Mouse Skin Carcinogenesis Model Research Articles

Abstract 1590: Taxifolin suppresses UV-induced skin carcinogenesis by targeting EGFR and PI3-K

Abstract Skin cancer is one of the most commonly diagnosed cancers in United States. Taxifolin (3, 3′, 4′, 5, 7-pentahydroxy-flavanone, (2R,3R)-dihydroquercetin), which is found in many citrus fruits such as grapefruits and oranges, has been studied for multiple biological effects. However, the molecular mechanisms and direct target of taxifolin in skin cancer chemoprevention are unclear. Here we show that taxifolin suppresses UV-induced skin carcinogenesis by directly targeting epidermal growth factor receptor (EGFR) and phosphoinositide 3-kinase (PI3-K). In silico computer screening results suggest that EGFR and PI3-K are potential targets for taxifolin. In vitro and ex vivo pull-down assay data revealed that taxifolin indeed directly interacts with EGFR and PI3-K at the ATP-binding pocket, and inhibited their kinase activities in vitro. UVB-induced phosphorylation of EGFR and Akt were substantially suppressed by taxifolin in mouse skin epidermal JB6 P+ cells. Taxifolin also suppressed UVB-induced phosphorylation of mitogen-activated protein kinases (MAPKs) and cyclooxygenase-2 (COX-2) expression. Importantly, topical treatment with taxifolin to the dorsal skin significantly suppressed tumor incidence, multiplicity and volume in a solar UV-induced skin carcinogenesis mouse model. Further analysis of the solar UV-exposed mouse skin showed that the taxifolin-treated group had a substantial reduction in solar UV-induced phosphorylation of EGFR and Akt. These results suggest that taxifolin exerts potent chemopreventive activity against UV-induced skin carcinogenesis by targeting EGFR and PI3-K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1590. doi:1538-7445.AM2012-1590

The Role of TGFβ Signaling in Squamous Cell Cancer: Lessons from Mouse Models.

TGFβ1 is a member of a large growth factor family including activins/inhibins and bone morphogenic proteins (BMPs) that have a potent growth regulatory and immunomodulatory functions in normal skin homeostasis, regulation of epidermal stem cells, extracellular matrix production, angiogenesis, and inflammation. TGFβ signaling is tightly regulated in normal tissues and becomes deregulated during cancer development in cutaneous SCC and many other solid tumors. Because of these diverse biological processes regulated by TGFβ1, this cytokine and its signaling pathway appear to function at multiple points during carcinogenesis with distinct effects. The mouse skin carcinogenesis model has been a useful tool to dissect the function of this pathway in cancer pathogenesis, with transgenic and null mice as well as small molecule inhibitors to alter the function of the TGFβ1 pathway and assess the effects on cancer development. This paper will review data on changes in TGFβ1 signaling in human SCC primarily HNSCC and cutaneous SCC and different mouse models that have been generated to investigate the relevance of these changes to cancer. A better understanding of the mechanisms underlying the duality of TGFβ1 action in carcinogenesis will inform potential use of this signaling pathway for targeted therapies.

Anti-tumor-Promoting Activities of Agaro-Oligosaccharides on Two-Stage Mouse Skin Carcinogenesis

We have previously reported that agaro-oligosaccharides (AGOs) suppressed the elevated levels of nitric oxide (NO), prostaglandin E₂(PGE₂), and pro-inflammatory cytokines in activated monocytes/macrophages, via heme oxygenase-1 induction. In this report, we initially demonstrated that AGOs intake inhibited NO production in activated peritoneal macrophages. Then, we tested for the ability of AGOs to prevent tumor promotion on the two-stage mouse skin carcinogenesis model. As a result, AGOs feeding led to delayed tumor appearance and decreased tumor number. It is known that PGE₂ is one of key players in carcinogenesis. Thus, we confirmed that PGE₂ production was suppressed by AGOs intake in TPA-induced ear edema model. We also demonstrated that cyclooxygenase-2 and microsomal PGE synthase-1, rate-limiting enzymes in PGE₂ production, were down-regulated by AGOs in human monocytes. Consequently, AGOs are expected to prevent tumor promotion by inhibiting PGE₂ elevation in chronic inflammation site.

