Progress 06/01/06 to 03/31/11
Outputs
OUTPUTS: TGFbeta in Skin Carcinogenesis was initiated in 2006 and in the spring of 2011 was merged into Project 4328, Cancer Molecular Pathways, Biomarkers and Targeted Therapeutics. Most human cancers in solid tissues develop through multiple stages from a premalignant lesion to invasive carcinoma. Understanding the molecular signals that drive this process is critical to develop new methods for detection, prevention and therapy of cancer. Transforming growth factor beta-1(TGFb1) is a potent growth inhibitory cytokine that is critical in the regulation of cancer development in many squamous epithelia such as the skin and oral mucosa. The goals of this project were to determine how TGFb1 produced by keratinocytes at different stages of tumor progression differentially affects tumor regression or tumor progression and to determine how TGFb1 signaling and oncogenic ras interact to cause tumor progression. We showed that in mice deficient for one copy of the Tgfb1 gene that skin tumor formation was reduced and this was linked to reduced inflammatory responses to tumor promoting chemicals. Similarly we showed that pharmacological inhibition of TGFb1 signaling blocked tumor formation. Together these results indicate that TGFb1 is a positive regulator of tumor formation rather than the accepted paradigm as a tumor suppressor. We also showed that TGFb1 is a major regulator of the inflammatory microenvironment in skin and skin cancers using a mouse model of TGFb1 overexpression. We found that in tumors, overexpression of TGFb1 caused Th17 differentiation and this was linked to cancer associated inflammation. Using an in vitro model of Ras oncogene transduced into primary keratinocytes we found that RAS does not block the early biochemical events of TGFb1 signal transduction but does alter global TGFb1 mediated gene expression in a gene specific manner. Expression of Smad3 dependent TGFb1 early response genes and growth arrest response genes were not altered by v-ras but TGFb1 and v-ras caused significant alteration in genes regulating matrix remodeling and specific matrix proteases associated with cancer progression. In addition to research publications (18) and review articles (2) and book editing (1) outputs from this project were through invited talks by the PI and poster presentation by graduate students at a number of scientific meetings, and hosting of the 9th International Skin Carcinogenesis Conference at Penn State in June 2010. PARTICIPANTS: Adam Glick TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major impact of this project and the research studies is new insight into the role of TGFb1 in cancer development. A previous paradigm for the action of TGFb1 at early stages of cancer is that this cytokine acts as a tumor suppressor, blocking outgrowth of premalignant cells. Our results have altered this paradigm and we now realize that TGFb1 can act as a significant tumor promoting stimulus, both through effects on extracellular matrix and on the tissue inflammatory microenvironment.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: Non-melanoma skin cancer (NMSC) is now the most common cancer in the white non-Hispanic population in the United States with the major risk factor being exposure to UV irradiation and subsequent mutation in the p53 tumor suppressor gene. Other chemical carcinogens present in the environment also contribute to human skin cancer. Most human cancers in solid tissues develop through multiple stages from a premalignant lesion to invasive carcinoma. Understanding the molecular signals that drive this process is critical to develop new methods for detection, prevention and therapy of cancer. Transforming growth factor beta-1(TGFb1) is a potent growth inhibitory cytokine that is critical in the regulation of cancer development in many squamous epithelia such as the skin and oral mucosa. Changes in expression of and response to TGFb1 are characteristic of cancer progression. The overall goals of this project are: To determine how TGFb1 produced by keratinocytes during multistage cancer development differentially affects tumor regression or tumor progression. The outputs for this project during 2010 were publications and the organizing and hosting of the 9th International Skin Carcinogenesis Conference at Penn State University June 24-27, 2010. The International Skin Carcinogenesis Conference is a biannual meeting, which promotes exchange of state-of-the-art ideas from a diverse group of experts from Europe, Asia, and the United States. The conference focuses on the most recent and relevant advances in the biological, cellular, and molecular changes that occur during skin tumor development and discussions of how the skin serves as a model system for the development and treatment of other tumors. At this meeting there were sessions covering squamous cell carcinoma, basal cell carcinoma and melanoma. Funding for this conference was successfully obtained form the NCI, NIEHS and other sources. Although