TPA-induced Epidermal Hyperplasia Research Articles

3′-Hydroxypterostilbene Inhibits 7,12-Dimethylbenz[a]anthracene (DMBA)/12-O-Tetradecanoylphorbol-13-Acetate (TPA)-Induced Mouse Skin Carcinogenesis

BackgroundA natural pterostilbene analogue isolated from the herb Sphaerophysa salsula, 3′-hydroxypterostilbene (HPSB), exhibits antiproliferative activity in several cancer cell lines; however, the inhibitory effects of HPSB on skin carcinogenesis remains unclear. PurposeThe aim of this study was to evaluate the inhibitory effects of HPSB on two-stage skin carcinogenesis in mice and its potential mechanism. Study Design and MethodsThis study investigated the anti-inflammatory and anti-tumor effects of HPSB in the 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated acute skin inflammation and 7,12-dimethylbenz[a]anthracene (DMBA)/TPA-induced two-stage skin carcinogenesis model. In addition, the effects of HPSB on the modulation of the phase I and phase II metabolizing enzymes in the DMBA-induced HaCaT cell model were investigated. ResultsThe results provide evidence that topical treatment with HPSB significantly inhibits TPA-induced epidermal hyperplasia and leukocyte infiltration through the down-regulation of cyclooxygenase-2 (COX-2), matrix metalloprotein-9 (MMP-9), and ornithine decarboxylase (ODC) protein expression in mouse skin. Furthermore, HPSB suppresses DMBA/TPA-induced skin tumor incidence and multiplicity via the inhibition of proliferating cell nuclear antigen (PCNA), Cyclin B1 and cyclin-dependent kinase 1 (CDK1) expression in the two-stage skin carcinogenesis model. In addition, pretreatment with HPSB markedly reduces DMBA-induced cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) gene expression in human keratinocytes; however, HPSB does not significantly affect the gene expression of the phase II enzymes. ConclusionThis is the first study to show that topical treatment with HPSB prevents mouse skin tumorigenesis. Overall, our study suggests that natural HPSB may serve as a novel chemopreventive agent capable of preventing carcinogen activation and inflammation-associated tumorigenesis.

Fucoxanthin-Containing Cream Prevents Epidermal Hyperplasia and UVB-Induced Skin Erythema in Mice.

Microalgae represent a source of bio-active compounds such as carotenoids with potent anti-inflammatory and antioxidant properties. We aimed to investigate the effects of fucoxanthin (FX) in both in vitro and in vivo skin models. Firstly, its anti-inflammatory activity was evaluated in LPS-stimulated THP-1 macrophages and TNF-α-stimulated HaCaT keratinocytes, and its antioxidant activity in UVB-irradiated HaCaT cells. Next, in vitro and ex vivo permeation studies were developed to determine the most suitable formulation for in vivo FX topical application. Then, we evaluated the effects of a FX-containing cream on TPA-induced epidermal hyperplasia in mice, as well as on UVB-induced acute erythema in hairless mice. Our results confirmed the in vitro reduction of TNF-α, IL-6, ROS and LDH production. Since the permeation results showed that cream was the most favourable vehicle, FX-cream was elaborated. This formulation effectively ameliorated TPA-induced hyperplasia, by reducing skin edema, epidermal thickness, MPO activity and COX-2 expression. Moreover, FX-cream reduced UVB-induced erythema through down-regulation of COX-2 and iNOS as well as up-regulation of HO-1 protein via Nrf-2 pathway. In conclusion, FX, administered in a topical formulation, could be a novel natural adjuvant for preventing exacerbations associated with skin inflammatory pathologies as well as protecting skin against UV radiation.

Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-κB-Cox2 Signaling.

