Andrzej A. Dlugosz - US grants

The skin has served as an extremely useful model for studying factors regulating normal epithelial growth and development and the perturbation of these processes that occurs during neoplasia. Although much previous work has centered on squamous cell carcinoma, there has been increased interest in basal cell carcinoma (BCC) following the discovery that deregulated Sonic hedgehog (Shh) signaling is linked to the development of these tumors. Shh pathway activation may be the result of loss-of- function mutations (involving the Shh receptor PTCH1), or gain- of-function mutations (involving SMO, which is normally repressed by PTCH1). While uncontrolled Shh pathway activation is associated with tumor development, we and others have shown that targeted disruption of Shh results in severe impairment of hair follicle morphogenesis. Although it is clear that Shh signaling has important functions in normal skin and BCC, the pivotal nuclear target(s) mediating keratinocyte responses to this pathway have yet to be identified. Much of what is known about this pathway is based on genetic analysis in Drosophila, where the transcription factor Cubitus interruptus (Ci) mediates responses to the Shh homolog Hedgehog. We will explore the notion that one or more of the vertebrate Ci homologs (Gli1, Gli2, Gli3) plays a central role in Shh signaling in keratinocytes. We propose a series of comprehensive studies focusing on the biological, biochemical, and molecular consequences of Gli protein overexpression in keratinocytes. Although there is substantial evidence implicating deregulated Shh signaling in BCC, there is little insight into how activation of this pathway leads to tumor formation. The results of the proposed studies will provide new information to fill in this gap in our knowledge. In addition to BCCs, several other neoplasms have been linked to the Shh pathway, including medulloblastomas and rhabdomyosarcomas. Moreover, precisely-controlled Shh signaling is essential for embryonic patterning in multiple tissues, with deregulation of this pathway leading to a variety of developmental abnormalities. Thus, the knowledge gained during the course of the proposed studies is likely to have relevance to a variety of clinical disorders, and may ultimately lead to improved treatments for BCC and other tumors.

The identification and characterization of signaling molecules regulating hair follicle morphogenesis remains a major challenge in cutaneous biology. The structure plays a critical role in normal skin function and disease: it is a source of stem cells required during wound-healing and other responses to cutaneous damage; a site of origin for several benign as well as malignant skin tumors; and it produces the hair shaft and sebaceous glands whose dysfunction forms the basis of a variety of dermatological disorders. Similar to other organs, the hair follicle develops through a series of inductive signals traveling between adjacent epithelial and mesenchymal cell primordia which ultimately give rise to the adult structure. While it is clear that an on-going dialogue between epithelium and mesenchyme is required for hair follicle morphogenesis, the nature of the messages being transmitted has remained obscure. Expression of Sonic hedgehog (Shh) mRNA, which encodes a secreted morphogen involved in multiple patterning events during development, is detected focally in epidermis that will give rise to hair follicle epithelium. The spatial and temporal expression of Shh are thus consistent with its playing a role in hair follicle morphogenesis, a concept that is supported by our analysis of Shh-/- mice, which fail to form normal hair follicles. Based on these finding and recent evidence demonstrating a pivotal role for Hedgehog proteins in the development of diverse vertebrate organ systems, we hypothesize that Shh functions as an epithelium-derived inductive signal regulating hair follicle morphogenesis, a concept that is supported by our analysis of Shh -/- mice, which fail to form normal hair follicles. Based on these findings and recent evidence demonstrating a pivotal role for Hedgehog proteins in the development of diverse vertebrate organ systems, we hypothesize that Shh functions as an epithelium-derived inductive signal regulating hair follicle development during embryogenesis and post-natal hair cycling. We will test this hypothesis using pharmacologic, immunologic, and genetic approaches to modulate the Shh pathway, both in vivo and in vitro, with the following specific aims: 1) to determine when Shh signaling is required for hair follicle development and cycling; 2) to identify target cell(s) responding to the Shh signal in hair follicles; 3) to identify and characterize the genetic targets of Shh in follicles; and 4) to define and exploit downstream signaling elements mediating the response to Shh in skin. The results of our studies will provide new insight into mechanisms underlying hair follicle morphogenesis. Given the fact that diverse organs use common signaling molecules to drive their development, our findings are likely to be relevant to understanding organogenesis in other systems. Given the fact that diverse organs using common signaling molecules to drive back their development, our findings are likely to be relevant to understanding organogenesis in other systems. In addition, since several components of the Shh pathway have been linked to a variety of human developmental disorders as well as neoplasia, particularly basal cell carcinoma and medulloblastoma, the proposed project will contribute new knowledge to further our understanding of pathological processes involving skin as well as other organs.

