Two-stage Skin Carcinogenesis Model Research Articles

Abstract 5266: Metabolic rewiring and epigenetic reprogramming by the environmental carcinogen benzo[a]pyrene in a two-stage skin carcinogenesis mouse model and cancer interception by triterpenoid ursolic acid

Abstract Nonmelanoma skin cancer (NMSC) is the most common skin cancer burden on the U.S. population. Environmental exposure to chemical carcinogens is one of the major causes of NMSC initiation, promotion, and progression. Ursolic acid (UA) is a naturally abundant pentacyclic triterpenoid showing anticancer potentials against diverse cancers. In the current study, we developed a two-stage skin carcinogenesis model in SKH1 hairless mice by administering cancer-initiating agent benzo[a]pyrene (B[a]P) and promoting agent 12-O-tetra-decanoylphorbol-13-acetate to study the epigenetic, transcriptomic, and metabolic changes at different stages (5, 20, and 26 weeks) during the development of NMSC, and investigated how UA regulates B[a]P-mediated alterations for NMSC interception. We found that UA protects against B[a]P-induced tumorigenesis at different phases of NMSC. Epigenetic CpG methyl-seq showed UA abrogated B[a]P-mediated alterations in differentially methylated regions (DMRs) profiles. Transcriptomic RNA-seq data exhibited UA reversed the differentially expressed genes (DEGs) of several inflammatory genes, such as chemokine ligand 8 (Ccl8) and interleukin 17F (Il17f), and epigenetic genes, such as DNA-methyltransferase 3-like (Dnmt3l) and protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (Pcmtd1) during different stages of NMSC. Association study between DEGs and DMRs showed that B[a]P promoted transcription of kallikrein-related peptidase 13 (Klk13) by promoter demethylation, while UA suppressed Klk13 expression through hypermethylation in the promoter during the initiation stage, indicating the early intervention of UA. Ingenuity pathway analysis further showed significant upregulation of macrophage-stimulating protein-recepteur d'origine nantais (MSP-RON) signaling pathway by B[a]P during the initiation stage while suppressed by UA treatment. The metabolomic study revealed UA modulated cancer-associated changes in metabolisms, including the TCA cycle and pyruvate metabolism/metabolites during the promotion phase, indicating UA plays a critical role in regulating B[a]P-regulated metabolic changes and intercepting NMSC progression. In summary, UA protects against the environmental carcinogen B[a]P-driven epigenetic, transcriptomic, and metabolic changes during the initiation, promotion, and progression of NMSC, potentially contributing to the anticancer effects of UA. (Supported by NIH R01 CA200129 to A.N.K) Citation Format: Md. Shahid Sarwar, Christina N. Ramirez, Hsiao-Chen Dina Kuo, Pochung Chou, Renyi Wu, Davit Sargsyan, Ahmad Shannar, Rebecca Mary Peter, Ran Yin, Yujue Wang, Xiaoyang Su, Ah-Ng Kong. Metabolic rewiring and epigenetic reprogramming by the environmental carcinogen benzo[a]pyrene in a two-stage skin carcinogenesis mouse model and cancer interception by triterpenoid ursolic acid. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5266.

Interleukin 17 Receptor C, but not Polo-like Kinase 4, Plays a Critical Role in the Development of Skin Papilloma and Psoriasis in Mouse Models

