Rozprawy doktorskie na temat „Skin cancer”

Thesis (Honors)--Ohio State University, 2006.
Title from first page of PDF file. Document formatted into pages: contains 45 p.; also includes graphics. Includes bibliographical references (p. 41-45). Available online via Ohio State University's Knowledge Bank.

In the medical field new technologies are incorporated for the sole purpose to enhance the quality of life for patients and even for the normal persons. Infrared technology is one of the technologies that has some applications in both the medical and biological fields. In this work, the thermal infrared (IR) measurement is used to investigate its potential in skin cancer detection. IR enjoys a non-invasive and non-contact advantages as well as favorable cost, apparently. It is also very well developed regarding the technological and methodological aspects. IR radiation, per se, is an electromagnetic radiation that all objects emit when their temperature is above the absolute zero. Human body is not different. The IR range extends, ideally, to cover wavelengths from 800 nanometer to few hundreds micrometer. Cancer, in modern life, has grown tangibly due to many factors apparently such life expectancies increase, personal habits, and ultraviolet radiation (UV) exposures among others. Moreover, the significant enhancement of technologies has helped identifying more types of cancers than before. The purpose of this work is to investigate further another method and application of IR technology not yet matured in detection of skin cancer to enhance detection ability that is accompanied with higher level of safety. An extensive research project was designed to use two laboratory animals injected with cancer cells subcutaneously and two IR radiation sensors able to detect wavelengths in the range 8 – 14 μm which proved to be a favorable range to measure the temperature of the skin. Data collection performed using two lab animals as subjects that formed a double blind investigation process. An analysis of the observations was conducted both in qualitative as well as quantitative approaches. The analysis and discussion revealed the potential of the thermal IR radiation in detecting skin cancer existence. The thesis was supported with significant evidence and achieved its target. Furthermore, it was clear that the functional nature of thermal IR detection constitutes another advantage for this method that can be used in the future to develop an objective and automated method for detection of skin cancer in a straight forward and cost effective manner.

Skin cancer is one of the most commonly diagnosed cancers in the world. Diagnosis of skin cancer is commonly performed by analysing skin lesions on the patient’s body. Today’s medical diagnostics use a established set of labels for different types of skin lesions. Another way of categorising skin lesions could be to let a computer perform the analysis without any prior knowledge of the data, where the data is a data set of skin lesion images. This categorisation could then be compared to the already existing medical labels assigned to each image. This categorisation and comparison could provide insight into underlying structures of skin lesion data. To investigate this, three unsupervised learning algorithms; K-means, agglomerative clustering, and spectral clustering, have been used to produce cluster partitionings on a data set of skin lesion images. We found no clear cluster partitionings and no connection to the already existing medical labels. The highest scoring partitioning was produced by spectral clustering when the number of clusters was set to two. Further investigation into the structure of this partitioning revealed that one cluster contained essentially every image. Although relatively low, the score does indicate that the underlying structure may be best represented by a single cluster.
Hudcancer är en av de mest förekommande typerna av cancer i världen. Det vanligaste sättet att diagnosticera hudcancer är för en dermatolog att analysera hudsår på en patients kropp. Dagens medicinsk diagnostik använder en etablerad mängd beteckningar för olika typer av hudsår. Ett alternativ till denna typ av diagnostisering skulle kunna vara att låta en dator utan förkunskap om datan (bilder på hudsår) sköta analysen. Denna katogorisering skulle sedan kunna jämföras med de existerande medicinska katogorierna som varje bild fått. För att undersöka detta användes tre algoritmer av typen oövervakat lärande för att producera kluster-indelningar på ett dataset innehållandes bilder på hudsår. Dessa algoritmer var K-means, agglomerative clustering, och spectral clustering. Vi fann inga uppenbara kluster-indelningar och ingen koppling mellan de nuvarande medicinska beteckningarna. Den indelning av kluster som fick högst poäng när den evaluaredes internt var den indelning av kluster genererad av spectral clustering. Detta skedde när antalet kluster som algoritmen skulle dela upp datan i var satt till två. En djupare undersökning i strukturen av denna indelning visade att ett av klustrerna i princip innehöll varje bild. Även fast Silhouette-värdet för denna indelning var låg, pekar värdet på att den underliggande strukturen bäst kan representeras av ett enda kluster.

Class of 2009 Abstract
OBJECTIVES: Skin cancer is particularly prevalent in Arizona, with incidence rates ranking number two worldwide. Pharmacists are useful advocates for educating patients about the risks of skin cancer and methods of prevention. This study was conducted to assess pharmacists’ knowledge of skin cancer and their demographics and to evaluate how these factors impact skin cancer prevention patient counseling. METHODS: Participants were recruited using a listserv from pharmacists that were members of the Arizona Pharmacy Alliance or preceptors of the University of Arizona College of Pharmacy. Subjects completed an online questionnaire consisting of knowledge- based questions, questions about patient counseling preferences and subject demographics. RESULTS: The average score by pharmacists on the Skin Cancer and Sun Exposure Knowledge Indicator was 5.8 + 1.9. Pharmacists living in Arizona for longer times were more likely to know the minimum recommended SPF of sunscreen for adults to use when outdoors (p=0.003) and the factors associated with malignant melanoma prognosis/survival (p=0.004), but were less likely to know the definition of ABCD acronym (p=0.027). Having a family or friend diagnosed with any form of skin cancer or precancerous skin condition led to more pharmacists knowing the risk factors for developing melanoma (p=0.046) and knowing how often to apply water resistant sunscreen (p=0.035). CONCLUSIONS: The length of pharmacy practice in Arizona and having a family member or close friend affected by skin cancer significantly impacted a pharmacists’ knowledge of skin cancer.

