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The cancers of the skin, melanoma and the keratinocyte cancers, basal cell andsquamous cell carcinomas (BCC and SCC), are among the most common cancersin white populations. While ultraviolet radiation (UVR) is their principal cause,links with non-UVR-related factors have also been noted. Ultimately, theinteraction of these elements results in malignancy however, understanding oftheir specific contributions remains incomplete. This thesis reports findings fromsix studies aiming to investigate gaps in current knowledge of the role of UVR andnon-UVR-related risk factors on skin cancer. The papers are groupedaccording to the aspects of skin cancer epidemiology and aetiology they address. The first two papers address the descriptive epidemiology of melanoma inEngland, a country with low ambient solar UVR. They arise from ecologicalstudies using national melanoma registration data and document rising trends inmelanoma incidence by anatomic site (Paper 1), and by region of residence andsocio-economic deprivation (Paper 2). Their findings were consistent with thesuggestion that increases in recreational UVR exposure are driving rises inmelanoma rates. These results emphasise both the need to closely monitor UVRexposure and melanoma trends and the importance of public health campaigns. The second group of three papers considers the assessment of associations ofnutritional factors with keratinocyte cancer. Two studies use data from aprospective cohort to evaluate the relationship between dietary intake (Paper 3)and blood concentrations (Paper 4) of omega-3 and omega-6 polyunsaturatedfatty acids (PUFA) in relation to BCC and SCC risk. Associations with both PUFAtypes were observed. In addition, Paper 5, a three-way correlational assessment,demonstrated that questionnaire and blood circulating levels of omega-3 PUFAwere highly correlated with measures of skin bioavailability. Collectively, thesestudies give evidence for associations of these nutrients with skin cancer and forthe utility of both intake and biomarker measures for assessing the relationships. The final paper explores the relationship between a widely cited non-UVR riskfactor, namely scars and cancers of the skin. It reports a systematic review of allpublished observational studies quantifying this association. While innumerablecase reports were found, quantitative analyses were rare. The review identifieda major gap in the literature where knowledge of scar malignancies is notevidence-based, but rather founded mainly on cumulative anecdotal reporting. Taken together, this body of published work highlights the largely unrecognisedcomplexity of the aetiology of cancers of the skin. Future research must bebroad in scope in order to advance understanding of the interaction betweenUVR and other risk factors and to provide a base for health messages aimed atreducing the burden of these malignancies.

The Hedgehog (Hh) family of lipid-modified signaling proteins directs embryonic development and tissue homeostasis, and dysregulated Hh signaling drives familial and sporadic cancers. Hh ligands bind to and inhibit the tumor suppressor Patched (PTCH) and allow the oncoprotein Smoothened (SMO) to accumulate in cilia, which in turn activates the GLI family of transcription factors. Recent work has demonstrated that oxysterol lipids bind and modulate SMO activity. This thesis explores the central theme that lipids regulate the Hh pathway which in turn regulates lipid metabolism. First, I review the myriad of sterols that activate or inhibit the Hedgehog pathway. I discuss the possibility utility of Smo ligands or inhibitors in sterol metabolism as cancer therapeutics. Next, I define the Hh gene expression program using RNA sequencing of cultured cells treated with ciliary ligands, human BCCs, and Hh-associated medulloblastomas from humans and mice. These results reveal Hh target genes such as the oxysterol synthase Hsd111 and the adipokine Retnla regulate lipid metabolism to drive cell fate decisions in response to Hh pathway activation. Lastly, I demonstrate that Hedgehog signaling can be co-opted to drive resistance to pharmacological blockade of CDK6, an important single-agent molecular therapy in cancer therapeutics. Through CRISPR screens and RNA-sequencing of a mouse model of HH-associated medulloblastoma with genetic deletion of Cdk6, I demonstrate that decreased ribosomal protein expression underlies resistance to CDK6 inhibition in HH-associated medulloblastoma, leading to endoplasmic reticular (ER) stress and activation of the unfolded protein response (UPR). I then demonstrate concurrent genetic deletion or pharmacological inhibition of CDK6 and HSD11ß2, an enzyme producing Smoothened-activating lipids, additively blocked cancer growth in multiple mouse genetic models of HH-associated medulloblastoma. In sum, this research program demonstrates that lipid signaling is an avenue to target Hh-dependent cancers and I demonstrate a proof-of-principle therapeutic to target lipid signaling in Hh signaling.

