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

#### **7.1. DNA markers**

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

**5. Current measures of ultraviolet radiation exposure and skin damage**

The current indicator of skin sun damage relies on the use of minimal erythema dose (MED), which refers to the amount of UVR that produces visible skin redness within 24 hours following exposure [86]. As an indicator of UV damage, MED is insensitive and inadequate because UV-induced molecular damage may occur at sub-MED UV doses [87, 88]. Other markers of UV exposure include clinical findings such as solar lentigines and solar elastosis. While these lesions are completely benign, they do have a positive association with NMSC, mainly due to the fact that they arise secondary to photodamage [89]. Although clinical findings of photodamage provide prognostic value, these lesions are neither sensitive nor specific as markers of skin cancer risk as many individuals with solar elastosis and lentigi-

While the association between UVR and skin cancer is well established, quantitative assessment of skin UV exposure and its effect on skin cancer development remains unknown. In a small case control study of 58 patients with cutaneous SCC, the risk was greatest in patients who had more than 30,000 hours of cumulative lifetime sun exposure [90]. This is in contrast to BCCs where studies suggest that intense, intermittent sun exposure resulting in sunburns may be more important for the development of BCC [91]. Quantifying the amount of sun exposed hours necessary to induce NMSC is technically challenging and is not practical for

**6. Biomarkers as a novel tool in monitoring ultraviolet skin damage to** 

Biomarkers are defined as measurable cellular, biochemical or molecular alternations in biological media such as blood or tissue. Several FDA-approved multi-gene panel tests are now approved for risk prediction and diagnosis of various cancers; however, no similar biomarker tests exist for patient risk stratification of NMSC [92]. At present, there are no skin cancer screening guidelines in the United States. Despite this, physicians are routinely confronted with the decision of who should receive total body skin exams and at what time interval. The United States Preventive Task Force (USPSTF) has repeatedly stated that in the absence of randomized controlled clinical trials, there is insufficient evidence to recommend skin cancer

growth, tumor initiation, and metastasis.

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

nes will never develop skin cancer [89].

**improve skin cancer prevention**

implementation as a risk measure at the population level.

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 have been encountered with this approach.

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 within clinically normal skin [99]. As another example, both copies of NOTCH1 are frequently inactivated in SCCs via point mutations and copy number alterations. While studies have demonstrated that up to 60% of SCCs have mutations in NOTCH1, approximately 20% of clinically normal skin cells also carry this mutation [100]. These instances suggest that DNA mutation alone is insufficient to drive skin carcinogenesis. Although this may signal that a significant number of keratinocytes are predisposed to developing skin cancer, the specific combination of events leading to malignant transformation is not well understood.

One of the more promising DNA-based UV biomarkers involves the use of mitochondrial DNA (mtDNA) as a biomarker of cumulative UVR exposure and oxidative stress [101]. Given that mitochondria lack classical NER pathways, UV signature mutations that form within mtDNA have limited capacity for repair [102]. Thus, the entirety of UV induced damage can be observed and correlated quantitatively with UV exposure. Since mutations in nuclear DNA (nDNA) are capable of repair via NER enzymes, the UV mutational burden in nDNA is less representative of lifetime UV exposure. Furthermore, individual variability in NER enzyme activity makes the assessment of nDNA mutations more complicated as repair rates are not uniform [39]. The use of mtDNA as a marker of UV damage is appealing; however, the most important question: whether there is a relationship between mtDNA mutations and actual disease: remains to be answered. Additional studies are required to establish a functional correlation between mtDNA mutation and skin cancer development.

#### **7.2. RNA markers**

Fundamental to the central dogma of molecular biology, RNA is a necessary intermediary between DNA and protein. The exploration of mRNA-based UV biomarkers is an exciting area of research that has been facilitated by recent advancements in next-generation sequencing technology [96]. While RNA only exists for a finite period of time before being degraded, it is easy to detect and quantify at very low levels [103]. There are currently numerous methods available by which RNA expression can be analyzed in a reliable and reproducible fashion. Compared to DNA biomarkers, RNA biomarkers provide a snapshot of temporal and spatial changes in regulatory pathways, which cannot be observed with singular DNA changes.