registration was required this conference was open to the public. Key research findings were presented at this meeting and the Annual Society of Toxicology Meeting in March 2010. Other outputs included publication of a paper in the journal Carcinogenesis, and in the Journal of Investigative Dermatology. A third manuscript has also been accepted for publication in Carcinogenesis. This project has generated significant new information on the role of TGFb in skin cancer and has disseminated this information through publication and presentation at scientific meetings. PARTICIPANTS: Individuals who worked on this project include Adam Glick, Ph.D. the principle investigator, two postdoctoral fellows, Dr. Rolando Perez-Lorenzo Ph.D. and Dr. Javed Mohammed, Ph.D. and five graduate students, Kelly Hogan, Nicholas Blazanin, Andrew Gunderson, Lauren Mordasky and Anand Ravindran. One student received her Ph.D. degree in October 2010. Training in laboratory methods and molecular biology techniques was given to three undergraduates in the laboratory, Kate Masiuk, Rich Koubeck and Anthony Ricco. TARGET AUDIENCES: The scientific target audience for this project is those in the field of cutaneous immunobiology, cutaneous cancer, and TGFb1. Health professionals who work with cancer issues in rural populations are also the target audience for this project. Outreach efforts include publication of data in scientific journals, presentation at scientific meetings and invited seminars, and presentation of data at meetings of the Penn State Cancer Institute, a forum that allows interaction with physicians and researchers concerned with health issues in rural and underserved communities in Pennsylvania. In addition a significant amount of was placed on coordinating the 9th International Skin Carcinogenesis Conference, which will be held at Penn State University in June 2010. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Several important findings were made and published in 2010. While it is a well accepted paradigm that TGFbeta1 is a tumor suppressor in early stages of cancer using TGFb1+/+ and TGFb1+/- we showed that reduced levels of cutaneous TGFb actually caused fewer skin tumors to appear in a chemical carcinogenesis bioassay. Additional results published in this article showed that physiological levels of TGFbeta could act as a tumor promoter for skin cancer development in part through effects on epidermal proliferation and on cutaneous inflammation. In our second publication we showed that expression of TGFb in tumors caused an inflammatory response that was linked to tumor regression and dependent on induction of a Th17 response in the tumors.
Publications
- Perez-Lorenzo, R., L. Mordasky Markell, K. A. Hogan, S. H. Yuspa, and A. B. Glick. 2010. Transforming growth factor beta1 enhances tumor promotion in mouse skin carcinogenesis. 2010. Carcinogenesis 31:1116-23.
- Mohammed, J., A. Ryscavage, R. Perez-Lorenzo, A. Gunderson, N. Blazanin, and A. B. Glick. 2010. TGFbeta1 induced inflammation in premalignant epidermal squamous lesions requires IL-17. J. Invest. Dermatol. 130:2295-303.
- Koesters, R., B. Kaissling, M. Lehir, N. Picard, F. Theilig, R. Gebhardt, A. B. Glick, B. Hahnel, H. Hosser, H. J. Grone, and W. Kriz. 2010. Tubular overexpression of transforming growth factor-beta1 induces autophagy and fibrosis but not mesenchymal transition of renal epithelial cells. Am J Pathol. 177:632-43.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Most human cancers in solid tissues develop through multiple stages from a premalignant lesion to invasive carcinoma. Understanding the molecular signals that drive this process is critical to develop new methods for detection, prevention and therapy of cancer. Transforming growth factor beta-1(TGFb1) is a potent growth inhibitory cytokine that is critical in the regulation of cancer development in many squamous epithelia such as the skin and oral mucosa. Changes in expression of and response to TGFb1 are characteristic of cancer progression. Additionally interactions of TGFb1 signaling with those of dominant oncogene pathways such as ras which is frequently mutated in many human cancers, is likely to be important determining how TGFb1 functions to inhibit or promote cancer development. The overall goals of this project are: To determine how TGFb1 produced by keratinocytes during multistage cancer development differentially affects tumor regression or tumor progression and to determine how TGFb1 signaling and oncogenic ras interact to cause tumor progression. The outputs for this project during 2009 were publications and participation in scientific meetings to disseminate findings. Key findings were presented at the American Association for Cancer Research Annual meeting, the Society of Toxicology Annual Meeting, and monthly meetings of the Penn state Cancer Institute Chemical Carcinogenesis and Chemoprevention group. The two publications Bae et al., and Vijayachandra et al., in the journal Molecular Carcinogenesis look at two different