**2. Anti-apoptotic signaling in skin tumor initiation**

#### **2.1 AKT signaling**

AKT (protein kinase B) is the human homolog of the viral oncogene *v-akt* and is associated with protein kinases A (PKA) and C (PKC) in humans [26, 27]. The three known AKT isoforms are derived from distinct genes (AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ). The N terminus of AKT contains a pleckstrin homology domain, which can mediate lipid-protein and/or protein–protein interactions [28–30]. The C terminus of AKT contains a hydrophobic and proline-rich domain [28, 29]. AKT is activated by various growth factors such as platelet-derived and fibroblast growth factors and is involved in the regulation of cell survival signaling [31, 32]. AKT activation depends on its phosphorylation and four different phosphorylation sites on AKT (Ser-124, Thr-308, Thr-450, and Ser-473) have been identified [33]. Studies showed that extracellular stimuli induce the phosphorylation of AKT at Thr-308 and Ser-473 residues while the phosphorylation of AKT at Ser-124 and Thr-450 residues appears to be basally maintained [33]. Mutagenesis studies have revealed that the phosphorylation of AKT at Thr-308 and Ser-473 is required for its activation [34].

It has been shown that AKT signaling inactivates several pro-apoptotic factors including BAD, procaspase-9 and Forkhead transcription factors [35, 36]. In contrast, AKT activates various transcription factors that are involved in the upregulation of anti-apoptotic genes, such as cyclic-AMP response element-binding protein (CREB). It also activates IκB kinase (IKK) to phosphorylate IκB (inhibitor of NF-κB), leading to its proteasomal degradation and NF-κB nuclear localization. In addition, AKT reduces the protein levels of p53 by promoting its degradation through MDM2 phosphorylation, which can contribute to centrosome hyperamplification and chromosome instability in cancer [37, 38]. Furthermore, AKT is involved in the regulation of subcellular localization of proteins. AKT can regulate the localization of various proteins and thereby their activity by phosphorylating specific binding sites for 14–3-3 proteins, which play a crucial role in the modulation of cellular location and degradation of proteins [39, 40].

Studies have shown that AKT is activated by environmental toxicants including UVB irradiation which protects keratinocytes against environmental attacks, implying its anti-apoptotic role in skin tumor initiation. It has been shown that UV radiation induces phosphorylation of AKT at Ser-473 and Thr-308 residues in mouse epidermal cell JB6 Cl41 in a time-dependent manner. These results were further confirmed by the observation that overexpression of AKT mutant, AKT-T308A/S473A, attenuated phosphorylation of AKT at Ser-473 and Thr-308 upon UVB irradiation [41]. The reactive oxygen species (ROS) generated by UV radiation acts as a mediator in UV-induced phosphorylation of AKT. It has been observed that pre-treatment of cells with either an antioxidant, N-acetyl-L-cysteine (NAC) or a specific antioxidant enzyme (catalase) inhibits phosphorylation of AKT in these cells, suggesting the link of ROS in UV radiation-induced activation of AKT [41]. Specific phosphorylation of Bad, a pro-apoptotic member of the Bcl-2 family, by AKT delayed the early activated apoptotic pathways in UVB-exposed human keratinocytes. AKT-mediated phosphorylation of Bad at serine 136 residue promoted its translocation from the mitochondria to the cytoplasm and subsequent cytoplasmic sequestration by 14–3-3ζ, resulting in the reduction of UVB-induced apoptosis in keratinocytes [42]. In addition to its anti-apoptotic function, studies using transgenic mice have shown that AKT can contribute to the development of skin cancer formation induced by environmental exposure through the regulation of epidermal proliferation. It has been shown that mice lacking *Akt1*−/−*;Akt2*−/− or *Akt1*−/−*;Akt3*+/− exhibit a hypoplastic epidermis due to decreased proliferation of keratinocytes while overexpression of wild type AKT1 (*wtAk*t) or constitutively active AKT (*myrAkt*) in the basal layer of mouse epidermis displays alterations in epidermal proliferation and differentiation [43–45]. Overexpression of either wtAKT or myrAKT in mouse epidermis displayed enhanced sensitivity to two stage skin carcinogenesis by promoting cell proliferation [45]. These studies suggest that AKT plays a critical role in the regulation of apoptosis and proliferation during skin carcinogenesis. Further detailed studies using transgenic mouse models will be helpful to elucidate the underlying molecular mechanisms and function of AKT in the initiation of skin carcinogenesis.