Over one million Americans are afflicted with skin cancer each year. Even though skin cancer has a 95% cure rate, approximately 10,000 people die in the United States each year of this disease. The current ABCDE(F) detection method is not sensitive enough to detect skin cancer in its early stages and requires a biopsy for any suspicious lesions. A lot of unnecessary biopsies, which are painful and costly to the patient, are taken. Therefore, a noninvasive technique is needed that can accurately detect the presence of skin cancer. In this thesis, an optical approach will be presented that has potential to be a noninvasive skin cancer detection technique. Several morphological and biochemical changes occur as tissue becomes cancerous, and therefore the optical properties of the tissue can be used to detect skin cancer. A Mueller matrix imaging system has been developed by our group that measures the 16 or 36-element Mueller matrix, which completely describes the optical properties of the tissue sample. The system is automated and can collect the Mueller matrix in less than one minute. This system will be used to image Sinclair swine, and data analysis techniques will be employed to determine if the system can distinguish between cancerous and noncancerous tissue. System software improvements will also be made, and a new calibration technique will be presented.

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.

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 5’ initiation codon variant (Fok I) and two 3’ 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.

Mutations in RHBDF2, the gene encoding inactive rhomboid protein iRHOM2, result in the dominantly inherited condition Tylosis with oesophageal cancer (TOC). TOC causes plamoplantar keratoderma, oral precursor lesions and up to a 95 % life-time risk of oesophageal squamous cell carcinoma (SCC). The role of iRHOM2 in the epidermis is not well characterised, although we previously showed dysregulated epidermal growth factor receptor (EGFR) signalling and accelerated migration in TOC keratinocytes, and a role for iRHOM2 was shown in trafficking the metalloproteinase ADAM17. Substrates of ADAM17 include EGFR ligands and adhesion molecules. iRHOM2 localisation and function were investigated in frozen sections and keratinocyte cell lines from control and TOC epidermis. Although iRHOM2 was predicted to be an ER-membrane protein, it showed cell-surface expression in control epidermis, with variable localisation in TOC. Increased processing and activation of ADAM17 was seen in TOC keratinocytes compared with control cells, suggesting that increased ADAM17-mediated processing of EGFR ligands may cause the changes in EGFR signalling. Downstream of iRHOM2-ADAM17, Eph/Ephrin and NOTCH signalling also appeared affected. Additionally, desmosomes in TOC epidermis lacked the electron-dense midline of the mature desmosomes seen in normal skin; this was accompanied by increased processing of desmoglein 2, a substrate of ADAM17. Expression and localisation of iRHOM2 was also investigated in TOC and sporadic SCC. iRHOM2 expression varied between SCC cell lines, and appeared to correlate with ADAM17 and NOTCH1 expression in oesophageal SCC and head and neck SCC cells. In summary, iRHOM2 mutations in TOC appear to be gain-of-function in nature, resulting in increased ADAM17 processing and enhanced EGFR signalling. Questions remaining include the reason why iRHOM2 is found at the plasma membrane. Future study of the iRHOM2-ADAM17 pathway may provide additional insight into the mechanism of epidermal wound healing and the pathogenesis of oesophageal SCC.

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.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

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.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Exposure to ultraviolet radiation and immunosuppression are the principal causes of non-melanoma skin cancer (NMSC). One of the skin’s main functions is to act as a barrier against environmental insults and transepidermal water loss (TEWL) is a marker of skin barrier function. Previous phase 2 studies have shown nicotinamide (NAM) to reduce premalignant actinic keratoses. Topical NAM has been shown to reduce TEWL. The effects of oral NAM were evaluated in two clinical trials. The Oral Nicotinamide To Reduce Actinic Cancer (ONTRAC) study was a multicentre phase 3, double-blind, randomised controlled trial in 386 immune-competent participants with at least 2 NMSCs in the past 5 years. Participants were randomised to receive 500mg of NAM or placebo twice daily for 12 months, with assessment at 3-monthly intervals for 18 months. TEWL measurements were taken 3-monthly for 12 months in 292 participants at a single study site. The primary end point was the number of new histologically-confirmed NMSCs during the 12-month intervention period. A second, phase 2 pilot study was undertaken in 22 immunosuppressed renal transplant recipients who were randomised to receive 500mg of NAM or placebo twice daily for 6 months, with assessments at 2-monthly intervals. The ONTRAC study found a 23% relative rate reduction in new NMSCs in the NAM group compared to the placebo group (p=0.02). The estimated relative reduction in TEWL with NAM at 12 months was 6% on the forehead (p=0.04) and 8% on the limbs (p=0.04). The nicotinamide renal transplant pilot study found new NMSCs to be 35% lower in the NAM group than the placebo group (p=0.4). Oral NAM was well-tolerated and safe. The work described in this thesis provides evidence that NAM is a new chemopreventive agent for NMSC in high-risk immune-competent patients. Phase 3 studies are now warranted in immunosuppressed organ transplant recipients. Nicotinamide is also a potential new systemic agent for improving skin barrier function.

