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

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

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 therapeutic targets in skin SCCs [85, 86].

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

**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.

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

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

and to develop metastases.

back to the epithelial CSC phenotype [74].

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

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 [87, 88]. In immunosuppressed HPV-infected patients, it was reported a notable HLA I—SCC connection, probably due to the fact that HLA I processes and presents intracellular peptide antigens, including viral proteins, and thus HPV could be a co-factor of tumorigenesis [87].

epithelial adhesion are currently studied regarding their metastatic involvement. Thus, collagen XVII, integrin α6β4 and especially their binding partner laminin 332 are mainly recognized to promote invasion and metastasis in various tumors. By tissue microarray analysis, it was registered that γ2 chain of laminin 332 has the highest expression in SCC samples, whereas the expression of collagen XVII and integrin β4 greatly differs in SCC and precursors lesions (actinic keratosis and Bowen's disease) [95] and moreover, integrin β4 knockdown would reduce the migration of keratinocytes and of malignant cells [96]. All these results suggest the contribution of collagen XVII, integrin α6β4 and laminin 332 to SCC tumorigenesis through their variable expression patterns translated in different migrations and invasion

Squamous Cell Carcinoma: Biomarkers and Potential Therapeutic Targets

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Thereby, the tumor microenvironment plays an important role in cSCC progression, offering a genuine reservoir for finding novel targets for both therapeutic purposes and risk assess-

Despite recent advances in diagnosis and therapy, OSCC is still one of the most difficult malignancies to handle due to its great invasive potential both locally and at lymphatic level (in the cervical lymph nodes) [98]. Its occurrence varies across the world as it is closely linked to diet and lifestyle choices (alcohol and cigarettes). OSCC occurs as a result of squamous cells genetic mutations, the new cells developing multiplicative and invasive characteristics [99]. Its genetic heterogeneity can be later highlighted by the fact that many tumors, at a similar stage and location, present significant clinical differences and they can react very differently to treatment. Although the therapeutic strategies are in a permanent development, the survival rate of OSCC patients remains low. It has also been found that predicting treatment outcome using conventional clinical and histopathological parameters carry a low success rate. It is clear that histopathology remains to this day the benchmark decision-making process as far as diagnosis and treatment are concerned. However, recent molecular studies have made significant progress in understanding and identification of those biomarkers best placed to predict OSCC aggression. Attempts have been made to refine histopathological analysis with immunohistochemistry; this detects gene composition at protein level and brings forward several prognostic tumor biomarkers associated with OSCC's clinical outcome. As such, tumor suppressor genes, oncogenes, angiogenic markers, cell adhesion molecules and cell proliferation markers have been discovered to be potential tools that could help to predict the outcome of OSCC patients [99]. Therapeutic management through molecular inhibition directed at those biomarkers associated with radiotherapy and/or adjuvant chemotherapy are

EGFR is a transmembrane cell-surface receptor that binds to ligands such as EGF and TGF-α and is one of the most studied OSCC biomarkers. It triggers the activation of the protein-tyrosine kinase system, which acts as a regulator of the signaling process linked to cell multiplication and differentiation [100]. It plays a significant role in OSCC's resilience to radiotherapy.

features [97].

ments in cSCC.

**8. Biomarkers of oral SCC**

promising treatments for OSCC patients.

Alterations in the composition of basement membrane and dermal extracellular matrix of premalignant lesions are early events in cSCC progression. An influx of inflammatory cells promotes the secretion of proteases, which in turn regulates the availability of growth factors, cytokines, and chemokines and thus influences the growth and invasion of cSCC. Later, the number of inflammatory cells increases with cSCC progression, and the expression of complement factors and inhibitors by tumor cells is induced (CFI, CFH, FHL-1) [89]. A fine interplay between matrix metalloproteinases (MMPs) and their inhibitors could settle the scene for discovering new targets and prognostic or monitoring predictors of the disease. As in cutaneous melanoma, where the role played by MMPs in the phenomenon of regression is an actual approach [90], in SCC, the cellular enzymatic portfolio is a good pool for emerging novel targets coupled to novel biomarkers. For instance, upregulation of MMP-7 expression has also been registered in cSCC, especially in the tumor invasive edge, and moreover activates heparin-binding epidermal growth factor-like growth factor (HB-EGF) promoting cellular proliferation [91] and thus suggesting a future therapeutic effect of HB-EGF antagonists in advanced cSCC [12].

Serine peptidase and their inhibitors (Serpins) are also considered useful for biomarker monitoringing of cSCC progression. Studies performed on serpin family gene expression levels in cSCC cell lines versus normal keratinocytes demonstrate a significantly raised Serpin-A1 expression correlated with the tumorigenic change of keratinocytes [92]. *In vivo* studies correlate Serpin-A1 expression with tumor progression in SCC tumor cells. By using a chemically induced skin carcinogenesis mouse model, as a valuable tool in completing cancer progression profile [23] it was checked the correlation of Serpin-A1 expression with progression of mouse skin SCC [92], suggesting that Serpin-A1 may serve as an useful biomarker for monitoring cSCC progression. Maspin is another member of serpin family—an inhibitor of mammary serine protease—reported as a tumor suppressor in various cancers. Real-time PCR and Western blotting analysis found that Maspin was downregulated in the cSCC tissues compared with the nearby normal tissues. Studies performed on A431 cell line revealed that overexpression of Maspin inhibits growth, cellular proliferation and enhances A431 cells apoptosis by increasing PARP and Bax expression, while decreasing Bcl-2 expression. Therefore, Maspin analysis may provide new insights in the diagnosis and therapy of cSCC [93].

New potential classes of agents for cSCC are also directed to counteract the metastatic feature of this tumor which represents a difficult challenge, knowing that metastatic cSCC has a mortality rate of over 70%. As a comprehensive chemotherapeutic approach in the metastatic form is still lacking, new molecular insights are to be done. Recently, expression of EGFR and nuclear active IκB kinase (IKK) was proved to have a role in metastatic prediction. Thus, a newer and more promising class of agents for metastatic cSCC therapy is represented by EGFR inhibitors. Other advances in finding novel treatments for metastatic cSCC are related to p53 studies, epigenetic approaches such as hypermethylation of specific genes, chromatin remodeling, and the RAS/RTK/PI3K pathway [94]. Molecules with well-established roles in epithelial adhesion are currently studied regarding their metastatic involvement. Thus, collagen XVII, integrin α6β4 and especially their binding partner laminin 332 are mainly recognized to promote invasion and metastasis in various tumors. By tissue microarray analysis, it was registered that γ2 chain of laminin 332 has the highest expression in SCC samples, whereas the expression of collagen XVII and integrin β4 greatly differs in SCC and precursors lesions (actinic keratosis and Bowen's disease) [95] and moreover, integrin β4 knockdown would reduce the migration of keratinocytes and of malignant cells [96]. All these results suggest the contribution of collagen XVII, integrin α6β4 and laminin 332 to SCC tumorigenesis through their variable expression patterns translated in different migrations and invasion features [97].

Thereby, the tumor microenvironment plays an important role in cSCC progression, offering a genuine reservoir for finding novel targets for both therapeutic purposes and risk assessments in cSCC.