## **2.2 STAT3 signaling**

Signal transducer and activator of transcription 3 (STAT3) is one of the family members of seven [STAT1 (α and β splice isoforms), STAT2 and STAT3 (α and β

#### *Regulation of Apoptosis during Environmental Skin Tumor Initiation DOI: http://dx.doi.org/10.5772/intechopen.97542*

isoforms), STAT4, STAT5a, STAT5b, and STAT6)] latent cytoplasmic transcription factors which are encoded by seven individual genes [46]. STATs are phosphorylated at their specific tyrosine residue and activated by a wide variety of stimuli including growth factors and cytokines, which act through intrinsic receptor tyrosine kinases [47]. Tyrosine phosphorylation of STAT induces its dimerization via reciprocal interaction of phospho-tyrosine with Src homology domain 2 (SH2) between two STAT molecules. The phosphorylated STATs then translocate to the nucleus and bind to the consensus promoter sequences of downstream target genes, resulting in the activation of their transcription [48].

Different tyrosine kinases, such as RTKs (receptor tyrosine kinases) and non-RTKs including JAKs (Janus kinases), can phosphorylate STAT proteins. STAT tyrosine phosphorylation is transient, which lasts from 30 minutes to several hours in normal cells. However, studies have shown that STATs (specifically STAT3) are persistently tyrosine phosphorylated either as a consequence of deregulation of positive regulators of STAT activation such as tyrosine kinases or deactivation of negative regulators of STAT phosphorylation, such as phosphatases, suppressor of cytokine signaling, or protein inhibitor of activated STATs, in numerous cancer-derived cell lines or in primary tumors [49].

Studies have revealed that STAT3 is associated with cell survival and oncogenic transformation among the seven members of STATs. It has shown that targeted inhibition of STAT3 activation by antisense, small interfering RNA, dominantnegative STAT3 constructs, and/or blockade of tyrosine kinases has been associated with growth arrest and induction of apoptosis in cancer cell lines [49]. Furthermore, overexpression of constitutively activated STAT3 into immortalized cell lines led to oncogenic transformation, indicating a potential role of STAT3 in carcinogenesis [50, 51].

The generation of epidermal-specific STAT3-dificient and STAT3-overexpressing transgenic mice led to the main discovery of functional role of STAT3 in the initiation stage of skin carcinogenesis [52, 53]. Epidermal-specific deficiency of STAT3 in mouse *(K5Cre.Stat3*fl/fl) significantly increased the sensitivity to apoptosis after 7,12-dimethylbenz[a]anthracene (DMBA) treatment both *in vitro* keratinocytes and *in vivo* epidermis compared with non-transgenic controls, as determined by increased caspase-3-positive cells. In particular, the significant increase in the number of keratinocyte stem cells (KSCs) undergoing apoptosis in the bulge region of hair follicles was observed in STAT3-deficient mice compared with non-transgenic littermates, indicating that STAT3 may be critical for maintaining the survival of KSCs during skin tumor initiation mediated by DMBA [54]. Similar with this observation, forced expression of a constitutively active form of STAT3 in mouse epidermis (*K5.Stat3C*) showed increased cell survival following DMBA exposure [55]. Further studies showed that STAT3 plays a critical role in the protection of damaged keratinocytes after UVB exposure. The epidermis of STAT3-deficient mice was highly sensitive to UVB-induced apoptosis compared with the epidermis of control mice, whereas the epidermis of K5.Stat3C mice was more resistant to UVB-induced apoptosis compared with the epidermis of control mice. UVB induces DNA damage and causes mutations in runs of tandemly located pyrimidine residues of DNA, resulting in the generation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6–4) pyrimidone photoproducts (6–4 PPs). The number of either 6–4 PP- or CPD-positive cells was greater in epidermis of K5.Stat3C mice compared with epidermis from STAT3-deficient mice after UVB exposure [56]. Additionally, generation of inducible STAT3-deficient mice (*K5.Cre-ER*T2 *x Stat3*fl/fl) clearly demonstrated that STAT3 has a critical role in the protection of keratinocytes during tumor initiation. Inducible deletion of STAT3 in mouse epidermis significantly increased apoptosis after DMBA treatment. Inducible STAT3 deletion in epidermis

