SOX9 Transcriptionally Regulates mTOR-Induced Proliferation of Basal Cell Carcinomas.

Currently available smoothened targeted therapies in patients with basal cell nevus syndrome are associated with substantial tumor recurrence and clinical resistance. Strategies bypassing smoothened and/or identifying additional downstream components of the Hedgehog pathway could provide novel antitumor targets with a better therapeutic index. Sry-related high mobility group box 9 (SOX9) is a Hedgehog/glioma-associated oncogene homolog-regulated transcription factor known to be overexpressed in basal cell carcinomas (BCCs). A sequence motif search for SOX9-responsive elements identified three motifs in the promoter region of mammalian target of rapamycin (mTOR). In murine BCC cells, SOX9 occupies the mTOR promoter and induces its transcriptional activity. Short hairpin RNA (shRNA)-mediated knockdown of SOX9, as well as smoothened inhibition by itraconazole and vismodegib, reduces mTOR expression and the phosphorylation of known downstream mTOR targets. These effects culminate in diminishing the proliferative capacity of BCC cells, demonstrating a direct mechanistic link between the Hedgehog and mTOR pathways capable of driving BCC growth. Furthermore, rapamycin, a pharmacologic mTOR inhibitor, suppressed the growth of UV-induced BCCs in Ptch1+/-/SKH-1 mice, a model that closely mimics the accelerated BCC growth pattern of patients with basal cell nevus syndrome. Our data demonstrate that Hedgehog signaling converges on mTOR via SOX9, and highlight the SOX9-mTOR axis as a viable additional target downstream of smoothened that could enhance tumor elimination in patients with BCC.

Proteomic profiling of extracellular vesicles released from vascular smooth muscle cells during initiation of phosphate-induced mineralization.

Purpose/Aim: Elevated serum phosphate is one of the major factors contributing to vascular calcification. Studies suggested that extracellular vesicles released from vascular smooth muscle cells significantly contribute to the initiation and progression of this pathology. Recently, we have demonstrated that elevated phosphate stimulates release of extracellular vesicles from osteogenic cells at the initiation of the mineralization process. Here, we used MOVAS cell line as an in vitro model of vascular calcification to examine whether vascular smooth muscle cells respond to high phosphate levels in a similar way and increase formation of extracellular vesicles. MATERIALS AND METHODS: Vesicles residing in extracellular matrix as well as vesicles released to culture medium were evaluated by nanoparticle tracking analyses. In addition, using mass spectrometry and protein profiling, protein composition of extracellular vesicles released by MOVAS cells under standard growth conditions and upon exposure to high phosphate was compared. RESULTS: Significant increase of the number of extracellular vesicles was detected after 72 h of exposure of cells to high phosphate. Elevated phosphate levels also affected protein composition of extracellular vesicles released from MOVAS cells. Finally, the comparative analyses of proteins in extracellular vesicles isolated from extracellular matrix and from conditioned medium identified significant differences in protein composition in these two groups of extracellular vesicles. CONCLUSIONS: Results of this study demonstrate that exposure of MOVAS cells to high phosphate levels stimulates the release of extracellular vesicles and changes their protein composition.

Basal cell carcinoma pathogenesis and therapy involving hedgehog signaling and beyond.

Basal cell carcinoma (BCC) of the skin is driven by aberrant hedgehog signaling. Thus blocking this signaling pathway by small molecules such as vismodegib inhibits tumor growth. Primary cilium in the epidermal cells plays an integral role in the processing of hedgehog signaling-related proteins. Recent genomic studies point to the involvement of additional genetic mutations that might be associated with the development of BCCs, suggesting significance of other signaling pathways, such as WNT, NOTCH, mTOR, and Hippo, aside from hedgehog in the pathogenesis of this human neoplasm. Some of these pathways could be regulated by noncoding microRNA. Altered microRNA expression profile is recognized with the progression of these lesions. Stopping treatment with Smoothened (SMO) inhibitors often leads to tumor reoccurrence in the patients with basal cell nevus syndrome, who develop 10-100 of BCCs. In addition, the initial effectiveness of these SMO inhibitors is impaired due to the onset of mutations in the drug-binding domain of SMO. These data point to a need to develop strategies to overcome tumor recurrence and resistance and to enhance efficacy by developing novel single agent-based or multiple agents-based combinatorial approaches. Immunotherapy and photodynamic therapy could be additional successful approaches particularly if developed in combination with chemotherapy for inoperable and metastatic BCCs.