Abstract PL02-02: BRAF and RAS signaling in cancer: From biology to therapeutics.

Abstract The RAS proteins are small G-protein that are activated downstream of cell surface receptors. The RAF proteins are protein kinases that are activated by RAS. RAF proteins then activate the MEK/ERK signalling pathway, which stimulates cell proliferation and drives tumorigenesis. There are three RAS proteins (HRAS, KRAS, NRAS) in humans and the NRAS isoform is mutated in about 25% of melanomas. There are also three RAF proteins in humans (ARAF, BRAF and CRAF) and BRAF is mutated in a further 50% of melanomas. Critically, drugs that target BRAF achieve impressive objective responses in patients with BRAF mutant melanoma, but are ineffective in patients whose melanomas express wild-type BRAF. These data validate mutant BRAF as a therapeutic target in melanoma. An unexpected property of RAF inhibitors is that they drive paradoxical activation of the MEK/ERK pathway in cells that express oncogenic RAS. This is because when they bind to RAF in cells in the presence of oncogenic RAS, these inhibitors drive the formation of CRAF:BRAF heterodimers and CRAF:CRAF homodimers in which one partner is drug-bound and the other is drug-free. Critically, the drug-bound partner acts as a scaffold, or induces a conformation change that hyper-activates the drug-free partner and this partner then drives pathway signalling. Notably, a quarter to a third of patients on BRAF inhibitors develop cutaneous squamous cell carcinomas (cuSCC) and keratoacanthomas. We have used a two-stage skin carcinogenesis model in mice that is driven by oncogenic HRAS to show that while BRAF inhibitors accelerate the development of RAS-driven cuSCC in mice, they do not initiate the formation of these lesions. We found that about 60% of BRAF-inhibitor driven cuSCC in melanoma patients harbour mutations in RAS genes and furthermore show that in mice cuSCC development driven by BRAF inhibitors can be blocked by co-administration of a MEK inhibitor. Thus, we conclude that RAS inhibitors do not act as tumor promoters per se; rather they accelerate the inevitable in susceptible patients and we provide a rational approach to blocking this side effect in BRAF mutant melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PL02-02.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

BackgroundMagnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development.MethodsUVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle.ResultsMagnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells.ConclusionsMagnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Protective role of cathepsin L in mouse skin carcinogenesis

Lysosomal cysteine protease cathepsin L (CTSL) is believed to play a role in tumor progression and is considered a marker for clinically invasive tumors. Studies from our laboratory using the classical mouse skin carcinogenesis model, with 7,12-dimethyl-benz[a]anthracene (DMBA) for initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) for promotion, showed that expression of CTSL is increased in papillomas and squamous cell carcinomas (SCC). We also carried out carcinogenesis studies using Ctsl-deficient nackt (nkt) mutant mice on three different inbred backgrounds. Unexpectedly, the multiplicity of papillomas was significantly higher in Ctsl-deficient than in wild-type mice on two unrelated backgrounds. Topical applications of TPA or DMBA alone to the skin of nkt/nkt mice did not induce papillomas, and there was no increase in spontaneous tumors in nkt/nkt mice on any of the three inbred backgrounds. Reduced epidermal cell proliferation in Ctsl-deficient nkt/nkt mice after TPA treatment suggested that they are not more sensitive than wild-type mice to TPA promotion. We also showed that deficiency of CTSL delays terminal differentiation of keratinocytes, and we propose that decreased elimination of initiated cells is at least partially responsible for the increased papilloma formation in the nackt model.