The contents of this article are the author’s original unpublished findings. Background: Each year, approximately 6 million Americans are treated for skin cancer, resulting in a total annual medical expense of $8.9 billion 1. The two most common types of skin cancer are basal cell carcinoma (BCC) followed by squamous cell carcinoma (SCC)2. The metastatic potential of these cancers is minimal; however, their treatment may involve severe scarring and disfigurement2. In contrast, melanoma is an aggressive skin malignancy that grows rapidly and can metastasize in a quick manner2. Risk factors of these skin cancers include UV exposure, fair skin complexions, and family history2. To prevent skin cancer, emphasis is placed on sun protection; however, this approach is suboptimal within the population. To combat this, alternatives such as niacinamide, a form of vitamin B3, may be used to reduce the risk of skin malignancies. Objectives: To evaluate the mechanisms and effects of oral niacinamide in the risk reduction of skin cancers among individuals with a history of sun exposure. Methods: A review was conducted by gathering randomized controlled trials and systematic reviews utilizing databases such as ScienceDirect, PubMed, Wiley Online Library, and SpringerLink. Research articles included in this paper have been published within the last eleven years and are inclusive of all races. Results: Niacinamide was associated with a significant reduction in the occurrence of BCCs and SCCs. However, concerning melanoma, its preventive effects were solely investigated through in vitro and in vivo studies. The lack of clinical trials makes it challenging to evaluate its effectiveness for melanoma. Conclusion: The research presented found compelling evidence supporting the potential of niacinamide in reducing the incidence of nonmelanoma skin cancers, particularly basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). Such results yield to the significance of larger clinical trials aimed at replicating the results obtained from the ONTRAC trial regarding SCCs and BCCs. Despite promising outcomes against nonmelanoma skin cancers, the evidence regarding niacinamide's efficacy in preventing melanoma remains inconclusive. While in vitro and in vivo studies suggest positive effects on melanoma cells, the lack of robust clinical trials specifically evaluating niacinamide's impact on melanoma prevention necessitates further investigation.

Basal cell carcinoma is the most common form of human malignancy, and as such there are varied methods for treating its various forms. Its more advanced and aggressive forms have required both the use of and advent of therapies which offer differing safety profiles, cost, and efficacy. Two therapies which differ substantially in these respects but have overlap in their recommended use are Mohs micrographic surgery and the pharmaceutical drug vismodegib. Few studies have sought to compare the two methods using these criteria, and as vismodegib has only received FDA approval in the past ten years, it is worthwhile to explore the limitations and advantages of each therapy. In exploring previous clinical trials and retrospective studies, the two therapies are put side by side to contrast their results with their shared intended use. The general findings were that Mohs micrographic surgery remains the gold standard for the treatment of locally advanced basal cell carcinoma, and there are few demonstrable instances in which vismodegib could be deemed a more appropriate therapy. The future of vismodegib appears to be in its use as a neoadjuvant therapy for locally advanced basal cell carcinomas for which a decrease in size by vismodegib would allow for better treatment outcomes.

Basal cell carcinoma (BCC) is the most common cancer in the US, but treatment of advanced tumors becomes complicated by drug resistance. Current targeted therapies for advanced BCC suppress inappropriate activation of the Hedgehog signaling pathway and result in potent tumor regression. Yet, they are only effective in 40% of the tumors, with 20% of the initial responders acquiring resistance to these drugs within one year. Therefore, there is an urgent need for alternative methods to treat advanced BCCs and possibly halt tumor development before they reach later stages. This thesis work investigates different mechanisms of modulating BCC tumor growth and progression to understand the mechanisms in the skin environment that dictate whether these tumors will form or regress. Understanding what dictates tumor progression versus intrinsic tumor control holds the potential to enable the development of novel and combination treatment strategies for BCC patients. We used a transgenic BCC mouse model that grows microtumors that spontaneously regress over time to study the role of immune system interactions in promoting tumor regression in BCC. The study aims to define the immune response against BCC tumors and determine the regulatory role of effector lymphocytes in regression. We use this inducible transgenic model coupled with varying degrees of immune cell perturbations, including genetic knockout of lymphocytes and antibody depletions of cell subtypes, to determine the effect on tumor burden. Single-cell RNA sequencing and immunofluorescence microscopy reveal an upregulated innate immunity cytokine response and a T cell Th1-driven response. The CellChat algorithm for inference of communication probability in single-cell data reveals IFNγ and CCL5 signaling from T cells to BCC cells, neutrophils, macrophages, or other T cells. There is also evidence of immune regulation by lymphocytes in the BCC tumor microenvironment, as lack of αβ T cells and adaptive immunity impairs efficient tumor growth (p