The increasing power of high throughput sequencing has allowed for detection of changes in both protein-coding RNA (i.e. mRNA) and non-coding RNA (i.e. small nuclear RNA, micro RNA, small nucleolar RNA, lncRNA) expression with high sensitivity and specificity [104]. Non-protein coding RNAs are well known to play regulatory roles in gene expression via post-transcriptional modification and there is a growing body of evidence that implicates non-coding RNA as key regulators of tumor pathways [105, 106]. Studies have demonstrated that specific UV induced miRNAs are differentially expressed in NMSCs (**Table 2**). While altered miRNA expression has been found within malignant tumors themselves, various cancers also demonstrate specific miRNA aberrations within serum and plasma [107]. Therefore, analysis of circulating miRNA expression levels can be potentially employed for use in a noninvasive biomarker test for NMSC. Plasma profiling for early detection of NMSC has been explored in one recent study, which showed significant changes in expression of miRNA-19a, miRNA-25, miRNA-30a, miRNA-145, miRNA-186 [108]. While these are interesting findings, extensive validation of the clinical utility of these miRNAs as NMSC biomarkers is required.

LncRNA is also being explored for its utility as a biomarker for cancer diagnosis and therapy. Following genome wide association studies, it is now known that 88% of trait associated single nucleotide polymorphisms (SNPs) are located in intergenic regions [109]. Since the majority of lncRNAs are transcribed from these regions, it has been suggested that SNPs of lncRNA may represent differential disease risk. While studies are limited, the potential role of circulating lncRNA for use as a biomedical tool is exciting. Dysregulation of lncRNAs has

**Table 2.** Aberrantly expressed microRNAs in NMSC tumors.

**microRNA Expression NMSC association Reference** miR-203 Downregulated BCC [133] Let-7a Downregulated BCC [124] miR-21 Upregulated BCC, SCC [124–129] miR-29c Downregulated BCC [130] miR-130a Upregulated BCC [126, 130] miR-124 Downregulated SCC 131] miR-203 Downregulated SCC [132] miR-184 Upregulated SCC [132] miR-30a Downregulated SCC [126] miR-387 Downregulated SCC [126] miR-135b Upregulated SCC [126] miR-424 Upregulated SCC [126] miR-766 Upregulated SCC [126] miR-145 Downregulated SCC [126] miR-140-3p Downregulated SCC [126] miR-26a Downregulated SCC [126] miR-31 Upregulated SCC [127, 134] miR-205 Upregulated SCC [135] miR-365 Upregulated SCC [136, 137] miR-1 Downregulated SCC [138, 139] miR-34a Downregulated SCC [140] miR-124/214 Downregulated SCC [131] miR-125b Downregulated SCC [127] miR-193b/265a Downregulated SCC [141] miR-199a Downregulated SCC [142] miR-361-5p Downregulated SCC [143] miR-483-3p Downregulated SCC [144]

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187


**Table 2.** Aberrantly expressed microRNAs in NMSC tumors.

within clinically normal skin [99]. As another example, both copies of NOTCH1 are frequently inactivated in SCCs via point mutations and copy number alterations. While studies have demonstrated that up to 60% of SCCs have mutations in NOTCH1, approximately 20% of clinically normal skin cells also carry this mutation [100]. These instances suggest that DNA mutation alone is insufficient to drive skin carcinogenesis. Although this may signal that a significant number of keratinocytes are predisposed to developing skin cancer, the specific

One of the more promising DNA-based UV biomarkers involves the use of mitochondrial DNA (mtDNA) as a biomarker of cumulative UVR exposure and oxidative stress [101]. Given that mitochondria lack classical NER pathways, UV signature mutations that form within mtDNA have limited capacity for repair [102]. Thus, the entirety of UV induced damage can be observed and correlated quantitatively with UV exposure. Since mutations in nuclear DNA (nDNA) are capable of repair via NER enzymes, the UV mutational burden in nDNA is less representative of lifetime UV exposure. Furthermore, individual variability in NER enzyme activity makes the assessment of nDNA mutations more complicated as repair rates are not uniform [39]. The use of mtDNA as a marker of UV damage is appealing; however, the most important question: whether there is a relationship between mtDNA mutations and actual disease: remains to be answered. Additional studies are required to establish a functional cor-

Fundamental to the central dogma of molecular biology, RNA is a necessary intermediary between DNA and protein. The exploration of mRNA-based UV biomarkers is an exciting area of research that has been facilitated by recent advancements in next-generation sequencing technology [96]. While RNA only exists for a finite period of time before being degraded, it is easy to detect and quantify at very low levels [103]. There are currently numerous methods available by which RNA expression can be analyzed in a reliable and reproducible fashion. Compared to DNA biomarkers, RNA biomarkers provide a snapshot of temporal and spatial changes in regulatory pathways, which cannot be observed with singular DNA changes.

