The pathogenesis of AD encompasses various immune pathways. Recent studies revealed that type 2 immune inflammation is the dominant pathway involved, driven by innate type 2 ILC and Th2 cells as well as their cytokines, such as IL-4 and IL-13. Itch is a sensation associated with AD. Previous studies revealed that neuroimmune communication is a key player in the development of itch in inflammatory skin diseases, such as AD. Therefore, targeting type 2 pathways in the neuroimmune interaction appears to be a reasonable therapeutic strategy for itch in AD. Recently developed biologic agents targeting type 2-associated cytokines have achieved promising outcomes. The mAb anti-IL-4Rα (dupilumab) and topical PDE4 inhibitor (crisaborole) have been approved by the FDA for moderate-to-severe and mild-to-moderate AD, while JAK inhibitors (baricitinib and delgocitinib) have been approved for AD in Japan. Based on the findings of recent clinical trials on the treatment of itch in AD, dupilumab appears to be the best option for moderate-to-severe AD, and crisaborole 2% for mild-to moderate AD. Further studies on

other agents will offer novel insights into the underlying pathogeneses and new targeted treatment alternatives for itch in AD.

**Funding:** This work was funded by the Foundation of Strategic Research Projects in Private Universities (Grant-in-Aid S1311011) and JSPS KAKENHI (Grant Numbers 18K07396 and 20H03568) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; and a grant from the Lydia O'Leary Memorial Pias Dermatological Foundation (2021).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Epidermolysis Bullosa—A Different Genetic Approach in Correlation with Genetic Heterogeneity**

**Monica-Cristina Pânzaru 1, Lavinia Caba 1,\*, Laura Florea 2,3,\*, Elena Emanuela Braha 4 and Eusebiu Vlad Gorduza 1**

**Abstract:** Epidermolysis bullosa is a heterogeneous group of rare genetic disorders characterized by mucocutaneous fragility and blister formation after minor friction or trauma. There are four major epidermolysis bullosa types based on the ultrastructural level of tissue cleavage: simplex, junctional, dystrophic, and Kindler epidermolysis bullosa. They are caused by mutations in genes that encode the proteins that are part of the hemidesmosomes and focal adhesion complex. Some of these disorders can be associated with extracutaneous manifestations, which are sometimes fatal. They are inherited in an autosomal recessive or autosomal dominant manner. This review is focused on the phenomena of heterogeneity (locus, allelic, mutational, and clinical) in epidermolysis bullosa, and on the correlation genotype–phenotype.

**Keywords:** epidermolysis bullosa; mutation; heterogeneity

#### **1. Introduction**

Epidermolysis bullosa (EB) is a heterogeneous group of rare genetic disorders characterized by mucocutaneous fragility and blister formation after minimal trauma [1]. EB presents a variable expression with a wide phenotypic spectrum ranging from localized, mild, acral blistering, and normal life expectancy, to generalized, severe blistering and extracutaneous involvement, which could lead to infections, electrolyte imbalances, or respiratory distress, as well as poor prognosis. Nail dystrophy, keratoderma, and atrophic scarring are common features. Major extracutaneous complications may develop in some subtypes of EB: laryngeal or esophageal stenosis, ectropion, corneal opacification, pseudosyndactyly, and microstomia. Pyloric atresia, nephropathy, muscular dystrophy, cardiomyopathy, and interstitial lung disease, are encountered in rare forms of EB. Some forms of EB (e.g., severe and intermediate recessive dystrophic EB) are associated with an increased risk of developing cutaneous squamous cell carcinoma [1,2].

Four major EB types are described based on the level of skin cleavage: EB simplex (EBS)—with changes at intraepidermal (epidermolytic) level, junctional EB (JEB)—with changes at the intra-lamina lucida (lamina lucidolytic) level, dystrophic EB (DEB)—with changes at the sub-lamina densa (dermolytic) level, and Kindler EB—with multiple changes at the cutaneous level [1]. Precise diagnosis requires the correlation of clinical data with immunofluorescence antigen mapping (IAM), transmission electron microscopy (TEM), and mutational analysis [2]. In EBS the structure and function of keratin intermediate filaments are altered and the intracellular components of the hemidesmosomes are mutated or

**Citation:** Pânzaru, M.-C.; Caba, L.; Florea, L.; Braha, E.E.; Gorduza, E.V. Epidermolysis Bullosa—A Different Genetic Approach in Correlation with Genetic Heterogeneity. *Diagnostics* **2022**, *12*, 1325. https:// doi.org/10.3390/diagnostics12061325

Academic Editor: Yasuhiro Sakai

Received: 7 April 2022 Accepted: 25 May 2022 Published: 27 May 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

missing [2]. In JEB, the transmembrane and extracellular proteins of the hemidesmosomes and the anchoring filaments are modified [2]. In DEB, the anchoring fibrils can be absent, reduced in number, or abnormal. In KS, there are multiple cleavage planes (intraepidermal, junctional, or dense sub-lamina) determined by abnormal fermitin family homolog 1, which is a component in the focal adhesion complexes [2].

Research in recent decades has deciphered some of the pathogenic changes in EB, mainly through histological and genetic studies. These researches proved an important genetic heterogeneity and changed the classification of disorders. Our paper is trying to present synthetically the gene mutations and their implications on the cellular level in connection with their clinical features.

#### **2. Genes and Proteins Involved in Epidermolysis Bullosa**

In EB, cell–matrix interactions are mainly altered. Normally cell–matrix interactions are achieved through two elements: hemidesmosomes and focal adhesion.

Hemidesmosomes (HD) are specialized structures that stably anchor the keratinocytes of the epidermis to the basement membranes. This is done by assembly between the intracellular and transmembrane proteins [2]. HD type I (the classic one) is present in the pseudo-stratified epithelium where interactions between intracellular proteins (plectin, dystonin) and transmembrane protein integrin (α6β4, collagen XVII) and CD151 antigen normally occur. HD type II is found in simple epithelial tissue and consists only of α6β4 integrin and plectin [3].

The focal adhesion is allowed by different proteins: integrin α3β1, transmembrane collagen XIII, and fermitin family homolog 1 (FFH1) [2,4].

Table 1 summarizes the main genes involved in the pathogenesis of EB and the encoded proteins.

**Table 1.** Genes and proteins involved in epidermolysis bullosa [1,5,6].

#### **Table 1.** *Cont.*

#### **Table 1.** *Cont.*

#### *2.1. Keratins*

Keratins are structural proteins. They form obligate heterodimers assembled into intermediate filaments and 3D cytoskeletons. There are two types of keratins: type I and type II. Keratin-5 (type II keratin protein) and keratin 14 (type I) have an α-helical "rod" domain which is flanked by the head and tail domain [7].

#### *2.2. Plectin*

Plectin is a cytoskeletal linker protein expressed in skin and skeletal muscle. It links intermediate filaments to hemidesmosomes and in this way functions as a mediator of keratinocyte mechanical stability in the skin [8].

#### *2.3. BTB Domain Containing Kelch-like Protein*

Unlike most EBS-associated proteins, the Kelch-like protein 24 (KLHL) is not a structural protein. KLHL proteins are expressed almost ubiquitously at low levels and are involved in cytoskeletal organization, the regulation of cell morphology, cell migration, protein degradation, and gene expression. They contain a BTB domain that binds to cullin 3, a scaffold protein required for the ubiquitination and proteasomal degradation of substrate proteins [7,9].

#### *2.4. Dystonin*

Dystonin is one of the largest human proteins and as a member of the plakin family is a structural component of hemidesmosomal inner plaques in basal keratinocytes, a key attachment point for keratin intermediate filaments and the location for other major plaque proteins, such as plectin [10]. The *DST* gene encodes dystonin; alternative splicing produces multiple tissue isoforms expressed in the central nervous system, skin, heart, and skeletal muscle [11,12].

#### *2.5. Exophilin*

*EXPH5* gene encodes exophilin-5, an effector protein of the Rab27B GTPase. This protein plays a role in intracellular vesicle trafficking and exosome secretion.

#### *2.6. Tetraspanins*

Tetraspanins form a protein superfamily widely distributed in the epidermis, renal glomeruli, and proximal and distal tubules. One of its members is the CD151 antigen. In the epidermis, the CD151 antigen participates in the formation of hemidesmosomes and forms very stable laminin-binding complexes with alpha-3beta-11 and alpha-6beta-4 integrins. This allows cell adhesion and the intracellular vesicular transport of integrins. In the kidney, CD151 forms complexes with integrins α3β1 and α6β1 and is essential for the proper assembly of the glomerular and tubular basement membranes [13].

#### *2.7. Laminin Subunits*

Laminin 332 is a heterotrimeric molecule that consists of alpha-3, beta-3, and gamma-2 subunits, and is essential for the formation and function of the basement membrane. Laminin 332 interacts with integrin alpha-6beta-4, alpha-3beta-1, and collagen VII, and

plays a pivotal role in epidermal adhesion, cell survival, migration, and regeneration. The *LAMA3*, *LAMB3*, and *LAMC2* genes encode the alpha-3, beta-3, and gamma-2 chains of laminin 332 [14,15].

#### *2.8. Collagen XVII*

Collagen XVII is expressed in the epithelial hemidesmosomes of the skin, mucous membranes, and eyes [16]. It consists of three identical α1 chains. It has three major domains: a globular intracellular domain, a transmembrane domain, and an extracellular domain. The extracellular domain consists of 15 collagenous domains (cell adhesion domains) and 16 noncollagenous domains (with roles in triple-helix folding) [16]. The collagen XVII binds intracellularly to plectin, dystonin, and β4 integrin, and extracellularly to α6 integrin (that binds to CD151) and laminin 332 [2].

#### *2.9. Collagen VII*

Type VII collagen is secreted by keratinocytes as procollagen VII. The procollagen VII is formed by three pro-alpha-1 chains that fold into one molecule. The molecule has an N-terminal noncollagenous 1 (NC1) domain, followed by an extended collagenous domain, and ends with the NC2 domain at the C-terminus. Its role is in anchoring the fibrils that form the cutaneous basement membrane zone adhesion complex [7].

#### *2.10. Integrins*

Integrins are transmembrane protein complexes, consisting of alpha and beta chain subunits. The alpha-6 beta-4 integrin is involved in hemidesmosome formation and stability and interacts with laminin, plectin, and dystonin. The activation of integrins mediates extracellular cell–matrix interactions and cytoskeleton organization [17]. *ITGA6* encodes the alpha-6, whereas *ITGB4* encodes the beta-4 subunit of the alpha-6beta-4 integrin. The *ITGA3* gene encodes the integrin alpha-3 subunit that is connected with a beta-1 subunit to form an integrin involved in interactions with extracellular matrix proteins including laminins. The integrin alpha-3 subunit is expressed in basal keratinocytes, podocytes, tubular epithelial cells, alveolar epithelial cells, and many other tissues [7].

#### *2.11. Fermitin Family Homolog 1*

The *FFH1* gene is expressed at the dermal–epidermal junction, oral mucosa, and in the gastrointestinal tract [7,18,19]. It is involved in the connection between the actin cytoskeleton and the extracellular matrix by focal adhesion [4]. Also, FFH1 participates in integrins' activation [2].

The main interactions between proteins are shown in Figure 1.

**Figure 1.** Main proteins involved in epidermolysis bullosa. Created with BioRender.com (accessed on 6 April 2022).

#### **3. Correlations Genotype–Phenotype**

#### *3.1. EB Simplex (EBS)*

EBS is the most common type of EB accounting for ~70% of all EB [7]. EBS has a prevalence of 6/1,000,000 individuals and an incidence of 7.87 per one million live births [20]. EBS is characterized by skin blistering due to intraepidermal cleavage (within the basal layer of keratinocytes) [7]. In general, blistering is caused by trauma, rarely occurs spontaneously, and tends to heal without scarring. EBS has a variable spectrum of severity ranging from mild blistering of the hands and feet to generalized forms with extracutaneous involvement and is sometimes fatal. Onset varies by subtype and occurs, usually, at birth or during infancy, although patients with localized EBS may not develop their first blisters until adolescence or early adulthood [21,22]. Mutations in the *KRT5* and *KRT14* genes occur in 75% of cases with EBS [23]. The most recent EB classification includes 14 EBS subtypes based on the distribution and severity of the blisters, specific cutaneous lesions, mode of inheritance, affected gene/protein, and extracutaneous manifestations [1].

#### 3.1.1. EBS, Localized

The most common and mildest subtype of EBS is localized EBS, previously known as Weber–Cockayne disease, with a reported incidence of 3.67 per one million live births [20], but probably a significant percentage of mild cases remain undiagnosed. Localized EBS is characterized by the formation of blisters usually limited to the palms and soles of the feet. The lesions can also appear in other regions of recurrent trauma, such as the knees and shins of a crawling toddler or flexures during hot weather. The age of onset is variable, and the lesions frequently develop in infancy/early childhood and are rarely present at birth or appear in adolescence/adulthood. Nail involvement is uncommon. Common complications are secondary infections, especially foot blisters. Lesions worsen in the warmer months and some patients develop focal palmoplantar keratoderma during adulthood [22,24]. Intraoral blisters or ulcerations are seen during infancy and usually are asymptomatic [22,25]. The disorder has an autosomal dominant inheritance and is produced by missense mutations in the *KRT14* and *KRT5* genes [26]. The mutations are located outside the highly conserved boundary motifs of the rod domain, usually in the head, tail, or non-helical portions, including the linker area of keratin. They are most frequently found in clusters including in the non-helical L12 linker motif, in the aminoterminal homologous domain (H1) of keratin-5, or in the 2B segment of keratin-14 [27,28]. Hut et al. proposed a genomic mutation detection system for exons 1, 4, and 6 of *KRT14* that encode the 1A, L1-2, and 2B domains containing the mutation hotspots [29]. Jiang et al. suggest that in localized EB sequences, coding for the head and the non-helical linker regions of *KRT5* should have propriety for the mutation screening [30]. However, mutations were also discovered in the conserved 1A and 2B helix hotspots but with conservative amino acid changes [28]. This is consistent with a report by Cho et al. regarding the influence of polarity on the severity of EBS [31].

