**3. Role of ultraviolet radiation in the development of NMSC**

The link between UVR exposure and NMSC is well established [32]. Sun light exists on the electromagnetic spectrum and is divided into ultraviolet, visible, and infrared light based on wavelength. UVR is further broken down into three subtypes: type A (UVA), type B (UVB), and type C (UVC), which, again, are distinguished from one another by wavelength [32]. Of the UVR that reaches earth, 97.5% is comprised of UVA (315–400 nm) and 2.5% is UVB (280– 315 nm) [33]. UVC is entirely absorbed by the atmosphere; however, UVC (200–290 nm) is emitted by man-made sources such as welding torches and mercury lamps. Thus, by and large, UVA and UVB are the most clinically relevant sources of UVR as it pertains to skin cancer [34].

While the daily dosage of UVB is significantly less when compared to UVA, UVB is considered far more dangerous. Based on the optical properties of skin, UVB is strongly absorbed within the stratum corneum and epidermis, therefore exerting a stronger effect on epidermal keratinocytes [34]. UVB is directly absorbed by proteins and nucleic acids and is responsible for the majority of sunburns. Melanin, found in the basal layer of the epidermis, is an important chromophore within the skin and primarily acts to absorb UVR. Upon stimulation by UVR, melanocytes undergo melanogenesis, which results in the increased production of melanin pigments that cause the skin to visibly tan [35]. Due to this property, melanin's photoprotective nature is believed to protect skin cells from UVB radiation damage. Furthermore, this reasoning has been used as evidence to support the observation that the incidence of skin cancer is much lower in individuals with higher levels of melanin at baseline (i.e. darker skin phenotypes) [36]. However, when effects of UVR exposure outpace the synthesis of melanin or its capacity to absorb harmful rays, skin damage will clinically appear as a sunburn.

The wavelength of UVB radiation primarily corresponds to the absorption spectrum of DNA. Upon skin exposure to UVB radiation, electromagnetic energy is absorbed by biologic molecules and transformed into chemical energy [34]. When UV photons are absorbed by DNA molecules, electrons are excited to a high energy state, which can result in formation of photoproducts [37]. The two major DNA lesions induced by UVB radiation are the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4PPs). These lesions are ultimately caused by the misbonding of two pyrimidines, either thymine or cytosine, within the same DNA strand. Both lesions can lead to genetic mutations including C→T and CC→TT transitions; however, CPDs are considered more carcinogenic because they are more prevalent and less efficiently repaired than 6-4PPs [34]. If left unrepaired, these dimers become mutagenic, which highlights the equally important role of the DNA repair system in skin carcinogenesis. Pyrimidine dimers are normally removed by nucleotide excision repair (NER) enzymes. In patients with xeroderma pigmentosum (XP), a disease characterized by defective NER enzymes, individuals exhibit a decreased ability to repair DNA mutations, especially those caused by UVR. In individuals with XP, this clinically manifests as the development of NMSC and melanoma skin cancers at a young age [38]. In building on the knowledge learned from patients with XP, studies have subsequently sought to uncover novel defects in NER enzymes within the general population. It has been suggested that polymorphisms within NER enzymes increase susceptibility to the development of NMSC [39]. While polymorphic variants of DNA repair genes may contribute to an individual's risk of developing NMSC following UV exposure, it is unclear at this time to the extent by which one is affected.

In contrast to UVB, the exact role of UVA in skin carcinogenesis is far more nebulous. Although UVA is partially absorbed by the epidermis, it also penetrates to the dermis where collagen fibers function to scatter light [34]. Until relatively recently, UVA has long been considered to play a minor role in the development of skin cancer as photons of UVA are not within the absorbable wavelength of DNA [34]. However, molecular studies have since illuminated the potentially significant function of UVA in photocarcinogenesis. Recently, research has shown that UVA causes indirect DNA damage via the generation of reactive oxygen species, and DNA-protein crosslinks, as well as direct DNA damage by formation of CPDs or singlestrand DNA breaks [40, 41]. While the role of UVA radiation in the formation of skin cancer is not yet clarified, epidemiologic studies also seem to support its harmful effects. Indoor tanning beds, which primarily emit UVA radiation, have been linked to the increased incidence of skin cancer among users [42, 43]. It has been reported that just one indoor tanning session can increase a user's risk of developing SCC by 67% and BCC by 29% [44]. There is thus compelling evidence to elevate the role of UVA in the formation of skin cancer by perhaps alternative pathways that lead to carcinogenesis.