The Hedgehog (Hh) signaling pathway is essential for instructing tissues to properly develop during vertebrate embryogenesis. Perturbations that cause constitutive activation of Hh signaling, primarily through loss-of-function mutations in PTCH1, drive the formation of basal cell carcinoma (BCC). While mutations in the Hh pathway play a necessary role for this disease, BCCs also possess high tumor mutational burdens, suggesting that additional mutations may collaborate with Hh signaling to promote oncogenesis. My thesis therefore examines whether other genetic factors can collaborate with Hh signaling to promote the formation of macroscopic BCC-like tumors in mice. Using BCC mouse models, I found that loss of Ptch1 is not sufficient to promote macroscopic tumor formation. Because NOTCH1, TRP53, and MYCN are frequently mutated in human BCCs, I investigated if these genetic factors can collaborate with Hh signaling to drive tumor progression. Indeed, ablating either Notch1 or Trp53 in our BCC mouse model system caused rare “successful” macroscopic tumors to form. Upon ablating Notch signaling, I found that microscopic BCC-like tumors persist and resist tumor regression. Losing Trp53 likely promotes tumor mutability and helps enable genomic amplification of Gli1 or Gli2. Finally, I found that overexpression of MYCN promotes proliferation and tumor progression. Collectively, these data suggest acquired secondary mutations that upregulate Gli1, Gli2 and/or Mycn levels enable tumors to expand indefinitely. Losing Notch1 and p53 also facilitates tumor progression. I also determined whether immune privilege extends to BCC and addressed if inhibiting CD200 suppresses tumor initiation. CD200 is thought to be a potential immunosuppressive molecule in the hair follicle and is expressed in BCC. I found that suppression of CD200 did not affect nascent tumor size during tumor initiation. Furthermore, suppression of CD200 did not affect immune cell numbers in tumor-containing skin. Finally, gene amplification is a common genetic alteration in cancer, and some amplified genes may cause cells to divide or become resistant to therapy. However, there are limited in situ hybridization approaches to detect and visualize copy number changes. I therefore optimized the new DNAscope® duplex assay to examine GLI1, GLI2, and MYCN copy number in human BCC samples. In summary, my findings expand our understanding of key aspects of BCC biology and define genetic factors that drive BCC progression. Furthermore, the tools and assays I have optimized can be leveraged in futures studies in skin cancer biology.

Background Gorlin syndrome is an autosomal dominant genodermatosis characterized by the early appearance of multiple basal cell carcinomas, odontogenic keratocysts and skeletal abnormalities. It is caused by mutations in the hedgehog signalling pathway, primarily in the tumour suppressor gene PTCH1 (9q22.1-q31). Case summary We present a 14-year-old girl who consulted for asymmetrical ears, in the context of multiple disorders such as mental retardation, snoring, non-specific coagulation abnormalities, retrognathia, pectus excavatum and scaphoid duplication. During the intervention, a retroauricular cystic tumour was found incidentally, reported by Pathology as a dermoid cyst. The syndrome is confirmed by a genetic study with the result of a new pathological variant in PTCH1. Conclusion We describe the coexistence of this entity with a dermoid cyst. Furthermore, it is exceptional in its retroauricular location and the pathological point mutation in the PTCH1 gene, consisting of the pCys56Gly variant.

Randomized studies to assess the efficacy of Mohs micrographic surgery in basal cell and squamous cell carcinomas are limited by methodological and ethical issues and a lack of long follow-up periods. This study presents the "real-life" results of a nationwide 7-years cohort on basal cell carcinoma and squamous cell carcinoma treated with Mohs micrographic surgery. A prospective cohort was conducted in 22 Spanish centres (from July 2013 to February 2020) and a multivariate analysis, including characteristics of patients, tumours, surgeries and follow-up, was performed. A total of 4,402 patients followed up for 12,111 patient-years for basal cell carcinoma, and 371 patients with 915 patient-years of follow-up for squamous cell carcinoma were recruited. Risk factors for recurrence included age, non-primary tumours and more stages or unfinished surgeries for both tumours, and immunosuppression for squamous cell carcinoma. Incidence rates of recurrence were 1.3 per 100 person-years for basal cell carcinoma (95% confidence interval 1.1-1.5) and 4.5 for squamous cell carcinoma (95% confidence interval 3.3-6.1), being constant over time (0-5 years). In conclusion, follow-up strategies should be equally intense for at least the first 5 years, with special attention paid to squamous cell carcinoma (especially in immunosuppressed patients), elderly patients, non-primary tumours, and those procedures requiring more stages, or unfinished surgeries.