DESCRIPTION (provided by applicant): Like other organs, developing hair follicles arise via a precisely-orchestrated series of secreted signals traveling between epithelial hair follicle progenitors and an adjacent mesenchymal condensate, which together ultimately generate a mature, hair-producing follicle. After birth, the follicle repeatedly cycles through periods of growth, regression, and rest, making it a unique model for studying organogenesis in postnatal life. Work from our lab and others has established that secreted Sonic hedgehog (Shh), which is produced by follicle epithelium and signals to both epithelial and mesenchymal follicle progenitors, is an indispensable regulator of embryonic hair follicle growth, but not differentiation. We have shown that the primary target for Shh in developing hair follicles is the epithelium, and that the proliferative response to Shh is mediated by the transcription factor GH2. We have also found that the Hedgehog (Hh) pathway is involved in the formation of oil-producing sebaceous glands, and that focal activation of Hh signaling is sufficient to drive epithelial bud development, a process previously shown to be controlled by Wnt signals. We hypothesize that epithelial Hh signaling can initiate epithelial bud formation in the absence of a mesenchymal signal, and that the Hh pathway normally drives cell proliferation during periods of active follicle growth after birth. In addition, we propose that Hh signaling has a crucial function in sebocyte development. To test these hypotheses, we have developed conditional mouse models that enable precise spatial and temporal control of Hh signaling activity. These models will be used to: 1) ascertain whether Hh signaling stimulates growth of follicle epithelium by acting directly on stem cells, their progeny, or both; 2) determine the precise function of Hh signaling in sebaceous gland development and maintenance; and 3) explore the mechanism by which ectopic, focal activation of Hh signaling leads to formation of epithelial buds. Given the widespread importance of Hh signaling in embryogenesis and cancer development, our findings in the hair follicle are likely to facilitate understanding of physiologic and pathologic Hh signaling in a variety of other organs.

[unreadable] DESCRIPTION (provided by applicant): Secreted Hedgehog (Hh) ligands play important roles in growth and patterning of various tissues during development, and recent studies have uncovered transient reactivation of Hh signaling during injury and repair of several adult tissues. There is compelling evidence that sustained Hh signaling, which may be initiated during an aberrant response to injury, is associated with a wide variety of human malignancies. Moreover, uncontrolled Hh pathway activity may be required for tumor maintenance, since blockade of Hh signaling inhibits the growth of several types of 'Hh-activated' tumor cells. It has been proposed that only those cell types which utilize the Hh pathway during embryogenesis or repair are prone to Hh pathway-associated tumorigenesis, but this concept has not been rigorously tested. Gastric cancer is the 2nd most common cause of cancer mortality worldwide, and the great majority of gastric tumors are associated with elevated Hh signaling activity, attributed to abnormally high expression of the Hh ligands Sonic hedgehog (Shh) and/or Indian hedgehog (Ihh). We have generated a robust model of gastric adenocarcinoma by constitutively activating Hh signaling in mouse stomach, pointing to a causal role for deregulated Hh signaling in the pathogenesis of these aggressive tumors in humans. However, there is presently little insight into how aberrations in Hh signaling contribute to gastric tumorigenesis, which progenitor cells give rise to 'Hh-activated' gastric cancers, and whether Hh signaling is required for maintenance of established tumors. Moreover, the precise functions of Hh signaling in normal stomach have not yet been established, and there is a little information regarding changes in Hh signaling activity during gastric injury and repair, which can predispose to cancer development. In Aim 1, we propose to identify the cell types expressing Hh pathway components, and determine the function of Hh signaling, in normal and pathologically altered stomach. In Aim 2, we will explore the function of Hh signaling in gastric tumor biology and maintenance. The proposed studies