**5. SCC progression and aggressiveness**

**3. Chemically induced keratinocyte proteome alterations**

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

thetic substances, etc.

**proteome pattern**

Chemically induced tumors in experimental models can mimic all the clinical cancer progress phases being useful in the evaluation of new drugs, studying biological context or in decoding molecular mechanisms responsible for tumor initiation and development. Among chemical carcinogens commonly applied in cancer models are the following: environmental contaminants, N-nitroso compounds, food additives, antineoplastic agents, natural and syn-

Combining chemically induced cancer models with innovative molecular imaging techniques may help to advance new anticancer diagnostics and therapeutics protocols [22]. By studying skin carcinogenesis, phases of early alterations in the skin layers and of the mechanisms beneath are highlighted. These mouse models share common mechanisms with human skin tumorigenesis, and moreover, there are similarities in terms of genetic milieu caused by carcinogens and pro-inflammatory cytokines and chemokines that favor tumor progression [23]. Different carcinogens imprint different changes on skin cells, including on the keratinocyte proteome pattern. Mancozeb—*ethylene (bis)dithiocarbamate*—is a fungicide and a multipotent carcinogen whose underlying mechanism of action is mostly unknown. By a two-dimensional gel electrophoresis and mass spectrometry analysis, a proteomic profile of mice skin exposed to mancozeb (200 mg/kg body weight) was generated. By comparison with control samples, two significantly upregulated proteins were found, Calcyclin (S100A6) and Calgranulin-B (S100A9); these two proteins are well-known markers of keratinocyte differentiation and proliferation, suggesting their role in neoplastic alterations induced by Mancozeb. The same approach in human keratinocyte carcinogenesis model with HaCaT cells revealed that upregulation of S100A6 and S100A9 confirms the neoplastic potential of Mancozeb. The authors conclude that S100A6 and S100A9 modulate the ERK1/2 signaling pathway underlying in this way the Mancozeb-induced neoplastic potential in human skin [24], and thus, a certain proteome milieu prescribe keratinocyte behavior in a chemically triggered carcinogenesis.

**4. Spotting differences between normal and inflammatory keratinocyte** 

The skin proteome has been the target of intense research in the last years, hence human epidermal keratinocytes, dermal fibroblasts, human epidermis, were characterized regarding their proteome pattern [25–28]. Ong et al. furthered these studies and published the specific proteomic markers in the normal skin and in the one subjected to inflammatory processes. In normal skin, there are several proteins that were reported as having high expression, such as carbonic anhydrase, HSP27, gelsolin, prostate binding protein, MnSOD, a1-antitrypsin, keratin 1 and keratin 10. On the contrary, in keloid scars, there is a low expression or even absence of carbonic anhydrase proving the maintenance of local inflammatory status of the skin. In this manner, the inflamed skin shows intense expression of the proliferative keratin 16 [29]. Other proteomic markers are found to be increased in the inflamed keratinocytes in Recent studies have compared non-advanced SCC to advanced SCC, in order to identify pathways that are activated in SCC progression. Considering the fact that SCC develops on skin areas exposed to sunlight, UV radiation is found to be responsible for activating signal transduction pathways in the processes of apoptosis, inflammation, proliferation, and differentiation, necessary for SCC development [2].

Molecular marker studies that reflect the initial changes in skin carcinogenesis have shown that the most important gene involved in the ultraviolet radiation effects is the p53 tumor suppressor gene, which plays an important role in apoptosis, cell proliferation, DNA differentiation and repairing process. Mutations of the p53 tumor suppressor gene result in the occurrence of other mutations in cascade with the loss of control of aberrant cell growth, leading to the formation of cancer cells [35, 36].

Also, several biomarkers, such as E-cadherin, Ki-67 and cyclin D1, have been shown to correlate with malignancy in NMSC [37]. Thus, designed to maintain the stability of epithelial tissues, E-cadherin is a Ca(2+) dependent intercellular adhesion molecule, whose downregulation is closely related to the increased potential for tumor invasiveness and metastasis. In SCC, a decrease in E-cadherin expression in the primary lesion is correlated with the development of regional lymph node metastases [38]. Ki-67 is a marker of the cell proliferation and a representative in fast and frequent recurrent aggressive tumors [39]. An important regulator of the cell cycle, cyclin D1 is a proto-oncogene which is essential in the development of skin cancer leading to the organization and abnormal differentiation of tissues [40].

It is known that main factors leading to tumor genesis are mutations in the tumor suppressor genes, such as the APC gene. Thus, mutations at this level lead to the synthesis of a non-functional APC protein that induces β-catenin destruction (the latter being a factor that activates transcription of oncogenes such as MYC and cyclin D1). In addition, 75% of patients diagnosed with cSCC were identified with mutations in NOTCH tumor suppressor genes [41].

There are many studies, both *in vitro* and *in vivo*, that investigate the involvement of epidermal stem cells in skin carcinogenesis, tumor invasion, metastasis [56, 57] as well as tumor recurrence [58, 59]. Post-initiation, CSCs can generate macroscopic tumors through self-renewal and processes leading to stem cell differentiation generating several cellular variants. Normal epithelial tissue continuously renews and is maintained through the action of proliferating stem cells. When their density decreases, stem cells generate proliferative colonies called holoclones. They bear different characteristics to the abortive colonies of differentiated cells, called paraclones [60]. It is worthwhile highlighting the limited ability of stem cells to renew, making them susceptible to carcinogenesis. This is relevant because it shows the impact of CSC in the development of SCC. It is therefore important to focus on any kind of CSC-related biomarkers that could provide insight into potential therapeutic SCC management schemes.

