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Cooper SJ, MacGowan J, Ranger-Moore J, Young MR, Colburn NH, Bowden GT.. Expression of dominant negative c-jun inhibits ultraviolet B-induced squamous cell carcinoma number and size in an SKH-1 hairless mouse model. Mol Cancer Res 1: 848-854
Abstract
UVB radiation is a complete carcinogen able to initiate, promote, and progress keratinocyte cells toward carcinogenesis. Exposure to UVB leads to the propagation of a number of signal transduction pathways resulting in increased DNA binding of transcription factors, including activator protein-1 (AP-1), and subsequent gene expression. To test the hypothesis that AP-1 activation plays a role in the promotion of UVB-induced skin tumors, a dominant negative c-jun (TAM67) mutant transgene was expressed in the epidermis of SKH-1 hairless mice and bred with mice expressing an AP-1 luciferase reporter gene. Single UVB exposure experiments showed a significant decrease in AP-1 activity, as measured by luciferase levels, in mice expressing TAM67 72 h postexposure. Transgenic and nontransgenic littermates were placed into a chronic UVB exposure experiment, three exposures per week for 25 weeks. Expression of TAM67 reduced the number of tumors per mouse by 58% and tumor sizes were 79% smaller than the tumors present in the nontransgenic study group. These tumors were histologically identified as squamous cell carcinomas. TAM67 had no effect on UVB-induced hyperplasia because comparable epidermal thickening was observed in both study groups over a 5-day period post-UVB exposure. Immunohistochemical analysis showed a reduction in the number of cyclin D(1)-expressing cells in squamous cell carcinoma samples removed from the TAM67 study group. These data show that TAM67 can inhibit UVB-induced squamous cell carcinoma formation, suggesting that AP-1 is a good candidate target for the development of new chemoprevention strategies to prevent sunlight-induced skin cancers.
... We investigated the use of EISO on cells irradiated with UVB light. Although UVB light comprises only 1-10% of solar UV light, UVB acts as a complete carcinogen capable of activating signaling pathways in HaCaT cells known to stimulate cell proliferation and survival, including p38, JNK, ERK and PI3-K upstream of activator protein-1 (AP-1) transcription factor activation. UVB-induced AP-1 activity has been linked to cellular proliferation and survival, and in mouse skin AP-1 activation has been demonstrated to be a major cause of skin cancer [13]. In addition, we investigated the effects of EISO on cell cycle progression and cell membrane integrity. ...
... Activation of AP-1 is critically important in UVB-induced skin carcinogenesis [13]. Treatment of HaCaT cells with EISO up to a concentration of 0.0005% resulted in a concentration-dependent reduction in AP-1 activity measured through the use of a luciferase reporter cell line, HCL-14 cells ( Figure 3A). ...
... However, consistent with our finding that signaling pathways upstream of AP-1 activity were not affected by EISO treatment, c-Fos promoter activity was not inhibited by EISO. These findings argue that EISO may also elicit chemopreventive action by direct inhibition of AP-1 activity, a major known causative factor in UV-induced skin cancer [13]. There is precedence for direct inhibition of UV-stimulated AP-1 by other natural products in the literature [25]. ...
α-Santalol, one of the primary components of the East Indian sandalwood oil (EISO), has been investigated for its potential use as a chemopreventive agent in skin cancer. Although there is some evidence that α-santalol could be an effective chemopreventive agent, to date, purified EISO has not been investigated. EISO is widely used for its health benefits in cultures around the world. In the current study, we show for the first time that EISO-treatment of cultured keratinocytes inhibits cell cycle progression and UV-induced AP-1 activity, two major cellular effects known to drive skin carcinogenesis. Unlike many chemopreventive agents, inhibition of signaling upstream of AP-1 was not a primary means of EISO-mediated AP-1 inhibition, as no effect of EISO was observed on UV-induced Akt, ERK, and p38 MAPK activity. EISO induced cell death at low concentrations, although caspase and PARP cleavage were not observed indicating that death was not due to apoptosis. Interestingly, plasma membrane integrity was severely compromised in EISO-treated cells and LC3 cleavage suggests the induction of autophagy. These effects were more pronounced in EISO-treated cells that were stimulated to proliferate than in quiescent cells. Together, these effects suggest that EISO has chemopreventive properties and may be useful as in preventing skin carcinogenesis.
Citation Format: Corey Levenson, Erik R. Olson, David S. Alberts, G. Tim Bowden. A novel chemopreventive mechanism for a traditional medicine: East Indian sandalwood oil induces autophagy and cell death in proliferating keratinocytes. [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 2254. doi:10.1158/1538-7445.AM2013-2254
... To investigate the potential chemopreventive activity of ISO, TPA/EGF-induced Cl41 cell transformation model was employed. Given that ISO could reduce cell viability in T24T bladder cancer cells with an approximate IC50 of 55 μM [12], we thus treated mouse epidermal Cl41 cells with ISO in concentrations of 30,40, and 50 μM with exposure to TPA/EGF. As shown in Figs. ...
... MKK7/JNK/AP-1 cascade is an important oncogenic signaling pathway [1,38]. Murine genetic loss-of-function studies have demonstrated that targets of JNK signaling cascade, particularly c-Jun and c-fos, may contribute to chemical-induced murine epidermal neoplasia [39][40][41]. JNK/AP-1 has been reported to play a role in inducing aberrant cell proliferation associated with human skin carcinogenesis [38][39][40][41][42][43]. Moreover, previous studies have also reported that TNFR1/MKK7/JNK/AP-1 cascade promoted human neoplasia and inhibition of this pathway represented a potential therapeutic approach for squamous cell carcinoma (SCC) [38]. ...
... Murine genetic loss-of-function studies have demonstrated that targets of JNK signaling cascade, particularly c-Jun and c-fos, may contribute to chemical-induced murine epidermal neoplasia [39][40][41]. JNK/AP-1 has been reported to play a role in inducing aberrant cell proliferation associated with human skin carcinogenesis [38][39][40][41][42][43]. Moreover, previous studies have also reported that TNFR1/MKK7/JNK/AP-1 cascade promoted human neoplasia and inhibition of this pathway represented a potential therapeutic approach for squamous cell carcinoma (SCC) [38]. ...
The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.
... Second, our use of the involucrin promoter permits targeting of TAM67 to the suprabasal epidermis [104][105][106] and alleviates problems that are observed with knockout mice where a specific AP1 transcription factor protein is lost from all layers. Third, a basal layer TAM67-targeted mouse model already existed [68,70,71,107,108] which permitted a direct comparison of the impact of basal versus suprabasal AP1 transcription factor inactivation. We will first discuss the impact of targeted expression of TAM67 in the epidermal basal layer. ...
... In vivo studies in mouse epidermis show that TAM67-dependent inactivation of AP1 transcription factor function in the basal epidermal layer does not produce obvious changes in keratinocyte proliferation or epidermal or dermal appearance [68,71,107]. However, basal layer TAM67 expression does reduce susceptibility of SKH-1 hairless mice to UVB-dependent cancer progression [68,71,107]. ...
... In vivo studies in mouse epidermis show that TAM67-dependent inactivation of AP1 transcription factor function in the basal epidermal layer does not produce obvious changes in keratinocyte proliferation or epidermal or dermal appearance [68,71,107]. However, basal layer TAM67 expression does reduce susceptibility of SKH-1 hairless mice to UVB-dependent cancer progression [68,71,107]. Both tumor number and size are reduced and this is associated with reduced numbers of cyclin D1 positive cells in the tumors [107]. ...
AP1 (jun/fos) transcription factors (c-jun, junB, junD, c-fos, FosB, Fra-1, and Fra-2) are key regulators of epidermal keratinocyte survival and differentiation and important drivers of cancer development. Understanding the role of these factors in epidermis is complicated by the fact that each protein is expressed, at different levels, in multiple cells layers in differentiating epidermis, and because AP1 transcription factors regulate competing processes (i.e., proliferation, apoptosis, and differentiation). Various in vivo genetic approaches have been used to study these proteins including targeted and conditional knockdown, overexpression, and expression of dominant-negative inactivating AP1 transcription factors in epidermis. Taken together, these studies suggest that individual AP1 transcription factors have different functions in the epidermis and in cancer development and that altering AP1 transcription factor function in the basal versus suprabasal layers differentially influences the epidermal differentiation response and disease and cancer development.
... Continued exposure to UVB drives clonal expansion of these initiated cells and ultimately gives rise to benign papillomas that can progress to invasive SCC. Our laboratory has demonstrated the important role that the AP-1 transcription factor complex plays in UV-induced skin tumor promotion [10]. We have demonstrated that UVB irradiation induces the expression of the c-fos proto-oncogene and that this is a major driver for AP-1 activity, as UVB-induced AP-1 consists primarily of JunD and c-Fos heterodimers [11]. ...
... We have demonstrated that UVB irradiation induces the expression of the c-fos proto-oncogene and that this is a major driver for AP-1 activity, as UVB-induced AP-1 consists primarily of JunD and c-Fos heterodimers [11]. The direct role of AP-1 in skin tumor development was also demonstrated through the use of a transgenic mouse (TAM67) that expresses a dominant negative c-Jun in the epidermis [10] . This dominant negative c-Jun inhibits UVBinduced AP-1 activation both in cultured keratinocytes and in mouse skin. ...
... Transgenic SKH-1 mice expressing an AP-1-luciferase reporter gene (AP-1-luc) have been described previously [10]. These mice were bred with p38DN mice to produce a double transgenic strain (p38DN/AP-1-luc). ...
UVB irradiation of epidermal keratinocytes results in the activation of the p38 mitogen-activated protein kinase (MAPK) pathway and subsequently activator protein-1 (AP-1) transcription factor activation and cyclooxygenase-2 (COX-2) expression. AP-1 and COX-2 have been shown to play functional roles in UVB-induced mouse skin carcinogenesis. In this study, the experimental approach was to express a dominant negative p38α MAPK (p38DN) in the epidermis of SKH-1 hairless mice and assess UVB-induced AP-1 activation, COX-2 expression, and the skin carcinogenesis response in these mice compared to wild-type littermates. We observed a significant inhibition of UVB-induced AP-1 activation and COX-2 expression in p38DN transgenic mice, leading to a significant reduction of UVB-induced tumor number and growth compared to wild-type littermates in a chronic UVB skin carcinogenesis model. A potential mechanism for this reduction in tumor number and growth rate is an inhibition of chronic epidermal proliferation, observed as reduced Ki-67 staining in p38DN mice compared to wild-type. Although we detected no difference in chronic apoptotic rates between transgenic and nontransgenic mice, analysis of acutely irradiated mice demonstrated that expression of the p38DN transgene significantly inhibited UVB-induced apoptosis of keratinocytes. These results counter the concerns that inhibition of p38 MAPK in a chronic situation could compromise the ability of the skin to eliminate potentially tumorigenic cells. Our data indicate that p38 MAPK is a good target for pharmacological intervention for UV-induced skin cancer in patients with sun damaged skin, and suggest that inhibition of p38 signaling reduces skin carcinogenesis by inhibiting COX-2 expression and proliferation of UVB-irradiated cells.
... TAM67 is a dominant-negative form of c-jun that interacts broadly with all AP1 factors to inhibit transactivation (Brown et al., 1994). In these interesting studies, basal layer AP1 factor inactivation did not produce obvious changes in keratinocyte proliferation or epidermal or dermal appearance; however, the mice were less susceptible to cancer progression (Young et al., 1999;Thompson et al., 2002;Cooper et al., 2003). ...