Basal cell carcinoma (BCC) is driven by overactivation of the Hedgehog signaling pathway whose current FDA-approved chemical inhibitors can efficiently suppress protein receptor Smoothened (SMOi) but have presented serious resistance problems in clinical settings. In SMOi-resistant BCCs, we documented an important correlation between the increase in mRNA level of the constitutively active isoform of atypical protein kinase C (aPKC-K) and the surge in mutational load of ciliary proteins. We demonstrated, for the first time, that aPKC protein expression was upregulated upon cilia loss and that chemical inhibition of aPKC increased both percent ciliation and cilia shaft length, both in cells and BCC mice. We also found that the Hedgehog pathway activity (a) was significantly diminished when ciliogenesis was disrupted by transient knockdown of important ciliary genes and (b) was adequately rescued when aPKC-K was simultaneously overexpressed. At the same time, cells that overexpressed aPKC-K displayed resistance to Vismodegib, a widely prescribed SMOi. We also recorded higher aPKC protein expression and lower ciliogenesis in human superficial BCC subtype in comparison to nodular BCC subtype, suggesting a connection between the interfollicular epidermal origin of superficial BCCs and the SMOi-resistance tendency, presumably via aPKC overactivation coupled with cilia loss. In a second project, we detailed the functional effects of phosphorylation by protein kinase A (PKA) at individual and combinatorial PKA-specific phosphosites on GLI1. Artificial loss of PKA-phosphorylation promoted growth in murine BCC cells and allografts; whereas overexpression of clinical mutations found in resistant BCCs near the PKA-sites on GLI1 altered GLI1 activity, localization, and stability. We also described, for the first time, a truncated protein product of GLI1 when PKA-phosphorylation was mimicked. These findings challenged the currently accepted notion that GLI1 could only be completely degraded by the βTrCP/proteasome complex. Instead, these results suggest that PKA may negate GLI1 activity in a graded fashion via specific phosphorylation at the three C-terminal PKA-phosphosites. Therefore, we propose two new mechanisms of SMOi-resistance in BCCs: (1) selecting for ciliary mutations that abolish the primary cilia-dependent canonical HH pathway while overactivating aPKC to non-canonically promote downstream GLI transcription factor, and (2) selecting for GLI1 mutations that disrupt the inactivating phosphorylation by PKA to maintain canonical HH signaling.

Background: The association of dietary pattern with the risk of basal cell carcinoma (BCC) is little understood and has scarcely been investigated. Objectives: We assessed the association of several complete dietary patterns [Mediterranean, Dietary Approaches to Stop Hypertension (DASH), and Pro-vegetarian dietary pattern] with the risk of BCC, conducting a nested case-control study (4 controls for each case). Methods: Cases and controls were selected from the SUN (Seguimiento Universidad de Navarra) cohort using risk set sam- pling. Cases were identified among subjects free of skin cancer at baseline but who later reported a physician-made BCC diagnosis during the follow-up period. In the cohort we identified 101 incident cases of BCC. Results: In multivariable-adjusted conditional logistic regression analyses, better adherence to the Mediterranean diet (highest compared with lowest quintile) was associated with a 72% relative reduction in the odds of BCC (OR: 0.28; 95% CI: 0.10, 0.77; Ptrend = 0.014); the DASH diet was associated with a 68% RR reduction (OR: 0.32; 95% CI: 0.14, 0.76; Ptrend = 0.013) for the comparison between extreme quintiles. No association was found between a Pro-vegetarian dietary pattern and BCC. Higher fruit consumption (highest compared with lowest quintile, OR: 0.27; 95% CI: 0.11, 0.64; Ptrend < 0.001) and low-fat dairy products (OR: 0.39; 95% CI: 0.16, 0.92; Ptrend = 0.014) were associated with a lower BCC risk. Conclusions: Our results suggest that Mediterranean and DASH dietary patterns may be associated with a lower risk of BCC, but confirmatory studies are required.

