Mammalian skin comprises a multi-layered epithelium, e.g., the epidermis, dermis, and underlying connective tissue. The epidermis primarily consists of epithelial keratinocytes interconnected with enforced cell junctions (16). The skin compartmentalization to the epidermis and dermis compartments is based on a BM, a highly specialized network of ECM proteins that separate and unify the two compartments. The architecture of the BM is involved in tumor progression (17). Thus, BCCs, dependent on SmoM2 constitutive activators of Sonic hedgehog signaling (Shh), bud inward into the stroma, retaining their BM and rarely metastasizing (18). In contrast, RAS/MAPK-dependent SCCs commence as bidirectional tissue folds and exhibit invasiveness (19). The generation of SmoM2 and Ras respective mutants show changes in the expression of collagen 4a1/2 (COL4a1/2), Nidogen 1 (Nid1), and Sparc BM components.

Furthermore, in SCC, the mechanical forces exerted by overlying differentiated cells are sufficiently strong to confer loss of membrane integrity. Therefore, the stiffness and assembly of BM and tensile forces affect tumor progenitors' invasion at the stratified epithelium's stromal border (5, 20). In a separate study, Nid1 and COL4 expressions were decreased around the tumor nest of SCC but increased around the nest of BCC, further supporting BM input to the carcinoma phenotype. The strong expression of Nid1 and COL4 by stromal cells surrounding BCC, in contrast to cSCC, may prevent BCC cells from degrading the BM and invading the dermis (21).

The dermis consists of an abundant ECM, into which blood vessels, skin appendages, fibroblasts, and other mesenchymal cells are embedded (16). Notably, the mechanical organization of the ECM surrounding NMKC cells significantly differs compared to normal skin ECM (22). In contrast to epithelial cells, mesenchymal cells exhibit extensive cell-matrix interactions, generating strong contractile forces with increased cytoskeletal activities (23). At the injury site, fibroblasts transform into highly contractile myofibroblasts, characterized by enhanced cytoskeleton allowing them to exert more pronounced mechanical force to the surrounding ECM, which enhances physiological goals such as wound closure but promotes carcinogenesis (23, 24).

Indeed, the stroma's mechanical properties, e.g., stiffness, are directly correlated to the pathogenesis of the NMKCs. Thus, patients with the highly disabling genodermatosis, the recessive dystrophic epidermolysis bullosa (RDEB), carry mutations in the COL7A1 gene encoding for type seven collagen, the main component of anchoring fibrils supporting cell binding to the BM. The majority of these patients progress to a highly aggressive type of cSCC (9). Thus, the resulting incompetence of the BM results in an inflammatory stroma profile where activated dermal fibroblasts promote cSCC development by creating a stiff, fibrotic ECM consisting of thick COL1 bundles, fibrinogen, and tenascin-C (25). Aspects of matrix-derived mechanotransduction signals are schematically depicted in Figure 1. Therefore, modifying the mechanotransduction signals could lead to novel therapeutic approaches for skin disease.

Hyaluronan (HA) is a significant component of skin ECM. This GAG is a linear, non-sulfated chain composed of repeating disaccharides of N-acetylglucosamine and glucuronic acid units (26). HA is synthesized by the three Hyaluronan Synthases (HAS1,2 and 3) isoforms as a high molecular weight product (HMWHA) (27). HASs are localized to the plasma membrane, and the newly synthesized HA chain is protruded to the ECM (26, 27). The degradation of HA to lower molecular weight HA (LMWHA) fragments is mediated by glycosidases, denominated hyaluronidases (HYALs).

HA exerts its biological functions via specific receptors, including the cluster of differentiation 44 (CD44) encoded by one gene but presenting several isoforms due to alternative splicing (28). HA also acts through the receptor for hyaluronan mediated motility (RHAMM), upregulated in cSCC (29). Toll-like receptor 4 (TLR4) is a transmembrane HA receptor whose signaling has been correlated to both skin inflammation (14) and cSCC progression (30). HA exerts strictly size-dependent biological effects. Thus, HMWHA exhibits anti-inflammatory, immunosuppressive, and anti-angiogenic properties, while LMWHA induces angiogenic, inflammatory, and tumorigenic impact (27).