The aims of the experiments presented in this thesis were to investigate some aspects of chemoprevention by a topically applied isoflavone, equol, its mechanisms and, in particular, its effect on solar-simulated ultraviolet radiation (SSUV)-induced immunosuppression and subsequent development of skin cancer in hairless mouse skin (SkthR-l). A dose-dependent efficacy of equol at micromolar (pM) concentrations has been demonstrated to protect against SSUV-induced skin damage and development of skin cancer.

Skin cancer is the most commonly diagnosed cancer in the United States; it is estimated that the number of Americans who have had a skin cancer in the last three decades is higher than the number for all other cancers combined. Fortunately, there are known prevention methods, effective treatments available for early-stage cases, and behavioral practices that can reduce the risk of secondary and recurrent cancer. However, in spite of these, skin cancer incidence continues to increase and mortality still exists, making skin cancer prevention of the utmost importance. Outlined in this dissertation are factors identify as associated with the development, diagnosis, and prognosis of skin cancer that could be targeted during primary, secondary, and tertiary skin cancer control and prevention interventions in Arizona. Utilizing the 2013 Arizona Behavior Risk Factor Surveillance System survey, aim one of this dissertation investigates factors associated with UVR exposure (as measured by sun protection use and sunburn history) that could be targeted during primary prevention efforts in order to reduce the disease burden. The results of this research are that approximately 20% of Arizona adults are protecting their skin with sunscreen or protective clothing every time they go outdoors and 28% of Arizona adults experienced one or more sunburns in the past 12 months. Compared with males, females were more likely to report that they protect their skin. Other factors associated with use of sun protection were higher education, higher income, good general health, and living in a more urban area. A recent history of sunburns was associated with being non-Hispanic white and a history of indoor tanning. Given that melanoma diagnosed in the earlier progression of the disease is associated with improved prognosis and significantly higher survival rates, secondary prevention interventions are essential to skin cancer control efforts. The second aim of this dissertation was to gain a better understanding of patient and community factors associated with late-stage melanoma diagnosis in Arizona. Based on Arizona Cancer Registry and community-level data, among melanoma patients there is evidence for significant associations between late-stage of diagnosis and being male (OR 1.22 [95%CI1.09-1.37]), non-white (OR 3.15 [95%CI 2.01-4.95]), and Hispanic (OR 2.13 [95%CI 1.61-2.81]). Additionally, access to care was found to influence stage of diagnosis. Residence in a rural area, compared to an urban area, was associated with late-stage melanoma diagnosis. Similarly, zip codes with a dermatologist density of less than 6 dermatologists per 100,000 persons, when compared to zip codes with greater than 12 dermatologists per 100,000 persons, were associated with late-stage melanoma diagnosis. A travel distance to the reporting hospital or clinic of over 40 miles, as compared to travel distance of 20 miles or less, was also associated with melanoma cases being diagnosed at a late-stage. Even after the progression of the disease, skin cancer survivors' prognosis and quality of life can be improved by following healthy lifestyle recommendations. The final aim of this study was to examine at what levels skin cancer survivors are meeting the recommended healthy lifestyle behaviors. Skin cancer survivors' behaviors were similar, with the exception of increased sun protection use, to behaviors among survivors of other non-skin forms of cancer. However, skin cancer survivors were more likely to practice healthy lifestyle behaviors than individuals without a reported history of cancer. Although skin cancer survivor did report better behaviors than non-cancer controls, there was still a considerable amount of survivors not practicing the recommended behaviors. Over 25% of skin cancer survivors only protected their skin during the summer or not at all. Additionally, low levels of other healthy lifestyle behaviors were noted among skin cancer survivors: slightly over half of skin cancer survivors met the physical activity recommendations, approximately half reported receiving their annual influenza vaccination, and less than 20% consumed 5 fruit or vegetable servings daily. This research suggests that there are opportunities for improved clinical and public health interventions targeted at increasing sun protection use, preventing sunburns, reducing disparities associated with late-stage melanoma, and improving healthy lifestyle behaviors among skin cancer survivors.

