Inducible Keratinocyte Specific FGFR2 Deficiency Inhibits UVB-Induced Signaling, Proliferation, Inflammation, and Skin Carcinogenesis.

A potential role for fibroblast growth factor receptor 2 (FGFR2) in cutaneous squamous cell carcinoma (cSCC) has been reported. To demonstrate the specific role of FGFR2 in UVB-induced skin carcinogenesis and development of cSCC, we generated a keratinocyte specific, tamoxifen inducible mouse model of FGFR2 deficiency. In this mouse model, topical application of 4-hydroxy tamoxifen led to the induction of Cre recombinase to delete FGFR2 in epidermal keratinocytes of both male and female transgenic mice. Analysis of epidermal protein lysates isolated from FGFR2 deficient mice exposed to UVB showed significant reductions of phospho-FGFR (pFGFR; Y653/654) and phospho-fibroblast growth factor receptor substrate 2α as well as downstream effectors of mTORC1 signaling. Phosphorylation of signal transducer and activators of transcription 1/3 was significantly reduced as well as levels of IRF-1, DUSP6, early growth response 1, and PD-L1 compared to the control groups. Keratinocyte-specific ablation of FGFR2 also significantly inhibited epidermal hyperproliferation, hyperplasia, and inflammation after exposure to UVB. Finally, keratinocyte-specific deletion of FGFR2 significantly inhibited UVB-induced cSCC formation. Collectively, the current data demonstrate an important role of FGFR2 in UVB-induced oncogenic signaling as well as development of cSCC. In addition, the current preclinical findings suggest that inhibition of FGFR2 signaling may provide a previously unreported strategy to prevent and/or treat UVB-induced cSCC.

Twist1 as a target for prevention of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) represents an important clinical problem requiring novel approaches for both prevention and treatment. The transcription factor, Twist-related protein 1 (Twist1), has been identified as having a key mechanistic role in the development and progression of cSCC. Studies in relevant mouse models of cSCC have shown that Twist1 regulates epithelial-mesenchymal transition (EMT) and stemness driving progression and metastasis of cSCC. In addition, further research has shown that Twist1 regulates the balance between keratinocyte proliferation and differentiation and therefore impacts earlier stages of cSCC development. Through use of keratinocyte specific Twist1 knockout models, a role for this gene in keratinocyte stem cell homeostasis has been revealed. As a transcription factor, Twist1 regulates a large number of genes both in a positive, as well as a negative manner across several interdependent pathways. Studies in keratinocyte specific knockout models have shown that Twist1 upregulates the expression of genes involved in proliferation, stemness, and EMT while downregulating the expression of genes associated with differentiation. Furthermore, a number of compounds, including naturally occurring compounds, have been identified that target Twist1 and can block its effects in cancer cells and in keratinocytes in vivo. Collectively, the current understanding of Twist1 function in cSCC development and progression suggests that it represents a potential target for prevention and treatment of cSCC.

Inhibition of Fibroblast Growth Factor Receptor Attenuates UVB-Induced Skin Carcinogenesis.

Altered fibroblast GF receptor (FGFR) signaling has been shown to play a role in a number of cancers. However, the role of FGFR signaling in the development and progression of UVB-induced cutaneous squamous cell carcinoma remains unclear. In this study, the effect of UVB radiation on FGFR activation and its downstream signaling in mouse skin epidermis was examined. In addition, the impact of FGFR inhibition on UVB-induced signaling and skin carcinogenesis was also investigated. Exposure of mouse dorsal skin to UVB significantly increased the phosphorylation of FGFRs in the epidermis as well as the activation of downstream signaling pathways, including protein kinase B/mTOR, signal transducers and activators of transcription, and MAPK. Topical application of the pan-FGFR inhibitor AZD4547 to mouse skin before exposure to UVB significantly inhibited FGFR phosphorylation as well as mTORC1, signal transducer and activator of transcription 3, and MAPK activation (i.e., phosphorylation). Moreover, AZD4547 pretreatment significantly inhibited UVB-induced epidermal hyperplasia and hyperproliferation and reduced the infiltration of mast cells and macrophages into the dermis. AZD4547 treatment also significantly inhibited mRNA expression of inflammatory genes in the epidermis. Finally, mice treated topically with AZD4547 before UVB exposure showed decreased cutaneous squamous cell carcinoma incidence and increased survival rate. Collectively, the current data support the hypothesis that inhibition of FGFR in the epidermis may provide a new strategy to prevent and/or treat UVB-induced cutaneous squamous cell carcinoma.