#### **4. Molecular mechanisms underlying skin carcinogenesis**

subtypes are recognized as high risk variants; however, traditionally low risk variants may achieve high risk status based on tumor features including size, location, and lesion depth,

The link between UVR exposure and NMSC is well established [32]. Sun light exists on the electromagnetic spectrum and is divided into ultraviolet, visible, and infrared light based on wavelength. UVR is further broken down into three subtypes: type A (UVA), type B (UVB), and type C (UVC), which, again, are distinguished from one another by wavelength [32]. Of the UVR that reaches earth, 97.5% is comprised of UVA (315–400 nm) and 2.5% is UVB (280– 315 nm) [33]. UVC is entirely absorbed by the atmosphere; however, UVC (200–290 nm) is emitted by man-made sources such as welding torches and mercury lamps. Thus, by and large, UVA and UVB are the most clinically relevant sources of UVR as it pertains to skin cancer [34]. While the daily dosage of UVB is significantly less when compared to UVA, UVB is considered far more dangerous. Based on the optical properties of skin, UVB is strongly absorbed within the stratum corneum and epidermis, therefore exerting a stronger effect on epidermal keratinocytes [34]. UVB is directly absorbed by proteins and nucleic acids and is responsible for the majority of sunburns. Melanin, found in the basal layer of the epidermis, is an important chromophore within the skin and primarily acts to absorb UVR. Upon stimulation by UVR, melanocytes undergo melanogenesis, which results in the increased production of melanin pigments that cause the skin to visibly tan [35]. Due to this property, melanin's photoprotective nature is believed to protect skin cells from UVB radiation damage. Furthermore, this reasoning has been used as evidence to support the observation that the incidence of skin cancer is much lower in individuals with higher levels of melanin at baseline (i.e. darker skin phenotypes) [36]. However, when effects of UVR exposure outpace the synthesis of melanin or its capacity to absorb harmful rays, skin damage will clinically appear as a sunburn.

which have been independently correlated with SCC risk [31].

Fanconi anemia

Xeroderma pigmentosum Ferguson-Smith syndrome Rothmund-Thomson syndrome Oculocutaneous albinism Epidermolysis bullosa

178 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

Epidermodysplasia verruciformis

Dyskeratosis congenita Bloom syndrome Werner syndrome

**3. Role of ultraviolet radiation in the development of NMSC**

**Table 1.** Hereditary syndromes predominantly associated with increased risk of cutaneous SCC.

Our understanding of cancer biology and the molecular pathology underlying malignant transformation has grown considerably within the past several decades. In particular, the advent of high-throughput sequencing technologies has enabled the detection of various mutational signatures. Dysregulation of proto-oncogenes and tumor suppressor genes, which are critical for controlling cellular growth, is the mechanistic basis of cancer development [45]. Gain of function mutations convert proto-oncogenes into oncogenes, which lead to unregulated cell growth [46]. Likewise, mutations in tumor suppressor genes, which normally inhibit cell growth, also lead to uncontrolled cellular proliferation due to loss of negative control [47].