#### 3.1.2. EBS, Severe, AD, KRT14/5

Severe EBS, with a reported incidence of 1.16 per one million live births, is characterized by generalized and severe blistering and birth-onset. A suggestive feature is the presence of multiple small blisters in a grouped or arcuate configuration, which explains the previous name "EB herpetiformis". Hemorrhagic blisters are also present. The involvement of the oral mucosa and nail dystrophy are common. Mucosal involvement may interfere with feeding, especially in neonates and infants. Inflammation can occur in hemorrhagic blisters followed by milia and hypo- and hyperpigmentation of the skin. The lesions tend to improve with age or paradoxically, in some cases, during periods of heat or fever. Progressive confluent palmoplantar keratoderma is common, precocious (childhood-onset), and more severe than in other subtypes. This subtype is frequently associated with marked morbidity and in a minority of cases with neonatal/infancy mortality [22,24,32,33]. Dominant-negative mutations in *KRT14* and *KRT5* have been clustered

in regions involved in the highly conserved ends of the rod domains or the helix boundary motifs of keratin. Substitutions are frequently reported and involve highly conserved amino acids within the helix initiation or termination motifs blocking the heterodimerization of keratin polypeptides. Common mutations change the glutamic acid from position 477 of keratin-5 (*KRT5* E477) or the arginine from position 125 of keratin-14 (*KRT14* R125). Both mutations cause the extensive formation of cytoplasmic protein aggregates, a hallmark of severe EBS [22]. Monoallelic in-frame deletion, splice-site, or nonsense mutations were also reported, leading to abnormal proteins with dominant-negative effects. Major changes in polarity or acidity are associated with this subtype [34–36]. Around 70% of cases with severe EBS are generated by one mutation in the *KRT5* gene (c.1429G > A; *p*.Glu477Lys or E477K) and three other in the KRT14 gene (c.373C > T [*p*.Arg125Cys or R125C]; c.374G > A [*p*.Arg125His or R125H]; c.368A>G[*p*.Asn123Ser or N123S]) [26]. Vahidnezhad et al. reported a case with digenic inheritance, an association of a mutation in the *KRT5* gene and others in the *KRT14* gene [37].

#### 3.1.3. EBS, Intermediate, AD, KRT14/5

This subtype, previously known as Koebner EBS, has an intermediate phenotype, between localized EBS and severe EBS. Blisters appear at birth or in the first few months with generalized distribution, which are milder than those in severe EBS but without a "herpetiform" configuration. The frequency of milia, scarring, nail dystrophy, and oral lesions is intermediate between that of localized EBS and severe EBS. Focal palmoplantar keratoderma can be observed. Lesions worsen in the warmer months. Lesions tend to improve in adolescence when they may become localized to the hands and feet [7,22,24]. Pathogenic variants in the 1A or 2B segments (except the beginning of 1A, 1B, and the end of 1B, which associate severe phenotype) of the rod domain of *KRT5* and *KRT14* are common in intermediate EBS cases. These mutations do not interfere with the elongation process during filament assembly, so filaments essentially appear normal upon ultrastructural examination but are structurally weakened [38,39].

#### 3.1.4. EBS, Intermediate or Severe, AR, KRT14/5

EBS due to *KRT14* or *KRT5* pathogenic variants is frequently inherited in an autosomal dominant mode but autosomal recessive cases were also reported. Most recessive cases are produced by *KRT14* variants and have a phenotype similar to previously described subtypes, but an improvement in blistering with age is not expected. Focal dyskeratotic skin lesions were also reported. Homozygous mutations in *KRT5* lead to severe phenotype, extracutaneous manifestations, and early mortality. Nonsense, missense, splice site, and deletions in *KRT14* have been associated with recessive inheritance. The unaffected parents of each patient were heterozygous for the respective mutations [40]. Rugg et al. consider that these mutations are likely to be associated with a nonsense-mediated messenger RNA decay leading to a functional "knockout" of keratin-14 [41]. Jonkman et al. suggested that increased expression of keratin-5 has a compensatory effect because keratin-14 knockout mice die within the first few weeks after birth [42].

#### 3.1.5. EBS with Mottled Pigmentation

EBS with mottled pigmentation (EBS-MP) with a reported incidence of 0.07 per one million live births, presents generalized blistering from birth but the severity of the lesions is intermediate. The hallmark feature is mottled or reticulate macular pigmentation typically of the neck, upper trunk, and acral skin. Small hyperpigmented macules appear in early childhood, progress over time, and coalesce into a reticulate pattern. Hypopigmented macules are interspersed. The pigmentation does not occur in areas of blistering and often disappears in adult life. Punctate palmar and plantar keratoderma and nail dystrophy may occur. The majority of cases (more than 90%) presented a missense mutation (c.74C > T [*p*.Pro25Leu or P25L]) in the *KRT5* gene [43]. The pigmentary anomalies observed in this EB form could be correlated with the modification of melanosome transport where the non-

helical head domain of keratin-5 is involved [44,45]. However, the pathogenic mechanism is incompletely deciphered and some modifiers could interfere with the function of keratin -5. For example, in some cases the pathogenic mutation c.356T > C (*p*.Met119Thr or M119T) in the *KRT14* gene was identified [46].

#### 3.1.6. EBS, Migratory Circinate

EBS, migratory circinate is a rare subtype, previously known as EBS with migratory circinate erythema. It is characterized by generalized blistering from birth with a background of inflammatory migratory circinate erythema that fades and heals with hyperpigmentation (sometimes with a mottled pattern) but without scarring. Nail dystrophy may occur. Some mutations were reported in this form of EB. For example, Gu et al. reported a heterozygous deletion c.1649delG (*p*.Gly550fs) in exon 9 of the *KRT5* gene which leads to a frameshift and delayed termination codon in two unrelated families with EBS, migratory circinate. Lee et al. identified a de novo in-frame 12-bp deletion in exon 7 of the *KRT5* gene, which alters the 2B domain of keratin-5 [47]. Mutations in the keratin-5 tail domain have been related to EBS with unusual features, such as mottled pigmentation and pigmentary disorders, suggesting a possible role of this domain in the regulation of inflammation and pigmentation [48,49].

#### 3.1.7. EBS, AD, with PLEC Mutations

Previously known as Ogna EBS, this subtype presents birth-onset and mild skin blistering, mainly acral and occasionally widespread. The characteristic features are easy skin bruising with the formation of violaceous and hypopigmented macules. Koss-Harnes et al. found the same mutation in exon 31 of the *PLEC* gene (c.6328C > T [*p*.Arg2110Trp or R2110W]) in two unrelated families with Ogna EBS. This mutation changes the plectin polypeptide, which connects the basal keratins to the hemidesmosomal plaque, and generates an aberrant ultrastructure of hemidesmosomes' attachment plates and a frequent fragmentation of hemidesmosomes [50]. Bolling et al. identified mutations in *PLEC* in 6/16 of individuals with biopsy-proven EBS who lack identifiable pathogenic variants in *KRT5* or *KRT14* genes. They suggest that *PLEC* mutations may be more common than previously realized [51].

#### 3.1.8. EBS, AR, with PLEC Mutations

This rare subtype with recessive inheritance has a more severe phenotype than the dominant form. EBS with AR *PLEC* mutations is characterized by generalized skin blistering that heals with scarring and hyperpigmentation. Nail dystrophy is severe. Mucous membranes and the heart and muscles are spared [7]. Gostynska et al. identified homozygosity for a nonsense mutation c.46C > T [*p*.Arg16X] in the first exon of the gene encoding plectin isoform 1a, in two sisters from a consanguineous family. Plectin has eight tissue-specific isoforms in humans, arising from the alternate splicing of the first exon. The isoform 1a is not expressed in striated or cardiac muscle tissue, so muscular dystrophy or cardiomyopathy are not expected to develop in these cases [52].

#### 3.1.9. EBS, Intermediate with Muscular Dystrophy

EBS, intermediate with muscular dystrophy (EBS-MD) is an autosomal recessive disorder characterized by early generalized blistering and variable (usually during childhood) onset of progressive limb-girdle type, muscular dystrophy. Considerable variability in the severity of the muscle weakness, sometimes not noticeable until the fourth decade of the patient's life, is reported. Onychodystrophy, focal plantar keratoderma, and mucosal involvement are common. Abnormal dentition (decay teeth), upper respiratory tract stenosis, urethral strictures, dilated cardiomyopathy, ventricular hypertrophy, and alopecia have been reported [53]. The majority of EBS-MD patients present compound heterozygous or homozygous truncation mutations in exon 31 of the *PLEC* gene, which encodes the rod domain of plectin. Natsuga et al. examined plectin expression in the skin

of patients with *PLEC* mutations. In EBS-MD, the expression of the N- and C-terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. The alternative splicing of exon 31, resulting in a rodless but still partially functional plectin, was suggested to account for the milder phenotype. Few EBS-MD cases have in-frame mutations in the N-terminal domain of plectin, where the actin-binding domain (ABD) and spectrin repeats are conserved. It is possible that the binding deficits with integrin beta-4 and the collagen alpha-1(XVII) chain, which may explain the phenotype [54–57].

#### 3.1.10. EBS, Severe with Pyloric Atresia

EBS, severe with pyloric atresia (EBS-PA) presents a severe phenotype with widespread generalized blistering or an absence of skin at birth and pyloric atresia. Antenatally, pyloric atresia can manifest with polyhydramnios. Additional features include failure to thrive, aplasia cutis, anemia, sepsis, intraoral blistering, urethral stenosis, and urologic complications. Death usually occurs in infancy [53]. Immunohistochemical studies showed an absent expression of plectin. In contrast to EBS-MD, EBS-PA patients typically have *PLEC* mutations, nonsense or frameshift, outside of exon 31, which leads to loss of both full-length and rodless plectin. Inheritance is autosomal recessive [58].

#### 3.1.11. EBS, Intermediate with Cardiomyopathy

This subtype is characterized by marked erosions in the limbs at birth, healing with dyspigmentation and cribriform atrophic scars, follicular atrophoderma, and late-onset dilated cardiomyopathy. Keratoderma, milia, nail and oral involvement, and progressive diffuse alopecia are reported [7,34,59]. All cases had a heterozygous gain-of-function in *KLHL24* gene start codon mutation, with c.1A-G being the most prevalent. This mutation produces a truncated KLHL24 protein lacking the initial 28 amino acids (KLHL24-ΔN28). The substrate of the KLHL24 protein is keratin-14 and the more stable KLHL24-ΔN28 due to gain-of-function variants inducing the excessive ubiquitination and degradation of keratin-14. Hee et al. consider that *KLHL24* gene mutations disturb the turnover and degradation of intermediate filaments [60]. Schwieger-Briel et al. showed that KHL24 is expressed at similar levels in keratinocytes and cardiomyocytes and may disrupt the degradation of the structural cytoskeletal proteins involved in mechanical resilience [61]. Hedberg-Oldfords et al. reported familial cases with cardiomyopathy due to *KHLH24* gene mutation with polyglucosan accumulation in some cardiomyocytes and with an accumulation of glycogen, desmin, and tubular structures in the cardiomyocytes and in skeletal muscle fibers. They suggest a pivotal role for KLHL24 during cardiogenesis, based on strong *KLHL24* gene expression in early ventricular myocytes and later in the established heart ventricle. As desmin is the cardiac homologue of keratin-14, Vermeer et al. hypothesized that KLHL24-ΔN28 leads to the excessive degradation of desmin, affecting tissue morphology and function. Also, dominant mutations in desmin are associated with a severe form of cardiomyopathy [9,62,63].

#### 3.1.12. EBS, Localized or Intermediate with Dystonin Deficiency

EBS, localized or intermediate with dystonin (BP230) deficiency presents an earlyonset with predominantly acral blistering, larger (several centimeters) than in localized EBS. The blisters appear in areas of mechanical trauma but also in non-pressure-prone sites. Blistering could heal without scarring or with post-inflammatory hypo- or hyperpigmentation. Asymptomatic plantar keratoderma was reported [64]. Loss-of-function mutations in the *DST* gene lead to a complete absence of hemidesmosomal plaques, a loss of adhesion, and increased cell spreading and migration. Reduced integrin beta-4 at the cell surface and increased levels of keratin-14 and integrin beta-1 were detected in abnormal cells. Mild phenotype, in contrast to autoimmune bullous pemphigoid who have autoantibodies to dystonin, could be explained by an upregulation of keratin-14 expression. The inheritance

is autosomal recessive, but a semi-dominant transmission mode is also plausible because some heterozygous recall some blistering in childhood [34,64,65].

#### 3.1.13. EBS, Localized or Intermediate with Exophilin-5 Deficiency

EBS, localized or intermediate with exophilin-5 deficiency is characterized by localized or generalized intermittent blistering with onset at birth or in early childhood. Skin fragility improves with age, but lesions could heal with hypopigmentation or mottled pigmentation, especially on the trunk and proximal limbs. Skin atrophy and acral blistering with hemorrhagic crusts are cited. Diociaiuti et al. consider that the lack of extracutaneous and adnexal involvement, together with the modest phenotype, differentiates this subtype from the common dominant EBS-MP due to keratin mutations [7,66]. McGrath et al. reported disruption of the keratin filament network, more cortically distributed F-actin, and significantly reduced cell adhesion in keratinocytes from patients with truncating mutations in *EXPH5*. Monteleon et al. demonstrated that exophilin-5 is involved in the delivery of lysosome-related organelles (LROs) to the plasma membrane and is essential for the differentiation of human keratinocytes. LROs are also involved in the packaging and trafficking of melanin, which may explain the pigmentation anomalies. Nonsense and frameshift mutations with autosomal recessive inheritance were reported [1,67–69].