Abstract 4290: The farnesyl transferase inhibitor SCH 66336 induces regression of Hras-driven tumors in mice with widespread endogenous expression of HrasG12V

Abstract We previously reported that mice with a knock-in HrasG12V allele (Caggs-Cre/FR-HrasG12V) develop skin and forestomach papillomas as well as angiosarcomas with a relatively long latency. In these mice, activation of a single Hras mutant allele was not sufficient for transformation. The two-stage 7,12-dimethylbenz(a)anthracene (DMBA) /12-o-tetradecanoylphorbol-13-acetate (TPA) model of mouse skin carcinogenesis has been used to study mechanisms of epithelial tumor development. Although DMBA-induced HrasQ61L mutations are implicated in tumor formation in this model, it is not clear whether Hras is a tumor initiator, and whether its activity is required for tumor maintenance. To further clarify the role of the Hras gene in the DMBA/TPA model, we treated the skin of HrasG12V mice with the TPA tumor promoter alone, and found that this was sufficient to trigger rapid papilloma development. Latency was shorter, and tumor burden ∼ 10-fold greater than in DMBA/TPA treated WT controls. The mutant Hras allele copy number was increased in all 10 papillomas induced by TPA alone. Interestingly, DMBA/TPA treatment of HrasG12V mice induced an even greater incidence of papillomas: whereas 7/10 randomly selected papillomas induced by DMBA/ TPA in HrasG12V mice had amplified the HrasG12V allele, the remaining 3 had acquired an HrasQ61L mutation in the second Hras allele. Laser-capture microdissection of normal, hyperplastic and papilloma tissues from TPA-treated HrasG12V mice showed that Hras mutant allele amplification occurred only at the papilloma stage. Moreover, HRAS allelic imbalance was also observed in several human cancer cell lines with HRAS mutation (Hth83, C643 and T24). These data indicate that oncogenic Hras is a tumor initiator, and that augmentation of oncogenic Hras signaling is likely required for tumor formation through increased mutant Hras copy number or through mutation in the second Hras allele. SCH66336 is a farnesyl transferase inhibitor (FTI) that blocks the farnesylation of Hras, resulting in Hras delocalization from the plasma membrane. All Ras isoforms are farnesylated, however, upon treatment with FTI, Nras and Kras, but not Hras, are alternatively prenylated by geranylgeranylprenyltransferase-1. When tested in human cell lines harboring HRAS, NRAS or KRAS mutations, the FTI SCH 66336 delocalized only the Hras protein, inhibited its downstream signaling, and preferentially inhibited growth of HRAS-mutant lines. Treatment of TPA-treated HrasG12V mice with SCH 66336 (80mg/kg, b.i.d PO) induced an almost complete regression of Hras-driven papillomas within 10 days. The mechanism by which SCH66336 induced dramatic papilloma regression is under investigation. These observations suggest that FTIs should be re-evaluated as a therapy for human HRAS-driven cancers, such as those of bladder, thyroid and other epithelial lineages. 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 4290. doi:10.1158/1538-7445.AM2011-4290