Abstract Skin is the largest organ in our body protecting us from ultraviolet radiation and xenobiotics. UV-radiation is a common cause of squamocellular carcinoma and melanoma of the skin that cause morbidity and mortality world wide. Reactive oxygen species are constantly formed by, for example, cellular respiration and UV-radiation, and they can readily react with virtually any macromolecule within cell structures causing damage to DNA, proteins and lipids. Oxidative stress (OS) is a homeostatic process that is dysregulated in cancer cells to their benefit. Nuclear factor erythroid-2-related factor 2 (Nrf2) is the main regulator of antioxidant response and it has been shown to be upregulated in various cancers enabling their survival and growth. By using immunohistochemistry we studied the change and prognostic significance of OS markers in melanoma from paraffin embedded patient samples. Nrf2 expression is increased in melanoma, associating with deeper invasion and a worse melanoma-specific outcome. In addition, epithelial-to-mesenchymal transition markers Slug, Twist and Zeb1 showed altered expression levels in relation to invasion and metastasis associating also with Nrf2. With the help of target inhibition molecules Vemurafenib and MEK-inhibitor CI-1040, In vitro study showed that BRAF- and NRAS-mutations might activate Nrf2. Furthermore, Nrf2-regulated antioxidant enzyme peroxiredoxin I showed decreased expression in malignant melanomas and metastases compared to benign naevi. Intriguing findings were made from the surrounding structures of melanomas e.g. loss of expression of an oxidative lesion marker 8-hydroxy-2’-deoxyguanosine in adjacent endothelial cells associated with worse melanoma-specific survival. Changes in the expression of adhesion molecules claudins 1-5 and 7 were studied in the progression of cutaneous squamous cell carcinomas and preneoplastic lesions. Change in claudin composition can alter epidermal permeability and cell polarity. Efficiency of oncological treatment modalities is frequently based on oxidative stress damage. Nrf2-inhibition could offer the means to increase the sensitivity of cancerous tissue to oxidative insults and hinder proliferative and survival signalling. Later research should focus on the relation of Nrf2 with other signalling and observations made from the tumour microenvironment
Tiivistelmä Iho on elimistön suurin elin, ja se suojaa meitä auringon ultravioletti (UV)-säteilyltä ja muilta ulkoisilta tekijöiltä. UV-säteily on yhteinen etiologinen tekijä ihon levyepiteelikarsinoomalle ja melanoomalle, jotka aiheuttavat maailmanlaatuisesti paljon sairastavuutta ja kuolleisuutta. Reaktiivisia happiradikaaleja muodostuu esimerkiksi soluhengityksestä ja UV-säteilystä, ja ne voivat reagoida minkä tahansa makromolekyylin kanssa aiheuttaen vaurioita solun perimäainekseen, proteiineihin ja lipidirakenteisiin. Oksidatiivisen stressin (OS) säätely on tärkeä homeostaattinen prosessi, joka vinoutuu syöpäsolujen hyödyksi. Nuclear factor erythroid-2-related factor 2 (Nrf2) on antioksidanttivasteen pääsäätelytekijä, ja sen ilmentyminen on lisääntynyt useissa syövissä lisäten syöpäsolun selviytymistä ja kasvua. Tutkimme potilasaineiston ja immunohistokemian avulla OS:n merkkiaineiden muutoksia melanoomassa ja niiden merkitsevyyttä taudin ennusteelle. Nrf2:n ilmentyminen on lisääntynyt melanoomassa liittyen syvempään invaasioon ja huonompaan tautispesifiseen ennusteeseen. Lisäksi epiteliaali-mesenkymaalitransition merkkiaineiden, Slug, Twist ja Zeb1 ekspression muutoksia havaittiin syvyyskasvun ja metastasoinnin yhteydessä assosioituen myös Nrf2 ilmentymiseen. In vitro- tutkimus osoitti spesifisten inhibiittoreiden avulla, että BRAF- ja NRAS-mutaatiot saattavat aktivoida Nrf2 melanoomassa. Myös Nrf2:n säätelemän entsyymin peroksiredoksiini I:n ilmentyminen on vähentynyt melanoomassa ja metastaaseissa verrattuna hyvänlaatuisiin pigmenttiluomiin. Merkittäviä muutoksia havaittiin myös melanoomaa ympäröivistä rakenteista, esimerkiksi OS:n vauriomarkkerin 8-hydroksi-2’-deoksiguanosiinin vähentynyt ilmentyminen endoteelisoluissa liittyi huonompaan tautispesifiseen ennusteeseen. Lisäksi tutkimme soluväliliitosproteiinien klaudiinien 1–5 sekä 7 ilmentymistä levyepiteelikarsinoomissa ja niiden esiasteissa. Klaudiinien muutokset voivat vaikuttaa ihon permeabiliteettiin ja solujen polarisaatioon. Onkologisten hoitomuotojen teho perustuu usein happiradikaalien aiheuttamiin vaurioihin. Nrf2-inhibitio voisi tarjota keinon lisätä syöpäkudoksen herkkyyttä näille vaurioille sekä estää syöpäsolun selviytymissignalointia. Tulevat tutkimukset tulisivat keskittyä Nrf2 signaloinnin ja muun solusignaloinnin välisiin suhteisiin sekä havaintoihin kasvaimen mikroympäristön muutoksista

The most important environmental risk factor for skin cancer is sunlight exposure. The genetic component is seen in the inherited genodermatoses, such as Xeroderma Pigmentosum (XP), where there is a 1000-fold increased risk of skin cancer. Nucleotide excision repair (NER), the pathway responsible for removal of UV-induced DNA damage, is defective in XP patients. The XPB and XPD helicases are essential components of the NER pathway. Frequent polymorphisms have been reported in NER genes and polymorphisms in ERCC2, ERCC1 and XPF have been investigated for association with various types of cancer, including melanoma, non-melanoma skin cancer and cancers of lung, brain, bladder. My study investigated the possibility of an association between two NER genes (XPB and XPD) and melanoma in a case control study on 28 cases and 33 controls. The study addressed the hypothesis that polymorphisms in NER genes leading to altered proteins might be a cancer risk factor because of altered interactions between repair proteins and the cell cycle control machinery. No variation was found in the XPB gene. Most of the 23 exons from the XPD gene have been screened and sequence variation has been observed in exons 6, 22, 23, 18 and 20. Analysis of the polymorphisms in exon 6, 22 and 23 showed that, in each case, one allele was over-represented in the melanoma group. The exon 6 (156Arg) and exon 22 (711Asp) changes were silent while the exon 23 change altered the protein sequence (Lys751Gln). The association did not extend to the closest flanking markers, suggesting that susceptibility to melanoma might be located within the XPD gene itself. Control human lymphocyte cultures with different exon 23 genotypes were studied to investigate the possibility that the XPD exon 23 alleles might interact differently with p53 to effect the response to UV-induced DNA damage. These was no association between the strength of the p53 response and the Lys751Gln polymorphism. These findings need confirmation from a larger study group and from additional functional assays.