Twist1 is required for the development of UVB-induced squamous cell carcinoma.

The transcription factor Twist1 has been reported to be essential for the formation and invasiveness of chemically induced tumors in mouse skin. However, the impact of keratinocyte-specific Twist1 deletion on skin carcinogenesis caused by UVB radiation has not been reported. Deletion of Twist1 in basal keratinocytes of mouse epidermis using K5.Cre × Twist1flox/flox mice led to significantly reduced UVB-induced epidermal hyperproliferation. In addition, keratinocyte-specific deletion of Twist1 significantly suppressed UVB-induced skin carcinogenesis. Further analyses revealed that deletion of Twist1 in cultured keratinocytes or mouse epidermis in vivo led to keratinocyte differentiation. In this regard, deletion of Twist1 in epidermal keratinocytes showed significant induction of early and late differentiation markers, including TG1, K1, OVOL1, loricrin, and filaggrin. Similar results were obtained with topical application of harmine, a Harmala alkaloid that leads to degradation of Twist1. In contrast, overexpression of Twist1 in cultured keratinocytes suppressed calcium-induced differentiation. Further analyses using both K5.Cre × Twist1flox/flox mice and an inducible system where Twist1 was deleted in bulge region keratinocytes showed loss of expression of hair follicle stem/progenitor markers, including CD34, Lrig1, Lgr5, and Lgr6. These data support the conclusion that Twist1 has a direct role in maintaining the balance between proliferation and differentiation of keratinocytes and keratinocyte stem/progenitor populations. Collectively, these results demonstrate a critical role for Twist1 early in the process of UVB skin carcinogenesis, and that Twist1 may be a novel target for the prevention of cutaneous squamous cell carcinoma.

Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin.

Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventative agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential combinatorial inhibition of skin tumor promotion using the mouse two-stage skin carcinogenesis model. In short-term experiments, the combination of UA + Curc given topically prior to 12-O-tetradecanoylphorbol-13-acetate (TPA) significantly inhibited activation of epidermal EGFR, p70S6K, NF-κB p50, Src, c-Jun, Rb, and IκBα. Levels of c-Fos, c-Jun, and Cox-2 were also significantly reduced by the combination compared to the TPA treated group. The alterations in these signaling pathways by the combination of UA + Curc were associated with decreased epidermal proliferation as assessed by measuring BrdU incorporation. Significant effects were also seen with the combination on epidermal inflammatory gene expression and dermal inflammation, with the greatest effects on expression of IL-1ß, IL-6, IL-22, and CXCL2. Furthermore, results from skin tumor experiments demonstrated that the combination of UA + Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival, tumor size, and tumor weight, although tumor incidence and multiplicity were also further reduced by the combination. These results demonstrate the potential cancer chemopreventive activity and mechanism(s) for the combination of UA + Curc.

Overexpression of PRAS40(T246A) in the Proliferative Compartment Suppresses mTORC1 Signaling, Keratinocyte Migration, and Skin Tumor Development.

The proline-rich Akt (v-akt murine thymoma viral oncogene homolog 1) substrate of 40 kDa (PRAS40), an inhibitory component of the mTORC1 complex, was identified as an Akt substrate through phosphorylation at Thr246. Phosphorylation at this site releases PRAS40 from the mammalian/mechanistic target of rapamycin complex 1 (mTORC1) complex allowing increased activity. Targeted expression of a mutant form of PRAS40 (PRAS40(T246A)) in basal keratinocytes of mouse epidermis (BK5.PRAS40(T246A) mice) has allowed further examination of mTORC1-specific signaling in epithelial carcinogenesis. BK5.PRAS40(T246A) mice were resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal hyperproliferation and skin tumor development. In transgenic mice, PRAS40(T246A) remained bound to raptor in keratinocytes even after treatment with TPA, consistent with reduced mTORC1 signaling and altered levels of cell cycle proteins. BK5.PRAS40(T246A) mice also displayed attenuated skin inflammation in response to TPA. Inhibition of mTORC1 in keratinocytes significantly inhibited their migration in vitro and, in addition, inhibited 12-O-tetradecanoylphorbol-13-acetate-induced proliferation and migration of bulge-region stem cells in vivo. Furthermore, targeted inhibition of mTORC1 in BK5.PRAS40(T246A) mice resulted in delayed wound healing. Decreased keratinocyte migration and impaired wound healing correlated with altered expression of epithelial-mesenchymal transition (EMT) markers and reduced smad signaling. Collectively, the current data using this unique mouse model provide further evidence that mTORC1 signaling in keratinocytes regulates key events in keratinocyte function and epithelial cancer development.