aspects of the interaction between TGFb1 signaling and oncogenic ras. While earlier studies with immortalized cells showed that oncogenic ras inhibited TGFb1 signaling this paper shows that in primary keratinocytes inhibition does not occur, rather oncogenic ras reprograms the TGFb1 genomic response. Expression of Smad3 dependent TGFb1 early response genes and growth arrest response genes were not altered by oncogenic ras but the normal induction of TGFb1 extracellular matrix genes was blocked by the ras oncogene and several metalloproteinases were synergistically induced. Smad3 deletion in keratinocytes repressed normal differentiation maker expression and caused expression of Keratin 8 a simple epithelial keratin and marker of malignant conversion. Smad3 was required for the TGFb1 cytostatic response in keratinocytes expressing oncogenic ras but also for protease induction, keratinocyte attachment and migration, additional markers of progression. These results show that pro-oncogenic activities of TGFb1 can occur early in carcinogenesis before loss of its tumor suppressive function and that selective regulation rather than complete inactivation of Smad3 function may be crucial for tumor progression. In the second manuscript, we showed that TGFb1 induced expression of the tumor suppressor p16ink4a and p19ARF in ras expressing keratinocytes and concluded that inactivation of cdkn2a locus during tumor development may alter cytostatic responses to TGFb1. PARTICIPANTS: Individuals who worked on this project include Adam Glick, Ph.D. the principle investigator, two postdoctoral fellows, Dr. Rolando Perez-Lorenzo Ph.D. and Dr. Javed Mohammed, Ph.D. and five graduate students, Kelly Hogan, Nicholas Blazanin, Andrew Gunderson, Lauren Mordasky and Anand Ravindran. Training in laboratory methods and molecular biology techniques was given to one undergraduate in the laboratory, Kate Masiuk. TARGET AUDIENCES: The scientific target audience for this project is those in the field of cutaneous immunobiology, cutaneous cancer, and TGFb1. Health professionals who work with cancer issues in rural populations are also the target audience for this project. Outreach efforts include publication of data in scientific journals, presentation at scientific meetings and invited seminars, and presentation of data at meetings of the Penn State Cancer Institute, a forum that allows interaction with physicians and researchers concerned with health issues in rural and underserved communities in Pennsylvania. In addition a significant amount of effort has been placed on coordinating the 9th International Skin Carcinogenesis Conference, which will be held at Penn State University in June 2010. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Describe findings, results, techniques, or other products that were developed or extended from the project generated or contributed to a change in knowledge, change in actions, or change in conditions. Several important findings were made that stem from work published in 2009 or currently submitted for publications. Following up on the Bae et al study we have begun to study the mechanism by which ras suppresses TGFb1 induced extracellular matrix gene expression. New results link this suppression to the increased expression of the transcriptional co-repressor TGIF (TG interacting factor). TGIF is a member of the TALE superfamily of homeodomain proteins that are important in brain and craniofacial development. TGIF competes with the coactivator p300 for binding to Smads and recruits mSin3 and HDACs to promoters to repress Smad dependent transcription. It is thought that the relative level of TGIF and p300 in a cell and the competition for Smads determines the degree to which TGFb1 can activate its transcriptional program. However, apart from these few studies in immortalized cell epithelial cell lines there is no direct evidence indicating whether TGIF plays a role in cancer development. Our preliminary data show that expression of oncogenic ras in primary mouse keratinocytes increases the level of total and phosphorylated TGIF. We developed SiRNA against TGIF and showed that reduction of TGIF protein in ras expressing keratinocytes had no effect on the induction of Smad7 by TGFb1 but restored the blocked induction of several ECM genes in ras keratinocytes. This is the first demonstration of gene specific suppression by TGIF but further study is needed to understand selectivity of TGIF suppression of Smad3 activated promoters. Since these preliminary data suggest selectivity of effect of TGIF towards a subset of TGFb1 regulated genes it is likely that TGIF could play an important role in modulating the phenotype of the premalignant cancer cell. It is possible that TGFb1 mediated increases in TGIF and altered patterns of gene regulation are relevant to later stages of cancer progression where increased TGFb1 levels frequently occur. Thus, we plan to use our in vitro and in vivo models to determine what functional role TGIF has in ras mediated skin carcinogenesis.
