Molecular and Cellular Mechanisms of Photocarcinogenesis[Dagger]

By Beissert, Stefan Loser, Karin

ABSTRACT Skin cancer constitutes one of the most frequent types of malignancies in humans with rapidly increasing incidences almost worldwide. UVR is an essential risk factor for the development of premalignant as well as malignant skin lesions. In this context UVR can function as a complete carcinogen by inducing “UV signature” DNA mutations and by suppressing protective cellular antitumoral immune responses. UV-induced DNA damage can result in impaired cutaneous cell cycle control if cell cycle regulators, such as the p53 gene, are affected. Besides interfering with cell cycle control genes, UV- induced DNA damage can result in the release of interleukin-10, a cytokine with known immunosuppressive effects on T-helper(h)-1 cells. For the development of antitumoral immune responses antigen- specific activation of effector T cells by antigen-presenting cells (APC) is required. It was demonstrated that UVR can inhibit antigen presentation both directly and indirectly via the induction of suppressive cytokines. In addition, subsets of T cells are induced upon UVR, which can actively suppress major histocompatibility complex class I/II-restricted immune responses. These UV-induced regulatory T cells appear to belong to the CD4^sup +^CD25^sup +^ T cell lineage and can express the characteristic transcription factor Foxp3, which programs for suppressor function. In mice UV-induced regulatory T cells can control the development of UV-induced skin cancer. Peripheral regulatory T cells are maintained by the expression of B7 molecules and can be expanded by APC of the skin. Recently, epidermal expression of CD254 (RANKL) has been shown to connect UVR with the expansion of regulatory CD4^sup +^CD25^sup +^ T cells. In the following, new molecular and cellular mechanisms of UV- induced skin tumor development will be described and discussed.

INTRODUCTION

UV irradiation is one of the most relevant risk factors for the development of skin cancer, such as basal-and squamous cell carcinoma or lentigo maligna-and superficial spreading melanoma (1- 3). These UV-induced tumors show a rapidly rising incidence not only in the United States but also in Europe and in Australia. These types of cancer can take a fatal course and cause significant disability besides increasing medical costs. Although the use of broad-spectrum sunscreens has proven protective effects on the development of skin cancer as well as on premalignant skin lesions, the rate of UV-induced skin malignancies is expected to rise significantly due to the cumulative nature of the factors inducing carcinogenesis, in addition to more outdoor activities and the longer life expectancy of the population (4-6). Furthermore, decreasing stratospheric ozone levels have led in the past decades to increasing exposure to short wave length UVB irradiation on the terrestrial surface (5). Especially, UVB irradiation accounts for most of the documented harmful biological effects of sunlight (reviewed in 1,2). Epidermal cells are considered to be the major target of UVB radiation as the vast majority of UVB is absorbed within the epidermis. Photons are the smallest carriers of nonionizing radiation and must be absorbed to induce biological effects. During photon absorption, energy is transferred to the chromophore and the photon ceases to exist. These photo-induced molecular changes can produce a cascade of events leading from inflammation to suppression of cellular immunity and the development of skin cancer (reviewed in 2).

The development of skin cancer appears to be controlled in part by the immune system. This hypothesis is supported by the observation of 30-fold increased numbers of cutaneous tumors within sun-exposed areas, such as the face, scalp or back of the hand, of therapeutically immunosuppressed organ transplanted patients (7-9). In murine models of photocarcinogenesis the growth of tumors seems to be regulated by T-cell-mediated immune responses (10). Murine UV- induced skin tumors are often immunogenic and accordingly rejected upon transplantation into immunocompetent hosts (10-16). These tumors grow only if the recipient animal is immunosuppressed by medication, single or chronic UV irradiation, and gamma irradiation. Also, UV-induced suppressor T cells have been detected in UV- treated mice, which apparently promote tumor growth by suppressing antitumor effector functions (17,18). Recent data suggest that UV- induced suppressor T cells belong to the CD4^sup +^CD25^sup +^ regulatory T cell lineage but little is known about the factors that regulate UV-induced suppressor T cell homeostasis or function (19). This is in contrast to the much better characterized naturally occurring CD4^sup +^CD25^sup +^ regulatory T cells.