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Cancer stem cells derive either through transformation of normal stem cells (NSCs) or through de-differentiation of tumor cells. Following initial transformations at the level of 17p (TP53) and 3p/9p (p16/FHIT), NSCs give rise to transformed transit-amplifying cells (TACs). These cells first multiply, then expand, and can lead to development of a neoplastic cell field. A further modification of the Rb 13q gene is followed by the inception of the main tumor. Tumor invasion can occur either in a monoclonal or polyclonal cancer inducing way. Thus, tumor CSCs can either spread through lateral migration (CD44h/ALDH1A1h) or they can plant and form a genetically similar tumor, as per the monoclonal model. On the other hand, successive modifications of normal stem cells in the epithelium can lead to the development of indepen-

Another important biomarker linked to proliferation and differentiation of skin cancers is CD133 [62, 63]. It is a transmembrane hematopoietic stem cell glycoprotein that correlates with an advanced stage of a poorly differentiated tumor, thus having a poor outcome in SCC [64–69]. Several studies have provided evidence that CD133+ CSCs exhibit resistance to apoptosis induced through action on TGF-β, or through tumor necrosis factor. This strengthens the conclusion that new therapeutic agents are required and they need to focus on CD133 being

An interesting aspect is that the phenotypic heterogeneity and plasticity of CSC has been associated with epithelial-to-mesenchymal transition (EMT), another important factor linked to both local and remote tumor invasiveness. Although directly responsible for many deaths caused by cancer, its role in SCC is still under debate. EMT is a process involved in embryogenesis and it is designed to create the mesoderm during gastrulation. This is a process through which epithelial cells acquire a migratory mesenchymal phenotype [71]. When the migratory mesenchymal cells mature, they may undergo a reverse process—mesenchymal-epithelial transition, to regain the epithelial phenotype. EMT and non-EMT CSC populations show a strong evidence of CD44 so much that they co-exist transitioning between the two phenotypic states through EMT and reverse mesenchymal-epithelial transition. To note, both cell types are present in oral squamous cell carcinoma (OSCC) generated cells. In addition, recent research that studied the expression of CD44 and epithelial-specific antigen (ESA) clarified that CD44(high)/ESA(low) EMT CSC has a mesenchymal phenotype, while CD44(high)/ESA(high) non-EMT-CSC has epithelial characteristics. To note, EMT CSC requires an ALDH + phenotype (aldehyde dehydrogenase 1) to evolve into non-EMT CSC and to develop metastasis [72].

dent clones, as per the polyclonal model (**Figure 1**) [61].

directed at stopping tumor recurrence and metastatic spread [70, 71].

Inhibition of squamous cell differentiation has been shown to be one of the most important mechanisms in the development of cSCC. Many therapeutic approaches have been proposed that have had at the forefront this mechanism and the molecules involved. Thus, S100 is a family of Ca-modulated proteins comprising the S100A7-psorasin-protein identified both in the keratinocytes in the psoriatic skin and in the various types of SCC (lung, oral cavity, bladder, skin) with an important role in metastasis and cellular differentiation [42–46]. Furthermore, the gene encoding the information required for the synthesis of this protein is located in the chromosome 1q21 containing the epidermal differentiation complex [47]. Recent studies have highlighted that overexpression of S100A7 is associated with increased cellular survival rates by decreasing cell differentiation, while poor expression of S100A7 correlates with significant cellular differentiation [48].

It has also been found that expression in tumor cells of a member of the signal transducer and activator of phosphorylated pSTAT3 transcription is closely correlated with the depth of tumor invasion and metastasis [49].

A fairly controversial issue has recently been the link between SCC and collagen VII, as a mortality of about 80% has been reported in patients with severe generalized recessive dystrophic epidermolysis bullosa (RDEB) associated with metastatic SCC. Currently, the link between SCC aggressiveness and collagen VII (Col 7) is being debated considering that mortality is high (more than 78%) in patients with severe generalized RDEB with metastatic squamous cell carcinoma. Mutations occurring in COL7AI, the gene encoding information for Col 7 synthesis, cause RDEB [50]. This disease is characterized by the fragility of skin and mucous membranes due to a decrease in Col 7 formation (the main component of the anchored fibrils) leading to formation of blisters and chronic skin trauma (a risk factor for SCC) [50]. There are many studies in progress that try to increase Col 7 synthesis by different methods but it has been observed that high levels of Col 7 are associated with activation of Phosphoinositide 3-kinase pathway which leads to an increase in SCC invasiveness, so there is no formal control of this process [51].

Moreover, tumor clinical factors such as size, anatomical location, tumor thickness, depth of invasion, histopathological subtypes, perineural invasion and inflammation [10, 37] correlate with an increased risk of developing metastatic lesions with significant impact on progression and aggressiveness of SCC. Immunocompromised patients have been shown to have a more aggressive course of SCC. In addition, there is evidence that age and sex can play a role in survival [52]. Although some of these factors provide a perspective on the prognosis and metastatic potential of SCC, they are less used in practice and have not been included in staging schemes [53].