Abstract 4047: TRIM32 facilitates cell growth, migration and anti-apoptosis

Abstract Introduction: Tripartite motif (TRIM) proteins are characterized by the presence of a RING finger, one or two zinc-binding motifs named B-boxes and an associated coiled-coil region. Most TRIM proteins have been reported to have a role in the ubiquitination process. Furthermore, several TRIM family members are involved in various cellular processes such as transcriptional regulation, cell growth, apoptosis, development and oncogenesis. TRIM32 also has a RING-finger domain and functions as an E3-ubiquitin ligase. In the previous study, it has been reported that TRIM32 protein expression is elevated in a mouse skin carcinogenesis model and in human skin squamous cell carcinoma and TRIM32 mRNA expression is elevated in human head and neck squamous cell carcinoma. However, the involvement of TRIM32 in carcinogenesis has not been fully elucidated. In this study, we studied the function of TRIM32 in the progression of carcinogenesis. Methods: We established the cell lines overexpressed TRIM32 wild type and the deletion mutant of the RING-finger domain by the infection of retrovirus vectors. Using these cell lines, we analyzed the effect of TRIM32 on cell growth, transforming activity, cell motility and apoptosis induced by cisplatin. Additionally, we investigated whether siRNA for TRIM32 effects on these functions. Furthermore, we examined the expression level of TRIM32 protein in oral carcinoma samples using immunohistochemical method and analyzed the relationship between TRIM32 and clinical presentation. Results: Overexpression of TRIM32 promoted cell growth, transforming activity and cell motility, whereas a dominant negative mutant of TRIM32 lacking the RING-finger domain inhibited these effects. In addition, we found that TRIM32 suppressed apoptosis induced by cisplatin in HEp2 cell lines, originating from human laryngeal squamous cell carcinoma. In contrast, siRNA for TRIM32 suppressed cell proliferation, cell motility and anti-apoptosis. In clinical research, a high-expression group of TRIM32 presented more advanced stage (clinical stage, T stage and neck lymph node metastasis) than a low-expression group. Conclusions: In this study, our experimental results in vitro were supported by clinical research. These findings suggest that TRIM32 is a novel oncogene that promotes tumor growth, metastasis and resistance to anti-cancer drugs. 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 4047. doi:10.1158/1538-7445.AM2011-4047

A novel role for the T‐box transcription factor Tbx1 as a negative regulator of tumor cell growth in mice

The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined. To study the effect of Tbx1 expression on growth and tumorigenic potential of carcinoma cells, we transfected mouse Tbx1 cDNA into a mouse spindle cell carcinoma cell line that did not express endogenous Tbx1. Following transfection, two cell lines expressing different levels of the Tbx1/V5 fusion protein were selected for further study. Intradermal injection of the cell lines into mice revealed that Tbx1 expression significantly suppressed tumor growth, albeit with no change in tumor morphology. In culture, ectopic Tbx1 expression resulted in decreased cell growth and reduced development into multilayered colonies, compared to control cells. Tbx1-transfectants exhibited a reduced proliferative rate compared to control cells, with fewer cells in S and G2/M phases. The Tbx1 transfectants developed significantly fewer colonies in soft agar, demonstrating loss of anchorage-independent growth. Taken together, our data show that ectopic expression of Tbx1 restored contact inhibition to the skin tumor cells, suggesting that this developmentally important transcription factor may have a novel dual role as a negative regulator of tumor growth. © 2011 Wiley Periodicals, Inc.

Podoplanin Associates with CD44 to Promote Directional Cell Migration

Podoplanin is a transmembrane glycoprotein up-regulated in different human tumors, especially those derived from squamous stratified epithelia (SCCs). Its expression in tumor cells is linked to increased cell migration and invasiveness; however, the mechanisms underlying this process remain poorly understood. Here we report that CD44, the major hyaluronan (HA) receptor, is a novel partner for podoplanin. Expression of the CD44 standard isoform (CD44s) is coordinately up-regulated together with that of podoplanin during progression to highly aggressive SCCs in a mouse skin model of carcinogenesis, and during epithelial-mesenchymal transition (EMT). In carcinoma cells, CD44 and podoplanin colocalize at cell surface protrusions. Moreover, CD44 recruitment promoted by HA-coated beads or cross-linking with a specific CD44 antibody induced corecruitment of podoplanin. Podoplanin-CD44s interaction was demonstrated both by coimmunoprecipitation experiments and, in vivo, by fluorescence resonance energy transfer/fluorescence lifetime imaging microscopy (FRET/FLIM), the later confirming its association on the plasma membrane of cells with a migratory phenotype. Importantly, we also show that podoplanin promotes directional persistence of motility in epithelial cells, a feature that requires CD44, and that both molecules cooperate to promote directional migration in SCC cells. Our results support a role for CD44-podoplanin interaction in driving tumor cell migration during malignancy.

