RESUMEN
The current study was designed to explore the role of signal transducer and activator of transcription 1 (Stat1) during tumor promotion using the mouse skin multistage carcinogenesis model. Topical treatment with both 12-O-tetradecanoylphorbol-13-acetate (TPA) and 3-methyl-1,8-dihydroxy-9-anthrone (chrysarobin or CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Y701) and serine (S727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. CHRY treatment also led to upregulation of interferon regulatory factor 1 (IRF-1) mRNA and protein, which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNγ) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNγ signaling. Stat1 deficient (Stat1(-/-) ) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1(-/-) mice and wild-type littermates with TPA as the promoter. Maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNγ signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1, IRF-1 and uStat1.
Asunto(s)
Antracenos/toxicidad , Queratinocitos/metabolismo , Factor de Transcripción STAT1/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/toxicidad , Animales , Transformación Celular Neoplásica , Células Cultivadas , Femenino , Humanos , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Interferón gamma , Queratinocitos/citología , Ratones , Neoplasias Experimentales , Fosforilación , Factor de Transcripción STAT1/genética , Transducción de Señal , Neoplasias Cutáneas/inducido químicamenteRESUMEN
Despite much recent progress, prostate cancer continues to represent a major cause of cancer-related mortality and morbidity in men. Prostate cancer is the most common nonskin neoplasm and second leading cause of death in men. 6-Shogaol (6-SHO), a potent bioactive compound in ginger (Zingiber officinale Roscoe), has been shown to possess anti-inflammatory and anticancer activity. In the present study, the effect of 6-SHO on the growth of prostate cancer cells was investigated. 6-SHO effectively reduced survival and induced apoptosis of cultured human (LNCaP, DU145, and PC3) and mouse (HMVP2) prostate cancer cells. Mechanistic studies revealed that 6-SHO reduced constitutive and interleukin (IL)-6-induced STAT3 activation and inhibited both constitutive and TNF-α-induced NF-κB activity in these cells. In addition, 6-SHO decreased the level of several STAT3 and NF-κB-regulated target genes at the protein level, including cyclin D1, survivin, and cMyc and modulated mRNA levels of chemokine, cytokine, cell cycle, and apoptosis regulatory genes (IL-7, CCL5, BAX, BCL2, p21, and p27). 6-SHO was more effective than two other compounds found in ginger, 6-gingerol, and 6-paradol at reducing survival of prostate cancer cells and reducing STAT3 and NF-κB signaling. 6-SHO also showed significant tumor growth inhibitory activity in an allograft model using HMVP2 cells. Overall, the current results suggest that 6-SHO may have potential as a chemopreventive and/or therapeutic agent for prostate cancer and that further study of this compound is warranted.
Asunto(s)
Catecoles/uso terapéutico , Proliferación Celular/efectos de los fármacos , FN-kappa B/antagonistas & inhibidores , Extractos Vegetales/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Factor de Transcripción STAT3/antagonistas & inhibidores , Zingiber officinale , Animales , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Alimentos en Conserva , Zingiber officinale/química , Humanos , Masculino , Ratones , FN-kappa B/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Metformina/farmacología , Neoplasias Cutáneas/prevención & control , Adenilato Quinasa/metabolismo , Adiponectina/metabolismo , Animales , Peso Corporal , Carcinogénesis , Carcinoma de Células Escamosas/prevención & control , Dieta , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Leptina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Obesos , Complejos Multiproteicos/metabolismo , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/prevención & control , Obesidad/complicaciones , Sobrepeso/complicaciones , Papiloma/prevención & control , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Sirolimus/farmacología , Neoplasias Cutáneas/inducido químicamente , Serina-Treonina Quinasas TOR/metabolismo , Acetato de TetradecanoilforbolRESUMEN
Obesity, an established risk factor for epithelial cancers, remains prevalent in the United States and many other countries. In contrast to positive energy balance states (overweight, obesity), calorie restriction (CR) has been shown to act as a universal inhibitor of tumorigenesis in multiple animal models of human cancer. Unfortunately, the mechanisms underlying the enhancing effects of obesity or the inhibitory effects of CR on cancer etiology remain elusive. Here, we evaluated the impact of dietary energy balance manipulation on epithelial carcinogenesis and identified several potential mechanisms that may account for the differential effects of obesity and CR on cancer. Obesity enhanced tumor promotion during epithelial carcinogenesis, in part, due to altered insulin-like growth factor-1 receptor (IGF-1R)/EGF receptor (EGFR) crosstalk and downstream signaling to effectors such as Akt/mTOR. Obesity-induced changes in cellular signaling subsequently led to altered levels of cell-cycle proteins that favored enhanced epidermal proliferation during tumor promotion. In contrast, CR reduced susceptibility to tumor promotion, attenuated IGF-1R/EGFR crosstalk and downstream signaling, and altered levels of cell-cycle proteins that favored reduced epidermal proliferation during tumor promotion. Collectively, these findings suggest potential targets for the prevention of epithelial cancers, as well as for reversal of obesity-mediated cancer development and progression. Cancer Prev Res; 5(10); 1236-46. ©2012 AACR.