p53 is a transcription factor that is responsible for controlling genes involved in cell cycle regulation, apoptosis, and DNA repair [50]. In skin cancer, many mutations in p53 are characterized by the C→T and CC→TT transitions, which are characteristic of the UV mutational signature. It is estimated that mutations in p53 occur in 58% of SCCs and 33% of BCCs [51, 52]. Mutations in p53 are found in many malignancies, which suggest its involvement in aberrant signaling pathways and subsequent DNA damage. However, in skin cancer, each p53 allele often carries different mutations at different locations along the gene. This is opposed to other cancers in which the p53 mutations occur within conserved regions [53]. Although p53 mutations in skin cancer do not consistently occur at the same locus, mutations do not occur randomly. Rather, these UV signature mutations accumulate in 'hot spots,' which, importantly, are different from regions of p53 that are mutated in internal malignancies [54]. This suggests that the proclivity for these mutations to occur within specific loci is perpetuated by a selective advantage. p53 mutations are believed to confer resistance to apoptosis in response to UVR,

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thereby leading to positive selection of p53 mutant cells and clonal expansion [55].

Patched (PTCH) is a transmembrane receptor protein that suppresses the hedgehog (HH) signaling pathway. While SCCs are believed to originate in the interfollicular epidermis, histologic evidence suggests that BCCs preferentially arise within stem cells of the hair follicle [56]. HH signaling through PTCH is critical for maintenance of skin stem cell populations, regulation of hair follicle, and sebaceous gland development. Binding of HH protein to the PTCH receptor inhibits the activation of smoothened (SMO) protein to dampen the expression of the HH pathway. Inactivating mutations in the PTCH gene or gain of function mutations in SMO can lead to constitutive expression of the HH pathway [57]. Aberrations in the HH signaling pathway are now recognized as major contributors in BCC tumorigenesis. Mutations in the PTCH gene were initially detected in patients suffering from basal cell nevus syndrome [58]. It was later discovered that a significant proportion of sporadic BCCs and BCCs arising in patients with XP also carried mutations in PTCH [59, 60]. High levels of mutant PTCH transcripts have been found in BCCs but not in normal skin or other types of tumors, making this a relatively specific genetic mutation observed in BCC [61]. The molecular link between PTCH mutations and BCC formation is regarded as a major scientific discovery; however, the exact mechanism by which over expression of the HH pathway leads to unrestricted proliferation

While mutations in the HH signaling pathway appears to be the most important genetic change leading to BCC oncogenesis, the search for a pathway of similar significance in SCC development is ongoing. Whole exome sequencing has revealed a very high mutational burden in cutaneous SCC with an average of one mutation per 30,000 base pairs [62]. Efforts to identify key driver mutations in SCC have thus been hindered by the high background mutation rate. Furthermore, when compared to BCCs, delineation of the mechanisms underlying SCC formation is somewhat more complex. While BCCs are largely believed to arise 'de novo,' SCCs can arise from clinically apparent precursor lesions, namely actinic keratoses [63].

*4.1.2. Basal cell carcinoma and PTCH mutation*

of skin basal cells is not known.

*4.1.3. Complexity of mutational patterns in squamous cell carcinoma*

Past studies on UV radiation in skin carcinogenesis have identified at least three pathways involved in skin cancer development (**Figure 1**): genetic mutations, epigenetic changes and alterations in gene expression. While these three pathways can act alone to cause cancer, they often interact with each other to trigger cancer development.

#### **4.1. Gene mutations**

UVR is recognized to induce a specific pattern of genetic mutations, namely C→T and CC→TT substitutions. To date, this specific mutation is found to be specifically enriched in skin cancers [48]. Since UVR, in particular UVB, is known to induce these types of mutations, C→T transitions are now widely referred to as 'UV signatures' [49]. Our understanding of these specific mutations as being UV dependent has enabled deduction of UV induced genetic mutations by backward inference and pattern recognition. While there have been many pathways implicated in skin carcinogenesis, there is strong evidence to support the impact of selected genetic mutations as being crucial for malignant transformation and tumorigenesis within clonal populations.

#### *4.1.1. P-53 and skin cancer*

Often described as the 'guardian of the genome,' the tumor suppressor protein p53 is arguably one of the most important regulatory proteins for its role in maintaining cellular integrity.