Twist1 regulates keratinocyte proliferation and skin tumor promotion.

In the present study, we evaluated the effect of deleting Twist1 on keratinocyte proliferation and on skin tumor development using the two-stage chemical carcinogenesis model. BK5.Cre × Twist1(flox/flox) mice, which have a keratinocyte-specific Twist1 knockout (Twist1 KO), developed significantly reduced numbers of papilloma (70% reduction) and squamous cell carcinoma (75% reduction) as well as delayed tumor latency compared to wild-type (WT) mice. Interestingly, knockdown of Twist1 in primary keratinocytes impeded cell cycle progression at the G1/S transition that coincided with reduced levels of the cell cycle proteins c-Myc, Cyclin E1, and E2F1 and increased levels of p53 and p21. Furthermore, ChIP analyses revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc at the canonical E-box binding motif suggesting a direct transcriptional regulation. Further analyses of Twist1 KO mice revealed a significant reduction in the number of label-retaining cells as well as the number of α6-integrin(+) /CD34(+) cells in the hair follicles of untreated mice compared to WT mice. These mice also exhibited significantly reduced epidermal proliferation in response to TPA treatment that again correlated with reduced levels of cell cycle regulators and increased levels of p53 and p21. Finally, Twist1 deficiency in keratinocytes led to an upregulation of p53 via its stabilization and nuclear localization, which is responsible for the increased expression of p21 in these cells. Collectively, these findings indicate that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation of keratinocytes, including keratinocyte stem cells during tumor promotion.

Evaluation of pentacyclic triterpenes found in Perilla frutescens for inhibition of skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

A series of pentacyclic tritperpenes found in Perilla frutescens (P. frutescens), including ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and compared to UA as the prototype compound. All compounds were given topically 30 min prior to each TPA application and significantly inhibited skin tumor promotion. 3-epiCA and MA were significantly more effective than UA at inhibiting tumor development. All of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds also reduced skin inflammation (assessed by infiltration of mast cells and T-cells) and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were again more effective than UA. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation (i.e., phosphorylation) of IGF-1R, STAT3 and Src. Further study of these compounds, especially 3-epiCA and MA, for chemopreventive activity in other cancer model systems is warranted.

Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis.

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.

Effect of Combined Treatment with Ursolic Acid and Resveratrol on Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate.

In this study, the effects of combining ursolic acid + resveratrol, for possible combined inhibitory effects on skin tumor promotion, were evaluated. Ursolic acid, resveratrol, and the combination of ursolic acid + resveratrol were applied topically prior to 12-O-tetracanoylphorbol-13-acetate (TPA) treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression, and skin tumor promotion. The combination of ursolic acid + resveratrol produced a greater inhibition of TPA-induced epidermal hyperproliferation. The combination of ursolic acid + resveratrol inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors, such as p21 and PDCD4, to a greater extent compared with the groups treated with the individual compounds. Ursolic acid + resveratrol also induced a dramatic increase of p-AMPK-α(Thr172). Combined treatment with ursolic acid + resveratrol resulted in a greater inhibition of expression of proinflammatory cytokines, including Il1a, Il1b, and Il22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of ursolic acid + resveratrol. Treatment with ursolic acid + resveratrol during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of ursolic acid + resveratrol to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation, and epidermal hyperproliferation induced by TPA treatment.

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.

Metformin inhibits skin tumor promotion in overweight and obese mice.