Skin cancer rates continue to rise affecting millions of individuals annually. While cutaneous malignant melanoma comprises a fraction of total skin cancers diagnosed, melanoma is associated with a poor prognosis and higher mortality rate when compared to other forms of skin cancer. The greatest risk factor for skin cancer is the amount of ultraviolet light exposure making skin cancer the most common preventable form of cancer. In conjunction with primary prevention, part of secondary prevention measures involves performing routine skin examinations. According to data from the National Health Interview Survey, only 8% of individuals who had seen a primary care provider in the previous 12 months had a skin examination performed (Johnson et al., 2017). A low rate of skin examination can largely be attributed to current professional guidelines from the United States Preventative Services Task Force (2016) not supporting routine skin screening of all patients. Despite the recommendation, primary care providers are consistently faced with the need to evaluate skin lesions. Other barriers identified include lack of training and practical screening methods. Dermoscopy is a noninvasive technique for identifying skin lesions. Based on the need for improved screening practices and identified barriers, a brief educational session and resource on skin cancer and dermoscopy was presented to primary care providers at an urban family practice clinic in eastern North Dakota. Following the educational session, a three-month implementation period provided time for providers to implement their knowledge and dermoscopy skills in practice. The purpose of the project was to increase knowledge, improve accuracy of identifying skin lesions, and increase provider confidence using dermoscopy. Evaluation using a pre-implementation survey of providers in the clinic found the primary care providers felt comfortable with their baseline knowledge of skin cancer but did not feel confident in their ability to use a dermoscope. Most of the participating providers deemed their level of knowledge regarding dermoscopy to be at a novice level. Results of the post-implementation found providers felt more comfortable using dermoscopy and knowledge in dermoscopy overall improved from novice to advanced beginner or competent.

Thesis (Honors paper)--Florida State University, 2008.
Advisor: Mary A. Gerend, PhD, Florida State University, College of Arts & Sciences, Dept. of Psychology. Includes bibliographical references.

Background: Skin cancer is the most common cancer worldwide and one of the most preventable cancers. Despite prevention efforts, skin cancer incidence continues to rise among adolescents. This is especially a challenge for the state of Arizona, which has a high incidence of skin cancer. The inconsistent evidence-based practice guidelines for skin cancer prevention create challenges for counseling adolescents. The knowledge, attitudes, and practices of primary skin cancer prevention by nurse practitioners who care for adolescents is unknown in Arizona or elsewhere in the U.S. Purpose: This doctor of nursing practice project investigates knowledge, attitudes, and practices of skin cancer primary prevention by Arizona nurse practitioners caring for adolescents on an outpatient basis and determines congruency of their counseling with primary prevention guidelines. Methods: The design is descriptive cross-sectional. An online survey using Qualtrics software was distributed via professional listservs to eligible Arizona nurse practitioners currently in practice. Participants' knowledge of skin cancer, skin cancer prevention, and current practice guidelines and recommendations were assessed using multiple choice items. Participants' attitudes regarding counseling for skin cancer prevention within the adolescent population and current nurse practitioner behaviors, in relation to current practice guidelines, were measured using Likert-type scales. Outcomes: Thirty-nine nurse practitioners responded to the online survey. Participant overall knowledge regarding skin cancer was moderate to low, and less was known about skin cancer in adolescents. Despite participants' overall positive attitudes toward skin cancer prevention, they reported low rates of skin cancer prevention counseling for adolescents in practice. Skin cancer prevention recommendations, identified by participants as used in practice, were not congruent with established clinical guidelines on counseling for primary prevention of skin cancer in adolescents.