... Second, use of the involucrin promoter permits targeting of TAM67 to the suprabasal epidermis and alleviates problems that are observed with knockout mice where the protein is lost from all layers. Third, a basal layer TAM67-targeted model already exists (Young et al., 1999;Thompson et al., 2002;Young et al., 2002;Cooper et al., 2003;Matthews et al., 2007) which permits a comparison of the impact of basal versus suprabasal AP1 factor inactivation. ...
... In an elegant series of experiments, Colburn and colleagues studied the impact of AP1 factor inactivation in the epidermal basal layer by targeting TAM67 to the epidermal basal layer using the keratin 14 promoter (K14-TAM67) and found no overt phenotype under resting conditions (Young et al., 1999). In these mice basal TAM67 produces no obvious changes in keratinocyte proliferation or epidermal morphology, nor does it block 12-Otetradecanoylphorbol-13-acetate (TPA)-, okadaic acid-, or UVB-dependent epidermal hyperplasia (Young et al., 1999;Thompson et al., 2002;Cooper et al., 2003). These findings suggest that reducing AP1 factor function in the basal compartment has no impact on the epidermis. ...
AP1 (jun/fos) factors comprise a family of transcriptional regulators (c-jun, junB, junD, c-fos, FosB, Fra-1 and Fra-2) that are key controllers of epidermal keratinocyte survival and differentiation, and are important drivers of cancer development. Previous studies indicate that targeted inactivation of epidermal basal layer AP1 factor function by expression of dominant-negative c-jun (TAM67) in the basal epidermis produces no overt phenotypic impact, but does inhibit UVB and carcinogen-dependent epidermal tumor formation. We hypothesized that the role of AP1 factors is likely to be different in basal versus suprabasal epidermis. To test this hypothesis, we targeted TAM67 to the suprabasal epidermis using a tetracycline-inducible construct. Induction of suprabasal TAM67 expression results in marked epidermal hyperproliferation and hyperkeratosis. This is characterized by expression of basal layer and hyperproliferation-associated keratins in the suprabasal epidermis, suggesting that the cells are undergoing incomplete differentiation. We further demonstrate global changes in the AP1 factor signaling including changes in level and subcellular distribution of selected AP1 factors. Surprisingly, these animals display reduced susceptibility to carcinogen-dependent tumor induction. These novel observations, which suggest that perturbing AP1 factor function in the basal versus suprabasal layer produces different biological outcomes, is cancer relevant and points to the complexity of AP1-associated regulation and that fact that AP1 factor function differs as the tissue differentiates.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4201.
... Characterization of the transcriptional activity of this mutant revealed that it can dimerize with either Jun or Fos AP-1 family members (27), has similar DNA binding kinetics as wild type c-Jun (28) and functions in a dominant negative fashion (28,29). Transgenic expression of TAM67 in mouse epidermal keratinocytes inhibited papilloma formation in a two stage skin tumorigenesis model (30) and also in UVB-induced skin tumor formation (31). Similarly, expression of TAM67 in breast epithelial cells inhibited mammary tumor formation (32). ...
... Previous studies demonstrated a tumor inhibitory effect of the dominant negative mutant Tam67 during mouse skin and breast tumorigenesis in vivo (30)(31)(32). Expression of Tam67 under control of an epidermal specific promoter inhibited skin papilloma formation in a two stage initiation/promotion model (30) and inhibited UVB-induced squamous cell carcinomas (31). Interestingly, in both cases early epidermal hyperplasia was unaffected although inhibition of AP-1 activity was confirmed using the AP1-luciferase reporter mouse suggesting that AP-1 transcriptional activation is important during the tumor promotion stage of skin tumors. ...
... Previous studies demonstrated a tumor inhibitory effect of the dominant negative mutant Tam67 during mouse skin and breast tumorigenesis in vivo (30)(31)(32). Expression of Tam67 under control of an epidermal specific promoter inhibited skin papilloma formation in a two stage initiation/promotion model (30) and inhibited UVB-induced squamous cell carcinomas (31). Interestingly, in both cases early epidermal hyperplasia was unaffected although inhibition of AP-1 activity was confirmed using the AP1-luciferase reporter mouse suggesting that AP-1 transcriptional activation is important during the tumor promotion stage of skin tumors. ...
Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non-small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1-dependent transcription as a potential target to prevent lung tumor progression.
... It has also been shown that transactivation of a subset of AP-1-dependent genes is required for skin tumor promotion in vivo (Young et al. 1999) and that pharmacological downregulation of AP-1 activity inhibits the tumorigenic potential of a neoplastic epidermal subline (Dong et al. 1995). AP-1 proteins have recently been shown to be involved in sun-light induced cancers in mouse models and therefore it has also been suggested to be a good candidate target for the development of new chemoprevention strategies to prevent sunlight-induced skin cancers (Cooper et al. 2003). ...
... AP-1 transcription factors have been known to be a critical modulator of proliferation of different cancer types including colorectal (Magrisso et al. 1993;Glinghammar et al. 1999), breast (Chen et al. 1996;Smith et al. 1999), skin Cooper et al. 2003) and NSCLC (Risse-Hackl et al. 1998). ...
... TAM67 is a dominant-negative form of c-Jun that interferes with all AP1 factors to inhibit transactivation [22]. AP1 factor inactivation by targeted expression of TAM67 in the suprabasal epidermis causes increased epidermal hyperproliferation and hyperkeratosis but reduced carcinogen-dependent tumor formation [23][24][25]. ...
... Mice lacking c-Jun die at mid-gestation and their embryonic lethality are associated with increased apoptosis in fetal liver cells [51][52][53]. A transactivation mutant of c-Jun (TAM67) expressed under the Keratin 14 promoter or an inducible system produces a thickened, hyperproliferative, parakeratotic epidermis, but the epidermis is resistant to DMBA/TPAdependent tumor formation [21,24,25,54]. Consistent with JunB expression status in squamous cell carcinoma [21], c-Jun is remarkably reduced in human ESCC. ...
Transcription factor c-Jun plays a key role in controlling epithelium cell proliferation, apoptosis and differentiation. However, molecular mechanism and biological functions of c-Jun in squamous differentiation and the progression of esophageal squamous cell carcinoma (ESCC) remain elusive. In this study, we found that c-Jun bound directly to the promoter region, and activated the transcription of differentiation-associated genes including cystatin A, involucrin and SPRR3 in vivo. Ectopic expression of c-Jun enhanced SPRR3 transactivation in KYSE450 cells. Conversely, TAM67, a dominant negative mutant of c-Jun, inhibited SPRR3 transactivation. c-Jun increased expression of SPPR3 mainly via a PKC/JNK pathway in response to TPA in KYSE450 cells. Furthermore, c-Jun was remarkably reduced in esophageal cancer. Interestingly, cystatin A, involucrin and SPRR3 were significantly downregulated as well, and associated with differentiation grade. Expression of c-Jun was correlated with the expression of these genes in normal epithelium and ESCC. Importantly, the expression of these genes was remarkably decreased during the malignant transformation from normal epithelium to low-grade intraepithelial neoplasia (LGIN) or high-grade intraepithelial neoplasia (HGIN). The expression of cystatin A and involucrin was significantly reduced from LGIN to HGIN. These results suggest c-Jun was involved in the regulation of differentiation-associated genes in ESCC. These genes might serve as the potential markers in distinguishing normal epithelium from esophageal squamous intraepithelial neoplasia.
... Both the c-Jun/AP-1 family of transcription factors [1], [2], [3], [4] and C/EBPs [5], [6], [7] play a pivotal role in the regulation of keratinocyte differentiation. Keratinocytes proliferation and oncogenic transformation is also dependent upon C/EBP's [8][9][10] and c-Jun/AP-1 [11][12][13][14][15][16][17]. C/EBPb, in particular, is known to be a positive regulator of keratinocyte proliferation [18], [8], [9]. ...
... Mice lacking both of these proteins in the epidermis show increased proliferation of basal keratinocytes and impaired commitment to differentiation [6]. c-Jun/AP-1 deficiency augments keratinocyte resistance to carcinogenesis by mechanisms associated with the repression of AP-1 targets that promote proliferation such as Cyclin D1 [15,16], [23] and EGFR [24]. ...
Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.
Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite "Half and Half" consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.
Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.
... AP1 has been generally characterized as a dominant promoter of skin cancer. AP1 inhibition through multiple genetic approaches, including epidermal expression of the dominantnegative mutant of c-Jun (DNc-Jun, also known as TAM67) in transgenic mice or genetic deletion of either c-Jun or c-fos, suppresses murine skin carcinogenesis induced by chemicals, UV radiation or viral oncogenes (Cooper et al., 2003; Dhar et al., 2004; Jochum et al., 2001; Saez et al., 1995; Thompson et al., 2002; Young et al., 2002; Zhang et al., 2007). In addition, expression of DNc-Jun inhibits tumorigenesis of murine squamous cell carcinoma (SCC) cell lines in nude mice (Domann et al., 1994). ...
... We predicted that, if the functional divergence between JunB and c-Jun was caused by the differences in their TA-domains, DNJunB and DNc-Jun would induce similar effects on epidermal cell growth and neoplasia. DNc-Jun has been previously shown to inhibit tumorigenesis in both mouse and human skin tumor models (Cooper et al., 2003; Dhar et al., 2004; Domann et al., 1994; Jochum et al., 2001; Thompson et al., 2002; Young et al., 2002; Zhang et al., 2007). To determine DNJunB effects on human SCC, we used a previously established human SCC model, in which coexpression of Ras and MKK7, an upstream activator of AP1, transforms normal human epidermal cells into malignancy (Zhang et al., 2007). ...
Deregulation of the activator protein 1 (AP1) family gene regulators has been implicated in a wide range of diseases, including cancer. In this study we report that c-Jun was activated in human squamous cell carcinoma (SCC) and coexpression of c-Jun with oncogenic Ras was sufficient to transform primary human epidermal cells into malignancy in a regenerated human skin grafting model. In contrast, JunB was not induced in a majority of human SCC cells. Moreover, exogenous expression of JunB inhibited tumorigenesis driven by Ras or spontaneous human SCC cells. Conversely, the dominant-negative JunB mutant (DNJunB) promoted tumorigenesis, which is in contrast to the tumor-suppressor function of the corresponding c-Jun mutant. At the cellular level, JunB induced epidermal cell senescence and slowed cell growth in a cell-autonomous manner. Consistently, coexpression of JunB and Ras induced premature epidermal differentiation concomitant with upregulation of p16 and filaggrin and downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). These findings indicate that JunB and c-Jun differentially regulate cell growth and differentiation and induce opposite effects on epidermal neoplasia.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub.
... Pharmacological studies have shown that the inhibition of JNK in human keratinocytes in vitro results in greater UV-induced apoptosis [34] . The same effect was shown in vivo when inhibiting AP-1 in dominant negative c-jun hairless mice [35] . ...