In the present study, the ability of metformin to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) was analyzed in mice maintained on either an overweight control diet or an obesity-inducing diet. Rapamycin was included for comparison, and a combination of metformin and rapamycin was also evaluated. Metformin (given in the drinking water) and rapamycin (given topically) inhibited development of both papillomas and squamous cell carcinomas in overweight and obese mice in a dose-dependent manner. A low-dose combination of these two compounds displayed an additive inhibitory effect on tumor development. Metformin treatment also reduced the size of papillomas. Interestingly, all treatments seemed to be at least as effective for inhibiting tumor formation in obese mice, and both metformin and rapamycin were more effective at reducing tumor size in obese mice compared with overweight control mice. The effect of metformin on skin tumor development was associated with a significant reduction in TPA-induced epidermal hyperproliferation. Furthermore, treatment with metformin led to activation of epidermal AMP-activated protein kinase (AMPK) and attenuated signaling through mTOR complex (mTORC)-1 and p70S6K. Combinations of metformin and rapamycin were more effective at blocking epidermal mTORC1 signaling induced by TPA consistent with the greater inhibitory effect on skin tumor promotion. Collectively, the current data demonstrate that metformin given in the drinking water effectively inhibited skin tumor promotion in both overweight and obese mice and that the mechanism involves activation of epidermal AMPK and attenuated signaling downstream of mTORC1.

Proteomic and pathway analyses reveal a network of inflammatory genes associated with differences in skin tumor promotion susceptibility in DBA/2 and C57BL/6 mice.

Genetic susceptibility to two-stage skin carcinogenesis is known to vary significantly among different stocks and strains of mice. In an effort to identify specific protein changes or altered signaling pathways associated with skin tumor promotion susceptibility, a proteomic approach was used to examine and identify proteins that were differentially expressed in epidermis between promotion-sensitive DBA/2 and promotion-resistant C57BL/6 mice following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). We identified 19 differentially expressed proteins of which 5 were the calcium-binding proteins annexin A1, parvalbumin α, S100A8, S100A9, and S100A11. Further analyses revealed that S100A8 and S100A9 protein levels were also similarly differentially upregulated in epidermis of DBA/2 versus C57BL/6 mice following topical treatment with two other skin tumor promoters, okadaic acid and chrysarobin. Pathway analysis of all 19 identified proteins from the present study suggested that these proteins were components of several networks that included inflammation-associated proteins known to be involved in skin tumor promotion (e.g. TNF-α, NFκB). Follow-up studies revealed that Tnf, Nfkb1, Il22, Il1b, Cxcl1, Cxcl2 and Cxcl5 mRNAs were highly expressed in epidermis of DBA/2 compared with C57BL/6 mice at 24h following treatment with TPA. Furthermore, NFκB (p65) was also highly activated at the same time point (as measured by phosphorylation at ser276) in epidermis of DBA/2 mice compared with C57BL/6 mice. Taken together, the present data suggest that differential expression of genes involved in inflammatory pathways in epidermis may play a key role in genetic differences in susceptibility to skin tumor promotion in DBA/2 and C57BL/6 mice.

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.

Growth factor signaling pathways as targets for prevention of epithelial carcinogenesis.

Growth factor receptor (GFR) signaling controls epithelial cell growth by responding to various endogenous or exogenous stimuli and subsequently activating downstream signaling pathways including Stat3, PI3K/Akt/mTOR, MAPK, and c-Src. Environmental chemical toxicants and UVB irradiation cause enhanced and prolonged activation of GFR signaling and downstream pathways that contributes to epithelial cancer development including skin cancer. Recent studies, especially those with tissue-specific transgenic mouse models, have demonstrated that GFRs and their downstream signaling pathways contribute to all three stages of epithelial carcinogenesis by regulating a wide variety of biological functions including proliferation, apoptosis, angiogenesis, cell adhesion, and migration. Inhibiting these signaling pathways early in the carcinogenic process results in reduced cell proliferation and survival, leading to decreased tumor formation. Collectively, these studies suggest that GFR signaling and subsequent downstream signaling pathways are potential targets for the prevention of epithelial cancers including skin cancer.

Deregulated activity of Akt in epithelial basal cells induces spontaneous tumors and heightened sensitivity to skin carcinogenesis.

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

Activation of epidermal akt by diverse mouse skin tumor promoters.

