse skin papilloma undergo extensive aberrant CpG island hypermethylation. Genes detected from Medip-chip assay may be utilized as a potential epigenetic tool to study skin cancer. Jones, P. A., & Baylin, S. B. (2002). The fundamental role of epigenetic events in cancer. Nature Reviews Genetics, 3, 415-428. Citation Format: Jong Hun Lee, Limin Shu, Mou-Tuan Huang, Allan H. Conney, Ah-Ng Tony Kong. Epigenetic regulation in skin cancer: global DNA methylation profiling in DMBA/TPA induced mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5371. doi:10.1158/1538-7445.AM2013-5371

Abstract 3699: Chemoprevention of skin cancer by benzophenone sunscreen agents in mice.

Abstract Our recent study demonstrated the skin cancer chemoprevention potential of commonly used benzophenone sunscreen agents using the Epstein-Barr virus early antigen in vitro assay (Int. J. Cosmet. Sci. 2012: doi:10.1111/ics.12015). We now report the in vivo anti-tumor activity of two of the benzophenone sunscreens tested positive in the above in vitro assay, octabenzone (UV-1) and dioxybenzone (UV-2), in the two-stage mouse skin carcinogenesis test model utilizing (±)-(E)-4-methyl-2-[-(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) as inducer and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as promoter. Studies were conducted in pathogen-free, female hairless mice of HOS-HR-1 strain, 15 animals per control and test groups. Tumor was induced by a single dose of NOR-1 (390 nmol in 100 μl of acetone). One week later, TPA (1.7 nmol in 100 μl of acetone) was applied to skin twice weekly for 20 weeks as a tumor promoter. The test compounds, 0.0025% of UV-1 or UV-2 were administered to the animals through drinking water ad libitum, starting 1 week prior to tumor initiation and stopping 1 week after tumor initiation. All animals were examined weekly for the development of skin papillomas and the following were recorded: the week papillomas appeared, number of mice bearing papillomas and number of papillomas per mouse. In both UV-1 and UV-2 treated mice, 2-week delay in tumor appearance and significant inhibition of tumor incidence (50% and 60%, respectively) and burden (papilloma inhibition/mouse, 50% and 70%, respectively) were observed when compared to the positive control group. Also, treatment with both benzophenone sunscreens produced marked reduction in the average number of tumors formed per mouse (5 and 3 papillomas/mouse for UV-1 and UV-2, respectively compared to 10 papillomas in positive control group) at the end of the 20-week experimental period. These in vivo results in mice further affirm the skin cancer chemoprevention potential of benzophenone sunscreen use in humans. Citation Format: Govind J. Kapadia, G Subba Rao, Midori Takasaki, Akira Iida, Nobutaka Suzuki, Takao Konoshima, Harukuni Tokuda. Chemoprevention of skin cancer by benzophenone sunscreen agents in mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3699. doi:10.1158/1538-7445.AM2013-3699

Imiquimod: the biochemical mechanisms of immunomodulatory and anti-inflammatory activity

Imidazoquinolins represent a new group of compounds that recently entered into clinical practice as anti-tumor and anti-viral immune modulators. They are low molecular weight synthetic guanosine-like molecules. Although imiquimod, the most widely used imidazoquinolin, is recommended for the treatment of several forms of skin cancer and papillomas, the molecular mechanisms of its action are not fully understood. In particular, imiquimod has been characterized as a specific agonist of Toll-like receptor 7 (TLR7) and is widely used in this capacity in a large number of experimental studies and clinical trials. However, detailed analysis of the published data with the use of imiquimod, suggests that its biological activity can not be explained only by interaction with TLR7. There are indications of a direct interaction of imiquimod with adenosine receptors and other molecules that regulate the synthesis of cyclic adenosine monophosphate. A detailed understanding of the biochemical basis of imiquimod immunomodulating and antitumor effect will increase its clinical effectiveness and accelerate the development of new drugs with similar but improved medical properties. This review summarizes the published data concerning the effects of imiquimod on a variety of intracellular biochemical processes and signaling pathways.

Anticarcinogenic effect of quercetin by inhibition of insulin-like growth factor (IGF)-1 signaling in mouse skin cancer

It has been shown that dysregulation of IGF-1 signaling is associated with tumor incidence and progression, whereas blockade of the signaling can effectively inhibit carcinogenesis. Although several mechanisms of anticancer activity of quercetin were proposed, molecular targets of quercetin have not been identified yet. Hence, we assessed the effect of quercetin on IGF-1 signaling inhibition in BK5.IGF-1 transgenic (Tg) mice, which over-expresses IGF-1 in the skin epidermis. A quercetin diet (0.02% wt/wt) for 20 weeks remarkably delayed the incidence of skin tumor by 2 weeks and reduced tumor multiplicity by 35% in a 7,12-dimethylbenz(a)anthracene (DMBA)-tetradecanoyl phorbol-13-acetate (TPA) two stage mouse skin carcinogenesis protocol. Moreover, skin hyperplasia in Tg mice was significantly inhibited by a quercetin supplementation. Further analysis of the MT1/2 skin papilloma cell line showed that a quercetin treatment dose dependently suppressed IGF-1 induced phosphorylation of the IGF-1 receptor (IGF-1R), insulin receptor substrate (IRS)-1, Akt and S6K; however, had no effect on the phosphorylation of PTEN. Additionally, the quercetin treatment inhibited IGF-1 stimulated cell proliferation in a dose dependent manner. Taken together, these data suggest that quercetin has a potent anticancer activity through the inhibition of IGF-1 signaling.