Melanoma and non-melanoma cutaneous malignancies are some of the leading causes of cancer-related death in the United States. Though melanoma is more known to have a high mortality rate, the total mortality per year is nearly equal for between melanoma and non-melanoma skin cancer. Moreover, the non-melanoma types of cutaneous malignancies have potential to become locally invasive and even metastasize with very little to no treatment options when advanced. The development of these malignancies involves various genetic pathways through the four hallmarks of cancer development: malignant cell growth, apoptosis evasion, the use of supporting stroma and vascularization, and modulating and promoting an inadequate immune response. The genetic signaling pathways of basal cell carcinoma, squamous cell carcinoma, verrucous carcinoma, basosquamous cell carcinoma, melanoma, and cutaneous T-cell lymphoma interact with each other through genetic predisposition as well as with environmental exposures. Furthermore, solar ultraviolet radiation and chronic inflammatory states are found to initiate the progression of many of these cutaneous malignancies. This paper includes validated models of genetic pathways, emerging pathways, and crosstalk between genetic pathways through the four hallmarks of cancer development. Moreover, unlike most reviews addressing oncogenetics of the well-recognized, as well as newly discovered, genetic pathway mutations, this review stresses that these pathways are not fixed but rather exist in dynamic, interrelated, interactive, complex, and adaptive flux states.
... The use of a dominant-negative c-jun (TAM-67) resulted in 58% reduction in the number of tumors per mouse and a 79% reduction in the size of tumors resulting from UVB (10 kJ/m 2 ) exposure. [54] UV-mediated tumor promotion and progression could involve angiogenic responses in the epidermis through enhanced expression of AP-1-regulated angiogenic factors. Further such increased vascularity could be required for early development and subsequent malignant tumor development. ...
Ultraviolet radiation (UVR) is a very prominent environmental toxic agent. UVR has been implicated in the initiation and progression of photocarcinogenesis. UVR exposure elicits numerous cellular and molecular events which include the generation of inflammatory mediators, DNA damage, epigenetic modifications, and oxidative damages mediated activation of signaling pathways. UVR‑initiated signal transduction pathways are believed to be responsible for tumor
promotion effects. UVR‑induced carcinogenic mechanism has been well studied using various animal and cellular models. Human skin‑derived dermal fibroblasts, epidermal keratinocytes, and
melanocytes served as excellent cellular model systems for the understanding of UVR‑mediated carcinogenic events. Apart from this, scientists developed reconstituted three‑dimensionalnormal human skin equivalent models for the study of UVR signaling pathways. Moreover, hairless mice such as SKH‑1, devoid of Hr gene, served as a valuable model for experimental carcinogenesis. Scientists have also used transgenic mice and dorsal portion shaved Swiss albino mice for UVR carcinogenesis studies. In this review, we have discussed the current progress in the study on ultraviolet B (UVB)‑mediated carcinogenesis and outlined appropriate experimental models for both ultraviolet A‑ and UVB‑mediated carcinogenesis.
... Once activated, JNK can phosphorylate c-Jun at Ser 63 and Ser 73 [120] and this phosphorylation increases AP-1 activity in response to UVR exposure [121]. Bowden and colleagues showed that pharmacological inhibition of JNK in human keratinocytes sensitizes cells to UVR-induced apoptosis in vitro [122], which correlates with a study by the same group showing that in vivo inhibition of AP-1 protected transgenic mice expressing a dominant negative c-jun from UVB-induced SCC [123]. Additionally, pharmacological inhibition of JNK inhibited the growth of xenograft studies using human head and neck SCCs in mice [124]. ...
Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide and the incidence continues to rise, in part due to increasing numbers in high-risk groups such as organ transplant recipients and those taking photosensitizing medications. The most significant risk factor for NMSC is ultraviolet radiation (UVR) from sunlight, specifically UVB, which is the leading cause of DNA damage, photoaging, and malignant transformation in the skin. Activation of apoptosis following UVR exposure allows the elimination of irreversibly damaged cells that may harbor oncogenic mutations. However, UVR also activates signaling cascades that promote the survival of these potentially cancerous cells, resulting in tumor initiation. Thus, the UVR-induced stress response in the skin is multifaceted and requires coordinated activation of numerous pathways controlling DNA damage repair, inflammation, and kinase-mediated signal transduction that lead to either cell survival or cell death. This review focuses on the central signaling mechanisms that respond to UVR and the subsequent cellular changes. Given the prevalence of NMSC and the resulting health care burden, many of these pathways provide promising targets for continued study aimed at both chemoprevention and chemotherapy. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
... As shown in Fig. 3D, ISO treatment had no observable effects on the expression levels of phospho-NFKB RELA, NFKB RELA, SP1 or E2F1, but profoundly increased JUN phosphorylation at Ser63, suggesting that JUN might be involved in the transcriptional induction of SESN2 by ISO treatment. To test this notion, we blocked JUN activation in UMUC3 cells by using a well-characterized JUN dominant negative mutant, TAM67 (Fig. 3E), 21 and treated these stable transfectants with 10 mM ISO for 12 or 24 h. As expected, ectopic expression of TAM67 successfully inhibited the induction of endogenous AP-1-dependent transcriptional activity by ISO (Fig. 3F), and further abolished the ISO-mediated transcriptional induction of SESN2 promoter activity (Fig. 3G). ...
Isorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM is able to trigger apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment is able to induce autophagy and inhibit bladder cancer growth through MAPK8-JUN dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.
... [13][14][15] Here, we describe the effects of red light exposure on expression patterns of select genes in the epidermis, in a well-established hairless SKH-1 mouse model. 16,17 In particular, we profiled an expression of genes involved in wound remodeling and healing, 18,19 and studied whether the exposure to red LLLT produced by light-emitting diode (LED) array was capable of attenuating the effects of UV light on gene expression in the live healthy epidermis. ...
Objective:
The purpose of this study was to investigate whether low-level light therapy (LLLT) was capable of modulating expression of ultraviolet (UV) light-responsive genes in vivo.
Materials and methods:
The effects of 670 nm light-emitting diode (LED) array irradiation were investigated in a hairless SHK-1 mouse epidermis model. Mice were given a single dose of UVA/UVB light, or three doses of red light (670 nm @ 8 mW/cm(2) x 312 sec, 2.5 J/cm(2) per session) spread over 24 h along with combinations of pre- and post-UV treatment with red light. Levels of 14 UV-responsive mRNAs were quantified 24 h after UV irradiation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Results:
The transcription of mRNAs encoding for cluster of differentiation molecule 11b (CD11b) (p < 0.05) and interferon (IFN)-γ (p < 0.012) increased after irradiation with red light alone, whereas expression level of cyclooxygenase (COX)-2 (p < 0.02) was downregulated. Genes unresponsive to UV did not change their expression levels after exposure to red light either. Pretreatment with red light significantly modified response of Fos to UV exposure (p < 0.01). A synergy of UV and post-treatment with red light in reducing the transcription levels of CD11b (p < 0.05) and inducible nitric oxide synthase (iNOS) (p < 0.05) was observed.
Conclusions:
This is an initial observation that in mouse red light LLLT more often than not causes opposite gene expression changes or reduces those caused by moderate UVA-UVB irradiation.
... Inspection of the proximal BRCA-1 promoter revealed the presence in close proximity to the exon-1B transcription start site of an element (CTGAG) homologous to a sequence that binds AP-1 factors (Weisz and Rosales, 1990). Several observations presented in this report support the notion that this site is important for basal and E2- (Brown et al., 1993;Cooper et al., 2003;Thompson et al., 2002). Second, the results of ChIP assays provided direct evidence that the BRCA-1 promoter region flanking the AP-1 site was targeted by cJun and FosB upon treatment of MCF-7 cells with E2. ...
... The level of cyclin D1 was increased in correlation with STAT3 activation in mouse skin (19). Cyclin D1 expression is known to increase in UVB-induced skin carcinogenesis (40,41). Similar to earlier studies, our current study showed that cyclin D1 expression is increased with STAT3 expression in TC-PTP-deficient mouse keratinocyte cell lines (Figs. 1B and 4A). ...
Chronic exposure of UV radiation can contribute to the development of skin cancer by promoting PTK signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces PTP inactivation. In the present work, we report that TC-PTP activity is stimulated during the initial response to UVB irradiation which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. In corroboration with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
... Indirect damage is caused by reactive oxygen species (ROS)initiated by UVB exposure, and results in the formation of 8-oxo-deoxyguanosine (8-oxo-dG) adducts125 . This is the initiation of the carcinogenic process.UVB is a complete carcinogen126 . Not only does it initiate the carcinogenesis process by causing DNA mutations, it also promotes the process. ...
Light in the UVB spectrum (280-320 nm) induces a number of changes in the epidermis and dermis of mice and humans, resulting in a robust inflammatory response. A standardized black raspberry extract (BRE) has been effective in reducing signaling pathways commonly initiated by inflammatory stimuli. In this study, we determined whether this extract could reduce cutaneous UVB-induced inflammation and carcinogenesis. In our carcinogenesis model, female SKH-1 hairless mice were exposed to one minimal erythemal dose of UVB thrice weekly on nonconsecutive days for 25 weeks. Immediately after each exposure, the mice were treated topically with either BRE dissolved in vehicle or with vehicle only. Beginning on week 19, mice treated with BRE had a significant reduction in tumor number and in average tumor size. This reduction correlated with a significant reduction in tumor-infiltrating CD3(+)foxp3(+) regulatory T-cells. In the acute model, mice were exposed to a single minimal erythemal dose of UVB and treated topically with BRE or with vehicle. At 48 hours post-UVB exposure, topical BRE treatment significantly reduced edema, p53 protein levels, oxidative DNA damage, and neutrophil activation. The ability of topical BRE to reduce acute UVB-induced inflammation and to decrease tumor development in a long-term model provides compelling evidence to explore the clinical efficacy of BRE in the prevention of human skin cancers.
... protein-1 (AP-1) (Cooper et al., 2003). And AP-1 protein, mostly controlling cell life and death through their ability to regulate the expression and function of cell cycle regulators such as Cyclin D1, p53, p21 cip1/waf1 , p19 ARF and p16, would trigger the programmed cell death of RPE (Eitan et al., 2001). ...
Green tea polyphenol (GTP) is water-soluble medical additives which possess particular significance as free radical scavengers or antioxidants in biological systems. The present study investigated the protective effect of green tea polyphenols against ultraviolet B (UVB)–induced damage to human retinal pigment epithelial (RPE) cells. Microstructure of RPE cells was examined by transmission electron microscopy and the expression of c-fos was examined in messenger ribonucleic acid (mRNA) as well as protein level by using real-time polymerase chain reaction (PCR) and western blot assay. The results indicated that UVB irradiation-induced injuries in RPE cells were markedly suppressed by GTP. The mechanism of GTP protected RPE cells from UVB damage might be related to signal pathway regulation and DNA restoration, suggesting GTP as a potential candidate for further development and aslo a chemoprotective material for prevention of UVB exposure induced eye diseases.
... A remarkable finding is that these mice display a phenotype that includes all of the hallmarks of keratoderma. As AP1 factor inactivation in the basal epidermis produces no such phenotype (Young et al., 1999(Young et al., , 2002Cooper et al., 2003), this implies that epidermal compartment-specific changes in transcription factor signaling can cause keratoderma pathogenesis. Early changes include a progressive increase in skin thickness at 48 to 72 hours after TAM67 expression, and a parallel increase in cell proliferation. ...
