Tatiana M. Oberyszyn - US grants

The skin has historically been considered to function as a barrier against chemical or physical insults from the external environment. In addition to these barrier functions, the skin is now recognized to be an organ with important immunological and biochemical functions that can impact the health of the entire organism. Ultraviolet B radiation (UVB, 290-320nm) is responsible for the majority of cutaneous damage following both acute and long term exposure and is believed to be the single most important etiologic agent in human skin cancer. UVB carcinogenesis has recently been associated with an inflammatory response that includes the production and release of prostaglandins, which may be critical to the observed damaging effects of UVB on skin, which include the formation of oxidative DNA adducts. The underlying Hypothesis for the proposed studies is that there is a link between selective induction of one of the isoforms of the gene responsible for prostaglandin production, i.e. cyclooxygenase-2 (COX-2), and the resulting formation of oxidative DNA damage within the cutaneous microenvironment which ultimately results in the epigenetic and genetic alterations observed during UVB induced mutagenesis and carcinogenesis. Studies in the first specific aim will take advantage of the development of knock-out mice that do not contain either the constitutive isoform of the COG gene, COX-1 or mice which are COX-2 null. These studies will determine the critical nature of the presence of prostaglandins derived from these genes on the formation of DNA adducts, the induction of focal p53 clustering within the hyperplastic skin and to the kinetics of development and growth of UVB- induced papillomas and carcinomas. Studies in specific aim 2 will examine the ability of NSAIDS, specific inhibitors of the cyclooxygenase isoforms including, NS-398 and indomethacin, to prevent UV induced tumor growth. These studies offer a unique opportunity to gain insight into the role that COX-1 and COX-2 derived prostaglandins play in UV induced squamous cell carcinoma development and provide a potentially important pharmacological approach to abrogate or inhibit the deleterious effects of UVB exposure.

DESCRIPTION (provided by applicant): Ultraviolet light B (UVB) is responsible for the majority of cutaneous damage following both acute and long term exposure and is believed to be the single most important etiologic agent in non-melanoma skin cancers (NMSC) development. These skin tumors are by far the most common form of cancer in humans, with over 1 million new cases identified in the United States each year. UVB carcinogenesis has recently been associated with an inflammatory response that includes increases in the cyclooxygenase-2 (COX-2) gene and the subsequent elevation of prostaglandin E2 (PGE2). The importance of COX-2 induction and subsequent PGE2 production in skin tumor formation has been demonstrated in both COX-2 knockout mice as well as in studies using the specific COX-2 inhibitor, celecoxib. The biological actions of PGE2 have been attributed to its signaling through four main receptors EP[1-4]. The recent development of specific EP receptor antagonists now makes it possible to evaluate the contribution of PGE2 to the carcinogenic process by blocking its binding to specific EP receptor(s). While studies in both breast and colon cancer suggest that specific EP receptors stimulate tumor development, the particular EP receptors important in UVB induced skin carcinogenesis have not been identified. The underlying Hypothesis for the proposed studies is that PGE2 signaling through the EP1 receptor contributes to both the acute changes that occur following UVB exposure, as well as to tumor development in response to chronic UVB exposure. Studies in the first specific aim will examine the effects of blocking EP1 receptor activity via administration of the specific EP1 receptor antagonist ONO-8713 alone or in combination with celecoxib, a specific COX-2 inhibitor, on the acute UVB-mediated cutaneous inflammatory response. Studies in specific aim 2 will examine the effects of blocking EP1 receptor activity via administration of the specific EP1 receptor antagonist ONO-8713 alone or in combination with celecoxib, a specific COX-2 inhibitor, on tumor promotion, progression and regression. Finally studies in specific aim 3 will demonstrate directly the importance of EP1 signaling in UVB-induced skin carcinogenesis by testing the susceptibility of EP1 KO mice backcrossed to Skh/hr hairless mice to UVBinduced skin carcinogenesis. These studies offer a unique opportunity to gain insight into the role of the EP1 receptor in the skin carcinogenesis process and potentially provide an important pharmacological approach to preventing or reversing the damaging effects of sunlight exposure.