The increasing power of high throughput sequencing has allowed for detection of changes in both protein-coding RNA (i.e. mRNA) and non-coding RNA (i.e. small nuclear RNA, micro RNA, small nucleolar RNA, lncRNA) expression with high sensitivity and specificity [104]. Non-protein coding RNAs are well known to play regulatory roles in gene expression via post-transcriptional modification and there is a growing body of evidence that implicates non-coding RNA as key regulators of tumor pathways [105, 106]. Studies have demonstrated that specific UV induced miRNAs are differentially expressed in NMSCs (**Table 2**). While altered miRNA expression has been found within malignant tumors themselves, various cancers also demonstrate specific miRNA aberrations within serum and plasma [107]. Therefore, analysis of circulating miRNA expression levels can be potentially employed for use in a noninvasive biomarker test for NMSC. Plasma profiling for early detection of NMSC has been explored in one recent study, which showed significant changes in expression of miRNA-19a, miRNA-25, miRNA-30a, miRNA-145, miRNA-186 [108]. While these are interesting findings, extensive validation of the clinical utility of these miRNAs as NMSC biomarkers is required.

combination of events leading to malignant transformation is not well understood.

relation between mtDNA mutation and skin cancer development.

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

**7.2. RNA markers**

LncRNA is also being explored for its utility as a biomarker for cancer diagnosis and therapy. Following genome wide association studies, it is now known that 88% of trait associated single nucleotide polymorphisms (SNPs) are located in intergenic regions [109]. Since the majority of lncRNAs are transcribed from these regions, it has been suggested that SNPs of lncRNA may represent differential disease risk. While studies are limited, the potential role of circulating lncRNA for use as a biomedical tool is exciting. Dysregulation of lncRNAs has been reported in a number of malignancies including colon, prostate, breast, and liver cancer where they act as tumor suppressors and oncogenes [110]. Similar to miRNAs, lncRNA can easily be detected by PCR in bodily fluids. Studies demonstrating the detection of lncRNA PCA3 in urine and lncRNA HULC in blood have been suggested as novel modes of cancer screening and diagnosis for prostate cancer and hepatocellular carcinoma respectively [111– 113]. With regard to skin cancer, the aberrant expression of lncRNA appears to be functionally important in skin carcinogenesis. In one study, lncRNA lincRNA-p21—a transcriptional target of p53 and HIF-1α—was found to be highly inducible by UVB radiation and crucial for p-53 mediated apoptosis of damaged keratinocytes [114]. The interaction between the vitamin D receptor and lncRNA also appears to play an important role in maintaining cellular homeostasis prevention of skin tumor formation [115]. In another study, differential expression analysis revealed 1516 lncRNAs were upregulated and 2586 lncRNAs were downregulated in cutaneous SCCs when compared to normal controls [116]. A similar study demonstrated analogous findings in BCCs that showed upregulation of 1851 lncRNAs and downregulation of 2165 lncRNAs when compared to normal skin [117]. While the prognostic, diagnostic, and therapeutic application of lncRNAs within skin cancer is not yet developed, their potential role in the molecular pathogenesis of NMSC warrants further analysis.

In addition to non-coding RNA, mRNA has been explored extensively for its use as a biomarker in numerous studies [118–123]. Similar to miRNA, multiple mRNA transcripts have been found to be differentially regulated in response to UV radiation. Despite the large number of differentially expressed genes identified so far, there is no consensus in terms of which genes are the most sensitive and specific markers of NMSC. Due to frequent inter-individual variations in the expression of many genes, it is unlikely that a singular mRNA will be sufficient as a reliable biomarker. There is thus a growing consensus that multi-gene biomarker panels will be required for the development of a robust and reliable screening test.

**7.3. Protein markers**

expression of UV induced mRNA.

human dendritic cells.