Asunto(s)
Transformación Celular Neoplásica/patología , Metabolismo Energético , Receptores ErbB/metabolismo , Papiloma/patología , Receptor IGF Tipo 1/metabolismo , Neoplasias Cutáneas/patología , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Western Blotting , Restricción Calórica , Carcinógenos/toxicidad , Ciclo Celular , Proliferación Celular , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Dieta , Receptores ErbB/genética , Femenino , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Ratones Endogámicos ICR , Obesidad , Papiloma/inducido químicamente , Papiloma/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor IGF Tipo 1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Male Hi-Myc mice were placed on three dietary regimens [30% calorie restriction (CR), overweight control (modified AIN76A with 10 kcal% fat), and a diet-induced obesity regimen (DIO) 60 kcal% fat]. All diet groups had approximately similar incidence of hyperplasia and low-grade prostatic intraepithelial neoplasia in the ventral prostate at 3 and 6 months of age. However, 30% CR significantly reduced the incidence of in situ adenocarcinomas at 3 months compared with the DIO group and at 6 months compared with both the overweight control and DIO groups. Furthermore, the DIO regimen significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared with the overweight control group (96% vs. 65%, respectively; P = 0.02) at the 6-month time point. In addition, at both 3 and 6 months, only in situ carcinomas were observed in mice maintained on the 30% CR diet. Relative to overweight control, DIO increased whereas 30% CR reduced activation of Akt, mTORC1, STAT3, and NFκB (p65) in ventral prostate. DIO also significantly increased (and 30% CR decreased) numbers of T-lymphocytes and macrophages in the ventral prostate compared with overweight control. The mRNA levels for interleukin (IL) 1α, IL1ß, IL6, IL7, IL23, IL27, NFκB1 (p50), TNFα, and VEGF family members were significantly increased in the ventral prostate of the DIO group compared with both the overweight control and 30% CR diet groups. Collectively, these findings suggest that enhanced growth factor (Akt/mTORC1 and STAT3) and inflammatory (NFκB and cytokines) signaling may play a role in dietary energy balance effects on prostate cancer progression in Hi-Myc mice.
Asunto(s)
Adenocarcinoma/patología , Restricción Calórica , Dieta , Metabolismo Energético , Próstata/patología , Neoplasias de la Próstata/patología , Adenocarcinoma/etiología , Adenocarcinoma/metabolismo , Animales , Western Blotting , Peso Corporal , Grasas de la Dieta/efectos adversos , Progresión de la Enfermedad , Ingestión de Energía , Técnicas para Inmunoenzimas , Masculino , Ratones , Obesidad/complicaciones , Obesidad/patología , Sobrepeso/complicaciones , Sobrepeso/patología , Próstata/metabolismo , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
In this report, we describe the development of a transgenic mouse in which a rat probasin promoter (ARR(2)Pb) was used to direct prostate specific expression of a constitutively active form of signal transducer and activator of transcription 3 (i.e., Stat3C). ARR(2)Pb.Stat3C mice exhibited hyperplasia and prostate intraepithelial neoplasia (PIN) lesions in both ventral and dorsolateral prostate lobes at 6 and 12 months; however, no adenocarcinomas were detected. The effect of combined loss of PTEN was examined by crossing ARR(2)Pb.Stat3C mice with PTEN(+/-) null mice. PTEN(+/-) null mice on an ICR genetic background developed only hyperplasia and PIN at 6 and 12 months, respectively. ARR(2)Pb.Stat3C x PTEN(+/-) mice exhibited a more severe prostate phenotype compared with ARR(2)Pb.Stat3C and PTEN(+/-) mice. ARR(2)Pb.Stat3C x PTEN(+/-) mice developed adenocarcinomas in the ventral prostate as early as 6 months (22% incidence) that reached an incidence of 61% by 12 months. Further evaluations indicated that phospho-Stat3, phospho-Akt, phospho-nuclear factor κB, cyclin D1, and Ki67 were upregulated in adenocarcinomas from ARR(2)Pb.Stat3C x PTEN(+/-) mice. In addition, membrane staining for ß-catenin and E-cadherin was reduced. The changes in Stat3 and nuclear factor κB phosphorylation correlated most closely with tumor progression. Collectively, these data provide evidence that Stat3 and Akt signaling cooperate in prostate cancer development and progression and that ARR(2)Pb.Stat3C x PTEN(+/-) mice represent a novel mouse model of prostate cancer to study these interactions.