Cancers of the human skin are divided into melanoma and non-melanoma. Being among the most commonly diagnosed cancer cases globally, non-melanoma skin cancers are comprised of basal and squamous cell carcinomas. In dermato-pathology, basal cell carcinomas (BCCs) are a frequently encountered diagnosis of skin cancer, and most cases are treated with surgical excisions. While sporadic BCC tumors appear primarily due to aging and ultra-violet exposure, the development of numerous BCCs from a young age is one of the main clinical signs in Gorlin syndrome patients. The critical driver of BCC tumor formation is the sonic hedgehog (SHH) pathway, a pivotal developmental signaling pathway that regulates organ development, cell proliferation, and tissue repair. The majority of all sporadic and syndromic BCCs exhibit mutations in two key components in this pathway, the tumor suppressor gene patched 1 (PTCH1) or the proto-oncogene smoothened (SMO), which result in aberrant pathway activation and continued transcription of SHH-dependent genes. In the last decade, SHH inhibitors have emerged as a novel treatment for advanced and metastatic BCCs. Systemic treatment with vismodegib, a potent SMO inhibitor, can effectively reduce BCC tumor burden in adult Gorlin syndrome patients. However, it is associated with chemotherapy-related adverse events, and treatment cessation results in cancer recurrence and formation of a subset of drug resistant BCCs. While aberrant SHH signaling is key, mechanisms that underlie epithelial–stromal crosstalk and reprograming of tumor metabolism can potentially converge with this pathway and promote BCC tumor development. In this study, we investigated the effects vismodegib on the morphology, behavior, and energy metabolism of human BCC cells and human dermal fibroblasts, in individual cultures as well as in co-cultures, that enabled the crosstalk between these two cell types. Computer-assisted bright-field microscopy was used to characterize cell morphology and behavior. Nuclear magnetic resonance (NMR) and metabolomics were used to determine the metabolic activity of these cells. We found that continuous crosstalk between the cells and different concentrations of vismodegib led to distinct changes in cell morphology and growth, as well as consumption of glucose, pyruvate, and glutamine and secretion of acetate, lactate, and glutamate by these cells. Deciphering tumor driver mechanisms that converge with SHH pathway and contribute to changes within the tumor microenvironment are important not only for better understanding of BCC pathobiology, but also for the development of new mechanism-based BCC therapies with improved clinical outcomes.

W terapii fotodynamicznej (PDT) wykorzystuje się fotouczulacze, które dopiero po naświetleniu światłem widzialnym o odpowiedniej długości fali, stają się toksyczne dla komórek nowotworowych. Obecnie, leczenie za pomocą PDT napotyka problemy związane między innymi ze zbyt niską selektywnością fotouczulaczy, niską rozpuszczalnością, czy występowaniem skutków ubocznych. Efektywność fotouczulaczy może być poprawiona przez zastosowanie dla nich nośników, np. dendrymerów. Celem pracy było zbadanie, czy wybrane dendrymery utworzą kompleksy z fotouczulaczami oraz czy wywołany przez kompleksy efekt fototoksyczny będzie silniejszy niż dla wolnych fotouczulaczy. W prowadzonych badaniach wykorzystano róż bengalski, błękit metylenowy oraz dwa dendrymery fosforowe posiadające przeciwnie naładowane grupy funkcyjne. Wykorzystując metody spektroskopowe, dokonano charakterystyki oddziaływań pomiędzy dendrymerami i fotouczulaczami. Pozwoliło to stwierdzić, że tworzą się dwa stabilne kompleksy, a za oddziaływanie odpowiedzialne są głównie siły elektrostatyczne. Kompleks róż bengalski-dendrymer kationowy oraz błękit metylenowy-dendrymer anionowy wykazały także zdolność do generowania tlenu singletowego po naświetleniu. W badaniach in vitro model stanowiły mysie linie komórkowe raka podstawnokomórkowego skóry. Otrzymane wyniki wykazały, że obydwa kompleksy wnikały do komórek w większym stopniu niż wolne fotouczulacze, nie były toksyczne w ciemności. Po naświetleniu kompleksy wykazały znacznie wyższą cytotoksyczność w porównaniu do wolnych fotouczulaczy. Lepszy efekt był jednak obserwowany dla kompleksu różu bengalskiego niż dla kompleksu błękitu metylenowego. Zbadano także poziom reaktywnych form tlenu w komórkach po naświetleniu. Zaobserwowano znaczny wzrost w przypadku kompleksów. Działanie kompleksów prowadziło do śmierci komórek głównie na drodze apoptozy. Zastosowanie dendrymerów jako nośników fotouczulaczy zwiększyło efektywność transportu fotouczulaczy do komórek, czego efektem była wyższa fototoksyczność. Dendrymery są zatem obiecującymi nośnikami fotouczulaczy.