Keratodermas comprise a heterogeneous group of highly debilitating and painful disorders characterized by thickening of the skin with marked hyperkeratosis. Some of these diseases are caused by genetic mutation, while other forms are acquired in response to environmental factors. Our understanding of signaling changes that underlie these diseases is limited. In the present study we describe a keratoderma phenotype in mice in response to suprabasal epidermis-specific inhibition of activator protein 1 transcription factor signaling. These mice develop a severe phenotype characterized by hyperplasia, hyperkeratosis, parakeratosis and impaired epidermal barrier function. The skin is scaled, constricting bands encircle the tail and digits, the footpads are thickened and scaled, and loricrin staining is markedly reduced in the cornified layers and increased in the nucleus. Features of this phenotype, including nuclear loricrin localization and pseudoainhum (autoamputation), are characteristic of Vohwinkel Syndrome. We confirm that the phenotype develops in a loricrin null genetic background, indicating that suppressed suprabasal AP1 factor function is sufficient to drive this disease. We also show that the phenotype regresses when suprabasal AP1 factor signaling is restored. Our findings suggest that suppression of AP1 factor signaling in the suprabasal epidermis is a key event in the pathogenesis of keratoderma.Journal of Investigative Dermatology accepted article preview online, 22 July 2014; doi:10.1038/jid.2014.310.
... Tam67, a transactivation deletion mutant of c-Jun, inhibits AP-1 activity by dimerizing with wild-type AP-1 proteins to yield low-activity dimmers [13,14]. Both in vitro and in vivo data showed that the expression of TAM67 inhibited AP-1 activation [14][15][16]. Our group recently demonstrated an induction of AP-1 in benzo(a)pyrene-induced mouse lung tumors as well as an inhibition of lung carcinogenesis following conditional expression of Tam67 [17], indicating that AP-1 is a key regulator of mouse lung tumorigenesis. ...
Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects. © 2013 Wiley Periodicals, Inc.
... A number of signaling pathways have been described as important in the development of SCCs, such as RAS, p53, Notch, and AP-1 (Guinea-Viniegra et al. 2012;Ratushny et al. 2012). Expression of a dominant-negative AP-1 transgene in the skin protects mice from UV-induced, chemically induced, and papillomavirus-induced tumor formation, indicating that AP-1 activity is essential for tumor development (Cooper et al. 2003). In skin physiology, c-Fos is required for the development of RAS-induced malignant papilloma or squamous cell lesions in the background of an epidermal-specific or complete c-fos knockout mouse, while it is dispensable for mouse skin development and homeostasis (Saez et al. 1995;Guinea-Viniegra et al. 2012). ...
Skin squamous cell carcinomas (SCCs) are the second most prevalent skin cancers. Chronic skin inflammation has been associated with the development of SCCs, but the contribution of skin inflammation to SCC development remains largely unknown. In this study, we demonstrate that inducible expression of c-fos in the epidermis of adult mice is sufficient to promote inflammation-mediated epidermal hyperplasia, leading to the development of preneoplastic lesions. Interestingly, c-Fos transcriptionally controls mmp10 and s100a7a15 expression in keratinocytes, subsequently leading to CD4 T-cell recruitment to the skin, thereby promoting epidermal hyperplasia that is likely induced by CD4 T-cell-derived IL-22. Combining inducible c-fos expression in the epidermis with a single dose of the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) leads to the development of highly invasive SCCs, which are prevented by using the anti-inflammatory drug sulindac. Moreover, human SCCs display a correlation between c-FOS expression and elevated levels of MMP10 and S100A15 proteins as well as CD4 T-cell infiltration. Our studies demonstrate a bidirectional cross-talk between premalignant keratinocytes and infiltrating CD4 T cells in SCC development. Therefore, targeting inflammation along with the newly identified targets, such as MMP10 and S100A15, represents promising therapeutic strategies to treat SCCs.
... Dysregulation of AP-1 signaling during cancer progression can lead to alterations in cellular proliferation, apoptotic responses and differentiation. Inhibition of AP-1 greatly reduces UVB-induced skin carcinogenesis in mice [6][7][8] . Recently, it has been shown that SFN inhibits AP-1 activity by directly binding to its DNA-binding domain, therefore preventing the protein from interacting with its response element, the TRE, in the promoters of AP-1-regulated genes 3 . ...
Context:
Sulforaphane (SFN) is a natural compound that has been investigated as a chemopreventive agent. SFN has been shown to inhibit the activator-protein-1 (AP-1) transcription factor and may be effective for inhibition of ultraviolet (UV) induced skin carcinogenesis. This study was designed to investigate the stability of SFN as a function of pH, temperature and in various solvents and formulations.
Materials and methods:
Stability was analyzed using high-performance liquid chromatography. A potential lead formulation was identified and evaluated in vivo.
Results:
SFN was determined to undergo apparent first-order degradation kinetics for the conditions explored. It was observed that SFN undergoes base catalyzed degradation. Buffer species and solvent type impacts stability as well. SFN was found to be very sensitive to temperature with degradation rate changing by a factor of nearly 3.1 for every 10 °C change in temperature (at pH 4.0). SFN completely degraded after 30 days in a conventional pharmaceutical cream formulation. Improved stability was observed in organic formulation components. Stability studies were conducted on two nonaqueous topical formulations: a polyethylene glycol (PEG) ointment base and an organic oleaginous base.
Conclusion:
Topically applied SFN in the PEG base formulation significantly reduced AP-1 activation after UV stimulation in the skin of a transgenic mouse model, indicating that SFN in this formulation retains efficacy in vivo.
... The mice were placed on a shelf 20 cm below the light tubes for irradiation. The cage order was rotated before each treatment to compensate for uneven lamp output [47,48]. UV radiation dosage was measured using a UVX radiometer (UVX-31) from UVP (Upland, CA). ...
The proprotein convertases (PCs) furin and PACE4 process numerous substrates involved in tumor growth, invasion, and metastasis. We have previously shown that PCs increase the susceptibility to chemical skin carcinogenesis. Because of the human relevancy of UV radiation in the etiopathogenesis of human skin cancer, we investigated whether or not transgenic mice overexpressing either furin alone or both furin and PACE4 show increased susceptibility to UV carcinogenesis. After backcrossing our previously described furin and PACE4 transgenic lines, targeted to the epidermis, into a SKH-1 background, we exposed both single and double transgenic mice to UV radiation for 34 weeks. The results showed an increase in squamous cell carcinoma (SCC) multiplicity of approximately 70% in the single furin transgenic mouse line SF47 (P < .002) and a 30% increase in the other single transgenic line SF49 when compared to wild-type (WT) SKH-1 mice. Interestingly, there was also an increase in the percentage of high histologic grade SCCs in the transgenic lines compared to the WT mice, i.e., WT = 9%, SF47 = 15%, and SF49 = 26% (P < .02). Targeting both furin and PACE4 to the epidermis in double transgenic mice did not have an additive effect on tumor incidence/multiplicity but did enhance the tumor histopathologic grade, i.e., a significant increase in higher grade SCCs was seen in the bigenic mouse line SPF47 (P < .02). Thus, we observed an increased susceptibility to UV in single furin transgenic mice that was not substantially enhanced in the double furin/PACE4 transgenic mice.
... In addition, the c-Jun protein activates several events required for tumor progression, including the expression of matrix metalloproteinases (MMPs), proteolytic enzymes that facilitate growth, invasion, and metastasis of cancer cells (2). Reduction of c-Jun/AP-1 activity using dominant-negative c-Jun (TAM67) or conditional inactivation of the c-Jun gene causes cell arrest (3), interferes with tumor development (4,5), suppresses the invasive ability in keratinocytes (6) and fibroblasts (7), and blocks papilloma-to-carcinoma conversion (8). Although the oncogenic activity of c-Jun has been convincingly demonstrated by multiple lines of evidence in human cell lines and mouse models, there are only few examples of elevated c-Jun expression in human cancer (9)(10)(11). ...
Although the protooncogene c-Jun plays a critical role in cell proliferation, cell death, and malignant transformation, DNA microarray screens have identified only a few human cancer types with aberrant expression of c-Jun. Here, we show that c-Jun accumulation is robustly elevated in human glioblastoma and that this increase contributes to the malignant properties of the cells. Most importantly, the increase in c-Jun protein accumulation occurs with no corresponding increase in c-Jun mRNA or the half-life of the c-Jun protein but, rather, in the translatability of the transcript. The c-Jun 5'UTR harbors a potent internal ribosomal entry site (IRES) with a virus-like IRES domain that directs cap-independent translation in glioblastoma cells. Accumulation of c-Jun is not dependent on MAPK activity but can be stimulated by a cytoskeleton-dependent pathway. Our findings provide evidence that human c-Jun is an IRES-containing cellular transcript that contributes to cancer development through translational activation. This previously undescribed mechanism of c-Jun regulation might also be relevant to other types of human cancer and offers unique potential targets for therapy.
... The majority of the research studies utilizing this SKH-1 cancer model use the size of the visible tumor as a function of time as a measure of tumor proliferation. 12,[34][35][36][37][38][39][40][41] The sensitivity of the model to detect small therapeutic effects is limited by the fact that the tumors in treatment and control groups are by their nature different genetically, as each tumor is a result of random mutagenic events. Although this difference is of no significance, because of large numbers of medium-sized tumors in both groups, the individual random mutations resulting in the induction of small numbers of large fast growing tumors potentially affected the overall statistical results. ...
Red light phototherapy is known to stimulate cell proliferation in wound healing. This study investigated whether low-level light therapy (LLLT) would promote tumor growth when pre-existing malignancy is present.
LLLT has been increasingly used for numerous conditions, but its use in cancer patients, including the treatment of lymphedema or various unrelated comorbidities, has been withheld by practitioners because of the fear that LLLT might result in initiation or promotion of metastatic lesions or new primary tumors. There has been little scientific study of oncologic outcomes after use of LLLT in cancer patients.
A standard SKH mouse nonmelanoma UV-induced skin cancer model was used after visible squamous cell carcinomas were present, to study the effects of LLLT on tumor growth. The red light group (n=8) received automated full body 670 nm LLLT delivered twice a day at 5 J/cm(2) using an LED source. The control group (n=8) was handled similarly, but did not receive LLLT. Measurements on 330 tumors were conducted for 37 consecutive days, while the animals received daily LLLT.
Daily tumor measurements demonstrated no measurable effect of LLLT on tumor growth.
This experiment suggests that LLLT at these parameters may be safe even when malignant lesions are present. Further studies on the effects of photoirradiation on neoplasms are warranted.
... We [35,36] and others [37][38][39][40][41][42][43] have used TAM67 to study AP1 factor function in keratinocytes. These studies show that TAM67 inhibits keratinocyte differentiation [35,36]. ...
Our previous study shows that inhibiting activator protein one (AP1) transcription factor function in murine epidermis, using dominant-negative c-jun (TAM67), increases cell proliferation and delays differentiation. To understand the mechanism of action, we compare TAM67 impact in mouse epidermis and in cultured normal human keratinocytes. We show that TAM67 localizes in the nucleus where it forms TAM67 homodimers that competitively interact with AP1 transcription factor DNA binding sites to reduce endogenous jun and fos factor binding. Involucrin is a marker of keratinocyte differentiation that is expressed in the suprabasal epidermis and this expression requires AP1 factor interaction at the AP1-5 site in the promoter. TAM67 interacts competitively at this site to reduce involucrin expression. TAM67 also reduces endogenous c-jun, junB and junD mRNA and protein level. Studies with c-jun promoter suggest that this is due to reduced transcription of the c-jun gene. We propose that TAM67 suppresses keratinocyte differentiation by interfering with endogenous AP1 factor binding to regulator elements in differentiation-associated target genes, and by reducing endogenous c-jun factor expression.
... Cyclooxygenase-2 (Cox-2/Ptgs2) and osteopontin (Opn/Spp1), which are known to be important contributors in driving carcinogenesis, were identified as TAM67-modulated genes [99]. Cooper et al. [100] expressed the TAM67 mutant transgene in the epidermis of SKH-1 hairless mice and bred with mice expressing an AP-1 luciferase reporter gene. Authors showed that expression of TAM67 efficiently inhibits UV-B-induced squamous cell carcinoma number and size in the SKH-1 hairless mouse, which correlated with a diminution in cyclin D1 expression. ...
Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer.
... It has also been well established that AP-1 plays a causative role in skin carcinogenesis, specifically in tumor promotion and/or progression steps both in vitro and in vivo [89][90][91][92] . As a result, inhibition of AP-1 by TAM67 (dominant negative version of c-Jun with a deletion of its transactivation domain) blocked skin carcinogenesis induced by the complete carcinogen, UVB light 93 . However, at this time we do not have similar direct evidence that AP-1 plays a functional role in UVA induced skin carcinogenesis. ...
There are more than two million new cases of non-melanoma skin cancers (NMSCs) diagnosed each year in the United States of America. The clear etiological factor is chronic exposure to solar radiation from the sun. The wavelengths of solar light that reach the earth's surface include UVB (280-320 nm), which accounts for 1-10%, and UVA (320-400 nm), which accounts for 90-99% of the radiation. While most published research has focused on the effects of UVB, little is known concerning UVA-mediated signal transduction pathways, and their role in skin tumor promotion and progression, giving rise to squamous cell carcinomas (SCCs). Here, we focus on UVA-mediated activation of p38 MAP kinase and c-Jun N-terminal kinase (JNK), and their roles in activator protein-1 (AP-1) mediated transcription, cyclooxygenase-2 (COX-2) and Bcl-XL expression. Since p38 MAP kinase and JNK play major roles in the expression of UVA-induced AP-1, COX-2 and Bcl-XL, pharmacological inhibitors of these kinases may be useful in the chemoprevention of SCC skin cancer.
... In particular, among the various members of the AP-1 family, c-fos acts as a tumor promoter, and c-fos upregulation causes cellular transformation that is characterized by colony formation in soft-agar and tumor formation in nude mice [7,8]. In addition, when c-fos binds to c-jun, it increases the gene expression of cyclinD1 and contributes to the potentiation of malignancy [9][10][11]. ...
To date, the major role of HPV16E6 in cancer has been considered to be its ability to inhibit the p53 tumor-suppressor protein, thereby thwarting p53-mediated cytotoxic responses to cellular stress signals. Here, we show that HPV16E6-dependent c-fos oncogenic protein expression contributes to AP-1 complex formation under oxidative stress in SiHa cells (HPV16-positive squamous cell carcinoma of the cervix). In addition, we examined the role of HPV16E6 in TGF-α-induced c-fos expression and found that the c-fos protein expression induced by TGF-α is HPV16E6 dependent. Thus, our results provide the first evidence that HPV16E6 contributes to AP-1 complex formation after both ligand-dependent and independent EGFR activation, suggesting a new therapeutic approach to the treatment of HPV-associated tumors.
... Since the initial discovery of v-JUN, solid evidence has accumulated linking AP-1 members and in particular c-JUN to tumor development. Using mouse models, c-JUN has been implicated in tumor formation in both skin and liver [32][33][34][35][36]. Inhibiting c-JUN activity in basal keratinocytes blocks chemically induced papilloma www.impactjournals.com/oncotarget ...
A novel way by which the AP-1 factor c-JUN interferes with tumorigenesis has recently been elucidated [1]. In a model of murine leukemia, c-JUN prevents the epigenetic silencing of the cell cycle kinase CDK6. In the absence of c-JUN, CDK6 is down-regulated and the 5’region of the gene is methylated. Down-regulation of CDK6 results in significantly delayed leukemia formation. Here we show that c-JUN is also involved in protecting the promoter region of the tumor suppressor p16(INK4a), which is consistently methylated over time in c-JUN deficient cells. In cells expressing c-JUN, p16(INK4a) promoter methylation is a less frequent event. Our study unravels a novel mechanism by which the AP-1 factor c-JUN acts as a “bodyguard”,and preventing methylation of a distinct set of genes after oncogenic transformation.
... In TAM-67 transgenic mice (TAM-67 under the control of keratin 14 promoter) skin tumor promotion induced by TPA or okadaic acid is suppressed [206,207]. Moreover, expression of TAM67 in epidermis also inhibits UV-induced SCC formation in hairless mice, demonstrating that AP-1 activity plays a role in UV-induced mouse skin carcinogenesis [208]. Recently, several specific AP-1 target genes were found to be required for carcinogenesis [209][210][211], suggesting that AP-1 regulated genes might also be targets for cancer prevention. ...
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.
... However, to the best of our knowledge, there are few reports regarding the regulatory role of AP-1 in hsp27 and hsp70 transcriptional control. Therefore, we tested whether c-Jun/AP-1 is involved in Hsp27 and Hsp70 induction in response to arsenite by applying TAM67, a dominant-negative mutant form of c-Jun with a deletion of the transactivation domain (18). The validation of TAM67 upon c-Jun activation is confirmed in Fig. 3A, which shows that overexpression of TAM67 blocked c-Jun phosphorylation at Ser 73 , the requisite for its activation. ...
p27 is an atypical tumor suppressor, which can regulate the activity of cyclin-dependent kinases (CDKs) and G0 to S phase transitions. More recent studies reveal that p27 may also exhibit its tumor suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anti-cancer effects of p27 are largely unknown. In current studies, we found that depletion of p27 expression by either gene knockout or knockdown approaches resulted in upregulation of both Hsp27 and Hsp70 expressions at mRNA and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provided a negative signaling for regulating the expressions of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knockout (p27-/-) and knockdown (shRNA p27) cells. Moreover, interfering the expression or function of JNK2, c-Jun and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expressions. Collectively, our studies demonstrated that p27 suppressed Hsp27 and Hsp70 expressions at transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provided additional important molecular mechanism for the tumor suppressive function of p27.
Key words: Arsenite, p27, Hsp27, and Hsp70
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1330. doi:10.1158/1538-7445.AM2011-1330
... AP-1 is a positive regulator of cell proliferation and transformation, and TPA stimulates its activity in the skin (12). The promotion step likely involves AP-1 because the oncogenic forms of c-jun and p21 ras cooperate in converting normal keratinocytes to SCCs (13) and, as shown more recently, expression of a dominant-negative form of c-jun in skin inhibits tumor formation in the SKH-1 model (14). Agents that suppress tumor formation, such as the vitamin A metabolite all-trans retinoic acid in the two-stage model and the isothiocyanate sulforaphane in the UVB model, can block AP-1 signaling in these model systems (15,16). ...
This perspective on Stratton et al. (beginning on p. 160), Kowalczyk et al. (beginning on p. 170), and Katiyar et al. (beginning on p. 179) highlights the common theme of translational investigation of natural substances and their molecular effects and mechanisms in preventing skin squamous cell carcinoma, which has potentially severe clinical consequences. These studies comprise results of naturally occurring phytochemicals and green tea polyphenols in mouse models of UV-induced and chemically induced skin carcinogenesis and results of perillyl alcohol in a phase IIa clinical trial-all pointing to the great promise of this exciting approach for better understanding of and preventing skin cancer.
... Inhibition of skin papilloma growth has also been observed in mouse models wherein the function of transcription factors that are not oncogenes by themselves is impaired either by gene knockout technology or dominant negative protein expression, including activator protein 1 (36,49), C/ EBPβ (34), signal transducer and activator of transcription 3 (41), C/EBP (35), heat shock protein 90 (50), and c-Myc (51). These results suggest that CREB falls into the class of genes that are involved in what has recently been described as non-oncogenic addiction (52). ...
We used a double transgenic tetracycline system to conditionally express A-CREB, a dominant negative protein that prevents the DNA binding and function of cAMP-responsive element binding protein (CREB) family members, in mouse basal epidermis using the keratin 5 promoter. There was no phenotype in the adult. However, following a 7,12-dimethylbenz(a)anthracene (DMBA)/phorbol-12-myristate-13-acetate two-stage skin carcinogenesis experiment, A-CREB-expressing epidermis develop 5-fold fewer papillomas than wild-type controls. However, A-CREB expression one month after DMBA treatment does not prevent papilloma formation, suggesting that CREB functions at an early stage of papilloma formation. Oncogenic H-Ras genes with A-->T mutations in codon 61 were found in wild-type skin but not in A-CREB-expressing skin 2 days after DMBA treatment, suggesting that A-CREB either prevents DMBA mutagenesis or kills oncogenic H-Ras cells. In primary keratinocyte cultures, A-CREB expression induced apoptosis of v-Ras(Ha)-infected cells and suppressed the expression of cell cycle proteins cyclin B1 and cyclin D1. These results suggest that inhibiting CREB function is a valuable cancer prevention strategy.
... Treatment with UVB increased AP-1:DNA binding 3, 12, 18, and 24 hours after irradiation of SKH-1 epidermis compared to mock treated AL-fed control animals at 0 hours. Other work has also shown that AP-1:DNA binding was increased following UVB treatment in both animal and cell culture models2122. The biphasic time course of UVB induced AP-1:DNA binding may have been, at least in part, a consequence of the changes in constituent proteins observed below, including the early increase in p-c-jun contributing to the first phase and fra-1 and jun- B alternations contributing in the second phase. ...
The study examined the timing of modulation of activator protein 1(AP-1):DNA binding and production of AP-1 constituent proteins by ultraviolet B (UVB) radiation and effect of dietary energy restriction [DER, 40% calorie reduction from fat and carbohydrate compared to control ad libitum (AL) diet] in SKH-1 mouse epidermis. AP-1:DNA binding by electromobility shift assay (EMSA) was increased in a biphasic manner after treatment with a tumor-promoting suberythemal dose (750 mJ/cm(2)) of UVB light (311-313 nm) with peaks at 3 and 18 h postirradiation. DER overall reduced AP-1:DNA binding in mock-treated and UVB-treated skin at 3 and 18 h after UVB treatment. The timing of modulation of production of AP-1 constituent proteins by Western blot analysis was examined at 0 h (mock treatment), 3, 9, 18, and 24 h. We found that c-jun (9 h), jun-B (9 and 18 h), phosphorylated c-jun (3 h), and fra-1 (18 h) protein levels were increased after UVB treatment compared to mock controls. In a follow-up diet experiment, animals were placed on DER or AL diet for 10-12 wk and treated with UVB as before. DER was found to completely block the UVB-induced increase in phosphorylated c-jun protein levels and decrease in fra-2 protein levels at 18 h. In addition, DER enhanced UVB-induced increase in jun B levels and lowered basal levels of c-fos seen 18 h after UVB. These data suggest that DER may be able to assist in the prevention of UVB-induced skin carcinogenesis by modulating AP-1:DNA binding and AP-1 constituent protein levels.
Laparoscopic liver resection (LR) for hepatocellular carcinoma (HCC) is usually applied to superficial and left-side small lesions. Therefore, well designed comparative studies about the results of LR versus open liver resection (OR) for HCC are difficult and still uncommon. The aim of this study was to compare the perioperative and long-term oncologic outcomes of LR versus OR for HCC between well-matched patient groups.
Between January 2000 and March 2012, 205 patients (43 with intent-to-treat with LR, 162 OR) underwent primary liver resection of less than three segments for HCC in our center. To select a comparison group, propensity score matching (PSM) was used at 1:1 ratio with covariates of baseline characteristics, including tumor characteristics. Outcomes were compared between the matched groups.