Publications
- Bae, D.S., N. Blazanin, M. Licata, J. Lee, and A.B. Glick. 2009. Tumor suppressor and oncogene actions of TGFbeta1 occur early in skin carcinogenesis and are mediated by Smad3. Mol. Carcinog. 48:441-53.
- Zheng, T., M.H. Oh, S.Y. Oh, J.T. Schroeder, A.B. Glick, and Z. Zhu. 2009. Transgenic Expression of Interleukin-13 in the Skin Induces a Pruritic Dermatitis and Skin Remodeling. J. Invest. Dermatol. 129:742-51.
- Jabbar, S.F., L. Abrams, A. Glick, and P.F. Lambert. 2009. Persistence of high-grade cervical dysplasia and cervical cancer requires the continuous expression of the human papillomavirus type 16 E7 oncogene. Cancer Research 69:4407-14.
- Rozenberg, J., V. Rishi, A. Orosz, J. Moitra, A. Glick, and C. Vinson 2009. Inhibition of CREB function in mouse epidermis reduces papilloma formation. Mol. Cancer Res. 7:654-64.
- Vijayachandra, K., W. Higgins, J. Lee, and A. Glick. 2009. Induction of p16(ink4a) and p19(ARF) by TGFbeta1 contributes to growth arrest and senescence response in mouse keratinocytes. Mol. Carcinog. 48:181-6.
- Ponnamperuma, R.M., K.E. King, T. Elsir, A.B. Glick, G.M. Wahl, M. Nister, and W.C. Weinberg. 2009. The transcriptional regulatory function of p53 is essential for suppression of mouse skin carcinogenesis and can be dissociated from effects on TGFb1 mediation growth regulation. J. Pathol. 219:263-74.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The goals of this project are: To determine how transforming growth factor beta-1 (TGFb1) produced by keratinocytes at different stages of tumor progression differentially affects tumor regression or tumor progression and to determine how TGFb1 signaling and oncogenic ras interact to cause tumor progression. A manuscript submitted to Cancer Research showed that TGFb1 can promote benign skin tumor formation through activation of PKC. In this paper we showed that TGFb1+/- mice had fewer numbers of benign papillomas, reduced tumor cell proliferation, reduced proliferative response to TPA compared to TGFb1+/+ mice, and no increase in epidermal TGFb1 or nuclear p-Smad2. TPA activation of PKCa correlated with TGFb1 gene dosage. Further data supported the idea that physiological levels of TGFb1 are required for maximal activation of PKC. However, the TPA-induced inflammatory response was greater in TGFb1+/- skin, but TGFb1+/+ papillomas had more tumor infiltrating neutrophils. A second manuscript examining the effect of TGFb1 at different cancer stages has been submitted to the journal Oncogene and is under review. Here, we showed that TGFb1 induction in papillomas increased tumor neutrophils, CD8+ T cells, and markers of activated Th1 and Th17 T cells. Premalignant keratinocytes expressing TGFb1 had elevated IL-6 and IL-23, likely polarizing the development of naive T cells towards a proinflammatory Th17 phenotype. These results show that immune responses to tumor cell derived TGFb1 are cancer stage specific and can promote either an inflammatory or immunosuppressed environment. For the second goal we have published 2 papers in the journal Molecular Carcinogenesis. In the first we showed that the v-ras oncogene does not block the early biochemical events of TGFb1 signal transduction but does alter global TGFb1 mediated gene expression in a gene specific manner. Expression of Smad3 dependent TGFb1 early response genes and growth arrest response genes were not altered by v-ras but TGFb1 and v-ras caused significant alteration in genes regulating matrix remodeling and specific matrix proteases associated with cancer progression. Current studies focus on ChIP analysis of Smad DNA binding at the promoters of key genes whose expression is differentially regulated by ras and TGFb1. In the second manuscript, we showed that expression of the tumor suppressors p16ink4a and p19ARF in v-ras expressing keratinocytes was dependent on TGFb1 signaling and concluded that inactivation of cdkn2a locus during tumor development may alter cytostatic responses to TGFb1. Additionally, to disseminate these outputs to the research community members of my laboratory and myself have presented data at several forums in 2008 including the American Association for Cancer Research Annual Meeting April 2008, the VBSC Pathobiology Retreat, The