BackgroundA recent focus in skin cancer prevention intervenes though modulating molecular links between inflammation and cell growth signaling, such as NF-κB. This study elucidates the effect of a non-tumor promoting phorbol ester, ingenol-3-angelate (I3A), on the growth of human melanoma cells and on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation and 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin carcinoma in mice.Material/MethodsCell viability was assessed by MTT assay, cell proliferation by clonogenic assay, apoptosis and cell cycle arrest was analyzed by flow cytometry, protein expression was studied by IHC and Western blotting, and gene expression by qPCR.ResultsI3A suppressed the survival and proliferation of human melanoma cells with estimated IC50 values around 38 and 46 μM for A2058 and HT144 cell, respectively. I3A activated the protein levels of PKCδ and PKCɛ, which induced apoptosis by activating caspase-9 and caspace-3 followed by lowering of mitochondrial membrane potential and enhancing DNA fragmentation. I3A induced G1 phase cell cycle arrest as well as G2/M phase arrest in both cell lines. I3A inhibited the levels of NF-κB p65 protein as well as phosphorylation of p65 and its nuclear translocation. I3A suppressed the gene expression of NF-κB, COX-2 and iNOS. I3A inhibited TPA-induced inflammation and epidermal hyperplasia in female ICR mice by downregulating NF-κB and iNOS. I3A suppressed the growth of skin tumor in DMBA-induced mice in dose-dependent manner.ConclusionsThe mechanism of I3A induces apoptosis in human melanoma cells and suppresses skin inflammation and carcinoma via downregulation of NF-κB-iNOS-COX-2 signaling.

Topical Application of Glycolipids from Isochrysis galbana Prevents Epidermal Hyperplasia in Mice.

Chronic inflammatory skin diseases such as psoriasis have a significant impact on society. Currently, the major topical treatments have many side effects, making their continued use in patients difficult. Microalgae have emerged as a source of bio-active molecules such as glycolipids with potent anti-inflammatory properties. We aimed to investigate the effects of a glycolipid (MGMG-A) and a glycolipid fraction (MGDG) obtained from the microalga Isochrysis galbana on a TPA-induced epidermal hyperplasia murine model. In a first set of experiments, we examined the preventive effects of MGMG-A and MGDG dissolved in acetone on TPA-induced hyperplasia model in mice. In a second step, we performed an in vivo permeability study by using rhodamine-containing cream, ointment, or gel to determinate the formulation that preserves the skin architecture and reaches deeper. The selected formulation was assayed to ensure the stability and enhanced permeation properties of the samples in an ex vivo experiment. Finally, MGDG-containing cream was assessed in the hyperplasia murine model. The results showed that pre-treatment with acetone-dissolved glycolipids reduced skin edema, epidermal thickness, and pro-inflammatory cytokine production (TNF-α, IL-1β, IL-6, IL-17) in epidermal tissue. The in vivo and ex vivo permeation studies showed that the cream formulation had the best permeability profile. In the same way, MGDG-cream formulation showed better permeation than acetone-dissolved preparation. MGDG-cream application attenuated TPA-induced skin edema, improved histopathological features, and showed a reduction of the inflammatory cell infiltrate. In addition, this formulation inhibited epidermal expression of COX-2 in a similar way to dexamethasone. Our results suggest that an MGDG-containing cream could be an emerging therapeutic strategy for the treatment of inflammatory skin pathologies such as psoriasis.