DESCRIPTION (provided by applicant): Ultraviolet light B (UVB) is responsible for the majority of cutaneous damage following both acute and long term exposure and is believed to be the single most important etiologic agent in non-melanoma skin cancers (NMSC) development. These skin tumors are by far the most common form of cancer in humans, with over 1 million new cases identified in the United States each year. UVB carcinogenesis has recently been associated with an inflammatory response that includes increases in the cyclooxygenase-2 (COX-2) gene and the subsequent elevation of prostaglandin E2 (PGE2). The importance of COX-2 induction and subsequent PGE2 production in skin tumor formation has been demonstrated in both COX-2 knockout mice as well as in studies using the specific COX-2 inhibitor, celecoxib. The biological actions of PGE2 have been attributed to its signaling through four main receptors EP[1-4]. The recent development of specific EP receptor antagonists now makes it possible to evaluate the contribution of PGE2 to the carcinogenic process by blocking its binding to specific EP receptor(s). While studies in both breast and colon cancer suggest that specific EP receptors stimulate tumor development, the particular EP receptors important in UVB induced skin carcinogenesis have not been identified. The underlying Hypothesis for the proposed studies is that PGE2 signaling through the EP1 receptor contributes to both the acute changes that occur following UVB exposure, as well as to tumor development in response to chronic UVB exposure. Studies in the first specific aim will examine the effects of blocking EP1 receptor activity via administration of the specific EP1 receptor antagonist ONO-8713 alone or in combination with celecoxib, a specific COX-2 inhibitor, on the acute UVB-mediated cutaneous inflammatory response. Studies in specific aim 2 will examine the effects of blocking EP1 receptor activity via administration of the specific EP1 receptor antagonist ONO-8713 alone or in combination with celecoxib, a specific COX-2 inhibitor, on tumor promotion, progression and regression. Finally studies in specific aim 3 will demonstrate directly the importance of EP1 signaling in UVB-induced skin carcinogenesis by testing the susceptibility of EP1 KO mice backcrossed to Skh/hr hairless mice to UVBinduced skin carcinogenesis. These studies offer a unique opportunity to gain insight into the role of the EP1 receptor in the skin carcinogenesis process and potentially provide an important pharmacological approach to preventing or reversing the damaging effects of sunlight exposure.

DESCRIPTION (provided by applicant): The past 30 years have seen unprecedented growth in the numbers of immunosuppressed patients. These include patients with disease-related immunosuppression, such as those infected with HIV, as well as those receiving long-term therapeutic immunosuppression including transplant patients and patients with inflammatory and autoimmune disorders. While the degree of immunosuppression varies considerably among these patients, common findings are a dampening of T-cell function and a dramatic increase in the development of ultraviolet light (UV)-induced cutaneous squamous cell carcinoma (SCC). Not only is the incidence of SCC higher in these patients than in the general population but the number of SCC that develop in each patient is increased. The SCC arising in immunosuppressed 90 patients are often very aggressive and are associated with substantial mortality. While it is clear that UV exposure and immunosuppression are major factors in the development of cutaneous malignancies, it is not clear how diminished T cell responses, either as a byproduct of disease or as a result of therapy, contributes to the generation of SCC. The following specific aims are designed to test the hypothesis that selective depletion of CD4 T-cells increases the UVB-induced inflammatory response in the skin and that this, in turn, results in an earlier onset of SCC, increased numbers of SCC, and increased aggressiveness of the tumors. These studies will also examine the importance of timing of immunosuppression relative to UV exposure on SCC development. Studies in specific Aim 1 will examine the effects of depleting CD4+ T-cells or decreasing function of these cells by treating with the therapeutically relevant immunosuppressive agent cyclosporine concurrently with DVB exposure on UVB-induced inflammation and tumor formation, in an SKH-1 hairless mouse model of UVB-induced SCC development. This aim examines the effects of T-cell dysregulation concurrently with UV exposure, as would be seen in children who are immunosuppressed early life, before they have accumulated substantial UVB exposure. Studies in specific Aim 2 will examine the effects of CD4* T-cell depletion or cyclosporine treatment begun after 10 weeks of UVB exposure on inflammation and tumor formation in SKH-1 hairless mice. This aim examines the effects of T-cell dysregulation following prior UVB exposure, as would be seen in adults who have had significant UVB exposure prior to immunosuppression. Studies in specific Aim 3 will determine the effects of CD4 T-cell depletion or cyclosporine treatment begun after 20 weeks of UVB exposure on the progression of papillomas to SCC in SKH-1 hairless mice. This aim examines the effects of T-cell dysregulation on the progression and aggressiveness of established UVB induced tumors. The studies in the present proposal will help to clarify the role of CD4* T cells in UVB induced inflammation as well as in the cutaneous carcinogenesis process.