Asunto(s)
Adenocarcinoma/patología , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/fisiología , Factor de Transcripción STAT3/fisiología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Proteína de Unión a Andrógenos/genética , Animales , Western Blotting , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosfohidrolasa PTEN/fisiología , Fosforilación , Reacción en Cadena de la Polimerasa , Próstata/metabolismo , Próstata/patología , Neoplasia Intraepitelial Prostática/genética , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Ratas , Transducción de SeñalRESUMEN
To establish a role for insulin-like growth factor-1 (IGF-1) in bladder cancer susceptibility, we tested the effect of p-cresidine, a potent bladder carcinogen, in transgenic (TG) mice with human IGF-1 expression in the bladder driven by the bovine keratin 5 promoter (referred to as BK5.IGF-1 TG mice). Indomethacin was also tested to determine if the cyclooxygenase (COX) pathway is a target for bladder cancer prevention in this model. Thirty-three female BK5.IGF-1 TG mice and 29 female nontransgenic littermates were randomized to the following treatments: (1) AIN-76A diet; (2) AIN-76A diet with 0.5% p-cresidine; or (3) AIN-76A diet with 0.5% p-cresidine + 0.00075% indomethacin. BK5.IGF-1 TG mice, with twofold greater total serum IGF-1 than nontransgenic mice, exhibited greatly increased susceptibility to p-cresidine-induced bladder tumors compared to nontransgenic mice. The most common type of bladder tumor in the BK5.IGF-1 TG mice was transitional cell carcinoma, which is the predominant type of bladder cancer observed in developed countries. Indomethacin inhibition of bladder tumor development in BK5.IGF-1 TG mice was not statistically significant. These results present further evidence for the role of IGF-1 in bladder cancer progression. In addition, these transgenic mice provide a useful model for studying the role of the IGF-1 pathway in bladder carcinogenesis and its prevention.
Asunto(s)
Carcinoma de Células Transicionales/inducido químicamente , Factor I del Crecimiento Similar a la Insulina/metabolismo , Papiloma/inducido químicamente , Neoplasias de la Vejiga Urinaria/inducido químicamente , Vejiga Urinaria/efectos de los fármacos , Compuestos de Anilina/toxicidad , Animales , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Bovinos , Femenino , Humanos , Queratina-5/genética , Ratones , Ratones Transgénicos , Papiloma/metabolismo , Papiloma/patología , Regiones Promotoras Genéticas , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Calorie restriction has been shown to inhibit epithelial carcinogenesis and this method of dietary restriction reduces many circulating proteins, including insulin-like growth factor I (IGF-I). Previously, we identified a relationship between elevated tissue IGF-I levels and enhanced susceptibility to chemically induced skin tumorigenesis. In this study, liver IGF-I-deficient (LID) mice, which have a 75% reduction in serum IGF-I, were subjected to the standard two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. We observed a significant reduction in epidermal thickness and labeling index in LID mice treated with either vehicle or TPA. A significant decrease in both tumor incidence and tumor multiplicity was observed in LID mice undergoing two-stage skin carcinogenesis relative to wild-type littermates. Western blot analyses of epidermal extracts revealed reduced activation of both the epidermal growth factor and IGF-I receptors in response to TPA treatment in LID mice. In addition, reduced activation of both Akt and the mammalian target of rapamycin (mTOR) was observed in LID mice following TPA treatment relative to wild-type controls. Signaling downstream of mTOR was also reduced. These data suggest a possible mechanism whereby reduced circulating IGF-I leads to attenuated activation of the Akt and mTOR signaling pathways, and thus, diminished epidermal response to tumor promotion, and ultimately, two-stage skin carcinogenesis. The current data also suggest that reduced circulating IGF-I levels which occur as a result of calorie restriction may lead to the inhibition of skin tumorigenesis, at least in part, by a similar mechanism.