Abstract Introduction/Objective Hyperproliferation of epidermis is a histopathological hallmark of skin cancers and psoriasis. As a master regulator of centriole replication, overexpression of Polo-like kinase 4 (PLK4) has been identified in skin cancers. The aberrant proliferation of epidermal keratinocytes provoked by interleukin 17 (IL17) leads to psoriasis. Thus, targeting centriole replication and IL17 signaling simultaneously has been speculated as a potential therapeutic strategy. We hypothesized that inhibition of centriole duplication enhances the blockade of epidermal proliferation through Il17rc knockout. Methods/Case Report To test our hypothesis, 37 mice were used to imbed a two-stage model of skin carcinogenesis using wild-type (WT), IL17 receptor A (T779A) knock-in (Il17ra(T779A)-KI), and IL17 receptor C knock-out (Il17rc-KO) C57BL/6J mouse strains. Furthermore, an imiquimod-induced psoriasis model was established using 69 mice with the same strains. Results (if a Case Study enter NA) In our two-stage skin carcinogenesis model, Il17ra(T779A)-KI mice showed significantly decreased tumor incidence, tumor multiplicity, and tumor volume compared to the WT mice. Il-17rc-KO mice didn’t develop any skin papilloma. The skin papilloma formed never progressed into squamous carcinoma. However, centrinone, a selective inhibitor of PLK4, didn’t affect skin papilloma formation or epidermal thickening. In our psoriasis model, the epidermis thickness of Il-17rc-KO mice was dramatically decreased compared to WT and Il17ra(T779A)-KI mice. There was no significant difference between WT and Il17ra(T779A)-KI mice in terms of skin lesion and thickening of the epidermis. Centrinone didn’t stall the thickening of the epidermis in the psoriasis model. IHC staining showed significantly increased Ki67+ basal keratinocytes in the untreated skin of Il17ra(T779A)-KI male mice compared to WT mice. In imiquimod-treated skin, the percentage of Ki67+ basal keratinocytes significantly decreased in the order from WT, Il17ra(T779A)-KI to Il17rc-KO mice. Conclusion Our data suggest that the proliferation of keratinocytes is not stalled by centrinone but is inhibited by Il17rc-KO. Il17ra(T779A)-KI significantly inhibits skin papilloma formation, but slightly decreases epidermal thickening in the psoriasis model. However, Il17ra(T779A)-KI increases keratinocyte proliferation based on Ki67 staining in the untreated normal skin.

Abstract 1313: IL-17RC, but not PLK4 inhibitor centrinone, plays a critical role in the development of skin papilloma and psoriasis in mouse models

Abstract Introduction: Hyperproliferation of epidermis is a histopathological hallmark of skin disorders, such as skin cancers and psoriasis. Polo-like kinase 4 (PLK4) is a master regulator of centriole replication and its overexpression has been identified in non-melanoma skin cancers. The aberrant proliferation of epidermal keratinocytes provoked by interleukin-17 (IL-17) leads to psoriasis. Thus, targeting centriole replication and IL-17 signaling simultaneously has been speculated as a potential therapeutic strategy. We hypothesized that inhibition of centriole duplication might enhance the blockade of epidermal proliferation through Il-17rc knockout. Methods: To investigate cooperation between IL-17 signaling and centriole duplication in the proliferation of epidermis, we used 37 mice to establish a two-stage model of skin carcinogenesis and 69 mice to establish imiquimod-induced psoriasis model in wild-type (WT), IL-17 receptor A (T779A) knock-in (Il-17ra(T779A)-KI) and IL-17 receptor C knock-out (Il-17rc-KO) C57BL/6J mouse strains. Results: During our 13-week monitor of the two-stage skin carcinogenesis model, we found that Il-17ra(T779A)-KI mice showed significantly decreased tumor incidence, tumor multiplicity, and tumor volume, compared to the wild-type mice. Il-17rc-KO mice didn’t develop any skin papilloma. The skin papilloma formed never progressed into squamous carcinoma based on our immunohistochemical (IHC) staining of laminin and cytokeratin 8. However, centrinone, a selective inhibitor of PLK4, didn’t affect skin papilloma formation or epidermal thickening. In our psoriasis model, we found that the thickness of the epidermis of Il-17rc-KO mice was dramatically decreased, compared to WT and Il-17ra(T779A)-KI mice. There was no significant difference between WT and Il-17ra(T779A)-KI mice, in terms of psoriasis formation and the thickness of the epidermis. Centrinone didn’t stall the thickening of the epidermis in the psoriasis model. IHC staining showed significantly increased Ki-67+ basal keratinocytes in the untreated skin of Il-17ra(T779A)-KI male mice, but not in female mice, compared to WT mice. In imiquimod-treated skin, the percentage of Ki-67+ basal keratinocytes significantly decreased in Il-17ra(T779A)-KI mice, compared to WT mice. The percentage of Ki-67+ basal keratinocytes was also dramatically decreased in Il-17rc-KO mice, compared to WT and Il-17ra(T779A)-KI mice. Conclusion: Our findings suggest that the proliferation of keratinocytes is not stalled by PLK4 inhibitor centrinone but is inhibited by Il-17rc-KO. Il-17ra(T779A)-KI significantly inhibits skin papilloma formation, but only slightly decreases epidermal thickening in the psoriasis model. However, in the untreated normal skin, Il-17ra(T779A)-KI increases keratinocyte proliferation based on Ki-67 staining. Citation Format: Ben Jin, Yongfeng Zhang, Haiyan D. Miller, Ling He, Zongbing You. IL-17RC, but not PLK4 inhibitor centrinone, plays a critical role in the development of skin papilloma and psoriasis in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1313.