will provide new insights into the functions of Hh signaling in normal and injured stomach, and the role of deregulated Hh signaling in the pathogenesis of gastric cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Merkel cell carcinoma (MCC) is a rare neuroendocrine skin tumor with a poor prognosis at advanced disease stages due to the unavailability of effective treatments. Most MCCs carry sequences from a novel polyomavirus, Merkel cell polyomavirus (MCPyV), and express two putative oncoproteins: MCPyV small t antigen (stAg) and tumor-specific truncated large T antigen (tLTAg). Like other viral oncoproteins, MCPyV transforming antigens target endogenous proteins that function as tumor suppressors: tLTAg targets RB1 while stAg targets PP2A and Fbw7, a component of the SCF ubiquitin ligase complex. Loss-of-function studies point to an important role for both tLTAg and stAg in MCC. Gain-of-function studies, on the other hand, show that stAg, but neither tLTAg nor full-length LTAg, has transforming potential in cultured cells. This stAg-driven transformation involves 4E-BP1 phosphorylation and resultant de-repression of cap-dependent translation, and is dependent on a novel stAg functional domain that binds Fbw7. The relevance of these in vitro findings to MCC tumor initiation, expansion, and progression, is currently unknown. More specifically, the role of MCPyV T antigens as oncogenic drivers of tumor development in vivo has not been addressed. In this exploratory grant application, we plan to develop and characterize mouse models testing the in vivo response of skin cells to expression of MCPyV st and LTAgs using both conventional and Cre-inducible transgenic mice. The proposed studies are highly significant since they will help fill a critical gap in our knowledge by defining the biological activity of MCPyV TAgs in intact animals, thus providing a much-needed set of tools for studying factors contributing to the development and maintenance of MCC. In addition, these exploratory studies will set the stage for identification of MCPyV TAg cellular targets whose deregulation via non-viral mechanisms may contribute more generally to cancer development. The proposed work is likely to have direct translational relevance to MCC patients as it may lead to the identification of new therapeutic targets and yield much-needed mouse models for functional assays and preclinical trials.

DESCRIPTION (provided by applicant): Taste is a vital sense that depends on taste bud receptor complexes in the gustatory epithelia to direct eating and food choices. Taste bud cells and supporting epithelia turn over, are renewed throughout life, and are susceptible to environmental and pharmacological agents. Taste organs therefore depend on tightly regulated proliferation and differentiation. The Hedgehog (HH) pathway regulates maintenance of adult stem and progenitor cells in many tissues. Our data implicate HH signaling as a principal regulator of maintenance and renewal of taste receptor organs. However, HH activity not only regulates tissue maintenance, but also uncontrolled HH signaling is the cause of basal cell carcinoma (BCC), a common skin tumor. Therefore, HH Pathway Inhibitors (HPIs) that block signaling by affecting the HH pathway effector, Smoothened, have been developed as targeted therapeutics for BCC. HPIs lead to regression of BCCs, but patients often discontinue treatment due to adverse effects including severe taste disturbances. Our preliminary data suggest that the taste alterations are an on-target effect reflecting a strict requirement for HH signaling in taste function. We hypothesize that HH signaling functions to control renewal of taste organs and that pharmacological disruption of this control is responsible for chemosensory disturbances in patients treated with HPIs. We use genetic models (mouse) and pharmacological treatment (mouse and human cancer patients) to study the taste system with altered HH signaling. Our Multi PI approach includes chemosensory and HH signaling biologists, and a clinician/scientist treating BCC patients with HPIs. In Aim 1 we hypothesize that HH signaling regulates taste bud and/or papilla maintenance and function through an essential role in epithelial tissue renewal. In mouse we analyze: Hh pathway gene expression pattern and signaling in taste organs throughout the oral cavity; taste bud receptor cell maintenance, renewal and function, during