before initiation with DMBA showed a significant delay in tumor development and a significantly reduced number of tumors compared with control groups in two-stage skin carcinogenesis [57].

Expression of *Ha-ras homolog* (*v-Ha-ras*) into cultured primary keratinocytes *in vitro* has been utilized to generate initiated keratinocytes. Studies showed that the introduction of a STAT3 decoy molecule into the v-Ha-ras–initiated keratinocytes increases apoptosis with a concomitant decrease in Bcl-xL expression levels. In general, inhibition of STAT3 activation can lead to increase apoptosis or growth arrest in cancer-derived cell lines containing high levels of phosphorylated STAT3. However, STAT3 inhibition was not relatively affected in cell lines containing low or no levels of detectable tyrosine phosphorylated STAT3. Therefore, how STAT3 can protect keratinocytes against DMBA-induced apoptosis remains to be unclear, because DMBA does not induce tyrosine phosphorylation of STAT3 in keratinocytes, nor is it likely that v-Ha-ras–containing keratinocytes contain abundant levels of phosphorylated STAT3 [58]. One possible explanation is that the low levels of phosphorylated STAT3 present in these keratinocytes are sufficient to drive transcription of anti-apoptotic genes such as *Bcl-xL*. Alternatively, it is also possible that non-phosphorylated STAT3 may play a role as a transcription factor as previously demonstrated for STAT1 [59]. There are a few notable examples where relatively low levels of phosphorylated STAT3 are sufficient to mediate protection from growth arrest or apoptosis [60, 61]. Therefore, determination of the relative levels of phosphorylated STAT3 required to impart a phenotype is likely to be cell-type specific and remains an important objective.

It suggests that KSCs, which are located mostly within the bulge region of the hair follicle, are the major target cells for two-stage carcinogenesis [62]. The labelretaining cells (LRCs) retain the label for a sustained period of time following continuous administration of nucleotide analogs such as bromodeoxyuridine (BrdU) or [3 H] thymidine, indicating a very slow cycling frequency. Studies showed that hair follicle KSCs are identified within the LRCs [63, 64]. It has been observed that the STAT3-deficient keratinocytes undergoing apoptosis following DMBA exposure were located primarily within the bulge region of the hair follicle in an area adjacent to the LRC population. It indicates that the DMBA-sensitive cells may be KSCs, given their proximity to the LRCs. However, given the lack of overlap between the LRCs and the apoptotic cells, the cell type most sensitive to DMBA-induced apoptosis remains to be identified. Studies using inducible bulge-region KSC-specific STAT3-deficient mice (*K15.CrePR1 x Stat3*fl/fl mice) have provided further evidence that STAT3 is required for survival of bulge-region KSCs during the initiation stage of skin carcinogenesis [65]. In these studies, the number of apoptotic KSCs in the bulge-region was significantly increased in *K15.CrePR1 x Stat3*fl/fl mice by inducible deletion of STAT3 prior to tumor initiation with DMBA compared with control littermates. In addition, the frequency of *Ha-ras* codon 61 A182 → T mutations was decreased in *K15.CrePR1 x Stat3*fl/fl mice compared to control mice [65]. Furthermore, the number of skin tumors that developed in a two-stage skin carcinogenesis protocol was dramatically reduced by targeted deletion of STAT3 in bulge region KSCs at the time of initiation [65]. Overall, these studies provide molecular basis of STAT3 involvement in the initiation of skin carcinogenesis.