Abstract 5345: Novel approach to attenuate melanoma initiation and progression

Abstract Background and Purpose: Melanocytic nevi are benign proliferations of melanocytic cells that are positively correlated with susceptibility to melanoma. Polyaromatic hydrocarbons (PAH) are ubiquitous environmental pollutants that are potent mutagens and carcinogens, and the investigators have taken advantage of these properties to investigate the mechanisms by which chemicals cause cancer of the skin and other organs. In comparison to C57Bl/6 strain, C3H/HeN mice are more susceptible to the development of PAH induced tumors. Therefore, a mouse model of nevus initiation and progression was developed in C3H/HeN mice using a modified chemical carcinogenesis protocol. Experimental Design: In the traditional two-stage skin carcinogenesis model, initiation is accomplished by the application of a subcarcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor promoting agent. Nevi develop due to DNA damage initiated by 7, 12-dimethylbenz[a]anthracene (DMBA) followed by chronic promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Results: Dysplastic pigmented skin lesions appeared in ~10 wk with 100% penetrance. Studies reported here demonstrating that inhibition or depletion of RLIP results in apoptosis and cancer regression in an exceptionally broad spectrum on neoplasia have suggested an intriguing possibility that RLIP is an anti-apoptotic mechanism required not only for the survival of cancer cells, but also for their very existence. In support of this assertion results presented here that the well-known carcinogens, DMBA and phorbol esters (PMA or TPA) are ineffective in causing neoplasia in C3H/HeN mice treated with RLIP-targeting agent’s (RLIP antibodies and RLIP antisense). Here we demonstrate that DMBA/PMA-induced skin carcinogenesis in C3H/HeN mouse was suppressed completely by depletion of RLIP by antisense or inhibition by antibodies. In addition, C3H/HeN mice treated with RLIP-targeting agent’s, p53, P38, and JNK activation did not occur in response to carcinogens. These findings demonstrate a fundamental role of RLIP in chemical carcinogenesis. Conclusions: We demonstrate that RLIP expression is significantly greater in cancer cells than in non-malignant cells, and RLIP-targeting agent’s (RLIP antibodies and RLIP antisense) treatment was significantly suppress the generation of melanocytic nevi and their progression to melanoma. (This work was supported in part by the Department of Defense grants W81XWH-16-1-0641 and W81XWH-20-1-0362. Funding from the Beckman Research Institute of City of Hope is also acknowledged). Citation Format: Sharad S. Singhal, Prakash Kulkarni, Jyotsana Singhal, David Horne, Sanjay Awasthi, Ravi Salgia. Novel approach to attenuate melanoma initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5345.

IL-13Rα1 Suppresses Tumor Progression in Two-Stage Skin Carcinogenesis Model by Regulating Regulatory T Cells

Type 2 inflammation‒related cytokine IL-13 plays a protective role in experimental papilloma induction in mice. To understand the mechanisms by which IL-13 contributes to papilloma formation, we utilized Il13rα1-knockout (KO) mice in a widely used 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoyl phorbol-13-acetate two-stage skin carcinogenesis protocol that mimics the development of squamous cell carcinoma. KO mice developed more papillomas and significantly faster than wild-type mice. Papilloma development reduced regulatory T cells in wild-type mice but substantially less in KO mice. In line with this, IL-2 and IL-10 levels decreased in wild-type mice but not in KO mice. Furthermore, systemic IL-5 and TSLP levels were elevated, whereas IL-22 was decreased during papilloma formation in the skin of KO mice. Polymorphonuclear myeloid‒derived suppressor cells were decreased in the KO mice at the early phase of papilloma induction. We show that IL-13Rα1 protects from papilloma development in chemically induced skin carcinogenesis, and our results provide further insights into the protective role of functional IL-4 and IL-13 signaling through type II IL-4 receptor in tumor development.