and after treatment with HPIs that target the signal transduction component Smoothened; and, in genetic models, effects of targeted deletion of Smoothened on taste organs. We study cell and tissue effects, and behavioral and neurophysiological taste function. In Aim 2 we propose that HH signaling acts to control taste organ maintenance and function in BCC patients, explaining why pharmacological inhibition of this pathway causes chemosensory disturbance. In patients receiving HPIs, we test predictions about the extent and time course of chemosensory disruption, before, during and after HPI treatment, with questionnaires and NIH Toolbox tests of taste and smell sensory function; and, we quantify the number and distribution of fungiform papillae to correlate with taste sensation tests. The project addresses mechanisms of HH signaling inhibition in altering taste organ dynamics and function. This knowledge contributes to explaining the poorly understood, taste disturbances in patients treated with HPIs, and could ultimately lead to dietary modifications or other approaches to ameliorate chemosensory disruption and improve quality of life.

DESCRIPTION (provided by applicant): Merkel cell polyomavirus T antigens and neoplastic transformation in vivo Project Summary Merkel cell carcinoma (MCC) is a rare neuroendocrine skin tumor with a poor prognosis at advanced disease stages due to the unavailability of effective treatments. Most MCCs carry sequences from a novel polyomavirus, Merkel cell polyomavirus (MCPyV), and express two putative oncoproteins: MCPyV small T antigen (sTAg) and tumor-specific truncated large T antigen (tLTAg). Like other viral oncoproteins, MCPyV transforming antigens are predicted to interact with multiple cellular proteins including tumor suppressors: tLTAg targets RB1 while sTAg targets the PP2A complex. At least some knock-down studies in cultured cells and xenografts support a requirement for tLTAg and sTAg antigens in MCC tumor cell maintenance. In addition, overexpression studies in cultured fibroblasts point to stAg as the predominant transforming oncogene which, surprisingly, operates in a PP2A-independent manner. In contrast, sTAg-driven fibroblast transformation is strictly dependent on a recently-described domain that binds the Fbxw7 component of the SCF E3 ubiquitin ligase complex and leads to accumulation of LTAg and several cellular oncoproteins that are Fbxw7 substrates. These reports have provided valuable insight into the transforming potential of sTAg in cultured fibroblasts, but studies assessing whether MCPyV TAgs can function as oncogenic drivers in vivo have not yet been reported. In preliminary studies we show that MCPyV sTAg, but neither tLTAg nor full-length LTAg, is a potent transforming oncogene in skin and oral epithelia of transgenic mice, leading to striking hyperplasia, impaired terminal differentiation, a DNA damage response, and apoptosis. We propose a series of experiments to further study the in vivo transformation potential of individual MCPyV TAgs, and to test their functional interaction when co-expressed in inducible mouse models. To investigate the MCC tumor cell of origin, we will compare MCPyV TAg transforming potential when targeted to 1) proliferating versus differentiating cellular compartments of epidermis, and 2) Merkel cell progenitors versus differentiated Merkel cells. We will also isolate defined cell populations from conditional MCPyV TAg transgenic mice and study their response to TAg expression in cell culture, enabling functional studies aimed at defining mechanisms of transformation which can then be verified in vivo. The proposed studies are highly significant since they will help fill a critical gap in our knowledge by defining the biological activity of MCPyV TAgs in intact animals, providing a much-needed set of tools for studying factors contributing to the development and maintenance of MCPyV-associated cancer. In addition, these studies will help identify MCPyV TAg cellular targets whose deregulation via non-viral mechanisms may drive the development of virus-negative MCC, and may contribute more generally to the development of other types of cancer. Finally, the proposed work is likely to have direct translational relevance to MCC patients, as it may lead to the identification of new therapeutic targets and yield much-needed mouse models for functional studies and preclinical trials.

