Molecular Genetic Mechanisms in Cancers of Keratinocytic Origin

Abstract

Keratinocytic cancers (KC) comprise a group of diseases that have a broad spectrum clinically and pathologically. At one end of the spectrum are benign proliferations (acanthomas), and at the other end are malignant tumors with aggressive growth and metastatic potential. Traditionally, about 80% of KC cases have basal cell carcinoma (BCC) and 20% have cutaneous squamous cell carcinoma (cSCC). Both tumors have different phenotypic features due to different oncogenic pathways. cSCC is biologically different and requires a different approach due to the higher risk of local recurrence, metastasis and death. Genetic factors play an important role in the development of KC. Family and family history studies, the presence of KC as a feature of rare hereditary syndromes, and genetic association studies give us clues in this regard. More than 20 genetic syndromes associated with KC have been described. Some syndromes are associated with multiple BCC, some with multiple cSCC, and some with both BCC and cSCC. Environmental risk factors include exposure to ultraviolet light radiation and immunosuppression in both tumors. Exposure to ionizing radiation is most common in BCC, while smoking and photosensitive drug use are among the environmental risk factors for cSCC. Molecular, epidemiological, and clinical studies will help better understand the cellular processes involved in tumorigenesis, and develop new strategies for treating and preventing KCs.

1. Introduction

Keratinocytic cancers (KC) comprise a group of diseases that have a broad spectrum clinically and pathologically. Keratinocytic cancers are very common and, despite their low mortality rate, represent a significant public health problem [1]. Traditionally, about 80% of KC cases have basal cell carcinoma (BCC) and 20% have cutaneous squamous cell carcinoma (cSCC) [2]. KC have a complex etiology involving environmental, phenotypical and genetic risk factors [3]. Molecular, epidemiologic, and clinical studies have led to a greater understanding of the cellular events that occur during tumorigenesis, epidemiologic risk factors, and have provided new strategies for treatment and prevention of keratinocyte carcinomas [4]. In the next part of the chapter, BCC- and cSCC-related risk factors and molecular mechanisms associated with tumorigenesis are discussed in detail.

2. Basal cell carcinoma

BCC is a skin tumor thought to arise from the pluripotent cells of the pilosebaceous unit, and there are several clinical types [5]. It is the most common malignancy in fair-skinned populations worldwide [6]. The mortality rate of BCCs is low, but it is an important cause of morbidity, mainly due to local destruction [7]. The incidence and prevalence of BCC increase with age, due to both increasing sun exposure and an aging population [8]. The highest incidence of BCC has been reported in the following countries: Australia, followed by the United States (US) and Europe [9, 10]. In the United States, an increase in an incidence of 4–8% per year has been noted [11]. BCC is more common in men than women, with a male-to-female ratio of approximately 2:1 [12]. The natural course of BCC is a lesion that usually begins as a small, barely visible papule and over the years grows into a nodule or plaque that sometimes ulcerates without showing aggression [13]. When the histologic subtypes of BCC were classified by the World Health Organization according to the risk of recurrence, they were divided into two groups: (1) those with lower risk: nodular, superficial, pigmented, infundibulocystic (a variant of BCC with adnexal differentiation), fibroepithelial; (2) those with higher risk: basosquamous carcinoma, BCC with sclerosing/morpheic, infiltrating, sarcomatoid differentiation, and micronodules [14]. The development of BCC is the result of the interaction of many genes and environmental factors. Most genes involved in BCC pathogenesis have a uniform mutational signature that results in the ultraviolet (UV)-induced Deoxyribonucleic acid (DNA) damage [15].

2.1 Etiopathogenesis

More than 99% of cases of basal cell carcinoma are sporadic. In the absence of an overtly inherited disease-causing mutation, both environmental factors and the sum of an individual’s genetic variations culminate in BCC development [16]. Among the most important risk factors for basal cell carcinoma is exposure to UV radiation [13]. Further risk factors include age, male gender, skin type I and II according to Fitzpatrick (individuals with genetically determined low skin pigmentation), history of BCC, pharmacological therapy, long-term exposure to arsenic, exposure to ionizing radiation, long-lasting immunosuppression, and genetic syndromes [15, 17]. Scarring and chronic ulceration are particularly significant for developing BCCs in non-chronic UV-exposed areas [17]. Chronic exposure to immunosuppressive agents due to organ transplantations linearly increases the risk of developing BCC over time [8]. The main carcinogenic factor is UV, which explains why most tumors are found in sun-exposed skin areas [13]. Indeed, BCC harbors the highest mutated human tumors (65 mutations/megabases) [18, 19] and a high percentage of UV-induced mutations (C:T or CC:TT transitions in dipyrimidine regions) [20]. Sunburns that occur after intense, episodic sun exposure increase the risk for BCC [21]. Both UVA and UVB play a role in skin carcinogenesis by causing DNA damage and immunosuppression. UVB is directly absorbed by the DNA molecule and causes UV-induced DNA damage. In one study, 75.7% of UV-induced DNA coding mutations resulted in the formation of cyclobutane dimers as a consequence of chronic UVB damage [22]. UVA induces cellular reactive oxygen species (ROS) that cause oxidative DNA damage [21, 22, 23, 24, 25]. Basal cell carcinomas are common in the elderly population given cumulative sun exposure and exogenous factors. Age-related deterioration of biological functions leads to chronic inflammation, decreased immune system function, genomic instability, and decreased DNA repair capacity. Therefore, aging skin is characterized by the accumulation and presence of DNA damage and senescent cells. The chronic inflammatory state leads to changes in the integrity of the dermal matrix. All these processes increase the rate of malignancy development with age [15]. The incidence of BCC is generally higher in men than in women, possibly due to occupational exposure to the sun and increased recreational activity in men. However, this difference is less pronounced today, possibly due to lifestyle changes-smoking or tanning beds [24, 25]. People with a previous history of BCC have a higher risk of developing both keratinocytic cancers (KC) and melanoma [15]. These patients have a 10-fold increased risk compared with the general population [26]. A prospective cohort study of 1426 patients showed that 40.7% of them developed a new KC within 5 years after the first lesion and 82% of them developed a new KC within 5 years after more than one lesion [27].

Medications taken by patients pose a risk for the development of BCC. Many drugs such as tetracyclines, thiazide diuretics, nonsteroidal anti-inflammatory drugs (NSAIDs), and retinoids are potentially photosensitizing. Therefore, these drugs elicit phototoxic, photoallergic skin reactions and, when combined with UV radiation, act as carcinogens that increase the risk of skin cancer [28]. Adults taking tetracycline who were exposed to UV light in their youth have an increased risk of BCC [29]. Angiotensin receptor blockers, an antihypertensive drug, have been shown to increase BCC risk by promoting angiogenesis and cancer progression [30]. A Dutch study found an increased risk of BCC in long-term users of loop diuretics, with no association with thiazides and potassium-sparing agents [31]. However, the available data are insufficient to draw firm conclusions about the association between the use of different types of antihypertensive drugs and skin cancer risk. Organ transplant patients have an increased risk of KC due to the immunosuppressive agents they receive. The incidence of BCC in transplant patients has increased tenfold. HIV seropositivity doubles the risk for BCC [32]. The fact that approximately 20% of lesions occur on skin sites not exposed to sunlight is an indication that extrinsic factors other than UV play a role in the pathogenesis of BCC. Other known extrinsic factors include ionizing radiation, arsenic, tar, psoralen and UVA (PUVA), and nitrogen mustard [33, 34]. Ionizing radiation (radiotherapy, X-rays, occupational exposure, whole-body irradiation treatments, atomic bombing) increases the risk of BCCs and possibly cSCCs in various carcinogenic ways. This occurs through various carcinogenic mechanisms such as DNA damage, genomic instability, and cell apoptosis [35, 36].