Loss of tumor progression locus 2 (tpl2) enhances tumorigenesis and inflammation in two-stage skin carcinogenesis

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase in the mitogen-activated protein kinase signal transduction cascade known to regulate inflammatory pathways. Previously identified as an oncogene, its mutation or overexpression is reported in a variety of human cancers. To address its role in skin carcinogenesis, Tpl2(-/-) or wild-type (WT) C57BL/6 mice were subjected to a two-stage dimethylbenzanthracene/12-O-tetradecanoylphorbol-13-acetate (TPA) mouse skin carcinogenesis model. Tpl2(-/-) mice developed a significantly higher incidence of tumors (80%) than WT mice (17%), as well as a reduced tumor latency and a significantly higher number of total tumors (113 vs 6). Moreover, Tpl2(-/-) mice treated with TPA experienced significantly higher nuclear factor kappaB (NF-κB) activation, edema, infiltrating neutrophils and production of proinflammatory cytokines than did WT mice. We investigated the role of the p38, JNK, MEK and NF-κB signaling pathways both in vitro and in vivo in WT and Tpl2(-/-) mice by using inhibitors for each of these pathways. We confirmed that the proinflammatory effect in Tpl2(-/-) mice was due to heightened activity of the NF-κB pathway. These studies indicate that Tpl2 may serve more as a tumor suppressor than as an oncogene in chemically induced skin carcinogenesis, with its absence contributing to both tumorigenesis and inflammation.

Potentiation of tumour promotion by topical application of argemone oil/isolated sanguinarine alkaloid in a model of mouse skin carcinogenesis

Several incidences of adverse effects on human health have been reported in many countries, due to consumption of edible oil adulterated with argemone oil (AO). The clinical manifestation of the disease is commonly referred to as epidemic dropsy. Our prior studies have shown that AO and isolated sanguinarine alkaloid (SANG) possess genotoxic and tumour initiating activity. In this study, the effect of AO/SANG was investigated on the development of tumour formation in mice using 7,12-dimethylbenz (a) anthracene (DMBA) initiated followed by tetradecanoyl phorbol acetate (TPA)-promoted skin tumour protocol. Single application of AO (300 μl) or SANG (4.5 μmol) when used during initiation phase in DMBA/TPA group did not reveal substantial difference in tumourigenic response. However, twice weekly application of AO (100 μl) or SANG (1.5 μmol) during promotion phase (25 weeks) resulted in enhanced tumourigenic response by ≥30% in DMBA/TPA treated group along with significant decrease in dermal tyrosinase (45–49%), histidase (30–32%), superoxide dismutase (53–56%), catalase (41%), GSH reductase (37–40%) and GSH-peroxidase activity (29–33%) compared to control. Furthermore, significant decrease of epidermal GSH (64–66%) content and enhanced formation of lipid peroxides (96–121%) was noticed following AO or SANG treatment during promotion phase to DMBA/TPA induced animals indicating the modified pro-oxidant status in skin. Although dermal biochemical parameters were also altered by AO or SANG when used during initiation phase in DMBA/TPA treated animals, nonetheless, the response in these parameters were relatively more when AO or SANG were used during promotion phase in DMBA/TPA treated animals. These results clearly suggest that AO and SANG have the ability to enhance the tumourigenic response, which may have relevance to its carcinogenic potential.

The chemopreventive effects of Protandim: modulation of p53 mitochondrial translocation and apoptosis during skin carcinogenesis.