Every year approximately 2800 Swedes are diagnosed with malignant melanoma, the form of cancer that is most rapidly increasing in incidence in the Western world. The earlier we can identify and diagnose a malignant melanoma, the better is the prognosis. In Sweden, 155 000 benign naevi, harmless skin tumours or moles, are surgically excised each year, many of them because melanoma cannot be dismissed by non-invasive methods. The excisions result in substantial medical costs and cause unrest and suffering of the individual patient. For untrained physicians, it is often difficult to make an accurate diagnosis of melanoma, thus a tool that could help to strengthen the diagnosis of suspected melanomas would be highly valuable. This thesis describes the development and assessment of a non-invasive method for early skin cancer detection. Using near infrared (NIR) and skin impedance spectroscopy, healthy and diseased skin of various subjects was examined to develop a new instrument for detecting malignant melanoma. Due to the complex nature of skin and the numerous variables involved, the spectroscopic data were analysed multivariately using Principal Component Analysis (PCA) and partial leas square discriminant analysis (PLS-DA). The reproducibility of the measurements was determined by calculating Scatter Values (SVs), and the significance of separations between overlapping groups in score plots was determined by calculating intra-model distances. The studies indicate that combining skin impedance and NIR spectroscopy measurements adds value, therefore a new probe-head for simultaneous NIR and skin impedance measurements was introduced. Using both spectroscopic techniques it was possible to separate healthy skin at one body location from healthy skin at another location due to the differences in skin characteristics at various body locations. In addition, statistically significant differences between overlapping groups of both age and gender in score plots were detected. However, the differences in skin characteristics at different body locations had stronger effects on the measurements than both age and gender. Intake of coffee and alcohol prior to measurement did not significantly influence the outcome data. Measurements on dysplastic naevi were significantly separated in a score plot and the influence of diseased skin was stronger than that of body location. This was confirmed in a study where measurements were performed on 12 malignant melanomas, 19 dysplastic naevi and 19 benign naevi. The malignant melanomas were significantly separated from both dysplastic naevi and benign naevi. Overall, the presented findings show that the instrument we have developed provides fast, reproducible measurements, capable of distinguishing malignant melanoma from dysplastic naevi and benign naevi non-invasively with 83% sensitivity and 95% specificity. Thus, the results are highly promising and the instrument appears to have high potential diagnostic utility.

This thesis investigates the problem of developing new computer vision techniques for early detection of skin cancer. The first part of this work presents a novel methodology to correct color reproduction in dermatological images when different cameras and/or dermoscopes are used. Next, the problem of automatic full body mapping is addressed by proposing a mosaicing method based on an on-the-shelf digital compact camera and a set of markers. This method increases the possibilities of total body photography by taking the low-resolution images of a whole body exploration and automatically combining them into a high-resolution photomosaic. The third contribution of this work consists of the development of a full body scanner for acquiring cutaneous images. On one hand, the scanner reduces the long time-consuming examinations done in dermoscopy explorations, and on the other hand, it increases the resolution of total body photography systems.
En aquesta tesi s'investiga el desenvolupament de noves tècniques de visió per computador per a la detecció del càncer de pell. La primera part del treball presenta una nova metodologia per a la correcció del color en imatges dermatològiques quan s'utilitzen diferents càmeres i/o els dermatoscops. A continuació és proposa una solució al problema del registre automàtic d'imatges de cos complert amb la proposta d’un mètode de mosaicing basat en l'ús de càmeres compactes i un conjunt de markers. Incrementant les possibilitats de la fotografia de cos complert mitjançant la combinació automàtica d’imatges de baixa resuloció per a l'obtenció d'un fotomosaic d’alta resolució. La tercera contribució d'aquest treball consisteix en el desenvolupament d'un escàner de cos complert per a l'adquisició d'imatges cutànies. D'una banda l'escàner redueix el llarg temps necessari per a les exploracions dermatoscòpiques, i de l'altre, incrementa la resolució de la fotografia de cos complet.

Mast cells (MCs) are long-lived immune cells, which were reported to play an important role in initiating innate and adaptive immune responses against various infections. MCs accumulate in high numbers in the stroma and at the invasion front of various human cancers, suggesting a possible contribution by MCs to tumour growth. Experimental studies using crosses of MC-deficient Kit-mutant mouse strains with mouse models of epithelial cancers have provided evidence for important MC tumour-promoting functions. However, the complex alterations of the immune system that characterize Kit-mutant mice in addition to their MC deficiency, limit the interpretation of these findings. Numerous key observations made in Kit mutant mice were not reproduced in novel, Kit-independent mouse models of MC deficiency. Thus, the impact of MCs on tumour biology remains unclear. The aim of this study is to clarify the contribution of MCs to the biology of Human papilloma virus (HPV)-induced skin cancer in a Kit-independent mouse model of MC deficiency. In K14-HPV16 transgenic mice, HPV oncogenes are constitutively expressed in the epidermis resulting in epidermal hyperplasia with 100% penetrance and squamous cell carcinoma in about 50% of the animals. A cross to a Kit-mutant line suggested that MCs are important tumour promoters in this model. We crossed K14-HPV16 mice to M5Cre R-DTA line, in which MCs are constitutively depleted with high efficiency and selectivity. Unexpectedly, the loss of MCs neither affected keratinocyte proliferation indices nor altered keratinocyte apoptosis at any stage of HPV-induced neoplasia. Furthermore, the loss of MCs did not result in any detectable changes in composition and gene expression of the inflammatory hematopoietic cell infiltrate in the tumour stroma. This shows that, contrary to current belief, MCs have no important function in orchestrating the tumour micro milieu. In keeping with this finding, MC deficiency resulted in no detectable difference in the incidence growth or grading of SSC in K14-HPV16 transgenic mice. Collectively, these results show that, despite their high density in HPV-induced neoplasia, MC have no role in cancerogenesis or neoplastic progression in the K14-HPV16 mouse model. Our findings also emphasize the importance of novel Kitindependent mouse models in the investigation of MC in vivo functions.