Relatively young mice are used for most biomedical research studies investigating human disease, even if the disease under study is much more common in aging patients. For example, basal cell carcinoma (BCC) is an extremely common skin cancer strongly associated with aging in humans, and yet mouse models of BCC examining the molecular basis and biology of these tumors routinely use young experimental animals. The goal of this proposal is to determine whether BCCs that develop in aged mice are different than BCCs arising in young mice; specifically, whether the tumors arising in aged mice are a more accurate model of human BCC. The age-related increase in human BCC incidence has been attributed to the gradual accumulation of mutations in genes encoding proteins in the Hedgehog pathway, which is deregulated in essentially all BCCs. However, multiple alterations take place both at the organismal level and in skin during the aging process, raising the possibility that aged skin responds differently than young skin to the same oncogenic signal. To produce BCCs in young as well as old mice, we will use a well-characterized, highly tractable, genetically- inducible mouse model that leads to uncontrolled activation of Hedgehog signaling, the oncogenic driver in BCC. During the first phase (UH2) of this project, we will breed mice to generate a sufficiently large cohort of experimental animals for tumor induction studies, which will be performed once these mice have aged to the equivalent of 55-60 years of age in humans. We will perform pilot studies to establish conditions needed to achieve transgene expression levels comparable to those measured in young mice that are usually used for these studies, and will obtain a preliminary assessment of tumor development in aged mice. During the second phase (UH3), we will perform transgene induction studies in aged as well as young mice and perform a detailed characterization of the resultant tumors. Since our mouse model allows for reversible activation of transgene expression, we will also assess tumor responses to shut-down of oncogenic Hedgehog signaling as an indicator of treatment response. The successful completion of the proposed studies will establish whether aging influences BCC tumor development in a genetic mouse model, and help determine whether the use of older animals provides a more faithful model of this common age-related human cancer. Our findings may have profound implications for the experimental design of studies using mouse models of BCC and potentially other skin cancers, and will provide a foundation for future work aimed at shedding light on how the aging process affects skin tumorigenesis.

PROJECT SUMMARY/ABSTRACT (CANCER BIOLOGY) Members of the Cancer Biology (CB) Program are engaged in basic and translational studies focused on cancer-associated alterations in cell and tissue biology that underlie tumor initiation, expansion, and progression. CB program members work on one or more of three interrelated themes (stem cells, tumor microenvironment, and signaling), and their research accomplishments have led to advances impacting on the diagnosis, prevention, and treatment of cancer. The CB Program comprises 64 members from 29 departments and four schools at the University of Michigan. Total annual direct research support is $21.7M, with $7.1M (33%) from the NCI and $6.6M (30%) from other NIH support, with a total of $16.4M (75%) representing peer-reviewed funding. Investigators are involved in intra- and inter-programmatic interactions and actively collaborate with UMCCC researchers within the Basic Science, Translational and Clinical Research, and Cancer Control and Population Sciences Programs. During the project period, CB Program members authored a total of 1038 cancer-relevant publications, of which 22% were intra-programmatic and 42% were inter-programmatic. The shared research interests of program members are reflected in the program?s overall scientific aims: 1) elucidate the role of stem cells and their niches in cancer, 2) determine functions of the tumor microenvironment in cancer, and 3) characterize deregulated signaling changes during tumorigenesis. Through focused effort centered on shared scientific interests and a commitment to mentorship, CB program members have generated a body of knowledge that has yielded a deeper understanding of the cellular and extracellular factors governing tumor behavior. The ultimate goal of the CB Program is to apply new insights towards improving cancer detection, prevention, and treatment through robust intra- and inter-programmatic interactions and collaborations, both at the University of Michigan and other institutions. 1