Protandim, a well defined dietary combination of 5 well-established medicinal plants, is known to induce endogenous antioxidant enzymes, such as manganese superoxide dismutase (MnSOD). Our previous studies have shown through the induction of various antioxidant enzymes, products of oxidative damage can be decreased. In addition, we have shown that tumor multiplicity and incidence can be decreased through the dietary administration of Protandim in the two-stage skin carcinogenesis mouse model. It has been demonstrated that cell proliferation is accommodated by cell death during DMBA/TPA treatment in the two-stage skin carcinogenesis model. Therefore, we investigated the effects of the Protandim diet on apoptosis; and proposed a novel mechanism of chemoprevention utilized by the Protandim dietary combination. Interestingly, Protandim suppressed DMBA/TPA induced cutaneous apoptosis. Recently, more attention has been focused on transcription-independent mechanisms of the tumor suppressor, p53, that mediate apoptosis. It is known that cytoplasmic p53 rapidly translocates to the mitochondria in response to pro-apoptotic stress. Our results showed that Protandim suppressed the mitochondrial translocation of p53 and mitochondrial outer membrane proteins such as Bax. We examined the levels of p53 and MnSOD expression/activity in murine skin JB6 promotion sensitive (P+) and promotion-resistant (P-) epidermal cells. Interestingly, p53 was induced only in P+ cells, not P- cells; whereas MnSOD is highly expressed in P- cells when compared to P+ cells. In addition, wild-type p53 was transfected into JB6 P- cells. We found that the introduction of wild-type p53 promoted transformation in JB6 P- cells. Our results suggest that suppression of p53 and induction of MnSOD may play an important role in the tumor suppressive activity of Protandim.

Topical application of valrubicin has a beneficial effect on developing skin tumors

Valrubicin is a second generation anthracycline characterized by an excellent safety profile presenting no skin toxicity or necrosis upon contact. In its current liquid formulation (Valstar; Indevus Pharmaceuticals, Lexington, MA), it is approved solely for the treatment of bladder cancer. Recently, valrubicin was incorporated in a cream formulation rendering this drug available for topical application. The cytostatic property of valrubicin can, thus, be employed for treating hyperproliferative skin diseases as was recently described for psoriasis. In the present study, the effect of topical application of valrubicin was investigated in skin tumor development; we hypothesized that valrubicin may be employed in treating actinic keratosis, a hyperproliferative skin condition that may transform into malignancy. A two-stage chemical mouse skin carcinogenesis model that represents the multistage etiology of human skin cancer-from developing papillomas to squamous cell carcinoma (SCC) was used. Moreover, two human skin SCC cell lines: DJM-1 and HSC-1 were cultured, to further investigate the effect of valrubicin in vitro. Cell viability was assessed by adenosine triphosphate presence, proliferation as proliferative cell nuclear antigen expression and apoptosis as cytokeratin 18 cleavage, caspase activation, poly-adenosine diphosphate-ribose-polymerase cleavage and bax and bcl-2 regulation. Valrubicin significantly inhibited tumor formation in the mouse skin carcinogenesis model and significantly decreased cell viability of the cultured human skin SCC cells. In both mouse skin and SCC cells, proliferation was significantly decreased. Apoptosis was significantly increased in SCC cells but unchanged in the treated mouse skin at study completion. This study demonstrated that topical application of valrubicin has a beneficial effect in treating developing skin tumors.

Abstract 3470: Effects of combined phytochemicals on skin tumorgenesis in SENCAR mice

Abstract The purpose of our study was to determine the effect of combined action of phytochemicals on early stages of skin tumorgenesis, i.e., initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (URA) were applied topically. The 7,12-dimethylbenz[a]anthracene (DMBA)-initiated, 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted multistage skin carcinogenesis model in SENCAR mice was used. Mice (6-7 weeks old) with shaved backs received one topical dose of DMBA (20 nmol), then after one month, two weekly doses (2 µg each) of TPA (tumor promoter) for 14 weeks until sacrifice. The phytochemicals RES (2.5 μmol) or URA (1 μmol) were applied topically 20 min prior to DMBA or TPA treatment and 2% dietary CG was given in the AIN-93G diet from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. We determined the tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) and using RT-PCR and immunochistochemical analyses, we checked the expression of some cell proliferation and inflammation related genes. URA applied alone and in combination with CG during the promotion stage was a strong inhibitor of tumor multiplicity and tumor incidence. Combinations of URA with dietary CG appeared to show some synergistic effects. All compounds and their combinations reduced epidermal proliferation, but only URA and a combination CG/URA applied during promotion significantly reduced hyperplasia, as determined by epidermal thickness measurements taken at 14 weeks of TPA treatment. DMBA/TPA treatments resulted in epidermal hyperplasia, characterized by significant increase in transcription factor AP-1 components and nuclear factor kappa B (NFkB) expression levels with a simultaneously increased expression of the inflammation markers COX-2 and IL-6, compared to untreated controls. All anti-promotion treatments (either single compounds or their combinations) caused a marked decrease of inflammation related genes levels compared to the DMBA/TPA control. On the other hand, only URA and URA with dietary CG diminished the induced expression of proto-oncogenes c-jun and c-fos (components of AP-1) and the expression of NFkB protein p50. These results show that URA is a potent inhibitor of skin tumor promotion and it may be useful in the prevention of skin cancer and other epithelial cancers in humans. Supported by NIH grants CA 102747 and P30 CA 54174-16S1. 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 3470.