Asunto(s)
Predisposición Genética a la Enfermedad , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Animales , Benzo(a)Antracenos/farmacología , Proliferación Celular , Transformación Celular Neoplásica , Epidermis/metabolismo , Femenino , Ratones , Modelos Biológicos , Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Serina-Treonina Quinasas TOR , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Aberrant activation of the Akt pathway has been implicated in several human pathologies including cancer. However, current knowledge on the involvement of Akt signaling in development is limited. Previous data have suggested that Akt-mediated signaling may be an essential mediator of epidermal homeostasis through cell autonomous and noncell autonomous mechanisms. Here we report the developmental consequences of deregulated Akt activity in the basal layer of stratified epithelia, mediated by the expression of a constitutively active Akt1 (myrAkt) in transgenic mice. Contrary to mice overexpressing wild-type Akt1 (Akt(wt)), these myrAkt mice display, in a dose-dependent manner, altered development of ectodermally derived organs such as hair, teeth, nails, and epidermal glands. To identify the possible molecular mechanisms underlying these alterations, gene profiling approaches were used. We demonstrate that constitutive Akt activity disturbs the bone morphogenetic protein-dependent signaling pathway. In addition, these mice also display alterations in adult epidermal stem cells. Collectively, we show that epithelial tissue development and homeostasis is dependent on proper regulation of Akt expression and activity.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Ectodermo/anomalías , Ectodermo/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Animales , Ectodermo/patología , Activación Enzimática , Epidermis/enzimología , Epidermis/patología , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Enzimológica de la Expresión Génica , Cabello/anomalías , Cabello/ultraestructura , Homeostasis , Ratones , Ratones Transgénicos , Uñas Malformadas/enzimología , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteínas Proto-Oncogénicas c-akt/genética , Células Madre/citología , Células Madre/enzimología , Anomalías Dentarias/enzimologíaRESUMEN
The prevalence of obesity, an established risk factor for several types of cancer, has increased steadily over the past several decades in the United States. New targets and strategies for offsetting the effect of obesity on cancer risk are urgently needed. In the present study, we examined the effect of dietary energy balance manipulation on steady-state signaling in multiple epithelial tissues, with a focus on the Akt and mammalian target of rapamycin (mTOR) pathways. For these experiments, male FVB/N and C57BL/6 and female ICR mice were maintained on a control (10 kcal% fat) diet, a diet-induced obesity (DIO; 60 kcal% fat) regimen, or a 30% calorie restriction (CR) regimen for 15 to 17 weeks. Relative to the control group, the DIO regimen increased, whereas CR decreased, circulating insulin-like growth factor-I (IGF-I) as has previously been reported. Western blot analyses showed that the DIO regimen enhanced, whereas CR inhibited, activation of Akt and mTOR, regardless of epithelial tissue or genetic background. In contrast, activation of AMP-activated protein kinase was modulated by dietary energy balance manipulation in the liver but not in the epidermis or dorsolateral prostate. Western blot analyses of epidermal extracts taken from ICR mice also revealed reduced activation of both the IGF-I receptor and epidermal growth factor receptor in CR mice, compared with control mice or mice maintained on the DIO regimen. Taken together, these novel findings suggest that dietary energy balance modulates signaling through cell-surface receptors (i.e., IGF-I receptor and epidermal growth factor receptor), affecting activation of multiple downstream pathways including Akt and mTOR, thus providing important dietary and pharmacologic targets for disrupting the obesity-cancer link.
Asunto(s)
Dieta , Metabolismo Energético/fisiología , Epitelio/metabolismo , Proteína Oncogénica v-akt/fisiología , Proteínas Quinasas/fisiología , Animales , Distribución de la Grasa Corporal , Peso Corporal , Ingestión de Alimentos/fisiología , Femenino , Homeostasis/fisiología , Factor I del Crecimiento Similar a la Insulina/análisis , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteína Oncogénica v-akt/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TORRESUMEN
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.
Asunto(s)
Epitelio/patología , Regulación Neoplásica de la Expresión Génica , Regulación de la Expresión Génica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patología , Animales , Bovinos , Epitelio/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Regiones Promotoras Genéticas , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol/toxicidadRESUMEN
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.