The two groups were well balanced by PSM and 29 patients were matched respectively. In LR, there was more non-anatomical resection (65.5 vs. 34.5 %; p = 0.012), less postoperative ascites (0.0 vs. 17.2 %; p = 0.025), and shorter hospital stay (7.69 ± 2.94 vs. 13.38 ± 7.37 days; p < 0.001). With the exception of these, there were no significant differences in perioperative and long-term outcomes. The 1-, 3- and 5-year survivals were 100, 100 and 92.2 % in LR, and 96.5, 92.2 and 87.7 % in OR (p = 0.267), respectively. The 1-, 3- and 5-year disease-free survivals were 81.7, 61.7 and 54.0 % in LR, and 78.6, 60.9 and 40.1 % in OR, respectively (p = 0.929).
The outcome of LR for HCC was technically feasible and safe in selected patients, and LR showed similar perioperative and long-term oncologic outcomes when compared with OR matched with PSM.
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. © 2013 Wiley Periodicals, Inc.
The litterbag technique was used to study the decomposition and nutrient dynamics of marsh litter in the four communities, Carex pseudocuraica (C.pa), C. lasiocarpa (C.la), Deyeuxia angustifolia (D.aa), and D. angustifolia-Shrub (D.aa-Srb), in Sanjiang Plain, Northeast China. Decomposition was divided into two periods in the first year, with the mass loss ranging from 11.7% to 31.4% of the initial mass during summer and autumn, accounting for more than 75% of the annual loss. The decomposition rates ranged from 0.000 612 to 0.000 945 d−1 depending on the depth of the flooding and its duration, and differed significantly in each community. The litter decomposed faster in communities with deeper and perennial flooding than in those with shallow and seasonal flooding. The initial ratios of C:N and C:P were also different among the four litter types, but these differences had no impact on the decomposition rates, suggesting that the main factor influencing the decomposition rates of marsh litter was the flooding status rather than the litter quality. The N concentrations in C.pa and C.la almost continuously increased over time, with their final values being 2.8 and 2.4 times higher than the initial ones, respectively. However, the nutrient dynamics in D.aa and D.aa-Srb offered another pattern, sharply falling in the first month and then gradually rising, with the values at the end of the experiment being close to those at the beginning. The litter accumulated substantial amounts of N in C.pa and C.la, while net N release from the litter was observed in both D.aa and D.aa-Srb. The difference may be caused by microorganisms' demand for nutrition, and then limited by the C:N ratios of litter and the availability of nitrogen from the soil and marsh water. In contrast with N dynamics, P concentrations of all the litter types apparently decreased during the first month, and then continued to decline in C.pa, remained constant in C.la and D.aa and increased slightly in D.aa-Srb. At the end of the experiment, the P concentrations decreased, respectively, by 56%, −5%, 47% and 24% of the initial values of C.la, C. pa, D.aa and D.aa-Srb. The net P release was observed in all marsh litter over 480 days of decomposition and the intensity of the P release was different amongst communities, which may be regulated by ratios of initial C:P. The results suggested that in the marsh with the N limitation, litter tended to accumulate N and release P during decomposition and the intensity of accumulation or release was closely related to the initial C:N and C:P ratios.
The chronic lung inflammatory activity and carcinogenicity of nickel compounds have been well documented by previous studies from epidemiology both in vitro and in vivo. However, the molecular mechanism involved in nickel- induced chronic lung inflammation is much less understood. The current study demonstrates that exposure of human bronchial epithelial cells (Beas-2B) to nickel compounds results in the induction of the inflammatory cytokine tumor ne- crosis factor-� (TNF-� ) and transactivation of nuclear factor of activated T cells (NFAT), nuclear factor-� B (NF-� B), and activator protein-1 (AP-1). Further studies show that neither overexpression of IKK� -KM, a kinase inactive mutant of IKK� , nor the ectopic expression of a dominant negative mutant of NFAT could inhibit the TNF-� induction by nickel exposure. Overexpression of TAM67, a dominant-negative mutant of c-Jun, dramatically reduced the TNF-� induction, suggesting that AP-1 is a mediator of TNF-� induction in nickel responses. Our results show that ERKs are AP-1 up- stream kinases responsible for TNF-� induction by nickel exposure; although JNKs, ERKs, and p38K were all activated in the Beas-2B cells exposed to nickel compounds. Our results demonstrate that inflammatory TNF-� could be induced by nickel exposure in Beas-2B cells specifically through an ERKs/AP-1-dependent pathway.
The X-linked inhibitor of apoptosis protein (XIAP) is a well known potent inhibitor of apoptosis; however, it is also involved in other cancer cell biological behavior. In the current study, we discovered that XIAP and its E3 ligase played a crucial role in regulation of cyclin D1 expression in cancer cells. We found that deficiency of XIAP expression resulted in a marked reduction in cyclin D1 expression. Consistently, cell cycle transition and anchorage-independent cell growth were also attenuated in XIAP-deficient cancer cells compared with those of the parental wild-type cells. Subsequent studies demonstrated that E3 ligase activity within the RING domain of XIAP is crucial for its ability to regulate cyclin D1 transcription, cell cycle transition, and anchorage-independent cell growth by up-regulating transactivation of c-Jun/AP-1. Moreover, we found that E3 ligase within RING domain was required for XIAP inhibition of phosphatase PP2A activity by up-regulation of PP2A phosphorylation at Tyr-307 in its catalytic subunit. Such PP2A phosphorylation and inactivation resulted in phosphorylation and activation of its downstream target c-Jun in turn leading to cyclin D1 expression. Collectively, our studies uncovered a novel function of E3 ligase activity of XIAP in the up-regulation of cyclin D1 expression, providing significant insight into the understanding of the biomedical significance of overexpressed XIAP in cancer development, further offering a new molecular basis for utilizing XIAP E3 ligase as a cancer therapeutic target.
Background: XIAP is involved in cancer cell proliferation.
Results: The deficiency of XIAP E3 ligase inhibits cancer cell anchorage-independent growth, cell cycle transition, and cyclin D1 transcription, which is mediated by its regulation of PP2A catalytic subunit phosphorylation, thereby activating AP-1.
Conclusion: XIAP E3 ligase mediates cyclin D1 transcription via PP2A-regulated AP-1 activation.
Significance: This study suggests that XIAP E3 ligase can serve as a cancer therapeutic target.
The transcription factor AP-1 (activator protein-1) regulates a number of genes that drive tumor promotion and progression. While basal levels of AP-1 activity are important for normal cell proliferation and cell survival, overactivated AP-1-dependent gene expression stimulates inflammation, angiogenesis, invasion, and other events that propel carcinogenesis. We seek to discover genes targeted by carcinogenesis inhibitors that do not also inhibit cell proliferation or survival. Transgenic TAM67 (dominant-negative c-Jun) inhibits mouse skin tumorigenesis and tumor progression without inhibiting cell proliferation or induced hyperproliferation. Expression profiling of wild-type and K14-TAM67 mouse epidermis has revealed a number of functionally significant genes that are induced by tumor promoters in wild-type mice but not in those expressing the AP-1 blocker. The current study now identifies Wnt5a signaling as a new target of TAM67 when it inhibits DMBA/TPA-induced carcinogenesis. Wnt5a is required to maintain the tumor phenotype in tumorigenic mouse JB6 cells and Ras-transformed human squamous carcinoma HaCaT-II4 cells, as Wnt5a knockdown suppresses anchorage-independent and tumor xenograft growth. The oncogenic Wnt5a-mediated pathway signals through activation of the protein kinase PKCα and oncogenic transcription factor STAT3 phosphorylation and not through the canonical Wnt/β-catenin pathway. Similar to Wnt5a knockdown, inhibitors of PKCα blocked STAT3 activation in both mouse JB6 and human HaCaT-II4 tumor cells. Moreover, expression of STAT3-regulated genes FAS, MMP3, IRF1, and cyclin D1 was suppressed with Wnt5a knockdown. Treatment of mouse Wnt5a knockdown cells with a PKCα-specific activator rescued phosphorylation of STAT3. Thus, Wnt5a signaling is required for maintaining the tumor phenotype in squamous carcinoma cells, Wnt5a targeting by the AP-1 blockade contributes to inhibition of skin carcinogenesis, and the signaling pathway traverses PKCα and STAT3 activation. Coordinate overactivation of Wnt5a expression and STAT3 signaling is observed in human skin and colon cancers as well as glioblastoma.
In order to identify important cell signaling derangements in squamous cell carcinoma (SCC) and its precursor actinic keratosis (AK) as a means to uncover important molecular targets and further characterize this disease, we analyzed two independent tissue study sets using Reverse Phase Protein Microarrays (RPMA). Set 1 served as a pilot as well as a means to generate candidate pathway alterations that could be further validated in a second independent sample set. Study Set 1 was comprised of 4 AK, 6 advanced SCC (≥ 2 cm), 7 non-advanced SCC, and 20 normal appearing skin samples [upper-inner arm, (UIA)]. Set 2 included15 AK, 5 advanced SCC, 4 non-advanced SCC and 20 UIA. LCM was used to isolate pure populations of epithelial cells. Fifty-one key signaling proteins and phosphorylated proteins were assessed for set 1 showing that the MEK-ERK pathway was activated in SCC compared to AK and normal skin, that EGFR was aberrantly activated in SCC, and that the mTOR pathway was highly activated in SCC. Unsupervised clustering revealed dramatic differences in SCC signaling architecture. Statistical analysis (p<0.01) found that pro-survival signaling through phosphorylation of ASK, and 4EBP1 as well as increased Bax and Bak expression were higher in AK vs UIA. We then expanded our pathway network activation mapping in set 2 to 108 key signaling proteins. We corroborated the MEK-ERK, EGFR and mTOR pathway activation and identified an expanded number of upstream and downstream signaling molecules within these pathways, and that SCC is indeed a pathway activation-driven disease.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2917. doi:10.1158/1538-7445.AM2011-2917
Quercetin (Qu) is currently being investigated as a chemopreventive agent for several cancers, including nonmelanoma skin cancer induced by UV light. We previously reported that Qu degradation has important consequences on signaling and cell biology. In the current study, we report that Qu induces c-Fos mRNA and protein expression through activation of p38 and cAMP-responsive element binding protein (CREB), and Qu potentiates UVB-induced c-Fos expression. Inclusion of ascorbic acid (AA) in cell culture medium stabilizes Qu and completely prevents both Qu- and UVB-induced p38 and CREB activation, leading to a blockade of c-fos gene expression through reduced CREB/cAMP-responsive element binding. AA stabilizes c-Fos mRNA, increasing steady-state levels even when c-fos gene expression is suppressed, but this has no effect on c-Fos protein levels in either mock- or UVB-irradiated cells. We report that Qu blocks mammalian target of rapamycin signaling and inhibits c-Fos protein expression directly through this mechanism because cotreatment with Qu and AA resulted in the complete suppression of UVB-induced c-Fos protein expression even in the presence of significantly increased mRNA levels. We further confirmed that this was not due to increased protein turnover because inhibition of proteasome activity with MG-132 did not raise c-Fos protein levels in Qu+AA-treated cells. Together, these data indicate that although Qu has been reported to have some beneficial properties as a chemopreventive agent, it is also capable of inducing c-fos expression, a cellular event important for the promotion phase of tumor development, if it is not stabilized.