**Figure 1.** Schematic illustration of major molecular mechanisms underlying UV-induced skin photocarcinogenesis.

p53 is a transcription factor that is responsible for controlling genes involved in cell cycle regulation, apoptosis, and DNA repair [50]. In skin cancer, many mutations in p53 are characterized by the C→T and CC→TT transitions, which are characteristic of the UV mutational signature. It is estimated that mutations in p53 occur in 58% of SCCs and 33% of BCCs [51, 52]. Mutations in p53 are found in many malignancies, which suggest its involvement in aberrant signaling pathways and subsequent DNA damage. However, in skin cancer, each p53 allele often carries different mutations at different locations along the gene. This is opposed to other cancers in which the p53 mutations occur within conserved regions [53]. Although p53 mutations in skin cancer do not consistently occur at the same locus, mutations do not occur randomly. Rather, these UV signature mutations accumulate in 'hot spots,' which, importantly, are different from regions of p53 that are mutated in internal malignancies [54]. This suggests that the proclivity for these mutations to occur within specific loci is perpetuated by a selective advantage. p53 mutations are believed to confer resistance to apoptosis in response to UVR, thereby leading to positive selection of p53 mutant cells and clonal expansion [55].

#### *4.1.2. Basal cell carcinoma and PTCH mutation*

are critical for controlling cellular growth, is the mechanistic basis of cancer development [45]. Gain of function mutations convert proto-oncogenes into oncogenes, which lead to unregulated cell growth [46]. Likewise, mutations in tumor suppressor genes, which normally inhibit cell growth, also lead to uncontrolled cellular proliferation due to loss of negative control [47]. Past studies on UV radiation in skin carcinogenesis have identified at least three pathways involved in skin cancer development (**Figure 1**): genetic mutations, epigenetic changes and alterations in gene expression. While these three pathways can act alone to cause cancer, they

UVR is recognized to induce a specific pattern of genetic mutations, namely C→T and CC→TT substitutions. To date, this specific mutation is found to be specifically enriched in skin cancers [48]. Since UVR, in particular UVB, is known to induce these types of mutations, C→T transitions are now widely referred to as 'UV signatures' [49]. Our understanding of these specific mutations as being UV dependent has enabled deduction of UV induced genetic mutations by backward inference and pattern recognition. While there have been many pathways implicated in skin carcinogenesis, there is strong evidence to support the impact of selected genetic mutations as being crucial for malignant transformation and tumorigenesis within

Often described as the 'guardian of the genome,' the tumor suppressor protein p53 is arguably one of the most important regulatory proteins for its role in maintaining cellular integrity.

**Figure 1.** Schematic illustration of major molecular mechanisms underlying UV-induced skin photocarcinogenesis.

often interact with each other to trigger cancer development.

180 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

**4.1. Gene mutations**

clonal populations.

*4.1.1. P-53 and skin cancer*

Patched (PTCH) is a transmembrane receptor protein that suppresses the hedgehog (HH) signaling pathway. While SCCs are believed to originate in the interfollicular epidermis, histologic evidence suggests that BCCs preferentially arise within stem cells of the hair follicle [56]. HH signaling through PTCH is critical for maintenance of skin stem cell populations, regulation of hair follicle, and sebaceous gland development. Binding of HH protein to the PTCH receptor inhibits the activation of smoothened (SMO) protein to dampen the expression of the HH pathway. Inactivating mutations in the PTCH gene or gain of function mutations in SMO can lead to constitutive expression of the HH pathway [57]. Aberrations in the HH signaling pathway are now recognized as major contributors in BCC tumorigenesis. Mutations in the PTCH gene were initially detected in patients suffering from basal cell nevus syndrome [58]. It was later discovered that a significant proportion of sporadic BCCs and BCCs arising in patients with XP also carried mutations in PTCH [59, 60]. High levels of mutant PTCH transcripts have been found in BCCs but not in normal skin or other types of tumors, making this a relatively specific genetic mutation observed in BCC [61]. The molecular link between PTCH mutations and BCC formation is regarded as a major scientific discovery; however, the exact mechanism by which over expression of the HH pathway leads to unrestricted proliferation of skin basal cells is not known.