DESCRIPTION (provided by applicant): Compared to the general population, transplant recipients are at greater risk for developing non-melanoma skin cancers, especially aggressive, life-threatening squamous cell carcinomas. In addition to ultraviolet light (UV) exposure, immunosuppression from multiple anti-rejection medications is implicated in the increased risk of skin cancer There is a clear need for treatments to prevent and treat skin cancer in transplant recipients. Ideally, such treatments could be administered after the damage of sun exposure has occurred, would not be administered as a pill or tablet and would have limited systemic effects. We hypothesize that topical application of black raspberry extracts (BRE) fulfill these criteria and should be explored as a treat- ment/preventive for non-melanoma skin cancer. Our preliminary data indicate that topical BRE applied after UVB exposure reduce DVB-induced inflammation and inhibit tumor development. This proposal will seek to determine the mechanisms by which inflammation is reduced. This proposal uses a murine model to test the novel hypothesis that BRE inhibit UV-induced skin inflammation and inhibit tumor formation through anti- oxidant properties, resulting in reduced keratinocyte activation, proliferation and DNA damage, reduced neutrophil infiltration and activity, and increased CD4+ T cell infiltration (Specific Aim 1). Further, we hypothesize that BRE will slow tumor progression to aggressive SCC when administered after tumors have formed (Specific Aim 2). Our team of investigators is uniquely able to test the hypothesis and perform these experiments due to complementary expertise in transplant and cellular immunology (VanBuskirk), photo- carcinogenesis and pathology (Kusewitt), skin inflammation and carcinogenesis (Oberyszyn), and the use of black raspberry extracts to prevent and treat cancer (Stoner and Hecht). The data obtained from our studies in this pre-clinical model will provide insights into the mechanisms by which BRE inhibit inflammation and tumor formation and will provide preliminary data for a more complete mechanism dissection and utilization in an R01 application. These studies are directly relevant to the RFA and the mission of NCCAM in that they begin dissection of the mechanisms by which topical application of a natural product, BRE, reduce inflammation after sun exposure and thereby inhibit skin tumor formation. These studies will also determine if BRE slow tumor progression, which will be important for future clinical applications

DESCRIPTION (provided by applicant): The past 30 years have seen unprecedented growth in the numbers of immunosuppressed patients. These include patients with disease-related immunosuppression, such as those infected with HIV, as well as those receiving long-term therapeutic immunosuppression including transplant patients and patients with inflammatory and autoimmune disorders. While the degree of immunosuppression varies considerably among these patients, common findings are a dampening of T-cell function and a dramatic increase in the development of ultraviolet light (UV)-induced cutaneous squamous cell carcinoma (SCC). Not only is the incidence of SCC higher in these patients than in the general population but the number of SCC that develop in each patient is increased. The SCC arising in immunosuppressed 90 patients are often very aggressive and are associated with substantial mortality. While it is clear that UV exposure and immunosuppression are major factors in the development of cutaneous malignancies, it is not clear how diminished T cell responses, either as a byproduct of disease or as a result of therapy, contributes to the generation of SCC. The following specific aims are designed to test the hypothesis that selective depletion of CD4 T-cells increases the UVB-induced inflammatory response in the skin and that this, in turn, results in an earlier onset of SCC, increased numbers of SCC, and increased aggressiveness of the tumors. These studies will also examine the importance of timing of immunosuppression relative to UV exposure on SCC development. Studies in specific Aim 1 will examine the effects of depleting CD4+ T-cells or decreasing function of these cells by treating with the therapeutically relevant immunosuppressive agent cyclosporine concurrently with DVB exposure on UVB-induced inflammation and tumor formation, in an SKH-1 hairless mouse model of UVB-induced SCC development. This aim examines the effects of T-cell dysregulation concurrently with UV exposure, as would be seen in children who are immunosuppressed early life, before they have accumulated substantial UVB exposure. Studies in specific Aim 2 will examine the effects of CD4* T-cell depletion or cyclosporine treatment begun after 10 weeks of UVB exposure on inflammation and tumor formation in SKH-1 hairless mice. This aim examines the effects of T-cell dysregulation following prior UVB exposure, as would be seen in adults who have had significant UVB exposure prior to immunosuppression. Studies in specific Aim 3 will determine the effects of CD4 T-cell depletion or cyclosporine treatment begun after 20 weeks of UVB exposure on the progression of papillomas to SCC in SKH-1 hairless mice. This aim examines the effects of T-cell dysregulation on the progression and aggressiveness of established UVB induced tumors. The studies in the present proposal will help to clarify the role of CD4* T cells in UVB induced inflammation as well as in the cutaneous carcinogenesis process.