Within the skin all the necessary cellular requirements are present to induce and elicit antitumoral immunity (20). Keratinocytes, the major cellular component of the epidermis, can produce a number of soluble mediators, which upon release into the circulation can affect the outcome of the ensuing immune response. Epidermal antigen-presenting cells (APC), the Langerhans cells (LC), can function as immune sentinels and can connect the epidermis with the immune system. Accordingly, LC are able to migrate directly from the epidermis to skin-draining lymph nodes to induce (tumor)antigen- specific T cell activation. UVR has been shown to suppress the APC function of LC to induce protective antitumoral immune responses (21- 23). Thereby, UVR inhibits APC function both directly via UV- induced LC cytotoxicity and indirectly via the release of suppressive mediators from neighboring keratinocytes. This article will focus on the current knowledge of the molecular and cellular mechanisms involved in the development of UV-induced skin tumors.

CD80/CD86-CD28/CD152 SIGNALING: KEY PATHWAYS FOR THE REGULATION OF PHOTOCARCINOGENESIS

Epidermal APC, such as LC, can present tumor-associated antigens both for the induction and elicitation of protective immunity (21- 23). For the induction of (tumor)antigen-specific immune responses by APC interaction of B7 family molecules is of great importance. The two initially discovered B7 family molecules CD80 and CD86 are expressed on APC and bind to their coreceptors CD28 and CD 152 (CTLA- 4) on T cells. It was previously suggested that CD80/CD86-mediated costimulation supports the development of effective cell-mediated immunity (24). Data to support this view include studies wherein treatment of mice with CTLA-4Ig, a soluble form of CTLA-4, which effectively blocks CD80/CD86 engagement with CD28, suppressed antitumor immunity, transplant rejection and autoimmune responses (25,26). However, a growing body of evidence suggests a more complicated role of CD80/CD86-mediated costimulation during immune responses (27-29). There are several reports showing that CD28 costimulation induced T cell activation, whereas ligation of CD 152 down-regulated T cell function (25,27,30). Thus, there are documented differences in response outcomes due to the differential effects of CD28 and CD152 ligation on immunity.

In previous investigations it was shown that functional blockade of CD80/CD86 costimulation, mediated by transgenic overexpression of soluble CTLA-4Ig in basal keratinocytes, inhibited significantly the development of UV-induced skin tumors (31). Functional CD80/CD86 blockade also impaired the development of UV-induced immunosuppression and a shift of immune responses upon UVR toward a T-helper 2-type as evidenced by the increased interferon-gamma production of T cells from transgenic mice. However, as CTLA-4Ig binding to both CD80 and CD86 could result in either CD28 or CD152 blockade during carcinogenesis, the relative contribution of each receptor could not be differentiated in this model. Moreover, the recent observations that CD28 and CD152 play important roles in regulatory T cell function made interpretation of the previous work complex.

Therefore the role of each coreceptor during tumor development has been determined. It was found that treatment of mice with neutralizing anti-CD152 (anti-CTLA-4) antibodies significantly reduced the development of UV-induced skin cancer suggesting that CD152 signaling is critically involved in incipient cutaneous malignancies (32). In addition, anti-CD152 treatment induced strong antitumoral memory responses as mice without skin tumors after chronic UVR and anti-CD152 injection were able to reject a tumor challenge after more than 1 year. Besides protecting from UV- induced skin tumor development, anti-CD152 antibodies were able to block the suppressor function of UV-induced regulatory CD4^sup +^CD25^sup +^ T cells. These findings suggest that the beneficial therapeutic effects of anti-CD152 treatment during photocarcinogenesis might be mediated by counteracting the function of UV-induced regulatory T cells.