2.2 Inherited susceptibility to BCC

Some individuals have an increased susceptibility to developing BCC due to genetic syndromes, germline single-nucleotide polymorphisms (SNPs), and genetic traits [37]. A recent study found an overall heritability of 14.0% for KC and 17.0% for BCC in data based on genome-wide array data and self-reported KC history [38]. In the absence of other predisposing factors, especially when multiple BCCs occur at a young age, a possible association with genodermatoses should be considered [17]. The genodermatoses most commonly associated with BCC include Gorlin-Goltz syndrome (also known as Nevoid Basal Cell Carcinoma Syndrome), Bazex-Dupre-Christol syndrome, Rombo syndrome, Generalized Follicular Basaloid Hamartoma syndrome, and Happle-Tinschert syndrome [5]. Nineteen rare syndromes have been described in BCC due to the inheritance of highly penetrant germline mutations. These syndromes, their associated mutations and molecular pathways are listed in Table 1 [37]. Genetic alterations associated with basal cell carcinoma were first described in Gorlin-Goltz syndrome, an inherited predisposition. In the pathogenesis of this syndrome, there are abnormalities associated with the long arm of the patched (PTCH1) gene on chromosome 9 (q22.3-q31) without significant heterogeneity [40]. The prevalence is reported to be approximately 1:56,000 [17]. Affected patients have multiple developmental anomalies, multiple BCCs, and odontogenic keratocysts of the jaw at an early age; the risk of developing medulloblastoma increases in early childhood [39, 41]. BCCs occur on average by age 25, typically in sun-exposed areas, with few to thousands of lesions [39]. Although, most cases are inherited in an autosomal dominant manner, approximately 26% (8–41) of cases develop as de novo [42]. In most cases, this syndrome is caused by mutations on chromosome 9q22.3 that inactivate the PTCH1-containing germline; these inactivating mutations lead to premature termination of the PTCH protein. A second somatic mutation, e.g., caused by UV radiation, can lead to malignancy by loss of the second copy (the loss of heterozygous expression) of the tumor suppressor gene [39]. Other less common mutated genes include PTCH2 and suppressor of fused (SUFU) gene, which play a role in Gorlin syndrome [43]. Bazex-Dupre-Christol syndrome is another genetic syndrome associated with the Hedgehog (HH) pathway. The development of multiple BCCs is associated with congenital hypotrichosis, follicular atrophoderma, and milia [44]. The prevalence is less than 1:1,000,000 [17]. The disease is inherited in an X-linked dominant manner, and mutations commonly occur in the actin-related protein T1 (ACTRT1) gene. The mutation in ACTRT1 ultimately leads to increased GLI transcription factors 1 (GLI1)-induced oncogenic transcription, responsible for the abnormal HH pathway [45]. Rombo syndrome is a very rare autosomal dominant syndrome. In addition to the development of BCCs, this syndrome is accompanied by acrocyanosis, keratosis pilaris, atrophoderma vermiculatum, hypotrichosis as well as hypohidrosis. The prevalence is less than 1: 1,000,000 and it is difficult to distinguish from Bazex-Dupré-Christol syndrome [17].

Syndrome	Gene(s)	Gene function(s)
Gorlin-Goltz syndrome (Nevoid Basal Cell Carcinoma syndrome)	PTCH1, SUFU, PTCH2	HH pathway members
Bazex-Dupré-Christol syndrome	UBE2A, ACTRT1	DNA repair and regulation of cell cycle, HH pathway
Rombo syndrome	Unknown	Rombo syndrome gene is not known; it is involved in DNA repair and/or cell cycle regulation and may play a role during hair follicle growth and differentiation
Generalized follicular basaloid hamartoma syndrome	Unknown	Unknown
Happle-Tinschert syndrome	Unknown	Unknown
Muir-Torre syndrome	MSH2, MLH1, MSH6, and PMS2	DNA mismatch repair
Cowden syndrome	PTEN	PI3K-AKT signaling pathway
Brooke-Spiegler syndrome	CYLD	NF-κB and EGFR pathways regulator
Cartilage-hair hypoplasia	RMRP	Immune response
Xeroderma pigmentosum	XPA-XPG, XPV, POLH	Nucleotide excision repair
Bloom syndrome	BLM (REC0L3)	Chromosomal stability
Werner syndrome	WRN (REC0L2), LMNA	Chromosomal stability
Rothmund-Thomson syndrome	REC0L4, C160rf57	Chromosomal stability
Schopf-Schulz-Passarge syndrome	WNT10A	WNT/β-catenin signaling pathway, cell proliferation and migration
Oculocutaneous albinism	TYR, OCA2, TYRP1, SLC45A2; (MATP), SLC24A5, C10orf11, 4q24	Melanin synthesis
Hermansky-Pudlak syndrome	HPS1-HPS8	Melanin synthesis
Epidermodysplasia verruciformis (Lewandowsky-Lutz dysplasia)	TMC6 (EVER1), TMC8 (EVER2	Immune response and signal transduction in the endoplasmic reticulum
Schimmelpenning syndrome	Unknown	Unknown
Phacomatosis pigmentokeratotica	Unknown	Unknown

Table 1.

Genetic syndromes associated with inherited susceptibility to BCC.

Adapted from Choquet et al. [37] and Kilgour et al. [39].

Susceptibility to BCC may be due to inherited deficiencies in DNA repair. Xeroderma pigmentosum (XP) is an autosomal recessive disorder caused by inherited mutations in any of 8 possible genes required for nucleotide excision repair (NER). Damaged DNA is recognized in actively transcribed genes via the transcription-dependent repair (TCR) pathway and in the rest of the genome via the slower global genome repair (GGR) pathway. Mutations in any of these proteins in the TCR, NER, or GGR pathways result in abnormalities in DNA repair. The classic phenotype of XP manifests in early childhood in the form of freckles before age 2, severe burns after minimal sun exposure, and early-onset skin cancer [46]. XP patients have a > 10,000-fold increased lifetime risk of non-melanocytic skin cancer and a > 2000-fold increased risk of melanoma compared to the general population [47]. Besides xeroderma pigmentosum, the other genetic abnormalities Bloom, Werner, and Rothmund-Thomson syndromes have a predominantly increased risk for BCC and an increased risk for cSCC. The genes involved in these syndromes impair nucleotide excision repair and chromosome stability [37]. In addition to genetic disorders, certain inherited phenotypic traits have also been shown to increase BCC risk [39]. People with fair skin, light eyes and hair, childhood freckles, inability to tan, and Northern European ancestry are known to have an increased risk of developing BCC [21, 48].