Abstract 4617: Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion

Abstract Considerable evidence indicates a crucial role for Akt and mTORC1 signaling in cancer. Previously, we discovered that the Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. The proline-rich Akt substrate of 40 kDa (PRAS40), a subunit of mTORC1 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246 and this phosphorylation is critical for mTORC1 activity. Moreover, PRAS40 has been identified to negatively regulate mTORC1 signaling. In this study, transgenic mice were generated that overexpress a mutant form of PRAS40 [PRAS40(T246A)] using the bovine keratin 5 (BK5) promoter to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40(T246A) transgenic mice did not have a discernable gross skin phenotype. However, these mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia. Overexpression of PRAS40(T246A) in the epidermis led to significant suppression of skin tumor development and significant reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis of epidermal protein lysates from BK5.PRAS40(T246A) mice showed that PRAS40(T246A) remained bound to raptor even after TPA treatment. BK5.PRAS40(T246A) mice also displayed reduced mTORC1 signaling, reduced levels of cell cycle proteins and increased autophagy signaling as well as the skin inflammatory response in the epidermis following treatment with TPA. Notably, TPA-induced proliferation and migration of bulge-region label retaining cells were also significantly inhibited in BK5.PRAS40(T246A) mice. In addition, expression of PRAS40(T246A) in basal keratinocytes significantly inhibited keratinocyte migration both in vivo and in vitro and led to delayed wound healing. Decreased migration and impaired wound healing may be attributed to altered expression of EMT markers as identified in PRAS40(T246A) epidermal cells. The current data using this novel mouse model provides further evidence that mTORC1 specific signaling in keratinocytes contributes significantly to the process of skin tumor promotion by regulating proliferation and migration of keratinocyte stem cells. In addition, the data demonstrate an important role for mTORC1 specific signaling in keratinocytes in regulating skin inflammation. Collectively, using this novel mouse model where mTORC1 signaling is selectively suppressed in keratinocytes has provided further evidence that this signaling pathway regulates several important mechanistic events involved in the process of skin tumor promotion. Research supported by NIH Grants CA129409, CA037111, and American Cancer Research Center and Foundation Citation Format: Okkyung Rho, Jaya Srivastava, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 by overexpression of PRAS40(T246A) in basal keratinocytes suppresses proliferation and migration of keratinocyte stem cells and skin inflammation during tumor promotion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4617.

Role of the Slug Transcription Factor in Chemically-Induced Skin Cancer

The Slug transcription factor plays an important role in ultraviolet radiation (UVR)-induced skin carcinogenesis, particularly in the epithelial-mesenchymal transition (EMT) occurring during tumor progression. In the present studies, we investigated the role of Slug in two-stage chemical skin carcinogenesis. Slug and the related transcription factor Snail were expressed at high levels in skin tumors induced by 7,12-dimethylbenz[α]anthracene application followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. TPA-induced transient elevation of Slug and Snail proteins in normal mouse epidermis and studies in Slug transgenic mice indicated that Slug modulates TPA-induced epidermal hyperplasia and cutaneous inflammation. Although Snail family factors have been linked to inflammation via interactions with the cyclooxygenase-2 (COX-2) pathway, a pathway that also plays an important role in skin carcinogenesis, transient TPA induction of Slug and Snail appeared unrelated to COX-2 expression. In cultured human keratinocytes, TPA induced Snail mRNA expression while suppressing Slug expression, and this differential regulation was due specifically to activation of the TPA receptor. These studies show that Slug and Snail exhibit similar patterns of expression during both UVR and chemical skin carcinogenesis, that Slug and Snail can be differentially regulated under some conditions and that in vitro findings may not recapitulate in vivo results.

Periplogenin induces necroptotic cell death through oxidative stress in HaCaT cells and ameliorates skin lesions in the TPA- and IMQ-induced psoriasis-like mouse models

Psoriasis is a multifactorial skin disease that inconveniences many patients. Considering the side effects and drug resistance of the current therapy, it is urgent to discover more effective and safer anti-psoriatic drugs. In the present study, we screened over 250 traditional Chinese medicine compounds for their ability to inhibit the cell viability of cultured human HaCaT keratinocytes, a psoriasis-relevant in vitro model, and found that periplogenin was highly effective. Mechanistic studies revealed that apoptosis and autophagy were not induced by periplogenin in HaCaT cells. However, periplogenin caused PI to permeate into cells, increased lactate LDH release and rapidly increased the number of necrotic cells. Additionally, the typical characteristics of necrosis were observed in the periplogenin-treated HaCaT cells. Notably, the necroptosis inhibitor Nec-1 and NSA were able to rescue the cells from necrotic cell death, supporting that necroptosis was involved in periplogenin-induced cell death. Furthermore, the ROS levels were elevated in the periplogenin-treated cells, NAC (an antioxidant) and Nec-1 could inhibit the ROS levels, and NAC could attenuate necroptotic cell death, indicating that the periplogenin-induced necroptotic cell death was mediated by oxidative stress. More importantly, in the murine models of TPA-induced epidermal hyperplasia and IMQ-induced skin inflammation, topical administration of periplogenin ameliorated skin lesions and inflammation. In sum, our results indicate, for the first time, that periplogenin is a naturally occurring compound with potent anti-psoriatic effects in vitro and in vivo, making it a promising candidate for future drug research.

