**8. The PI3K/Akt pathway**

cycle progression arrest through G1-S; while p14 favors apoptosis and blocks oncogenic transformation by stabilizing p53 levels through the inhibition of Mdm2-mediated p53 ubiquitination [27,28,29,30]. Loss of p16 promotes hyper-phosphorylation of pRb resulting in its inactivation while the loss of p14 inactivates p53 — leading to unrestricted cell cycle progression. Germline mutations in CDKN2A have been found in 40% of families with 3 or more family members affected by melanoma [31]. Not all individuals carrying germ‐ line CDKN2A mutations develop mutations. Individuals with large numbers of pigment lesions or nevi have familial atypical mole-melanoma syndrome (FAMS) are associated with

The other melanoma susceptibility gene, CDK4 is located at chromosome 12q14 [34,35,36]. Mutations in CDK4 abrogate binding of cdk4 to p16 have been associated with melanoma pathogenesis [32]. This is evidence that links the entire p16Ink4A-cdk4/cdk6-pRb pathway to melanoma indicating that hereditary retinoblastoma patients with germline inactivation of

Understanding the regulating pathways involved in melanoma development and progression has advanced significantly in recent years. The discovery of genetic alterations that aids in the formation of various cancers has aided in the development of numerous molecularly targeted therapies for individuals with metastatic disease [39,40,41]. These genes are known to be key molecular driver in melanoma; >70% cases harbor activating mutations in these genes. The molecule that is most commonly found to be mutated in melanomas is BRAF (~50% of all cancers) followed by NRAS (20%) and c-kit (1%) [42,43,44]. Melanoma is the result of complex changes in multiple signaling pathways affecting growth, cell mobility, metabolism and the ability to escape cell death progression. The Ras-Raf-Mek-Erk pathway followed by PI3K/Akt

In 2002, a breakthrough study found that Braf to be mutated in a large percentage of melanomas – triggering new studies that focus on MAPK (mitogen activated protein kinase) signaling in melanomas. Braf is mutated in upto 82% of cutaneous nevi [45,46], 66% of primary melanomas [44] and 40-68% of metastatic melanomas [47,48]. A specific muta‐ tion substitution of valine with glutamic acid at residue 600 (BRAF V600E), account for 90% + BRAF mutation. Raf, a downstream effector of RAS is a serine-threonine specific protein kinase that activates Mek, which inturn activates Erk. Humans have 3 Raf genes: A-raf, Braf and C-raf. The occurrence of mutation in Nras or Braf is 80-90% of all melanomas suggests that constitutive activation of extracellular signal regulated protein kinase (Ras-Raf-Mek-Erk). Most Ras mutations are present in codon 61 of N-Ras with K-Ras and H-

pathway is constitutively activated in a significant number of melanoma tumors.

increased risk to developing melanoma [32,33].

48 Highlights in Skin Cancer

retinoblastoma (Rb1) are predisposed to melanoma [37,38].

**6. Acquired genetic alterations in melanoma**

**7. The Ras-Raf-Mek-Erk**

The PI3K/Akt pathway is activated in various cancers, mostly on accont of mutations in tumor suppressor PTEN (phosphatase and tensin homolog) [62]. In melanoma, los of PTEN on chromosome 10q 23-24 was first reported by Parmiter et al [63]. The PTEN gene encodes a phosphatase that degrades products of PI3K by dephosphorylating phosphatidylinositol 3,4,5 triphosphate and phosphatidylinositol 3,4-biphosphate at 3 positions [64]. Loss of PTEN increases AKT phosphorylation and activity leading to increased mitogenic signaling and decreased apoptosis [65]. Various studies suggest that 30-40% of melanoma cell lines and 5-15% of uncultured melanoma specimens carry inactivating mutations or homozygous deletions of Pten [63,66,67].