#### *4.1.3. Complexity of mutational patterns in squamous cell carcinoma*

While mutations in the HH signaling pathway appears to be the most important genetic change leading to BCC oncogenesis, the search for a pathway of similar significance in SCC development is ongoing. Whole exome sequencing has revealed a very high mutational burden in cutaneous SCC with an average of one mutation per 30,000 base pairs [62]. Efforts to identify key driver mutations in SCC have thus been hindered by the high background mutation rate. Furthermore, when compared to BCCs, delineation of the mechanisms underlying SCC formation is somewhat more complex. While BCCs are largely believed to arise 'de novo,' SCCs can arise from clinically apparent precursor lesions, namely actinic keratoses [63]. Histologically characterized by atypical keratinocytes, AKs give rise to approximately 65% of SCCs [25]. In addition to clinical observation, this notion has been further supported by genomic analysis. While AKs and SCCs exhibit similar karyotypes, AKs demonstrate less genotypic complexity, suggestive of an earlier stage of tumor development [64]. While the genetic relationship between AKs and SCCs has not yet been clearly defined, it appears that AKs and SCCs exist on a continuum in which certain mutations drive progression from premalignant to malignant forms [65]. Thus, mutations in both AKs and SCCs have been explored for their potential roles as drivers of carcinogenesis.

development of skin cancer is multifactorial, epigenetic alterations are believed to be among the earliest detectable changes in UVR exposed skin [70]. Over exposure of the skin to UVR leads to oxidative stress, inflammation, and DNA damage, factors that are known to significantly alter epigenetic pathways. In particular, chronic inflammation has been shown to accelerate the formation of DNA methylation changes [71]. Thus, environment-induced aberrant expression of epigenetic regulators can trigger widespread epigenetic changes that may subsequently disturb a variety of downstream target genes, potentially increasing the risk of

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Despite the increasing recognition that abnormal DNA methylation is a crucial factor in skin carcinogenesis, histone modifications serve as another dynamic epigenetic pathway that is frequently altered in cancer. Histone modifying enzymes can be divided into multiple families [72, 73]. Among these, proteins involved in histone acetylation and deacetylation have been shown to play important roles in both normal development and disease states. Limited data suggest that silencing of tumor suppressor genes in UV-induced skin leads to photocarcinogenesis and aberrant epigenetic modifications including alterations in DNA methylation and histone acetylation at specific loci [74, 75]. UV-induced differential gene expression has also been linked to changes in global H3 lysine 27 acetylation (H3K27ac), an epigenetic marker of active promoter and enhancer regions. Using ChIP-seq analysis, one study revealed that UVR induced genome-wide loss of H3K27ac, as well as regional gains in H3K27ac levels. Upon further analysis, UV-induced differential H3K27ac acetylation was functionally correlated with differential gene expression was observed [76]. The genome wide loss of H3K27ac may be attributable to the suppression of histone acetyltransferases activities, whereas the regional gain of H3K27ac may occur secondary to the binding of UV-responsive transcription factors, such as JUN/FOS or TP53, which subsequently recruit HATs to their target regions [76, 77]. Though the epigenetic mechanisms underlying the effects of UVR in promoting skin cancer warrant more extensive studies, the use of histone biomarkers for clinical diagnosis and/or prognosis is an interesting approach that is also being investigated for use in other

There is a growing body of evidence to suggest that non-coding RNA (ncRNA) is involved in the development of many malignancies, including skin cancer [80]. ncRNAs are RNA transcripts transcribed from DNA that are not translated into protein and classified based on size: small non-coding RNA (<200 nucleotides) and long non-coding RNA (>200 nucleotides). While there are various different subtypes of ncRNA, microRNA (miRNA), a subtype of small non-coding RNA, is perhaps the most widely studied. miRNAs are single stranded ncRNA molecules that modulate gene expression by binding to the 3′ untranslated region of target mRNA. This ultimately causes mRNA instability and eventual degradation, leading to subsequent alterations in cell differentiation, metabolism, apoptosis, and signal transduction [81]. Studies have demonstrated that UV irradiation of human keratinocytes modulates the expression of numerous miRNAs. In addition to a common miRNA response, UVA and UVB were also shown to induce wavelength specific miRNA expression signatures [82]. Although interpretation of changes in UV-responsive miRNA expression is complex, miRNA mediated gene silencing likely has important downstream effects, which contribute

cancer development.

malignancies [78, 79].

to the development of skin cancer.