Penn State Cancer Institute Chemical Carcinogenesis Program meetings, Seminar series on the University Park Campus, Invited seminars that include the Vaccine Branch in the National Cancer Institute, and the 8th International Skin Carcinogenesis Meeting, October 2008, and the Toxicology Gordon Conference. PARTICIPANTS: Individuals who worked on this project include Adam Glick, Ph.D. the principle investigator, two postdoctoral fellows, Dr. Rolando Perez-Lorenzo Ph.D. and Dr. Javed Mohammed, Ph.D. and four graduate students, Kelly Hogan, Nicholas Blazanin, Frank Horvath and Lauren Mordasky. During this time Frank Horvath graduated with a Masters degree, and he was replaced by another graduate student Andrew Gunderson. Training in laboratory methods and molecular biology techniques was given to four undergraduates in the laboratory: Kristen Holler, Samuel Zonshayn TARGET AUDIENCES: The scientific target audience for this project is those in the field of cutaneous immunobiology, cutaneous cancer, and TGFb1. Health professionals who work with cancer issues in rural populations are also the target audience for this project. Outreach efforts include publication of data in scientific journals, presentation at scientific meetings and invited seminars, and presentation of data at meetings of the Penn State Cancer Institute, a forum that allows interaction with physicians and researchers concerned with health issues in rural and underserved communities in Pennsylvania. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A change in knowledge for the PI occurred during the last year as a direct result of research conducted to accomplish these research objectives. In 2007 it was determined that overexpression of TGFb1 in the normal epidermis acts as a negative regulator of tumor promoter-induced inflammatory responses, as mice that were hemizygous for a TGFb1 allele had a more pronounced and sustained inflammatory response to TPA. This was observed at the histological level, by Immunohistochemistry and by mRNA analysis. This year we have made significant progress using a tumor induction protocol and have shown that mice that express less TGFb1 are less susceptible to tumor formation. This goes against current paradigms which consider TGFb1 a potent tumor suppressor. We have found however, that mice deficient in TGFb1 have lower epidermal proliferation, and reduced tumor cell proliferation compared to mice with normal TGFb1 levels. In 2008 we have identified that crosstalk between TGFb1 and protein kinase C (PKC) signaling pathways is essential for the positive impact of TGFb1 on cell proliferation. These studies have been submitted for publication and are under review. In contrast, blocking TGFb1 signaling with a pharmacological agent completely blocked the cutaneous inflammatory response to TPA suggesting that the influence of TGFb1 on cutaneous inflammation is complex and may depend on what cells are making TGFb1 and where the response is blocked. In benign tumors expressing TGFb1 immunophenotyping analysis suggests that TGFb1 induces a pro-inflammatory TH17 phenotype. The implications of this are significant since a TH17 response has been observed in other inflammatory conditions linked to TGFb1 but not so far to tumor regression. Furthermore it has become apparent that tumors generated in the skin of mice by overexpressing a ras oncogene have a similar immunological profile as those generated by chemical carcinogens. This suggests that different levels of ras may be important in generating low or high risk benign tumors. We have also made significant progress in identifying TGFb1 as an important component of the UV response. Our results show that mice deficient in Smad3 a critical intermediate in the TGFb1 pathway do not undergo the normal growth arrest that occurs in normal mouse skin that occurs after exposure to UVB irradiation. Since this growth arrest is associated with repair of UV induced mutations we predict that Smad3 deficient mice will be more sensitive to UV induced skin cancer.
Publications
- Bae, D. S., N.Blazanin, M. Licata, J. Lee and A. B. Glick. 2008. Tumor suppressor and oncogene actions of TGFbeta1 occur early in skin carcinogenesis and are mediated by Smad3. Mol. Carcinog. (In Press).