2.3 Genetic polymorphism

Genome-wide association studies (GWAS) have played a key role in identifying polygenic effects that mediate susceptibility to BCC [16]. Over the past decade, GWAS has accelerated the discovery of genetic determinants [37]. GWAS studies of SCC and BCC in a European population have identified 33 loci associated with susceptibility to BCC. Taken together, these 33 loci account for 10.98% of the heritability of BCC [37, 39]. In addition to specific mutations of BCC, germline polymorphisms in genes that determine pigmentary characteristics, such as the melanocortin-1 receptor (MC1R), the human homolog of agouti signaling protein (ASIP), and tyrosinase (TYR), have also been associated with increased risk of BCC [49, 50, 51]. MC1R encodes a G-protein-coupled transmembrane receptor that activates adenylate cyclase to produce intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by α-melanocyte-stimulating hormone (α-MSH). Signal transduction by cAMP induces the maturation of phenomelanosomes to eumelanosomes and is responsible for the darker pigmentation and thus the increased UV resistance. The MC1R gene is highly polymorphic in fair-skinned individuals [51]. Several studies have found that pigmentation-independent mechanisms, even after controlling for skin phototype and hair color, significantly increase BCC risk in some of the common variants [50, 51]. GWAS studies have also identified the IRF4, HERC2, LPP, BNC2, EXOC2, RALY, and SLC45A2 genes as important risk loci for BCC, along with other pigmentation genes [39]. While most of these loci are associated with increased risk, ORs of less than one has been reported for SLC45A2, BNC2, and HERC2, suggesting a lower risk [52]. Polymorphism studies on tumor suppressor genes have mainly focused on the tumor suppressor gene p53 (TP53). One polymorphism encoding the TP53 gene, rs78378222, is highly significant for BCC with an overall OR of 2.16. The s78378222 polymorphism affects the AATAAA polyadenylation of the signal of the 30 untranslated regions of the TP53 gene, changing it to AATACA. These results are related to an interrupted polyadenine tail of TP53 mRNA, which is required for stabilization and nuclear export [53]. Genes that determine epidermal differentiation and cytoskeletal organization have also been identified as carrying polymorphisms associated with increased BCC susceptibility [17, 39]. The keratin5 (KRT5) gene, together with its heterodimeric partner keratin14 (K14), produces the K5 protein. These proteins are essential proteins for the cytoskeletal intermediate filament network in the basal keratinocyte. The rs11170164 polymorphism results in a G138E substitution in the KRT5 gene and increases BCC risk [54]. Recently, attention has focused on gene polymorphisms affecting the NOTCH signaling pathway. The NOTCH signaling pathway plays an essential role in regulating keratinocyte proliferation and differentiation; this pathway is associated with skin abnormalities and skin cancer [55]. P53 is thought to induce NOTCH signaling and further promote NOTCH by inhibiting AP-1, a p53 inhibitor. NOTCH signaling regulates keratinocyte proliferation through two mechanisms: first, it inhibits p63, a transcription factor essential for epidermal growth, and second, it increases the expression of cyclin-dependent kinase inhibitor 1A (CDKN1A), a cell cycle inhibitor [39]. NOTCH signaling regulates keratinocyte differentiation through increased expression of transcriptional regulators such as the IRF6 and Hes/Hey genes [55, 56]. Polymorphism of genes (FOXP1 and IRF4) in transcription factor regions that repress NOTCH signaling causes increased susceptibility to BCC [39].

Chromosomal instability is a known risk factor for KC, including BCC [37]. Chromosomal instability is believed to increase tumor adaptation and survival through genetic variation [57]. In addition, several studies have identified polymorphisms in telomere length-related genes that pose a risk for BCC [39]. Studies of gene polymorphisms affecting DNA repair also provide information. Lin et al. examined SNPs in 165 genes of the DNA repair pathway, identified no variants of XPA, MUS81, and NABP2 in three major repair genes, and associated these three variants with a significantly increased risk of BCC risk. The former variant decreased BCC risk (OR 0.93), whereas the latter two increased risk (ORs 1.06 and 1.11, respectively) [58]. Immunity and inflammation of the skin are known to influence the risk of skin cancer, including BCC [39]. Polymorphisms in the human leukocyte antigen (HLA) region have been associated with increased BCC susceptibility, as have IRF4 and UBAC2, which play a role in immune regulation [37]. In 2009, Welsh et al. examined the CT60 GG genotype of the cytotoxic lymphocyte-associated antigen-4 (CTLA-4) gene. They showed that this genotype reduced BCC risk. CTLA-4 is known to play a critical role in UV-induced skin immunosuppression, as it is expressed on t-regulatory cells that are upregulated by UV radiation. The CT60 GG genotype has been shown to result in decreased t-regulatory cell function, UV-induced immunosuppression, and consequently increased antitumor capacity [59]. Moreover, gene polymorphisms are associated with different phenotypes of BCC and modulation of BCC risk. Such a phenotype was observed in young men, in patients with multiple BCC clusters localized to the trunk and not exposed to the sun [60]. This phenotype was associated with germline polymorphisms of genes encoding the liver detoxification enzymes cytochrome p450 2D6 and glutathione S-transferase, as well as with germline polymorphisms of the vitamin D receptor and TNF-α [39].

2.4 Somatic mutations implicated in BCC tumorigenesis

Hedgehog is a signaling pathway in the skin that maintains the stem cell population and controls the development of hair follicles and sebaceous glands [39]. Abnormal activation of this pathway controls many aspects of tumorigenesis, including stages of initiation, progression, and relapse, in part by directing a cancer stem cell phenotype.

2.4.1 Hedgehog signaling pathway

Abnormal activation of the HH signaling pathway is a hallmark of the pathogenesis of BCC. The HH pathway is a highly conserved signaling pathway that plays a critical role in embryogenesis, cell differentiation, and cell proliferation. During embryogenesis, this signaling pathway regulates the morphogenesis of the epidermis and its appendages. Hedgehog is responsible for maintaining and controlling the growth of swollen stem cells. In addition, it is responsible for hair follicle growth and epidermal regeneration in the postnatal period, as well as the protection of bulge stem cells [15]. The HH family includes many intracellular signaling proteins and was first described in Drosophila melanogaster (fruit fly). The HH mutation in the fruit fly causes the embryo to have a spiny appearance reminiscent of a curled hedgehog and is called a hedgehog [61]. The HH signaling pathway consists of several key components: HH ligands, the transmembrane receptor proteins PTCH1 and PTCH2, smoothened proteins (SMO), and GLI1, GLI2, and GLI3 [62]. The PTCH gene encodes a receptor that mediates HH signaling. The intracellular PTCH1/Hh signaling pathway is required for cell growth, regulation, and differentiation. PTCH mutations have been detected not only in Gorlin-Goltz syndrome but also in sporadic BCC cases [5]. Activation of the HH pathway begins with the binding of the HH ligand to a transmembrane receptor complex consisting of the PTCH and SMO proteins. When the HH ligand binds to PTCH, the HH-PTCH complex is cleaved by lysosomes, which suppress SMO and preregulate the pathway’s downstream signaling cascade through several proteins, including the SUFU. As a result, GLI family proteins are released, which are normally sequestered from the cytoplasm. GLI acts as a transcription factor. When secreted, it migrates to the nucleus and triggers the transcription of genes involved in cell renewal, cell fate and survival, and angiogenesis [20, 63]. In addition, GLI1 forms a feedback loop that automatically regulates HH signaling via modulation of PTCH1 [64]. PTCH protein has a negative regulatory effect on HH and suppresses PTCH SMO in the resting state. Oncogenic mutations affecting PTCH and SMO proteins lead to activation of the HH signaling pathway, which in turn causes epidermal hyperplasia and basal keratinocyte proliferation [65]. The PTCH-1 mutation plays a role in BCC carcinogenesis; PTCH-2 is effective in BCC pathogenesis only when together with the PTCH-1 mutation [5]. Activation of the noncanonical HH signaling pathway via GLI transcription factors occurs independently of the aforementioned signaling pathway. In this pathway, the binding of HH-PTCH1 and SMO is passed on. GLI activation is positively regulated by KRAS, TGF-β, PI3K-AKT, and PKC-α and negatively regulated by p53, PKA, and PKC-δ [66]. HH pathway activation is regulated at the genetic level by inactivating PTCH1 mutations identified in 90% of sporadic BCCs and by SMO activating mutations in about 10%. In about half of PTCH1 mutations, the “UV signature” includes C-T and tandem transitions CC-TT. However, the source of UV radiation needs to be adjusted for these mutations, as other factors, such as oxidative stress, are involved in the mutagenesis of this gene. Both point mutations and somatic copy number aberrations (SCNAs) in the PTCH1 gene have been frequently reported in BCC [13] In summary, like PTCH tumor suppressor genes, SMO serves as a proto-oncogene and inactivation of PTCH or activation of SMO plays a role in the pathogenesis of BCC [5].

