Squamous cells carcinoma (SCC) is the second most frequent of the keratinocyte-derived malignancies after basal cell carcinoma and is associated with a significant psychosocial and economic burden for both the patient himself and society. Reported risk factors for the malignant transformation of keratinocytes and development of SCC include ultraviolet light exposure, followed by chronic scarring and inflammation, exposure to chemical compounds (arsenic, insecticides, and pesticides), and immune-suppression. Despite various available treatment methods and recent advances in noninvasive or minimal invasive diagnostic techniques, the risk recurrence and metastasis are far from being negligible, even in patients with negative histological margins and lymph nodes. Analyzing normal, dysplastic, and malignant keratinocyte proteome holds special promise for novel biomarker discovery in SCC that could be used in the future for early detection, risk assessment, tumor monitoring, and development of targeted therapeutic strategies.
Squamous cell carcinoma (SCC) is responsible for 20% of skin malignancies [
Considering the increasing incidence in cSCC and the risk of metastasis and recurrence, even in patients with negative histological margins and lymph nodes, it is necessary to identify circulating molecules that can help predict the prognosis/evolution of this pathology [
Generally all epithelia play a paramount protective role for their underlying tissue, being the first line of defense against many harmful exogenous agents and at the same time acting as a permeable barrier that prevents loss of body fluids. Epithelia are usually composed of several layers of cells, each with their own specific phenotypes, being characterized by different degrees of differentiation according to their placement within the thickness of the tissue [
Studying the proteins present in normal keratinocytes and the changes of their pattern that occur in inflammation or carcinogenesis may lead to identification of new therapeutic targets or new biomarkers valuable in early diagnosis and prognosis of skin cancer [
A recent
The same study revealed a change in keratinocyte pattern of proteins in inflammatory conditions induced by stimulation with IL-1 beta. Thus, the level of proteins with roles in keratinocyte differentiation, such as alpha-2 macroglobulin-like protein-1, and the level of proteins involved in motility of keratinocytes such as integrin beta 4 were reduced. On the other hand, the presence of proinflammatory cytokines, such as IL-1F9 and IL-18, was observed. Moreover, stimulation with IL-1 beta increased the level of proteins involved in nuclear factor kappa B (NF-
UV exposure is one of the most important risk factors of skin cancer. Several studies using proteomic approaches have highlighted the alterations of protein expression induced by UV radiation on skin cells. UV exposure of skin induces suppression of cell-mediated immune responses, DNA damage, and formation of reactive oxygen species which can lead to oxidative stress and cellular damage (see Figure
UV-induced skin carcinogenesis. UV radiation alters the normal immune responses, induces DNA damage and oxidative stress, and may lead to development of skin cancer.
Chronic exposure to low doses of UV radiation also impacts the skin pattern of proteins by activation of different cellular signalling pathways, such as the mitogen-activated protein kinases (MAPK) pathway, the phosphoinositide 3-kinase (PI-3K) pathway, and the nuclear factor NF-
Exposure to carcinogenic chemicals is another factor that increases the risk of developing SCC. One of the main environmental factors with a strong link to skin carcinogenesis is arsenic [
Mouse models are the most commonly used animal models for the study of skin cancer, because in many aspects they mirror the mechanisms of human carcinogenesis [
However, there are numerous differences between distinct strains and different experimental models and there is hope that proteomic techniques will allow highlighting of the intimate mechanisms underlying these differences. Proteomic analysis in animals C57BL/6-resistant and DBA/2 sensitive, following 12-O-tetradecanoylphorbol-13-acetate (TPA) administration, demonstrated 19 different expressed proteins, such as S100 calcium binding proteins A8, A9, and A11 as well as parvalbumin
Inflammation is involved in many types of pathologies, from AK and Bowen’s disease (BD) to cutaneous SCC (cSCC) and other kind of cancers; thus the involvement of inflammatory markers, such as the complement factor H (CFH) and FHL-1 (factor H-like protein-1) in the development of cSCC has attracted an increasing interest [