- Zheng, T., M. H. Oh, S. Y. Oh, J. T. Schroeder, A. B. Glick and Z. Zhu. 2008. Transgenic Expression of Interleukin-13 in the Skin Induces a Pruritic Dermatitis and Skin Remodeling. J. Invest. Dermatol. (In Press).
- Bility, M. T., M. K. Devlin-Durante, N. Blazanin, A. B. Glick, J. M. Ward, B. H. Kang, M. J. Kennett, F. J. Gonzalez and J. M. Peters. 2008. Ligand activation of peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) inhibits chemically-induced skin tumorigenesis. Carcinogenesis. (In Press).
- Vijayachandra, K., W. Higgins, J. Lee, and A. B. Glick. 2008. Induction of p16(ink4a) and p19(ARF) by TGFbeta1 contributes to growth arrest and senescence response in mouse keratinocytes. Mol. Carcinog. (In Press).
- Traykova-Brauch, M., K. Schonig, O, Greiner, T. Miloud, A. Jauch, M. Bode, D. W. Felsher, A. B. Glick, D. J. Kwiatkowski, H. Bujard, J. Horst, M. von Knebel Doeberitz, F. K. Niggli, W. Kriz, H. J. Grone, and R. Koesters. 2008. An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice. Nat. Med. 14:979-84.
- Hoseong, Y. S., T. Andl, V. Grachtchouk, A.Wang, J. Liu, L. J. Syu, J. Ferris, T. S. Wang, A. B. Glick, S. E. Millar, and A. A. Dlugosz. 2008. Pathological responses to oncogenic Hedgehog signaling in skin are dependent on canonical Wnt/beta-catenin signaling. Nat Genet. (In Press).
- Shukla, A., Y. Ho, X. Liu, A. Ryscavage, and A. B. Glick. 2008. Cripto-1 alters keratinocyte differentiation via blockade of transforming growth factor-beta1 signaling: role in skin carcinogenesis. Mol. Cancer Res. 6:509-16.
- Horvath, F. J. 2008. Neutrophil depletion during skin-restricted ras expression reduces hyperplasia and inflammation. M.S. Thesis. The Pennsylvania State University, University Park, PA. 102 pp.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: The goals of this project are as follows: 1) To determine how transforming growth factor beta-1 (TGFb1) produced by keratinocytes at different stages of tumor progression differentially affects tumor regression or tumor progression. 2) To determine how TGFb1 signaling and oncogenic ras interact to cause tumor progression. To address the first goal we have begun to examine changes in the cutaneous immune system in response to TGFb1 produced by keratinocytes. We are using mice that are hemizygous for the TGFb1 gene and treating them with a tumor promoter (TPA) to examine how TGFb1 levels in the epidermis regulate proliferative responses and inflammatory responses. In addition we are blocking TGFb1 signaling in the epidermis using a small molecule inhibitor or the receptor and assessing effects on proliferative and inflammatory responses to a tumor promoter. Since UV light from the sun is a major source of cutaneous cancer in humans we are examining how TGFb signaling affects
cutaneous responses to UV using mice wildtype or deficient for Smad3 an intracellular mediator of TGFb signaling. To address how TGFb1 production by benign or malignant squamous alters the immune response to the tumor we have examined changes in immune markers by RT-PCR and by flow cytometery to determine if a TH17 or Treg phenotype predominates in either tumor type after induction of TGFb1. To address the second goal we have developed a chromatin immunoprecipitation (CHIP) assay to examine binding of specific transcription factors regulated by ras and TGFb1 at the promoters of key genes whose expression is differentially regulated by ras and TGFb1 as determined by microarray analysis. We are also examining changes in epidermal T cells and neutrophil infiltration following induction of oncogenic ras in the skin. To disseminate these outputs to the research community members of my laboratory and myself have presented data at several forums in 2007 including the American Association for
Cancer Research Annual Meeting April 2007, International Conference on Immunology, August 2007, Pathobiology Retreat, Penn State Cancer Institute Chemical Carcinogenesis Program meeting, Invited seminar Maestro laboratory UP, Invited seminar Pegg laboratory Hershey Medical Center, Invited seminar Hershey Dermatology Department.
PARTICIPANTS: A. Glick
TARGET AUDIENCES: Scientific, Academic, Government and Agricultural Professionals.