**Author UV type & dose (mJ/cm2 )**

> 1000 (UVB)—rat 3 MED (UVB)—human

300 (UVB) + 3700

5 (UVA) + 12.5 (UVB) + 1.2 (UVC)

(UVA)

Rieger and Chu [118]

Dazard et al. [119]

Takao et al. [120]

Dawes et al. [121]

de la Fuente et al. [122]

Yang et al. [123]

Koch-Paiz et al. [145]

Enk et al. [146]

The majority of genes function through their protein products. Therefore, it is generally accepted that gene activity and function are positively correlated with their protein expression level. Characteristics of protein expression such as variation in quantity, timing of synthesis, and interaction with other proteins has been studied as a means to correlate molecular mechanisms with clinical pathology. Although there are benefits to directly measure changes in protein expression, the use of protein biomarkers has several significant challenges. The main limitation with the use of protein markers lies within currently available detection methods. Protein biomarker panels can be expensive because each protein requires a specific antibody for detection. Secondly, it remains technically challenging and laborious to standardize the quality and specificity of antibodies in their research and clinical applications. Synthetically produced protein antibodies often exhibit great variability between manufacturers, increasing the likelihood of inconsistent results [148]. Moreover, there is a significant number of proteins whose activity depends on specific forms of posttranslational modifications, thus making it more complex in interpreting their clinical relevance solely based on their expression levels [149]. For these reasons, no studies have focused on the role of aberrantly expressed proteins in NMSC. Unless improvements in technology are able to overcome the aforementioned issues, the use of protein biomarkers for patient risk stratification of NMSC is unlikely to be practical.

Abbreviations: NHEK, normal human epidermal keratinocytes; NHM, normal human melanocytes; NHDC, normal

**Table 3.** Comparison of study design and methods of previous gene expression profiling studies analyzing differential

**Transcriptional analysis: time post UVR (hours)**

1 (UVC) 4 Lymphoblastoid cell lines U95A-v2 chips

20–80 (UVB) 0, 0.5, 3, 6, 12, 24 NHEK vs. SCC U95A-v2 chips

10 (UVB) 6 NHEK HuGene FL

48 Human vs. rat epidermal cells

25 (UVB) 24 NHM U133+-v2 chips

150–250 (UVB) 2, 24, 72 NHEK U95A-v2 chips

6, 12 Differentiated mammary

6 NHM, MHEK, NHDC Whole genome

epithelium

**Cell type Platform**

http://dx.doi.org/10.5772/intechopen.70879

Molecular Mechanisms and Biomarkers of Skin Photocarcinogenesis

chips (6800)

189

RNA-Seq

chips

7684 probes cDNA array

Analysis of combined transcriptomic data from previous studies has been complicated by large variations in experimental design including cell type, UV exposure, dose, and time points of analysis [118–123, 145, 146] (**Table 3**). Therefore, development of a validated consensus UV biomarker panel has not yet been achieved. However, a recent transcriptomic profiling study has begun to shed light on the viability of UV biomarker panel for clinical application [147]. In this study, rigorous bioinformatics and statistical analyses were performed to identify UV-responsive genes that are conserved among different donors, in response to various UVR doses, and at different time points after UV exposure. Through this comprehensive transcriptomic analysis, 401 conserved UV-responsive genes were identified out of approximately 4000 U-induced differentially expressed genes detected following each specific UVR condition. Through RNA-seq analysis, this study also generated a SCC-specific signature based on differential gene expression analysis of five pairs of human SCC tumor tissue and adjacent normal skin tissue. Moreover, there is a significant similarity between the conserved UV responsive genes and the SCC signatures genes. The conserved UV-responsive gene set has little similarity to gene sets dysregulated in other human cancers, highlighting their specificity for skin cancer gene expression signatures [147]. Additional validation studies will be needed to select which of these conserved UV-responsive genes may be used to develop a consensus UV biomarker panel.


Abbreviations: NHEK, normal human epidermal keratinocytes; NHM, normal human melanocytes; NHDC, normal human dendritic cells.

**Table 3.** Comparison of study design and methods of previous gene expression profiling studies analyzing differential expression of UV induced mRNA.