#### **2.3 MAPK signaling**

Mitogen-activated protein kinases (MAPKs) are essential signaling components that are vital in converting extracellular stimuli into intracellular responses through transcriptional regulation of various regulatory genes. MAPK signaling is activated by sequential protein kinase cascades including three enzymes: a MAPK kinase

#### *Regulation of Apoptosis during Environmental Skin Tumor Initiation DOI: http://dx.doi.org/10.5772/intechopen.97542*

kinase (MAPKKK), a MAPK kinase (MAPKK) and a MAPK [66]. MAPK signaling pathways are involved in the regulation of a wide variety of cellular processes including cell proliferation, differentiation, development, stress responses and apoptosis. Therefore, MAPK signaling is considered as one of potential therapeutic targets for many signaling-related diseases including cancer and diabetes. Three structurally related but biochemically and functionally distinct MAPKs are identified and named as extracellular signal-regulated kinases (ERKs), c-Jun N terminal kinases (JNKs) and p38 MAPKs [67–70].

ERK was the first MAPK identified and contains two isoforms: ERK1 and ERK2. ERK plays a critical role in the signaling of a variety of extracellular stimuli, such as growth factors and phorbol esters. ERK signaling is involved in the regulation of cell cycle progression and cell proliferation as one of major checkpoint signaling pathways for cellular mitogenesis [66, 71]. JNK was initially identified as a regulator of transcription factor c-Jun and consisted of three isoforms: JNK1, JNK2, and JNK3. JNK is also known as a stress-activated protein kinase (SAPK) as it is stimulated by various intra- or extracellular stresses. JNK signaling is involved in many cellular processes including immune response, neuronal activity, and insulin signaling [70, 72]. Studies have also shown that JNK signaling is critical in the promotion of apoptosis in response to a variety of harmful external stimuli through p53 activation [73, 74]. p38 MAPK contains four isoforms: p38α, p38β, p38δ, and p38γ. p38 MAPK is another SAPK and is activated by stress related stimuli. Similar with JNK, p38 MAPK is also involved in many cellular processes including apoptosis, inflammation, migration, differentiation, and cell cycle checkpoints [68, 75, 76].

In skin keratinocytes, both ERK and JNK are activated by UVB irradiation and protect cells against UVB-induced apoptosis [77, 78]. JNK is significantly activated by UVB exposure and pretreatment of antioxidant N-acetylcysteine reduced its activation, implying UVB-induced oxidative stress plays an important role in the activation of JNK [77]. Furthermore, UVB-mediated generation of reactive oxygen species significantly increased the activation of both JNK and ERK in human keratinocytes. Activated JNK and ERK then induced the upregulation of Bcl-2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3) expression, which is known to protect keratinocytes from UVB-induced apoptosis through autophagy. Pretreatment with the antioxidant N-acetylcysteine, the JNK inhibitor SP600125, or the ERK inhibitor U0126 significantly reduced the expression of BNIP3 upon UVB exposure and decreased cell survival by inducing apoptosis [78]. p38 MAPK is also activated by UVB irradiation [79]. However, in contrast to JNK and ERK, p38 MAPK promotes epidermal apoptosis following UVB exposure in mouse skin. The epidermis of transgenic mice that express a dominant negative p38α MAPK (p38DN) showed a significant reduction in UVB-induced apoptosis compared with the epidermis of control mice. The p38DN mice also showed a significant reduction of tumor number and growth compared to wild-type mice in UVB skin carcinogenesis assay [80]. Overall, it implies that JNK and ERK may protect damaged keratinocytes by reducing apoptosis during the initiation of skin carcinogenesis induced by environmental carcinogens, while p38 MAPK may contribute to remove damaged keratinocytes by promoting apoptosis in response to environmental exposure.
