Protein Kinase D1 in normal skin and skin cancer - International

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International Journal of Contemporary Research and Review ISSN 0976 – 4852

Research

CrossRef DOI: https://doi.org/10.15520/ijcrr/2018/9/02/428 February, 2018|Volume 09|Issue 02| Section: Biochemistry

Comparison analysis of Basocellular Carcinoma and Spinocellular Carcinoma - Protein Kinase D1, Wnt/β-catenin and Epithelial to Mesenchymal Transition (markers) Petya V. Ivanova1, Ana I. Maneva2 1,2

Department Chemistry and Biochemistry, Pharmaceutical Faculty, Medical University of Plovdiv, 15A Vasil Aprilov Blvd, 4002 Plovdiv, Bulgaria 1 Email id: [email protected] 2 Email id: [email protected] Received 2018-01-04; Accepted 2018-02-15

Abstract: Understanding cancer biology is crucial to the successful diagnosis and application of personalized therapies. Skin carcinogenesis is a multi-step process. The first step is the development of potentially malignant disorders (PMDs) known as leukoplakia, erytroplakia, lichen planus, probably trough mutation in the proteins regulated cell cycle (p16INK4A, p21Waf1/Cip1/Sdi1, p27 kip1, Cyclin D1) and, second in p53 (TP53), Ras, hTert, EGFR, will reverse benign phenotype of late precancer lesions (PMDs) into benign cancer lesion. Third mutation probably in PI3K, PKD1 or E-cadherin, will increase malignant potential of SCCs (Spinocellular Carcinomas), activating EMTransition, invasion and metastasis, e.g. aggressive phenotype. In BCC (Basocellular Carcinoma) mutations of p53 are known to be late events (UV signature), whereas silencing of 14-3-3 takes place early in tumor progression, concomitant with increased activity of Snail. Early increased expression of PKD1 and down-regulation of c-myc mRNA are also events in pathogenesis of BCC (Basocellular Carcinoma). There is no data for detected mutations in PKD1 gene in both cancers, although the kinases is with high expression in BCC (Basocellular Carcinoma) and lack of expression in SCCs (Spinocellular Carcinomas). There is no data concerning PKD1 expression in the PMDs leading to SCCs (Spinocellular Carcinomas), nor to BCC (Basocellular Carcinoma), such data were recently published concerning PKD1 expression in pancreatic oncogenesis. Curently adequate question is whether lack of PKD1 expression in SCCs (Spinocellular Carcinomas), despite its PMDs origin, is a consequence of its spinous layer origin, or is a consequense of PKD1 gene mutations (downregulation). Identification of mutations as markers for early malignant transformation could be useful for early diagnosis and treatment of skin head and neck cancers. Keywords: PKD1, Wnt/β-catenin, EMT, SCCs (Spinocellular Carcinomas), Carcinoma), skin cancer

BCC (Basocellular

Abbreviations: PKD1 - Protein Kinase D1 PRKD1- gene of Protein Kinase D1 International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20193

Petya V. Ivanova et al. Comparison analysis of Basocellular Carcinoma and Spinocellular Carcinoma - Protein Kinase D1, Wnt/β-catenin and Epithelial to Mesenchymal Transition (markers)

PMDs - Potentially Malignant Disorders ERK1/2 - Extracellular signal Regulated Kinase 1/2 EMT - Epithelial to Mesenchymal Transition HIF-1α - Hypoxia-Inducible Factor 1α LOX - Lysyl Oxidase BCC – BasoCellular Carcinoma (Basal Cell Carcinoma) SCC - SpinoCellular Carcinoma (Squamous Cell Carcinoma) HNSCC – Head and Neck SCC HPV - Human Papilloma Virus LOH - Loss Of Heterozygosity MMP – Matrix Metallo-Proteinase COX-2 –: COX-2 – Cyclooxygenase-2 (PGHS-2 - Prostaglandin (G)/H Synthase) EGF - Epidermal Growth Factor EGFR - Epidermal Growth Factor Receptor ROS - Reactive Oxygen Species HDAC - Histone Deacetylase I. Introduction: Protein kinase D1 (PKD1), a ubiquitous serine/threonine kinase, was originally described as a novel κ isoform of the protein kinase C (PKC) family, as it shares two cysteine-rich domains (C1a and C1b) that bind phorbol esters and diacylglycerol as in the PKC family. Unlike other members of the PKC family, PKD1 also has a unique pleckstrin homology (PH) domain, differentiating them from other members of the PKC family, and the catalytic domain of PKD1 is most closely related to calcium calmodulin– dependent kinases (CaMK) [1,2,3,4]. Protein Kinase D1 (PKD1), has been implicated in numerous cellular functions, including Golgi organization, oxidative stress responses (including UVB), immune regulation, angiogenesis, cell survival, proliferation, differentiation, migration, cell-cell adhesion and epithelial mesenchymal transition (EMT). It is implicated in pathological states like hypertrophic cardiomyopathy, human myeloid leukemia, diabetes, and cancer. PKD1 has been reported to be downregulated in advanced prostate, breast and gastric cancers, shown to play a role in tumorigenesis and metastasis, and upregulated in BCCs (basocellular carcinoma) and pancreatic cancer. Embryonic deletion of PKD1 in mice is lethal, suggesting PKD1 plays a crucial role

cancer which resembles alterations in SCC (SpinoCellular Carcinoma) and pancrearic cancer, resembling alterations in BCC (BasoCellular Carcinoma), at least in alterations in PKD1 expression: Breast cancer: Analysis of invasive human breast tumors has revealed that PKD1 expression is downregulated in infiltrative ductal carcinoma compared to normal breast tissue. Supporting these results, several transcriptional microarray studies of gene expression in normal breast tissue and of early and advanced-stage breast tumors have shown reduction of the PRKD1 (PKD1) gene correlating with increased invasiveness and cancer progression. The highly invasive cell lines SKBR3, T47D, and MDA-MB-231 have shown to express little or no PKD1, while normal breast cell lines and very lowinvasive breast cancer cell lines such as MCF-7 and BT-474 have been shown to express PKD1 in moderate levels, though considerable variation exists in the reported literature when different antibodies for PKD1 are utilized [1,3]. Furthermore, the authors showed that that the gene promoter of PRKD1 (the gene of PKD1) is aberrantly methylated and silenced in its expression in invasive breast cancer cells and during breast tumor progression, increasing with the aggressiveness of tumors. Using an animal model, they showed that reversion of PRKD1 promotor methylation with the FDA-approved DNA methyltransferase inhibitor decitabine restores PKD1 expression and block tumours spread and metastasis (decrease MMP-9 (matrix metalloproteinase-9) and COX-2

in development, which cannot be replaced by other PKD family members, PKD2 and PKD3 [1,2,3,4] Reviews of PKD1 as a potential new target for cancer therapy were published [1,3]. Here in brief will be discussed alterations in breast and prostate International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20194


(cyclooxygenase 2) expression) to the lung in a PKD1-dependent fashion. However, treatment with decitabine induces the reexpression of multiple genes, including tumour supressors such as TP53 (gene of p53) and CDKN1A (gene of cyclindependent kinase inhibitor 1A (p21Cip1)) or the gene encoding the ER (Estrogen-Receptor) [5]. Studies investigating the role of PKD in breast cancer progression have focused on the processes of invasion and adhesion. As early as 1999, Mueller and colleagues described an interaction between PKD1, paxillin, and cortactin at sites of invadopodia in MDA-MB-231 breast cancer cells. Invadopodia are actin-containing protrusions that extend outward into the extracellular matrix (ECM) and participate in degradation of the ECM. This interaction, present in invasive breast cancer cells but not in non-invasive lines, suggested that PKD1 may regulate the function or formation of the paxillin/cortactin complex to promote invasion. Furthermore, cortactin was recently determined to be a PKD1 substrate, though it remains to be determined whether this phosphorylation event and the PKD1/cortactin/paxillin association does indeed promote invasion [1]. Expression of constitutivelyactive PKD1 in invasive tumour cells enhanced phosphorylation of cofilin and effectively blocked the formation of free actin filament barbed ends. The inhibition of barbed end formation by PKD1 directly translated to decreased directed cell migration of tumour cells [6]. Multiple separate studies have strongly supported an opposing role for PKD in breast cancer cell invasion and adhesion. Based on mechanistic argumentation, it has been suggested that the regulation of adhesion and invasion by PKD1 may be related to MMP (Matrix Metallo-Proteinase) expression. Activated PKD1 caused reduced expression of metalloproteinases MMP-2, MMP-7, MMP-9, MMP-10, MMP-11, MMP-13, MMP-14, and MMP-15, while upregulating expression of MMP-3 [7]. As described earlier, MMPs are endopeptidases involved in the degradation of the ECM (extracellular matrix), a process that is required for the development of malignant tumors. Invasive behaviour in breast cancer cell lines has been linked to matrix metalloproteinases (MMPs) [5]. In breast cancer, MMP-2 in particular has been identified as an indicator of potential malignancy. Thus, it is possible that PKD1 may inhibit breast cancer invasion and metastasis through regulation

of MMP expression. Another critical target through which PKD1 exerts its inhibitory effect on cell migration is SSH1L, which was first shown in MTLn3 rat breast carcinoma cells. Later, a separate study by Hausser and colleagues showed that both PKD1 and PKD2 negatively regulate cell migration through direct phosphorylation of SSH1L in MCF-7 and MDA-MB-231 breast cancer cells [1]. PKD1 was recently implicated in EMT (Epithelial to Mesenchymal Transition) via inhibition of Snail functions and modulation of mesenchymalepithelial markers, such as vimentin, fibronectin and E-cadherin in breast cancers [3,5,6,7] (see below). Collectively, these studies suggest a potential significant role for PKD1 in the suppression of breast cancer metastasis [1,5]. Pancreatic cancer: Pancreatic cancer is one of the most aggressive cancers, and it is highly resistant to cancer chemotherapy. The overexpression of PKD1 has been shown to play a role in pancreatic cancer progression3. In normal pancreas, only PKD3 is expressed in acinar cells, whereas PKD1 is only expressed in islets of Langerhans and pancreatic ducts. This expression pattern changes when pancreatic acinar cells acquire an oncogenic Kras mutation or aberrant EGFR activation. Approximately 95% of all pancreatic ductal adenocarcinoma (PDAC) express either somatic activating mutations of Kras or show increased epidermal growth factor receptor (EGF-R) signaling [8,9]. It is thought that oncogenic KRas is an initial event leading to pancreatic cancer. Oncogenic KRas upregulates the epidermal growth factor receptor (EGF-R) and its ligands TGFα and EGF, which leads to additional activation of wildtype KRas; and activity of both pathways are needed for pancreatic tumorigenesis. Major downstream signaling cascades activated by active KRas in pancreatic cancer are the PI3-K/ PDK1/Akt (known also as PKB) and Raf/MEK1/2/ERK1/2 (Extracellular signal Regulated Kinase) pathways [10] . In response to such signaling acinar cells undergo acinar-to-ductal metaplasia (ADM). During ADM cells down-regulate expression of PKD3 and upregulate expression of PKD1, whereas PKD2 expression remains unchanged. In addition to increased expression, PKD1 activity is also elevated in presence of a mutant Kras, or after EGFRmediated activation of endogenous wildtype Kras.