#### 3.1.14. EBS, Localized with Nephropathy

EBS, localized with nephropathy presents early-onset blistering, particularly on pretibial areas associated with nephropathy. Early alopecia, poikiloderma, and nail dystrophy may occur. Involvement of the ocular, oral, gastrointestinal (including esophageal webbing), and urogenital mucosal membranes is reported. Nephropathy manifests with proteinuria and progression to end-stage renal disease [7,34,70]. Homozygous frameshift and splicesite mutations in exon 5 of the *CD151* gene leading to truncated proteins without an integrin-binding domain, were reported [13,53,70,71].

#### *3.2. Junctional EB*

Junctional EB (JEB) is a disease with different prevalence in different geographic areas. The USA National EB Registry reported a prevalence of 0.49 per one million population, whereas the Dystrophic Epidermolysis Bullosa Research Association of America showed a prevalence of 3.59 per million per one million population. In Germany, the prevalence of disease was estimated at 6.7 per one million population [20,72,73]. Early lethality of severe forms could explain the differences. The incidence is higher in the Middle East, due to the high inbreeding coefficient. The inheritance is autosomal recessive and germline mosaicism and uniparental isodisomy were reported [74–76]. In JEB skin cleavage occurs within the lamina lucida of the basement membrane zone. The severity of cutaneous and mucosal fragility varies considerably ranging from forms with early lethality to milder phenotypes. A characteristic feature is represented by mature dental enamel anomalies ranging from small pits in the enamel surface to generalized hypoplasia. Impaired adhesion of the odontogenic epithelium from which ameloblasts are derived is involved in abnormal enamel formation [7,22,25]. On a clinical basis, JEB was divided into several categories.

#### 3.2.1. JEB, Severe

In severe JEB, previously known as Herlitz JEB, extensive mucocutaneous blistering with early-onset (at birth or in the neonatal period) may lead to large erosions with extensive loss of proteins, fluids, and iron, which increases susceptibility to infection and electrolyte imbalance. Sometimes, at birth, blisters may be mild and localized to periungual, buttock, or elbow regions [7,22]. The pathognomonic feature is an exuberant granulation tissue located in orofacial (which produces microstomia), periungual, or friction regions, Accumulation of subglottic granulation tissue may lead to a weak, hoarse cry, stridor, and respiratory distress. Alopecia and mature dental enamel defects are common. Involvement of the mucous membranes of the upper respiratory tract, esophagus, bladder, urethra, rectum, and cornea has been reported. Scarring pseudosyndactyly of the hands and feet with severe loss of function has been cited. JEB has the highest risk of infant mortality among the EB subtypes, and the major causes are sepsis, failure to thrive, or tracheolaryngeal obstruction [7,22,24,25,72]. Biallelic mutations in the *LAMA3*, *LAMB3*, and *LAMC2* genes were identified in severe JEB. Varki et al. reported a high proportion of *LAMB3* mutations. The majority lead to premature stop codons, mRNA decay, and synthesis of no protein or to truncated unstable polypeptides. The most frequent mutation in the *LAMB3* gene (45–63%) is c.1903C > T (*p*.Arg635Ter or *p*.R635X) [77]. The distribution of laminin 332 in multiple epithelial basement membranes, including those of the cornea, kidney, lung, thymus, brain, gastrointestinal tract, and lung explains the extracutaneous features [14,15]. However, Abu Sa'd et al. reported a case of severe lethal JEB caused by a homozygous mutation in the *COL17A1* gene [76].

#### 3.2.2. JEB, Intermediate

*JEB*, intermediate previously called JEB non-Herlitz, presents a less severe clinical phenotype than JEB severe, with a reduced tendency to develop exuberant granulation tissue. Generalized blisters (that predominate in sites exposed to friction, trauma, or heat), heal with atrophy and pigmentation anomalies. Alopecia, enamel defects, and dystrophy or absence of nails are common. Also, a milder involvement of the mucous membranes of the upper respiratory tract (with a lower risk of upper airway occlusion), bladder, and urethra was reported, and adult patients have an increased risk of developing squamous cell carcinoma on their lower extremities in areas of chronic blistering, long-standing erosions, or atrophic scarring [22,24,78]. Specific mutations in the *LAMA3*, *LAMB3*, and *LAMC2* genes (missense or splice-site or compound heterozygosity) that lead to partially functional laminin 332 are reported in this subtype. The intermediate JEB phenotype is also associated with mutations in the *COL17A1* gene. The hallmark of these phenotypes was the total lack of collagen XVII in the skin due to different mutation mechanisms (nonsense/insertions and deletions predicted to result in premature termination/splice site with the production of truncated unstable molecules). The majority of mutations were located in exons 51 and 52. Notably, splice-site mutations occurred preferentially in intron 51 [79]. In this subtype, Jonhman and Pasmooji reported the first cases with revertant mosaicism, both in collagen and laminin deficiency, sustained by the reexpression of the deficient protein on skin specimens. Revertant mosaicism has since been documented in EB forms, implicating the *COL17A1*, *KRT14*, *LAMB3*, *COL7A1*, and *FERMT1* genes [80,81]. Cases with self-improving JEB and milder than expected phenotypes were also reported. The possible underlying molecular mechanisms are an alternative modulation of splicing, a spontaneous readthrough of premature termination codons, or a skipping of exons containing stop codons [1,82–84].

#### 3.2.3. JEB, with Pyloric Atresia

JEB, with pyloric atresia, presents an association between generalized blistering at birth and pyloric atresia. Other gastrointestinal anomalies, such as duodenal and anal atresia, are rarely reported. Aplasia cutis congenita, atrophic scarring, enamel anomalies, oral involvement, and nail dystrophy with patulous nail folds are common. Exuberant granulation tissue in the perioral, neck, and upper back regions may occur. This disorder is associated with a significant risk of genitourinary anomalies (polypoid bladder lesions, urethral stricture, dysplastic kidney, hydronephrosis, ureterocele) and infantile or neonatal death [14,85,86]. JEB with pyloric atresia is associated with mutations in the *ITGA6* or *ITGB4* genes. The majority of the mutations reside in the *ITGB4* gene, being nonsense (with the formation of a premature stop codon) or missense mutations in the amino-terminal extracellular domain that facilitate the association of the alpha-6 or beta-4 subunits. Loss of function mutations in the *ITGA6* gene have been identified in some cases [14,53,87]. The absence of alpha-6 integrins modifies the adhesion of the collecting duct cells to the basal membrane and makes the kidney-collecting system susceptible to degeneration and injury, which explains its renourinary features [88]. DeArcangelis et al. demonstrated by alfa-6 integrin ablation in mice that loss of intestinal epithelial cells/basal membrane interactions initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma [89].

#### 3.2.4. JEB, Localized

Localized JEB is characterized by mild blistering, often acral, variable nail dystrophy, enamel defects, and a tendency to develop cavities. In contrast to the other JEB subtypes, alopecia, extensive atrophic scars, and extracutaneous findings are rarely reported [24,79]. Mutations in the *LAMA3*, *LAMB3*, *LAMC2*, *COL17A1*, *ITGB4*, and *ITGA3* genes were reported. Mutations allowing the expression of a residual protein (usually missense or splicing) lead to this mild phenotype. Condrat and Has stated that as little as 5–10% of residual protein, even if truncated and putatively partially functional, significantly alleviates the phenotype. Some mutations in *COL17A1* that are predicted to lead to a premature stop codon (associated with severe phenotype) escape this outcome because of alternative splicing. Out of the 56 exons of *COL17A1*, 54 are in-frame and can be skipped without shifting the reading frame [57,90,91].

#### 3.2.5. JEB, Inversa

Congenital blistering and erosions confined to flexural areas are suggestive of this rare form of JEB. Blistering is usually severe and may heal with atrophic scarring and milia formation. Nail dystrophy, enamel anomalies and dental caries, oral, esophageal, and vaginal involvement are common. Reduced expression of laminin 332 due to biallelic mutations in the *LAMA3*, *LAMB3*, and *LAMC2* genes was reported [32,92].

#### 3.2.6. JEB, Late-Onset

In contrast to the other subtype of JEB with early-onset, in JEB with late-onset (JEB-lo) the blistering starts in childhood and affects the hands and feet and, to a lesser extent, the elbows, knees, and oral mucosa. Other clinical features are palmoplantar hyperhidrosis, enamel defects, and progressive skin atrophy. The disappearance of dermatoglyphs because of scarring is reported [7]. Yuen et al. reported cases with mutations located in the fourth noncollagenous domain (NC4) of the alpha-1(XVII)-chain gene (c.3908G > A, [*p*.R1303Q or *p*.Arg1303Gln]), which are predicted to affect protein folding and laminin 332 binding. They suggested that missense mutations located in the NC4 domain may be specific for JEB-lo [78].

#### 3.2.7. Laryngo–Onycho–Cutaneous Syndrome

Laryngo–onycho–cutaneous syndrome (LOC), previously called Shabbir syndrome, is characterized by a hoarse cry in the neonatal period, by marked exuberant granulation tissue, in particular affecting the larynx, conjunctiva, and periungual/subungual sites, and by skin blistering and erosions. In contrast to the excessive blistering and erosions described in severe JEB, patients with LOC have minimal blistering but more extensive granulation tissue. Ocular granulation tissue may extend leading to symblepharon and corneal opacification (suggestive features of LOC). Progressive laryngeal granulation can lead to severe respiratory compromise and premature death. Aberrant granulation tissue could also develop on the face, neck, epiglottis, trachea, and main bronchi. Nail dystrophy and enamel anomalies are common [7,32,93,94]. In the affected members of 15 families, McLean et al. identified a homozygous single nucleotide insertion in the *LAMA3* gene (c.151dup; [V51fs]), predicting a stop codon in exon 39 that is specific to laminin alpha-3A, a protein secreted only by the basal keratinocytes of stratified epithelia. They suggested that LOC may be caused by the dysfunction of keratinocyte–mesenchymal communication and hypothesized that the laminin alpha-3A N-terminal domain may be a key regulator of the granulation tissue response. All cases reported are of Punjabi origin, suggesting a

possible founder effect. Prodinger et al. reported 3 new mutations in the *LAMA3* gene, outside exon 39 and underscores that molecular diagnostics can be challenging [93,95].

#### 3.2.8. JEB, with Interstitial Lung Disease and Nephrotic Syndrome

The association of congenital nephrotic syndrome, interstitial lung disease, and skin fragility is suggestive of JEB with interstitial lung disease and nephrotic syndrome (ILNEB). The respiratory and renal features predominate and rapid progression usually leads to death in early infancy. The renal anomalies occurring in patients with ILNEB include congenital nephrotic syndrome, focal-segmental glomerulosclerosis, bilateral renal cysts, unilateral kidney hypoplasia, and ectopic conjoint kidney. Patients present variable degrees of cutaneous involvement, nail dystrophy, and sparse hair. Cases with mild phenotypes (without renal anomalies or without lung disease) were reported [34,96,97]. Biallelic mutations (missense, frameshift, or in splice sites) in the *ITGA3* gene were identified. Has et al. reported cases with functionally null mutations and a severe course of disease. Mutations allowing expression of a residual, truncated, or dysfunctional protein may lead to a milder phenotype and improved survival. Lin et al. stated that the phenotype of the *ITGA3* gene mutation may be determined by the residual function of the mutant integrin alpha-3 strain [98,99].

#### *3.3. Dystrophic EB*

In dystrophic EB (DEB) the plane of skin cleavage is below the lamina densa in the most superficial portion of the dermis. DEB may be inherited in a dominant (DDEB) or recessive (RDEB) pattern. The prevalence of DDEB and RDEB is quite similar: 1.49 and 1.35 per one million live births respectively [20]. In DEB, blisters, and ulcerations heal with significant scarring and milia formation. Generally, the recessive form is more severe than DDEB; however, there is significant phenotypic overlap between subtypes. All subtypes of DEB are caused by mutations in the *COL7A1* gene, the gene coding collagen VII, the main constituent of the anchoring fibrils at the cutaneous basement membrane zone [1,24,57]. Hovnanian et al. stated that the nature and location of these mutations are important determinants of the phenotype [100]. Mariath et al. suggested that the DEB phenotype is determined by the expression and residual function of collagen VII [24].

#### 3.3.1. DDEB

The majority of DDEB cases result from dominant-negative mutations. Missense substitutions that replace glycine in the collagenous triple-helical domain (frequently in exons 73, 74, and 75) are reported in over 75% of cases. The most common DDEB-causing mutations are c.6100G>A(*p*.Gly2034Arg or G2034R) and c.6127G>C(*p*.Gly2043Arg or G2043R) [101,102]. The conservation of glycine residues in every third position of the amino acid sequence is required for the tied packing of the triple helix and these substitutions highly destabilize the triple helix [103]. Other substitutions, insertions, deletions, and splicesite variants have also been described. These mutations involve amino acids essential for the structure of the triple helix and the stability of the anchoring fibrils. However, an interand intrafamilial phenotypic variability is reported [86,104,105].

#### Intermediate DDEB

This subtype presents with generalized blisters from birth or early infancy, milia, albopapuloid lesions, atrophic scarring, and nail dystrophy. Acral sites, elbows, and knees are commonly affected. Mucous membranes may also be involved leading to microstomia, ankyloglossia, and esophageal stenosis, although less commonly than in severe RDEB [1,24].