Lymphatic Dysfunction Exacerbates Cutaneous Tumorigenesis and Psoriasis-Like Skin Inflammation through Accumulation of Inflammatory Cytokines

Lymphatic transport plays an important role in coordinating local immune responses. However, the biologic effects of impaired lymphatic flow invivo are not fully understood. In this study, we investigated the roles ofthe lymphatic system in skin carcinogenesis and psoriasis-like inflammation using k-cyclin transgenic (kCYC+/-) mice, which demonstrate severe lymphatic dysfunction. kCYC+/- mice showed augmented tumor growth in the two-stage skin carcinogenesis model and severe clinical scores in imiquimod-induced psoriasis-like skin inflammation compared with wild-type mice. Although mRNA levels of inflammatory cytokines in skin after topical application of 12-O-tetradecanoylphorbol-13-acetate or imiquimod were comparable between kCYC+/- and wild-type mice, protein levels of inflammatory cytokines, such as IL-17A, IL-22, and IL-23, were significantly upregulated in kCYC+/- mice in both models. Consistently, signal transducer and activator of transcription 3 pathway and NF-κB signaling were augmented in epidermal keratinocytes in kCYC+/- mice. These results suggest that lymphatic dysfunction in kCYC+/- mice caused accumulation of inflammatory cytokines, leading to the exacerbation of two-stage skin carcinogenesis and imiquimod-induced psoriasis-like skin inflammation. These findings add insight into the clinical problems of secondary malignancies and inflammatory dermatoses that may occur with extremity lymphedema.

Gene Deletion of Microsomal Prostaglandin E Synthase-1 Suppresses Chemically Induced Skin Carcinogenesis.

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) is a terminal enzyme in PGE2 synthesis and highly expressed in several cancers. In this study, to reveal the involvement of mPGES-1 in skin carcinogenesis, the effect of mPGES-1 deficiency on two-stage skin carcinogenesis in mice was investigated. A two-stage skin carcinogenesis model using 7,12-dimethylbenz[a]anthracene (DMBA) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter was applied on mPGES-1 knockout (KO) mice and littermate wild-type mice of a Balb/c genetic background. DMBA/TPA-induced skin carcinogenesis was suppressed in mPGES-1 KO mice. The induction of IL-17 and other inflammatory cytokines by TPA was also suppressed by mPGES-1 deficiency, although DMBA-induced apoptosis was not affected. mPGES-1 promotes chemically induced skin carcinogenesis and might play an important role in the TPA-induced promotion phase of the two-stage skin carcinogenesis model. mPGES-1 inhibition may be a therapeutic target for skin cancer.

A wild-derived inbred mouse strain, MSM/Ms, provides insights into novel skin tumor susceptibility genes.

Cancer is one of the most catastrophic human genetic diseases. Experimental animal cancer models are essential for gaining insights into the complex interactions of different cells and genes in tumor initiation, promotion, and progression. Mouse models have been extensively used to analyze the genetic basis of cancer susceptibility. They have led to the identification of multiple loci that confer, either alone or in specific combinations, an increased susceptibility to cancer, some of which have direct translatability to human cancer. Additionally, wild-derived inbred mouse strains are an advantageous reservoir of novel genetic polymorphisms of cancer susceptibility genes, because of the evolutionary divergence between wild and classical inbred strains. Here, we review mapped Stmm (skin tumor modifier of MSM) loci using a Japanese wild-derived inbred mouse strain, MSM/Ms, and describe recent advances in our knowledge of the genes responsible for Stmm loci in the 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage skin carcinogenesis model.

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

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

Chemical-Induced Skin Carcinogenesis Model Using Dimethylbenz[a]Anthracene and 12-O-Tetradecanoyl Phorbol-13-Acetate (DMBA-TPA).

Cancer is one of the most devastating human diseases. Experimental cancer models are important to gain insight into the complex interplay of different cell types and genes in promoting tumor progression and to provide a platform for testing the efficacy of different therapeutic approaches. One of the most commonly used experimental inflammatory cancer models is the DMBA-TPA two-stage skin carcinogenesis model. Tumor formation is induced in this model by the topical application of two different chemicals, 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA), that together cause papilloma formation in the skin. As the primary outcome is papilloma formation in the skin, the model is an ideal, reliable, and reproducible way to address both tumor initiation (tumor-free survival) and tumor progression (number and size of visible tumors). The effects of the DMBA-TPA treatment are transmitted via an inflammatory mechanism, which makes this model especially suitable for studying the role of the immune system in tumor formation. However, this model is restricted to the skin and other surfaces where the chemicals can be applied on. A detailed protocol is provided in this article to use the model successfully.

Loss of Rab25 promotes the development of skin squamous cell carcinoma through the dysregulation of integrin trafficking.