Pten encodes a negative regulator of extracellular growth signals that are transcended via PI3K-Akt pathway. Akt/protein kinase B (PKB), a serine-threonine kinase, is a core component of the PI3K signaling cascade and is activated through the phosphorylation of Ser 473/474 and Thr 308/309 [68,69]. Activated Akt regulates a network of factors that control cell proliferation and survival and this pathway is hyperactive in most metastatic melanomas [70,71,72]. Akt activates the transcription of a wide variety of genes involved in a wide range of cellular activities – those involved in immune activation, cell proliferation, apoptosis and cell survival [69]. Several studies have documented Akt activation in melanoma. Dai et al undertook a 292 sample study of pAkt levels using tissue microarray & immunohistochemistry strategies and identified strong pAkt expression in 17%, 43%, 49% and 77% of the biopsies in normal nevi, dysplastic nevi, primary melanoma and melanoma metastasis respectively. An important cell adhesion protein MelCAM that plays critical roles in melanoma development was increased upon active Akt expression [73,74]. PI3K and Akt is known to increase the expression of MMP2 and MMP9 by a mechanism involving Akt activation of NF-kappaB binding to the MMP promoter [75,76]. Akt overexpression led to upregulation of VEGF, increased production of superoxide ROS. Akt can suppress apoptosis by phosphorylating and inactivating many proapoptotic proteins like caspase 9 and Bad [77,78]. PI3K pathway emerges as the central axis that is deregulated in melanoma and along with constitutively active MAPK pathway makes an important role in melanoma development progression. Thus targeting PI3K is expected to be an important therapeutic target modality for melanoma treatment.

expression may contribute to the progression of melanoma [86]. ICAT mRNA expression analysis in two-third melanoma specimens revealed a 20% or less decrease in ICAT transcrip‐ tion [86]. However the mechanism behind the reduced ICAT mRNA level in melanoma is

An Overview of Important Genetic Aspects in Melanoma

http://dx.doi.org/10.5772/55257

51

Identification of Wnt target genes is also important towards the study of melanoma progres‐ sion. Brn1, the POU domain transcription factor is directly controlled by Wnt signaling in transgenic mouse models and melanoma cell lines [90]. Studies indicate that overexpression of Brn2 is associated with increased melanoma progression and tumorigenicity [90,91].MITF (microphthalmia-associated transcription factor), a Wnt target gene, is essential for the development of the melanocyte lineage and has an important role in the control of cell proliferation, survival and differentiation [54,92,93]. The regulation of MITF expression by βcatenin significantly influences the growth and survival behavior of treatment resistant melanoma [94]. A study by Schepsky A et al demonstrated that MITF can directly interact with β-catenin and redirect transcriptional activity away from canonical Wnt signaling-regulated target gene specific for MITF [95]. Induction of Wnt signaling can be blocked by 5 different proteins – Dkk, Wise, sFrp (secreted Frizzled related protein), Wif (wnt inhibitory factor), and Cerebrus that compete for the Wnt ligand or the Lrz-Frp receptor [96]. Interestingly, Dkk1 (Dickkopf 1) expression is negligible in melanomas [97]. Studies by Kuphal et al have dem‐ onstrated a downregulated or loss of Dkk-1, -2 and-3 in all melanoma cell lines and most of the melanoma tumor samples that were analyzed [98]. In xenograft mouse model, overex‐

Activation of Jnks is usually in response to diverse stresses. These kinases play an important role in the regulation of cell proliferation, cell survival, cell death, DNA repair and metabolism. A variety of extracellular stimuli by cytokines, growth factors, hormones, UV radiation and tumor promoters are known to activate Jnks [101]. Sequential protein phorphorylation through a MAP-kinase module (MAP3K-MAP2K-MAPK) is responsible for Jnk activation [102]. Depending upon the cellular context, Jnk has been shown to elicit both positive and negative effects on tumor development [103]. Activation of Jnk is required for Ras-mediated transfor‐ mation and mediate proliferation and tumor growth [104,105]. These observations are consistent with the findings of constitutively active Jnk in tumor samples and cell lines [103,106]. Jnk mediated the phosphorylation at serine 63 & 73 residues enhancing the ability of transcription factor c-Jun, a component of the AP-1 transcription complex [107]. The activation of Jnk leas to the induction of AP-1 dependent target genes that play important roles in cell proliferation, cell death and inflammation. Other members of the AP-1 transcription complex include c-Jun, Jun B, Jun D, c-Fos, Fra1 and Fra 2. The role of Jnk in oncogenesis is emerging; however c-Jun is a well defined oncogene in cancer. c-Jun is amplified and overex‐ pressed in undifferentiated and aggressive sarcomas [108], breast and lung cancer [109,110]. Since the 1990s, the role of Jnk pathway in melanomas was recognized [111,112]. c-Jun, Jun B, c-fos genes play a role in the transformation of melanocytes into malignant melanomas [111].

pression of Dkk-1 and Wif-1inhibited melanoma tissue growth [99,100].