International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20195


As a result of such signaling, PKD1 expression and activity can be detected in regions of ADM, PanIN1 and PanIN2 pre-neoplastic lesions, while the 2 other PKD isoforms are not involved in these processes. Questions remaining are i) how PKD1 expression is upregulated by both, mutant Kras and EGFR signaling?; and ii) how both pathways can mediate activation of PKD1? Since PKD1 activity downstream of Kras was determined by measuring nPKC-mediated activating phosphorylations, an involvement of the novel PKCs PKCε and/or PKCδ is most likely [10]. Yuan J. and Pandol J. (2016) identified PKD as a novel early convergent point for PKCδ and ε in the signaling pathways mediating NF-θB activation in pancreatitis [11]. In response to ROS (Reactive Oxygen Species), the occurrence of increased oxidative stress in tumor cells requires ROS-sensing signaling, activation through the ROS/Src/Abl/PKCδ pathway, PKD1 induces canonical NF-θB signaling through IθB kinase β and subsequent downregulation of inhibitor of kappa- light-chain-enhancer of activated B cells alpha [12]. Further more, Döppler et al.; Eiseler T. et al.; demonstrate that KRas-induced activation of the canonical NF-θB pathway is one mechanism of how PRKD1 (gene of PKD1) expression is increased and identify the binding sites for NF-θB in the PRKD1 promoter [10]. The authors have shown that PKD1 regulates ADM by activating the Notch pathway, which previously had been established as a driver of acinar cell re-programming. On one hand active PKD1 downregulated the expression of known suppressors of Notch (e.g. Cbl, Sel1l). On the other hand, active PKD1 also upregulated the expression of Notch target genes (e.g., Hes-1, Hey-1), molecules that are involved in Notch signaling (e.g. MAP2K7), stem cell markers (e.g., CD44), as well as proteinases, including Adam10, Adam17 and MMP7, that mediate Notch activation by S2 cleavage. PKD1 has been shown to activate nuclear factor θ-B (NF- θB); and NFθB and Notch both cooperate in some signaling pathways (reviews [8,9]). A crosstalk between Notch and canonical NF-θB signaling pathways is needed for progression of pancreatic cancer and PKD1 is a key enzyme linking KRas to Notch and NF-θB. Therefore, PKD1 could be a promising new target to treat pancreatitis, prevent precancerous lesions and tumor formation, but also progression of tumors [10,11,12].

In PANC-1 cells (human pancreatic ductal adenocarcinoma cell line), PKD stimulated DNA synthesis and mitogenic signaling and was activated by neurotensin (NT), a mitogenic neuropeptide that has been implicated in the autocrine/paracrine growth stimulation of human pancreatic cancer1. In vitro and in vivo assays following NT treatment revealed rapid PKC-dependent activation of PKD1, which in turn led to rapid activation of MAPK/ERK kinase 1 (MEK1/2), activation and nuclear translocation of extracellular signal regulated kinase 1/2 (ERK-1/2), and eventually increased DNA synthesis. PKD1, ERK-1, and ERK-2 activation was inhibited by the specific PKC (Protein Kinase C) inhibitors bisindolylmaleimide 1 (GF109203X, also called Go6850) and bisindolylmaleimide IX (Ro31-8220), suggesting the involvement of a PKC/PKD signaling process in human ductal pancreatic carcinoma cells. IHC analyses of a small cohort of tissue samples revealed the overexpression of PKD1 in pancreatic cancer compared with normal pancreatic tissues. The overexpression of PKD1 has been suggested to confer enhanced proliferation and higher antiapoptotic activity to the pancreatic cancer cells. The overexpression of PKD1 in the Colo357 pancreatic cancer cell line, which shows low PKD1 expression, not only decreased the sensitivity of the cells to CD95-mediated apoptosis but also enhanced cell growth and telomerase activity, suggesting a correlation between PKD1 expression and resistance to apoptosis. A recent in vitro and in vivo animal study involving the use of a new PKD1specific, small-molecule inhibitor (CRT0066101) showed inhibition of pancreatic cancer growth, attenuated PKD1-mediated NF-θB activation, abrogated the expression of NF-θB – dependent proteins including cyclin D1, survivin, and cIAP-1, blocks pancreatic cancer growth in vivo and suggests the development of PKD1 inhibitors as a novel therapeutic target for treatment of pancreatic cancer [3,13]. Furthermore, Eiseler T. et al.; indicate that PKD1, as opposed to PKD2, regulates (up-regulates) the expression of marker proteins involved in a hyperproliferative phenotype such as Cyclin D1 and -D2 [14,3] as well as Ajuba via phosphorylation of Snail1 at serine 11 in pancreatic cancer cells. Their data also suggest, that phosphorylation at this site is necessary for efficient binding of vital co-repressors to Snail1, such as HDAC1/2 (modulating Snail1dependent HDAC (Histone Deacetylase) activity. In



contrast to Balaji et al. (prostate cancer-see below) Snail1 phosphorylation at S11 did not affect nucleocytoplasmic shuttling of the protein. This may be explained by 14-3-3σ (stratifin) down-regulation in many tumor cells by different mechanisms, including promotor methylation or inhibition downstream of p53 mutations, thereby facilitating cancer formation by many routes. Snail1 is therefore required and sufficient for PKD1driven proliferation and anchorage-independent growth of pancreatic tumor cells [14]. Prostate cancer: Prostate cancer is one of the leading causes of cancer-related deaths in men in the United States. Although early detection of prostate cancer has greatly decreased the mortality rate, this cancer is often fatal at later stages because it can progress from an androgen-dependent (AD) to androgenindependent (AI) phenotype, for which there is no effective treatment. The highest expression of PKD1 is observed in prostate tissues, suggesting a crucial role of this protein in normal prostate functions [1]. In prostate cancer, somatic mutation in AR (androgen-receptor) results in progression of tumor from an androgensensitive (AS) stage to an androgen-insensitive (AI) stage that is refractory to androgen-depletion treatment. Recently, it was shown that PKD1 exists in a transcription complex along with AR and a promoter sequence for prostate specific antigen (PSA) in prostate cancer cells. PKD1 negatively regulates the function of AR in prostate cancer cells, because the overexpression of wildtype PKD1 or kinase-dead PKD1 attenuates ligand-dependent AR function. Alternatively, PKD1 knockdown enhances ligand-dependent AR activity. Studies have also revealed that PKD1 interacts and phosphorylates the Ser-82 residue of heat-shock protein 27 (Hsp27, a molecule that is necessary for nuclear translocation of AR) and represses AR functions in prostate cancer cells. The AR function is also modulated by interaction with other proteins, such as β-catenin, which augments AR functions. Because PKD1 interacts with and downregulates both nuclear β-catenin and AR transcription activity, deregulated expression of PKD1 may play a critical role in the initiation and progression of prostate cancer [3].

(IHC) analyses revealed severe downregulation of PKD1 in prostate cancer cells and an incremental decrease in PKD1 expression following progression from AD to AI prostate cancer. Additionally, an AI C4-2 (derived from LNCaP cell line) cell line showed significantly lower PKD1 expression compared with AD LNCaP prostate cancer cells (the prostate cancer cell lines LNCaP, which has less metastatic potential). These data suggest a potential role of PKD1 in the progression of prostate cancer from an AD to an AI state. Furthermore, their studies show that PKD1 interacts, phosphorylates, and positively regulates the functions of E-cadherin and β-catenin (cadherincatenin complex), enhancing cell-cell adhesion and decreasing cellular motility, strongly suggesting a major role for PKD1 in prostate cancer progression and metastasis [15,16,17,18]. Activated PKD1 also decreases nuclear β-catenin levels, resulting in the attenuation of oncogenic signaling by β-catenin‘s cotranscription factor activity. Additionally, while overexpression of PKD1 and E-cadherin suppresses the cancer cell phenotype, simultaneous coexpression of PKD1 and E-cadherin in prostate cancer cells results in a cumulative decrease in cancer phenotypes. PKD1 suppresses transcription factor Snail, a known E-cadherin repressor by inhibitory phosphorylation, induces expression of E-cadherin and up-regulates βcatenin (see below) [16,17,18]. These data strongly support a pivotal role of PKD1 in prostate cancer. Recent studies in xenograft mouse models with PKD1 overexpressing prostate cancer cells revealed significantly reduced tumor growth compared with control, supporting a tumor suppressor role of PKD1[3,17]. Luef et al.; demonstrate for the first time that the AR/NCOA1 (Androgen Receptor/ AR coactivator nuclear receptor coactivator) complex stimulates migration of prostate cancer cells through suppression of PRKD1. Furthermore, PRKD1 was negatively regulated by AR and PRKD1 knockdown could significantly enhance the migratory potential of the two cell lines tested. A strong decrease in migration and invasion upon NCOA1 knockdown, independently of the cell line‘s AR status, was observed. The authors suggest that specifically targeting NCOA1 could restore PRKD1 expression and reduce the migratory capability of tumor cells. Inhibition of PRKD1 reverted the reduced migratory potential caused by NCOA1 knockdown. Immunohistochemical

Studies from Balaji et al.; and Jaggi et al.; suggest important roles for PKD1 in prostate cancer development and metastasis. Immunohistochemical International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20197


staining of prostate cancer patient samples revealed a strong increase in NCOA1 expression in primary tumors compared with normal prostate tissue, while no final conclusion could be drawn for PRKD1 expression in tumor specimens. Thus, the authors‘ findings directly associate the AR/NCOA1 complex with PRKD1 regulation and cellular migration and support the concept of therapeutic inhibition of NCOA1 in prostate cancer [19]. According Zhang L. et al.; androgen deprivation gradually up regulated PKD1 protein expression. In LNCaP cells, inhibition of AR by bicalutamide also upregulated PKD1 protein expression in a concentration-dependent manner. These data suggested that AR was required for the transcriptional repression of PKD1 gene expression caused by androgen stimulation in androgensensitive prostate cancer cells. However the AREs (androgen response elements - two) in PKD1 promoter did not play an active role in regulating PKD1 transcription in response to androgen stimulation. The involvement of AR and an androgen-induced repressor protein prompted the authors to conduct an esiRNA screen that targeted 23 AR corepressors and other related proteins. They identified FRS2 (Fibroblast Growth Factor Receptor substrate 2) as a potential repressor of androgeninduced PKD1 repression [20]. The adaptor protein FRS2 is a major mediator of the FGFR signaling in normal and malignant cells. FGFR stimulation by FGF leads to the tyrosine phosphorylation of FRS2, which then forms a complex with Grb2 and Sos to activate the downstream Ras/Raf/MEK/ERK signaling pathway. Androgen-sensitive LNCaP cells express low levels of FGF2, and its expression is upregulated in response to androgen stimulation. Current data support an AR-mediated indirect mechanism involving the cell surface adaptor protein FRS2 in the repression of PKD1 by androgen-induced AR/FGFR/FRS2/Ras/Raf/MEK/ERK pathway. As a well-documented prosurvival signaling protein, PKD1 upregulation in response to androgen deprivation and anti-androgen treatment may have significant implications in therapy resistance and progression to CRPC. These data did not completely exclude the potential involvement of other pathways in the regulation of PKD1 expression by androgen, because the binding of FGF to FGFR leads to the recruitment of multiple adaptor proteins, including FRS2, Grbs, Sos, and