Abstract 5357: Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice

Abstract Considerable evidence indicates a crucial role of Akt and mTORC1 signaling in cancer growth and progression. Previously, we discovered that Akt and mTORC1 signaling pathways play an important role in epithelial multistage carcinogenesis using the mouse skin model, especially during the tumor promotion stage. Targeting mTORC1 with rapamycin effectively blocked mTORC1 downstream signaling, epidermal hyperproliferation induced by TPA in a dose-dependent manner in both wild-type and BK5.AktWT transgenic mice. Rapamycin effectively inhibited TPA skin tumor promotion in TPA-hypersensitive BK5.AktWT transgenic mice although higher doses were required to achieve similar levels of inhibition compared to wild-type mice. Elevated Akt expression led to a significant expansion of the label retaining cells (LRCs) as well as a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K and phospho-PRAS40 compared to wild-type mice. Furthermore, TPA-mediated proliferation and migration of LRCs to the interfollicular epidermis from the hair follicle were significantly inhibited by rapamycin. PRAS40 was originally identified as a novel Akt substrate through Akt-mediated phosphorylation at Thr246. Recently, PRAS40 has been identified as a negative regulator when bound to mTORC1. We have used the bovine keratin 5 (BK5) promoter to generate transgenic mice that overexpress a mutant form of PRAS40 (PRAS40T246A) to further examine the importance of mTORC1 signaling in epithelial carcinogenesis. BK5.PRAS40T246A transgenic mice were significantly less sensitive to TPA-induced epidermal hyperproliferation and hyperplasia measured by both epidermal thickness and labeling index. Overexpression of mutant PRAS40T246A in the epidermis led to a significant suppression of skin tumor development by 63% as well as reductions in tumor weight and tumor size compared to wild-type mice. Immunoprecipitation analysis showed that PRAS40T246A in BK5.PRAS40T246A mice remained bound to raptor even after TPA treatment. In addition, BK5.PRAS40T246A mice displayed reduced TPA-induced mTORC1 signaling and cell cycle progression, but increased autophagy signaling and elevated apoptotic cells in the epidermis following TPA treatment. Furthermore, TPA-induced proliferation and migration of LRCs was also effectively inhibited in BK5.PRAS40T246A mice. Collectively, the current data demonstrate that mTORC1 downstream signaling pathways contribute significantly to the process of skin tumor promotion leading to a potential target for chemoprevention of cancer. Supported by CA037111 and CA0129409. Citation Format: Okkyung Rho, Jiyoon Cho, John DiGiovanni. Targeting mTORC1 suppresses proliferation of keratinocyte stem cells and inhibits skin tumor promotion in mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2014-5357

Epidermal p65/NF-κB signalling is essential for skin carcinogenesis.

The nuclear factor kappa B (NF-κB) signalling pathway exhibits both tumour-promoting and tumour-suppressing functions in different tissues and models of carcinogenesis. In particular in epidermal keratinocytes, NF-κB signalling was reported to exert primarily growth inhibitory and tumour-suppressing functions. Here, we show that mice with keratinocyte-restricted p65/RelA deficiency were resistant to 7, 12-dimethylbenz(a)anthracene (DMBA)-/12-O-tetra decanoylphorbol-13 acetate (TPA)-induced skin carcinogenesis. p65 deficiency sensitized epidermal keratinocytes to DNA damage-induced death in vivo and in vitro, suggesting that inhibition of p65-dependent prosurvival functions prevented tumour initiation by facilitating the elimination of cells carrying damaged DNA. In addition, lack of p65 strongly inhibited TPA-induced epidermal hyperplasia and skin inflammation by suppressing the expression of proinflammatory cytokines and chemokines by epidermal keratinocytes. Therefore, p65-dependent NF-κB signalling in keratinocytes promotes DMBA-/TPA-induced skin carcinogenesis by protecting keratinocytes from DNA damage-induced death and facilitating the establishment of a tumour-nurturing proinflammatory microenvironment.

