**6. Cancer stem cells in SCC**

Cancer stem cells (CSC) represent a pluripotent population of tumor cells with self-renewal properties playing an important role in tumor initiation, growth and maintenance [54, 55]. 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.

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].

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

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

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].

Cancer stem cells (CSC) represent a pluripotent population of tumor cells with self-renewal properties playing an important role in tumor initiation, growth and maintenance [54, 55].

increase in SCC invasiveness, so there is no formal control of this process [51].

tumor invasion and metastasis [49].

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

**6. Cancer stem cells in SCC**

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

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

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].

therapeutic resistance but also in tumor recurrence [59, 77, 78] being associated with resistance to epithelial growth factor receptor (EGFR) inhibitors [79]. In epithelial tissues, some stromal signals may induce EMT, leading to downregulation of epithelial processes and upregulation

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**7. Current and future molecular therapeutic targets in cutaneous SCC**

acts critical for promoting SCC [83] or UVB-induced cutaneous neoplasia [84].

apeutic targets in skin SCCs [85, 86].

The high level of gene mutations in UV-exposed skin has hampered the search for novel hints of disease invasiveness and metastatic potential. In SCC, metastasis to regional lymph nodes occurs in roughly 5% of cases and is associated with significant morbidity. Clinical biomarkers of SCC metastasis are currently missing and histological assessment could be unreliable [82]. Cutaneous SCC typically manifests gradually, ranging from a precursor actinic keratosis (AK) to *in situ* SCC, invasive SCC, and finally metastatic SCC. Molecular inquiring into SCC could be done by different experimental models. Although the differences in skin structure amid mice and humans would limit somewhat the correlation with human disease, transgenic mice models have revealed that upregulation of the EGFR/Fyn/Src/Erk pathway

Latest studies associated omics approaches with humoral immune systems components in SCC involvement; thus recent approaches discern the expression of complement system components in SCC. SCC cell lines and human normal keratinocytes were profiled with an Affymetrix platform and then subjected to quantitative real-time PCR revealing upregulation of complement factor H (CFH) and factor H-like protein-1 (FHL-1) mRNA in cancer cell lines and were proven significantly higher in tumors compared to normal skin. Moreover, immunohistochemistry analysis of CFH and FHL-1 in invasive SCCs, *in situ* SCCs and premalignant lesions (actinic keratoses) showed a specific and stronger expression in SCCs compared with *in situ* carcinoma and actinic keratoses. Not surprisingly, it was found that the level expression of complement factor I (CFI) was higher in the aggressive transformed cell line (RT3) than in less tumorigenic HaCaT cell lines. In addition, by knocking down CFH and FHL-1 expression, proliferation and migration of SCC cells were inhibited, suggesting a role of CFH and FHL-1 in cSCC progression and spotting them as progression markers and potential ther-

Huge costs related to skin cancers therapies in general, including SCC became another request for defining reliable biomarkers and better understanding a pathogenesis with significant public health impact. Host immune system influences SCC risk as its incidence is considerably higher in patients with compromised immunity. Very recent studies assign a role for HLA system in SCC risk. Unlike BCC and cutaneous melanoma, SCC often displays partial expression of HLA I proteins, also exhibiting aberrant surface expression of HLA II proteins as a defense mechanism for immune evasion. Analyzing allelic variation and cellsurface protein expression germline of HLA I and II antigens in SCC patients and healthy controls, it was suggested that HLA pattern differs between immunocompetent and immunosuppressed patients regarding the risk for developing SCC. This difference may be owed to some viruses (HIV, HPV) that potentiate tumorigenesis in immunosuppressed patients

of EMT-inducing transcription factors such as Twist and Snail [80, 81].

**Figure 1.** Model for cancer stem cell field onset the process is initiated by a carcinogenic injury producing an alteration (p53/p16/FHIT) in the epithelial normal stem cell (NSC). The cancer stem cell (CSC) will proliferate and form a patch of transit amplifying cells (TAC) which then extends to form a field. At this point, the cells are still in a dysplastic, premalignant stage. It is only after another assault (Rb), one of the field's cells forms the primary tumor. Field cancerization progression takes place through either the monoclonal or polyclonal models. In the monoclonal model, CSCs extend the field by lateral migration (CD44h/ALDH1A1h) or implant at a new site ultimately forming a genetically similar tumor. In the polyclonal model, multiple assaults to epithelial NSCs lead to the evolution of independent clones.

A large number of ALDH1 cells have been detected in lymph node metastases, more than the corresponding primary tumors—indicating the CSC capacity to complete metastatic cascade and to develop metastases.

It has also been shown that a CD44-regulated signaling pathway mediated by the phosphorylation of glycogen synthase kinase 3 (GSK3) can influence CSC phenotypes [73]. Hence, the inhibition of GSK3 reduces the expression of stem cell markers and upregulation of the differentiation markers found in the CD44(high)/ESA(high) cell fraction reversing from EMT and back to the epithelial CSC phenotype [74].

EMT's involvement in several types of cancers such as OSCC [74], breast cancer [75] and others is variable, affecting both tyrosine kinase receptors as well as Wnt signaling pathways [76].

It has been shown that cell lines derived from oral and dermal SCC contain a new population of CSC that influences EMT. It has also been established that EMT is involved not only in therapeutic resistance but also in tumor recurrence [59, 77, 78] being associated with resistance to epithelial growth factor receptor (EGFR) inhibitors [79]. In epithelial tissues, some stromal signals may induce EMT, leading to downregulation of epithelial processes and upregulation of EMT-inducing transcription factors such as Twist and Snail [80, 81].