Gab1, and induces the activation of multiple downstream signaling pathways, including not only MEK/ERK, but also PI3K/Akt, PLCγ/PKC, and Stat3 pathways [20]. Studies have shown that PKD1 and PKD3 expression levels are elevated in human prostate carcinoma tissues compared to normal prostate epithelial tissue, and advanced-stage tumors were found to have increased PKD3 nuclear accumulation. In contrast, androgen-independent tumors showed reduced PKD1 expression [1,15]. The studies not only highlight the significance of PKD signaling in prostate cancer progression, but also strongly suggest isoform-specific functions and contrasting roles for PKD1 and PKD3 in prostate cancer cells. Further studies are needed to determine whether indeed there are distinct and opposing roles for the PKD isoforms in prostate cancer and what implications this might have in the development of PKD inhibitors for potential use as chemotherapeutic agents in the treatment of prostate cancer [1]. EMT (Epithelial to Mesenchymal Transition): There are tree types of cells in advanced cancers– transformed (modified, mutated) cancer cells, EMT cells (epithelial-to-mesenchymal transitional cells) and CSCs (cancer stem cells). An essential thrust for the future is understanding whether the CSCs exhibit this property on their own or acquire it during transitions, such as epithelial-tomesenchymal transition (EMT) and mesenchymalto-epithelial transition (MET). Through the process of EMT, molecular alterations in the form of loss of apical polarity, loss of epithelial cell-cell junctions, reorganization of actin cytoskeleton, acquisition of more spindle-shaped morphologic features, and upregulation of mesenchymal markers (fibronectin, N-cadherin, vimentin) and downregulation of epithelial markers (E-cadherin - epithelial-specific antigen) are acquired by epithelial cells [21,22]. Epithelial-Mesenchymal transition (EMT), which is known as the probable first step in the complex process of metastasis, is a distinctive morphological change, in which the series of events converting the epithelial cancer cells switch from a welldifferentiated, adherent phenotype to an individual, invasive migratory mesenchymal cell. Based on the biological context, EMT has been classified into three types. Type 1 EMT involves embryonic development. Type 2 EMT is associated with



wound healing, tissue regeneration, and organ fibrosis. Type 3 EMT occurs in epithelial cancer cells and involves cancer progression and metastasis [23]. EMT is mainly characterized by loss of Ecadherin (CDH1) expression and other special molecular changes that promote architectural changes, followed by the loss of cell-cell junction, cell-matrix adhesion, and modulation loss of apical polarity, reorganization of actin cytoskeleton, resulting in acquisition of mesenchymal features (fibroblast-like cells), such as spindle shape and increased migratory and invasive capacity [21]. EMT is triggered through extracellular signaling of collagen or from growth factors such as fibroblast growth factors (FGFs), epidermal growth factors (EGFs), and platelet-derived growth factors (PDGFs) such as PDGF-A, PDGF-B, and PDGF-D. Iinflammatory signals (e.g., transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNFα)/nuclear factor θB (NF-θB), cytokines) which induce EMT, also induce TG2 (transglutaminase-2) expression, suggests a possible link between TG2, inflammation, and cancer progression [24,25]. Through the process of EMT, upregulation of mesenchymal markers (fibronectin, N-cadherin, vimentin) and downregulation of epithelial markers (E-cadherin - epithelial-specific antigen) are acquired by epithelial cells [21,22]. Other typical markers of EMT are cadherin-11 and fibroblastspecific protein (FSP)-1 which are associated with an increased motility. Transcription factor Snail (Snai1), Slug (Snai2), Twist, ZEB1/ZEB2 and E47 are mainly involved in EMT transition (Twist repress the transcription of E-cadherin), through activation of TGF-β, Wnt, Notch, NF-θB, ERK/MAPK pathways, and activation of PDGF, EGFR [23,24,25,123]. Snail also downregulates the expression of E-cadherin. Wnt and EGFR, HH and EGFR signaling pathways crosstalk and transactivate one another in cancer. Furthermore, EGFR has been shown to form a complex with βcatenin and increase the invasion and metastasis of cancer cells [23]. The molecular mechanisms underlying the initiation of EMT consist, in part, in the constitutive activation of survival signaling pathways such as the nuclear factor (NF)-θB pathway. The NF-θB pathway has been implicated directly in the regulation of EMT and indirectly through the transcription and expression of several gene products that participate in the EMT cascade, such

as Snail, the metastasis-inducer and E-cadherin suppressor transcription factor. In turn, Snail represses the metastasis-suppressor gene product Raf-kinase inhibitor protein (RKIP) that inhibits both the Raf-1/MEK/ERK and NF-κB survival pathways implicated in EMT. Consequently, tumor cells normally exhibit a dysregulated NFθB/Snail/RKIP circuitry that is intimately involved in the initiation of EMT and maintenance of drug resistance. Additional deregulated gene products in this circuit, such as the metastasis-suppressor phosphatase and tensin homologue (PTEN; repressed by Snail) and the putative-metastasis inducer Yin Yang (YY) 1 (target of NF-θB) also have been associated in the regulation of EMT [26]. Hypoxia has received considerable attention as an inducer of tumor metastasis. In this process, Notch signaling controls Snail expression by two distinct but synergistic mechanisms, including direct transcriptional activation of Snail and an indirect mechanism operating via lysyl oxidase (LOX). Notch increases LOX expression by recruiting hypoxia-inducible factor 1α (HIF-1α) to LOX promoter, which stabilizes the Snail protein, resulting in up-regulation of EMT and migration and invasion of cancer cells [23]. EMT may be histologically represented by the presence of tumor budding which is described as the occurrence of single tumor cells or small clusters ( 0.05). The expression of p63 was found strongly and diffuse in 72.3% of solid undifferentiated and 82.1% differentiated and in 77.8% of superficial type BCCs. p63 is consistently expressed in epidermal basal/suprabasal and adnexal basal cells. Most BCCs have higher homogeneous p63 expression than nontumoral epidermis, which is not changed according to histological differentiation subtypes. Thus, overexpression of p63 in all histological subtypes may confirm that basaloid progenitor cells are linked tumor-cell lineage and have a role in the tumorigenesis of BCC [76]. Thus, early increase of PKD1 expression in BCC [35] is probably also connected with phosphorylation of Snail (Ser11, as in other cancer types), but it recieds nucleous in consequence of 14-3-3 mutations (methylation, down-regulation) and probably followed by upregulation of expression of Cyclin D1 and Ajuba (see Figure 1). PKD1 is also known activator of ERK 1/2 and NFθB signal pathways [4], which both could additionally activate Snail promoter activity [70]. Increased PKD1 expression together with 14-3-3 mutation could be one of the main reasons for early increased of Snail activity and cancer development in BCCs, similarly to pancreatic cancer [14,17], with exception of 14-3-3ζ downregulation (mutation) [63], early overexpression of Gli [77,78] and low-frequency of Ras mutations in this cancer type [8,9]. 1.2. Other EMT Transcriptional Factors: Snail is expressed, in a transient manner, in hair placodal cells, during budding morphogenesis of the hair follicle, but is not detectable in the IFE (interfollicular epidermis). Slug (Snai2, Snail2) is expressed in all epidermal layers at mid-gastattion but becomes gradually restricted to the basal layer that harbors epidermal precursor cells and hair placode that harbors hair follicle precursor cells, and progressively disappears after birth. Overexpression of Snail in skin basal cells leads to loss of E-cadherin, epidermal hyperproliferation and expansion of the basal compartment. Additionally, Slug knock-down mice show a thinner epidermis and delayed hair follicle development (normal multipotent mammary epithelial stem cells express EMT markers) [79] . Activation of Snail by other signal pathways оr activation of other EMT transcriptional factors Slug, Twist, Zeb1, could also induce EMT

phenotype in BCCs. The transcription factors Slug and Twist except Snail are known to bind to the Ebox regulatory regions of E-Cadherin and inhibited also its expression [80]. Recently Majima et al. reported a case of morphoeic and metastic BCCs showing the induction of Twist 1 and the EMT convertion of cadherins in association of multiple organ metastases. The tumour cells were positive for Twist1 and N-cadherin at the invasive front of the primery tumour, whereas the tumour cells centrally were negative for Twist1. Control representative tumour cells of nodular BCCs showed no Twist1 expression in the nuclei and no N-cadherin expression in the front [81]. Aberrant epidermal growth factor receptor (EGFR) signaling (EGFR and/or its ligands) is a major cause of tumor progression and metastasis; the underlying mechanisms, however, are not well understood. Lo Hui-W. et al. showed that EGF induces EGFR-expressing cancer cells to undergo a transition from the epithelial to the spindle-like mesenchymal morphology. EGF reduced Ecadherin expression and increased that of mesenchymal proteins. In search of a downstream mediator that may account for EGF-induced EMT, the authors focused on transcription repressors of E-cadherin, TWIST, SLUG, and Snail and found that cancer cells express high levels of TWIST and that EGF enhances its expression. EGF significantly increases TWIST transcripts and protein in EGFR-expressing lines. Forced expression of EGFR reactivates TWIST expression in EGFR-null cells. TWIST expression is suppressed by EGFR and Janusactivated kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) inhibitors, but not significantly by those targeting phosphoinositide-3 kinase and MEK/ERK. Furthermore, constitutively active STAT3 significantly activates the TWIST promoter, whereas the JAK/STAT3 inhibitor and dominant-negative STAT3 suppressed TWIST promoter. Deletion/mutation studies further show that a 26-bp promoter region contains putative STAT3 elements required for the EGFresponsiveness of the TWIST promoter. Chromatin immunoprecipitation assays further show that EGF induces binding of nuclear STAT3 to the TWIST promoter. Immunohistochemical analysis of 130 primary breast carcinomas indicates positive correlations between non-nuclear EGFR and TWIST and between phosphorylated STAT3 and



TWIST. Together, the authors report that EGF/EGFR signaling pathways induce cancer cell EMT via STAT3-mediated TWIST gene expression [82]. 2 Signal pathways in BCC (HH, Wnt and EGFR): BCC are thought to be caused by uncontrolled activation of the hedgehog (HH) signaling pathway. In the majority of BCCs cases, activation of HH pathway is due to inactivating mutations in the HH receptor and tumor suppressor gene PTCH. PTCH mutations in BCC were first observed in basal cell nevus syndrome (also known as nevoid basal cell carcinoma syndrome or Gorlin-Goltz syndrome), which is a rare familial autosomaldominant disorder that predisposes the affected individual to developing this tumor. Only a minority of BCC are caused by activating mutations in Smoothened (Smo) [77]. The receptor for Hhs is a transmembrane protein called Patched (PTC (PTCH)). In the absence of Hh ligands, PTC is bound to another transmembrane protein, smoothened (SMO), and functions as an inhibitor of SMO. The binding of Hh ligands to PTC (or inactivating PTCH mutations) releases SMO from the inhibitory effect of PTC and allows SMO to transduce signals leading to the activation of transcription factor, called glioma associated (Gli), zinc finger TFs GLI2 and GLI3, and the expression of genes involved in regulating embryonic and postnatal development, and the transformation of cancerand metastasis-initiating cells [83]. Activation of GLI2 and GLI3 leads to transcription of GLI1. Thus, the expression level of GLI1 is considered as a reliable indicator of the pathway‘s activity. Another HH target is PTCH itself, which regulates its expression in a negative feedback. Indeed, nearly all BCC express GLI1 and PTCH, which demonstrates the important role of aberrant HH signaling in these tumors [77]. Both Gli1 and Gli2 are highly expressed in human BCCs. In particular, Gli2 directly regulates the expression of Gli1, further activating the Hh pathway. Gli2 silencing reduces growth of BCC xenografts by decreasing vascularization and increasing apoptosis. Activation of the Hh pathway might exert its mitogenic effect on keratinocytes by activation of several targets. For instance, Gli1 can induce expression of platelet-derived growth factor receptor alpha and FOXM1, and Gli2 activates