Dietary energy restriction, in part through glucocorticoid hormones, mediates the impact of 12‐O‐tetradecanoylphorbol‐13‐acetate on jun D and fra‐1 in sencar mouse epidermis

Dietary energy restriction (DER, 40% calorie reduction from fat and carbohydrate) inhibited mouse skin carcinogenesis and decreased 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced activator protein-1 (AP-1):DNA binding previously. This study measured protein levels of c-jun, jun B, jun D, c-fos, fra-1, and fra-2 and examined their contribution to AP-1:DNA binding by electrophoretic mobility shift assay (EMSA) with supershift analysis in the epidermis of control and DER Sencar mice exposed to TPA. TPA significantly increased c-jun, jun B, c-fos, fra-1, and fra-2 and decreased jun D within 3-6 h after treatment. AP-1:DNA binding reached a maximum 2.5-fold induction over controls 4 h after TPA treatment and antibodies to jun B, jun D, and fra-2 in the EMSA binding reaction resulted in supershifts in both acetone- and TPA-treated mice 1-6 h after treatment. The effect of corticosterone (CCS) and DER on the AP-1 proteins and on the composition of the AP-1:DNA complex was measured in adrenalectomized (adx) mice. DER reduced the TPA impact on jun D and enhanced the induction of fra-1. In addition, CCS-supplemented groups had significantly lower jun D and higher fra-2 than adx groups and sham groups. While sham animals treated with either acetone or TPA contained jun B, jun D, and fra-2 proteins in the AP-1:DNA complex by supershift analysis, fra-2 was no longer seen in adx DER animals. In summary, our study supports potential roles for jun D, jun B, and fra-1 in the DER regulation of AP-1 function in the Sencar mouse skin carcinogenesis model.

Elevated Phosphate Activates N-ras and Promotes Cell Transformation and Skin Tumorigenesis

Recent results suggest a paradigm shift from viewing inorganic phosphate as a passive requirement for basic cell functions to an active regulator of cell behavior. We have previously shown that elevated concentrations of phosphate increased cell proliferation and expression of protumorigenic genes such as Fra-1 and osteopontin in a preosteoblast cell line. Therefore, we hypothesized that elevated phosphate concentrations would promote cell transformation in vitro and tumorigenesis in vivo. Supplementation of medium with phosphate increased anchorage-independent transformation and proliferation of BALB/c mouse JB6 epidermal cells, activation of N-ras, ERK1/2, and activator protein-1, and increased gene expression of Fra-1, COX-2, and osteopontin in a dose-dependent manner. These in vitro results led to the hypothesis that varying the levels of dietary inorganic phosphate would alter tumorigenesis in the mouse model of skin carcinogenesis. Female FVB/N mice were treated with 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate and fed high- or low-phosphate diets (1.2% versus 0.2% of the diet) for 19 weeks. The high-phosphate diet increased skin papilloma number by approximately 50% without changing feed intake and body weights. High dietary phosphate increased serum concentrations of phosphate, parathyroid hormone, and osteopontin and decreased serum concentrations of calcium. Thus, we conclude that elevated phosphate promotes cell transformation and skin tumorigenesis partly by increasing the availability of phosphate for activation of N-ras and its downstream targets, which defines reducing dietary phosphate as a novel target for chemoprevention.