In another photocarcinogenesis study it was shown that CD86- deficient mice developed more rapidly skin tumors compared with CD80- deficient or wild-type mice (32). As CD80 and CD86 expression is important for efficient antigen presentation by APC to induce antigen-specific priming of naive T cells, the T cell allostimulatory capacity of CD80-, CD86-deficient and wild-type APC was analyzed. In contrast to wild-type or CD80-deficient APC, which induced strong T cell proliferation, CD86-deficient APC showed impaired allostimulatory capacity (32). These results suggest that the reduced APC function of CD86-deficient dendritic cells might contribute to the reduced latency period during UV-induced skin tumor development. Similar to mice with a functional blockade in CD80/CD86-mediated costimulation induced by soluble CTLA-4Ig, mice deficient in both CD80 and CD86 demonstrated reduced skin tumors upon chronic UVR. As CD80/CD86-mediated costimulation has been shown to be important for the peripheral maintenance of CD4^sup +^CD25^sup +^ regulatory T cells, UV-induced regulatory T cells have been scrutinized in CD80-, CD86- and CD80/CD86-double deficient animals. Whereas CD80- and CD86-deficient mice showed normal peripheral numbers of CD4^sup +^CD25^sup +^ regulatory T cells, their number was markedly reduced in mice deficient for both CD80 and CD86 (32). These data indicate that functional blockade of CD80/CD86-mediated costimulation protects from UV-induced skin tumor development possibly by interfering with the peripheral maintenance of UV- induced CD4^sup +^CD25^sup +^ regulatory T cells. These results furthermore point to new treatment strategies for the management of skin tumor patients by modulating CD80/CD86-CD28/CD152 signaling.

INTERLEUKIN-10: A KEY MEDIATOR OF UV-INDUCED SKIN TUMOR DEVELOPMENT

Several lines of evidence suggest that UV-induced expression of interleukin (IL)-10 contributes to the development of photocarcinogenesis by suppressing protective cellular immune responses. This view was supported by findings showing that invasively growing basal cell carcinomas secreted IL-10 (33). Expression of IL-10 has also been reported in human melanoma cells and IL-10 production of melanomas correlates with a poorer prognosis (34-36). However, injection of IL-10-overexpressing tumor cells into mice did not result in enhanced tumor growth kinetics but rather in tumor rejection (37). Furthermore, transgenic mice overexpressing viral IL-10 under control of a skin-specific keratin-14 promotor resulting in increased IL-10 serum concentrations developed significantly reduced skin tumors upon chronic UV irradiation (38). These unexpected findings were explained by the fact that the activation of NK. cell function may contribute to impaired photocarcinogenesis in viral IL-10 transgenic mice. According to these discrepancies, the exact role of UV-mediated IL-10 expression for the development of UV-induced skin tumors was not understood.

A recent photocarcinogenesis study in IL-10-deficient mice showed a significantly reduced skin tumor development after chronic UVR (39). Wheras all wild-type mice developed UV-induced skin tumors none was documented in the IL-10-deficient animals indicating an important role of IL-10 for the generation and growth of cutaneous malignancies. The tumors from wild-type mice were mostly immunogenic as they were rejected upon inoculation into immunocompetent recipient mice.

Furthermore, IL-10 deficiency induced strong protective major histocompatibility complex (MHC) class I- and II-mediated antitumoral immunity. It was demonstrated that mice treated with CD8^sup +^ T cells from UV-irradiated IL-10^sup -/-^ mice rejected a UV tumor challenge significantly faster compared with controls suggesting strong antitumoral CD8-mediated effector functions (39). Moreover, in draining lymph nodes of IL-10^sup -/-^ mice that had been challenged with UV tumor cells and were UV irradiated increased expression of T cell activation and cytotoxic markers was detected in CD8^sup +^ T cells. In addition, enhanced granzyme A^sup +^ cells could be identified within the tumor tissue of IL-10^sup -/-^, which was not further increased upon UV treatment pointing to increased CTL activity against the injected UV-tumor cells in IL-10^sup -/-^ mice compared with IL-10^sup +/+^ controls. In inoculated UV tumors in IL-10^sup -/-^ mice more CD4^sup +^TIM-3^sup +^ T cells were detectable compared with controls. TIM-3 is a specific molecular marker for Th1 cells (39). This number was not increased by additional UV treatment. These findings are suggestive of a marked Th1-mediated antitumoral immune response during IL-10 deficiency.