Project Summary/Abstract Human polyomaviruses have been implicated in disease arising in multiple organs. In skin, polyomaviruses have been linked to both cancer (Merkel cell carcinoma) and a hair follicle disorder (trichodysplasia spinulosa) which is seen almost uniformly in immunosuppressed patients, and is strongly linked to trichodysplasia-associated polyomavirus (TSPyV). The clinical findings in trichodysplasia spinulosa include numerous minute follicular papules, most commonly on the face, with prominent hyperkeratotic spicules in place of hair shafts. The affected follicles are distended and comprised almost exclusively of hair matrix cells, inner root sheath-like cells, and masses of abnormally keratinizing cells, with a poorly formed or inapparent dermal papilla. Essentially nothing is known about how TSPyV infection leads to these profound alterations in hair follicle biology. In this exploratory proposal, we will use mouse models to gain insight into the mechanisms underlying development of trichodysplasia spinulosa. Specifically, we will test the hypothesis that one or more TSPyV transforming antigens (TAgs) drive aberrant proliferation of hair matrix cells, leading to disproportionate expansion of inner root sheath cells at the expense of cell lineages comprising the hair shaft. We propose to generate Cre-inducible mouse models to express TSPyV TAgs in a spatially and temporally restricted manner in adult mice. We will characterize resultant changes in proliferation, cell death, and lineage marker expression; and screen for alterations in candidate signaling pathways. The proposed studies will yield novel insight into the molecular and cellular basis of trichodysplasia spinulosa and may identify new targets for therapeutic intervention.

There is an unmet need for a centralized Core providing expertise and training specifically focused on mouse models relevant to cutaneous biology and disease. Capitalizing on over 18 years of intensive mouse modeling expertise by the Core Director and Associate Director, the Animal Modeling Core (AMC) of the University of Michigan Skin Biology and Diseases Resource-based Center (UM-SBDRC) will serve as a shared resource to facilitate the design, development, and characterization of mouse models aimed at gaining deeper insight into skin biology and disease. The Core will also provide technical expertise and guidance needed to properly perform a variety of in vivo procedures and assays. Conventional and inducible genetically-engineered mouse models (GEMMs) provide powerful tools for functional analyses and preclinical studies, but it would be costly and inefficient for all investigators to independently acquire the knowledge and expertise needed to successfully design and carry out these studies. Thus, the overarching, long-term goal of this Core is to facilitate the development and use of state-of-the-art mouse models and provide relevant consultation, training, and troubleshooting for Center members interested in pursuing skin-related studies in mice. We will provide hands-on assistance with the following services and training. 1) Consultation on the design and development of project-specific GEMMs, including conventional, Cre-inducible, doxycycline-inducible, and conditional mutant mice. 2) Guidance for producing and validating mouse models, including transgene construction, verification, genotyping, and GEMM production; screening; breeding, strain establishment, and validation. 3) In vivo manipulation, including transgene induction protocols, UV irradiation, induction of skin inflammation by exogenous agents, bleomycin-induced fibrosis, and orthotopic xenografts and allografts. 4) GEMM phenotyping, including proper tissue collection and processing; morphologic, biochemical, and molecular characterization; cross-species validation; and establishment of GEMM-derived primary cultures and immortalized cell lines, taking into consideration key experimental variables including body site, gender, and age. Work performed with the assistance of this Core will 1) greatly facilitate the in vivo, functional validation of key inflammatory mediators and interacting pathways identified by Center Members through work done in the Functional Analytics Core; 2) ensure consistent and reproducible phenotype characterization across the range of mouse models used by Center Members; 3) yield novel mouse models of human skin disease that will be of value to the skin research community; 4) promote the development of new mouse modeling technology that will be of use to multiple disciplines; and 5) provide powerful GEMMs and tissues for detailed multi-omics analysis in the Functional Analytics Core.