HA metabolism is correlated to the pathogenesis of NMKCs. Thus, SSC tumors cells express alternative isoforms of CD44, associated with enhanced cell growth and cancer development (31). Furthermore, CD44 receptors can bind HA of a broad molecular weight range resulting in discrete downstream signaling. Thus, HMWHA through CD44/Rac-signaling in UV exposed mice promoted keratinocyte survival, normal differentiation, and DNA repair. In contrast, LMWHA-CD44 interactions facilitated a RhoA activation/NFκB/Stat-3 signaling/miR-21 production axis, leading to inflammation, abnormal keratinocyte proliferation, and SCC progression (31). Moreover, exposure of mice to chronic UV irradiation, the most common cause of cSCC, increased CD44 expression in mouse epidermis, while 20% of the exposed animals developed SCC tumors. Indeed, in this study, UV also upregulated HAS levels and HA synthesis, indicating the existence of a CD44/HA signaling axis (32). A similar upregulation of HAS2 and 3 was observed upon UV treatment in primary NHEK(F) cells. However, blocking interleukin-1β (Il-1β) activity minimized this effect, implying an interaction of HA metabolism with inflammatory signaling (33).

Interestingly, a combination of CD29 and CD44 was suggested to identify cancer stem-like cells (CSC) undergoing EMT in SCC. Thus, CD29high/CD44high expressing SCC cells were found to exhibit molecular markers of EMT, indicating that CSC-associated mechanisms contribute to the process of EMT (³⁴). Moreover, in a separate study, a high CD44 phenotype in SCC is suggested to identify candidates for implementing targeted therapy aiming at CSCs (34).

Even though early studies did not detect a correlation between the CD44v isoform expression and SCC metastatic potential, recent reports show an enhanced expression of CD44v3-10, CD44v6-10, and CD44v8-10 isoforms co-expressed with CD44s and podoplanin in human SCC cell lines (35). Moreover, CD44 and the cell membrane glycoprotein, podoplanin, facilitated directional motility in epithelial cells and were shown to enhance SCC cell's directional migration.

Early studies implementation immunohistology show that BCCs displayed very low expression of CD44s, whereas the expression of CD44v6 exhibited a heterogeneous distribution pattern enhanced in the peripheral palisading tumor cells. In superficial BCCs, the labeling intensity for CD44v6 increased with the size of the tumor nests. These authors suggested that CD44v6 does not correlate with BCC metastatic potential, while the low CD44s is indicated as attenuating for BCCs metastasis (36). Moreover, in BCC, CD44 expression was reduced in metastasizing compared to non-metastasizing tumors. Together with increased Twist1 expression, this may suggest a critical EMT input in metastasizing tumors and characterize a metastatic phenotype (37). On the other hand, CD44 was one of the markers upregulated in BCC tumors compared to margins and controls (38). These data highlight the need for further study on the implications of the CD44/HA signaling axis and especially the determination of the expression of specific CD44 isoforms (39).

Notably, RHAMM has been related to keratinocyte motility, essential for wound healing and metastasis (40).

TLR4 is widely known for its implication in skin inflammatory responses, including allergic dermatitis (10). Moreover, in NMSC, it is overexpressed (41), whereas; in TLR4 knock-out mice, the progression of NKCS tumors was attenuated (42). The above data highlight the critical role of HA and its receptors in the mechanisms regulating the onset of NKCSs (Figure 2).

Collagens (COLs) are a primary group of ECM fiber proteins consisting of twenty-eight different types. COL participation is shown in various types of cancer, including NMSC (43). The skin content of collagen is reduced with age and is also affected by sunlight, especially UV exposure.

Indeed, in actinic keratosis, a loss of COL14 and 15 from the BM suggests that the ECM remodeling is an early sign of cSCC progression. Furthermore, the actinic keratosis stroma is negative for COL14 and 15, whose expression is evident later in the stroma of established cSCC tumors (44). Moreover, COL18 is detected in SCCs from grade one onwards, while normal keratinocytes in premalignant lesions were negative for this COL type (44).

Recessive dystrophic epidermolysis bullosa (RDEB) is a skin fragility disease caused by mutations that affect the function and/or the amount of COL 7, the primary component of anchoring fibrils. Hallmarks of RDEB are unremitting blistering and chronic wounds leading to tissue fibrosis and scarring. Nearly all patients with severe RDEB develop highly metastatic cSCC, the leading cause of death. Accumulating evidence from a murine RDEB model and human RDEB cells demonstrates that the lack of COL 7 also directly alters the wound healing process. Non-healing RDEB wounds are characterized by increased inflammation, high transforming growth factor-beta 1 (TGF-β1) levels and activity, and are heavily populated by fibroblast-derived, myofibroblasts responsible for enhanced fibrogenesis and matrix stiffness. In the next stage, fibroblasts are transformed into cancer-associated fibroblasts (CAF), contributing to cancer invasion and progression (45). Moreover, the in vitro downregulation of COL 7, which is crucial for the attachment of basal keratinocytes to the dermis, has been related to enhanced invasion in SCC (46). COL7 also suppresses TGF-β mediated downregulation of vascular endothelial growth factor (VEGF), responsible for SCC vascularization (47).