DESCRIPTION (provided by applicant): Non-melanoma skin cancers (NMSC) are the most common form of cancer in humans, with over 1 million new cases identified in the United States each year. In fact, more Americans will be diagnosed with some form of skin cancer than all other cancers combined. Epidemiological studies have shown that there are gender differences in the development of NMSC, with men being as twice as likely to NMSC in general and three times as likely to develop squamous cell carcinomas as women. This disparity has been attributed to lifestyle choices, since males historically have had professions requiring more time out in the sun and are less likely to use sun protection. However, no studies had determined if gender differences in skin tumor development occur when males and females receive equivalent cumulative UV exposure. Recent studies from our laboratory suggest that the pathways by which UV exerts its damaging effects in male and female skin are not the same and that blocking UV-induced inflammation in male skin may not be as effective in decreasing tumor development as in female skin. The primary goals of the proposed studies are to determine the underlying mechanistic differences in the UVB response of male and female skin and to identify differences between the genders in initiation, promotion and progression of UV-induced SCC. Two specific aims are proposed to test the hypothesis that decreased levels of antioxidant activity, increased reactive oxygen species (ROS) production and increased oxidative DNA damage in male skin not only influence the acute cutaneous response to UVB exposure but also contribute to the observed gender disparity in the development of NMSC. Studies in Specific Aim 1 will determine the differential contribution of antioxidants and inflammation generated reactive oxygen species to DNA damage in unirradiated skin and during an acute UVB-induced inflammatory response in male and female Skh-1 murine skin. Studies in Specific Aim 2 are designed to determine the differential chemopreventive effects of topical treatment with an antioxidant (vitamin E), an anti-inflammatory drug (celebrex), a natural antioxidant/anti-inflammatory agent (Black Raspberry extract) or a combination of the two on UV induced tumor initiation, promotion and progression in male vs. female Skh-1 mice. A clearer understanding of the differences in the skin of males and females will ultimately allow for the development of more appropriately targeted prevention and treatment strategies.

DESCRIPTION (provided by applicant): Americans live in a culture that glorifies youth. According to market researcher FIND/SVP, the anti- aging products market is expected to hit $56 billion by 2007. Studies in post-menopausal women have found that hormone replacement therapy is effective at reversing the dryness and wrinkling that affects aging skin. Based on these studies, there is increasing interest in the use of topical creams containing hormones such as estrogen to prevent or reverse some of the normal cutaneous aging processes in younger pre-menopausal women. While exposure to these creams may be beneficial cosmetically, the effect of applying estrogen to sun exposed sites for prolonged periods of time, on skin cancer development is not known. Our preliminary studies using female Skh-1 hairless mice found a significant increase in the number of tumors in mice treated topically with estrogen immediately following UVB exposure compared to mice treated with vehicle control. These data indicate that increased levels of estrogen in the skin combined with UV exposure may act to enhance initiation and promotion of UV-induced skin cancers. These findings also suggest that the use of lotions and creams containing estrogenic compounds on sun exposed sites by younger women may be contributing to the increase in the number of skin tumors being diagnosed in women under the age of 40. Most studies have examined the effects of topical or systemic estrogen on the skin in post-menopausal women, however the reality is that younger pre-menopausal women are applying topical estrogen containing creams on their faces previously exposed to UV light to prevent/reverse the signs of aging. Two specific aims are proposed to test the hypothesis that topical estrogen application to previously UVB exposed skin accelerates skin carcinogenesis. Studies in specific aim 1 will use the Skh-1 hairless mouse murine model of UVB induced skin carcinogenesis to determine the effects of clinically used topically applied estrogen (Estrogel(R)) on UVB induced skin tumor development in previously UVB exposed female skin of intact (pre-menopausal) and ovariectomized (post-menopausal) mice. Studies in specific aim 2 will determine the effects of topically applied estrogen (Estrogel(R)) on UVB induced skin tumor progression in female Skh-1 skin of intact and ovariectomized mice. The studies carried out in these aims will determine whether topical estrogen increases the number of UVB induced skin tumors that develop and also whether it differentially enhances the progression of benign UVB-induced tumors to malignant squamous cell carcinomas in intact (pre-menopausal) and ovariectomized (post-menopausal) mice. PUBLIC HEALTH RELEVANCE: There is an increase societal pressure in the US to remain young looking. Several studies carried out in post-menopausal women demonstrate the effectiveness of topical estrogen in reversing the signs of aging including thinning, dryness and wrinkling. As a result younger pre- and peri-menopausal women are turning to topical creams containing estrogen as anti-aging lotions. Our preliminary studies using female Skh-1 hairless mice found a significant increase in the number of tumors in mice treated topically with estrogen immediately following UVB exposure compared to mice treated with vehicle control. These data indicate that increased levels of estrogen in the skin combined with UV exposure may act to enhance initiation and promotion of UV- induced skin cancers. These findings also suggest that the use of lotions and creams containing estrogenic compounds on sun exposed sites by younger women may be contributing to the increase in the number of skin tumors being diagnosed in women under the age of 40. The current studies are designed to determine the effect of topical estrogen treatment of previously UVB exposed skin on tumor development and progression from benign lesions to frank malignant squamous cell carcinomas.