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that frequently carries an integrated copy of Merkel cell polyomavirus (MCPyV) and expresses viral transforming antigens (TAgs) that likely play a key role in tumorigenesis. MCC tumor cells also express transcripts detected in normal, post- mitotic Merkel cells residing in skin, including a set of mRNAs encoding lineage-specific transcription factors implicated in neuroendocrine cell fate. Work from our lab and others established that MCPyV small T antigen (sTAg), +/- truncated large T antigen (tLTAg), is sufficient to drive transformation in vivo, but MCC-like tumors were not detected in any of these models despite TAg targeting to various potential tumor progenitor. The lack of a viable mouse model of MCC has been a major impediment to progress in this field. Normal Merkel cells arise from epidermal progenitors in specialized cellular compartments called touch domes, but the cell of origin of MCC is unknown. In an effort to override the apparent resistance of multiple cell types to MCPyV TAg-driven MCC development in genetically-engineered mice, we generated mice co- expressing the Merkel cell transcription factor Atoh1, together with MCPyV TAgs, in epidermal keratinocytes. These mice developed small collections of proliferating MCC-like tumor cells, and when coupled with deletion of one copy of p53, yielded gross tumors strikingly similar to human MCCs based on multiple criteria. Given the pivotal role of Atoh1 in MCC tumorigenesis we performed complementary loss-of-function studies, and discovered that Atoh1 knock-down converts human MCC cells from their typical neuroendocrine phenotype and growth in suspension, to cells with adherent growth, loss of neuroendocrine markers, and more aggressive behavior in vivo. We thus hypothesize that Atoh1 and other transcription factors governing Merkel cell fate play a pivotal role in MCC pathogenesis as well as maintenance of the neuroendocrine tumor phenotype. We propose the following aims to test this hypothesis. 1) Generate and characterize MCC-like tumors driven by MCPyV TAg expression targeted to candidate MCC progenitor cells. 2) Determine whether MCPyV TAg expression is required for mouse MCC maintenance. 3) Examine the role of Merkel cell lineage transcription factors in governing neuroendocrine cell fate and biological behavior of human MCC cell lines. These studies will yield new insights into the molecular basis of MCC and validate a much-needed mouse model of MCC.

ABSTRACT Cancer is clearly a disease of aging. This connection between aging and cancer incidence is especially true in the case of skin cancer, which is the most common form of human cancer, accounting for more than all other cancers combined in USA. The major objective of this application is to gain understanding of cellular and molecular mechanisms that link age-related skin changes to the initiation of skin cancer. This application is based on our findings that fibroblasts in aged human skin express elevated levels of a matricellular protein named CCN1, and that elevated CCN1 acts to deleteriously alter the dermal compartment of skin to create a microenvironment that enhances cancer initiation. Based on these observations, we have created genetically modified mice that express elevated levels of CCN1 selectively in dermal fibroblasts (source of elevated CCN1 in aged human skin). These mice exhibit strikingly accelerated dermal aging and display multiple hallmarks of aging that are seen in human skin. Importantly, mice that express elevated levels of CCN1 in the dermis also have a high propensity for skin tumor initiation. These results provide direct support for the overarching hypothesis of this application; that age- related changes in the dermal microenvironment, driven by fibroblast expression of CCN1, create a dermal microenvironment that enhances initiation of keratinocyte cancer. We propose to test this hypothesis with the following specific aims. Aim 1: define the impact of CCN1-induced accelerated dermal aging on keratinocyte cancer initiation. Aim 2: test the hypothesis that activation of the hepatocyte growth factor pathway by the CCN1-induced dermal aging microenvironment drives keratinocyte cancer initiation. Aim 3: using targeted gene deletion, test the requirement for CCN1 expression in dermal fibroblasts for the development of an aging-related dermal microenvironment and initiation of keratinocyte cancer. The aims of this proposal directly address the objectives of the National Cancer Institute/National Aging Institute Funding Opportunity Announcement to understand mechanisms by which age-related alterations in the cellular niche/microenvironment contribute to cancer initiation.