Collagen 17 (COL17), a dermo-epidermal junction structural component, is a type II hemidesmosomal transmembrane protein that stabilizes the hemidesmosome complex and basal keratinocyte binding to the BM (48). In addition to basal keratinocytes, the squamous keratinocytes around invasive tumor fronts, the adjacent tumor stroma, endothelium, and histiocytes exhibit upregulated COL7. Furthermore, aberrant shedding of COL7 ectodomain in SCC patients correlates to metastasis (49, 50). Indeed, the released COL17 endodomain enhances proliferation and survival, whereas the shedded ectodomain facilitates the invasiveness of SC cells (51).

An in vitro study with murine COL17a-/-keratinocytes revealed PI3K signaling activation. The upstream activators are the β4 integrin subunit of hemidesmosomal a6b4 integrin and the focal adhesion kinase (FAK), resulting in consequent Rac1 activation. This signaling pathway attenuated the ability of keratinocytes to migrate (52). Indeed, these authors suggest that COL17 facilitates keratinocyte adhesion and directed motility by interfering with the integrin-dependent PI3K activation and stabilizing lamellipodia in invasive cSCC. Moreover, the activity of integrin-β1 and COL17 expression at the intercellular site promotes contact following during the collective invasion of an SC cell population (53).

Reports mention the downregulation of total collagen deposition in NMSC patients (54, 55). Indeed, the total collagen levels are not significantly altered in a precancer state, suggesting that collagen downregulation could be considered a biomarker for cSCC occurrence. A possible explanation is the enhanced release of the collagen-degrading MMPs (55).

PGs are complex molecules whose protein core is glycosylated with one or more GAG chains (56). These versatile molecules regulate both the mechanical properties of tissues and act as key mediators of biological function. PGs are either secreted to the ECM or located to the cell membrane with the exception of serglycin, an intracellular PG (56). Due to their mediation of the cell/matrix interface PGs are involved in the pathogenesis of a multitude of cancer types (57–59).

Syndecans are a family of four transmembrane heparan sulfate PGs (HSPGs) exhibiting related structures. Indeed, all syndecan members consist of a variable N-terminal ectodomain, and highly conserved transmembrane and C-terminal cytoplasmic domains (60). Syndecans have been directly implicated in various tumor development (60, 61).

Syndecan-1 was shown to regulate intercellular and cell/matrix adhesion (60). Early studies on cutaneous biopsies of SCC detected a decrease of syndecan-1 expression with decreased adhesion of SCC cells suggesting that its' expression is inversely correlated to cSCC invasion into the dermis (62). This was supported by following studies showing that in invasive cSCC both syndecan-1 and cadherin E expressions were decreased with the loss of keratinocyte differentiation, especially in the acantholytic areas of tumors where interestingly these molecules were localized to the cytoplasm and not to the cell membrane (63), Indeed, syndecan-1 expression was identified to be discrete between keratoacanthoma and invasive SCC. Thus, syndecan-1 staining was diminished in invasive SCC compared to in situ SCC and keratoacanthoma (63). However, in a separate study, it was determined that syndecan-1 expression was decreased in BCC, cSCC, and metastatic human skin cancers compared to normal human skin (64). Indeed, Stepp et al. indicate that syndecan-1 is a significant factor in the early development and progression of skin cancers, suggesting that the expression of this PG could be utilized as a progression marker for cancerous skin lesions (64).

Interestingly, pioneer studies of the field demonstrated that basement membrane HSPGs and chondroitin sulfate PGs (CSPGs) were significantly decreased in cSCC compared to the normal epidermis (65).

Collectively, these data indicate that PGs, well established to regulate the cell to matrix adhesion as well as intercellular contacts important for epithelial cohesion, are important factors in keratinocyte carcinoma development and invasion to the dermis. Further studies defining their roles could improve the assessment of specific skin lesion prognoses.

MMPs are zinc-containing endopeptidases that exhibit a wide range of substrate specificities, meditating, among others, the degradation of the different components of the ECM (66). However, the MMPs also act on membrane receptors, signaling molecules, and ligands modulating their activities and can thus be defined as cell signal regulators (67). Keratinocytes and dermal fibroblasts secrete these enzymes in response to various stimuli, including UV radiation, oxidative stress, and cytokines (68). For example, the exposure of keratinocytes to UV-radiation initiates critical cellular signaling pathways, including the nuclear factor-kappa beta (NF-kB), the mitogen-activated protein kinase (MAPK), the JAK/STAT (signal transduction and activation of transcription), and the nuclear factor erythroid 2-related factor 2 (Nrf2), critical regulators of inflammation and cancer (69). In continuation, exposed keratinocytes secrete active regulators that, in turn, activate the release of MMPs by dermal fibroblasts (70). The content of dermis ECM being remodeled by the MMPs facilitates an intricate balance that maintains the skin's homeostasis or its' response to injury. However, aberrant MMPs expression and activity in cancer tissues promote essential tumor-related functions (71).