Positron emission tomography imaging of DMBA/TPA mouse skin multi-step tumorigenesis

Many tumor cells have elevated rates of glucose uptake that can be measured quantitatively, noninvasively and repeatedly by positron emission tomography (PET) with 2-deoxy-2-[ 18F]-fluoro-D-glucose ( 18F-FDG). Clinical imaging with 18F-FDG PET has been used for detection and staging of primary and metastatic tumors. High-resolution microPET scanning and murine cancer models make it possible to analyze longitudinally glucose metabolism during the appearance, development and progression of individual experimental tumors. In this study, we used 18F-FDG microPET and micro computerized tomography (microCT) to investigate glucose uptake in the DMBA/TPA chemically-induced multistage mouse skin carcinogenesis model. 18F-FDG uptake is significantly higher in all papillomas than in surrounding skin. Elevated 18F-FDG uptake is observed when tumors can be identified morphologically, but not before. Although 18F-FDG uptake is high in all fully invasive, malignant skin squamous cell carcinomas, uptake in papillomas and microinvasive malignant squamous cell carcinomas is variable and does not exhibit any correlation with tumor stage.

Cancer-Associated Fibroblasts Are Activated in Incipient Neoplasia to Orchestrate Tumor-Promoting Inflammation in an NF-κB-Dependent Manner

Cancer-associated fibroblasts (CAFs) support tumorigenesis by stimulating angiogenesis, cancer cell proliferation, and invasion. We demonstrate that CAFs also mediate tumor-enhancing inflammation. Using a mouse model of squamous skin carcinogenesis, we found a proinflammatory gene signature in CAFs isolated from dysplastic skin. This signature was maintained in CAFs from subsequent skin carcinomas and was evident in mammary and pancreatic tumors in mice and in cognate human cancers. The inflammatory signature was already activated in CAFs isolated from the initial hyperplastic stage in multistep skin tumorigenesis. CAFs from this pathway promoted macrophage recruitment, neovascularization, and tumor growth, activities that are abolished when NF-kappaB signaling was inhibited. Additionally, we show that normal dermal fibroblasts can be "educated" by carcinoma cells to express proinflammatory genes.

Synergistic Effects of Combined Phytochemicals and Skin Cancer Prevention in SENCAR Mice

The purpose of our study was to determine the inhibitory effect of combined phytochemicals on chemically induced murine skin tumorigenesis. Our hypothesis was that concurrent topical and dietary treatment with selected compounds would lead to more efficient prevention of skin cancer. We tested ellagic acid and calcium D-glucarate as components of diets, while resveratrol was applied topically; grape seed extract was applied topically or in the diet. The 4-week inflammatory-hyperplasia assay based on the 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin carcinogenesis model in SENCAR mice was used. We have found that all the selected combinations caused a marked decrease of epidermal thickness compared with the DMBA-treated group and also with groups treated with a single compound and DMBA. All combinations of resveratrol with other compounds showed a synergistic effect on hyperplasia and Ha-ras mutations. Skin tissue of mice receiving the combinations showed decreased cell proliferation and Bcl2 expression; decreased p21, a regulator of cell cycle; and decreased marker of inflammation cyclooxygenase-2. All the selected combinations diminished the DMBA-induced mRNA expression of the CYP1B1 level, and also caused a marked decrease of proto-oncogenes c-jun and c-fos, components of transcription factor activator protein. In conclusion, all combinations showed either additive or synergistic effects and their joint actions allowed for decreasing the doses of the compounds. Especially, resveratrol combinations with ellagic acid, grape seed extract, and other phytochemicals are very potent inhibitors of skin tumorgenesis, based on the suppression of epidermal hyperplasia as well as on the modulation of intermediate biomarkers of cell proliferation, cell survival, inflammation, oncogene mutation, and apoptosis.