2.4.2 TP53 gene

The second most common event associated with BCC pathogenesis is the inactivation of the TP53 gene. The TP53 tumor suppressor gene is involved in cell cycle arrest and activation of programmed cell death [67]. In a mouse model study investigating the pathogenesis of BCC, it was shown that in X-ray-induced BCCs, inactivation of the P53 gene in interfollicular keratinocytes upregulates HH pathway activity through increased SMO expression [68]. Somatic mutations in TP53 are common in all human cancers, and non-synonymous mutations in BCC occur sporadically in approximately 61% of cases. Similar to PTCH1, most mutations in TP53 are UV-signed in the majority of BCCs [39].

2.4.3 Hippo-YAP signaling genes

The Hippo signaling pathway is critical for limiting tissue growth. It consists of a cascade of kinases that repress a downstream transcriptional co-activator, Yes-associated protein (YAP), by phosphorylation. The YAP1 protein is the major downregulatory effector of the Hippo pathway [69]. Genetic studies in mouse models suggest that the Hippo pathway plays a role in stabilizing skin growth and differentiation [70]. Moreover, elevated nuclear YAP1 levels lead to the massive proliferation of proliferative basal epidermal cells [71]. Premature stop mutations in the LATS1 gene, one of the kinases required for coding in the Hippo pathway, were reported in 16% of BCCs. Similarly, loss of functional mutations in the TPN14 gene, a tumor suppressor gene that acts as a negative regulator of YAP, was observed in 23% of BCC patients [72].

2.4.4 MYCN/FBXW7 signaling

bHLH transcription factor (MYCN) is a member of the MYC family of transcriptional activators and functions as a potential downstream effector of the HH pathway [73]. Alterations in the levels of MYC family transcription factors affect cell growth, proliferation, differentiation and apoptosis [74]. MYCN missense mutations have been identified in 30% of BCCs. Most mutations are located in the MYC box 1 domain, which is involved in the interaction with the tumor suppressor FBXW7. Functional studies have shown that there are four common N-MYC substitutions (T58A, P59L, P60L and P63L) that impair binding to FBXW7 and result in excess N-MYC protein expression [72].

2.4.5 TERT-promoter

Telomerase is a ribonucleoprotein complex. It maintains telomere length by telomere DNA repeats (TTAGGG) synthesized at the 30 ends of chromosomes to reverse the progressive shortening of DNA with each cell division. Telomerase consists of a reverse transcriptase protein (TERT) encoded by the hTERT gene and an RNA component (hTERC) that serves as a template for DNA telomere synthesis. Activation of the promoter-mediated hTERT gene has been shown to generate de novo binding sites for the family of E26 transformation-specific (ETS) transcription factors, thereby increasing telomerase expression and preventing cancer cell senescence or apoptosis [75]. TERT promoter mutations have been detected in high frequency in many different cancers such as melanoma, non-melanoma skin cancer, bladder cancer, and glioma, as they negatively affect TERT gene expression [76]. UV-related mutations of the TERT gene affecting the promoter region are found in 39–74% of patients with BCC [20].

2.4.6 DPH3-OXNAD1 bidirectional promoter

Similar to TERT, recurrent mutations were also found in the bidirectional promoter of the diphthamide biosynthesis protein 3 (DPH3) and NAD-binding domain containing 1 (OXNAD1) genes at non-coding positions close to the transcription start site. The DPH3 gene diphthamide, a modified histidine residue found in eukaryotic elongation factor 2, is required for protein synthesis [39]. Inactivation of DPH3 is characterized by the loss of a tumor cell’s ability to metastasize; thus, the gene has a tumor-suppressive effect [77]. UV-signed genetic somatic mutations in the DPH3 and OXNAD1 genes have been detected in 42% of BCC [78].

2.4.7 Other potential BCC-associated genes

In a large cohort study, mutations in two cancer-associated genes, PPP6C and STK19, were observed to be associated with BCC tumorigenesis at high frequency [72]. PPP6C regulates cell cycle progression in human cells by controlling cyclin D1. STK19 is a kinase with an unknown function involved in transcriptional regulation [20, 77]. The PPP6C mutation was observed in 15% of patients with BCC and the STK19 mutation in 10%. Other genes commonly mutated in BCC include ARID1A, CASP8, CSMD1, GRIN2A, KRAS, NOTCH1, NOTCH2, NRAS, PIK3CA, PREX2, and RAC1. However, no statistically significant association was demonstrated between these genes and BCC. Two independent exome sequencing studies reported a high frequency of loss-of-function mutations in NOTCH1 (29% and 50%, respectively) and NOTCH2 (26% and 67%, respectively) genes, suggesting that they play a tumor-suppressive role in BCC [22, 72].

2.5 Epigenetic modifications

MicroRNAs (miRNAs) are small regulatory RNAs that act at multiple transcriptional, posttranscriptional, and epigenetic levels. Expression levels of altered miRNAs are associated with BCC progression and non-coding RNA (ncRNA) regulation and play a role in tumor promotion [15]. Studies have found that the expression levels of miRNAs differ in different BCC subtypes. For example, the expression of miR-183, a miRNA that inhibits metastasis to other organs, is significantly lower in infiltrative BCC than in nodular BCC [79]. In nodular BCC, the upregulated miR-141 is associated with the 200a and 200c, C-MYC-, WNT and beta-catenin signaling pathways [79]. MiR-203 and miR-451a function as tumor suppressors. HH and epidermal growth factor receptor (EGFR) signaling suppress the effect of miR-203 on c- JUN and thus cell proliferation [80]. The expression of miRNA-451a has been shown to decrease significantly in both human and mouse BCC patients. Inhibition of primary miRNA-451a increases cell growth via its target TBX1. Conversely, overexpression of miRNA-451a in tumor cells leads to cell cycle arrest by suppressing cell growth [81]. The OncomiR-1 cluster (miR-17-92) shows a regulatory role in SHH signaling in a mouse model of PTCH1, and the corresponding miRNAs were overexpressed in human BCCs [82]. Some long ncRNAs (lncRNAs) (such as ANRIL) are differentially expressed in BCCs [83].