Capsiate, one of the major capsaicinoids, is nonpungent and present in sweet pepper. We investigated the effects of capsiate on the ultraviolet B (UVB)-induced inflammatory response in skin and its molecular mechanisms. Capsiate-pretreated human keratinocytes inhibited intracellular reactive oxygen species (ROS), which activate the mitogen-activated protein kinase and nuclear factor-kappaB (NF-kappaB) pathways. Therefore, we determined the effects of capsiate on these pathways. Capsiate inhibited UVB-induced cyclooxygenase-2 (COX-2) expression, extracellular signal-related kinase 1/2 phosphorylation, nuclear translocation of NF-kappaB, and the expression of proinflammatory cytokines and potent angiogenic factors, including vascular endothelial cell growth factor and matrix metalloproteinase-2 (MMP-2) and MMP-9. In addition, capsiate inhibited UVB-induced epidermal growth factor receptor (EGFR) activation, which reduces the levels of proinflammatory cytokines and angiogenic factors. We also investigated the photoprotective effects of capsiate in vivo. Topical treatment with capsiate significantly decreased UVB-induced skin damage and inhibited the expression of COX-2, proinflammatory cytokines, and angiogenic factors, including platelet/endothelial cell adhesion molecule-1 and intercellular adhesion molecule-1. Inhibition of Src kinase activity and ROS may inhibit the EGFR activation. Therefore, capsiate may protect the skin from UVB-induced adverse effects and these results provide a molecular basis for understanding its effects on inflammation and angiogenesis.
The chemopreventive and antitumor properties of perillyl alcohol (POH) that were studied preclinically indicate that topical POH inhibits both UVB-induced murine skin carcinogenesis (squamous cell tumor models) and 7,12-dimethylbenz(a)anthracene-induced murine melanoma (transgenic models involving tyrosinase-driven Ras). A previous phase 1 clinical trial in participants with normal-appearing skin showed that topical POH cream was well tolerated at a dose of 0.76% (w/w). Here, we performed a 3-month, double-blind, randomized, placebo-controlled phase 2a trial of two different doses of topical POH in individuals with sun-damaged skin. Participants applied POH cream twice daily to each dorsal forearm. Baseline and end-of-study biopsies were taken from each participant to evaluate whether the topical application of POH was effective in reversing actinic damage as evidenced by normalization of quantitative skin histopathologic scores and change in nuclear chromatin pattern as measured by karyometric analysis. There was a borderline reduction in the histopathologic score of the lower-dose POH group compared with the placebo (P = 0.1), but this was not observed in the high-dose group. However, in the high-dose group, a statistically significant reduction in the proportion of nuclei deviating from normal was observed by the use of karyometric analysis (P < 0.01). There was no statistical significance shown in the lower-dose group. No changes were observed in p53 expression, cellular proliferation (by proliferating cell nuclear antigen expression), or apoptosis in either treatment group compared with the placebo group. These results suggest that whereas our karyometric analyses can detect a modest effect of POH in sun-damaged skin, improved delivery into the epidermis may be necessary.
Sulforaphane is an isothiocyanate derived from cruciferous vegetables that has been linked to decreased risk of certain cancers. Although the role of sulforaphane in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor activator protein-1 (AP-1) may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that sulforaphane is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinoma in a mouse model using cotreatment with the compound and the carcinogen. We also show that sulforaphane pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by sulforaphane. Electrophoretic mobility shift assay analysis of nuclear proteins also shows that sulforaphane and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun, we show that mutation of critical cysteines in the DNA-binding domain of these proteins (Cys(154) in cFos and Cys(272) in cJun) results in loss of sensitivity to both sulforaphane and diamide in electrophoretic mobility shift assay analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of squamous cell carcinoma by sulforaphane.
Healthy cells strictly regulate gene transcription to control crucial cellular regulatory pathways. Membersof the Jun protein family, c-Jun, JunB, and JunD are key subunits of the transcription factor AP-1 thatcontrols transcription from various gene promoters. The genes targeted by Jun affect essential lifeprocesses, such as cell cycle progression, differentiation or programmed cell death. Therefore, the loss ofproper Jun function is often associated with cancer. This review summarizes recent advances inunderstanding of function of the Jun proteins in healthy and cancer cells.
Inositol hexaphosphate (IP6) is a naturally occurring polyphosphorylated carbohydrate that is abundant in many plants and in various high-fiber foods, such as cereals and legumes. IP6 has a striking, broad-spectrum anticancer activity in various in vitro and animal models, in which it interferes with key pathways in malignancy to inhibit cell proliferation, cell-cycle progression, metastasis, invasion, and angiogenesis and to induce apoptosis. In this study, we investigated the protective effects of IP6 in drinking water on the incidence of UVB-induced skin cancer in the SKH1 (Crl: SKH1-hr) mouse model. One group of 15 mice received 2% IP6 in drinking water and UVB exposure, and the other group (n = 15) received UVB exposure only. All mice in both groups were fed an IP6-deficient diet (AIN 76A). The treatment group started receiving 2% IP6 in the drinking water 3 d before irradiation. Mice were irradiated 3 times each week, starting at a dose of 1.5 kJ/m2, with weekly increases in increments of 1.5 kJ/m2 to a final dose of 7.5 kJ/m2. Tumor formation was monitored until the week 31. IP6 in drinking water significantly decreased tumor incidence by 5-fold and tumor multiplicity by 4-fold. These results show that IP6 has an antiphotocarcinogenic effect and can protect against UVB-induced tumor formation.
Tumor promoters may bring about events that lead to neoplastic transformation by inducing specific promotion-relevant effector genes. Functional activation of the transacting transcription factor AP-1 by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) may play an essential role in this process. Clonal genetic variants of mouse epidermal JB6 cells that are genetically susceptible (P+) or resistant (P-) to promotion of transformation by TPA were transfected with 3XTRE-CAT, a construct that has AP-1 cis-enhancer sequences attached to a reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected JB6 P+, but not P- variants, showed TPA-inducible CAT synthesis. Epidermal growth factor, another transformation promoter in JB6 cells, also caused P+ specific induction of CAT gene expression. These results demonstrate an association between induced AP-1 function and sensitivity to promotion of neoplastic transformation.
Cyclin D1 is part of a cell cycle control node consistently deregulated in most human cancers. However, studies with cyclin D1-null mice indicate that it is dispensable for normal mouse development as well as cell growth in culture. Here, we provide evidence that ras-mediated tumorigenesis depends on signaling pathways that act preferentially through cyclin D1. Cyclin D1 expression and the activity of its associated kinase are up-regulated in keratinocytes in response to oncogenic ras. Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumors generated through either grafting of retroviral ras-transduced keratinocytes, phorbol ester treatment of ras transgenic mice, or two-stage carcinogenesis.
NF-kappaB activation in response to UV irradiation of HeLa cells or of primary human skin fibroblasts occurs with two overlapping kinetics but totally different mechanisms. Although both mechanisms involve induced dissociation of NF-kappaB from IkappaBalpha and degradation of IkappaBalpha, targeting for degradation and signaling are different. Early IkappaBalpha degradation at 30 min to approximately 6 h is not initiated by UV-induced DNA damage. It does not require IkappaB kinase (IKK), as shown by introduction of a dominant-negative kinase subunit, and does not depend on the presence of the phosphorylatable substrate, IkappaBalpha, carrying serines at positions 32 and 36. Induced IkappaBalpha degradation requires, however, intact N- (positions 1-36) and C-terminal (positions 277-287) sequences. IkappaB degradation and NF-kappaB activation at late time points, 15-20 h after UV irradiation, is mediated through DNA damage-induced cleavage of IL-1alpha precursor, release of IL-1alpha and autocrine/paracrine action of IL-1alpha. Late-induced IkappaBalpha requires the presence of Ser32 and Ser36. The late mechanism indicates the existence of signal transfer from photoproducts in the nucleus to the cytoplasm. The release of the 'alarmone' IL-1alpha may account for some of the systemic effects of sunlight exposure.
The effects of UVB irradiation on transcription factor activator protein-1 (AP-1) DNA binding and AP-1 transactivation were
studied in a human keratinocyte cell line, HaCaT. UVB-induced AP-1 binding to a consensus AP-1 binding site was observed by
gel shift assays with maximum stimulation at 12 h after UVB irradiation. A promoter region of the human collagenase-1 gene
containing the same AP-1 binding sequence linked to a luciferase reporter gene was stably transfected into HaCaT cells. UVB
irradiation significantly increased luciferase activity in these stably transfected cells, with maximum activity observed
at 24 h after UVB irradiation. c-Fos and Jun D were identified by antibody clearing assays as the main components of the bound
AP-1 complexes. Inhibition of transcription with actinomycin D and inhibition of protein synthesis with cycloheximide significantly
abrogated the effect of UVB on AP-1 DNA binding, indicating that transcription and translation were required for AP-1 activation.
Northern and Western analyses revealed a correlation between increased AP-1 activity and accumulation of c-fos mRNA and c-Fos protein after UVB irradiation. UVB irradiation increased c-fos transcription in HaCaT cells stably transfected with a plasmid containing the human c-fos promoter driving a luciferase reporter gene. These results suggest that increased c-fos expression may play an important role in UVB-induced AP-1 activation in HaCaT cells.
c-Jun is a component of the transcription factor AP-1, which is activated by a wide variety of extracellular stimuli. The regulation of c-Jun is complex and involves both increases in the levels of c-Jun protein as well as phosphorylation of specific serines (63 and 73) by Jun N-terminal kinase (JNK). We have used fibroblasts derived from c-Jun null embryos to define the role of c-Jun in two separate processes: cell growth and apoptosis. We show that in fibroblasts, c-Jun is required for progression through the G1 phase of the cell cycle; c-Jun-mediated G1 progression occurs by a mechanism that involves direct transcriptional control of the cyclin D1 gene, establishing a molecular link between growth factor signaling and cell cycle regulators. In addition, c-Jun protects cells from UV-induced cell death and cooperates with NF-kappaB to prevent apoptosis induced by tumor necrosis factor alpha (TNFalpha). c-Jun mediated G1 progression is independent of phosphorylation of serines 63/73; however, protection from apoptosis in response to UV, a potent inducer of JNK/SAP kinase activity, requires serines 63/73. The results reveal critical roles for c-Jun in two different cellular processes and show that different extracellular stimuli can target c-Jun by distinct biochemical mechanisms.
The c-jun proto-oncogene encodes a component of the mitogen-inducible immediate-early transcription factor AP-1 and has been implicated as a positive regulator of cell proliferation and G1-to-S-phase progression. Here we report that fibroblasts derived from c-jun-/- mouse fetuses exhibit a severe proliferation defect and undergo a prolonged crisis before spontaneous immortalization. The cyclin D1- and cyclin E-dependent kinases (CDKs) and transcription factor E2F are poorly activated, resulting in inefficient G1-to-S-phase progression. Furthermore, the absence of c-Jun results in elevated expression of the tumor suppressor gene p53 and its target gene, the CDK inhibitor p21, whereas overexpression of c-Jun represses p53 and p21 expression and accelerates cell proliferation. Surprisingly, protein stabilization, the common mechanism of p53 regulation, is not involved in up-regulation of p53 in c-jun-/- fibroblasts. Rather, c-Jun regulates transcription of p53 negatively by direct binding to a variant AP-1 site in the p53 promoter. Importantly, deletion of p53 abrogates all defects of cells lacking c-Jun in cell cycle progression, proliferation, immortalization, and activation of G1 CDKs and E2F. These results demonstrate that an essential, rate-limiting function of c-Jun in fibroblast proliferation is negative regulation of p53 expression, and establish a mechanistic link between c-Jun-dependent mitogenic signaling and cell-cycle regulation.