#### **7.3. Protein markers**

been reported in a number of malignancies including colon, prostate, breast, and liver cancer where they act as tumor suppressors and oncogenes [110]. Similar to miRNAs, lncRNA can easily be detected by PCR in bodily fluids. Studies demonstrating the detection of lncRNA PCA3 in urine and lncRNA HULC in blood have been suggested as novel modes of cancer screening and diagnosis for prostate cancer and hepatocellular carcinoma respectively [111– 113]. With regard to skin cancer, the aberrant expression of lncRNA appears to be functionally important in skin carcinogenesis. In one study, lncRNA lincRNA-p21—a transcriptional target of p53 and HIF-1α—was found to be highly inducible by UVB radiation and crucial for p-53 mediated apoptosis of damaged keratinocytes [114]. The interaction between the vitamin D receptor and lncRNA also appears to play an important role in maintaining cellular homeostasis prevention of skin tumor formation [115]. In another study, differential expression analysis revealed 1516 lncRNAs were upregulated and 2586 lncRNAs were downregulated in cutaneous SCCs when compared to normal controls [116]. A similar study demonstrated analogous findings in BCCs that showed upregulation of 1851 lncRNAs and downregulation of 2165 lncRNAs when compared to normal skin [117]. While the prognostic, diagnostic, and therapeutic application of lncRNAs within skin cancer is not yet developed, their potential

In addition to non-coding RNA, mRNA has been explored extensively for its use as a biomarker in numerous studies [118–123]. Similar to miRNA, multiple mRNA transcripts have been found to be differentially regulated in response to UV radiation. Despite the large number of differentially expressed genes identified so far, there is no consensus in terms of which genes are the most sensitive and specific markers of NMSC. Due to frequent inter-individual variations in the expression of many genes, it is unlikely that a singular mRNA will be sufficient as a reliable biomarker. There is thus a growing consensus that multi-gene biomarker

Analysis of combined transcriptomic data from previous studies has been complicated by large variations in experimental design including cell type, UV exposure, dose, and time points of analysis [118–123, 145, 146] (**Table 3**). Therefore, development of a validated consensus UV biomarker panel has not yet been achieved. However, a recent transcriptomic profiling study has begun to shed light on the viability of UV biomarker panel for clinical application [147]. In this study, rigorous bioinformatics and statistical analyses were performed to identify UV-responsive genes that are conserved among different donors, in response to various UVR doses, and at different time points after UV exposure. Through this comprehensive transcriptomic analysis, 401 conserved UV-responsive genes were identified out of approximately 4000 U-induced differentially expressed genes detected following each specific UVR condition. Through RNA-seq analysis, this study also generated a SCC-specific signature based on differential gene expression analysis of five pairs of human SCC tumor tissue and adjacent normal skin tissue. Moreover, there is a significant similarity between the conserved UV responsive genes and the SCC signatures genes. The conserved UV-responsive gene set has little similarity to gene sets dysregulated in other human cancers, highlighting their specificity for skin cancer gene expression signatures [147]. Additional validation studies will be needed to select which of these conserved UV-responsive genes may be used to develop a

panels will be required for the development of a robust and reliable screening test.

role in the molecular pathogenesis of NMSC warrants further analysis.

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

consensus UV biomarker panel.

The majority of genes function through their protein products. Therefore, it is generally accepted that gene activity and function are positively correlated with their protein expression level. Characteristics of protein expression such as variation in quantity, timing of synthesis, and interaction with other proteins has been studied as a means to correlate molecular mechanisms with clinical pathology. Although there are benefits to directly measure changes in protein expression, the use of protein biomarkers has several significant challenges. The main limitation with the use of protein markers lies within currently available detection methods. Protein biomarker panels can be expensive because each protein requires a specific antibody for detection. Secondly, it remains technically challenging and laborious to standardize the quality and specificity of antibodies in their research and clinical applications. Synthetically produced protein antibodies often exhibit great variability between manufacturers, increasing the likelihood of inconsistent results [148]. Moreover, there is a significant number of proteins whose activity depends on specific forms of posttranslational modifications, thus making it more complex in interpreting their clinical relevance solely based on their expression levels [149]. For these reasons, no studies have focused on the role of aberrantly expressed proteins in NMSC. Unless improvements in technology are able to overcome the aforementioned issues, the use of protein biomarkers for patient risk stratification of NMSC is unlikely to be practical.