Serpin A1 or 1-antitrypsin is included in the serine peptidase inhibitors (Serpins) family which has a very large distribution in the human body and has various functions (coagulation, inflammation, and turnover of extracellular matrix). Serpins are divided into two groups: A which includes extracellular molecules and B formed by intracellular molecules [
A factor which may promote tumor genesis is represented by the mutations in tumor suppressor gene. APC gene is such an example; mutations occurring in this gene conduct to the synthesis of a short nonfunctional APC protein. This gene was identified in patients with familial adenomatous polyposis (FAP) as well as in patients with sporadic colorectal carcinomas [
Dysregulation of cellular signalling in SCC. Aberrant activation of EGFR induces phosphorylation of
The development of cSCC is influenced by many other modifications induced by UV radiation such as the presence of melanocortin-1 receptor (associated with fair skin and red hair) which represents a risk factor for developing cSCC as well as melanoma [
Studies of molecular markers reflecting initial changes in skin carcinogenesis showed that, in sun-exposed skin, in which AK or SCC develops, the main molecular mutation is of gene p53. Considering the fact that this mutation is found in AK as well as in SCC represents the proof that this alteration is produced early in the development of cancer (AK is considered a precursor of SCC) [
Using reverse phase protein microarray (RPMA) samples from normal skin, AK, nonadvanced SCC, and advanced SCC were analyzed in order to identify which pathways were activated in the progression of SCC. The study showed that UV radiation activates numerous signal transduction pathways, such as p38, MAPK, and PI3K-AKT. These alterations may further influence apoptosis, proliferation, inflammation, and differentiation which may result in SCC development. It was demonstrated that in samples of skin from SCC and AK the percentage of phosphorylated AKT was significantly higher than in normal skin and in skin samples from metastatic SCC this protein value was the highest. The same results were obtained for mTOR (Ser2448), 4EBP1 (Ser65), 70S6K1 (Thr421), p70S6K1 (Thr421/Ser424), and S6 (Ser6) [
It appears that the inhibition of squamous cell differentiation is the most important mechanism that increases the invasiveness of cSCC; thus identifying molecules that can counteract this mechanism may help instate a more efficient treatment [
The link between aggressive SCC and type VII collagen (Col7) is debated considering the fact that mortality is high (more than 78%) in patients with severe generalized recessive dystrophic epidermolysis bullosa (RDEB) from metastatic squamous cell carcinoma. Mutations occurring in COL7AI, the gene which encodes the information for Col7 synthesis, cause RDEB. This disease is characterized by skin and mucosal fragility due to a decrease in Col7 formation (the main component of anchoring fibrils) which leads to blister formation and chronic skin traumatisms (risk factor for SCC) [
It was discovered that SCC in mice is determined by Pam212, a keratinocyte cell population which does not have the ability of metastasizing, although cells that drift from Pam212 (LY lines) were found in lymph nodes metastases [
Tyrosine kinase receptor family contains a larger group of receptors named erythropoietin-producing hepatocellular (Eph) receptors divided into two smaller subclasses A and B. The molecule that serves as ligand is ephrins [
Cancer stem cells (CSC) represent a population of cells with the unique characteristic of being solely responsible for initiating and maintaining tumor growth [
Available treatment options for skin SCC [
Nr. crt. | Type of cSCC | Therapy | Adjuvant |
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(1) | Low risk cSCC | Electrodessication |
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(2) | Invasive cSCC | Surgical excision |
Radiation therapy provides good locoregional control and can also be used as primary therapy for lesions that cannot be surgically excised |
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(3) | Metastatic cSCC | Chemotherapy | |
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(4) | Prevention | Decreased UVR exposure |
Molecular pathways governing epidermal stem cells homeostasis and tumorigenesis.