Chemopreventive effect of a novel, selective TACE inhibitor on DMBA- and TPA-induced skin carcinogenesis

Context: Tumor necrosis factor (TNF)-α, a potent proinflammatory cytokine, plays a major role in the pathogenesis of cancer. TNF-α converting enzyme (TACE) mediates processing and release of biologically active TNF-α.Objective: We aimed to investigate the effect of a novel, selective TACE inhibitor (compound 11p) on skin inflammation and associated tumorigenesis in mice.Methods: Skin edema was induced in mice by dermal application 12-O-tetradecanoylphorbol-13-acetate (TPA) solution in acetone on to the ear and the effect of post-treatment of compound 11p (topical application) was evaluated. Edema and inflammation was assessed by measuring ear thickness, weight of skin punch and cytokine levels. Skin cancer in mice was initiated by single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by repeated TPA application for 20 weeks. The effect of compound 11p on papilloma incidence and multiplicity was evaluated.Results: Treatment with compound 11p strongly suppressed TPA-induced elevation in skin thickness and weight. A dose-dependent suppression in TPA-mediated TNF-α, IL-6, IFN-γ, IL-17 and PGE2 levels which was associated with a decrease in infiltration of inflammatory cells was also observed with the treatment. Moreover, compound 11p treatment delayed the onset, markedly reduced the papilloma incidence and multiplicity persuaded by DMBA and TPA.Discussion and conclusion: These findings suggest that selective blockade of TACE suppresses TPA-induced epidermal hyperplasia, inflammatory cell infiltration and cytokine level. Inhibition of inflammatory events related to tumor growth might have led to the anti-tumor effect in mouse skin cancer model induced by DMBA and TPA.

Impact of mTORC1 inhibition on keratinocyte proliferation during skin tumor promotion in wild‐type and BK5.AktWT mice

In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.Akt(WT) mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2-4 h) was mediated by activation of EGFR and Akt. BK5.Akt(WT) transgenic mice, where Akt1 is overexpressed in basal epidermis, are highly sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 with rapamycin effectively inhibited TPA-induced epidermal hyperplasia and hyperproliferation as well as tumor promotion in a dose-dependent manner in both wild-type and BK5.Akt(WT) mice. A significant expansion (∼threefold) of the label retaining cell (LRC) population per hair follicle was observed in BK5.Akt(WT) mice compared to FVB/N mice. There was also a significant increase in K15 expressing cells in the hair follicle of transgenic mice that coincided with expression of phospho-Akt, phospho-S6K, and phospho-PRAS40, suggesting an important role of mTORC1 signaling in bulge-region keratinocyte stem cell (KSC) homeostasis. After 2 weeks of TPA treatment, LRCs had moved upward into the interfollicular epidermis from the bulge region of both wild-type and BK5.Akt(WT) mice. TPA-mediated LRC proliferation and migration was significantly inhibited by rapamycin. Collectively, the current data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of the target cells for tumor development.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Abstract 1302: Targeting mTORC1 by epidermal overexpression of PRAS40(T246A) suppresses TPA-induced epidermal hyperplasia during tumor promotion.