Increasing evidence suggests that the underlying pathogenesis of cutaneous SCC involves mutations in several genes and pathways. Besides p53, published research has mainly focused on a handful of key mutations frequently found in cutaneous SCC including NOTCH, RAS, EGFR, TGFΒ, NF-KΒ, and most recently, KNSTRN [66]. KNSTRN gene, one of the newest genetic mutations reported in SCC, encodes a kinetochore associated protein that modulates anaphase onset and chromosome segregation during mitosis. Recurrent UV signature point mutations in KNSTRN at codon 24 (p.Ser24Phe) have been observed in 19% of SCCs and 13% of AKs [67]. Functionally, this specific mutation in KNSTRN results in disruption of chromatid cohesion in normal cells, which can lead to aneuploidy and chromosomal aberrations. Since KNSTRN mutations occur rarely in other malignancies, this may represent a previously unrecognized oncogene in skin tumorigenesis; however, studies to clarify its clinical applicability are needed.

#### **4.2. Epigenetic alterations**

Apart from the mutagenic effects of UVR on the genome, the role of epigenetic changes induced by UVR in skin cancer remains underappreciated. Epigenetics refers to heritable changes in gene expression that are not due to alterations within the DNA sequence itself. This includes changes in DNA methylation, histone modification, and miRNAs. The human genome encodes a subset of genes that function in epigenetic modifications of the genome and thus regulate the activities of other genes. While the list of such epigenetic regulators continues to grow, DNA methyltransferases (DNMTs) and histone modifying enzymes are among the best studied epigenetic regulators [68]. DNA methylation is a process catalyzed DNMTs by which a methyl group is added to the 5′ carbon of a cytosine ring of DNA. These modified cytosine residues are often adjacent to guanine bases, resulting in the formation of a CpG dinucleotide. These CpGs are often concentrated in short CpG-rich DNA segments, known as CpG islands, the majority of which are found in the promoter region of genes [68]. Methylation events in promoter regions can have profound effects via the secondary effects on transcription.

Both DNA hypomethylation and hypermethylation have been implicated in tumorigenesis of skin cancers. In particular, DNA hypermethylation in CpG islands is an important mechanism by which tumor suppressor genes are silenced. This in turn leads to downstream effects on gene expression that may eventually lead to a neoplastic phenotype [69]. Like other human malignancies, the development of skin cancer involves a complex interplay between environmental factors and alterations in gene expression within skin cells. While the development of skin cancer is multifactorial, epigenetic alterations are believed to be among the earliest detectable changes in UVR exposed skin [70]. Over exposure of the skin to UVR leads to oxidative stress, inflammation, and DNA damage, factors that are known to significantly alter epigenetic pathways. In particular, chronic inflammation has been shown to accelerate the formation of DNA methylation changes [71]. Thus, environment-induced aberrant expression of epigenetic regulators can trigger widespread epigenetic changes that may subsequently disturb a variety of downstream target genes, potentially increasing the risk of cancer development.

Histologically characterized by atypical keratinocytes, AKs give rise to approximately 65% of SCCs [25]. In addition to clinical observation, this notion has been further supported by genomic analysis. While AKs and SCCs exhibit similar karyotypes, AKs demonstrate less genotypic complexity, suggestive of an earlier stage of tumor development [64]. While the genetic relationship between AKs and SCCs has not yet been clearly defined, it appears that AKs and SCCs exist on a continuum in which certain mutations drive progression from premalignant to malignant forms [65]. Thus, mutations in both AKs and SCCs have been