2.6 Tumor microenvironment

Most BCC tumors develop from a surrounding fibromyxoid stroma. It likely provides a permissive tumor microenvironment (TME) by protecting the tumor from the immune system [84]. Indeed, an enhanced tumor-stromal response and local host immunosuppression have been noted in BCCs with high-risk histopathological subtypes [85]. Previous studies have shown a shift toward a Th2 response, an increase in T-regulatory lymphocytes, and the presence of cancer-associated fibroblasts in BCC TME [86]. However, high-throughput sequencing of T-cell receptors has not identified clonal tumor-specific populations of tumor-infiltrating lymphocytes in BCC [87]. The recent study by Lefrançois et al. [86] supports the above information. This study also found that tumor inflammation induced by macrophage activity is associated with advanced BCCs and lymphocytic infiltration plays an important role in nonadvanced tumors, possibly related to an adaptive antitumor response. In TMJ studies related to HH, the major pathway in the pathogenesis of BCC, the paracrine HH pathway is thought to be a complex mechanism involving cancer-related fibroblasts, leading to angiogenesis, fibrosis, immune evasion, and neuropathic pain [88]. Further studies are needed to clarify this issue. In summary, the cancer-associated genes and the various pathways contributing to BCC carcinogenesis suggest a heterogeneous genetic origin. Understanding the molecular genetics of BCC carcinogenesis is important for developing new targeted therapies, increasing treatment efficacy, and overcoming tumor resistance.

3. Cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma is the second most common KC [89]. Data from the Rochester Epidemiology Project, conducted by the Mayo Clinic, show an overall 263% increase in cSCC incidence between 1976 and 1984 and 2000–2010 [90]. In the United Kingdom, the age-standardized incidence of primary cSCC was 77 per 100,000 between 2013 and 2015, with an average annual increase of 5% [91]. This increase has been associated with higher sun exposure levels, tanning bed use, an increase in the aging population and advances in skin cancer detection [92, 93]. The incidence of cSCC is higher in men than in women (3:1 ratio) and increases significantly with age [75, 94]. cSCC results from the uncontrolled proliferation of atypical epidermal keratinocytes due to mutations in genes involved in epidermal homeostasis. It is well known that tumor development is a gradual process that defines different histological and pathological stages, from a premalignant lesion, actinic keratosis (AK), to invasive cSCC [95]. However, similar mutations can also be found in normal keratinocytes, especially in chronically sun-exposed skin [96]. Therefore, other factors-including epigenetic alterations, viral infections, or microenvironmental changes-promote the development and progression of cSCC [97]. cSCC lesions typically occur on chronically sun-exposed sites such as the face, lips, ears, and bald scalp and are characterized by hyperkeratotic, often ulcerated plaques or nodules [75]. The histopathologic classification of cSCC includes keratoacanthomas, acantholytic, spindle cell, verrucous, adenosquamous, and clear cell cSCC [14]. Tumors classified as desmoplastic, acantholytic, and de nova are at higher risk of metastasis to the skin. In addition, less differentiated tumors are associated with a poorer prognosis [98]. In addition to histopathological features, other features determine the high risk of cSCC and the risk of metastasis: 1. subclinical metastasis, 2. depth of invasion (>2 mm), 3. high-risk anatomical localization (face, ear, pre/postauricular, genital, hands and feet), 4. perineural involvement, 5. recurrence, 6. multiple cSCC tumors, 7. immunosuppression [98, 99]. It is well known that the mortality rate for cSCC in the south and the central United States is similar to that for renal and oropharyngeal carcinomas and melanomas [94].

3.1 Etiopathogenesis

The etiology of cSCC is multifactorial. Environmental, immunologic, and genetic factors all play a role [95]. Cumulative exposure to ultraviolet radiation under the influence of the sun and/or tanning devices is the most important causative factor [100, 101]. Epidemiological studies suggest that cumulative sun exposure (mainly UVB radiation) is the most important environmental cause of cSCC. In contrast, intense, intermittent sun exposure (e.g., sunburn, childhood exposure) is the most important risk factor for BCC and melanoma [102, 103]. Although, ionizing radiation is considered a potential risk factor for cSCC, studies have not fully proven this [37]. One study found that therapeutic radiation increased the total number of BCCs but had no effect on SCCs [36]. Another study found an increased risk of both BCC and SCC at sites with prior radiation [104]. Chronic immunosuppression due to organ transplant medications, chronic leukemias and lymphomas, and HIV infection has been shown to be a major risk factor for cSCC and, to a lesser extent, BCC [105, 106, 107]. In contrast to the general population, heart and lung transplant recipients, in particular, tend to develop cSCC more frequently than BCC [108]. The tumorigenic effect of immunosuppression is thought to be related to weak immune surveillance of keratinocytes that do not clear precancerous lesions [109]. While class I HLA proteins are expressed in patients with cSCC, these proteins are not expressed in patients with BCC; therefore, potential immunosuppression has been reported to lead to the development of more cSCC [110]. Patients with chronic lymphocytic leukemia who lack competent cell-mediated and humoral immunity also have an 8- to 10-fold increased risk of developing cSCC [111]. Chronic inflammation increases the risk of cSCC development and progression [37]. 1% of all skin cancers and 95% of patients with cSCC develop on chronically inflamed skin such as scars, burns, and ulcers [112]. Chronic inflammation produces ROS and reactive nitrogen intermediates that lead to DNA damage, which leads to genomic instability and tumorigenesis, resulting in the development of cSCC [113].

Human Papillomavirus (HPV) is a double-stranded DNA virus that infects the squamous epithelium. HPVs are classified into 5 genera (alpha, beta, gamma, mu, and nu) [95]. The β-subtype of HPV has been associated with an increased risk of cSCC [114]. A meta-analysis study reported increased rates of cSCC with HPV types 5, 8, 15, 17, 20, 24, 36, and 38 [115]. However, a study comparing HPV viral load and HPV mRNA expression in tumorous and normal tissues found no difference in HPV viral load between tumors from individuals with cSCC and healthy tissues, and HPV mRNA expression was not detected in tumorous tissues [116]. Nevertheless, HPV is not transcriptionally active in cSCC. HPV likely plays an important role in the pathogenesis of cSCC, during the onset of the disease, not during its progression [117]. In HIV-infected individuals, the development of cSCC depends on the number of CD4+ T cells and viral load. A 2017 study found that the risk of developing SCC increased by 222% in patients with a CD4+ T-cell count <200 cells/mL and a viral load ≥10,000 copies/mL [107].