UV-induced CD4^sup +^CD25^sup +^ T cells can inhibit antitumoral immunity against incipient UV-induced skin cancer (18,32,39). In mice, lineage development of naturally occurring CD4^sup +^CD25^sup +^ regulatory T cells is controlled by the transcription factor Foxp3 (40,41). Interestingly, slightly higher peripheral numbers of CD4^sup +^Foxp3^sup +^ regulatory T cells were detectable in IL- 10^sup -/-^ mice; however, these cells had an impaired suppressor function in vitro and in vivo. Adoptive transfer of UV-induced CD4^sup +^CD25^sup +^ T cells from IL-10^sup -/-^-into recipient animals with inoculated UV-regressor tumors failed to inhibit antitumoral immunity as evidenced by the increased tumor rejection. In contrast, mice injected with UV-regressor tumors that were treated with UV-induced regulatory T cells from wild-type mice failed to reject the tumor challenge (39). Hence, IL-10 is critically involved in the suppressor function of UV-induced CD4^sup +^CD25^sup +^ T cells. Together, these results indicate that IL-10 plays a central role as a mediator for inhibiting antitumoral immunity during photocarcinogenesis. These data demonstrate the crucial role of Th1 responses and UV-induced regulatory T cell function in the protection against development of UV-induced skin cancer.

UROCANIC ACID: A CHROMOPHORE WITH RELEVANCE FOR SKIN CARCINOGENESIS

For the induction of biological effects, photons have to be absorbed by chromophores. Basically, two major cutaneous chromophores for UVB have been identified in the epidermis, DNA and urocanic acid (UCA). Upon absorption of photons, DNA forms cyclobutane pyrimidine dimers and (6-4)-photo-products (21). UV- induced dimer formation in particular leads to the release of immunosuppressive cytokines from keratinocytes, such as IL-10 (42).

Urocanic acid, a histidine derivative synthesized by keratinocytes, accumulates within the epidermis in significant amounts as catabolic enzymes are absent from that site (43). Two isoforms exist, trans- and cis-UCA. trans-VCA, the major cutaneous isomer of UCA, isomerizes to cis-UCA upon exposure to UV. Increased amounts of cis-VCA can be detected for several weeks after UV exposure in the skin as well as more transiently in the blood. cis- UCA has been shown to suppress cellular immune reactions, such as delayed-type hypersensitivity responses to herpes simplex virus (43). In addition, systemic application of cis-UCA prolonged allograft survival in mice (43). Topically applied trans-VCA together with chronic UVR treatment resulted in enhanced photocarcinogenesis in mice, indicating that cis-VCA may be involved in the generation of UV-induced skin cancer probably by causing immunosuppression (44). To this end, previous investigations by our group have shown that cis- but not trans-VCA inhibits the ability of LC, the primary APC in the skin, to present tumor antigen, both for the induction and elicitation of antitumoral immune responses in mice (45). As LC are important in the generation of antitumoral immunity, these findings are suggestive of a role of UCA in the development of UV-induced skin cancer.

To directly investigate the role of endogenously produced cis- UCA after UV exposure for the development of skin cancer, mice were chronically irradiated and treated with neutralizing anti-cis-UCA antibodies. It was found that neutralizing cis-VCA significantly reduced the probability of tumor development during UV irradiation (46). Together, these data indicate an important role of cis-VCA in the development of UV-induced skin cancer and point to a therapeutic alternative in the prevention of photocarcinogenesis by inhibiting the effects of cis-UCA.