Rab25 can function as both a tumor suppressor and a tumor promoter across different tissues. This study sought to clarify the role of Rab25 as a tumor suppressor in skin squamous cell carcinoma (SCC). Rab25 loss was closely associated with neoplastic transition in both humans and mice. Rab25 loss was well correlated with increased cell proliferation and poor differentiation in human SCC. While Rab25 knockout (KO) in mice did not induce spontaneous tumor formation, it did significantly accelerate tumor generation and promote malignant transformation in a mouse two-stage skin carcinogenesis model. Xenografting of a Rab25-deficient human keratinocyte cell line, HaCaT, also elicited neoplastic transformation. Notably, Rab25 deficiency led to dysregulation of integrins β1, β4, and α6, which matched well with increased epidermal proliferation and impaired desmosome-tight junction formation. Rab25 deficiency induced impairment of integrin recycling, leading to the improper expression of integrins. In line with this, significant attenuation of integrin β1, β4, and α6 expression was identified in human SCCs where Rab25 was deficient. Collectively, these results suggest that loss of Rab25 promotes the development and neoplastic transition of SCC through dysregulation of integrin trafficking. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pten loss in Lgr5+ hair follicle stem cells promotes SCC development

Accumulating data support that tissue stem cells give rise to cancer cells. Hair follicle stem cells (HFSCs) undergo cyclic quiescence and activation and may sever as the origin of cutaneous squamous cell carcinoma (SCC). Pten is a tumor suppressor gene that is frequently mutated in hereditary cancer syndromes such as Cowden disease, which is featured with papillomatosis in cutaneous tissues and hyperkeratosis in the acral region of the skin. Additionally, mice with keratinocyte-specific Pten deficiency (k5-Pten-/- mice) show epidermal hyperplasia and spontaneous tumor formation. However, the impact of Pten mutation in HFSCs, such as in Lgr5+ HFSCs, on SCC formation is unclear.Methods: We established experiments with wildtype and Lgr5-CreER; Ptenflox/flox mice, and used DMBA/TPA two-stage skin carcinogenesis model to explore the effect of Pten loss in Lgr5+ HFSCs of 3 weeks old mice in skin carcinogenesis. In vitro experiments (cell culture and protein expression analysis) are employed to investigate molecular mechanisms involved.Results: Pten loss in Lgr5+ HFSCs promoted SCC formation, which was attenuated in TNF-/- mice. Notably, β-catenin loss in Lgr5+ HFSCs decreased the formation of SCC. In addition, Pten loss in cultured epidermal stem cells upregulated the levels of both phospho-Akt and β-catenin.Conclusion: Pten loss in Lgr5+ cells induced Akt/β-catenin signaling, and SCCs can subsequently be raised as progeny from these primed Lgr5+ stem cells.

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

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

Abstract 278: Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin

Abstract Despite the many advances that have been made in understanding cancer development and the discovery of new therapies, cancer still remains the second leading cause of death in the US. In addition, approximately two out of five people are expected to be diagnosed with an invasive cancer in their lifetime. Prevention remains an important strategy to reduce the burden of cancer. One approach to prevent cancer is the use of phytochemicals in various combinations as safe and effective cancer preventive agents. The purpose of this study was to examine the effects of the combination of ursolic acid (UA) and curcumin (Curc) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc alone have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due primarily to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), the combination of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and Stat3 to a greater extent than either compound alone. Additionally, these compounds increased levels of programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Furthermore, results from skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) indicate that the combination of UA and Curc given topically significantly inhibited mouse skin tumor promotion by TPA to a greater extent than the individual compounds given alone. The greatest effects were seen on tumor free survival and on tumor size. These results suggests the potential for the combination of UA and Curc to be used topically for chemopreventive strategies in non-melanoma skin cancer. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. Inhibition of skin tumor promotion by TPA using a combination of topically applied ursolic acid and curcumin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 278.