Impacts
A change in knowledge for the PI occurred during the last year as a direct result of research conducted to accomplish these research objectives. It was determined that overexpression of TGFb1 in the normal epidermis acts as a negative regulator of tumor promoter-induced inflammatory responses, as mice that were hemizygous for a TGFb1 allele had a more pronounced and sustained inflammatory response to TPA. This was observed at the histological level, by Immunohistochemistry and by mRNA analysis. In contrast, blocking TGFb1 signaling with a pharmacological agent completely blocked the cutaneous inflammatory response to TPA suggesting that the influence of TGFb1 on cutaneous inflammation is complex and may depend on what cells are making TGFb1 and where the response is blocked. In benign tumors expressing TGFb1 immunophenotyping analysis suggests that TGFb1 induces a pro-inflammatory TH17 phenotype. The implications of this are significant since a TH17 response has been
observed in other inflammatory conditions linked to TGFb1 but not so far to tumor regression. Furthermore it has become apparent that tumors generated in the skin of mice by overexpressing a ras oncogene have a similar immunological profile as those generated by chemical carcinogens. This suggests that different levels of ras may be important in generating low or high risk benign tumors. The laboratory mastered a new technique (CHIP) although clearcut results have not yet been obtained
Publications
- Glick, A., R. Perez-Lorenzo, J. Mohammed. 2007. Context Dependent Regulation of Cutaneous Immunological Responses by TGFbeta1 and its Role in Skin Carcinogenesis. Carcinogenesis (In Press).
- Hogan, K. A. and A. B. Glick. 2007. TGF-beta1 induces Smad3-dependent NFkappaB activation in primary mouse keratinocytes. In Proc 100th AACR Annual Meeting, Los Angeles, CA. Abstract #2419 bhttp://www.aacr.org/home/scientists/meetings--workshops/previous-ann ual-meetings/annual-meeting-2007.aspx
- Perez-lorenzo, R. and A. B. Glick. 2007. Skin tumor formation is associated with TGF-beta1 induced alterations in proliferative and inflammatory responses following phorbol ester promotion. Proceedings of the 13th International Congress of Immunology, Rio di Janiero, Brazil. April 14-18, 2007. p. 722 (Abstract).
- Darwiche, N., A. Ryscavage, R. Perez-Lorenzo, L. Wright, D. Bae, H. Hennings, S.H. Yuspa, and A. B. Glick. 2007. Expression profile of skin papillomas with high cancer risk displays a unique genetic signature that clusters with squamous cell carcinomas and predicts risk for malignant conversion. Oncogene. 26:6885-6895.
- Sainte Marie, Y., A. Toulon, R. Paus, E. Maubec, A. Cherfa, M. Grossin, V. Descamps, M. Clemessy, J.M. Gasc, M. Peuchmaur, A. Glick, N. Farman, and F. Jaisser. 2007. Targeted skin overexpression of the mineralocorticoid receptor in mice causes epidermal atrophy, premature skin barrier formation, eye abnormalities, and alopecia. Am J Pathol. 171:846-860.
- Glick, A., A. Ryscavage, R. Perez-Lorenzo, H. Hennings, S. Yuspa, and N. Darwiche. 2007. The high-risk benign tumor: evidence from the two-stage skin cancer model and relevance for human cancer. Mol Carcinog. 46:605-610.
- Garcia, R. J., A. Ittah, S. Mirabal, J. Figueroa, L. Lopez , A. B. Glick and L. Kos. 2007. Endothelin 3 Induces Skin Pigmentation in a Keratin-Driven Inducible Mouse Model. J Invest Dermatol (In Press).