Medications also increase the risk of developing cSCC. We can examine drugs in three categories: immunosuppressive drugs, B-Raf proto-oncogene, serine/threonine kinase (BRAF) inhibitors, and photosensitive drugs. Several immunosuppressive agents increase the risk of KC through direct mutagenic effects, regardless of their immunosuppressive role [37]. Because of the increasing effect of azathioprine on UVA photosensitivity, the KC risk is increased by increased oxidative DNA damage [118]. In a large whole-exome sequencing study of 40 primary cSCCs from immunosuppressed and immunocompetent patients, a novel signature mutation (signature 32) associated with chronic exposure to azathioprine was identified in 27 of the tumor samples. The calcineurin inhibitor cyclosporine has a direct tumorigenic effect [119]. Cyclosporine has been observed to increase tumor growth in mice with severe combined immunodeficiency [120]. Another study showed that cyclosporine-mediated inhibition of calcineurin (and thus a nuclear factor of activated T cells) prevents p53-dependent cellular senescence [121]. Patients receiving BRAF inhibitors, targeted therapies for the treatment of melanoma, have been found to have an increased development of cSCC, approximately 15–30%. The mechanism of carcinogenesis for these inhibitors is likely to be those pre-existing mutations in RAS or RTK that lead to the proliferation of cancer cells, secondary to activation of the MAPK pathway [122]. cSCC has been associated with photosensitive drug use. Long-term treatment with voriconazole, an antifungal drug, leads to SCC development in immunocompromised patients, including children [123]. Phototoxic eruptions due to voriconazole have been documented in almost all patients who develop SCC [123, 124]. The use of thiazide, an antihypertensive drug among photosensitive drugs, and cSCC development should be discussed. Although, Gandini et al. [125] found no association between this treatment and SCC in their meta-analysis, Tang et al. [126] defined a possible association in their meta-analysis. In addition, treatments with PUVA are known to increase cSCC risk due to their mutagenic and immunosuppressive effects. The risk of SCC increases moderately in those receiving fewer than 150 PUVA treatment sessions, whereas the risk of SCC increases greatly in those receiving more than 350 treatment sessions [127]. Epidemiologic studies have yielded conflicting results regarding the role of smoking as a risk factor for SCC. A 2012 systematic review and meta-analysis found that smoking was associated with a 50% increased risk of SCC in never smokers [128]. In 2017, an Australian cohort study reported that the rate of SCC was twice as high in smokers compared with never smokers after adjusting for factors such as age, gender, education, skin color, tanning ability, number of freckles, childhood sunburn, and cumulative sun exposure [129]. Further studies are needed to investigate the effects of cigarette smoking on KC risk.

3.2 Inherited susceptibility to cSCC

A large cohort study in Sweden concluded that people whose siblings or parents had invasive cSCC had a two- to three-fold higher risk of receiving the same diagnosis [130]. Similar skin phenotypes, shared environmental exposures, and genetic factors may contribute to familial risk [131]. In a population-based study, after adjusting for phenotypic and environmental risk factors for cSCC, individuals with a family history of KC were found to have a 4-fold higher risk of cSCC [132]. The genetic syndromes associated with cSCC are summarized in Table 2. Epidermolysis bullosa syndromes (EBS) are a group of mechanobullous skin diseases characterized by bullae that occur with little or no trauma. EBS-associated SCCs usually develop over chronic wounds or scars, tend to be multiple and aggressive, and increase with age [133]. Oculocutaneous albinism (OCA) is a group of autosomal recessive melanin biosynthesis disorders characterized by generalized pigment reduction of the skin, eyes, and hair. Patients with OCA are at increased risk for early-onset skin cancers, most commonly SCC [134]. Epidermodysplasia verruciformis is a rare disease characterized by hypersensitivity to HPV strains 5 and 8 and SCC [135].

Syndrome	Gene(s)	Gene function(s)
Epidermolysis bullosa	KRT5, KRT14, LAMB3, COL17A1, COL7A1, FERMT1 KIND1	Keratinization, collagen formation, cell junction organization, extracellular matrix organization
Fanconi anemia	BRAC1, BRAC2, BRIP1, ERCC4, FAAP20, FAN1, FANCA-FANCM, MAD2L2, PALB2, RAD51, RAD51C, SLX4, UBE2T, and XRCC2	Fanconi anemia pathway
Dyskeratosis congenita (Zinsser-Engman-Cole syndrome)	ACD, CTC1, DKC1, NHP2, NOP10, PARN, RTEL1, TERC, TERT, TINF2, and WRAP53	Telomere maintenance and trafficking
Multiple self-healing squamous epithelioma (Ferguson-Smith disease)	TGFBR1	TGF-β signal transduction
Huriez syndrome	Unknown	Unknown
Xeroderma pigmentosum	XPA-XPG, XPV, POLH	Nucleotide excision repair
Bloom snydrome	BLM (RECQL3)	Chromosomal stability
Werner syndrome	WRN (REC0L2), LMNA	Chromosomal stability
Rothmund-Thomson syndrome	REC0L4, C160rf57	Chromosomal stability and telomere maintenance and trafficking
Schöpf-Schultz-Passarge syndrome	WNT10A	WNT/β-catenin signaling pathway and cell proliferation and migration
Epidermodysplasia verruciformis (Lewandowsky-Lutz dysplasia)	TMC6 (EVER1), TMC8 (EVER2	Immune response and signal transduction in endoplasmic reticulum
Oculocutaneous albinism	TYR, OCA2, TYRP1, SLC45A2 (MATP), SLC24A5, C10orf11, 4q24	Melanin synthesis
Hermansky-Pudlak syndrome	HPS1-HPS8	Melanin synthesis

Table 2.

Genetic syndromes associated with inherited susceptibility to cSCC.

In 2016, three GWAS cSCC risk studies of European origin were published, identifying 16 genetic risk loci [136, 137, 138]. In the first GWAS study, 11 loci associated with cSCC risk were reported at the genome-wide significance level (P < 5 × 10^–8). These included FOXP1, TPRG1/TP63, SLC45A2, IRF4, HLA-DQA1, BNC2/CNTLN, TYR, OCA2, HERC2, DEF8, and ASIP/RALY [136]. The second study focused on the DEF8 locus [138]. The third and most recent GWAS study determined SLC45A2, IRF4, BNC2/CNTLN, TYR, OCA2, HERC2, and ASIP/RALY, as well as the intergenic region on chromosome 2p22, AHR, SEC16A, CADM1, and MC1R [137]. As a result of these studies, cSCC-related nucleotide polymorphisms were identified in MC1R, ASIP/RALY, TYR, SLC45A2, and OCA2 genes, CADM1 in the metastasis suppressor gene, and AHR, a transcription factor regulating cell proliferation, in SEC16A’a gene associated with secretion and cellular proliferation [101]. SLC45A2, IRF4, TYR, HERC2, DEF8, and RALY identified for cSCC are known to be multiple risk loci near the pigmentation gene, supporting the role of lighter pigmentation and exposure to UV radiation in the developmental risk of cSCC [137]. The relationship between genetic variants in the FOXP1 and IRF4 regions is also associated with Notch signaling in cSCC [37].

3.3 Somatic mutations

Recent studies have shown that somatic mutations are much more common in cSCC than in other SCCs and melanomas [139]. cSCC has 5 times more mutations than other malignancies, such as lung mutations, and 4 times more mutations than melanomas [139, 140]. The accumulation of these mutations and other cellular alterations transforms the skin into cSCCs with an increased degree of dysplasia [94]. Most cSCCs carry a UV mutational signature with characteristic C > T or CC > TT dinucleotide mutations, although some may be passenger mutations also found in surrounding photoaged skin [141]. Genes altered in cSCC include mutations in UV-induced TP53, CDKN2A, NOTCH1, and NOTCH2, which are involved in cell cycle control, and in epigenetic regulators KMT2C, KMT2D, TET2, and TGF-β receptors, leading to their inactivation [101]. The most frequently altered tumor suppressor genes in cSCC are TP53, CDKN2A, NOTCH1, and NOTCH2. In addition, a network of dysregulated molecular signaling pathways, including EGFR signaling cascades affecting RAS/RAF/MEK/ERK and PI3K/AKT/mTOR signaling pathways, has been shown to play a critical role in the carcinogenesis of cSCC [75].