DESCRIPTION (provided by applicant): Ultraviolet light B (UVB) is a major environmental carcinogen that has been implicated in the development of non-melanoma skin cancers (NMSC). These skin tumors are the most common form of cancer in humans, with over 1 million new cases identified in the United States each year. In fact, more Americans will be diagnosed with some form of skin cancer than all other cancers combined. Epidemiological studies have shown that there are gender differences in the development of NMSC, with men being twice as likely to develop Basal Cell Carcinoma (BCC)and three times as likely to develop squamous cell carcinoma (SCC) as women. Studies from our laboratory suggest that the pathways by which UV exerts its damaging effects in male and female skin are not the same and that blocking UV-induced inflammation in male skin may not be as effective in decreasing tumor development as we have shown it to be in female skin. Recent studies have described a correlation between tomato consumption and a decrease of a number of different cancers; however, the effects of tomato consumption on UVB-induced skin cancer development have not been evaluated. The studies in this application are designed to test the hypothesis that the dietary consumption of tomatoes containing the carotenoids lycopene, phytoene, or phytofluene will reduce skin damage caused by UV radiation as compared to no tomato consumption, ultimately resulting in decreased tumor development. We also hypothesize that there will be a dose-response relationship such that diets containing freeze dried tangerine tomato products containing high levels of phytoene and phytofluene will confer a greater level of protection against UV damage than red tomato products containing an intermediate level of these carotenoids. Furthermore, based on our preliminary studies, we hypothesize that there will be gender-dependent differences in the response to the tomato diets. Two specific aims are proposed. Specific aim 1 will determine whether the dietary consumption of phytoene, phytofluene, and lycopene in tangerine tomatoes and/or red tomatoes reduces the inflammatory and DNA-damaging effects of acute UVB exposure differentially in male and female Skh-1 murine skin. Studies in specific aim 2 will determine the differential chemopreventive effects of the dietary consumption of phytoene, phytofluene, and lycopene in tangerine tomatoes and red tomatoes on UVB-induced tumor promotion and progression in male and female Skh-1 murine skin. Ultimately, we hope to translate our pre-clinical findings to nutritional interventions in humans in collaboration with clinical investigators at OSU.

? DESCRIPTION (provided by applicant): Ultraviolet light B (UVB; 290-320 nm) is a major environmental carcinogen that has been implicated in the development of both basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), collectively known as nonmelanoma skin cancers (NMSC). As highlighted by the recent Surgeon General's Call to Action, these skin tumors are the most common form of cancer in humans, with over 3.5 million new cases identified in the United States each year. It was recently estimated that between 3932 and 8791 individuals died from cutaneous SCC in the United States in 2012, just slightly fewer than deaths from melanoma. The mortality rate greatly increases in the immunosuppressed population such as solid organ transplant recipients (OTR). In these patients SCC is vastly over-represented compared with its frequency in non-immunosuppressed patients. Organ transplantation increases the risk of development of SCC by 65-200 fold. In fact the term catastrophic cutaneous carcinomatosis has been coined to describe the development of SCCs in these patients. Cases of catastrophic cutaneous carcinomatosis may require decreasing immunosuppressive therapy risking the loss of the transplanted organ. As seen in the immunocompetent population, sex plays a strong role, with immunosuppressed men having greater risk of developing SCC than women. Clearly more effective preventive and/or treatment strategies are needed to prevent the morbidity and mortality resulting from SCC development in these patients. Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine that is overexpressed in several cancers including melanoma and head/neck SCC in humans. We have demonstrated in immune competent MIF KO and wild type mice treated with a pharmacological MIF inhibitor (MIFi) following chronic UVB exposure that this cytokine likewise contributes to the development of UVB- induced SCC of the skin. To date we are not aware of any studies examining the effects of immunosuppressive therapies on cutaneous levels of MIF. The studies in the current application will use this pre-clinical model to test the hypothesis that increases in UVB-induced SCC observed in OTR are mediated at least in part by elevated cutaneous MIF levels. Studies in Aim 1 will use the preclinical Skh-1 murine model to determine the efficacy of therapeutic intervention with a MIFi prior to the initiation of immunosuppressive therapy on UVB induced tumor development in the sexes. Studies in Aim 2 will determine the efficacy of MIFi treatment in inducing regression of established tumors in the sexes under an immunosuppressive environment. Completion of these studies will provide important information about a novel intervention/treatment strategy for catastrophic cutaneous carcinomatosis in OTR.