Abstract Akt activation is an important event during the early stages of skin tumor promotion by diverse tumor promoting stimuli. Transgenic mice overexpressing either wild-type Akt1 (Aktwt) or a constitutively active form of Akt1 (Aktmyr) exhibit dramatically enhanced susceptibility to two-stage skin carcinogenesis and skin tumor promotion. Several downstream Akt targets are modulated (i.e., increased phosphorylation) in keratinocytes during tumor promotion, including the mammalian target of rapamycin (mTORC1). Recent experiments show that inhibiting mTORC1 by using topical rapamycin in both wild-type and BK5.Aktwt transgenic mice can effectively block TPA-induced activation of mTORC1 signaling, epidermal hyperproliferation and skin tumor promotion in a dose-dependent manner. Treatment with rapamycin also inhibited TPA-induced proliferation and migration of labeling retaining cells (LRCs) of the bulge region from the hair follicles to upper epidermal compartments. To further examine the importance of mTORC1 signaling in epithelial carcinogenesis, we generated BK5.PRAS40mt (PRAS40T246A) mice using the BK5 vector that targets expression to the basal layer of epidermis. PRAS40 was originally identified as a novel Akt substrate with a phosphorylation site at Thr246 and acts as a negative regulator when bound to mTORC1. PRAS40T246A expressed in BK5.PRAS40mt mice remained bound to raptor even after TPA treatment as determined by immunoprecipitation analysis. BK5.PRAS40mt mice displayed reduced phosphorylation of both S6K (T389) and 4EBP1 (S65) in epidermis following treatment with TPA. Furthermore, these transgenic mice were significantly less sensitive to TPA-induced epidermal proliferation (measured by both epidermal thickness and labeling index) compared to nontransgenic siblings. TPA-mediated migration and proliferation of LRCs was also inhibited in BK5.PRAS40mt mice. BK5.PRAS40mt mice also expressed lower levels of cell cycle proteins in epidermis after TPA treatment. Collectively, the current data further support the hypothesis that downstream signaling pathways through mTORC1 contribute significantly to the process of skin tumor promotion through effects on proliferation. Supported by CA37111 and 129409. Citation Format: Okkyung Rho, John DiGiovanni. Targeting mTORC1 by epidermal overexpression of PRAS40(T246A) suppresses TPA-induced epidermal hyperplasia during tumor promotion. [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 1302. doi:10.1158/1538-7445.AM2013-1302

Abstract 2559: Targeting mTORC1 may provide selectivity for inhibiting proliferation of hair follicle stem/progenitor cells during tumor promotion

Abstract Activation of Akt occurs during tumor promoter treatment of mouse skin and plays an important role in skin tumor promotion through multiple downstream pathways, including mTORC1. In this study, we examined the impact of rapamycin on mTORC1 signaling in relation to its effect on TPA-induced epidermal proliferation and skin tumor promotion. TPA activates mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and an immortalized mouse keratinocyte cell line. Early activation (15-30 min) of mTORC1 signaling induced by TPA was mediated in part, by PKC activation, whereas the later activation (2-4 hr) of mTORC1 was mediated by activation of EGFR and Akt. BK5.AktWT transgenic mice where Akt1 is overexpressed in basal epidermis were significantly more sensitive to TPA-induced epidermal proliferation and two-stage skin carcinogenesis. Targeting mTORC1 effectively inhibited TPA-induced epidermal hyperplasia as well as tumor promotion in epidermis of both wild-type and BK5.AktWT mice in a dose-dependent manner. We also examined the effect of rapamycin on the proliferation and migration of bulge-region KSCs from the hair follicles induced by TPA. Ten-day old mice were injected with BrdU every 12h over a 48h time period and then 70 days later mice were treated with acetone, TPA or rapamycin and TPA. Seventy days after the last injection of BrdU, only the label retaining cells (LRCs) were seen in the bulge region of the hair follicles. BK5.Akt1WT transgenic mice, which overexpress Akt1 in the K5 compartment of mouse epidermis (which includes bulge region KSCs) led to significant expansion of LRCs (∼3-fold) per hair follicle compared with wild-type FVB mice. There was also a significant expansion of the K15 expressing cell population, especially in the hair follicle that coincided with expression of phospho-Akt. In addition, a significant increase in phospho-S6K and phospho-PRAS40 was seen in the hair follicles, suggesting an important role of mTORC1 signaling in the alterations seen in terms of LRCs and K15 expressing cells. After two weeks of TPA treatment, LRCs were reduced in the bulge region and moved upward into the interfollicular epidermis. Treatment with rapamycin inhibited the migration and proliferation of the LRCs from the hair follicles to upper epidermal compartments induced by TPA. Furthermore, LRCs remained in the bulge region of hair follicles. Since the bulge-region KSCs are considered to be the key target population for tumor development in the two-stage skin carcinogenesis model, these data indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion through effects on proliferation of multiple keratinocyte populations including bulge-region KSCs. Thus, blocking this pathway may be an effective strategy for prevention of epithelial carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2559. doi:1538-7445.AM2012-2559

Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate

Aberrant activation of phosphoinositide-3-kinase (PI3K)/Akt signaling has been implicated in the development and progression of multiple human cancers. During the process of skin tumor promotion induced by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), activation of epidermal Akt occurs as well as several downstream effectors of Akt, including the activation of mTORC1. Rapamycin, an established mTORC1 inhibitor, was used to further explore the role of mTORC1 signaling in epithelial carcinogenesis, specifically during the tumor promotion stage. Rapamycin blocked TPA-induced activation of mTORC1 as well as several downstream targets. In addition, TPA-induced epidermal hyperproliferation and hyperplasia were inhibited in a dose-dependent manner with topical rapamycin treatments. Immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin also significantly decreased the number of infiltrating macrophages, T cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Using a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as the promoter, rapamycin (5-200 nmol per mouse given topically 30 minutes prior to TPA) exerted a powerful antipromoting effect, reducing both tumor incidence and tumor multiplicity. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited further growth. Overall, the data indicate that rapamycin is a potent inhibitor of skin tumor promotion and suggest that signaling through mTORC1 contributes significantly to the process of skin tumor promotion. The data also suggest that blocking this pathway either alone or in combination with other agents targeting additional pathways may be an effective strategy for prevention of epithelial carcinogenesis.

Tumor Necrosis Factor-Alpha-Nuclear Factor-Kappa B-Signaling Enhances St2b2 Expression during 12-O-Tetradecanoylphorbol-13-acetate-Induced Epidermal Hyperplasia

The mouse cholesterol sulfotransferase St2b2 contributes to epidermal differentiation by biosynthesizing cholesterol sulfate (CS) from cholesterol in the epidermis. 12-O-Tetradecanoylphorbol-13-acetate (TPA) causes epidermal hyperplasia, an abnormal increase in epidermal cell numbers resulting from aberrant cell differentiation and an increase in St2b2 protein levels. The mechanisms underlying enhanced St2b2 expression and the pathophysiologic significance of the increased expression are unclear, however. To verify whether increased St2b2 levels are necessary for TPA-induced epidermal hyperplasia, the effects of St2b2-specific small hairpin RNA (St2b2-shRNA) on hyperplasia were examined in mice. St2b2-shRNA clearly suppressed TPA-induced epidermal hyperplasia and the expression of a marker of epidermal differentiation, involucrin (INV). Interestingly, treating mouse epidermal cells with tumor necrosis factor-alpha (TNFα) increased St2b2 expression. Furthermore, treatment with TNFα-siRNA or anti-TNF receptor antibodies reduced the TPA-induced enhancement of St2b2 expression. Treatment with BAY 11-7082, a specific inhibitor of nuclear factor-kappa B (NF-κB), diminished TPA-induced St2b2 expression. These results suggested that enhancement of St2b2 expression by TPA treatment occurs mainly through the TNFα-NF-κB inflammatory signaling pathway, which in turn leads to increased CS concentrations in epidermal cells and hyperplasia.

Topical N-acetyl-S-farnesyl-L-cysteine Inhibits Mouse Skin Inflammation, and Unlike Dexamethasone, its Effects Are Restricted to the Application Site

N-acetyl-S-farnesyl-L-cysteine (AFC), a modulator of G protein and G-protein coupled receptor signaling, inhibits neutrophil chemotaxis and other inflammatory responses in cell-based assays. Here, we show topical AFC inhibits in vivo acute inflammation induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and arachidonic acid using the mouse ear model of inflammation. AFC inhibits edema, as measured by ear weight, and also inhibits neutrophil infiltration as assayed by direct counting in histological sections and by measuring myeloperoxidase (MPO) activity as a neutrophil marker. In addition, AFC inhibits in vivo allergic contact dermatitis in a mouse model utilizing sensitization followed by a subsequent challenge with 2,4-dinitrofluorobenzene. Unlike the established anti-inflammatories dexamethasone and indomethacin, AFC's action was restricted to the site of application. In this mouse model, both dexamethasone and indomethacin inhibited TPA-induced edema and MPO activity in the vehicle-treated, contralateral ear. AFC showed no contralateral ear inhibition for either of these end points. A marginally significant decrease due to AFC treatment was seen in TPA-induced epidermal hyperplasia at 24 hours. This was much less than the 90% inhibition of neutrophil infiltration, suggesting that AFC does not act by directly inhibiting protein kinase C.