#### Localized DDEB

Blistering is confined to the hands, feet, and milia and atrophic scars can also occur. There is no extracutaneous involvement. Rare cases with progressive nail dystrophy and

without any other sign of skin fragility are reported [106]. A pretibial form with the development of lesions predominantly in the anterior lower legs is described [107].

### 3.3.2. RDEB

Severe RDEB

The most severe subtype of DEB, formerly known as Hallopeau-Siemens RDEB, is associated with generalized blistering at birth, progressive extensive scarring, and development of microstomia, ankyloglossia, esophageal stenosis, flexion contractures of limbs, and pseudosyndactyly. Alopecia, milia, and permanent loss of nail plates are common. Eye involvement with corneal erosions, symblepharon, ectropion, and loss of vision is also observed [32,108,109]. The lifetime risk of aggressive squamous cell carcinoma is greater than 90% [110]. Biallelic nonsense or frameshift *COL7A1* gene mutations (insertions/deletions, substitutions, or splice sites) that result in premature termination codons were reported. The consequences for the protein are severe: the absence of or a markedly reduced collagen VII [101,105,110,111].

#### Intermediate RDEB

Phenotype is similar to intermediate DDEB, but with greater severity of joint contractures and pseudosyndactyly in some cases. Extracutaneous involvement is milder than in severe RDEB. The risk of developing squamous cell carcinomas is also increased (47.5% by age 65) but less common than in severe RDEB and neoplasia occurs later in adulthood [7,108,110]. Many patients are compound heterozygous for a premature stop codon and a glycine substitution within the collagenous domain. The mutations may affect the association of polypeptides and the stability of the triple helix or may cause conformational change [14,86,105].

#### RDEB, Inversa

This rare subtype is characterized by a peculiar course. Generalized blistering of intermediate severity occurs in the neonatal period, improves with age, and tends to localize to flexure sites in adults. Mucosal involvement (oral, esophageal, anal, genitourinary) is similar but milder than in severe RDEB [32,112,113]. Van den Akker et al. reported specific glycine or arginine substitutions in the carboxyl portion of the triple-helical domain caused by a missense mutation in the *COL7A1* gene. Patients were homozygotes or compound heterozygotes (missense mutation/loss of function mutation). The localization of the amino acid substitutions in specific domains correlates with the synthesis of a thermolabile collagen VII that is specifically less stable in the warm flexural regions [112,113].

#### RDEB, Localized

The phenotype is similar to localized DDEB. Splice-site mutations and other amino acid (non-glycine) substitutions were reported. In localized RDEB, splice-site mutations result in exon skipping, without altering the remaining protein sequence. This abnormal collagen VII allows the assembly of the anchoring fibrils with small functional defects, which explains the phenotype [61,100,111].

#### 3.3.3. DEB, Pruriginosa

DEB, pruriginosa (*DEB-Pr*) is an unusual subtype that presents blistering in infancy and late-onset (adolescence/adulthood) of intense pruritus and linear cords of lesions (papules, nodules), especially on the extensor surfaces of the limbs (initially on the lower legs). Nail dystrophy, milia, and atrophic scarring are common [7]. Cases with autosomal dominant and autosomal recessive inheritance have been described and glycine substitutions in the collagenous domain, splice-site mutations, and small deletions have been reported. Some of these mutations have been reported in cases with other subtypes of DEB, without pruritus. No specific correlation of the genotype–phenotype has been established. Patients were shown to synthesize a normal or variably reduced amount of type VII

collagen, which was correctly deposited at the dermal–epidermal junction [104,114–117]. Studies have excluded other triggering factors, including atopy, elevated IgE levels, matrix metalloproteinase 1 gene polymorphisms, filaggrin gene mutations, and interleukin 31 gene haplotypes [117–120].

#### 3.3.4. DEB, Self-Improving

Previously known as transient bullous dermolysis in a newborn, this rare subtype is characterized by generalized blistering at birth followed by significant improvement within the first 2 years of life [22]. Both dominant and recessive inheritance have been reported in cases of self-improving DEB. The most frequently reported mutations are glycine substitutions and splice-site variants resulting in the skipping of exons (e.g., exon 36). The immunofluorescence shows the accumulation of granular intraepidermal deposits of collagen VII, which regresses with time [121,122]. Christiano et al. suggested that with advancing age, the abnormal polypeptides become degraded at an increasing rate, thus diminishing their dominant-negative effects. The genotype–phenotype relationship remains unclear because of the limited number of cases [123–125].

#### 3.3.5. DEB, Severe, Dominant, and Recessive (Compound Heterozygosity)

The phenotype is indistinguishable from severe RDEB, with severe mucocutaneous involvement from birth. Compound heterozygosity for dominant *COL7A1* glycine substitution mutation and recessive mutation (frameshift leading to a premature termination codon) on the second allele has been reported [14,126–128].

#### *3.4. Kindler EB*

In contrast to other types of EB, Kindler EB (KEB) presents a blister formation at different levels of the dermal–epidermal junction: below the lamina densa, within the lamina lucida, or within basal keratinocytes. A single or multiple cleavage planes may be seen within the same sample of skin. KEB manifests with generalized blistering (more prominent on extremities) at birth followed by the development of photosensitivity and progressive poikiloderma. Palmoplantar keratoderma and skin atrophy may occur. Extracutaneous findings include chronic gingivitis, periodontitis, esophageal strictures, ectropion, anal stenosis, and colitis. Pseudosyndactyly has been reported. Patients with KEB have an increased risk of developing cutaneous squamous cell carcinoma (66.7% in those >60 years of age), usually occurring in the fourth to fifth decade of life [22,129]. KEB is caused by a homozygous mutation in the *FERMT1* gene. Zhang et al. suggested that fermitin family homolog 1 is also important for the suppression of UV-induced inflammation and DNA repair [130]. The protein is predominantly expressed in the epithelial cells in the skin, oral mucosa, and the gastrointestinal tract, explaining the distribution of manifestations [131,132]. Deletions, insertions, nonsense, splice-site, and missense mutations (majority loss-of-function) have been reported. Has et al. suggested that mutations compatible with the expression of an abnormal protein (e.g., in-frame) will translate into mild phenotypes, whereas null mutations cause severe forms [133].

#### **4. Current Molecular Approach in Therapeutics**

Molecular therapies for EB are conducted in correlation with the mutant genes and specific mutations. They are represented by gene-replacement therapies, gene editing, natural gene therapy, exon skipping, protein therapy, read-through therapies, and small molecules repurposed to relieve symptoms [134]. However, all these treatment methods are still in the phase of therapy trials. Has et al. summarize these gene therapy trials. Mainly recessive dystrophic EB and the type VII collagen and type XVII collagen proteins are targeted. There are ongoing trials in phase I/II in which interventions consist of the ex vivo grafting of gene-corrected epidermal sheets with a gamma-retroviral vector carrying *COL17A1* cDNA or *COL7A1* cDNA [134,135]. The gene-editing strategies are in the preclinical phase and use gene correction in keratinocytes or fibroblasts from patients

with RDEB and the skin grafts are transplanted into immunocompromised mice. There are also studies for the JEB and *LAMB3* genes and the EBS and *KRT14* genes [134].

RNA-based therapies use antisense oligonucleotides (ASO) for in-frame exon skipping in the *COL7A1* gene. There are preclinical studies with good results in the skipping exons 13, 70, 73, 80, or 105 in the *COL7A1* gene. A clinical trial testing the ASO-targeting exon 73 in *COL7A1* is currently ongoing [134].

Protein therapy uses recombinant type VII collagen and a phase I/II clinical trial is ongoing in order to evaluate its safety and tolerability in adults with RDEB [134].

The read-through therapies use small molecular-weight compounds, which incorporate an amino acid in a place of a stop codon and in such a way as to suppress the nonsense mutations. Gentamicin was used in clinical studies for RDEB and JEB and also amlexanox to induce the read-through of *COL7A1* [134].

A small molecule used in clinical trials for the reduction of fibrosis (a major complication of RDEB) is losartan [134,136].

#### **5. Conclusions**

Epidermolysis bullosa is characterized by high-clinical, allelic, and locus heterogeneity. These features could be explained by the multitude of proteins that are involved in communication and signaling at the basal layers of the skin. In addition, the phenotypes are overlapping and different mutations in the same genes produce the different forms of the disease. The deciphering of pathogenic mechanisms corroborated with the discovery of the genotype–phenotype correlations and will form the basis of personalized management and the prevention of complications.

**Author Contributions:** All authors contributed equally to this article-type review. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **A Positive Dermcidin Expression Is an Unfavorable Prognostic Marker for Extramammary Paget's Disease**

**Shun Ohmori, Yu Sawada \*, Natsuko Saito-Sasaki, Sayaka Sato, Yoko Minokawa, Hitomi Sugino, Hikaru Nanamori, Kayo Yamamoto, Etsuko Okada and Motonobu Nakamura**

> Department of Dermatology, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-Ku, Kitakyushu, Fukuoka 807-8555, Japan<EMAIL_ADDRESS>(S.O<EMAIL_ADDRESS>(N.S.-S<EMAIL_ADDRESS>(S.S<EMAIL_ADDRESS>(Y.M<EMAIL_ADDRESS>(H.S<EMAIL_ADDRESS>(H.N<EMAIL_ADDRESS>(K.Y<EMAIL_ADDRESS>(E.O<EMAIL_ADDRESS>(M.N.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +81-093-691-7445

**Abstract:** Extramammary Paget's disease is recognized as an apocrine-origin cutaneous tumor and is localized in the intraepithelial skin lesion. However, its advanced form is intractable, and there is currently no therapeutic option with a satisfactory level of clinical outcome. Therefore, it is of great importance to identify a potential biomarker to estimate tumor advancement in extramammary Paget's disease. Dermcidin is an antimicrobial peptide derived from the eccrine gland and is identified as a biomarker in various malignancies. To investigate the potential of dermcidin in extramammary Paget's disease, we investigated dermcidin expression in tumors using the immunostaining technique. Although previous studies have reported that extramammary Paget's disease has no positive staining against dermcidin, 14 out of 60 patients showed positive staining of dermcidin in our study. To clarify the characteristics of positive dermcidin in extramammary Paget's disease, we investigated the clinical characteristics of positive dermcidin extramammary Paget's disease patients. Positive dermcidin patients showed a significantly high frequency of lymph node metastasis. We next investigated the impact of positive dermcidin on overall survival. Univariate analysis identified that positive dermcidin showed a significantly increased hazard ratio in overall survival, suggesting that dermcidin might be a prognostic factor for extramammary Paget's disease.

**Keywords:** extramammary Paget's disease; dermcidin; prognosis; lymph node metastasis; survival

#### **1. Introduction**

The skin is a large surface organ in the human body and is complicatedly organized with various unique cells and glands to adjust to external environmental changes [1–3]. Due to this unique characteristic of skin as a peripheral organ, various types of tumors, including malignancies, have the chance to emerge in it. In the early phase of tumor development, the tumor is localized in the skin, which can be adequately targeted through skin-focused local treatment, such as surgical resection [4]. However, once it progresses to an advanced form and causes distant organ metastasis, it is intractable due to the limited number of therapeutic options against these metastatic skin cancers [5]. Despite recent advancements in immune checkpoint and molecular targeted therapy, this therapeutic approach has still not reached a satisfactory level to obtain positive clinical outcomes [6].

The origin of extramammary Paget's disease is believed to be the apocrine glands. This is because the disease usually arises in the genital, perianal, and axillary regions, where the apocrine glands are located [7]. In agreement with this, previous histological studies have identified that extramammary Paget's disease showed positive immunoreactivity against gross cystic disease fluid protein (GCDFP)-15, carcinoembryonic antigen (CEA), and cytokeratin (CK) 7, all of which receive the same response from the apocrine glands [8]. In general, patients with extramammary Paget's disease have a good prognosis, with a 5-year

**Citation:** Ohmori, S.; Sawada, Y.; Saito-Sasaki, N.; Sato, S.; Minokawa, Y.; Sugino, H.; Nanamori, H.; Yamamoto, K.; Okada, E.; Nakamura, M. A Positive Dermcidin Expression Is an Unfavorable Prognostic Marker for Extramammary Paget's Disease. *Diagnostics* **2021**, *11*, 1086. https:// doi.org/10.3390/diagnostics11061086

Academic Editor: Yasuhiro Sakai

Received: 8 May 2021 Accepted: 11 June 2021 Published: 14 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

overall survival of 75% to 95%, because the disease is localized in the epidermis [4,9–12]. In contrast, dermal invasion is closely associated with lymph node metastasis and poor prognosis [8]. However, there is a limited number of biomarkers that can help toward the prognosis of extramammary Paget's disease.

A unique antimicrobial peptide was identified in 2001, i.e., dermcidin., which is constitutively produced by sweat glands. Additionally, an abundance of dermcidin was detected in sweat, showing a beneficial impact on antimicrobial action against microorganisms [13]. In contrast to its beneficial effect on the human body, however, dermcidin can also play an important role in the development of malignant tumors and other diseases. Based on this property of dermcidin, its usefulness as a biomarker for various diseases has been evaluated. Because extramammary Paget's disease is believed to be a malignant tumor derived from the apocrine glands, representing a negative expression of dermcidin in normal tissue, the possible role of dermcidin as a biomarker in patients with extramammary Paget's disease has not been investigated.

In this study, we investigated the potential of dermcidin as a biomarker for the prognosis of extramammary Paget's disease. We identified that there are two groups, dermcidin-positive and -negative extramammary Paget's disease. In addition, dermcidinpositive patients showed an unfavorable clinical behavior and a high frequency of lymph node metastasis. Our results suggest that dermcidin might be an independent prognostic factor in patients with extramammary Paget's disease.