Abstract 5262: The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA

Abstract The purpose of this study was to examine the effects of combinations of ursolic acid (UA), curcumin (Curc), and metformin (Met) for potential synergistic chemopreventive activity to inhibit tumor promotion using the mouse two-stage skin carcinogenesis model. UA and Curc have been shown to inhibit tumorigenesis in several mouse models, and both have been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). The anti-cancer activity of these phytochemicals is due to their anti-inflammatory, anti-oxidant, and anti-proliferative effects. These effects are accomplished by acting on a broad spectrum of signaling pathways. Met has also been shown to have anti-cancer activity. In retrospective studies, type II diabetes patients taking Met have been shown to have a decreased cancer incidence. Met has also been shown to inhibit mouse skin tumor promotion by TPA. Met activates AMP-activated protein kinase (AMPK), which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. mTORC1 is a key kinase involved in the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, one of the most commonly altered pathways in human tumors. In initial short-term experiments (mice treated twice weekly for two weeks with TPA), combinations of UA and Curc pretreated topically inhibited TPA-induced activation of epidermal NF-κB, Cox-2, epidermal growth factor receptor (EGFR) and JNK to a greater extent than either compound alone. Additionally, these compounds increased levels of AMPK and programmed cell death protein 4 (Pdcd4). The alterations in these signaling pathways by the combination of UA and Curc were associated with decreased epidermal proliferation and skin inflammation as assessed by measuring BrdU incorporation and inflammatory gene expression, respectively. Further studies using dietary administration of a combination of UA and Curc, as well as combinations of UA or Curc (in diet) with Met administered in the drinking water were conducted. These combinations produced greater inhibition of TPA-induced activation of epidermal NFκB, Cox-2, and EGFR than the compounds given alone. These combinations also decreased epidermal proliferation, again shown by an inhibition of BrdU incorporation. In addition to these data, the results from ongoing skin tumor experiments (mice initiated with DMBA and promoted twice weekly with TPA) using both topical administration and dietary administration of the various combinations will be presented. The current results indicate that certain combinations of UA, Curc and Met may inhibit mouse skin tumor promotion by TPA to a greater extent than the compounds alone. The ability of the combinations to work when given both topically as well as in the diet suggests the potential for broad applications for chemopreventive strategies. Citation Format: Lisa Tremmel, Okkyung Rho, John DiGiovanni. The inhibitory effect of combinations of curcumin, ursolic acid, and metformin on skin tumor promotion by TPA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5262. doi:10.1158/1538-7445.AM2017-5262

Abstract 2106: Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation

Abstract Twist1 is a basic helix-loop-helix transcription factor and is known to play a role in tumor progression through regulation of genes involved in epithelial-mesenchymal transition. Twist1 is a known transcriptional target of signal transducer and activator of transcription-3 (Stat3). In previous studies, Stat3 was shown to play a role in skin tumor progression in the two-stage skin carcinogenesis model, at least in part, by regulating the levels of Twist1. Recent studies using keratinocyte-specific knockout (KO) of Twist1 indicate that Twist1 regulates keratinocyte proliferation during skin tumor promotion. Western blot analysis of lysates from both Twist1 deficient mouse primary keratinocytes and from epidermis isolated from BK5.Cre x Twist1flox/flox mice indicated that Twist1 KO leads to reduced levels of the cell cycle proteins Cyclin E1, E2F1, and Cdk2 and increased expression of p21 following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). ChIP analysis of epidermal lysates from BK5.Cre x Twist1flox/flox mice revealed that Twist1 bound to the promoter regions of Cyclin E1, E2F1, and c-Myc, indicating direct transcriptional regulation of these cell cycle regulators. Moreover, Twist1 KO in keratinocytes impeded cell cycle progression by reducing the number of cells that advanced to S-phase. Further analyses have demonstrated that deletion of Twist1 either in primary keratinocytes or in vivo leads to an upregulation of p53, which is responsible for the increased expression of p21 observed in these cells. Keratinocyte specific deletion of Twist1 in vivo suppressed epidermal proliferation induced by TPA treatment compared to that observed in wild-type (WT) mice. To further characterize the potential role of Twist1 during epithelial carcinogenesis, we analyzed the impact of Twist1 deletion in vivo on bulge region keratinocyte stem cells (KSCs). Keratinocyte specific deletion of Twist1 in vivo led to a significant reduction in the number of label-retaining cells as well as the number of α6-integrin+/CD34+ cells in the hair follicles of untreated mice. An ongoing two-stage skin carcinogenesis experiment shows that BK5.Cre x Twist1flox/flox mice have significantly reduced tumor development as observed by decreased tumor multiplicity and delayed latency compared WT mice. Furthermore, tumors that developed in BK5.Cre x Twist1flox/flox mice have significantly reduced size compared to tumors on WT mice. Collectively, these findings suggest that Twist1 has a novel role in epithelial carcinogenesis by regulating proliferation and migration of keratinocytes and KSCs. Research supported by RP140108, CA76520, and T32 ES007247. Citation Format: Jaya Srivastava, Okkyung Rho, John DiGiovanni. Twist1 regulates keratinocyte stem cell proliferation and migration and is required for skin tumor formation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2015-2106

Resveratrol synergistically augments anti-tumor effect of 5-FU invitro and invivo by increasing S-phase arrest and tumor apoptosis.