3.3.1 TP53, CDKN2A, NOTCH1, NOTCH2 molecular alterations

TP53 mutations occur in 54–95% of cSCC and often show a consistent C > T or CC > TT UV signature. Resistance to apoptosis and clonal growth in keratinocytes in the TP53 gene as a consequence of UV-mediated DNA damage [142, 143]. Variants in the TP53 gene frequently occur in AKs and precancerous lesions such as cSCC in situ and thus represent the first event of carcinogenesis. The CDKN2A gene encodes the alternatively spliced proteins p16INK4a and p14ARF. These proteins regulate cell cycle progression and are involved in retinoblastoma and proliferation by TP53, respectively. Loss of this locus with heterozygous or point mutations has been detected in 21–62% of cSCC patients [119, 139, 144, 145, 146]. Hypermethylation of the CDKN2A promoter was reported in 35–78% of patients [147].

NOTCH receptor mutations are an important tumor suppressor mechanism in cSCC and appear to play an important role in HRas proto-oncogene, GTPase (HRAS)-induced skin carcinogenesis [146]. The NOTCH family includes the single-pass transmembrane receptor. This receptor consists of an extracellular ligand-binding domain containing multiple EGF-like repeats (EGF-LR) and an intracellular domain that mediates transcription of the target gene. Loss of NOTCH signaling leads to impaired differentiation, increased stem cell activity, and the development and progression of cSCC [56, 139, 146]. NOTCH1 or NOTCH2 mutations have been identified in approximately 80 percent of cSCC and occur early in cSCC carcinogenesis [146, 148]. Activating HRAS mutations, NOTCH1 mutations, and CDKN2A mutations have been found in high frequency in cSCC from patients treated with BRAF inhibitors [146].

3.3.2 Overexpression of EGFR and MET tyrosine kinases receptors

The EGFR gene encodes the ErbB transmembrane glycoprotein receptor, which belongs to the tyrosine kinase receptor family. Ligand binding to EGFR causes the receptor to downregulate several signaling pathways such as MAPK/ERK and PI3K/AKT/mTOR. These signaling pathways control cell maturation, proliferation, inhibition of angiogenesis, and apoptosis [75]. The overall incidence of EGFR mutations in cSCC varies from 2.5 to 5% [144, 149, 150]. Overexpression of EGFR protein has been associated with poor prognosis and lymph node metastases in cSCC [151]. In recent studies, overexpression of this protein has been associated with a reduction in degradation and dephosphorylation mechanisms [152]. MET is a tyrosine kinase receptor that upregulates the RAS/RAF/MEK/ERK signaling pathway and its overexpression [75]. This signaling pathway has been shown to contribute to proliferation, invasion, migration, drug resistance, and angiogenesis in a variety of human tumors [153]. A recent study pointed out that treatment with capmatinib, a selective MET inhibitor, was able to prevent the upregulation of hepatocyte growth factor- stimulated EGFR protein, demonstrating the cooperation of MET and EGFR in cSCC carcinogenesis [154].

3.3.3 RAS-RAF-MEK-ERK pathway

RAS molecules are a family of GTP-binding proteins and are among the most frequently mutated genes in human cancers. RAS is an up-activator of the RAF/MEK/ERK1/ERK2 kinase pathway, and activating mutations in RAS promote SCC formation [155]. The active GTP-RAS complex promotes the formation of RAF dimers, which in turn activate the MEK-ERK cascade through phosphorylation. ERK interacts with multiple cytosolic targets and many nuclear substrates, including kinases, cytoskeletal proteins, and transcription factors. Signals for cell proliferation, differentiation, survival, migration, angiogenesis, and chromatin remodeling are triggered [75]. Recycling cycles are controlled by the MAPK cascade [156]. The MAPK pathway has been shown to play a role in cSCC development, and the MAPK inhibitor sorafenib has been associated with cSCC development in patients using BRAF inhibitors such as vemurafenib and dabrafenib [157, 158]. As for RAS genes, harbor activating RAS mutations (HRAS) are associated with cSCC. According to recent data, 21% of cSCC-associated somatic mutations were associated with HRAS [159].

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that contributes to cell proliferation by inducing ERK phosphorylation and MAPK pathway activation [160]. MIF also functions as a negative regulator of P53 [161]. MIF has been determined to be overexpressed in cSCC lesions compared to normal tissue [162]. In addition, ERK is involved in regulating the transcriptional activity of E-twenty-six 1 (ETS) 1 [163]. ETS-1 belongs to the family of ETS transcription factors, which are characterized by the presence of a conserved ETS DNA-binding domain. ETS-1 transcribes numerous target genes, including metalloprotease family members critical for extracellular matrix remodeling and angiogenesis, and regulates genes required for cell proliferation and survival [4]. ETS-1 has been detected to be immunohistochemically overexpressed in its poorly differentiated and metastatic form in SCC [164].

3.3.4 PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway has common inputs and interacts with the MAPK pathway. Once activated, PI3K converts PIP2 to PIP3, which leads to activation of the serine/threonine kinase AKT, also known as protein kinase B, which in turn activates mTOR [75]. mTOR functions as a physiological sensor for nutrients and cell division and regulates growth by promoting RNA translation and protein synthesis [75, 165]. The PI3K/AKT/mTOR signaling cascade is negatively regulated by PTEN [166]. Activation of this signaling pathway results in SCC development and progression [167]. This condition is mainly controlled by loss of function of the PTEN gene or activation or amplification of mutations in the PIK3CA gene, which encodes the catalytic subunit of PI3K [75]. In the PI3K/AKT/mTOR pathway, molecular alterations of PTEN are observed in 7–25% of patients with cSCC due to inactivating mutations and resulting hyperactivation and homozygous loss [119, 145].

3.3.5 Telomerase pathway

Telomerase consists of reverse transcriptase (TERT) protein component encoded by the hTERT gene. The activation of the hTERT gene that occurs through promoter mutations has been shown to create de novo binding sites for the ETS transcription factors family, thus promoting telomerase expression and preventing senescence or apoptosis of cancer cells [75]. TERT promoter mutations were found in 31.6% of cSCC and are frequently associated with recurrent (OR = 6.75) and metastatic (OR = 15.89) lesions [75, 168].

3.4 Genetic polymorphisms

In cSCC, nearby loci, including the human leukocyte antigen locus at 6p21, and loci containing genes involved in germline pigmentation pathways with SNPs, GWAS studies identified an intronic SNP in non-pigment loci such as FOXP1 (3p13), an intergenic SNP at 3q28 near T63, an intergenic SNP at 9p22, and the SNP rs12203592 in IRF4 [136, 137, 169, 170]. In a study using 21 published SNPs based on GWAS, the risk attributable to the multilocus population was calculated to be 62%, suggesting that the risk for cSCC would be reduced by 62% if the effects of all risk alleles were removed from a population [171]. In the new GWAS meta-analysis conducted in 2020, eight new susceptibility loci for cSCC were discovered. These loci included genes involved in cancer progression (SETDB1: rs10399947, CASP8/ALS2CR12: rs10200279, WEE1: rs7939541), immune regulation (BACH2: rs10944479), keratinocyte differentiation (TRPS1:rs7834300, KRT5: rs11170164 and rs657187) and pigmentation: rs1325118) [170]. Another polymorphism that increases cSCC development is methylenetetrahydrofolate reductase polymorphisms related to the folate mechanism [172]. In a cohort of renal transplant recipients, the risk of developing cSCC was statistically significantly increased in those carrying the common MTHFR:C677T polymorphism in the folate pathway [173]. In a cohort of 694 transplant patients, cSCC was reported to develop earlier in these patients due to a polymorphism in the rs12203592 T allele of the gene encoding IRF4, which plays a role in the activation of melanin synthesis via tyrosinase [169]. Polymorphisms in thiopurine S-methyltransferase, which regulates azathioprine inactivation, have not been found to increase cSCC risk in studies [172].