#### **2. Materials and Methods**

#### *2.1. Patient Population*

In total, 60 patients who underwent surgery as an initial form of treatment for extramammary Paget's disease at the Department of Dermatology, University of Occupational and Environmental Health, were enrolled in this study from December 1979 to August 2017. The diagnosis was based on histopathological analysis carried out by two independent pathologists. Tissue specimens of the tumor were obtained from patients who underwent surgery at our institution. Because of the rarity of this malignant cutaneous tumor [14], it is sometimes difficult for a diagnosis of extramammary Paget's disease to be made, especially as it is hard to distinguish the disease from the pagetoid phenomenon. To exclude a pagetoid phenomenon, perianal extramammary Paget's disease was evaluated via GCDFP15+ and CK20- to determine the correctness of the diagnosis of extramammary Paget's disease. Patients were categorized according to the degree of dermcidin expression, age, sex, and the presence of depigmentation in the skin lesion.

#### *2.2. Immunostaining for Dermcidin*

Immunostaining was performed as reported previously [15,16]. In brief, immunochemical staining for dermcidin was conducted using two dermcidin monoclonal antibodies (mAbs) (A-20 and N-20) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on formalinfixed, paraffin-embedded specimens. In brief, specimens were cut into 4 μm thick sections and then deparaffinized in xylene and dehydrated through graded alcohol solutions. Antigen retrieval was achieved via boiling in citrate buffer, pH 6.0, using a microwave treatment. All sections were treated with methanol containing 0.3% H2O2 for 15 min to block endogenous peroxidase activity. Immunoglobulin G was treated using normal rabbit serum (Nichirei, Tokyo, Japan) to avoid nonspecific antibody binding. After overnight incubation at 4 ◦C with mouse anti-dermcidin mAb (Lifespan BioSciences, Inc., Seattle, Washington, DC, USA), the sections were incubated with biotinylated rabbit–anti-mouse secondary antibody (Nichirei, Tokyo, Japan) followed by incubation in a streptavidin–peroxidase complex solution for 30 min. Signals were generated via incubation with 3-amino-9-ethyl carbazole to visualize the immunostaining. The expression of dermcidin was classified into 2 groups: dermcidin-positive patients and dermcidin-negative patients. Negative dermcidin indicated absolutely no immunostaining reaction to anti-dermcidin antibody in both specific antibodies.

#### *2.3. Statistical Analyses*

Fisher's exact test for unpaired data was used to analyze the association between dermcidin expression and various clinicopathologic factors. Univariate analyses of overall survival were conducted using the log-rank test, and Kaplan–Meier curves were generated. Overall survival was calculated from the date of first diagnosis to the date of death or latest contact with the patient. Univariate analysis was performed using the SPSS software (IBM Corp., Armonk, NY, USA). Kaplan–Meier survival analyses and Fisher's test were performed using GraphPad Prism 4.0. The senser on the survival curve means still alive or discontinuation of follow-up observation during this study period.

#### *2.4. Microarray Data Analysis*

For microarray data analysis, dermcidin mRNA expression in healthy subject tissues was obtained from a public data set deposited in the National Center for Biotechnology Information (NCBI) obtained from the Gene Expression Omnibus (GEO) database (GEO accession no. GDS3834) [17]. mRNA was extracted from human tissues, which were purchased from commercial vendors and subjected to microarray analysis.

#### *2.5. Study Approval*

Our retrospective study was approved by the Institutional Review Board at the University of Occupational and Environmental Health following the Declaration of Helsinki. Because this study was a retrospective cohort study, the opt-out method of obtaining informed consent was adopted, and informed consent was waived by the Institutional Review Board at the University of Occupational and Environmental Health.

#### **3. Results**

#### *3.1. The Finding of Dermcidin-Positive Extramammary Paget's Disease*

Dermcidin is produced by the eccrine glands, which are located in the skin. In agreement with this, microarray data set analysis showed that dermcidin expression was highest in the skin from healthy human tissues (Figure 1A). In addition, we confirmed that two antibodies against dermcidin showed a specific positive reaction to the eccrine glands (Figure 1B), suggesting that these antibodies reflect the positivity of dermcidin in the skin.

It has previously been reported that the expression of dermcidin was not identified in epithelial tumors, melanoma, and extramammary Paget's disease [18]. Although there were cases with no staining of dermcidin (Figure 1C), these antibodies showed that several patients diagnosed with extramammary Paget's disease had different expression patterns of dermcidin in the tumor, such as minuscule, average, and strong expression (Figure 1D). These unexpected results prompted us to investigate the characteristics of extramammary Paget's disease with or without dermcidin-positive reaction in further detail.

#### *3.2. The Different Characteristics of Extramammary Paget's Disease Depending on the Expression Degree of Dermcidin*

Although extramammary Paget's disease is usually characterized by no expression of dermcidin, as reported previously, we speculated that there were differences in clinical characteristics in extramammary Paget's disease between positive and negative expression of dermcidin.

To clarify this issue, we investigated the differences in age, sex, depigmentation as the manifestation of extramammary Paget's disease, and lymph node metastasis between dermcidin high- and low-expressing groups (Tables 1 and 2). Although there was no significant difference in age, sex, and depigmentation of the tumor, we noticed that the dermcidin-positive group showed a significantly high frequency of nodules and erosion of the tumor upon physical examination. In addition, the dermcidin-positive group also showed a high frequency of dermal invasion and lymph node metastasis. Dermal invasion cases enrolled in this study showed an unfavorable 5-year survival rate of 68.8% (*p* < 0.0001)

(Figure 2). These findings suggest that the positive expression of dermcidin might reflect the extension of tumor development.

**Figure 1.** Dermcidin expression in healthy tissue and in the skin and positive staining in extramammary Paget's disease. (**A**) Microarray dataset analysis of dermcidin gene expression; (**B**) representative dermcidin immunostaining for eccrine glands in healthy subjects using two different immunostaining antibodies; (**C**,**D**) representative negative and (**C**,**D**) positive staining patterns of dermcidin were observed in extramammary Paget's disease tumor in both intraepithelial and dermal invasive tumors. (**C**) Scale bar: 100 μm; (**D**) scale bar: minuscule expression and average expression were determined at 100 μm, and strong expression was determined at 50 μm.

**Figure 2.** Difference of overall survival curve with or without dermal invasion in extramammary Paget's disease. The overall survival curves were drawn using the Kaplan–Meier method and were compared with the log-rank test.

**Table 1.** Clinical characteristics of extramammary Paget's disease patients in this study.

**Table 2.** Difference in clinical characteristics in dermcidin expression.

#### *3.3. The Different Prognosis in Extramammary Paget's Disease*

We next investigated the prognostic impact of dermcidin in extramammary Paget's disease. The mean survival times were different between positive and negative dermcidin expression in the tumor. Kaplan–Meier curves of overall survival are shown in Figure 3. The overall survival rate in dermcidin-positive patients was significantly lower than that in dermcidin-negative patients. Therefore, a high expression of dermcidin is associated with the poorest prognosis.

Finally, we conducted univariate analyses of dermcidin expression in comparison with clinical variables (Table 3). Univariate analysis showed significantly increased hazard ratios in nodules upon physical examination and dermal invasion and lymph node metastasis in the histological examination, consistent with previous studies [4,19]. In addition, a high expression of dermcidin leads to a significantly increased hazard ratio. Although the impact of dermcidin on prognosis might be limited, dermcidin might become a tool for estimating prognosis in patients with extramammary Paget's disease in some cases.

**Figure 3.** Differences in overall survival curve with or without dermcidin expression in extramammary Paget's disease. The overall survival curves were drawn using the Kaplan–Meier method and were compared with the log-rank test.

**Table 3.** Univariate analysis of clinical variables.

#### **4. Discussion**

This study revealed that positive dermcidin expression reflects unfavorable clinical behavior in extramammary Paget's cell tumors. Cancer cell migration into lymph nodes is an important step in the progression toward the advanced stage of malignant tumors. However, the detailed molecular mechanism determining whether dermcidin promotes such tumor cell migration remains unclear.

Our study showed that dermcidin-positive extramammary Page's disease exhibited a high frequency of nodules upon physical examination. One of the reasons behind this might be that dermcidin contributes to the development of the tumor. High dermcidin expression is associated with tumor growth in gastric [20] and breast cancer [21].  As regards the mechanisms, dermcidin has been found to modulate the HER-2-mediated signal pathway [21], which is one of the major pathways in breast cancer [22]. Because HER-2 signaling is also involved in the pathogenesis of extramammary Paget's disease [23], it is assumed that dermcidin might also activate HER-2 signaling in extramammary Paget's disease and subsequently lead to the development of tumor growth. Because there was no commercially available cell line of extramammary Paget's disease, however, further investigation will be required to clarify the detailed molecular mechanisms.

Several studies have already shown the potential of dermcidin as a biomarker for malignancies. A high expression of dermcidin was identified in approximately 10% of breast cancer patients and has been found to be closely associated with the advanced clinical stage and unfavorable clinical behavior due to regulation of tumor cell growth [24]. Serum dermcidin levels were significantly increased in hepatocellular carcinoma patients and were positively correlated with metastasis [25]. Dermcidin expression in gastric cancer reflects overall survival and is positively correlated with lymph node metastasis [20]. Dermcidin expression is higher in lung cancer patients compared with that in healthy subjects [26]. Among cutaneous malignancies, having high serum levels of dermcidin at the moment of melanoma diagnosis has been associated with the metastatic progression of melanoma among melanoma patients [27,28].

Dermcidin has also been reported as a biomarker in various diseases in addition to malignant tumors. Dermcidin has been identified as a biomarker for Alzheimer's disease (AD) [29], asthma [30], acne vulgaris [31,32], severe obstructive sleep apnea [33], and facioscapulohumeral muscular dystrophy [34]. For example, an abundance of dermcidin was identified in exhaled breath condensate in asthma patients [30]. Therefore, dermcidin may also be a potential biomarker in a variety of skin diseases.

The reason that positive dermcidin expression was observed in clinical patients with unfavorable outcomes who suffered from extramammary Paget's disease remains unclear. A previous study suggested the possibility that one of the characteristics of the eccrine glands might be linked to extramammary Paget's disease. The expressions of histoblood group A type 1, 2, and 3 antigens in normal human skin and extramammary Paget's disease were examined via the immunohistochemical technique [35]. The eccrine glands expressed these antigens, while a negative expression of these antigens was observed in apocrine glands, suggesting that extramammary Paget's disease might be an apocrinegland-derived tumor stemming from a negative reaction to these antigens. However, 7 out of 16 cases were positive for these antigens, and 6 out of 7 positive cases were associated with dermal invasion. Meanwhile, 5 cases without dermal invasion were negative against these antigens. Although there has been a limited number of studies focusing on this issue to date, the possibility still exists that eccrine gland characteristics include the development of extramammary Paget's disease.

A previous study showed a negative dermcidin expression in patients with extramammary Paget's disease [18]. However, we speculated that the reason behind this result might be that this study did not include any unfavorable clinical cases to show a representative tumor phenotype of extramammary Paget's disease, which is generally located in the epidermis. This previous study may have selected noninvasive extramammary Paget's disease samples to visualize a representative sample of an indolent cutaneous tumor from an extramammary Paget's disease patient.

One possible limitation of our study was that the number of patients involved might not be sufficient to investigate the more detailed characteristics of dermcidin-positive patients with extramammary Paget's disease. Additionally, the detailed molecular role of dermcidin in extramammary Paget's disease for the invasion and metastasis of tumors still needs to be clarified, especially how the degree of dermcidin is associated with the activation of metastatic factors and tumor development, which are involved in the molecular mechanism mediated by HER-2 signaling.

The reason that many of the censored cases are in the dermcidin-negative survival curve may be related to the characteristics of indolent-type cutaneous malignancy. Patients with the nondermal invasion type had their clinical observation follow-up in other hospitals after surgical resection in our department. By contrast, dermal invasion cases are known to have an unfavorable clinical behavior, as shown in Figure 2, and thus, careful follow-up was needed in our hospital or in another hospital where skin oncologists are available.

In conclusion, dermcidin has the potential to help toward the prognosis of extramammary Paget's disease at the moment of surgical resection of the tumor. It is therefore urgently needed to further investigate the actual impact of dermcidin on the molecular mechanism of the development of extramammary Paget's disease.

**Author Contributions:** S.O. and Y.S. designed, analyzed, and wrote the manuscript. N.S.-S., S.S., Y.M., H.S., H.N., K.Y., and E.O. were extramammary Paget's disease patients treated at our hospital and enrolled in this study who conducted a critical review of this manuscript. M.N. organized this study and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This study was conducted with the approval of and in accordance with the guidelines of the Ethics Committee of the University of Occupational and Environmental Health (approved code H29-211, approved date 20 November 2017), and in accordance with the Declaration of Helsinki. This study was conducted using the opt-out method of obtaining a waiver of informed consent, which was adopted with the Ethics Committee's approval.

**Informed Consent Statement:** Because this study was a retrospective cohort study, the opt-out method of obtaining informed consent was adopted, and informed consent was waived by the In-stitutional Review Board at the University of Occupational and Environmental Health.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**

## *Article* **Mechanical Intermittent Compression Affects the Progression Rate of Malignant Melanoma Cells in a Cycle Period-Dependent Manner**

**Takashi Morikura 1 and Shogo Miyata 2,\***

**Abstract:** Static mechanical compression is a biomechanical factor that affects the progression of melanoma cells. However, little is known about how dynamic mechanical compression affects the progression of melanoma cells. In the present study, we show that mechanical intermittent compression affects the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results suggest that intermittent compression with a cycle of 2 h on/2 h off could suppress the progression rate of melanoma cells by suppressing the elongation of F-actin filaments and mRNA expression levels related to collagen degradation. In contrast, intermittent compression with a cycle of 4 h on/4 h off could promote the progression rate of melanoma cells by promoting cell proliferation and mRNA expression levels related to collagen degradation. Mechanical intermittent compression could therefore affect the progression rate of malignant melanoma cells in a cycle period-dependent manner. Our results contribute to a deeper understanding of the physiological responses of melanoma cells to dynamic mechanical compression.