RAGE signaling sustains inflammation and promotes tumor development

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

Antipsoriatic effects of avarol‐3′‐thiosalicylate are mediated by inhibition of TNF‐αgeneration and NF‐κB activation in mouse skin

Avarol is a marine sesquiterpenoid hydroquinone with anti-inflammatory and antipsoriatic properties. The aim of this study was to evaluate the in vitro and in vivo pharmacological behaviour of the derivative avarol-3'-thiosalicylate (TA) on some inflammatory parameters related to the pathogenesis of psoriasis. Human neutrophils and monocytes as well as the human keratinocyte cell line HaCaT were used to study the effect of TA on oxidative stress, the arachidonic acid pathway, tumour necrosis factor-alpha (TNF-alpha) release and nuclear factor-kappaB (NF-kappaB) activation. All these parameters were also determined in vivo using the zymosan induced mouse air pouch model and the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced mouse epidermal hyperplasia model. TA showed antioxidant properties in human neutrophils and in the hypoxanthine/xanthine oxidase assay. This compound reduced, in a concentration-dependent manner, leukotriene B(4), prostaglandin E(2) and TNF-alpha production in activated leukocytes. Oral and intrapouch administration of TA in the mouse air pouch model produced a dose-dependent reduction of all these inflammatory mediators. TA also inhibited secretory phospholipase A(2) activity and NF-kappaB DNA-binding in HaCaT keratinocytes. In TPA-induced mouse epidermal hyperplasia, topical administration of TA reduced oedema, leukocyte infiltration, eicosanoid levels and TNF-alpha in skin. In addition, interleukin (IL)-1beta and IL-2 production were also inhibited. Finally, TA was also capable of suppressing NF-kappaB nuclear translocation in vivo. TA inhibited several key biomarkers up-regulated in the inflammatory response of psoriatic skin and this compound could be a promising antipsoriatic agent.

Growth stimulation of UV-induced DNA damage retaining epidermal basal cells gives rise to clusters of p53 overexpressing cells

Ultraviolet (UV) radiation induces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4)PPs) in DNA, which may give rise to clusters of cells expressing mutant p53 (‘p53 patches’) and eventually to skin carcinomas. We have previously reported that some basal cells in murine skin accumulate CPDs upon chronic low-level UV exposure and that these CPD-retaining basal cells (CRBCs) encompass epidermal stem and progenitor cells. Through replication of their damaged DNA CRBCs may become mutagenic foci from which tumors might form. We therefore investigated whether CRBCs may give rise to p53 patches after forced proliferation by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). CRBCs, induced in SKH-1 hairless mice by chronic low-level UV exposure (70J/m2 daily for 40 days), disappeared in the TPA-induced epidermal hyperplasia within 2 weeks and numerous clusters of epidermal cells with overexpressed p53 appeared after 4 weeks. Neither mutant p53 patches nor any foci of pErk1/2-overexpressing cells that could have caused reactive wild type p53 expression were found. In skin exposed to a single high UV dose (2.8kJ/m2) no CRBCs occurred, and no p53 clusters were observed after TPA treatment. These experiments suggest that CRBCs are a prerequisite for the formation of clusters of p53-overexpressing cells. The high frequency of these clusters (about 1 for every 3 CRBCs) precludes mutations in p53 as a likely cause. We surmise that forced proliferation of CRBCs gives rise to genomic instability that is propagated in daughter cells and evokes wild type p53 overexpression, signifying a potentially oncogenic process different from classic UV carcinogenesis involving mutant p53.