Nucleotide excision repair (NER) is one of the major repair systems for removal of DNA lesions. The NER pathway has evolved mainly to repair UV-induced DNA damage and is also active against a broad range of endogenously generated oxidative lesions. Defects in NER result in the human inherited disorder xeroderma pigmentosum (XP), which is characterised by UV hypersensitivity and a 1000-fold increased risk of skin cancer. ERCC1 is essential for the NER pathway where it acts in a complex with the XPF protein to make the incision 5' to the DNA lesion. The normal 1.1kb Ercc1 transcript is expressed in all tissues. Our group has discovered a second larger 1.5 kb transcript, which initiates from an alternative promoter, and is the most abundant Ercc1 transcript in mouse skin. The aims of this project were: 1, To investigate the role of ERCC1 and of the 1.5kb skin specific Ercc1 transcript in protecting the skin against UV-induced DNA damage. 2, To study the importance of ERCC1 in melanoma skin cancer and investigate ERCC1as a possible target for therapy against melanoma. Using a panel of Ercc1 wild-type and deficient cells, we established a quantitative western blotting system to study the expression of ERCC1 in a range of mouse tissues and mouse and human cell types. Although the skin-specific Ercc1 transcript was found to be present at much higher levels in the skin of albino compared to pigmented mouse strains, this did not result in an elevated level of ERCC1 protein. We were also unable to demonstrate that UV-irradiation, or other stress-inducing treatments resulted in increased levels of ERCC1 protein in cultured mouse keratinocytes. We investigated the DNA methylation status of the normal Ercc1 promoter and that of two potential upstream promoter regions that were candidates for the source of the 1.5kb skin-specific Ercc1 transcript. We found no evidence that they were the source and, instead, used 5' RACE analysis to locate the skin-specific promoter to a polymorphic region 500bp upstream of the normal initiation site. In albino strains this region contains a SINE element, which we hypothesize could be involved in the production of the skin-specific Ercc1 transcript. We also investigated the protein level of ERCC1 and other DNA repair proteins, including XPF, MSH2, MSH6 and MLH1 in human melanoma cells and ovarian tumour cells. Significantly elevated protein levels of ERCC1 and XPF, as well as the mismatch repair protein MLH1 were found in melanoma cells. This could possibly contribute to the higher resistance to chemotherapy in melanoma, although the melanoma cell lines we tested did not show increased resistance to UV and cisplatin compared to the ovarian cancer cells tested. When Ercc1 proficient mouse melanoma cells were xenografted into nude mice the xenografts grew rapidly. Cisplatin treatment caused an initial shrinkage of the tumours, but re-growth rapidly followed. Cells re-isolated into culture from cisplatin treated xenografts had significantly higher levels of ERCC1 protein than either input cells, or cells re-isolated from untreated xenografts. An isogenic Ercc1 deficient derivative of the Ercc1 proficient mouse melanoma cell line grew as rapidly as the parent line in vitro, but grew much more slowly as xenografts. In addition, the xenografts shrank completely following cisplatin treatment and did not recover. This suggests that ERCC1 could be a drug target for melanoma therapy.

Human cancer stem cells are proposed to play a critical role in tumour initiation and maintenance by their exclusive ability to regenerate the tumour. Thus cancer stem cells share many of the properties of normal stem cell including self-renewal and ability to give rise to progeny which undergo tissue-specific differentiation. Thus we hypothesised that by determining the normal patterns of tissue differentiation within cancer we could identify tumour type specific factors that promote differentiation, for therapeutic development. Therefore the aim of this study is to define patterns of human hair follicle differentiation in human basal cell carcinoma (BCC) in order to elucidate potential drug-able targets that can promote tumour specific differentiation. To test this hypothesis we analysed 20 different hair follicle specific differentiation markers, which define distinct layers within the normal adult hair, in six different human BCC samples using RT-PCR with normal hair follicle tissue as control. For the 12 specific keratin genes expressed in the BCC, we analysed expression by immunofluorescence on 20 different BCC samples, using hair follicle samples as positive controls. Our findings suggest that human BCC demonstrates both inward and upward differentiation patterns similar to the hair follicle, with expression of: outer root sheath (K5,14,16,and k17), companion layer (K75), inner root sheath (K26,27,28,71,72,and k74), and cuticle (K32,35,82,and k85); but not hair shaft (K31) markers. Consistent with these findings we observed the mutually exclusive relationship between expression of the early differentiation marker K19 and cell proliferation in the hair follicle and BCC. Similarly, expression of the outer root sheath keratins coincided with nuclear translocation of both GLI1 and NFIL-6, suggesting that BCC also share normal hair follicle tissue regulatory pathways. To further test the hypothesis that normal tissue factors observed in the hair follicle regulate BCC differentiation we have developed an in vitro BCC assay. Using this tissue culture model we hypothesised that BCC’s are stuck in the telogen part of the hair follicle cycle, resulting from autocrine expression of bone morphogenic proteins 2 and 4. Inhibition of BMP signalling by addition of noggin as well as addition of TGF-β to BCC colonies in tissue culture led to further induction of inner root sheath, cuticle and medulla keratins. In summary we have shown that BCC exhibit hair follicle differentiation, which is similarly regulated, but is stuck in telogen arrest and can be rescued by addition of noggin and TGF- β2.