Activator protein-1 (AP-1) is a transcription factor that consists of either a Jun-Jun homodimer or a Jun-Fos heterodimer. Transactivation of AP-1 is required for tumor promoter-induced transformation in mouse epidermal JB6 cells and for progression in mouse and human keratinocytes. Until now, the question of whether AP-1 transactivation is required for carcinogenesis in vivo has remained unanswered, as has the issue of functionally significant target genes. To address these issues we have generated a transgenic mouse in which transactivation mutant c-jun (TAM67), under the control of the human keratin-14 promoter, is expressed specifically in the basal cells of the epidermis where tumor induction is initiated. The keratin-14-TAM67 transgene was expressed in the epidermis, tongue, and cervix, with no apparent abnormalities in any tissue or organ. TAM67 expression blocked 12-O-tetradecanoylphorbol 13-acetate (TPA, phorbol 12-tetradecanoate 13-acetate) induction of the AP-1-regulated luciferase in AP-1 luciferase/TAM67 mice, but did not inhibit induction of candidate AP-1 target genes, collagenase-1 or stromelysin-3. More interestingly, TAM67 expression did not inhibit TPA-induced hyperproliferation. In two-stage skin carcinogenesis experiments, the transgenic animals showed a dramatic inhibition of papilloma induction. We conclude that transactivation of a subset of AP-1-dependent genes is required for tumor promotion and may be targeted for cancer prevention.
Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor κB (NFκB) transcription factors. The present study addresses the question of whether elevated NFκB in addition to elevated AP-1–dependent gene expression is necessary for maintaining the tumor cell phenotype. When a tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1- and NFκB- but not p53-dependent reporter activity was inhibited by 40–60%. Tumor phenotype, as measured by anchorage-independent growth, was inhibited by 90%. Neither AP-1/NFκB activation nor expression of tumor phenotype was inhibited in TAM67-harboring keratinocytes under noninducing conditions. Electrophoretic mobility shift analysis showed that induction of TAM67 expression slightly increased AP-1- but reduced NFκB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFκB p65, suggesting that inhibition of NFκB by TAM67 is mediated by direct protein-protein interactions, possibly producing decreased binding to DNA or inactivating p65. To analyze the putative effector genes that may be targeted by TAM67, expression of genes responsive to AP-1 or NFκB was measured by reverse transcriptase–polymerase chain reaction in TAM67 transfectants with or without TAM67 induction. Induction of TAM67 inhibited or reduced the expression of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6 (NFκB responsive). These results indicate that genes controlled by NFκB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFκB activation through direct interaction with NFκB p65. Moreover, the findings provide proof for the principle of using inducible TAM67 as a gene therapy to suppress tumor phenotype in human carcinoma cells. Mol. Carcinog. 29:159–169, 2000. © 2000 Wiley-Liss, Inc.
Abstract— Hairless mice were irradiated repeatedly by exposure to unfiltered black-light (FR74T12 PUVA) fluorescent lamps and the time to development of skin tumors was determined. For several groups of animals the treatment variable was the size of the weekly dose. A similar approach had been used previously to determine dose-response characteristics for other ultraviolet radiation emitting sources: a xenon arc solar simulator (with a series of five cut-off filters producing five source spectra), and a fluorescent (FS40T12) “sunlamp”. The median tumor latent period (time period for just more than one half of the animals to develop at least one tumor each) was accurately predicted for all these ultraviolet radiation emitting sources by a mathematical equation incorporating the spectral source description and a spectral weighting function. The weighting function judged most appropriate for ultraviolet radiation-induced photocarcinogenesis was the action spectrum, determined previously, for acute (single dose) skin edema in hairless mice. The mathematical equation assigns no effectiveness to wavelengths greater than 330 nm. There was no evidence for wavelength interaction in the spectral range of 26MW nm. Our data, combined with results of others, lead us to conclude that radiation with wavelength greater than 330 nm has an average relative efficacy (297 nm =1.0) less than 0.0002, and that this efficacy is not detectable with sources in which at least 2% of the UV radiation is in the UV-B range.
The mouse epidermal cell line 308 contains an activated Ha-ras gene and forms benign papillomas when transplanted to the skin of athymic nude mice. A radiation-associated malignant variant of this cell line, 308-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in malignant conversion of 308-10Gy5, we assessed the activator protein-1 (AP-1) binding and transactivating ability of 308 and 308-10Gy5. In nuclear protein extracts of 308, AP-1 sequence-specific binding to an oligonucleotide containing a single high-affinity AP-1 binding site was induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as determined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in malignant versus benign tumor cells, but so was transactivation of a unique AP-1-responsive chloramphenicol acetyltransferase reporter construct, pTiCTaK. Constitutive transactivation of this AP-1-responsive reporter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-associated AP-1-responsive genes stromelysin, urokinase-type plasminogen activator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells than in benign 308 cells. These results suggest that acquisition of constitutive AP-1 DNA binding and transactivation can result in sustained deregulation of gene expression. While malignant progression in keratinocytes is probably not due solely to the acquisition of constitutive cellular AP-1 activity, the effect of deregulated expression of AP-1-regulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion.
AP-1 transcriptional activity is stimulated by the transformation promoters phorbol 12-myristate 13-acetate ("12-O-tetradecanoylphorbol 13-acetate," TPA) and epidermal growth factor (EGF) in promotion-sensitive (P+) but not in promotion-resistant (P-) JB6 mouse epidermal cell lines. Although TPA stimulates expression of the jun and fos family genes, only c-jun expression shows higher elevation in P+ cells than in P- cells. The present study tests the hypothesis that induced AP-1 activity is required for tumor promoter-induced transformation in JB6 P+ cells. Both retinoic acid and the glucocorticoid fluocinolone acetonide inhibited basal and TPA-induced AP-1 activities that were tested with a stromelysin promoter-chloramphenicol acetyltransferase reporter gene in P+ cells. Since both retinoic acid and fluocinolone acetonide are active in inhibiting TPA-induced anchorage-independent transformation of P+ cells in the dose range that blocks TPA-induced AP-1 activity, their antipromoting effects may occur through inhibition of AP-1 activity. To test the hypothesis with a more specific inhibitor, stable clonal transfectants of P+ cells expressing dominant negative c-jun mutant encoding a transcriptionally inactive product were analyzed. All transfectants showed a block in TPA and EGF induction of AP-1 activity. All transfectants also showed inhibition of TPA-induced transformation, and most transfectants showed a block in EGF-induced transformation. These results indicate that AP-1 activity is required for TPA- or EGF-induced transformation. This work demonstrates that a specific block in induced AP-1 activity inhibits tumor promoter-induced transformation.
We previously reported that induced activator protein-1 (AP-1) transcriptional activity appears to be required for tumor promoter-induced transformation in mouse epidermal JB6 cells. To extend this investigation to a keratinocyte culture model and a transgenic mouse model, we constructed K14TAM67, a keratin 14 promoter-controlled version of the dominant negative jun mutant to directly block AP-1 activity and possibly indirectly block NF kappa B activity in basal squamous epithelia. This study was directed at characterizing TAM67 expression and biological activity in the mouse cell line 308, a keratinocyte model for studying carcinogenesis. Cotransfection of K14TAM67 with luciferase plasmid reporter DNAs produced inhibition of basal and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced AP-1 and NF kappa B activity but had no effect on p53-dependent transcriptional activity. In an in vitro invasion assay, stable expression of TAM67 in 308 cells blocked TPA-induced Matrigel invasion. This suggests that blocking TPA-induced AP-1- or NF kappa B-regulated gene expression by TAM67 inhibits TPA-induced progression. Recombinant tissue inhibitor of metalloproteinase 1 reduced TPA-induced in vitro invasion, thus implicating metalloproteinases at least in part in the transcription factor-dependent process. Analysis of mRNA levels for members of the matrix metalloproteinase (MMP) family, however, revealed that the expression of any single MMP family member did not correlate with regulation of AP-1 or NF kappa B activity. However, the combination of substantial levels of mRNA for stromelysin-1, stromelysin-2, collagenase, membrane type 1 MMP, and gelatinase A occurred only in TPA-treated cells in the absence of TAM67. These results suggest that the action of the dominant negative jun mutant on AP-1 and NF kappa B gene regulation results in complex alterations in the levels of downstream effector genes, such as the metalloproteinases, that effect TPA-induced cellular invasion.
The vertebrate transcription factor NF-kappaB is induced by over 150 different stimuli. Active NF-kappaB, in turn, participates in the control of transcription of over 150 target genes. Because a large variety of bacteria and viruses activate NF-kappaB and because the transcription factor regulates the expression of inflammatory cytokines, chemokines, immunoreceptors, and cell adhesion molecules, NF-kappaB has often been termed a 'central mediator of the human immune response'. This article contains a complete listing of all NF-kappaB inducers and target genes described to date. The collected data argue that NF-kappaB functions more generally as a central regulator of stress responses. In addition, NF-kappaB activation blocks apoptosis in several cell types. Coupling stress responsiveness and anti-apoptotic pathways through the use of a common transcription factor may result in increased cell survival following stress insults.
Genetically modified mice have provided important insights into the biological functions of the dimeric transcription factor complex AP-1. Extensive analyses of mice and cells with genetically modified Fos or Jun proteins provide novel insights into the physiological functions of AP-1 proteins. Using knock-out strategies it was found that some components, such as c-Fos, FosB and JunD are dispensable, whereas others, like c-Jun, JunB and Fra-1 are essential in embryonic development and/or in the adult organism. Besides the specific roles of AP-1 proteins in developmental processes, we are beginning to obtain a better molecular understanding of the cell-context dependent function of AP-1 in cell proliferation and apoptosis, in bone biology as well as in multistep tumorigenesis.
A plethora of physiological and pathological stimuli induce and activate a group of DNA binding proteins that form AP-1 dimers. These proteins include the Jun, Fos and ATF subgroups of transcription factors. Recent studies using cells and mice deficient in individual AP-1 proteins have begun to shed light on their physiological functions in the control of cell proliferation, neoplastic transformation and apoptosis. Above all such studies have identified some of the target genes that mediate the effects of AP-1 proteins on cell proliferation and death. There is evidence that AP-1 proteins, mostly those that belong to the Jun group, control cell life and death through their ability to regulate the expression and function of cell cycle regulators such as Cyclin D1, p53, p21(cip1/waf1), p19(ARF) and p16. Amongst the Jun proteins, c-Jun is unique in its ability to positively regulate cell proliferation through the repression of tumor suppressor gene expression and function, and induction of cyclin D1 transcription. These actions are antagonized by JunB, which upregulates tumor suppressor genes and represses cyclin D1. An especially important target for AP-1 effects on cell life and death is the tumor suppressor p53, whose expression as well as transcriptional activity, are modulated by AP-1 proteins.
Expression of the human papillomavirus (HPV) type 16 E6 and E7 gene products is a risk factor for human cervical carcinogenesis as well as skin and oral carcinogenesis. Expression of the HPV-16 E7 gene in mouse skin induces hyperplasia and enhances tumor promotion. Expression of dominant-negative c-jun (TAM67) in the mouse skin protects mice from 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced papillomagenesis without blocking mitogen-induced hyperproliferation. To determine the role of activator protein-1 (AP-1) in HPV-induced cancer, we crossed HPV-16 E7 mice with TAM67 mice and analyzed the effects of DMBA/TPA on tumor promotion. We showed that expression of TAM67 protected mice from HPV-16 E7-enhanced tumorigenesis, suggesting AP-1 as a target for prevention of HPV-induced cancer.