FOXE1. Gli2 has been shown to activate the antiapoptotic factor Bcl-2 and to counteract death ligand-mediated apoptosis by inducing expression of the caspase-8 inhibitor c-FLIP, suggesting an antiapoptotic role in BCC. Hh signaling enhances ribosomal RNA transcription in BCC by increasing basonuclin gene expression. In addition, insulinlike growth factor binding protein 2 is upregulated in both murine and human BCCs and has been shown to play a role in Hh-mediated expansion of epidermal progenitor cells in K14-Cre; Ptch1fl/fl mouse model skin explants with BCC-like lesions, an inducible K14 promoter used of skin-specific Cre strains to drive homozygous Ptch1 ablation [78]. The biological activity of GLI proteins can be modulated by phosphinositide-3 kinase (PI3K)/AKT, MEK/ extracellular signal-regulated kinase (ERK), Protein Kinase Cα (PKCα) [84], PKCδ [85], PKCη [86], transforming growth factor β/SMAD, and VDR (1,25-dihydroxyvitamin D3 receptors) [83], which affect stability, subcellular localization, or expression of GLI proteins. Protein kinase A (PKA) can retain Gli1 in the cytoplasm, inhibiting its transcriptional activity. Atypical protein kinase C (aPKC) η/ι and the downstream effector of the mammalian target of rapamycin (mTOR) pathway ribosomal protein S6 kinase 1 activate Gli1. Deacetylation of Gli1 and Gli2 by histone deacetylase 1 increases their transcriptional activity. Upon genotoxic stress, p53 induces the acetyltransferase p300/CREB-binding protein (CBP)-associated factor (PCAF), identified as a novel E3 ubiquitin ligase targeting Gli1 for proteasomal degradation [78]. One of the earliest molecular changes during the reprogramming process includes the activation of Wnt/β-catenin signaling, on which the development of BCCs critically depends. In addition, activation of canonical Wnt/ β-catenin signaling seems to play a role in specific histological BCC subtypes. These subtypes include early stages of superficial BCC, pilomatricoma (a tumor of the hair follicle) as well as infiltrative BCC variants. Indeed, nuclear β-catenin is found in infiltrative BCC and in superficial BCC, but only rarely in human nodular BCC. Conditional overexpression of the Wnt pathway antagonist Dkk1 results in inhibition of Hh-driven benign hamartomas [78]. EGFR signaling seems to be an essential in vivo requirement in HH-driven BCC because EGFR signaling cooperates with the HH/GLI pathway to



induce genes (e.g., JUN, SOX9, and FGF19) critical for the determination of the oncogenic BCC phenotype [77]. On transcriptional level cooperative interactions of the GLI activator forms (GLI-A) (transcriptionally active form [78]) and JUN/AP1 transcription factor, downstream effector of the EGFR-mediated activation of the RAS/RAF/MEK/ERK cascade [85, 87]. The epidermal growth factor receptor/MEK/ERK pathway has been shown to modulate Gli-dependent transcription in human keratinocytes and to synergize with Hh signaling in inducing oncogenic transformation of human keratinocytes through activation of c-Jun. In accordance with these studies, an increase of c-Jun upon Hh or epidermal growth factor receptor activation inhibits the tumor suppressor miR-203, which in turn represses c-Jun, creating a negative regulatory loop. Interestingly, Hh and epidermal growth factor receptor signaling synergistically activate a number of cooperation response genes, including SOX2, SOX9, JUN, CXCR4, and FGF19, which are required for growth of BCC in vivo[78]. Although BCC are with basal origin, EGFR is expressed at a significantly higher level in SCC than in BCC [48]. EGFR activity maintains proliferation levels of the basal layer and its inhibition causes differentiation of the keratinocytes and induction of suprabasal markers K1 and K10 [85]. By immunohistochemical analysis of a panel of 20 human BCC using a clinically approved diagnostic anti-EGFR antibody, Schnidar H et al. can show that all BCC tested expressed EGFR. Five BCCs showed EGFR expression at levels slightly lower than normal skin, eight tumors gave signals comparable with normal skin, and seven tumors showed higher EGFR levels than normal skin. This was partially confirmed by qPCR analysis of EGFR mRNA expression in BCC samples compared with normal human keratinocytes or N/TERT-1 keratinocytes. Similarly, Ptch _/_ (ASZ001) and Ptch _/_; p53_/_(BSZ2, CSZ1) mouse BCC cell lines express elevated levels of EGFR mRNA when compared with Ptch+/+ keratinocytes. In agreement with the mRNA expression data, all BCC cell lines express significant levels of total and activated tyrosine–p-EGFR [66]. Additionally, ERK1/2 is one of the major kinases lying downstream of EGFR and transdusing EGFR signal into the cells. Only 5/12 (48%) BCC

examined stained positive for pERK more than in normal skin, in comparision with 100% positivity of cSCC (N=11) [88]. Schnidar H et al. have previously shown that activated ERK is not highly expressed throughout the entire tumor mass of BCC, although it can be readily detected in small subregions of the tumors, such as the peripheral palisading cells with high proliferative activity, as well as in infiltrating cells. The authors speculate that the synergistic interaction of EGFR/MEK/ERK and GLI act in distinct subpopulations of BCC cells is associated with tumor growth and a more aggressive phenotype [57]. Finally, ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular), indicating that GLI1 does not routinely co-operate with ERK to induce the formation of this common skin tumour [87]. Just like keratinocytes, BCC cells also express VDR, and furthermore, peripheral cells forming BCC tumours show even higher expression of VDR than neighbouring, unaffected epidermal cells. It was demonstrated that vitamin D suppresses a key tumour pathway in BCCs development — hedgehog signalling pathway. It was shown that VDR-knockout mice, after the exposure to a carcinogen, were more prone to BCCs skin tumours development than the wild type animals. Another studies on mice showed that topical application of vitamin D3 reduces BCC cell proliferation and also inhibits the hedgehog signalling pathway, both in vitro and in vivo [89]. Recent studies indicate a cross-talk between vitamin D3 and Hh signaling mediated by at least two mechanisms. First, PTC has been shown to stimulate the secretion of a vitamin D3-related compound, which is likely responsible for the inhibitory action of PTC on SMO. Second, 1α,25(OH)2D3 can down regulate the expression of some members of the Hh pathway genes, including PTC, SMO and Gli in an epidermal explants culture system, suggesting a direct regulation by 1α,25(OH)2D3. These results are in agreement with the increased expression of Shh in the keratinocytes of the VDRnull animal and hyperactivation of the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms. More interestingly, Uhmann et al. demonstrated that 1α,25(OH)2D3 was capable of inhibiting Hh signaling at the level of SMO in the absence of VDR. Similar conclusion was obtained by Tang et



al. who studied murine basal cell carcinomas (BCC) in vitro and in vivo, and found that the effect of 1α,25(OH)2D3 induced Gli expression is likely independent of VDR. The results provide strong evidence of the non-genomic and rapid nonVDR action of 1α,25(OH)2D3 on cell growth and differentiation mediated by Hh/Gli signaling pathway [83]. What is more, 20(OH)D3, 20,22(OH)D3 and 20,23(OH) D3, novel vitamin D3 analogues produced by P450scc, show prodifferentiation, anti-proliferative and anticancer properties. Although the in vitro and animal studies suggested that vitamin D may prevent development of BCCs and SCCs, additional studies on humans are needed to assess the suitability of topical or oral vitamin D3 supplementation in chemoprevention of non-melanoma skin cancers [89]. Chronic inflammatory mediators exert pleiotropic effects in the development of cancer. On the one hand, inflammation favors carcinogenesis, malignant transformation, tumor growth, invasion, and metastatic spread; on the other hand inflammation can stimulate immune effect or mechanisms that might limit tumor growth. The link between cancer and inflammation depends on intrinsic and extrinsic pathways. Both pathways result in the activation of transcription factors such as NF-θB (Nuclear Factor-θB), STAT-3 (Signal Transducer and Activator of Ttranscription), and HIF-1[24], which can be also activated in response of EGFR activation. NFkB is activated by the phosphorylation and activation of inhibitory k kinase (IkK). This results in the degradation of inhibitory protein IkB and the release of NF-kB from the inhibitory complex, followed by accumulation in the nucleus and induction of downstream target genes, which in turn causes cell survival, cell proliferation, and inflammation [4], inflammation- driven carcinogenesis and antitumor immunity [24] . Ming LK have screened IθKα, p-IθBα, NF-θB/p65 expression by immunohistochemistry in SCC, BCC and normal skin tissue to investigate the relationship between expression of these proteins and formation, growth of SCC and BCC. The positive rate of IθKα was 77.80% (21/27) in SCC, 46.20% (12/26) in BCC and 15.00% (3/20) in normal skin respectively. The expression of IθKα in SCC was no significant relationship with pathological grade (P>0.05). Second: the positive expression of p-IθBα was localized in the

cytoplasm of tumor cells. The positive rates of pIθBα was 70.40% (19/27) in SCC, 42.31% (11/26) in BCC respectively. 17 normal skin tissues were negative. The positive rates of NF-θB/p65 was 74.10% (20/27) in SCC, 42.31% (11/26) in BCC respectively. 18 normal skin tissues were negative. The expression of NF-θB/p65 in SCC was no significant relationship with pathological grade (P>0.05). Conclusions: 1. Comparing with the nomal skin, overexpression of NF-θB/p65, p-IθBα and IθKα in SCC, BCC suggest that the NF-kB signal transduction pathway may play an important role in carcinogenesis of SCC and BCC; 2. The expression of NF-θB/p65, p-IθBα and IθKα in BCC are significantly lower than in SCC may be related to the slower growth and rare metastasis of BCC; 3. The expression of p-IθBα is significantly correlated with IθKα in SCC and BCC; the expression of NF-θB/p65 is significantly correlated with p-IθBαin SCC and BCC. Above those suggest that the three factors consisting in the NF-θB signal transduction pathway induces the formation and growth of SCC and BCC by cooperating with each other; 4. NF-θB/p65 may become a new target to cure SCC and BCC because it is the key point in the NF-θB signal transduction pathway [90] . Activation of the PI3K/AKT signalling pathway has been reported both in squamous cell carcinoma (SCC) of the skin and in basal cell carcinoma (BCC). It has been demonstrated that UVR, the major etiologic factor for BCC, suppresses PTEN expression, and that this decrease is required for enhanced cell survival in transformed human keratinocytes, indicating that PTEN might be the critical target for UV-induced skin tumorigenesis. Deletion of 10q23, where PTEN is located, was found to be an infrequent event in human BCC [91] Several other signaling pathways are presumably involved in BCC tumorigenesis. Mutations of the tumor suppressor gene p53 have been shown in 40% of sporadic BCC and were correlated with aggressive behavior. P53 mutations are present in approximately 56% of all types of BCCs, also frequent in SCCs, mutated in 79% of the head and neck cancers and overexpressed in 47% of precancerous lesions [78]. p53 mutations were detected in 33 of 50 aggressive BCCs (66%), 37 of 98 nonaggressive BCCs (38%), 28 of 80 aggressive SCCs (35%), 28 of 56 nonaggressive SCCs (50%), and 3 of 29 samples of sun-exposed skin (10%). About 71% of the p53 mutations



detected in aggressive and nonaggressive BCCs and SCCs were UV signature mutations. The frequency of CC to TT mutations in aggressive (36%) and nonaggressive SCCs (39%) was 2-fold higher than in aggressive (18%) and nonaggressive (14%) BCCs. In contrast, aggressive BCCs had a higher frequency (24%) of transversions than nonaggressive BCCs (8%) and aggressive (14%) and nonaggressive (11%) SCCs did [192]. In addition, loss of p53 has been shown to accelerate tumorigenesis of BCC in Ptch1+/− mice, likely through Gli1 activation [78]. Another study showed dysregulation of Ras in 100% and mutations in 10% to 50% of BCCs [92]. 3. Early alterations in p21Waf1/Cip1/Sdi1, p27 kip1, wif1) 

SCC

(p16INK4A,

HPV-positive squamous cell carcinoma:

HPV-positive tumors represent a different clinicopathological and molecular entity compared to HPV-negative cases (see below). HPV infection is now recognized as one of the primary causes of oropharyngeal SCC (especially SCC of the tonsils and the base of the tongue) [58].

carcinoma in situ. Hence, p16 positivity is currently regarded as a reliable surrogate marker for HPV-related oropharyngeal carcinoma [58]. Positive HPV/p16 status has been consistently found to be a favourable prognostic factor in terms of locoregional control and overall survival irrespective of treatment modality [59]. HPVnegative HNSCC are typically characterised by TP53 and RB genetic alterations resulting in genomic instability and resistance to apoptosis. No TP53 mutations were seen in HPV-positive HNSCC on exome sequencing, and the overall mutation rate was approximately half of that seen in HPV-negative samples. In addition, in contrast to HPV-negative tumours, the expression of CDKN2A, encoding p16INK4A, is highly upregulated, but often inactivated and and cyclin D1 is often overexpressed in HNSCC contributing to increased proliferation [93] , but its amplification is infrequent [61]. Previously it was detected that expression of total and nuclear EGFR was higher in p16-negative tumors compared to p16-positive tumors [94]. Additionally Doorslaer and Burk showed that oncogenic types papilloma virus (HPV) specifically activate the hTERT promoter, while non-oncogenic types do not [95].