Naproxen Inhibits UVB-induced Basal Cell and Squamous Cell Carcinoma Development in Ptch1+/- /SKH-1 Hairless Mice.

Naproxen possesses anti-proliferative and pro-apoptotic effects besides its known anti-inflammatory functions. Here, we demonstrate the anticancer effects of naproxen against UVB-induced basal cell carcinoma (BCCs) and squamous cell carcinoma (SCCs) in a highly susceptible murine model of UVB carcinogenesis. Naproxen significantly inhibited UVB-induced BCCs and SCCs in this model. Tumor number and volume were significantly decreased (P < 0.005 and P < 0.05, respectively). Inhibition in UVB-induced SCCs and BCCs was 77% and 86%, respectively, which was associated with reduced PCNA and cyclin D1 and increased apoptosis. As expected, inflammation-related iNOS, COX-2 and nuclear NFκBp65 were also diminished by naproxen treatment. Residual tumors excised from naproxen-treated animal were less invasive and showed reduced expression of epithelial-mesenchymal transition (EMT) markers N-cadherin, Vimentin, Snail and Twist with increased expression of E-cadherin. In BCC and SCC cells, naproxen-induced apoptosis and activated unfolded protein response (UPR) signaling with increased expression of ATF4, p-eIF2α and CHOP. Employing iRNA-based approaches, we found that naproxen-induced apoptosis was regulated by CHOP as sensitivity of these cutaneous neoplastic cells for apoptosis was significantly diminished by ablating CHOP. In summary, these data show that naproxen is a potent inhibitor of UVB-induced skin carcinogenesis. ER stress pathway protein CHOP may play an important role in inducing apoptosis in cancer cells.

Retinoid X Receptor Agonists Upregulate Genes Responsible for the Biosynthesis of All-Trans-Retinoic Acid in Human Epidermis.

UAB30 is an RXR selective agonist that has been shown to have potential cancer chemopreventive properties. Due to high efficacy and low toxicity, it is currently being evaluated in human Phase I clinical trials by the National Cancer Institute. While UAB30 shows promise as a low toxicity chemopreventive drug, the mechanism of its action is not well understood. In this study, we investigated the effects of UAB30 on gene expression in human organotypic skin raft cultures and mouse epidermis. The results of this study indicate that treatment with UAB30 results in upregulation of genes responsible for the uptake and metabolism of all-trans-retinol to all-trans-retinoic acid (ATRA), the natural agonist of RAR nuclear receptors. Consistent with the increased expression of these genes, the steady-state levels of ATRA are elevated in human skin rafts. In ultraviolet B (UVB) irradiated mouse skin, the expression of ATRA target genes is found to be reduced. A reduced expression of ATRA sensitive genes is also observed in epidermis of mouse models of UVB-induced squamous cell carcinoma and basal cell carcinomas. However, treatment of mouse skin with UAB30 prior to UVB irradiation prevents the UVB-induced decrease in expression of some of the ATRA-responsive genes. Considering its positive effects on ATRA signaling in the epidermis and its low toxicity, UAB30 could be used as a chemoprophylactic agent in the treatment of non-melanoma skin cancer, particularly in organ transplant recipients and other high risk populations.

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro.

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

Shh and p50/Bcl3 signaling crosstalk drives pathogenesis of BCCs in Gorlin syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder that is due, in large measure, to aberrant Shh signaling driven by mutations in the tumor suppressor gene Ptch1. Here, we describe the development of Ptch1+/-/ SKH-1 mice as a novel model of this disease. These animals manifest many features of NBCCS, including developmental anomalies and are remarkably sensitive to both ultraviolet (UVB) and ionizing radiation that drive the development of multiple BCCs. Just as in patients with NBCCS, Ptch1+/-/SKH-1 also spontaneously develops BCCs and other neoplasms such as rhabdomyomas/rhabdomyosarcomas. Administration of smoothened inhibitors (vismodegib/itraconazole/cyclopamine) or non-steroidal anti-inflammatory drug (sulindac/sulfasalazine) each result in partial resolution of BCCs in these animals. However, combined administration of these agents inhibits the growth of UVB-induced BCCs by >90%. Employing small molecule- and decoy-peptide-based approaches we further affirm that complete remission of BCCs could only be achieved by combined inhibition of p50-NFκB/Bcl3 and Shh signaling. We posit that Ptch1+/-/SKH-1 mice are a novel and relevant animal model for NBCCS. Understanding mechanisms that govern genetic predisposition to BCCs should facilitate our ability to identify and treat NBCCS gene carriers, including those at risk for sporadic BCCs while accelerating development of novel therapeutic modalities for these patients.

Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch.

Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15±2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8±1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in tumors induced in these two murine models suggesting the specificity of Notch pathway in this regard. These data provide a novel role of ODC in augmenting tumorigenesis via negatively regulated Notch-mediated expansion of stem cell compartment.

Hair follicle disruption facilitates pathogenesis to UVB-induced cutaneous inflammation and basal cell carcinoma development in Ptch(+/-) mice.

Hairless mice carrying homozygous mutations in hairless gene manifest rudimentary hair follicles (HFs), epidermal cysts, hairless phenotype, and enhanced susceptibility to squamous cell carcinomas. However, their susceptibility to basal cell carcinomas (BCCs), a neoplasm considered originated from HF-localized stem cells, is unknown. To demonstrate the role of HFs in BCC development, we bred Ptch(+/-)/C57BL6 with SKH-1 hairless mice, followed by brother-sister cross to get F2 homozygous mutant (hairless) or wild-type (haired) mice. UVB-induced inflammation was less pronounced in shaved haired than in hairless mice. In hairless mice, inflammatory infiltrate was found around the rudimentary HFs and epidermal cysts. Expression of epidermal IL1f6, S100a8, vitamin D receptor, repetin, and major histocompatibility complex II, biomarkers depicting susceptibility to cutaneous inflammation, was also higher. In these animals, HF disruption altered susceptibility to UVB-induced BCCs. Tumor onset in hairless mice was 10 weeks earlier than in haired littermates. The incidence of BCCs was significantly higher in hairless than in haired animals; however, the magnitude of sonic hedgehog signaling did not differ significantly. Overall, 100% of hairless mice developed >12 tumors per mouse after 32 weeks of UVB therapy, whereas haired mice developed fewer than three tumors per mouse after 44 weeks of long-term UVB irradiation. Tumors in hairless mice were more aggressive than in haired littermates and manifested decreased E-cadherin and enhanced mesenchymal proteins. These data provide novel evidence that disruption of HFs in Ptch(+/-) mice enhances cutaneous susceptibility to inflammation and BCCs.

Erb-041, an estrogen receptor-ß agonist, inhibits skin photocarcinogenesis in SKH-1 hairless mice by downregulating the WNT signaling pathway.

Estrogen receptors (ER), including ER-α and ER-ß, are known to regulate multiple biologic responses in various cell types. The expression of ER-ß is lost in various cancers. ER-ß agonists were shown to modulate inflammation, cancer cell proliferation, and differentiation. Here, we investigated the cancer chemopreventive properties of Erb-041, an ER-ß agonist, using a model of UVB-induced photocarcinogenesis in SKH-1 mice. Erb-041 significantly reduced UVB-induced carcinogenesis. Tumor numbers and volume were reduced by 60% and 84%, respectively, in the Erb-041-treated group as compared with UVB (alone) control. This inhibition in tumorigenesis was accompanied by the decrease in proliferating cell nuclear antigen (PCNA), cyclin D1, VEGF, and CD31, and an increase in apoptosis. The lost ER-ß expression in squamous cell carcinomas (SCC) was significantly recovered by Erb-041 treatment. In addition, the UVB-induced inflammatory responses were remarkably reduced. Myeloperoxidase activity, levels of cytokines (interleukin (IL)-1ß, IL-6, and IL-10), and expression of p-ERK (extracellular signal-regulated kinase) 1/2, p-p38, p-IκB, iNOS, COX-2, and nuclear NF-κBp65 were diminished. The number of tumor-associated inflammatory cells (GR-1(+)/CD11b(+) and F4/80(+)) was also decreased. Tumors excised from Erb-041-treated animal were less invasive and showed reduced epithelial-mesenchymal transition (EMT). The enhanced expression of E-cadherin with the concomitantly reduced expression of N-cadherin, Snail, Slug, and Twist characterized these lesions. The WNT/ß-catenin signaling pathway, which underlies pathogenesis of skin cancer, was found to be downregulated by Erb-041 treatment. Similar but not identical changes in proliferation and EMT regulatory proteins were noticed following treatment of tumor cells with a WNT signaling inhibitor XAV939. Our results show that Erb-041 is a potent skin cancer chemopreventive agent that acts by dampening the WNT/ß-catenin signaling pathway.