**Keywords:** mechanical intermittent compression; malignant melanoma; in vitro model; cancer progression

#### **1. Introduction**

Malignant melanoma is a melanocyte-derived cutaneous skin tumor, which is known as one of the most aggressive cancers and intractable disease with a poor prognosis [1]. The incidence of malignant melanoma is increasing worldwide [2–4], but there are few effective pathological diagnostic techniques to find melanoma [5]. Although a classical "ABCDE" approach is generally considered useful for pathological evaluation of major melanoma subtype such as superficial spreading melanoma (SSM), the pathological evaluation of specific minor subtype is difficult, such as acral lentiginous melanoma (ALM) because the lesions are often heterogeneous and unique [6–8]. The difficulty in diagnosing delays the early detection of lesions, and the associated mortality rate is high because the stage of the disease is often advanced at the time of detection [9,10].

In addition to the lack of effective diagnostics, establishing an effective therapeutic strategy without adverse events remains challenging [11]. Most of the currently available clinical therapies have been developed for major melanoma subtypes, such as SSM, which often occurs in the UV-exposed skin, and there are very few effective treatments for minor subtypes such as ALM, which often occurs on the plantar surface [12,13]. For instance, the molecular targeted drugs and immune checkpoint inhibitors that target mutation, such as BRAF and NRAS gene, are effective for SSM, but ALM has a poor response to these treatments because there are relatively few above-mentioned genetic mutations [14–17]. Therefore, the development of effective treatment and diagnostic strategy for minor melanoma subtypes, such as ALM, is also vitally important.

**Citation:** Morikura, T.; Miyata, S. Mechanical Intermittent Compression Affects the Progression Rate of Malignant Melanoma Cells in a Cycle Period-Dependent Manner. *Diagnostics* **2021**, *11*, 1112. https:// doi.org/10.3390/diagnostics11061112

Academic Editor: Yasuhiro Sakai

Received: 18 May 2021 Accepted: 16 June 2021 Published: 18 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

To establish effective new therapies and diagnostic techniques for cancer, it is important to elucidate the progression mechanisms of the targeted cancer. The development and progression of superficial spreading melanoma, which is common in Caucasoid patients, is correlated with UV exposure, which can cause genetic mutations, such as BRAF and NRAS mutations, which lead to the development and progression of malignant melanoma [18–20]. However, UV exposure of the plantar surface, where ALM commonly occurs, is limited, and some studies have suggested that there is a different mechanism in the progression of melanoma than the genetic mutations caused by UV exposure [12,21]. Recently, the physical environment surrounding malignant melanomas and mechanical stimulus have attracted attention, and the relationship between mechanical stimulation and the progression of malignant melanoma cells has been highlighted.

During tumor growth, cancer cells invade the surrounding interstitial tissue and distantly metastasizes to other tissues by passing through the extracellular matrix (ECM) to infiltrate blood vessels or lymphatics [22–24]. During interstitial tissue invasion, cancer cells are exposed to a variety of mechanical stimuli, such as compression, tension, and shear stimuli. Interestingly, some studies have reported that the behavior of cancer cells changed to adapt to external mechanical stimuli as a biochemical response. Cheng et al. reported that microenvironmental mechanical stimuli regulate tumor size and morphology by inhibiting cell proliferation and promoting apoptosis [25], while Janet et al. showed that mechanical compression contributes to the acquisition of invasive capabilities by cancer cells [26]. Similarly, several previous studies on malignant melanoma have also reported a relationship between the mechanical environment and cancer progression. Importantly, there seems to be a correlation between the area of the plantar surface, where strong mechanical stimuli are applied, and the site of malignant melanoma development, with malignant melanoma size being more expanded in areas under more intense mechanical stimuli [27,28]. We previously reported that static mechanical compression promotes melanoma cell invasion [29]. Those reports suggest that mechanical compression lead a biochemical response associated with progression of melanoma cells. However, little is known about how dynamic mechanical compression affects the progression of melanoma cells. Therefore, in the present study, we investigated the effect of mechanical intermittent compression on the progression of melanoma cells as fundamental research.

The aim of the present study was to elucidate how mechanical intermittent compression affects the progression of malignant melanoma cells in a cell culture model simulating physiological conditions. We established an in vitro cell culture model and cell culture device to apply the mechanical intermittent compression with temporal observation. After the establishment of the cell culture system, the effect of mechanical intermittent compression on the progression of melanoma cells was evaluated.

#### **2. Materials and Methods**

*2.1. In Vitro Malignant Melanoma Model to Enable Mechanical Intermittent Compression and Temporal Observation of Cell Behavior*

A mouse malignant melanoma cell line (B16F10, RIKEN BioResource Center, Tsukuba, Japan) was used to establish an in vitro malignant melanoma model. B16F10 cells were thawed from cryopreserved stock and subcultured twice in Dulbecco's modified Eagle's medium (DMEM)-high glucose, supplemented with 10% fetal bovine serum and 1% antibiotics/antimycotics. The cells were maintained in a 5% CO2 atmosphere at 37 ◦C and passaged once in 2–3 days to avoid reaching confluence, which inhibited cell-cell contact.

An in vitro malignant melanoma model was established in our previous study [29]. Briefly, the model was established by seeding B16F10 cells under a type I collagen gel layer, simulating dermal tissue. B16F10 cells were seeded at 1.6 × 105 cells/cm2 in a 1.5 mm cylindrical area on an f 60 mm cell culture dish (Figure 1a). A type I collagen neutral solution was prepared at a final concentration of 2.4 mg/mL from acid-soluble collagen (I-AC30, KOKEN, Tokyo, Japan). Type I collagen solution (2 mL) was poured into f 60 mm cell culture dishes to cover the B16F10 cells. After polymerization at 37 ◦C for 20 min, a Cell Culture Insert (pore size; f 8.0 mm, BD Falcon Inc, Franklin Lakes, NJ, USA) was mounted

on the gel layer to permit oxygen and nutrient diffusion toward the B16F10 cell-seeded area. The malignant melanoma model was maintained in DMEM-high glucose with 10% FBS and 1% antibiotics/antimycotics at 37 ◦C in 5% CO2 for 72 h.

**Figure 1.** Schematic of the in vitro malignant melanoma model. (**a**) Fabrication of the in vitro malignant melanoma model. B16F10 cells were seeded in a 1.5 mm cylindrical area in the PDMS mold on a cell culture dish. After one day of culture, the mold was removed from the dish and covered with neutralized type I collagen gel. (**b**) Photograph of the experimental set-up. (**c**) Schematic side view of experimental set-up for imposing intermittent mechanical compression and monitoring the cell behavior. (**d**) Mechanical intermittent compression pattern of T = 4 groups. (**e**) Mechanical intermittent compression pattern of T = 8 groups.

A cell culture device was also established to enable intermittent mechanical compression with temporal observation (Figure 1b). To impose mechanical compression onto the gel-covered cells, a cell culture insert with a cylindrical SUS304 weight was mounted on the gel layer (Figure 1c). B16F10 cells were compressed through the collagen gel layer using the Cell Culture Insert with a ring-shaped weight. The melanoma model was subjected to a mechanical intermittent compression of 7.7 × 102 Pa with a cycle of 2 h on/2 h off (T = 4 groups) (Figure 1d) or 4 h on/4 h off (T = 8 groups) (Figure 1e).

Gene expression related to cellular behavior, such as invasion and cell proliferation, fluctuates over time. Gene expression in response to sustained mechanical stimulation is transient, and stabilizes within a few hours. Therefore, we prepared two sample groups that switched mechanical stimuli at the level of several hours. A malignant melanoma model without weights was also prepared similarly for use as the control.

#### *2.2. Creep Phenomenon of Collagen Gel in the Cell Culture Device during Application of Continuous Mechanical Compression*

For evaluating the creep in our experimental system, a type I collagen gel containing f 20 mm polystyrene microspheres (Polybead; 18329, Polysciences Inc., Warrington, PA, USA) was prepared. Briefly, a type I collagen neutral solution was prepared at a final concentration of 2.4 mg/mL from I-AC30 acid-soluble collagen. The collagen-neutral solution was mixed with 20 mm polystyrene microspheres to yield a final concentration of 5 *v*/*v*% of microspheres. The microspheres were used as markers to evaluate the creep phenomenon of the collagen gel under compression. The type I collagen solution (2 mL) was poured into f 60 mm cell culture dishes, and polymerized at 37 ◦C for 20 min, and the

cell culture insert and ring-shaped weight were mounted on the gel layer, similar to the cell culture experiments.

The prepared creep test specimens were subjected to compressive stimulation for 30 min. Time-lapse images were acquired every minute using a phase contrast microscope (CKX41, Olympus Inc., Tokyo, Japan) equipped with a CCD camera (DP73, Olympus Inc., Tokyo, Japan). Using these images, the temporal strain change in the collagen gel was measured using digital image correlation (DIC), which is a contact-free measurement of material deformation [30,31]. The strain in the collagen gel was measured according to the DIC algorithm at each time point before and after deformation as follows: (1) an interrogation window was set in an arbitrary search area at each time point (Figure 2e). (2) The cross-correlation coefficients of the pixel value pattern in the interrogation window before and after deformation were calculated. (3) The location of the interrogation window where the cross-correlation was maximum was measured as the location after deformation. (4) The displacement of the location between the set interrogation window before and after deformation was calculated as the deformation. (5) Using the measured deformation magnitude, the Green-Lagrange strain was calculated, which contains normal strain and shear strain variables. The normal strain and shear strain in the collagen gel were measured according to the DIC algorithm at each time point before and after deformation, and given as the Green-Lagrange strain. The temporal strain change was measured using the open-source software package Ncorr [32] in the numerical analysis software MATLAB (<IP_ADDRESS>0001 (R2020b), MathWorks, Natick, MA, USA).

**Figure 2.** Image analysis and evaluation of creep phenomenon: (**a**) Quantification of cell-occupied area to evaluate cell progression. (**b**) Enumeration of live cells using the ITCN plugin in ImageJ. (**c**) Enumeration of nuclei using binarization and segmentation (**d**) Quantification of total F-actin length using binarization and skeletonization. (**e**) Schematic of digital image correlation method (DIC) (**f**) Three-element generalized Kelvin-Voigt model.

After the representative strain value, defined as the squared norm of the median value of the normal strain in the analyzed area, was calculated, a creep curve was generated. The creep phenomenon of biomaterials, such as biological tissue and collagen gel, is generally described using the generalized Kelvin-Voigt model [33,34]. Nonlinear regression of the creep curve of each sample was performed using a three-element model (Figure 2f). The three-element generalized Kelvin-Voigt model is described as follows:

$$
\pi = \frac{\eta}{E\_2} \tag{1}
$$

$$\gamma = \frac{\sigma\_0}{E\_1} + \frac{\sigma\_0}{E\_2} \left( 1 - e^{-\frac{t}{\tau}} \right) \tag{2}$$

where γ is the strain, *σ*0 is the applied constant stress, *Ei* (*i* = 1, 2) is the elastic modulus for each component, and *η* is the viscosity. The delay time *τ* of the model, which is defined as Equation (1), was estimated using the Levenberg-Marquardt method in the open-source statistical analysis software R. The fit index between the creep curve and the estimated nonlinear curve using the three-element model was evaluated using Pearson's correlation coefficient.

#### *2.3. Quantification of Cell Progression*

The cell behavior was observed for 24 h using a phase-contrast microscope (CKX41, Olympus Inc., Tokyo, Japan) equipped with a CCD camera (DP73, Olympus Inc., Tokyo, Japan). Phase contrast images were continuously acquired at 0 h, 8 h, 16 h, and 24 h under mechanical intermittent compression. Progression was evaluated using the progression distance (*l*) in the phase-contrast images, which was measured using ImageJ software. To remove the noise within the phase-contrast images, pre-processing was conducted, including filtering and binarization (Figure 2a). The progression distance at each time point (*lt*) was calculated as follows:

$$d\_t = \sqrt{\frac{a\_t}{\pi}} - \sqrt{\frac{a\_0}{\pi}} \tag{3}$$

where *at* is the cell-occupied area at each time and *a*0 is the area at 0 h. The radius of the approximate perfect circle, which is equivalent to the cell-occupied area, was calculated, and the difference between the radius of the perfect circle approximating the cell-occupied area at each time and that at the start of culture was defined as the cell progression distance (*lt*).

#### *2.4. Cell Viability and Cell Proliferation Assay*

To determine the effect of mechanical intermittent compression on cell viability and cell proliferation rate in the malignant melanoma model, a fluorescence live/dead assay was performed after 24 h of culture. The cells were characterized using calcein AM/propidium iodide (PI) double fluorescence staining.

Cell viability was defined as the dead cell rate (*DCR*), which was calculated as follows:

$$DCR = \frac{N\_L}{N\_L + N\_D} \tag{4}$$

where *NL* is the number of live cells and *ND* is the number of dead cells at the end of the culture duration. The number of viable cells (*NL*) was measured using the ITCN plugin in ImageJ (Figure 2b). The number of dead cells (*ND*) was measured using ImageJ according to the following: (1) grayscale images were binarized using the *Otsu* algorithm, and (2) the nucleus area was segmented using the *watershed* algorithm (Figure 2c).

The cell proliferation rate (*CPR*) was calculated as follows:

$$CPR = \frac{N\_L + N\_D}{(N\_L + N\_D)\_{control}} \tag{5}$$

where *NL* and *ND* were calculated using the same measurement method as the cell viability assay, and (*NL* + *ND*)*control* was defined as the sum of *NL* and *ND* in the control group.