HPV-related oropharyngeal squamous cell carcinoma is characterized by the expression of the E6 and E7 viral oncoproteins. The E6 oncoprotein PIK3CA gene, encoded one of the isoforms of causes substantial loss of p53 activity by PI3K the 110 kDa catalytic subunit, p110α is degradation of p53 at the protein level. mutated in 6–20% of HNSCC, especially through Consequently, normal p53 function, such as G1 the mechanisms of gene amplification and lowcell cycle arrest or induction of apoptosis, which level copy number increase. It has been found to be are important steps to allow for adequate cellular particularly common in HPV-positive HNSCC response to DNA damage, is hampered resulting in cases, and specific mutations, such as H1047R in a higher susceptibility to genomic instability. The exon 20, may predict higher response rates to E7 oncoprotein causes degradation and treatment with PI3K pathway inhibitors (mTOR inactivation of the retinoblastoma tumour inhibitors). In addition, PTEN mutations have been suppressor gene product (pRb), preventing it from reported in 7% of HNSCC, and the mTOR binding to the E2F transcription factor and thereby pathway is frequently activated, independent from promoting cell cycle progression. As pRb normally INK4A activation of EGFR or the presence of mutant p53, functions as a negative regulator of p16 (p16) particularly in HPV-positive tumours [61] . expression, a tumour suppressor gene located on chromosome 9p21, the functional inactivation of There are three classes of PI3Ks, each with its own pRb results in a reciprocal overexpression of p16 substrate specificity, and class 1A is most protein. As a consequence, HPV-associated frequently associated with cancer. Class 1A PI3Ks oropharyngeal squamous cell carcinoma shows are heterodimers and composed of a 110-kDa nuclear and cytoplasmatic p16-overexpression, catalytic subunit and an 85-kDa regulatory subunit, which is predominantly absent in HPV-negative both of which exist in several isoforms. PI3Ks are oropharyngeal squamous cell carcinoma. On the activated by RTKs, such as EGFR, and the contrary, according to the molecular progression catalytic subunit phosphorylates model for tobacco-induced HNSCC, loss of 9p21 phosphatidylinositol 4,5-bisphosphate (PIP2) to is the most frequent event and is also present in the form phosphatidylinositol 3,4,5-triphosphate earliest definable lesions, including dysplasia and International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20215


(PIP3). Interaction of PIP3 with the PH (Pleckstrin Homology) domain of AKT results in a conformational change causing phosphorylation of AKT/PKB by PDK1 and mammalian target of rapamycin complex 2 (mTORC2). This activates AKT that then phosphorylates proteins involved in cell growth and survival. The tumour-suppressor phosphatase and tensin homology (PTEN) mediates the conversion of PIP3 to PIP2, counteracting the activation of AKT. mTOR is a protein kinase that acts downstream of PI3K and AKT and plays an important role in cell growth, survival and protein synthesis regulation. There are two mTOR complexes: mTORC1 activates ribosomal protein S6 kinase 1 (p70S6K, which directs the translation of cell cycle regulatory proteins such as Cyclin D1 and myc [93] and inactivates eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), resulting in protein translation and cell growth, whereas mTORC2 activates AKT. Ras can also activate the PI3K signalling cascade [61]. Loss of TRAF3, activating mutations of PIK3CA, and amplification of E2F1 in HPV(+) oropharyngeal cancers point to aberrant activation of NF-kB, other oncogenic pathways, and cell cycle, as critical in the pathogenesis and development of new targeted therapies for these tumours [42]. 

HPV-negative squamous cell carcinoma

Tobacco and alcohol-induced HNSCCs are characterized by TP53 mutation. The excess of G to T transversions and the codons more frequently affected were attributed to the carcinogenic effect of tobacco smoking. Other genes are involved in the pathogenesis of HPV-negative tumors. CCND1, which encodes cyclin D1, is amplified or gained (overexpressed) in more than 80% of HPV-negative HNSCCs [96]. CDKN2A (encoding p16) can be inactivated by (point) mutation, homozygous deletion, and/or transcriptional suppression associated with promoter hypermethylation in cancer cell lines and primary tumors [97,60]. TP53 mutation, loss of p16INK4A and overexpression of cyclin D1 are all associated with reduced survival [61]. Carcinogenesis is a multi-step process. One possible step is the development of potentially malignant disorders (PMDs) known as leukoplakia, erytroplakia, lichen planus, submucous fibrosis, actinic cheilitis (cheratosis) and palatal keratosis

associated with inverted smoking, discoid lupus erythematosus, Marjolin ulcer, immunodeficiency in relation to cancer predisposition and some inherited cancer syndromes [98,52]. The loss of p16 may be an early event in cancer progression, because deletion of at least one copy is quite high in some premalignant lesions. p16INK4a is a major target in carcinogenesis, rivaled in frequency only by the p53 tumorsuppressor gene [99]. Alterations of p16 have been described in a wide variety of histological types of human cancers including astrocytoma, melanoma, leukemia, breast cancer, head and neck squamous cell carcinoma, malignant mesothelioma, and lung cancer. Methylation of p16INK4a was observed in 44% of 34 patients with oral leukoplakia lesions, and hypermethylation in 76% of OSCCs [52]. These findings demonstrate that methylation is an early event in oral carcinogenesis and that its study may be useful to detect precancerous lesions [52]. Previously it was detected that expression of total and nuclear EGFR was higher in p16-negative tumors compared to p16-positive tumors [53]. The p16 gene (also known as CDKN2A) encodes p16INK4A, which inhibits (inactivating) the CDK4:cyclin D and CDK6:cyclin D complexes. These complexes mediate phosphorylation of the Rb protein and allow cell cycle progression beyond the G1-S-phase checkpoint [97,57]. The INK4a locus encodes another structurally and functionally independent protein, p14ARF, which is also believed to be a potent tumor suppressor. p14ARF activates the p53 pathway in response to oncogenic signals, such as the c-myc or ras oncogene, by binding to the p53 negative regulator Mdm2 and preventing p53 degradation thereby inducing cell cycle arrest or apoptosis. Since p53 is often mutated in skin carcinomas and is likely to be an early event, elimination of p16INK4 rather than p14ARF seems to be involved in skin cancer development. Accordingly, p16INK4 mutations were detected, although at a low frequency, in the general population and also in patients suffering from xeroderma pigmentosum, whereas p14ARF mutations were not yet reported [100]. Whereas the Rb gene is inactivated in a narrow range of tumor cells, the pattern of mutational inactivation of Rb is inversely correlated with p16 alterations, suggesting that a single defect in the p16/CDK4:cyclin D/Rb pathway is sufficient for tumorigenesis [97].



p21WAF1 is transcriptionally regulated by p53 and is a downstream mediator of p53-induced growth arrest after DNA damage through its interaction with proliferating cell nuclear antigen, cyclins, and cyclin-dependent kinases. p21WAF1 may also be regulated through p53-independent pathways in response to diverse stimuli including growth factors, and experiments in p53-null cells indicate that increased expression of p21WAF1 during terminal differentiation occurs through p53independent pathways [101]. p21Cip1, which is induced in tumors by the activated Ras–ERK pathway, is repressed by c-Myc. Acute elimination of c-Myc in established tumors leads to the upregulation of p21Cip1, and epidermis lacking both p21Cip1 and c-Myc reacquires normal sensitivity to DMBA/TPA-induced tumorigenesis. This identifies c-Myc-mediated repression of p21Cip1 as a key step for Ras-driven epidermal tumorigenesis [102]. p27 kip1, wif1 was originally discovered as a Cdk inhibitory activity induced by extracellular antimitogenic signals. It accumulates in serumstarved and density-arrested cells, and its overexpression causes cell cycle arrest in G1. Furthermore, depletion of p27 by antisense oligonucleotides prevents cell cycle arrest in serum-deprived cells. Notably, some reports indicate that perturbation of EGFR signaling with tyrosine kinase inhibitors or bivalent antibodies against the receptor‘s ectodomain results in stabilization of p27 and G1 arrest. Low levels of p27 and increased proteasome-dependent degradation of p27 have both been reported in several epithelial neoplasias, suggesting an association between loss of p27 and oncogenesis or tumor progression [103]. p27 was positive in 23.4%, 26.2%, 25.9% and 4.5% of specimens in the normal skin, AK (Actinic Keratosis), BD (Bowen Disease) and SCC groups, respectively [104]. In a complementary study, it was observed that assembly of cyclin D1/D2–CDK4 complexes was impaired in primary mouse embryo fibroblast (MEF) strains taken from animals lacking the p21 gene, the p27 gene, or both. In MEFs fromp21/p27 double-null mice, nuclear import of cyclin D1 is inefficient, and overexpressed D cyclins remain predominantly cytoplasmic. The half-life of unassembled cyclin D1 is significantly reduced from 25 to l0 min [105]. Mutations in both p21 gene and the p27 gene are also often observed in

premalignant lesions and it seems that these mutations could ocure as a protective cell mechanism against hyperproliferative signals. Additionally, although constitutive expression of p16, p21 and p27 in keratinocytes causes cell cycle arrest, terminal diferentiation is not stimulated [106]. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations (premature termination of the protein by nonsense, frameshift or splice-site mutations) of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. In HPV(-) HNSCCs, mutually exclusive subsets containing amplicons on 11q with CCND1, FADD, BIRC2 and YAP1, or concurrent mutations of CASP8 with HRAS, also target cell cycle, death, NF-kB and other oncogenic pathways [42]. 4. Signal pathways in SCC (EGFR, ras, p53 and Notch): SCC typically exhibits a broad spectrum of progressively advanced malignancies, ranging from premalignant actinic keratosis (AK) (precursor lesions) to squamous cell carcinoma in situ (SCCIS), invasive cSCC and finally metastatic cSCC. The primary risk factor for AK is chronic UV exposure, and the estimated rate for an individual lesion to progress to cSCC is between 0.025% and 16% per year. In patients with metastatic cSCC, however, the prognosis is very poor, with only a 10%–20% survival rate over 10 years. Histologically, AKs are characterized by dysplasia of the keratinocytes in the basal layer, often accompanied by parakeratosis and thinning of the granular layer. This localized epidermal atypia reflects a partial disruption of the differentiation program, whereas a more complete loss of differentiation is associated with cSCCs [85]. Genetically, AKs and cSCCs are associated with amplifications and activating mutations of the Ras oncogene indicate that 11% of cSCCs harbor



activating Ras mutations (6% HRAS, 3% NRAS, 2% KRAS; n = 371 cases). In cSCCs Ras is frequently activated, but with low frequency of mutations [85]. According previous data up to 30% of all human tumors harbor mutations in canonical RAS genes (KRAS, HRAS, NRAS). Activating HRAS mutations have been found in 4–5% of HNSCC cases [61], and the presence of HRAS mutations in HPV-driven tumors, suggesting potential cooperativity in tumor promotion [62]. K-ras is a downstream mediator of EGFR-induced cell signaling, and Ras mutations confer constitutive activation of the signal pathways without EGFR activation [107]. Ras mutations are infrequent in Western patients and detected in fewer than 5% of oral cancers. In contrast, 55% of lip cancers have H-ras mutation, which is also present in 35% of oral cancers in Asian populations in association with betel nut chewing [52]. K-ras mutations seem to be resistant to EGFR targeting agents (Gefitinib or Erlotinib (EGFR kinase inhibitors) [108,46], and are reported to be mutually exclusive to EGFR or HER2 gene mutations [107]. Whereas only benign tumors were observed after KRasG12D expression alone (mouse strain, which expresses physiological levels of an activated KRas (KRasG12D) allele along the midline epidermis and hair follicles (Msx2-cre; KrasG12D)), combined p53 deletion and oncogenic KRas expression initiated invasive cSCCs. Consistent with this finding, around 40% of human cSCCs harbor p53 mutations, indicating that p53 loss might be tightly associated with cSCC progression [85].

detected mutations in 35% of OSCCs, Balz et al. in 79% of HNCs (head and neck carcinomas) [52]. In addition to p53, mutations in the retinoblastoma (Rb) gene are involved in the pathogenesis of HNSCC. p16INK4A, a major target of the Rb pathway, is a tumor suppressor gene; its function is inhibited through a variety of pathways including loss of heterozygosity (LOH) of chromosome 9p21 where it is located. LOH of 9p21 is seen in 30% of premalignant lesions and up to 80% of malignant lesions. Absence of p16 expression has been detected in 59% of premalignant lesions and in 63% of OSCCs, 9p21/p16 (CDKN2A) hypermethylation in 76% of OSCCs. Hypermethylation of the cell cycle-regulating gene promoters p16INK4a and p15INK4b was found to be common in OSCC (p16: 76%; p15: 30%), although no significant correlation was observed with clinico-pathological characteristics or with the prognosis [52]. It has been previously shown that CDKN2A inactivation, the gene encoding not only the cell cycle regulators p16/INK4A but and p14/Arf/INK4B, by mutation is significantly more rare than deletion or epigenetic inactivation, which together account for inactivation of the gene in up to 75% of HNSCCs [62].