Nr crt | Molecular pathway | Roles |
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(1) | p63 | Proliferation, self-renewal, development, |
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(2) | SRF/MAL | Differentiation, development, cytoskeletal regulation |
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(3) | mTOR | Senescence, cell size, tumorigenesis |
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(4) | p75 | Apoptosis, communication, differentiation |
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(5) | Hippo | Organ size, antiproliferative, apoptosis |
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(6) | Notch | Differentiation, morphogenesis, suprabasal switch [ |
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(7) | FOXM1 | Proliferation, genome instability, tumorigenesis |
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(8) | p38 MAPK | Proliferation, wound healing, differentiation, cell migration, invasivity, tumorigenesis [ |
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(9) | BMP | Proliferation, differentiation, plasticity, wound |
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(10) | TGF |
Proliferation, immortalization, tumorigenesis |
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(11) | TGF |
Proliferation, hyperplasia, immortalization, |
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(12) | EGFR | Proliferation, maintenance, tumorigenesis [ |
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(13) | c-myc | Proliferation, differentiation, tumorigenesis |
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(14) | Shh | Development, morphogenesis, proliferation |
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(15) | Wnt | Proliferation, self-renewal, wound healing, morphogenesis, tumorigenesis |
cSCC biomarkers.
Nr. crt. | Biomarker | Roles |
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( |
CFH |
(i) Inhibiting one of the three pathways that activate the complement C3 |
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( |
Serpin A1 | (i) Coagulation |
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( |
APC | (i) Inducing the destruction of |
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( |
Phosphorylated AKT, |
(i) Influencing apoptosis, proliferation, inflammation, and differentiation [ |
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( |
S100A7 | (i) Role in metastasis [ |
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( |
Col7A1 | (i) Encoding the information for Col7 formation [ |
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( |
MMP-7 | (i) Maintaining homeostasis of many tissues including skin, by proteolysis of extracellular matrix [ |
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( |
Krt8 |
(i) Together they induce a higher rate of invasiveness in a cell population [ |
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( |
CD133 | (i) Proliferation |
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( |
CYFRA 21-1 | (i) Component of structural proteins involved in epithelial intermediary filaments formation [ |
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( |
mtDNA | (i) Mitochondrial functions [ |
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( |
Hsp70 | (i) it is presumed that it may help tumorous cells survive apoptosis and necrosis [ |
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( |
Plectin | (i) Cytolinker of plakins family which forms the links between filaments [ |
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( |
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(i) Vulvar carcinogenesis |
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( |
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(i) Invasiveness |
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( |
EphB2 | (i) Determining proliferation, migration, and invasion [ |
In the last decades cancer therapy studies have focused on targeted molecular treatments (monoclonal antibody, small molecule tyrosine kinase inhibitor); therefore scientists have developed a great interest for EGFR (epidermal growth factor receptor) which is a tyrosine kinase receptor and two of its most important ligands are epidermal growth factor and transforming growth factor-
The incidence of
Another molecule that can help identify patients with a high mortality risk is CRP (inflammation marker), the low survival rate and cancer invasiveness being demonstrated for oSCC (inflammation provides the circumstances for proliferation and angiogenesis); also studies showed that elevated CRP was correlated with bone, skin, and lymph node invasion [
In oral squamous cell carcinoma, measuring mitochondrial DNA (mtDNA) may be useful for postoperative monitoring considering the fact that an important number of patients with head and neck SCC (HNSCC) that had histological negative margins had mtDNA mutations [
Considering the significant risk of recurrence and metastasis of SCC, there is a high necessity to discover novel molecules harvested from various biological samples that could explain the occurrence and evolution of this keratinocyte-derived tumor. In this regard, protein-focused research based on high-throughput proteomic technologies has evolved rapidly to identify unique biosignature of skin cancer.
Analyzing differences between normal, inflammatory, and malignant keratinocyte proteome holds special promise for novel biomarker discovery in SCC that could be used in the future for early detection, risk assessment, and tumor monitoring. Furthermore, identification of novel potential biomarkers for SCC development and progression will aid the discovery of individualized targeted therapies for these patients.
The authors declare that there is no conflict of interests regarding the publication of this paper.
All authors have equally contributed to the conception and preparation of the manuscript.
This paper is partly supported by Grant PNII-PT-PCCA-2013-4-1386 (Project 185/2014) financed by Executive Agency for Higher Education, Research, Development and Innovation and by Young Researchers Grant 33891/2014 financed by Carol Davila University of Medicine and Pharmacy, Bucharest. Bogdan Calenic acknowledges that this work was supported by a Grant from the Romanian National Authority for Scientific Research and Innovation, CNCS-UEFISCDI, Project no. PN-II-RU-TE-2014-4-1879.