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Progress 01/01/06 to 12/31/06
Outputs
To determine if the transcriptional response to TGFbeta1 overexpression is similar or distinct between a benign squamous papilloma and a malignant squamous carcinoma we have used mice with a doxycyline regulated TGFbeta1 transgene to express this cytokine in chemically-induced papillomas and squamous cell carcinomas. Induction of TGFbeta1 expression increased nuclear p-Smad2 in the benign and malignant tumor cells, but caused a 3-fold reduction in proliferating tumor cells and reduction in tumor volume over 10 days only in the benign papillomas but not squamous cell carcinomas. There was a shard increase in apoptotic cells in the benign papillomas as well following induction of TGFbeta1. RNA has been isolated from these tumors and microarray comparisons of gene expression done in the benign and malignant tumors with and without TGFbeta1 overexpression. Current studies are focused on understanding the significance and validating the differentially expressed genes
between these two tumor types. To test if TGFbeta1 is required for skin tumor formation, we compared the response of TGFbeta1+/+ and +/- mice to a DMBA-TPA carcinogenesis protocol. TGFbeta1+/- mice had reduced papilloma numbers but higher frequency of conversion to SCC than TGFbeta1+/+ littermates. After 5 weeks of TPA alone, the epidermis of TGFbeta1 +/- mice was 2 times more hyperplastic than WT mice. However, the proliferation index of the basal layer was 15.6% in the TGFbeta1+/+ epidermis but only 2.9% in the TGFbeta1+/- epidermis (p<0.05). Additionally, the TPA-induced increase in keratinocytes apoptosis was blocked in the TGFbeta1+/- mice. Inflammatory infiltrates composed mainly of neutrophils were greater in the TGFbeta1 +/- TPA-treated skin, but there was no difference in infiltrating CD3+ cells. Our results suggest that TGFbeta1 is involved in upregulating the cell turnover in the epidermis and suppressing the inflammatory response to TPA treatment and this may be important
in papilloma outgrowth. To model the interaction between oncogenic ras and TGFbeta signaling and the impact on skin tumor development in vivo we generated a triple transgenic mouse expressing the K5/rTA transgene and the dox inducible transgenes tetOTGFbeta1 and tetOrasV12G. Addition of doxycycline to triple transgenic mice co-induced TGFbeta1 and oncogenic ras in the epidermis and within 48 hrs produced a rapid increase in vascularity and disorganization of the epidermis without blocking ras induced hyperplasia. The epidermis from triple transgenic mice had increased staining for CD31 and VWF confirming the angiogenic response and decreased expression of E-cadherin in the basal layer. Quantitative-PCR and immunohistochemistry showed that urokinase expression was induced 4 fold in the epidermis of triple transgenic mice.
Impacts
TGFbeta is a member of a large family of structurally related secreted cytokines that play an important role in normal tissue homeostasis and tumor development. This cytokine plays a dual role in cancer development acting both as a tumor suppressor and prometastatic factor, but the molecular basis of this switch remains unresolved. These studies will identify mechanisms by which TGFbeta1 regulates skin cancer development in mice as a model for multistage cancer development in humans. The molecular targets of TGFbeta signaling identified in this project will potentially new therapeutic targets for cancer therapy.
Publications
- Abdel-Fattah, R., Glick, A., Rehman, I., Maiberger, P., and Hennings, H., 2006. Methylation of the O6-methylguanine-DNA methyltransferase promoter suppresses expression in mouse skin tumors and varies with the tumor induction protocol. Int J Cancer. 118:527-531.
- Gerdes, M. J., Myakishev, M., Frost, N.A., Rishi, V., Moitra, J., Acharya, A., Levy, M.R., Park, S.W., Glick, A., Yuspa, S.H., and Vinson, C. 2006.b Activator protein-1 activity regulates epithelial tumor cell identity. Cancer Res. 66:7578-7588.
- Kirshner, J., Jobling, M.F., Pajares, M.J., Ravani, S.A., Glick, A.B., Lavin, M.J., Koslov, S., Shiloh, Y., and Barcellos-Hoff, M.H. 2006. Inhibition of transforming growth factor-beta1 signaling attenuates ataxia telangiectasia mutated activity in response to genotoxic stress. Cancer Res. 66:10861-10869.
- Bae, D.S., Blazanin, N., and Glick, A. 2006. Angiogenesis and disrupted epidermal structure in mice coexpressing TGFbeta1 and oncogenic ras. J. Invest. Derm 126 (S4), 21.
- Darwiche, N., Ryscavage, A., Nolan, L., Hennings, H., Yuspa, S.H. and Glick, A. 2006. Identification of a gene expression signature for benign precursor lesions to cutaneous squamous cell carcinoma. Proceedings Society for Investigative Dermatology. J. Invest Derm 126 (S4), 21
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