P53, a tumor suppressor gene, has been implicated in the early pathogenesis of HNSCC, as it controls cell growth through regulation of the cell-cycle and apoptosis. P53 acts as transcription factor of cell cycle inhibitors such as p21Waf1/Cip1/Sdi1 and prevents the cell from going beyond phase G1 of the cell cycle, permitting DNA repair. If this is not possible, p53 induces apoptosis of these cells to avoid the transmission of potentially carcinogenic information [52] .

The tumor suppressor genes p53 and Rb, and regulatory proteins like SphK1 and EphB4/EphrinB2 offer both prognostic value and a means with which to detect malignant cells. HNSCC is a tumor that carries with it a significant morbidity and a very poor prognosis, especially in advanced disease. Therefore, the development of biomarkers that can play a role in the earlier detection of tumor cells, offer prognostic information and can be used as targeted therapies is crucial [57].

In a study analyzing HNSCC patients with a history of tobacco and alcohol use, Brennan et al. found a significantly higher proportion of patients with mutations of p53 and other distinct sites when compared to nonsmokers and nondrinkers. p53 mutations have been found in up to 50% of HNSCC patients and have been shown to be associated with decreased survival. Kuo et al.

LOH in other locations, including LOH of chromosome 3p, has also been associated with tumorigenesis. Lee et al. examined mutations in eight different HNSCC cell lines and found that three candidate oncogenes encoded on chromosome 3p (ALS2CL, EPHA3, and CMYA1) were mutated, implying that LOH of chromosome 3p is also associated with HNSCC [67].

Although the reported percentage of patients with overexpression of EGFR varies, recent data shows that from 80% to 100% of patients with premalignant or malignant oral lesions have high EGFR expression [109]. EGFR itself is overexpressed, amplified, or constitutively



activated by ligand interaction or mutation. EGFR gene is often amplified (30% of OSCCs [52] and/or with activating mutations in cancer cells [57]. Amplification of the EGFR is particularly common in human squamous cell carcinomas. Constitutively active EGFR mutants can transform cultured cells. Conversely, dominantnegative constructs for EGFR can reverse the transformed phenotype in vitro. Transgenic targeting of transforming growth factor-α to the mammary gland, skin, and liver enhances tumor formation. In these models, there is a strong correlation between EGFR and EGFR ligand-induced hyperproliferation and tumorigenesis [110]. Human and mouse squamous cell carcinomas of the skin overexpress EGFR ligands [110]. EGFR TK mutations produce activation of the signaling pathways downstream and preferentially activated antiapoptotic pathways (ERK/MAPK, PI3K/AKT, and JAK-STAT). These mutations are correlated with the clinical response of patients to tyrosine kinase inhibitors (gefinitib and erlotinib), because the tumor cells are addicted to the constant activation of specific signaling pathways [111]. EGFR mutation (EGFRvIII) [112], corresponds to a deletion of the extracellular domain (lack of aminoacids 6-273). This variant has been found in 42% of HNSCC, related to the poor response to monoclonal antibody Cetuximab (competitively inhibits EGFR) [113]. In contrary mutations in Tyrosine Kinase domain , a case of a patient with an exon 19 mutation, P753S (splice site 748-1G>T) in EGFR was reported, documented an excellent response with the use of cetuximab. It is unclear what effects an isolated EGFR P753S mutation has on the expressed EGFR protein product, but the location of this mutation in the splice site acceptor of exon 19 may lead to exon skipping or protein truncation, which could activate the EGFR kinase domain and have an increased sensitivity to monoclonal antibody inhibition, although the exact mechanism is unclear. Studies from lung cancer indicate that small deletions in the region, including del747P753insS, can activate the EGFR kinase domain and also confer sensitivity to inhibition by tyrosine kinase inhibitors [114]. A case of locally advanced cutaneous SCC of the periocular skin with orbital extension was reported to showed improvement in motility and sensation after 4 weeks of treatment with cetuximab. This patient‘s tumor was also

analyzed for 182 cancer-related genes and found to have EGFR P753S mutation along with CDKN2A mutation, MYC amplication and TP53 mutation [115]. In organotypic 3D culture of human esophageal cells (keratinocytes) EGFR overexpression and mutant p53 resulted in transformation and invasive growth [116]. Overexpression of inactivated or mutated forms of p53 in oral epithelial dysplasia has been associated with high risk for transformation to early stage OSCC [100]. RKIP is a scaffolding protein capable of binding to and inhibiting Raf kinase. Raf kinase classically participates in the Ras/Raf/MEK/ERK kinase cascade that transfers mitogenic signals from the cell membrane to the nucleus in response of EGFR activity. Therefore, RKIP has the capacity to interrupt cell differentiation and growth, depending on the cell signal being received and is considered as an important tumor suppressor [117,118]. RKIP inhibits also NF-kappaB activity through direct interaction with NIK and TAK1 [119]. Additionally, RKIP activity in cervical and stomach cancer is inversely correlated with endogenous levels of the Notch1 intracellular domain (NICD), which stimulates the epithelial to mesenchymal transition (EMT) and metastasis. Overexpression of RKIP in several cell lines resulted in a dramatic decrease of NICD and subsequent inhibition of several mesenchymal markers, such as vimentin, N-cadherin, and Snail. In contrast, knockdown of RKIP exhibited opposite results both in vitro and in vivo using mouse models. Nevertheless, knockdown of Notch1 in cancer cells had no effect on the expression of RKIP, suggesting that RKIP is likely an upstream regulator of the Notch1 pathway [120]. A circuitry is developed in which overexpression of Snail in tumors inhibits RKIP and induces EMT. In addition, NF-kappaB, Snail, and RKIP have been shown to regulate tumor-cell resistance to apoptotic stimuli. Inhibition of NFkappaB and Snail and induction of RKIP sensitize resistant tumor cells to apoptosis by various chemotherapeutic and immunotherapeutic drugs. RKIP is poorly expressed in primary cancers and absent in various metastatic cancers [119]. Studies report significant down-regulation of RKIP expression in differentiated gastric cancer cells and a number of solid tumors including prostate, breast, colorectal, and melanoma [117].



RKIP-expression was investigated retrospectively in 321 esophageal cancers [179 adenocarcinomas (ACs), 142 squamous cell carcinomas (SCCs)]. RKIP-expression was further investigated in 41 precursor lesions consisting of 14 cases of nondysplastic Barrett‘s mucosa, 5 low grade dysplasias (LGD), and 12 high grade dysplasias (HGD) as well as, 4 cases with low grade and 6 cases with high-grade squamous cell dysplasia. Corresponding lymph node metastases were investigated in 140 patients, distant metastases in 29, and local recurrences in 12. High RKIPexpression was significantly more common in Barrett‘s mucosa without dysplasia and in LGD compared to HGD and invasive AC. In 187 primary esophageal cancers (58.3 %) RKIP was downregulated (AC: 51.4 %; SCC: 66.9 %). RKIP status of primary tumors influenced RKIP expression in corresponding lymph node and distant metastases. Downregulation of RKIP was associated with shorter overall survival (OS) and disease free survival (DFS) in all tumors. In AC, downregulation of RKIP was an independent prognostic factor for OS and DFS, while in SCC it reached significance only in univariate analysis. In conclusion, downregulation of RKIP is associated with shorter survival in esophageal cancers, and RKIP status of tumor cells seems to be preserved at the formation of metastases. Inhibition of RKIPdownregulation might reduce the ability of esophageal cancers to establish disseminated disease [118]. Analysis on tissue biopsies of oral cancers collected from patients reduced RKIP expression and increased phosphorylated RKIP was showen. RKIP phosphorylation, caused by PKC activation, is associated with poor outcomes in certain cancers including colon [117]. We could not find data concerning RKIP expression in BCC. Notch signaling, which is also mutated in 75% of cutaneous SCCs [121], has been linked to multiple biological functions, including regulation of selfrenewal capacity, cell cycle exit (in part through upregulation of p21/CDKN1A expression), and cell survival. In the stratified epithelium, Notch has a central role in promoting terminal differentiation, negatively regulated by EGFR, which is mediated through both direct effects (e.g., on activation of suprabasal keratins) and indirect effects on the Wnt, hedgehog, and interferon response pathways. Additionally, Notch activity has been linked to suppression of HPV E6 and E7 protein expression, potentially providing additional selective pressure

for loss of Notch in HPV+ HNSCC [62]. In mature epithelium, expression of p63 (p53 family member) is highest in basal epithelial cells, where it functions as an inhibitor of NOTCH1 expression, and becomes downregulated during terminal differentiation coincident with NOTCH1 upregulation. Reactivation of p63 expression is observed in the suprabasal layers of dysplastic mucosa, and overexpression and/or genomic amplification of the TP63 locus is observed in the majority of invasive HNSCCs [62]. Genetic aberrations of the PI3K pathway are common in HNSCC. One of the isoforms of the 110 kDa catalytic subunit, p110a, is encoded by the PIK3CA gene. This gene is mutated in 6–20% of HNSCC, especially through the mechanisms of gene amplification and low-level copy number increase. It has been found to be particularly common in HPV-positive HNSCC cases, and specific mutations, such as H1047R in exon 20, may predict higher response rates to treatment with PI3K pathway inhibitors. In addition, PTEN mutations have been reported in 7% of HNSCC, and the mTOR pathway is frequently activated, independent from activation of EGFR or the presence of mutant p53, particularly in HPVpositive tumours [61]. PIK3CA missense mutations in exons 9 and 20 were identified in 21.4% (3/14) of OSCC cell lines and 7.4% (8/108) of OSCC tumors by genomic DNA sequencing. An increase in the copy number of PIK3CA, although small, was detected in 57.1% (8/14) of OSCC lines and 16.7% (18/108) of OSCC tumors using quantitative real-time PCR. A significant correlation between somatic mutations of PIK3CA and disease stage was observed: the frequency of mutations was higher in stage IV (16.1%, 5/31) than in a subset of early stages (stages I-III) (3.9%, 3/77) [122]. One of the earliest molecular changes during the reprogramming process includes the activation of Wnt/β-catenin signaling, with increase β-catenin staining, and alterations in EGFR pathways. Systematic reviews for signal pathways in SCC [61,62]. 1α,25(OH)2D3 also inhibits the growth of SCCs in vivo as well as in vitro. Studies on animals showed that mice lacking VDR and exposed to high and prolonged doses of UVB are predisposed to SCC tumour formation. In addition, as in BCCs, topically applied 1α,25(OH)2D3 inhibits formation