Increasing evidence suggests that the underlying pathogenesis of cutaneous SCC involves mutations in several genes and pathways. Besides p53, published research has mainly focused on a handful of key mutations frequently found in cutaneous SCC including NOTCH, RAS, EGFR, TGFΒ, NF-KΒ, and most recently, KNSTRN [66]. KNSTRN gene, one of the newest genetic mutations reported in SCC, encodes a kinetochore associated protein that modulates anaphase onset and chromosome segregation during mitosis. Recurrent UV signature point mutations in KNSTRN at codon 24 (p.Ser24Phe) have been observed in 19% of SCCs and 13% of AKs [67]. Functionally, this specific mutation in KNSTRN results in disruption of chromatid cohesion in normal cells, which can lead to aneuploidy and chromosomal aberrations. Since KNSTRN mutations occur rarely in other malignancies, this may represent a previously unrecognized oncogene in skin tumorigenesis; however, studies to clarify its clinical applica-

Apart from the mutagenic effects of UVR on the genome, the role of epigenetic changes induced by UVR in skin cancer remains underappreciated. Epigenetics refers to heritable changes in gene expression that are not due to alterations within the DNA sequence itself. This includes changes in DNA methylation, histone modification, and miRNAs. The human genome encodes a subset of genes that function in epigenetic modifications of the genome and thus regulate the activities of other genes. While the list of such epigenetic regulators continues to grow, DNA methyltransferases (DNMTs) and histone modifying enzymes are among the best studied epigenetic regulators [68]. DNA methylation is a process catalyzed DNMTs by which a methyl group is added to the 5′ carbon of a cytosine ring of DNA. These modified cytosine residues are often adjacent to guanine bases, resulting in the formation of a CpG dinucleotide. These CpGs are often concentrated in short CpG-rich DNA segments, known as CpG islands, the majority of which are found in the promoter region of genes [68]. Methylation events in promoter regions can have profound effects via the secondary

Both DNA hypomethylation and hypermethylation have been implicated in tumorigenesis of skin cancers. In particular, DNA hypermethylation in CpG islands is an important mechanism by which tumor suppressor genes are silenced. This in turn leads to downstream effects on gene expression that may eventually lead to a neoplastic phenotype [69]. Like other human malignancies, the development of skin cancer involves a complex interplay between environmental factors and alterations in gene expression within skin cells. While the

explored for their potential roles as drivers of carcinogenesis.

182 Human Skin Cancers - Pathways, Mechanisms, Targets and Treatments

bility are needed.

**4.2. Epigenetic alterations**

effects on transcription.

Despite the increasing recognition that abnormal DNA methylation is a crucial factor in skin carcinogenesis, histone modifications serve as another dynamic epigenetic pathway that is frequently altered in cancer. Histone modifying enzymes can be divided into multiple families [72, 73]. Among these, proteins involved in histone acetylation and deacetylation have been shown to play important roles in both normal development and disease states. Limited data suggest that silencing of tumor suppressor genes in UV-induced skin leads to photocarcinogenesis and aberrant epigenetic modifications including alterations in DNA methylation and histone acetylation at specific loci [74, 75]. UV-induced differential gene expression has also been linked to changes in global H3 lysine 27 acetylation (H3K27ac), an epigenetic marker of active promoter and enhancer regions. Using ChIP-seq analysis, one study revealed that UVR induced genome-wide loss of H3K27ac, as well as regional gains in H3K27ac levels. Upon further analysis, UV-induced differential H3K27ac acetylation was functionally correlated with differential gene expression was observed [76]. The genome wide loss of H3K27ac may be attributable to the suppression of histone acetyltransferases activities, whereas the regional gain of H3K27ac may occur secondary to the binding of UV-responsive transcription factors, such as JUN/FOS or TP53, which subsequently recruit HATs to their target regions [76, 77]. Though the epigenetic mechanisms underlying the effects of UVR in promoting skin cancer warrant more extensive studies, the use of histone biomarkers for clinical diagnosis and/or prognosis is an interesting approach that is also being investigated for use in other malignancies [78, 79].