Inhibiting cycloxygenase and ornithine decarboxylase by diclofenac and alpha-difluoromethylornithine blocks cutaneous SCCs by targeting Akt-ERK axis.

Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser(473) & thr(308)) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.

Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption.

Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses.

Nitric oxide-releasing sulindac is a novel skin cancer chemopreventive agent for UVB-induced photocarcinogenesis.

Nitric oxide (NO)-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) which have been synthesized to reduce gastro-intestinal and cardiovascular toxicities of NSAIDs, possess anti-proliferative, pro-apoptotic and anti-cancer activities. Here, we show that NO-sulindac inhibited UVB-induced skin tumorigenesis in SKH-1 hairless mice. Topical application of NO-sulindac reduced tumor incidence, number (p<0.05) and volume (p<0.005) as compared to UVB (alone)-irradiated vehicle-treated mice. An increase in TUNEL-positive cells in skin lesions was accompanied by the enhanced Bax:Bcl-2 ratio. The expression of pro-apoptotic Bax was increased whereas anti-apoptotic Bcl-2 reduced. However, proliferation was identified as the major target of NO-sulindac in this study. A reduced expression of PCNA and cyclin D1 associated with the dampening of cell cycle progression was observed. The mechanism of this inhibition was related to the reduction in UVB-induced Notch signaling pathway. UVB-induced inflammatory responses were diminished by NO-sulindac as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases Erk1/2, p38 and JNK1/2. In this regard, NO-sulindac also inhibited NFκB by enhancing IκBα as evidenced by the reduced expression of iNOS and COX-2, the direct NFκB transcription target proteins. NO-sulindac significantly diminished the progression of benign lesions to invasive carcinomas by suppressing the tumor aggressiveness and retarding epithelial-mesenchymal transition. A marked decrease in the expression of mesenchymal markers such as Fibronectin, N-cadherin, SNAI, Slug and Twist and an increase in epithelial cell polarity marker E-cadherin were noted in NO-sulindac-treated tumors. Our data suggest that NO-sulindac is a potent inhibitor of UVB-induced skin carcinogenesis and acts by targeting proliferation-regulatory pathways.

Vorinostat, an HDAC inhibitor attenuates epidermoid squamous cell carcinoma growth by dampening mTOR signaling pathway in a human xenograft murine model.

Histone deacetylase (HDAC) inhibitors are potent anticancer agents and show efficacy against various human neoplasms. Vorinostat is a potent HDAC inhibitor and has shown potential to inhibit growth of human xenograft tumors. However, its effect on the growth of skin neoplasm remains undefined. In this study, we show that vorinostat (2 µM) reduced expression of HDAC1, 2, 3, and 7 in epidermoid carcinoma A431 cells. Consistently, it increased acetylation of histone H3 and p53. Vorinostat (100mg/kg body weight, IP) treatment reduced human xenograft tumor growth in highly immunosuppressed nu/nu mice. Histologically, the vorinostat-treated tumor showed features of well-differentiation with large necrotic areas. Based on proliferating cell nuclear antigen (PCNA) staining and expression of cyclins D1, D2, E, and A, vorinostat seems to impair proliferation by down-regulating the expression of these proteins. However, it also induced apoptosis. The mechanism by which vorinostat blocks proliferation and makes tumor cells prone to apoptosis, involved inhibition of mTOR signaling which was accompanied by reduction in cell survival AKT and extracellular-signal regulated kinase (ERK) signaling pathways. Our data provide a novel mechanism-based therapeutic intervention for cutaneous squamous cell carcinoma (SCC). Vorinostat may be utilized to cure skin neoplasms in organ transplant recipient (OTR). These patients have high morbidity and surgical removal of these lesions which frequently develop in these patients, is difficult.

Metformin, an antidiabetic agent reduces growth of cutaneous squamous cell carcinoma by targeting mTOR signaling pathway.