of chemically induced tumour in a dose-dependent manner [89]. Wnt/β-catenin is an evolutionarily conserved signaling pathway that plays an essential role in a diverse array of biologic processes, including organogenesis, tissue homeostasis and, in some instances, pathogenesis of diseases, including cancers [83]. Expression of E-cadherin and βcatenin (proteins forming intracellular junctions and participating in Wnt/β-catenin pathway) is also decreased in skin malignant tumours, including basal cell carcinoma, squamous cell carcinoma and melanoma. Earlier studies indicate that 1α,25(OH)2D3 and its analogs are able to promote the differentiation of colon cancer cells by inhibiting Wnt/β-catenin signaling pathway mediated by VDR, competing with transcription factor TCF-4 for β-catenin binding, increasing E-cadherin expression, inhibiting cadherin ―switch‖, decreasing β-catenin nuclear staining, inhibiting SNAIL1 and SNAIL2 expression and secretion of MMP2, MMP9, and MMP13 in particular cancer types (inhibiting EMT) [123,83,89]. One of the studies revealed, that 4-day incubation of human keratinocytes with 1α,25(OH)2D3 caused the assembly of adherens junctions, upregulating E-cadherin expression, but not of desmosomes. The same study demonstrated that 1α,25(OH)2D3 may induce formation of intracellular junctions by protein kinase C (PKC) activation [89]. Many epidermal genes induced by WNT/β-catenin contain VDR response elements and were activated independently of TCF/LEF, implying that it is part of a TCF/LEF-independent aspect of WNT signaling [124]. Thus, it is speculated that 1α,25(OH)2D3-induction of cell– cell junctions formation may be a novel, promising mechanism of the anti-neoplastic and antiproliferative cancer treatment [89]. V. PKD1, Wnt/β-catenin and MMPs in skin and oral cancer: Evaluation of an asymptomatic patient for earlystage cancer, based on its physical features alone, is frequently compromised because malignant and late benign lesions may not be clinically distinguishable. Consequently, approximately 60% of oral cancers are advanced by the time they are detected, and approximately 15% of patients have another cancer in a nearby area such as the larynx, esophagus or lungs. Therefore, there is a

need to identify and use molecular biomarkers to evaluate individuals with potentially malignant disorders who are at a high risk of developing OSCC and those with early-stage malignant lesions [125]. 1. PKD1 and Wnt /β-catenin in normal skin: Normal human epidermis demonstrated predominant PKD protein expression in the stratum basalis, the proliferative epidermal compartment, with decreased relative expression throughout the suprabasal strata [39]. Our results also showed expression of PKD1 in proliferating subconfluent normal human keratinocytes, which recembles basal keartinocytes, although in very low mRNA and protein levels [126]. The expression of the kinases decreases when keratinocytes reached confluency. The estimated function of the kinase in human keartinocytes is proproliferative and antidifferentiative, since knock-down of PKD1 using siRNA showed decrease mRNA expression of proliferative marker PCNA and increased expression of keratinocytes differentiave markers – K10 and Involucrin [126]. Uninvolved psoriatic skin showed a similar pattern of predominant PKD protein expression in the stratum basalis, but in contrast, involved (lesional) psoriatic epidermis demonstrated a diffuse pattern of staining with ectopic foci of increased staining intensity in the suprabasilar layers was observed. This result is consistent with altered PKD protein expression in the epidermis potentially mediating the antidifferentiative phenotype characterizing psoriasis [39]. More and more evidence supports that Wnt/βcatenin signaling functions in skin stem cells through cooperation with Cdc42, Notch, and vitamin D. Evidence supports that theWnt/βcatenin signal controls the fate of the bulge stem cells and plays an essential role in hair but not epidermal differentiation [68]. β -catenin is not required for keratinocyte proliferation but has been shown to regulate keratinocyte stem cells and hair follicle morphogenesis. In normal skin tissues, positive staining for E-cadherin and β-catenin was detected in all layers of the normal epidermis at the sites of cell-cell junctions, and E-cadherinmediated adhesion plays a key role in maintaining the tissue integrity and differentiation of epidermal keratinocytes. Whereas distribution of P-cadherin is restricted to the SB (stratum basale), E-cadherin is the predominant AJ cadherin in supra basal



layers. In epithelial cells, raising [Ca2+]o stimulates the binding of E-cadherin to its counterpart on the surface of neighboring cells, and its interactions with β- (or γ-), α-, p120-catenins, and the actin cytoskeleton to form AJs (adherens junctions). These interactions stabilize AJs and support cell stratification [127,128]. Downregulation of E-cadherin and β-catenin expression was found in the granular layer and basal layer of the psoriatic lesions. Downregulation of E-cadherin and β-catenin expression and increased nuclear β-catenin cyclin D1 overexpression in psoriatic skin are probably involved in keratinocyte hyperproliferation in psoriasis vulgaris compared with uninvolved or normal skin [129,130]. Increased active unphosphorylated β-catenin was also detected within the differentiating compartment of involved psoriatic epidermis. Expression of Tgase-1 overlapped with β-catenin in suprabasal lesional psoriasis. The TGase 1 promoter was positively regulated by activated β-catenin and by the glycogen synthase kinase binding protein, suggesting that β-catenin and glycogen synthase kinase 3β may regulate TGase 1 expression [130]. Disregulation of E-cadherin/ β-catenin expression is observed also in both BCCs and SCCs (see below). The results suggest an association between loss of expression of E-cadherin and increase βcatenin and a lower degree of differentiation in SCC [128]. 2. PKD1 in BCC: Analysis of BCC (basal cell carcinoma) lesions of Ristich et al. showed increased expression of PKD1 compared with normal epidermis, but not in SCC lesions (squamous cell carcinoma). For BCCs samples in which comparison was possible, the tumours exhibited elevated PKD immunoreactivity relative to the basal layer of the normal epidermis. This result suggests that BCCs possess greater amounts of PKD than normal basal keratinocytes. So as the authors wrote, the question remain: are the enhanced PKD1 levels in BCCs are simply a marker of their basal origin or does this elevated PKD1 contributes to the pathogenesis of BCCs [39]. Thus, another question is curently adequate, lack of PKD1 expression in SCCs despite increase expression of EGFR, is a consequence of its spinous layer origin, or is a consequense of PKD1 gene mutation(s) (silencing, methylation) as a result of gene alterations (including down-

regulation) connected with the progression of precancer to cancer. Using only immunohistochemical analysis authors showed that expression of PKD1 in normal epidermis was primarily restricted to the stratum basalis, the proliferative compartment of the epidermis, supporting the concept that PKD1 promotes proliferation of normal keratinocytes and that this kinase is probably connected with hyperproliferative disorders of the skin (increased expression of PKD1 was detected also in involved psoriatic lesions) [39]. Our results supported these hypotesis and proved the proproliferative and antidifferentiating role of the kinase in human normal keratinocytes, mediated probably by a EGFR/PKC/PKD1/ERK1/2 dependent pathway [131,132]. Knock-down of PKD1 in human keratinocytes using antisense oligonucleotides led to increas in ERK1/2 activity (unpublished result), and increase in suprabasal markers Involucrin and K10 [126] and inhibited proliferation marker PCNA. In contrast, in hTert keratinocytes, obtained from normal human epidermal keratinocytes infected with amphotropic retroviral vectors encoding hTert (catalytic subunit ot telomerase), the expression of PKD1 was increased near 9-fold. However, the function of the kinase was reversed (prodifferentiating role), connected with induction of EGFR expression and activity, ERK1/2 expression and activity and induction of suprabasal markers involucrin and K10. The received results suppose that PKD1 posses different function in normal and hTert (N/Tert-1) keratinocytes and probably in premalignant diseases (N/Tert-1 keratinocytes possess mutation only in p16INK-4A, 72) [133,134]. Additionally, Bertrand-Vallery V. et al observed alternative differentiation in hTert keratinocytes after repeated exposures to sublethal doses of UVB, lacking functional p16INK-4a, immortalized with telomerase and retaining their differentiation capacities (called N-hTERT cells). Keratinocytes are more resistant to UV than other cell types due to specialized responses. However, repeated exposures to UV can lead to epidermal malignancies. While expression of telomerase does not abolish UVB-induced premature senescence in human diploid fibroblasts, nor in human keratinocytes, absence of functional p16INK-4a does. In such sublethal conditions alternative differentiation is observed. The authors identified TRIM29 (TRIpartite Motif Protein 29), which



expression is increased after the exposures to UVB. Knocking down the expression of TRIM29 by short-hairpin RNA interference decreased the viability of keratinocytes after UVB exposure. The abundance of involucrin mRNA, a marker of late differentiation, increased concomitantly. In TRIM29-knocked down reconstructed epidermis, the presence of picnotic cells revealed cell injury. Increased abundance of TRIM29 was also observed upon exposure to DNA damaging agents and PKC activation. The UVB-induced increase of TRIM29 abundance was dependent on a PKC signaling pathway, likely PKCδ. These findings suggest that TRIM29 allows keratinocytes to enter a protective alternative differentiation process rather than die massively after stress [135]. TRIM29 played a crucial role in the progression and malignancy of TC (Thyroid cancer), and silencing of TRIM29 exerted its antitumor effect by suppressed cell proliferation; enhanced chemosensitivity to cisplatin; inhibited cell invasion and migration; caused cell cycle arrest at G0/G1 phase by decreasing cyclin B1, cyclin D1 and CDK2, while increasing p21 and p27; and induced cell apoptosis by enhancing the activities of caspase-3, caspase-9, and Bax, while decreased Bcl-2. Notably, decreased TRIM29 expression significantly inhibited the activation of P13K/AKT signaling pathway as well [136]. Furthurmore, it is well known that UV irradiation is the main cause for BCCs, and one of the etiological cause for SCCs. In particular UVB (approximately 280-320 nm wavelength light), activates mouse keratinocyte PKD1 [48]. PKD1 activation in response to UVB involved tyrosine phosphorylation mediated by a Src family kinase cascade, rather than via a protein kinase Cmediated transphosphorylation, and was downstream of UVB-elicited oxidative stress [48]. It is well known that UV activates EGFR in human skin, which blocks cell cycle arrest, increases cells proliferation, suppresses apoptotic cell death, and increase skin tumorogenesis [46]. Thus, at least some of the UV induced EGFR effects could be mediated by PKD1 in skin [48]. Bollag et al. detected that PKD1 activation after UV irradiation, may allow survival of UVdamaged cells. This ability of PKD1 to promote survival would be beneficial in preventing excessive apoptosis with low levels of UVB exposure, causing minimal DNA damage that can be repaired. However, if PKD1 allows survival of

cells that have suffered irreparable UV-induced DNA damage, opposing action of pro-apoptotic PKCδ-p38δ signal, these keratinocytes with DNA mutations could continue to proliferate and form skin BCCs. Thus, either a pro-proliferative or prosurvival mechanism could provide a means by which PKD1 could contribute to epidermal tumorigenesis [48]. One possible explanation of PKD1 action could be explained with the fact that PKD1 influences the activity of two important signal pathways in kerainocytes ERK1/2 and NF-θB4, pathways lining downsream and targeted by EGFR. ERK activation in suspension has been implicated in both cell death and survival depending on the type and duration of the stimulus and cellular context. Crowe DL et al. report that long-term ERK1 activation as a result of anchorage deprivation results in cell cycle arrest and telomerase inhibition in human stratified squamous epithelial cells [137]. To determine if inhibiting ERK1 activation could block suspension-induced loss of telomerase activity, Crowe DL et al. examined the effect of the MEK inhibitory drug, PD98059, on telomerase activity in suspension differentiating culture. PD98059 treatment protected cells from loss of telomerase expression and activity in these cultures for up to 24 h. To confirm these results, the authors created stable SCC4 clones which expressed a dominant negative ERK1 construct. SCC4 cells undergo loss of telomerase in suspension culture similar to NHEK cells. dnERK1 expression preserved telomerase expression in anchoragedeprived SCC4 cells for up to 24 h, while suspension culture and wild-type ERK1 overexpression inhibited telomerase activity. These data suggest that when attached to ECM, one of the functions of α2β1 integrins is to modulate growth factor-induced MAPK activity to prevent terminal differentiation. Constitutive activation of MAPK in cultured human keratinocytes has been shown to result in cell cycle progression and terminal differentiation [137] . The author‘s data suggest that, in keratinocytes, anchorage deprivation induces S phase entry followed by G2 arrest associated with cyclin B expression. Suspension culture of normal keratinocytes has been shown to induce growth arrest in both G1 and G2 phases. Interestingly, myc overexpression promotes G2 phase arrest of keratinocytes. This cell cycle block was associated