CBL-B: A NEW PLAYER FOR IMMUNOSURVEILLANCE AGAINST SKIN CANCER

The recognition and elimination of cancer by T cells has been termed immunosurveillance. However, in many cases growth of tumors can be ascribed to the fact that malignant cells do not provide enough T cell stimulatory surface molecules or escape recognition by cytotoxic T cells by induction of tolerance or active suppression (47). Intensive research has focused on mechanisms to break established immunotolerance and to direct T cell responses against cancer. The general difficulty with such attempts is the development of severe autoimmunity, the requirement of large numbers of T cells and the difficulties with genetic manipulation of effector T cells. A “tolerogenic” factor that has received large attention in this context is the Casitas B cell lymphoma-b (Cbl-b) protein of the family of Cbl E3 ligases (48). Analysis of T cells from Cbl-b^sup -/ -^ mice has demonstrated increased activation in the absence of costimulation and the stimulation of Cbl-b^sup -/-^ T cells by “weak” antigens (49-51). Recently, it was shown that Cbl-b^sup -/-^ mice rejected a tumor challenge, and transfer of CD8^sup +^ T cells from Cbl-b^sup -/-^ mice into recipient animals with established tumors resulted in tumor rejection (52,53). Therefore, Cbl-b appears to be an important factor for the immunosurveillance against cancer. To investigate the role of Cbl-b during photocarcinogenesis, Cbl- b^sup -/-^ mice were chronically irradiated with UVB. Interestingly, Cbl-b^sup -/-^ mice developed significantly fewer skin tumors compared with controls indicating that Cbl-b signaling protects against UV-induced skin cancer (53). This protective effect was MHC class I dependent as small UV-induced skin tumors in Cbl-b mice grew progressively after depletion of CD8^sup +^ T cells. In addition, more activated CD8^sup +^ T cells were detectable in skin tumors in Cbl-b^sup -/-^ mice (53). These data indicate that Cbl-b is an important T cell signaling molecule that regulates antitumoral immunity against incipient UV-induced cancer. Furthermore, blocking Cbl-b appears to be an interesting alternative for the treatment of cutaneous malignancies. CUTANEOUS RANK-RANKL INTERACTION: A NEWLY DISCOVERED SIGNAL THAT CONNECTS UV IRRADIATION WITH THE IMMUNE SYSTEM

UVR results in the induction of regulatory T cells and local as well as systemic immunosuppression (54). Furthermore, UVR can lead to immunotolerance, which can be adoptively transferred by UV- induced regulatory T cells. However, the molecular mechanisms underlying the expansion of regulatory T cells after UV exposure are largely unknown. Recently, we have identified the expression of the tumor necrosis factor (TNF) family member RANKL (CD254) within the epidermis of humans and mice (55). RANKL expression was found to be up-regulated in lesional psoriatic skin and was not expressed in lesional skin of patients with cutaneous forms of lupus erythematosus. To study the role of cutaneous RANKL signaling transgenic (tg) mice were generated, which overexpress RANKL under control of the keratin-14 promoter in basal keratinocytes of the epidermis (55). Interestingly, RANKL tg mice showed two- to three- fold increased numbers of peripheral CD4^sup +^ CD25^sup +^ regulatory T cells and a markedly inhibited contact hypersensitivity reaction. As LC are within the epidermis, the only cell type which express the RANKL co-receptor RANK, it was suggested that RANKL- stimulated LC induced the peripheral expansion of regulatory T cells. Indeed, isolated LC from RANKL tg mice induced strong proliferation when cocultured with isolated regulatory T cells. LC from RANKL tg mice expressed CD205 and CD86, two surface molecules which had been associated with the induction or maintenance of regulatory T cells (55). In addition, RANKL-stimulated LC produced significant amounts of IL-6, IL-10 and TNF-alpha. As UVR is able to induce RANKL expression in murine skin, wild-type mice were grafted with skin from RANKL^sup -/-^ animals. Subsequently, UVR plus immunization to haptens was performed through RANKL^sup -/-^ skin and no significantly impaired immunosuppression was detectable (55). These findings suggest that RANKL within the epidermis is an important mediator of UV-induced immunosuppression. We speculate that the cutaneous RANKL expression is also involved in the development of UV-induced skin tumors. In support of this hypothesis RANKL expression has been associated with tumor growth and metastasis (56). Together, these findings suggest that RANK-RANKL signaling might play a role during skin tumor development and metastasis.

Acknowledgements-Many references could not be cited due to space limitations for which we apologize to their authors. This work was supported by grants from the German Cancer Foundation (Krebshilfe 107891), the German Research Association (DFG SFB293 B8), and a grant from the Interdisciplinary Clinical Research Center. Munster (IZKF; Lo2-017-07).

[dagger] This paper is dedicated to Professor Margaret L. Kripke on the occasion of her retirement from the University of Texas, MD Anderson Cancer Center.

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Stefan Beissert* and Karin Loser

Department of Dermatology, University of Munster, Munster, Germany

Received 1 August 2007, accepted 18 September 2007, DOI: 10.1111/ j.1751-1097.2007.00231.x

* Corresponding author email: beisser@uni-muenster.de (Stefan Beissert)

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