There is a growing body of evidence to suggest that non-coding RNA (ncRNA) is involved in the development of many malignancies, including skin cancer [80]. ncRNAs are RNA transcripts transcribed from DNA that are not translated into protein and classified based on size: small non-coding RNA (<200 nucleotides) and long non-coding RNA (>200 nucleotides). While there are various different subtypes of ncRNA, microRNA (miRNA), a subtype of small non-coding RNA, is perhaps the most widely studied. miRNAs are single stranded ncRNA molecules that modulate gene expression by binding to the 3′ untranslated region of target mRNA. This ultimately causes mRNA instability and eventual degradation, leading to subsequent alterations in cell differentiation, metabolism, apoptosis, and signal transduction [81]. Studies have demonstrated that UV irradiation of human keratinocytes modulates the expression of numerous miRNAs. In addition to a common miRNA response, UVA and UVB were also shown to induce wavelength specific miRNA expression signatures [82]. Although interpretation of changes in UV-responsive miRNA expression is complex, miRNA mediated gene silencing likely has important downstream effects, which contribute to the development of skin cancer.

While less studied than short non-coding RNA, long non-coding RNA (lncRNA) has recently become an area of significant interest within cancer research. It is now known that lncRNA can effect gene expression through interactions with transcription factors and can interact with miRNAs to regulate mRNA stability [83, 84]. Though thousands of lncRNA transcripts have been identified, the majority remain uncharacterized with unknown functions [85]. Nonetheless, a growing number of lncRNAs are being investigated for their role in cancer growth, tumor initiation, and metastasis.

screening in the general population [93]. While the majority of the USPSTF skin cancer screening report focuses on melanoma, one of the main arguments made against screening for NMSC is that there is limited evidence regarding the diagnostic accuracy of primary care physicians to correctly identify BCCs and SCCs. Given the lack of rigorous dermatology training in most medical school and primary care residencies, this argument is not necessarily unfounded [94]. Although there are several other arguments to be made against the USPSTF recommenda-

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It is unlikely the USPSTF will obtain a satisfactory level of evidence in the near future to recommend skin cancer screening in the United States given the large number of patients required to adequately power a clinical trial [95]. It thus behooves physicians and scientists to search for alternative measures as a means to quantify skin cancer risk. The presence of an objective UV biomarker test could facilitate patient triage by identifying high risk individuals for dermatology referral. Identification of susceptible patients in the primary care setting via the use of an accessible genetic screening test would thus provide physicians with an evidence based method to make informed decisions regarding which patients should receive

The development of a UV biomarker panel has the potential to have a profound impact on patient care. Breakthroughs in next generation sequencing technology have provided a powerful tool for identifying biomarkers of a given physiological status or exposure [96]. Many studies have attempted to identify biomarkers that correlate UV exposure and skin damage with variable success; however, no consensus UV biomarkers have been established to date. Various types of biomarkers have been investigated including DNA, RNA, and protein.

Given the potent mutagenic effect of UVR and its role in skin carcinogenesis, skin cancer research in the past several decades has primarily focused on mapping cancer-related mutations. Such efforts have been greatly enhanced by recent advances in whole-genome and whole-exome sequencing studies, which have identified recurrent genomic aberrations that underlie the development of BCC and SCC [97]. As such, these particular gene mutations have been suggested for use as markers of skin cancer risk. Unfortunately, numerous issues

Based on our current understanding of cancer development, tumor growth is initiated by the presence of driver mutations, which lead to clonal expansion of mutant cells. This increases the total number of cells that are at risk to develop further mutations and malignant transformation [98]. Given the high prevalence of p53 mutations observed in NMSC, this had previously been suggested as a potential genetic marker of patient risk. Upon further investigation, it was found that mutant clones with p53 UV signature mutations were found in high numbers

**7. Candidate biomarkers for assessing UV damage: strengths and** 

tions and reasoning, the lack of clear skin cancer screening guidelines is problematic.

regular skin cancer screening.

**weaknesses**

**7.1. DNA markers**

have been encountered with this approach.