Purpose: Basal cell carcinoma of the skin (BCC) is the most common subsequent malignant neoplasm among childhood cancer survivors. Ionizing radiation is an established risk factor in this population. We assessed the impact of genetic sensitivity to radiotherapy as an additional risk factor for BCC. Patients and Methods: Participants in the Childhood Cancer Survivor Study (CCSS), who were treated with radiation therapy and who provided biospecimens, were included in the study. Subsequent BCC cases (n=321) and controls without BCC (n=825) were screened for 128 SNPs in radiation-related DNA repair genes. Results: The most important risk factors for BCC were radiation dose, age at original cancer diagnosis, and time at risk (p

Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5Â’ initiation codon variant (Fok I) and two 3Â’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.

Genetic changes are hallmarks of cancer development involving the activation and/or inactivation of oncogenes and tumour suppressor genes, respectively. In non-melanoma skin cancer (NMSC) development, the initiation of genetic mutations results from exposure to solar ultraviolet radiation. Non-melanoma skin cancers are comprised of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Several related cutaneous lesions also exist, of which solar keratoses (SK) are widely accepted as a precursor dysplasia to SCC development. The study of recurrent genetic changes present within NMSC and SK should help reveal causative mutations in skin cancer development. Such analysis could also elucidate links in the genetic similarity of these dysplasia. The rapid screening of numerical changes in DNA sequence copy number throughout the entire genome has been made possible by the advent of comparative genomic hybridisation (CGH). This technique enables the identification of net gains and loss of genetic material within a tumour DNA sample. Chromosomal regions of recurrent gain or loss identify loci containing putative oncogenes and tumour suppressor genes, respectively with potential roles in NMSC tumourigenesis. Used in conjunction with tissue microdissection and universal degenerate PCR techniques this can enable the elucidation of aberrations in small histologically distinct regions of tumour. Such a technique can utilize archival material such as paraffin embedded tissue, which is the major source of neoplastic material available for cancer research. This study used the CGH technique to investigate aberrations in BCC, SCC and SK samples. The screening of copy number abnormalities (CNAs) in BCC revealed that although these tumours were close to diploid and generally genetically stable, they did contain several recurrent aberrations. The loss of genetic material at 9q was identified in a third of BCC tumours studied. This is characteristic of inactivation of the PTCH tumour suppressor gene, a known attribute in some sporadic BCC development. Validation of this loss was performed via loss of heterozygosity, demonstrating good concordance with the CGH data. In addition the over-representation of the 6p chromosome arm was revealed in 47% of biopsies. This novel CNA is also commonly observed in other cutaneous neoplasias, including Merkel cell carcinoma and malignant melanoma. This suggests a possible common mechanism in development and or promotion in these cutaneous dysplasias, the mechanisms of which have yet to be clearly defined. In contrast to BCC, numerical genetic aberrations in SCC and SK were much more frequent. Several regions of recurrent gain were commonly shared between both dysplasias including gain of 3q, 4p, 5p, 8q, 9q, 14q, 17p, 17q and 20q. Common chromosomal regions of loss included 3p, 8p, 9p, 11p, 13q and 17p. In addition loss of chromosome 18 was significantly observed in SCC in comparison to SK, a possible defining event in SK progression to SCC. The identification of shared genetic aberrations suggests a clonal and genetic relationship between the two lesions. This information further supports the notion for re-classification of SK to an SCC in situ or superficial SCC. Finally, the CNAs detected have been similarly observed in other squamous cell-derived tumours, for example cervical and head and neck SCC. This provides further evidence to common mechanisms involved in the initiation, development and progression of SCC neoplasia. This study has identified a number of recurrent chromosomal regions, some of which are novel in NMSC development. The further delineation of these loci should provide additional evidence of their significance and degree of involvement in NMSC tumourigenesis. The identification of the cancer-causing genes mapped to these loci will further demarcate the genetic mechanisms of BCC and SCC progression. An understanding of the events involved in skin cancer formation and progression should shed additional light on molecular targets for diagnostics, management and therapeutic treatment.