Asunto(s)
Carcinógenos/farmacología , Epidermis/enzimología , Epidermis/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias Cutáneas/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Antracenos/farmacología , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Epidermis/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Heparina/metabolismo , Heparina/farmacología , Indoles/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Queratinocitos/patología , Maleimidas/farmacología , Ratones , Ratones Endogámicos ICR , Ácido Ocadaico/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/patologíaRESUMEN
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.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Cromonas/farmacología , Epidermis/metabolismo , Femenino , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Ratones Transgénicos , Morfolinas/farmacología , Proteínas Proto-Oncogénicas c-aktRESUMEN
Recent work from our laboratory has shown that elevated src kinase activity enhances tumor promotion, malignant progression, and metastasis during multistage skin carcinogenesis. In this study, we have generated "gene-switch" src(530) transgenic mice to further analyze the role of this nonreceptor tyrosine kinase in multistage carcinogenesis. Target transgenic mice that have an activated form of the human c-src (src(530)) gene fused with GAL4 binding sites upstream of the thymidine kinase (TK) promoter were generated. Two lines of epidermis-specific transactivator mice were used that targeted the expression of GLVPc or GLp65 transactivators, fusion molecules containing a truncated progesterone receptor with a GAl4-DNA binding domain, with either a mouse loricrin (ML) or human keratin 14 (HK14) promoter, respectively. The transactivator mice (ML.GLVPc or HK14.GLp65) and the target mice (TK.src(530)) were mated to generate bitransgenic mice, and src(530) transgene expression was induced by topical application of RU486 (mifepristone, a progesterone receptor antagonist). In both ML.GLVPc/TK.src(530) and HK14.GLp65/TK.src(530) bitransgenic mice, histological analysis revealed that only the bitransgenic mice had marked epidermal hyperplasia and hyperkeratosis after treatment with RU486. Neither the nontransgenic mice nor the mice hemizygous for either the transactivator transgene or the target transgene alone showed any response to treatment with RU486. In addition, no differences were observed in the skin of the bitransgenic mice versus nontransgenic littermates without treatment of RU486. Interestingly, in HK14.GLp65/TK.src(530) bitransgenic mice, squamous cell carcinomas (SCCs) arose along the periphery of the area of the punch biopsies in 25% of the bitransgenic mice several weeks after taking the biopsy and subsequent to RU486 treatment. Collectively, the data support a role of c-src activation in epidermal hyperproliferation. Furthermore, the data support the conclusion that src activation can substitute for an initiating event in the presence of a tumor promoting stimulus (i.e., wounding). Finally, inducible src(530) transgenic mice provide a new tool for dissecting the role of src activation in multistage carcinogenesis by allowing temporal control of the expression of this oncogene.
Asunto(s)
Epidermis/metabolismo , Proteínas Tirosina Quinasas/genética , Animales , Proteína Tirosina Quinasa CSK , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Epidermis/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica/fisiología , Antagonistas de Hormonas/farmacología , Ratones , Ratones Transgénicos , Mifepristona/farmacología , Proteínas Tirosina Quinasas/metabolismo , Familia-src QuinasasRESUMEN
In this study, we generated transgenic mice that overexpressed either a constitutively active human c-src mutant (src(530)) or a wild-type human c-src (src(wt)) in epidermal basal cells driven by human keratin 14 (HK14) or bovine keratin 5 (BK5) promoters, respectively. HK14.src(530) transgenic mice developed severe epidermal hyperplasia and hyperkeratosis, and did not survive beyond 3 weeks of age. Four transgenic founders were obtained after injection of a BK5.src(wt) construct with variable phenotypes, and three lines (lines A-C) were established. BK5.src(wt) founder D exhibited a severe skin phenotype similar to HK14.src(530) transgenic mice and died 5 days after birth. Line C transgenic mice also exhibited significant epidermal hyperplasia and hyperkeratosis, and developed spontaneous squamous cell carcinomas (SCCs) of the skin beginning at approximately 3 months of age (70% incidence at 1 year). Mice from lines A and B did not show a marked phenotype; however, elevated human src(wt) protein in the epidermis of line B mice was clearly evident. Additional analyses of line B transgenic mice showed an enhanced responsiveness to 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and cell proliferation. Analysis of the susceptibility of line B mice to two-stage skin carcinogenesis revealed that papillomas and SCCs arose earlier and in greater numbers compared with nontransgenic littermates. In addition, malignant conversion occurred more rapidly, and the SCCs that developed in line B transgenic mice had a greater propensity to metastasize to peripheral lymph nodes and other organs. These observations support the hypothesis that c-src plays an important role in skin tumor promotion. In addition, the data show that elevated c-src activity enhances malignant progression and metastasis in this model system.
Asunto(s)
Carcinoma de Células Escamosas/etiología , Proteínas Tirosina Quinasas/fisiología , Neoplasias Cutáneas/etiología , 9,10-Dimetil-1,2-benzantraceno , Secuencia de Aminoácidos , Animales , Proteína Tirosina Quinasa CSK , Carcinoma de Células Escamosas/patología , Femenino , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Proteínas Tirosina Quinasas/análisis , Piel/patología , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/farmacología , Familia-src QuinasasRESUMEN
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.