3.5 Epigenetic modifications

Environmental factors can alter the epigenetic state of cells. Epigenetic changes include DNA methylation and histone modification (i.e., methylation, acetylation, phosphorylation, ubiquitination, and chromatin remodeling) [174]. A link between cSCC and gene-specific promoter hypermethylation has been established. Specifically, the CDKN2A gene is involved in positive cell cycle regulation, ASC is associated with apoptosis in G0S2 and the DAPK1 gene, and SFRPs and FRZBs are associated with Wnt signaling in the transcription factor and the adhesion molecules cadherin CDH1 and CDH13 [175, 176, 177, 178]. PE-cadherin (CDH1) promoter hypermethylation was found in cSCC (85%), in situ cSCC (50%), AK (44%), and normal skin (22%). Downregulation of E-cadherin has been associated with increased tumor invasion, increased metastatic potential, and advanced stage of cSCC [178, 179]. Several enzyme families catalyze different types of histone modifications. The best known are the modifications, acetylation and methylation of H3 histones; it is H4 that directly alters chromatin condensation and gene transcription. Acetylation is catalyzed by histone acetyltransferases (HATs); histone deacetylases (HDACs) remove the acetyl group, allowing chromatin condensation and thus gene inactivation [95]. HAT up-regulation of p300 plays an important role in the development and progression of cSCC [180].

Methylation of the amino acids lysine and arginine occurs by histone methyltransferases, and the effect of the modification depends on which residue is modified. For example, trimethylation of histone H3 on lysine 4 (H3K4me3) activates transcription, whereas lysine 27 or lysine 9 on histone H3 represses transcription [181]. Polycomb group proteins (PcG) are an important family of histone modifiers that have been extensively studied in skin cancer. The PcG enzyme EZH2 is the primary histone methyltransferase and controls the proliferative potential of self-renewing keratinocytes by suppressing the CDK2A locus. EZH2 is frequently mutated in cancer and its overexpression is associated with cSCC progression [182, 183, 184]. Type 2 lysine methyltransferase, together with the enzymes KMT2C and KMT2D, forms a transcriptional core complex and provides histone H3 modification. Mutations in KMT2C and KMT2D have been found in both primary cSCC (36% and 31%, respectively) and metastatic tumors (43% and 62%, respectively) [185].

3.6 Tumor microenvironment

The tumor microenvironment, age-associated secretory phenotype, or SASP (i.e., cytokines, growth factors, and metalloproteinases) is a complex molecular system composed of a heterogeneous population of cells (tumor cells and adjacent stromal cells (fibroblasts, endothelial cells, inflammatory and immune cells) [95]. The TME plays an important role in the carcinogenesis of cSCC. The newly developing neoplastic keratinocytes of cSCC interact with other stromal cell types. It interacts with the local microenvironment and regulates the cell-cell relationship by acting as a tumor promoter and via epigenetic reprogramming, DNA damage, promotion of hypoxia, angiogenesis, activation of cancer-associated fibroblasts (CAFs), recruitment of regulatory immune cells, and inhibition of antitumor immune surveillance [97, 186, 187]. Organotypic culture studies have shown that the epidermal component formed by epidermal hyperplasia of aged epidermal keratinocytes requires the presence of CAF to penetrate the matrix [188].

Human leukocyte antigen variants and the programmed cell death protein 1/programmed cell death ligand-1 axis (PD-1/PD-L1) also play a role in the tumor microenvironment [189, 190]. Expression of PD-L1 is also involved in primary cSCC. PD-L1 has been detected in approximately 26% of primary cSCC and 50% of metastatic lesions [101]. In cSCC, p63 regulates the transcriptional expression of the proinflammatory cytokines IL -1, IL -6, IL −8, tumor-associated angiogenesis, and lymphangiogenesis by altering the expression of human beta-defensins. All these findings suggest that p63 contributes globally to cSCC development by regulating the tumoral environment [191, 192, 193].

4. Conclusion

In conclusion, the development and progression of BCC and cSCC are associated with various alterations such as genetic mutations, epigenetic modifications, viral infections, or microenvironmental changes that affect epidermal hemostasis. Future studies will better explain the etiopathogenesis of BCC and cSCC, and thus contribute to the development of disease prevention and targeted therapies.

Abbreviations

AHR	aryl hydrocarbon receptor
AKT	serine/threonine-protein kinase
ARID1A	AT rich interactive domain 1A
ASC	apoptosis-associated speck-like protein containing a CARD
BNC2	basonuclin 2
CADM1	cell adhesion molecule 1
CASP8	caspase-8
CDKN2A	cyclin dependent kinase inhibitor 2A
C-MYC	transcriptional regulator Myc-like
CNTLN	centlein
CSMD1	CUB and Sushi multiple domains 1
DAPK1	death-associated protein kinase 1
DEF8	differentially expressed in FDCP 8
EXOC2	exocyst complex component 2
EZH2	enhancer of zeste 2 polycomb repressive complex 2 subunit
FBXW7	F-box and WD repeat domain containing 7
FOXP1	forkhead box P1
FRZB	frizzled-related protein
GRIN2A	glutamate ionotropic receptor NMDA type subunit 2A
HERC2	HECT and RLD domain containing E3 ubiquitin protein ligase 2
HES	hairy/enhancer of split
HEY	HES-related genes such as hairy/enhancer of split related with YRPW motif
HLA-DQA1	major histocompatibility complex, class II, DQ alpha 1
IRF4	interferon regulatory factor 4
IRF6	interferon regulatory factor 6
KMT2C	lysine methyltransferase 2C
KMT2D	lysine methyltransferase 2D
KRAS	KRAS proto-oncogene, GTPase
LATS1	large tumor suppressor kinase 1
LPP	LIM domain containing preferred translocation partner in lipoma
MC1R	melanocortin 1 receptor
MET	MET proto-oncogene, receptor tyrosine kinase
MUS81	MUS81 structure-specific endonuclease subunit
NABP2	nucleic acid binding protein 2
NOTCH1	Notch receptor 1
NOTCH2	Notch receptor 2
NRAS	NRAS proto-oncogene, GTPase
PI3K/AKT/Mtor	phosphatidylinositol 3-kinase/serine/threonine-specific protein kinases/mammalian target of rapamycin
PI3K	phosphatidylinositol 3-kinase
PIK3CA	phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha
PIP2	phosphatidylinositol (4,5)-bisphosphate
PIP3	phosphatidylinositol (3,4,5)-trisphosphate
PKA	cAMP dependent protein kinase
PKC-α	protein kinase C-alpha
PKC-δ	protein kinase C-delta
PREX2	phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2
RALY	RALY heterogeneous nuclear ribonucleoprotein
RAS/RAF/MEK/ERK	Ras/Raf/mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK)
RTK	receptor tyrosine kinase
SEC16A	SEC16 homolog A, endoplasmic reticulum export factor
SFRP	secreted frizzled-related protein
SLC45A2	solute carrier family 45, member 2
TBX1	T-box transcription factor 1
TET2	tet methylcytosine dioxygenase 2
TGF-β	transforming growth factor beta
TNF-α	tumor necrosis factor alpha
TPRG1	tumor protein p63 regulated 1
UBAC2	UBA domain containing 2
WNT/β-catenin	wingless signaling transduction beta catenin
XPA	Xeroderma pigmentosum complementation group A (XPA, DNA damage recognition and repair factor)