with endoreplication and polyploidy similar to treatment with the M phase blocking agent nocodazole. Crowe DL et al. data suggest that increased ERK activity as the result of anchorage deprivation can result in forced S phase entry, G2 growth arrest, and telomerase inhibition in stratified squamous epithelial cells. Telomerase inhibition in stratified squamous epithelial cells occurs in suspension culture via formation of an Rb/HDAC1 repressor complex on E2F1 site of the hTERT promoter [137]. These results lead to the proposal that hTert expression could be regulated by ERK1/2 in proliferating keratinocytes, depending on duration and strength of its activation. Тhe estimated proproliferative ERK1/2 function in keratinocytes could be conneted with regulation of hTert expression (there is no data), detected only in basal proliferating layer of epidermis. It is well known that NF-θB, also activated by PKD1, is the kinase participating in the regulation of hTert promoter [4,138]. Thus, increase in hTert expression in keratinocytes whether by PKD1/ERK1/2/hTert (proproliferative) or whether by PKD1/NFκB/hTert (prosurvival, pro-inflammational) pathway could explain mechanism of PKD1 action after UV-exposure. From the above data concerning regulation of hTert expression in keratinocytes, we can speculate that increase of its expression is proproliferative and probably prosurvival for keratinocyte since N/Tert-1 keratinocytes are immortalized cells [72]. Our results showed that increased PKD1 expression as a consequence of forced hTert expression [126]. But the current question is whether increased PKD1 activity/expression influences hTert expression. PKD1 is expressed also only in the basal layer of epidermis, posessing proproliferative activity, inhibites ERK1/2 activity to basal proliferating levels and do not changes c-Myc expression/activity in normal keratinocytes [126, our unpublished results]. Increased expression of GLI1 is a common feature of BCC and is linked to the induction of epidermal SC (stem cells) markers in immortalized N/Tert-1 keratinocytes. Neil et al. demonstrated that GLI1 over-expression is linked to additional SC characteristics in N/Tert-1 cells including reduced epidermal growth factor receptor (EGFR) expression and compact colony formation that is associated with repressed extracellular signalregulated kinase (ERK) activity. Colony formation

and repressed ERK activity remain evident when EGFR is increased exogenously to the basal levels in GLI1 cells revealing that ERK is additionally inhibited downstream of the receptor. Exposure to epidermal growth factor (EGF) to increase ERK activity and promote migration negates GLI1 colony formation with cells displaying an elongated, fibroblast-like morphology. However, as determined by Snail messenger RNA and Ecadherin protein expression this is not associated with epithelial–mesenchymal transition (EMT), and GLI1 actually represses induction of the EMT marker vimentin in EGF-stimulated cells. Instead, live cell imaging revealed that the elongated morphology of EGF/GLI1 keratinocytes stems from their being ‗stretched‘ due to migrating cells displaying inefficient cell–cell detachment and impaired tail retraction. Taken together, these data suggest that GLI1 opposes EGFR signalling to maintain the epithelial phenotype [87]. PKD1 is activated in responce of EGFR activation in normal human kerainocytes, and the opposite of Gli effect of PKD1 on ERK1/2 expression/activity and EGFR expression /activity in N/Tert-1 keratinocytes is difficult to be interpretated. However, increased expression of Gli could be consider as a consequence of second mutation. BCCs posess both PKD1 and Gli increased expression (see above) and ERK activity was predominantly negative in 13/14 BCCs (superficial/nodular) [87,133,134]. However this is not the exact model for studing BCCs, since N/Tert-1 keratinocytes possess mutation in p16 INK4A, which is not typical for BCCs. 3. Wnt/ β-catenin, MMPs and COX-2 in BCC: Various immunohistological markers have been investigated to assess the aggressive characteristics of basal cell carcinoma (BCC), what as it was allready mentioned is of importance when a surgical procedure is planned [50] (BCC rarely metastasizes, but it is often locally aggressive). Methylation commences in UV exposed skin at a relatively early event and occurs in skin prior to the onset of recognizable preneoplastic changes [139]. Among the 12 genes studied, for the cadherin genes CDH1 (gene of Ecadherin) and CDH3 (gene of P-cadherin) and for two of the laminin 5 encoding genes LAMA3 (laminin alpha 3, subunit of laminin 5) and LAMC2 (laminin gamma 2, subunit of laminin 5) methylation frequencies greater than 30% were noted in one or more specimen types. Methylation



was highly significantly related to sun exposure, and sun protected specimens had little or no methylation [139]. E-Cadherin is expressed in all the living layers, whereas P-Cadherin expression is restricted to the basal layer of normal epidermis. A severe reduction of E-Cadherin expression with a disordered distribution of cells with different immunostaining intensity was observed in most specimens of infiltrative BCC. In contrast, P-CD expression was preserved in all cases of infiltrative BCC [140]. Moreover E-CD expression was preserved in all specimens of superficial and nodular BCC, and was reduced in 10 of 15 infiltrative BCCs. A heterogeneous distribution of cells with different immunostaining intensity was more frequently observed in specimens of infiltrative BCC. These results support the idea that E-CD might play a role as an invasion-suppressor molecule in vivo [141] . Paraffin-embedded tissue sections from 100 human BCC cases were processed by immunohistochemistry for the expression of ILK (Integrin-linked kinase), E-cadherin, Snail, βcatenin and alpha-smooth muscle actin (α -SMA). ILK overexpression was observed in 100% of cases and strongly correlated with tumour invasion and infiltrative BCC. Loss of membranous Ecadherin was found in 71% of cases while nuclear immunoreactivity for E-cadherin was also observed in 90% of the tumours. Snail, nuclear βcatenin and α-SMA expression was detected in 100%, 99% and 97% of tumours, respectively. Aberrant expression of E-cadherin, nuclear β catenin and α -SMA correlated with BCC tumour invasion. Interestingly, there was a significant correlation between ILK expression and all the EMT markers examined. ILK overexpression in BCC is implicated in tumour progression probably through the induction of an EMT-related molecular profile. Nuclear localization of E-cadherin in BCC is also associated with aggressive tumour features [142]. Saldanha G. et al.found nuclear β-catenin in 20 of 86 paraffin-embedded sections of BCCs using immunohistochemistry [143]. Nuclear localization was most notable in the infiltrative and morphoeic variants, followed by the superficial variant, and seen least in nodular BCC. All micronodular BCCs showed strong membranous staining, weak cytoplasmic and no nuclear staining. In the infiltrative and morphoeic BCCs membranous staining was completely lost at the advancing

margins of the invading cell strands, with a marked increase in cytoplasmic staining; nuclear staining was observed in all these tumours. Its prominence at tumour margins suggests that this may be associated with more aggressive types of invasion [144]. The study of El-Bahrawy M. et al. also aimed to investigate the presence of differences in the immunoprofile of β-catenin among histological variants of BCC. Methods Eighty BCCs were studied (32 nodular, 7 micronodular, 24 superficial and 17 infiltrative and morphoeic). Formalin-fixed, paraffin-embedded tissue sections were stained for β-catenin using the avidin/biotin immunodetection technique. All the nodular BCCs showed membranous and weak cytoplasmic staining. Nuclear staining was seen in 15 of 32 (47%) cases, being stronger at the periphery of the nodules in 11 of 15 (73%) of these cases. In superficial BCCs the membranous staining was variable and cytoplasmic staining was increased. Nuclear staining was seen in 16 of 24 (67%) cases, being more notable at the periphery in 8 of 16 (50%) of these cases. All micronodular BCCs showed strong membranous staining, weak cytoplasmic and no nuclear staining. In the infiltrative and morphoeic BCCs membranous staining was completely lost at the advancing margins of the invading cell strands, with a marked increase in cytoplasmic staining; nuclear staining was observed in all these tumours. The expression of β-catenin varied between different types of BCC. Nuclear localization was most notable in the infiltrative and morphoeic variants, followed by the superficial variant, and seen least in nodular BCC. Its prominence at tumour margins suggests that this may be associated with more aggressive types of invasion [144,145]. According Brinkhuizen et al. increased canonical WNT activity was visualized by β-catenin staining, showing nuclear β –catenin in only 28/101 (27.7%) of BCC. Absence of nuclear β -catenin in some samples may be due to high levels of membranous E-cadherin (in 94.1% of the samples), although significantly lowered when compared to adjacent normal epidermis, which would also be consistent with the general inability of BCC to metastasize [146]. Oh ST. et al. detected that β-Catenin and MT1MMP immunoreactivity was increased in the highrisk BCCs compared with the low-risk (nodular) BCC (P < 0·001). Nuclear β-catenin



Liu P. et al.found that β-catenin in MDCK noncancer cells inhibited the cell surface localization of MT1-MMP, and thus its proteolytic activity on pro-MMP2 activation, via direct interaction with the 18-amino-acid cytoplasmic tail of MT1-MMP in the cytoplasm. In contrast, β-catenin in HT1080 cancer cells enhanced the activity of MT1-MMP by entering the nucleus and activating transcription factor Tcf-4/Lef, and elevating the level of MT1MMP protein. They also found that enhancement of cell growth in three-dimensional (3-D)/twodimensional (2-D) type I collagen gels and of cell migration by MT1-MMP were inhibited by βcatenin in MDCK cells, whereas these functions were enhanced in HT1080 cells. In addition, regulation of MT1-MMP by β-catenin involved Ecadherin in MDCK cells and Wnt-3a in HT1080 cells. Taken together, their results present a differential effect of cytoplasmic and nuclear βcatenin on MT1-MMP activity in non-cancer cells versus cancer cells [149]. As it was already mentioned analysis of BCC (basal cell carcinoma) lesions of Ristich et al. showed increased expression of PKD1 when compared with normal epidermis and SCC [34], which may lead to decrease expression of MMPs with exception for MMP-3 [7]. Majmudar G. et al. detected that three of three BCC tumors obtained from NBCCS (Nevoid basal cell carcinoma syndrome) patients overexpressed MMP-3 mRNA. In contrast, only 25% of BCC specimens in patients without NBCCS demonstrated overexpression of MMP-3 mRNA. Moreover, fibroblasts isolated and cultured from all nine uninvolved skin specimens of NBCCS patients overexpressed MMP-3 mRNA. MMP-3 mRNA was not detected or was detected at very low levels in normal skin and fibroblast cultures isolated from normal skin in nonsyndrome patient [150]. The data of Karahan N. et al. confirm previous findings that COX-2 and MMP-9 (MMP-2) expressions are increased in BCC. In addition, MT1-MMP mRNA was detected in 19/21 SCCs COX-2 expression was significantly higher in both in epithelial cancer cells and stromal the infiltrating pattern of BCC compared with fibroblasts and in 14/18 BCCs only in fibroblasts. the nodular and superficial subtypes in the MMP-10 was expressed in 13/21 SSCs and 11/19 primary BCC group. There was not a BCCs only in epithelial laminin-5 positive cancer significant difference between nodular and cells, while premalignant lesions were entirely superficial BCCs for COX-2 expression. And negative. The level of MMP-10 was upregulated in finally, COX-2 expression was significantly a cutaneous SCC cell line (UT-SCC-7) by higher in the recurrent BCC group than in the transforming growth factor-alpha and keratinocyte primary BCC group. There was no statistically growth factor [148]. significant difference between the histological International Journal of Contemporary Research and Review, Vol. 9, Issue. 02, Page no: BC 20193-20252 doi: https://doi.org/10.15520/ijcrr/2018/9/02/428 Page | 20226 immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesion (P< 0·01). For the mixed BCC (P < 0·01), infiltrative BCC (P < 0·001), morphoeiform BCC (P < 0·001), micronodular BCC (P < 0·001) and basosquamous (P < 0·001) carcinoma, β-catenin immunoreactivity was increased at the invading front compared with nodular BCC. MT1-MMP (membrane-anchored type metalloproteinase) immunoreactivity was increased in the high-risk BCCs compared with the low-risk (nodular) BCC (P < 0·01). The membranous MT1-MMP immunoreactivity was increased at the invading front of mixed BCC tumour islands compared with the upper portion of the lesions (P < 0·01). For the mixed (P