#### *2.5. Fluorescence Staining of F-Actin and Nuclei*

To determine the effect of mechanical intermittent compression on the morphological changes in F-actin filaments in the cell-occupied area, the morphology of F-actin filaments was observed by rhodamine-phalloidin/DAPI fluorescence double staining at 24 h of culture. Briefly, cells in the malignant melanoma model were fixed with 4% paraformaldehyde for 10 min. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min. To

stain F-actin filaments, the cells were incubated with 0.7% rhodamine-phalloidin (PHDR1, Cytoskeleton Inc., Denver, CO, USA) for 30 min at 37 ◦C. After rhodamine-phalloidin staining, 300 nM DAPI solution was added and incubated for 5 min. After removing the DAPI solution, the cells were rinsed with PBS + 1% antimycotic/antibiotic for 5 min three times. F-actin and DAPI were visualized using an inverted fluorescent microscope (CKX41, Olympus Inc., Tokyo, Japan) equipped with a CCD camera (DP73, Olympus Inc., Tokyo, Japan) and fluorescent equipment (U-LH50HG, Olympus Inc., Tokyo, Japan). The length of F-actin filaments was measured to quantitatively evaluate morphological changes in the cytoskeleton. The length of F-actin filaments per single cell (*LFC*) was calculated as follows:

$$LFC = \frac{L\_f}{N\_n} \tag{6}$$

where *Lf* and *Nn* are the total actin fiber length and number of cell nuclei per acquired image at the end of the culture duration, respectively. The number of cell nuclei per acquired image (*Nn*) was measured using ImageJ according to the following: (1) grayscale images were binarized using the *mean* algorithm, and (2) the nucleus area was segmented using the *watershed* algorithm (Figure 2c). The total actin fiber length per acquired image (*Lf*) was measured using ImageJ according to the following: (1) acquired images were pre-processed by a bandpass filter for noise removal and edge-enhancement, (2) grayscale images were binarized using the *Otsu* algorithm, (3) pixels were repeatedly removed from the edges of objects in the binary image until they were reduced to single-pixel-wide shapes, (4) the sum of grayscale in the skeletonized images was equivalent to the total actin fiber length per acquired image (Figure 2d).

#### *2.6. Relative Quantification of mRNA Expression Levels*

The relative mRNA expression levels in the cell culture model were quantified by RT-qPCR for matrix metalloproteinase-14 (*Mmp-14*) and glyceraldehyde 3-phosphate dehydrogenase (*Gapdh*), encoding MMP14 and GAPDH, respectively. *MMP14* is a key ECM-degrading enzyme, and also a regulator that activates proteins that promote the progression of melanoma cells [35]. GAPDH is a crucial factor in glycolysis and is one of the most commonly used reference genes [36].

Relative mRNA expression levels were measured using total RNA extracted from the cell culture model collected after 24 h of culture. Total RNA was extracted using NucleoSpin RNA kits (740955.50; Takara Bio Inc., Shiga, Japan) and quantified using a Thermal Cycler Dice Real Time System Lite (TP700; Takara Bio Inc., Shiga, Japan). RNA was reverse transcribed into cDNA using the PrimeScript Master Mix (Perfect Real Time) (RR036A; Takara Bio Inc., Shiga, Japan) with an oligo (dT) primer and random hexamer primer for 15 min at 37 ◦C and 5 s at 85 ◦C. The concentration of cDNA was quantified using a Biophotometer (6131; Eppendorf, Hamburg, Germany), and then diluted with RNase-free water (9012; Takara Bio Inc., Shiga, Japan) to 10 ng/mL of cDNA. RT-qPCR was conducted in a Thermal Cycler Dice Real Time System Lite using the PCR program 30 s at 95 ◦C, followed by 60 cycles of 5 s at 95 ◦C and 30 s at 60 ◦C. The RT-qPCR reaction mix contained 12.5 μL of TB Green Premix Ex Taq II (Tli RNaseH Plus) (RR820A; Takara Bio Inc., Shiga, Japan), 20 ng of cDNA, 0.4 mM of each forward and reverse primer, and 8.5 μl of RNase-free water. The primer sequences are listed in Table 1. RT-qPCR was performed in technical triplicates for each primer pair and cDNA sample. In addition, the reactions were conducted in biological triplicates under similar conditions. To verify that primer dimers were not responsible for the obtained fluorescence signals, melting curve analysis of the amplicons was performed for each primer pair. Negative control reactions without templates were also included to ensure data quality. Relative mRNA expression was normalized to GAPDH and then calibrated to that of the control group. The fold change was calculated using the 2−ΔΔ*Ct* method, where *Ct* is the threshold cycle.

#### **Table 1.** RT-qPCR primer sequences.

#### *2.7. Statistical Analysis*

The statistical significance of the differences between experimental groups was evaluated using Dunnett's test. Statistical significance was set at *p* < 0.05 and *p* < 0.001.

#### **3. Results**

*3.1. Establishment of a Cell Culture Device to Apply Mechanical Intermittent Compression with Temporal Observation*

We established a cell culture device to apply mechanical intermittent compression with temporal observation. Prior to observing the progression of melanoma cells in the cell culture device, we evaluated the deformation of collagen gel during applying mechanical compression; the creep phenomenon of the gel.

Representative images of the deformation and strain distribution in the collagen gel are shown in Figure 3a,b. Figure 3c shows the creep curve during mechanical compression and an estimated nonlinear curve fitted by the three-element Kelvin-Voigt model. Regarding the fit index of nonlinear regression, the median of the Pearson correlation coefficients between the creep curves and the estimated nonlinear curves using the generalized Kelvin-Voigt model was 0.968 (Figure 3d). This indicates that the estimation of the creep curve using the models fitted well. As a result of nonlinear regression, the median of the estimated delay time was 7.74 min, which is very small compared to the application time of compression, which was 120 min (Figure 3e). Based on these results, the creep phenomenon horizontal to the surface of the cell culture dish during temporal compression was negligible.

#### *3.2. Progression Rate of Cells in Melanoma Model Was Regulated by Mechanical Intermittent Compression in a Cycle Period-Dependent Manner*

Representative microscopic images of B16F10 cells in the control, T = 4, and T = 8 groups are shown in Figure 4a. The white dotted line indicates the cell-occupied area at 0 h of culture, and the yellow dotted line indicates the cell-occupied area at 24 h of culture. The cell-occupied area in the established cell culture model increased during the cultivation period. Figure 4b shows the progression distance at each time, (*lt*), during the cultivation period. The slope of the progression distance in the T = 4 group was lower than that in the control group. In contrast, the slope in the T = 8 group was higher than that in the control group. In other words, the progression rate in the T = 4 group decreased, whereas that in the T = 8 group increased. This suggests that intermittent compression with a cycle of 2 h on/2 h off could suppress the progression rate of melanoma cells, while a cycle of 4 h on/4 h off could promote the progression rate.

**Figure 3.** Creep estimation in the collagen gel during mechanical compression: (**a**) Representative time-lapse images acquired by phase contrast microscopy. The black dot object in the image indicates a polystyrene microsphere. The white arrow indicates the direction of displacement. (**b**) Representative images of Green-Lagrange strain. *Sxx* indicates a horizontal normal strain, *Syy* indicates a vertical normal strain, and *Sxy* indicates a shear strain. (**c**) Representative creep curve and estimated nonlinear curve fitted by the three-element Kelvin-Voigt model. (**d**) Boxplot of Pearson's correlation coefficients. The median of the Pearson's correlation coefficients between the creep curves and the estimated nonlinear curves was 0.968 (n = 6). (**e**) Boxplot of estimated delay time. The median estimated delay time was 7.74 min (n = 6).

**Figure 4.** Progression of B16F10 cells in a malignant melanoma model under mechanical intermittent compression. (**a**) Representative phase contrast image. The white dotted lines indicate the cell-adhered area at 0 h of culture, and the yellow dotted lines indicate the cell-adhered area at 24 h of culture. (**b**) Quantification of progression distance. The green circles indicate the progression distance in the control group, the blue triangles indicate the T = 4 group, and red rectangles indicate the T = 8 group. The green, blue, and red dashed lines indicate a regression line to the progression distance in the control, T = 4, and T = 8 groups, respectively (n ≥ 12, data represents the mean ± S.E).

#### *3.3. Cell Viability and Cell Proliferation Rate*

Representative fluorescence double staining images using calcein-AM/PI in the control, T = 4, and T = 8 groups are shown in Figure 5a. Most cells in all groups were alive after 24 h of culture. Figure 5b shows the quantitative cell viability, defined as the dead cell rate (*DCR*). Figure 5c shows the quantitative cell proliferation rate (*CPR*). There was no

significant difference in *DCR* between the control groups and the T = 4 and T = 8 groups. There was no significant difference in *CPR* between the control and T = 4 groups, while the *CPR* in the T = 8 group increased significantly compared to that in the control group. These findings suggest that intermittent compression with a cycle of 2 h on/2 h off did not affect cell viability and proliferation. In contrast, intermittent compression with a cycle of 4 h on/4 h off did not affect cell viability, but promoted cell proliferation.

**Figure 5.** Cell viability and cell proliferation assay. (**a**) Representative fluorescent images stained by calcein AM/PI at 24 h culture duration. The green fluorescence indicates live cells, and the red fluorescence indicates dead cells. (**b**) Quantification of cell viability (*DCR)* (n = 3, mean ± S.D.). (**c**) Quantification of cell proliferation rate (*CPR)* (n = 3, mean ± S.D.). Dunnett's test was used to compare groups. \* indicates a significant difference compared to the control group (*p* < 0.05).

#### *3.4. Cell Migration Capacity*

Representative rhodamine-phalloidin/DAPI fluorescence staining images in the control, T = 4, and T = 8 groups are shown in Figure 6a. Figure 6b shows the value of the *LFC*, which was defined as the length of F-actin filaments. The *LFC* in the T = 4 group decreased significantly compared to that in the control group, and there was significant decrease between the *LFC* values in the control and T = 8 groups. The LFC in the T = 4 group tended to decrease compared to that in the T = 8 group. In general, elongation of F-actin filaments is correlated with cell motility [37–39]. These results suggest that intermittent compression with a cycle of 2 h on/2 h off could suppress the cell migration capacity rather than a cycle of 4 h on/4 h off.

#### *3.5. Relative mRNA Expression Levels*

The relative mRNA expression levels of *Mmp-14* are shown in Figure 6c. The mRNA expression of *Mmp-14* in the T = 4 group was lower than that in the control group, while that in the T = 8 group increased compared to in the control group. Mechanical intermittent compression with a cycle of 2 h on/2 h off might suppress the invasion ability of melanoma cells by regulating the expression of *Mmp-14*. In contrast, compression with a cycle of 4 h on/4 h off might activate the invasive ability of melanoma cells.

**Figure 6.** Quantification of cell migration and invasion capacity. (**a**) Representative fluorescence images stained by rhodamine-phalloidin/DAPI at 24 h of culture. The red fluorescence indicates F-actin filaments, and the blue fluorescence indicates nuclei. (**b**) Quantification of the length of F-actin filaments (*LFC*) (n ≥ 3, mean ± S.D.). (**c**) Relative quantity of *Mmp-14* (n = 3, mean ± S.D.). Dunnett's test was used to compare groups. Asterisks indicate a significant difference compared to the control group (\*: *p* < 0.05, \*\*\*: *p* < 0.001).

#### **4. Discussion**

Pathological diagnosis of minor melanoma subtype, which often occurs on the soles of feet, is difficult compared to other melanoma types [6–8]. In addition, the minor subtype responds poorly to current therapy strategies [14–16]. For these reasons, it is important to elucidate the mechanisms by which these minor melanoma progress to establish new pathological diagnostic strategies and therapies. Interestingly, although ultraviolet light is generally thought to be a factor in the development of melanoma, mechanical stimuli may affect the development and progression of melanoma as well as genetic damage caused by UV exposure [12,21]. We previously reported that static mechanical compression promotes the progression of melanoma cells [29]. However, little is known about how dynamic mechanical compression, such as intermittent compression, affects the progression of melanoma cells. The aim of the present study was therefore to elucidate the mechanisms by which mechanical intermittent compression affects the progression of melanoma cells.

We established a cell culture model simulating the physiological conditions of melanomas, and a cell culture device to apply intermittent mechanical compression with temporal observation. In general, it is known that creep occurs when a continuous mechanical force is applied to a viscoelastic material. As a result of the creep phenomenon, the material deforms gradually under continuous force. In the established cell culture device, creep deformation occurred horizontal to the surface of the cell culture dish when a compressive stimulus was applied. The horizontal deformation in response to mechanical compression could apply shear stress to the melanoma cells. If the shear stress is not negligible, it may be a confounding factor in the elucidation of the effects of mechanical intermittent compression on the progression of melanoma cells. Therefore, we evaluated the creep phenomenon of collagen gel in cell culture devices under compression. When the delay time of the creep phenomenon was negligible compared to the observation time, we assumed that the shear stress that the cells were subjected to was also negligible. We measured creep in the collagen gel, and showed that the creep phenomenon in the horizontal direction of the culture dish that was caused by continuous applied compression was negligible. Based on this, we can assume that the effect of intermittent compressive stimulation on melanoma

cells can be measured because shear stimulation caused by gel creep can be ignored in the established cell culture device.