Indeed, the invasion of tumor cells, a complex, multifaceted process, is initiated by MMPs degrading the BM and the surrounding ECM (72, 73). It is well established that the gelatinase MMP members, including MMP-2,−7, and−9, participate in BM degradation due to their capability to cleave key BM components such as COL4 (74–76). In addition, cancer-stroma interactions are essential at this point as MMP-2 is secreted chiefly by the surrounding fibroblast-like stromal cells and seldom by keratinocytes and BCC cells (77). Likewise, MMP-9 is secreted mainly by tumor-associated macrophages (TAMs), not BCC tumor cells (78). Interestingly, a higher number of macrophages, able to initiate BCC cells' MMP-9 secretion, utilizing the p38 MAPK/NF-kB/COX-2 cascade, was detected in aggressive forms of BCC compared to lees aggressive phenotypes (79). Furthermore, CAFs secrete MMP-1, MMP-3, MMP-7, MMP-9, and MMP-13, releasing ECM-bound growth factors such as VEGF supporting tumor angiogenesis (80).

Notably, MMP-1, MMP-7, MMP-9, MMP-13, and MMP-14 overexpression correlated to intensive remodeling of the BCC matrix was identified (81). Interestingly, a correlation between the MMP-2 and MMP-9 expression between the stroma and tumor suggests that tumor cells affect stroma MMPs deposition in BCC (82). However, in this study, no correlation between the MMP expression and the presence of the peritumoral cleft was shown (82). In chronic wounds, MMP-7, MMP-12, and MMP-13, but not MMP-1, MMP-3, MMP-8, MMP-9, and MMP-10 expressions, were identified as diagnostic clues for discerning SCCs from nonmalignant wounds (83).

A separate study suggested that MMP-1 has the highest COL1-degrading activity generating high molecular weight collagen fragments. There is an accumulation of these fragments as MMP-2 and MMP-9 have lower activities. The deposition of high molecular weight collagen fragments attenuates stromal fibroblasts' functions correlated with BCC progression (84). MMP-13 and MMP-1 also played a significant role in tumor progression of eyelid BCC in a clinical study, as immunoreaction positivity was identified in most patients (85). Likewise, MMP-11 was overexpressed in the biopsies of both cSCC and BCC patients compared to normal skin (86).

Interestingly the complement system seems to be involved in the MMP-dependent progression of non-melanoma skin cancers. Thus, the knock-out of the serine proteinase C1r, a complement system constituent, in cSCC led to these cells attenuating proliferation, migration, and invasion through collagen type I. Moreover, the knock-out of C1r downregulated the expression of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. In addition, knock-out of C1r decreased the expression of MMP-13, inhibited the invasion of cSCC, and decreased collagen cSCC xenografts (87). Therefore, the authors suggest that the complement facilitates SCC cells' invasion by enhancing MMPs' expression.

Intriguingly, in NMSC patients, antibodies against MMP-7 were detected. As these autoantibodies are being utilized as an early marker for other malignancies, it was suggested that they could also be a biomarker for NMSCs. Notably, the expression of MMP7 autoantibodies was not followed by changes in the MMP-7 mRNA levels in both BCC and cSCC patients (88). On the other hand, at the protein level, the expression of MMP-7 and CD44 was found to be inversely correlated in cSCC and BCC sections. Indeed, in well-differentiated SCC, the MMP-7 staining was weak, while CD44 expression was high. In poorly differentiated SCC, increased MMP-7 staining was identified while the expression of CD44 was weak (89).

MMPs expression is correlated to the progression and recurrence of these tumor types. cSCC patients' tumor tissues exhibited a notably higher level of MMP-13 compared to control tissues. In addition, these patients showed significantly increased MMP-13 levels compared to healthy controls. Moreover, patients with invasive SCC had increased serum MMP-13 levels compared to in situ cSCC patients (90). Therefore Wang et al. suggest that serum MMP-13 is a relevant diagnostic marker for cSCC (90). In cSCC and BCC biopsies, MMP-2 expression was correlated to the depth of invasion, whereas the expression of MMP-2, MMP-9, TIMP-1 with inflammation and microvessel density (91). Likewise, Gozdzialska et al. show that MMP-2 protein expression in the tumor stroma was higher in high-risk BCCs than low-risk BCCs, whereas MMP-2 deposition was enhanced in both the tumor tissue and stroma in SCC when compared to BCC (92). These data point to a crucial role of MMP-2 in skin cancer invasion (92).