The biguanide metformin is widely used for the treatment of Type-II diabetes. Its antiproliferative and pro-apoptotic effects in various tumor cells suggest its potential candidacy for cancer chemoprevention. Herein, we report that metformin significantly inhibited human epidermoid A431 tumor xenograft growth in nu/nu mice, which was associated with a significant reduction in proliferative biomarkers PCNA and cyclins D1/B1. This tumor growth reduction was accompanied by the enhanced apoptotic cell death and an increase in Bax:Bcl2 ratio. The mechanism by which metformin manifests antitumor effects appears to be dependent on the inhibition of nuclear factor kappa B (NFkB) and mTOR signaling pathways. Decreased phosphorylation of NFkB inhibitory protein IKBα together with reduced enhancement of NFkB transcriptional target proteins, iNOS/COX-2 were observed. In addition, a decrease in the activation of ERK/p38-driven MAP kinase signaling was seen. Similarly, AKT signaling activation as assessed by the diminished phosphorylation at Ser473 with a concomitant decrease in mTOR signaling pathway was also noted as phosphorylation of mTOR regulatory proteins p70S6K and 4E-BP-1 was significantly reduced. Consistently, decreased phosphorylation of GSK3ß, which is carried out by AKT kinases was also observed. These results suggest that metformin blocks SCC growth by dampening NFkB and mTOR signaling pathways.

Nitric oxide donor exisulind is an effective inhibitor of murine photocarcinogenesis.

NO-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) have been shown to have anti-inflammatory, antiproliferative and apoptosis-inducing effects in tumor cells. Herein, we have investigated the effects of NO-exisulind on the growth of UVB-induced skin tumor development in a murine model. We found that the topical treatment with NO-exisulind significantly reduced UVB-induced tumors in SKH-1 hairless mice. The tumors/tumor bearing mouse, the number of tumors/mouse and tumor volume/mouse decreased significantly (P < 0.05) as compared with vehicle-treated and UVB-irradiated positive controls. Consistently, NO-exisulind-treated animals showed reduced expression of proliferation markers, such as PCNA and cyclin D1. These mice also manifested increased expression of proapoptotic Bax and decreased expression of antiapoptotic Bcl2 with an increase in the number of TUNEL-positive cells in tumors. We also investigated whether NO-exisulind-treated tumors are less invasive and progress less efficiently from benign to malignant carcinomas. For this, tumors were stained for various epithelial-mesenchymal transition (EMT) markers. NO-exisulind decreased the expression of mesenchymal markers, such as Fibronectin, N-cadherin, SNAI, Slug and Twist and enhanced the epithelial marker E-cadherin. Similarly, UVB-induced phosphorylation of Erk1/2 and p38 was decreased in NO-exisulind-treated animals. These data suggest that NO-exisulind reduces tumor growth and inhibits tumor progression by blocking proliferation, inducing apoptosis and reducing EMT.

Unfolded protein response signaling and MAP kinase pathways underlie pathogenesis of arsenic-induced cutaneous inflammation.

Arsenic exposure through drinking water is a major global public health problem and is associated with an enhanced risk of various cancers including skin cancer. In human skin, arsenic induces precancerous melanosis and keratosis, which may progress to basal cell and squamous cell carcinoma. However, the mechanism by which these pathophysiologic alterations occur remains elusive. In this study, we showed that subchronic arsenic exposure to SKH-1 mice induced unfolded protein response (UPR) signaling regulated by proteins, inositol-requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). Arsenic activated all three UPR regulatory proteins in the skin. Arsenic induced IRE1 phosphorylation which resulted in augmented splicing of X-box binding protein 1 (XBP-1) leading to its migration to the nucleus, and also enhanced transcriptional activation of downstream target proteins. Hyperphosphorylation of PERK which induces eukaryotic translation initial factor 2α (eIF2α) in a phosphorylation-dependent manner enhanced translation of ATF4, in addition to augmenting proteolytic activation of ATF6 in arsenic-treated skin. A similar increase in the expression of CHOP was observed. Enhanced XBP-1s, ATF4, and ATF6 regulated downstream chaperones GRP94 and GRP78. In addition, arsenic induced inflammation-related p38/MAPKAPK-2 MAPK signaling and alterations in Th-1/Th-2/Th-17 cytokines/chemokines and their receptors. Antioxidant N-acetyl cysteine blocked arsenic-induced reactive oxygen species, with a concomitant attenuation of UPR and mitogen-activated protein kinase (MAPK) signaling and proinflammatory cytokine/chemokine signatures. Our results identify novel pathways involved in the pathogenesis of arsenic-mediated cutaneous inflammation which may also be related to enhanced cancer risk in arsenic exposed cohorts.