Akt is a serine/threonine kinase involved in a variety of cellular responses, including cell proliferation and cell survival. Recent studies from our laboratory suggest that Akt signaling may play an important role in skin tumor promotion. To explore this premise, we examined epidermal Akt activation and signaling in response to chemically diverse skin tumor promoters. Mice received single or multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, or chrysarobin. All three tumor promoters were able to activate epidermal Akt as early as 1 h after treatment. Activation of Akt following tumor promoter treatment led to enhanced downstream signaling, including hyperphosphorylation of glycogen synthase kinase-3beta and Bad. Structure activity studies with phorbol ester analogues revealed that the magnitude of activation paralleled tumor-promoting activity. In cultured primary keratinocytes, TPA treatment also led to activation of Akt. Activation of the epidermal growth factor receptor (EGFR) seemed to underlie the ability of TPA to activate Akt as both PD153035, an inhibitor of EGFR, and GW2974, a dual-specific inhibitor of both EGFR and erbB2, were able to effectively reduce TPA-induced Akt phosphorylation as well as TPA-stimulated EGFR and erbB2 tyrosine phosphorylation in a dose-dependent manner. Furthermore, inhibition of protein kinase C (PKC) activity blocked TPA-stimulated heparin-binding EGF production and EGFR transactivation. Inhibition of PKC also led to a decreased association of Akt with the PP2A catalytic subunit, leading to increased Akt phosphorylation. However, combination of EGFR inhibitor and PKC inhibitor completely abrogated TPA-induced activation of Akt. Collectively, the current results support the hypothesis that elevated Akt activity and subsequent activation of downstream signaling pathways contribute significantly to skin tumor promotion. In addition, signaling through the EGFR via EGFR homodimers or EGFR/erbB2 heterodimers may be the primary event leading to Akt activation during tumor promotion in mouse skin.

Role of PI3K/Akt signaling in insulin-like growth factor-1 (IGF-1) skin tumor promotion.

Overexpression of human IGF-1 with the bovine keratin 5 (BK5) promoter (BK5.IGF-1 transgenic mice) induces persistent epidermal hyperplasia and leads to spontaneous skin tumor formation. In previous work, PI3K and Akt activities were found to be elevated in the epidermis of BK5.IGF-1 transgenic mice compared to nontransgenic littermates. In the present study, we examined the importance of the PI3K/Akt signaling pathway in mediating the skin phenotype and the skin tumor promoting action of IGF-1 in these mice. Western blot analyses with epidermal lysates showed that signaling components downstream of PI3K/Akt were altered in epidermis of BK5.IGF-1 mice. Increased phosphorylation of GSK-3 (Ser(9/21)), TSC2(Thr(1462)), and mTOR(Ser(2448)) was observed. In addition, hypophosphorylation and increased protein levels of beta-catenin were observed in the epidermis of BK5.IGF-1 mice. These data suggested that components downstream of Akt might be affected, including cell cycle machinery in the epidermis of BK5.IGF-1 mice. Protein levels of cyclins (D1, E, A), E2F1, and E2F4 were all elevated in the epidermis of BK5.IGF-1 mice. Also, immunoprecipitation experiments demonstrated an increase in cdk4/cyclin D1 and cdk2/cyclin E complex formation, suggesting increased cdk activity in the epidermis of transgenic mice. In further studies, the PI3K inhibitor, LY294002, significantly blocked IGF-1-mediated epidermal proliferation and skin tumor promotion in DMBA-initiated BK5.IGF-1 mice. In addition, inhibition of PI3K/Akt with LY294002 reversed many of the cell cycle related changes observed in untreated transgenic animals. Collectively, the current results supported the hypothesis that elevated PI3K/Akt activity and subsequent activation of one or more downstream effector pathways contributed significantly to the tumor promoting action of IGF-1 in the epidermis of BK5.IGF-1 mice.

Overexpression of a constitutively active form of c-src in skin epidermis increases sensitivity to tumor promotion by 12-O-tetradecanoylphorbol-13-acetate.

Transgenic mice were developed to study the role of c-src in epithelial tumorigenesis through targeted expression of a constitutively active form of murine c-src (src(529)). Src(529) was targeted to the interfollicular epidermis with the human keratin 1 (HK1) promoter. The skin phenotype of these mice was characterized by exaggerated epidermal hyperplasia and hyperkeratosis within the first week after birth. The severity of this phenotype correlated with overall src kinase activity, both of which subsided with age. Treatment of adult HK1.src(529) transgenic mice with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate resulted in an increase in epidermal hyperplasia and labeling index significantly greater than that seen in nontransgenic littermates. In addition, HK1.src(529) transgenic mice developed papillomas earlier and in significantly greater numbers compared with nontransgenic littermates in a standard initiation-promotion experiment. The data support the hypothesis that activation of c-src kinase plays a role in skin tumor promotion.