Many studies have shown that natural dietary agents, in combination with chemical agents, can improve the therapeutic response of cancers to chemotherapy and reduce the associated side-effects. In the present study, we investigated the therapeutic potential and mechanisms of anticancer effects for the combination of 5-fluorouracil (5-FU) and resveratrol (Res). In these studies, we employed the cancer cell lines TE-1 and A431 and an animal model of skin cancer. The presented results provide the first evidence that Res can enhance the anti-tumor potency of 5-FU by inducing S-phase arrest. The combination of Res and 5-FU demonstrates synergistic efficacy, causing tumor regression in a two-stage model of mouse skin carcinogenesis induced by DMBA and TPA. There was clear evidence of Res augmenting the growth inhibitory effect of 5-FU on the TE-1 and A431 cancer cells invitro. In the invivo studies, the tumor regression rate in the combination group increased significantly after four weeks of treatment (P < 0.01). The combination of 5-FU and Res significantly increased the percentage of apoptotic cells and the level of activated caspase-3, cleaved PARP and p53 proteins as well as increased the Bax/Bcl-2 ratio. In conclusion, the 5-FU/Res combination enabled a more effective inhibition of cell growth and the induction of apoptosis in cancer cells than 5-FU alone. The results of this study suggest that chemotherapy using natural dietary agents with chemical agents represents a superior cancer treatment option.

Abstract 5355: Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion

Abstract A number of studies have established the role of receptor tyrosine kinases (RTKs) and their downstream signaling targets, including PI3K/Akt/mTOR and Stat3, as important regulatory factors of epidermal proliferative capacity during both chemical (two-stage) and UV skin carcinogenesis. We have previously shown that total protein levels of Twist1 can be regulated by Stat3 in the two-stage skin carcinogenesis model and that nuclear localization of Twist1 was restricted to the proliferative compartment. Twist1, a protein commonly associated with the induction of EMT, has previously been shown to associate with cell cycle effectors, such as p53 and p21, as well as impact cell cycle progression. The cell cycle regulatory functions of Twist1 have been linked to p53 via phosphorylation of Twist1 at Ser42 by Akt. To investigate the role of Twist1 in cellular proliferation, primary keratinocytes were infected with lentiviral particles to create Twist1 knockdown or overexpressing cells, and were used, together with wild-type primary keratinocytes, to analyze the role of Twist1 during cell cycle progression. Western blot analysis of lysates from primary keratinocytes indicated that the expression of cell cycle regulatory proteins Cyclin E1, NF-κB, and c-Myc, coincided with the decrease or increase of Twist1. Protein levels of p21 exhibited an inverse correlation to Twist1 expression. Additionally, ChIP analysis identified Cyclin D1, Cyclin E1, c-Myc, and NF-κB as novel transcriptional targets of Twist1. To further investigate the role of Twist1, we have established transgenic mice with deletion of Twist1 in the basal compartment of the epidermis using BK5.Cre (i.e. BK5.Cre x Twistflox/flox mice). BK5.Cre x Twistflox/flox mice exhibited a significant reduction in epidermal thickness and labeling index (LI) compared to wild-type mice following treatment with TPA. In comparison to wild-type mice, acetone treated Bk5.Cre x Twistflox/flox mice also displayed a significant reduction of epidermal LI. Western blot analysis of epidermal lysates collected from BK5.Cre x Twistflox/flox mice treated with TPA demonstrated a reduction of Cyclin E1 and reduced phosphorylation of Rb at Ser795 compared to wild-type mice, further implicating the regulatory capacity of Twist1 during keratinocyte proliferation. Twist1 Ser42 phosphorylation also correlated with expression of the aforementioned cell cycle proteins. Collectively, these results, combined with other data to be presented, suggest that Twist1 plays a key role in keratinocyte proliferation during skin tumor promotion. Research supported by CA76520 and T32 ES007247. Citation Format: Jaya Srivastava, Everardo Macias, Kaoru Kiguchi, John DiGiovanni. Twist1 regulates the cell cycle and proliferative capacity of keratinocytes during tumor promotion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5355. doi:10.1158/1538-7445.AM2014-5355

Bile acid accelerates erbB2-induced pro-tumorigenic activities in biliary tract cancer.