It is widely known that skin cancer is a significant health concern. Studies show that farmers and ranchers are at increased risk of skin cancer, presumed to be secondary to the increased time they spend outdoors and their increased exposure to the sun. This study examined the current sun protection techniques utilized by a sample of North Dakota individuals, who spend the majority of their occupational time outdoors. After collecting information reflecting demographics and current sun protection measures practiced by the sample, educational material focusing on skin cancer prevention and healthy sun behaviors was distributed to the individuals and the primary researcher gave an educational power-point presentation. A post-survey was then given to the sample, identifying the effectiveness of the education, as well as the intentions of the individuals to change. It was found that 74% of participants had never received previous instruction on sunscreen use. The computed odds ratio showed that the intent of participants to observe sunscreen use after the presentation was 3.47 times than before. An encouraging 88% of the participants reported increased intent to complete a self-skin examination post-intervention. The research showed areas for improvement from numerous aspects, including provider and patient education, encouraging preventative techniques while working outdoors, and encouraging regular self-skin examinations. The findings support the importance and effectiveness of verbal communication of health care providers in the family practice setting to verbally discuss skin cancer and sun protection behaviors with their patients, as well as provide patients with written educational information. By identifying the benefits, barriers, and intent of the participants to change, interventions may be implemented.

Non-melanoma skin cancer (NMSC), including basal and squamous cell carcinoma (BCC and SCC, respectively), is the most common malignancy among populations of European ancestry. It is estimated that over 2 million cases of NMSC occur each year in the United States, with the incidence continues to increase. This disease imposes a growing burden on healthcare system, making it an important public health issue. However, understanding of its etiology and biological mechanisms remains incomplete. In Chapter 1 of this dissertation, we applied a novel approach that integrates skin expression-related single-nucleotide polymorphisms (eSNPs) and pathway analysis to identify potential novel biological pathways that are associated with BCC risk. We evaluated the associations of skin eSNPs with BCC among 2,323 cases and 7,275 controls of European ancestry, and assigned them to the pathways defined by KEGG, GO, and BioCarta databases. Three KEGG pathways (colorectal cancer, regulation of actin cytoskeleton, and basal cell carcinoma) and two GO pathways (cellular component disassembly involved in apoptosis, and nucleus organization) showed significant association with BCC risk. Our results indicate that genes that are undetectable by conventional genome-wide association studies (GWASs) are significantly associated with risk of BCC as groups. In Chapter 2, we tested gene-caffeine consumption interaction on BCC risk in a genome-wide analysis. We determined that SNP rs142310826 shows a genome-wide significant interaction with caffeine consumption (p = 1.78x10-8 for interaction, p = 0.64 for heterogeneity between genders) on BCC risk. We also found several loci that modify the caffeine-BCC association differently in men and women. This study is proof of concept that inclusion of environmental factors can help identify genes that are missed in conventional GWASs. In Chapter 3, we prospectively examined the risk of SCC and BCC in relation to adult height. After controlling for potential confounding factors, the hazard ratios were 1.09 (95% CI: 1.03, 1.16) and 1.10 (95% CI: 1.07, 1.12) for the associations between every 10cm increase in height and risk of SCC and BCC respectively. However, no significant association was observed between height-related SNPs and risk of these diseases.

One way sunlight causes skin cancer is by suppressing the anti-tumor immune response. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4-C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. I have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-alone developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. The results presented in this thesis provide important evidence that the CXCR4-CXCL12 chemokine axis plays multiple roles in the development of UV-induced skin cancer. It makes a significant and unique contribution to our understanding of the effects of antagonising CXCR4 in chronic UV-induced skin damage, mast cell migration, UV- immune suppression and melanin production. These studies provide important pre-clinical proof of principle evidence that antagonising CXCR4 will be of therapeutic benefit to high risk, chronically sun-damaged patients.

The folates are a family of structurally similar, water-soluble, B vitamins, documented to play prominently in human health and disease. The potential impact of folate nutrition has been demonstrated by large-scale epidemiological and nutritional studies indicating that decreased folate intake increases the risk of cancer development. Human skin is particularly prone to the development of carcinomas and it is established that skin cancer risk correlates with exposure to the complete carcinogen ultraviolet radiation (UVR) in the form of sunlight. Recently a link between skin, sunlight, and folate has emerged from studies demonstrating that folate species are degraded by exposure to wavelengths of UVR contained within the solar spectrum. It is hypothesized that the unique physiology, function, and environment of skin combine to make skin tissue prone to folate deficiencies and that folate supplementation is a promising strategy for the prevention of skin cancer. However, many questions regarding folate nutrition within human skin must be answered before strategies to modulate folate nutrition may be rationally designed and safely implemented. This work presents novel means to examine skin-specific folate nutrition, including an analytical method to quantify individual folate species in human keratinocytes adaptable for the analysis of intact skin tissue and innovative cultured keratinocyte models of both acute and chronic folate deficiencies. It is demonstrated that folate deficiencies in skin tissue are possible and even likely as proliferating human keratinocytes are unable to maintain intracellular folate concentrations when nutrient conditions are limited and exposure to UVR results in biologically relevant folate degradation. Folate deficiency in human keratinocytes is observed to have potential pro-carcinogenic consequences including S-phase proliferation arrest, increased inherent DNA strand breaks, increased uracil misincorporation into DNA, and deficiencies in DNA damage repair, which are reversed when folate nutrient levels are optimized. The presented work characterizes the relationship between intracellular folate species and environmental carcinogens known to induce skin cancer and addresses challenges facing supplementation strategies for specifically improving folate nutriture in human skin. In total, this report broadens our understanding of folate nutrition in human skin and demonstrates that optimization of folate nutrition holds promise as a cancer preventive strategy.