We showed that mechanical intermittent compression affects the progression rate of melanoma cells in a cycle period-dependent manner. Interestingly, we found that intermittent compression with a cycle of 2 h on/2 h off suppressed the progression rate of melanoma cells. Under these conditions, the length of F-actin filaments decreased and the mRNA expression level of *Mmp-14*, which is related to collagen degradation, decreased. In general, the cytoskeleton, including F-actin filaments, is reorganized and elongated in the direction of cell migration [40–43]. In other words, the suppression of F-actin filament length correlates with decreased cell motility. In addition, the gene expression level of *Mmp-14*, which promotes collagen degradation, correlates with the invasive ability of melanoma cells in collagen gel [35]. Taken together, these findings suggest that intermittent compression with a cycle of 2 h on/2 h off reduced the progression rate by decreasing the cell migration capacity and invasive ability of melanoma cells through the inhibition of F-actin elongation and collagen degradation, respectively. Here, we should note that the morphological analysis algorithm developed for F-actin has some advantages and limitations compared to conventional analysis approaches. In general, Evaluation of the single-cell level is required to quantify the change of the cytoskeletal morphology. However, in cell culture model simulating biological tissue with high cell density such as our model, it is extremely difficult to segment them even with the advanced mathematical models and machine learning techniques because the cells overlap each other [44]. Hence, to evaluate the morphological changes of the cytoskeleton in the high cell density area, such as our established model, we developed an algorithm to extract the bulk morphological features of the cytoskeleton at the multi-cell level. It has the advantage of being able to measure changes in the cytoskeleton even in regions of high cell density, and the analysis results using our algorithm are sufficient to evaluate the effects between the different stimulus conditions as a fundamental study. On the other hand, the algorithm does not allow for a detailed evaluation of various actin morphologies, such as the filamentous and globular actin. To gain a deeper understanding of cytoskeletal responses under the dynamic mechanical compression, immunofluorescence staining and protein expression analysis are necessary, which is our future work.

In contrast, intermittent compression with a cycle of 4 h on/4 h off could promote the progression rate of melanoma cells. The increasing of the cell-occupied area in the established cell culture model, which indicates the progression of melanoma cells, is caused by the synergistic interaction between cell proliferation, migration, and invasion. The combined effect of those factors causes an increase in the cell-occupied area. As shown in the results from the molecular biological evaluation and image analysis, the cell proliferation and mRNA expression level of *Mmp-14* increased in T = 8 groups. It is also known that the increased mRNA expression level of *Mmp-14* correlates with the promotion of invasion via collagen degradation [45]. Some studies have reported that the collagen degradation by *Mmp-14* is crucial for cancer cells to proliferate and invade in the ECM [46–48]. Shaverdashvili et al. showed that *Mmp-14* is directly contributed to the metastasis of melanoma [49]. In addition, although *Mmp-2* and *Mmp-9* are known to play important roles in the migration and invasion processes of melanoma, *Mmp-14* can activate both [50,51]. Thus, *Mmp-14* is a critical factor in the progression process of melanoma. The result that the expression of *Mmp-14* increased in our melanoma model suggests promoting the progression of melanoma cells. To elucidate the physiological mechanisms of the melanoma progression under the conditions in more detail, the gene expression analysis of other mRNA and protein, such as gene related to cytoskeleton reconstruction, and the metabolic measurements such as glucose consumption are required. In summary, intermittent compression with a cycle of 4 h on/4 h off might promote the progression rate of melanoma cells by accelerating the increase in cell number and invasive ability through the promotion of cell proliferation and collagen degradation, respectively.

We showed for the first time that mechanical intermittent compression affects melanoma cell invasion in a cycle period-dependent manner in this study. However, we should notice that the cell line used in this study is a mouse melanoma cell line, not a human cell line. It is necessary to determine whether the mechanical intermittent compression can affect human melanoma similarly in future work, such as human melanoma cell line and primary melanoma cells collected from the patients. Also, to understand deeply the molecular biological mechanisms more, it is required to conduct comprehensive gene expression analysis and metabolic measurement, and cell culture experiments under cyclic compressive stimulation with different time resolutions in the future.

It may be possible to regulate the invasion of melanoma cells by applying mechanical compressive stimuli with appropriate cycle periods. Mechanical stimuli can be controlled less invasively and more precisely than pharmacokinetic or electromagnetic field control methods. Thus, our results may contribute to establish new therapies that are less invasive and more locally effective than conventional therapies, such as drug therapy, surgery, and radiotherapy. In addition, if a unique relationship between the mechanical stimulation pattern and the progression rate of melanoma is found, new criteria for pathological diagnostic techniques could be established. Our results have the potential to contribute to the establishment of new diagnostic and therapeutic methods.

#### **5. Conclusions**

We established an in vitro cell culture model using melanoma cells to simulate the physiological conditions of malignant melanoma, and a cell culture device to apply intermittent mechanical compression with temporal observation.

In the present study, mechanical intermittent compression with a cycle of 2 h on/2 h off could suppress the progression rate of melanoma cells, by suppressing the elongation of F-actin filaments and regulating the levels of mRNA related to collagen degradation. In contrast, mechanical intermittent compression with a cycle of 4 h on/4 h off could promote the progression rate of melanoma cells by promoting cell proliferation and regulating the levels of mRNA related to collagen degradation.

In conclusion, our study revealed that the mechanical intermittent compression affected the progression of melanoma cells in a cycle period-dependent manner. The result will lead to a deeper understanding of melanoma cell behavior under dynamic mechanical compression and could contribute to the establishment of new diagnostics and therapy.

**Author Contributions:** Conceptualization, T.M. and S.M.; methodology, T.M.; validation, T.M. and S.M.; investigation, T.M.; resources, S.M.; data curation, T.M.; writing—original draft preparation, T.M.; writing—review and editing, S.M.; visualization, T.M.; supervision, S.M.; project administration, S.M.; funding acquisition, T.M. and S.M. Both authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partially supported by the JSPS KAKENHI (Grant numbers: 17K01369 and 26560222), the Translational Research Network Program from Japan Agency for Medical Research and Development (AMED), and Mori Manufacturing Research and Technology Foundation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **STAT3 Activation in Psoriasis and Cancers**

**Megumi Kishimoto 1, Mayumi Komine 1,\*, Miho Sashikawa-Kimura 1, Tuba Musarrat Ansary 1, Koji Kamiya 1, Junichi Sugai 1, Makiko Mieno 2, Hirotoshi Kawata 3, Ryutaro Sekimoto 4, Noriyoshi Fukushima 3 and Mamitaro Ohtsuki 1**

**Abstract:** Activation of signal transducer and activator of transcription (STAT)3 has been reported in many cancers. It is also well known that STAT3 is activated in skin lesions of psoriasis, a chronic skin disease. In this study, to ascertain whether patients with psoriasis have a predisposition to STAT3 activation, we examined phosphorylated STAT3 in cancer cells of psoriasis patients via immunohistochemistry. We selected patients with psoriasis who visited the Department of Dermatology, Jichi Medical University Hospital, from January 2000 to May 2015, and had a history of cancer. We performed immunostaining for phosphorylated STAT3 in tumor cells of five, four, and six cases of gastric, lung, and head and neck cancer, respectively. The results showed that there was no significant difference in STAT3 activation in any of the three cancer types between the psoriasis and control groups. Although this study presents limitations in its sample size and inconsistency in the histology and differentiation of the cancers, results suggest that psoriasis patients do not have a predisposition to STAT3 activation. Instead, STAT3 activation is intricately regulated by each disorder or cellular microenvironment in both cancer and psoriasis.

**Keywords:** psoriasis; STAT3; cancer; immunohistochemistry

#### **1. Introduction**

Psoriasis is a chronic inflammatory skin disease associated with musculoskeletal symptoms in about 25% of patients [1]. Psoriasis is thought to be triggered by environmental factors such as trauma and infection in addition to genetic background, with both innate and acquired immunity involved in its pathogenesis. The pathogenesis of psoriasis has been intensively investigated, but its enigmatic nature has yet to be defined [2]. The relationship between psoriasis and STAT3 was first described by Sano et al. in 2005, when they reported that STAT3 was activated in keratinocytes of psoriasis lesions [3]. Since then, STAT3 hyperactivation has been reported in the cell types involved in psoriasis, including Th17 cells and keratinocytes [4].

STAT3 was first identified in 1993 [5] and known to be an important transcription factor and mediator in a number of different cell biological processes including proliferation, survival, differentiation, and angiogenesis under both physiological and pathological conditions [6,7]. It is one of the members of the seven STAT proteins, STAT 1, 2, 3, 4, 5A, 5B, and 6 [8]. Activation of STAT3 usually occurs through phosphorylation, in response to all IL-6 family members and various other cytokines, growth factors, oncoproteins, and hormones such as leptin [4,5,9]. STAT3 has two phosphorylation sites, namely a

**Citation:** Kishimoto, M.; Komine, M.; Sashikawa-Kimura, M.; Ansary, T.M.; Kamiya, K.; Sugai, J.; Mieno, M.; Kawata, H.; Sekimoto, R.; Fukushima, N.; et al. STAT3 Activation in Psoriasis and Cancers. *Diagnostics* **2021**, *11*, 1903. https://doi.org/ 10.3390/diagnostics11101903

Academic Editor: Yasuhiro Sakai

Received: 31 August 2021 Accepted: 13 October 2021 Published: 15 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tyrosine residue (Tyr705) and a serine residue (Ser727) [10]. The canonical function of STAT3 as a transcription factor is mainly through the phosphorylation of tyrosine [7]. When a ligand binds to its cognate receptor, phosphorylation of Tyr705 occurs, resulting in dimerization of STAT3 and its translocation into the nucleus to exert its function as a transcription factor [5,11]. Non-receptor tyrosine kinases, such as c-Src, MAPK, and Abl are also involved in the activation of STAT3 through Tyr 705 phosphorylation [12]. Phosphorylation of Tyr705 plays a main and important role in the transcriptional function of STAT3, while phosphorylation of Ser727 also has various functions [12]. STAT3 is found in mitochondria, acting as a modulator of mitochondrial respiration and regulator of complex I activity and ROS production. These activities are related to the phosphorylation of Ser727 [13,14].

In recent years, there have been many reports of STAT3 overexpression has been found in cancer cells. Activation of STAT3 in malignant tumors has been implicated in poor prognosis, metastasis, and proliferation of cancers, and several STAT3 inhibitors are currently under development [5].

STAT3 is also involved in inflammation and immunity [15–17]. As mentioned above, Sano et al. reported in 2005 that STAT3 activation was observed in human epidermal keratinocytes in more than 90% (*n* = 19 of 21) of psoriatic lesions and some adjacent uninvolved epidermis by immunohistochemical analyses [3]. They also reported that transgenic mice expressing a constitutively active form of STAT3 in keratinocytes developed skin lesions that closely resembled human psoriasis [3]. STAT3 has recently emerged as a key player in the development and pathogenesis of psoriasis and psoriasis-like inflammatory conditions [4].

The risk of malignancy in patients with psoriasis is thought to be slightly increased compared to that in the normal population; thus, we speculated that STAT3 activation in psoriasis patients is related to malignancy risk.

In this study, we investigated the rate of active STAT3 tumors in patients with psoriasis compared to that in patients with eczema. To the best of our knowledge, very few studies have focused on STAT3 expression in the tumor cells of patients with psoriasis.

#### **2. Materials and Methods**

#### *2.1. Patients*

We selected Japanese patients with psoriasis and Japanese patients with eczema without psoriasis who presented to the Department of Dermatology in Jichi Medical University Hospital between 1 January 2000 and 31 May 2015. Among these patients, those with a medical history of non-skin cancers were selected for statistical analyses of malignancy risk. For the STAT3 immunohistochemical study, we extracted those who had undergone biopsy or surgery of their tumor at our hospital with a sufficient quantity of paraffin-embedded samples.

All patients with psoriasis and eczema were clinically diagnosed by experienced dermatologists with or without histological examination. All malignant tumors underwent histopathological diagnosis by pathologists. All patients were aged 20 years or above. All protocols were approved by the ethics committee of the Jichi Medical University.

#### *2.2. Immunohistochemical Staining*

Formalin-fixed, paraffin-embedded samples were sliced to a thickness of 5 mm. Antigen retrieval was performed by autoclaving sample slides at 120 ◦C for 10 min in citrate buffer (pH 6.0) and incubated with the primary antibody, rabbit monoclonal antiphosphorylated STAT3 (Tyr 705) antibody (Cell Signaling Technology, Danvers, MA, USA), at a dilution of 1:100 overnight at 4 ◦C. Peroxidase staining was then performed with VECTASTAIN®ABC Kit (Vector Laboratories, Inc., Burlingame, CA, USA) following the manufacturer's protocol, with diaminobenzidine (DAB, Dojindo, Kumamoto, Japan) as a chromogenic substrate.

#### *2.3. Immunohistochemical Analysis*

The outcomes of the staining were assessed by two factors: the staining intensity and the proportion of positive cells among cancer cells. Since phosphorylated STAT3 usually localizes to the nucleus, the staining of the nucleus was evaluated with anti-phosphorylated STAT3 antibody. The staining intensity was graded with a score of 0 to 3 (0: no staining, 1: mild staining, 2: moderate staining and 3: strong staining), and the proportion of positive cells was graded with a score of 0 to 3 (0: <1%, 1: 1–33%, 2: 34–66% and 3: 67–100%) by three independent experienced researchers under an optical microscope (BX53, Olympus, Tokyo, Japan).

The final score for each specimen was defined as the sum of the intensity score and proportion score.

#### *2.4. Statistical Analysis*

Chi-squared test and Student's *t*-test were used to compare the groups. The Mantel-Haenszel method was used to determine the difference in the frequency of cancer in each organ between the psoriasis and control groups.

The Mann–Whitney U test was used to compare the expression of phosphorylated STAT3 between the groups. Statistical significance was set at *p* < 0.05. Statistical analysis was performed using IBM SPSS software (version 22.0).

#### **3. Results**

There was no significant difference in the frequency of malignant tumors in each organ between the psoriasis and eczema groups, but the frequency of patients with multiple malignant tumors was higher in the psoriasis group than in the eczema group.