Although very few studies have addressed the molecular and cellular mechanisms underlying the development of biliary tract cancer (BTC), several lines of evidence suggest a role for the erbB receptor family. Overexpression and activation of erbB2 has been reported in a significant percentage of human BTC. Further, we previously reported that overexpression of erbB2 basal epithelial cells of the biliary tract (BK5.erbB2 mouse) led to the development of BTC. However, the mechanisms by which erbB2 overexpression led to the spontaneous development of tumors specifically in the biliary tract are not completely understood. The goals of the current study were to (1) determine whether a cooperative relationship between bile acid exposure and erbB2 activation exists during biliary tract carcinogenesis and (2) to characterize the mechanism(s) underlying bile acid-mediated biliary tract carcinogenesis in cells with activated erbB2. In this study, we demonstrated that the secondary conjugated bile acid, taurochenodeoxycholic acid (TCDC), increased proliferation of primary cultured gallbladder epithelial cells from BK5.erbB2 mice and human BTC cells. TCDC treatment activated EGFR/erbB2 and downstream signaling molecules in both primary cultured cells and human BTC cells. TCDC also increased the expression of epidermal growth factor receptor (EGFR) ligands and TACE activity in human BTC cells. Inhibition of src activation led to attenuation of bile-induced upregulation of TACE activity as well as signaling through the EGFR/erbB2, suggesting that during the development of BTC erbB2 overexpression/activation accelerates the bile acid-induced signaling cascade: bile acid → src → TACE → EGFR/erbB2 → downstream signaling. We also provide direct evidence that bile acids possess tumor promoting capacity in epithelial cells overexpressing erbB2 using the two-stage skin carcinogenesis model. Collectively these findings suggest cooperative roles for bile acid and erbB2 activation in epithelial cell proliferation; bile acid appears to accelerate erbB2-induced pro-tumorigenic activities in the biliary tract and skin.

Abstract 1290: Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis.

Abstract Recent studies have identified the involvement of receptor tyrosine kinases (RTKs) and their downstream signaling pathways, including MAPK, PI3K/Akt/mTOR, and Stat3, as key regulatory factors of epidermal proliferative capacity during skin carcinogenesis. Through development and study of transgenic mice with overexpression of a constitutively active form of Stat3 (Stat3C) in the basal cell layer, previous studies in the laboratory discovered that constitutive Stat3 activation accelerates tumor progression in the two-stage skin carcinogenesis model, which utilizes the tumor initiator 7,12-dimethylbenz[α]anthracene (DMBA) and tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Twist1, a well-known inducer of EMT, has been shown to be regulated by Stat3 in a number of systems. Western blot analysis of epidermal protein lysates from Stat3C transgenic mice indicated a two-fold induction of Twist1 expression as compared to wild type mice. In addition, immunofluorescence staining analysis of tumors from wild type mice [papillomas and squamous cell carcinomas (SCCs)] and Stat3C mice (SCCs) showed nuclear co-localization of Twist1 and Stat3. In the present study, we investigated the activation and subsequent downstream signaling of Twist1 during TPA-induced epidermal hyperproliferation. Western blot analysis of epidermal lysates from mice treated topically with multiple applications of TPA showed an initial decrease (4-6 h) followed by a subsequent rebound of Twist1 protein at 18 hours post TPA treatment. This initial decrease and then rebound coincided with that of the cell cycle regulatory proteins cyclin D1, cyclin E1, E2F-1, and c-myc. Interestingly, basal levels of Twist1 were reduced in epidermis specific Stat3 deficient mice. Furthermore, Stat3 deficient mice treated with TPA failed to show a rebound in expression of Twist1 in the epidermis. Immunofluorescence experiments of TPA treated epidermis revealed that nuclear localization of Twist1 was restricted to the proliferative compartment. Collectively, these results, combined with other data to be presented, suggest that Stat3 regulates basal levels of Twist1. This protein is localized in the nucleus of cells found primarily in the proliferative compartment of hyperplastic epidermis and skin tumors. The current results further suggest that Twist1 may play a role in cell cycle progression in keratinocytes during tumor promotion. Research supported by CA76520. Citation Format: Jaya Srivastava, Everardo Macias, Dharanija Rao, Kaoru Kiguchi, John DiGiovanni. Investigations to evaluate the role of Twist1 during tumor promotion and epithelial carcinogenesis. [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 1290. doi:10.1158/1538-7445.AM2013-1290