**10. The JNK/c-Jun pathway**

unclear.

### **9. Wnt/β-catenin pathway**

Βeta-catenin (β-catenin) is a key component of the Wnt signaling pathway. Signaling through this pathway controls a wide range of cellular functions and aberrant Wnt/β-catenin signaling can lead to cancer development and progression [79]. Wnts are glycoproteins that act as ligands to stimulate receptor-mediated signal transduction pathways involved in cell survival, proliferation, behavior and fate. Wnt proteins are known to activate 3 different extracellular pathways – Wnt/β-catenin, Wnt/planar-polarity and Wnt/Ca2+ pathways [80]. The Wnt/βcatenin also known as the canonical Wnt pathway plays an important role in melanoma development. In the absence of Wnt ligands, free β-catenin binds to the destructive complex of Axin, adenomatous polyposis coli (APC) and glycogen synthase kinase-3β (GSK-3β). GSK-3β mediates the phosphorylation of β-catenin at specific regulatory sites on the Nterminal side marking β-catenin for ubiquitination and subsequent proteosomal degradation. Upon the binding of Wnt ligand, GSK--catenin for ubiquitination and subsequent proteosomal degradation. Upon the binding of Wnt ligand, GSK-3β activity is inhibited resulting in accumulation of β-catenin in the cytoplasm and shuttles into the nucleus where it serves as an essential co-activator of the Tcf/Lef (T-cell factor / lymphoid enhancer factor) family [81]. Numerous genes implicated in the tumorigenic process like c-myc and cyclinD1 have been identified as targets of the canonical Wnt signaling.

Increased nuclear localization of β-catenin – an important indication of activated Wnt signaling pathway is observed in over a third of melanoma specimens [82,83,84]. Mutations in β-catenin have been observed in about 23% of melanoma cancer cell lines and these mutations affect phosphorylation sites at Ser33, Ser37, Thr41 and Ser45 [85] at the N-terminal domain. These mutations render β-catenin resistant to phosphorylation and subsequent degradation. Low rates of β-catenin mutation have been observed in primary melanomas and metastasis indicating that activating mutations is a rare event in melanoma tumorigenesis [82,83,84,86,87,88]. Mutations in APC were observed sporadically in primary melanomas [82, 85,88]. While APC promoter 1A hypermethylation was observed in 17% of melanoma biopsies and 13% of melanoma cell lines. Wnt signaling pathways is activated in tumors through aberration in other genes. ICAT (inhibition of β-catenin & T-cell factor), a gene that negatively regulates the association of β-catenin with TCF4 thus repressing the transactivation of βcatenin-Tcf4 target genes [89]. A study by Reifenberger J et al suggests that loss of ICAT expression may contribute to the progression of melanoma [86]. ICAT mRNA expression analysis in two-third melanoma specimens revealed a 20% or less decrease in ICAT transcrip‐ tion [86]. However the mechanism behind the reduced ICAT mRNA level in melanoma is unclear.

Identification of Wnt target genes is also important towards the study of melanoma progres‐ sion. Brn1, the POU domain transcription factor is directly controlled by Wnt signaling in transgenic mouse models and melanoma cell lines [90]. Studies indicate that overexpression of Brn2 is associated with increased melanoma progression and tumorigenicity [90,91].MITF (microphthalmia-associated transcription factor), a Wnt target gene, is essential for the development of the melanocyte lineage and has an important role in the control of cell proliferation, survival and differentiation [54,92,93]. The regulation of MITF expression by βcatenin significantly influences the growth and survival behavior of treatment resistant melanoma [94]. A study by Schepsky A et al demonstrated that MITF can directly interact with β-catenin and redirect transcriptional activity away from canonical Wnt signaling-regulated target gene specific for MITF [95]. Induction of Wnt signaling can be blocked by 5 different proteins – Dkk, Wise, sFrp (secreted Frizzled related protein), Wif (wnt inhibitory factor), and Cerebrus that compete for the Wnt ligand or the Lrz-Frp receptor [96]. Interestingly, Dkk1 (Dickkopf 1) expression is negligible in melanomas [97]. Studies by Kuphal et al have dem‐ onstrated a downregulated or loss of Dkk-1, -2 and-3 in all melanoma cell lines and most of the melanoma tumor samples that were analyzed [98]. In xenograft mouse model, overex‐ pression of Dkk-1 and Wif-1inhibited melanoma tissue growth [99,100].
