Melanoma Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

Fig.1 Melanoma signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in green) and those in preclinical or early phase development (colored in red) for the treatment of melanoma.

An Introduction to Melanoma

Melanoma is the most serious type of skin cancer developing in the skin, eye, inner ear, and leptomeninges. Main signs and symptoms of melanomas include a change of an existing mole, a new pigmented or unusual-looking growth on the skin, such as an increase in size, irregular edges, change of mole color, itchiness, or skin breakdown. The main cause of most of the cancer cases is the exposure to ultraviolet (UV) radiation from the sun and from tanning lamps and beds. Other melanoma cases occur in the body that don't receive exposure to UV light mainly attribute to some risk factors including fair skin, a history of sunburn, excessive ultraviolet (UV) light exposure, living closer to the equator or at a higher elevation, having many moles or unusual moles, a family history of melanoma and weakened immune system. Currently, the treatment options for the melanoma include surgery to remove affected lymph nodes, chemotherapy, radiation therapy, biological therapy and targeted therapy.

1 Main Signaling Pathways in Melanoma Therapy

1.1 RAS-RAF-MEK-ERK signaling cascade

The breakthrough discovery of B-Raf mutation in most melanoma in 2002 has triggered numerous new studies focusing on mitogen-activated protein kinase (MAPK) signaling in melanoma. These studies revealed that constitutive activation of the extracellular signal-regulated protein kinase (Ras-Raf-MEK-ERK) signaling cascade is a hallmark of cutaneous malignant melanoma. In this pathway, Ras is mutated in approximately 15-20% of human melanomas. The Ras proteins plays a critical role in regulating cell proliferation, survival and differentiation by activating a variety of effector proteins, including the Ral guanine nucleotide dissociation stimulator (GDS) exchange factors, the phosphatidylinositol-3 kinase (PI3Ks), and the three Raf protein kinases (A-Raf, B-Raf and C-Raf). Constitutive activation of the signaling cascade has been shown to contribute to melanoma tumorigenesis by increasing cell proliferation, tumor invasion and metastasis, and by inhibiting apoptosis. The importance of constitutive activation of this pathway for the maintenance of melanoma phenotypes has been confirmed by specific targeting of the BRaf and MEK pathways using kinase inhibitors in in vitro and xenotransplantation models.

1.2 PI3K/AKT signaling cascade

The PI3K/Akt pathway has been found to be activated in various cancers including melanoma, mostly due to mutations in the tumor suppressor gene PTEN. The PTEN gene encodes a phosphatase that functions to degrade the products of PI3K by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate at the 3 position. Once PTEN loses function in tumor cells, which can result in the accumulation of these critical second messenger lipids. This process then increases Akt phosphorylation and activity, leading to decreased apoptosis and/or increased mitogenic signaling. A PTEN mutation was found in 30-50% melanoma cell lines, and 57-70% showed homozygous deletion of the PTEN gene. Importantly, PTEN protein levels were found to be altered in metastatic melanoma in the absence of genetic alterations. In addition to PTEN mutation, other mechanisms, such as epigenetic silencing, altered subcellular localization or ubiquitination also can cause the PTEN inactivation. Ectopic expression of PTEN was demonstrated to suppress melanoma cell growth and melanoma tumorigenicity and metastasis. All study results revealed that PTEN and PI3K play an important role in melanoma tumorigenesis.

1.3 WNT signaling cascade

Wnts are secreted glycoproteins acting as ligands to stimulate receptor-mediated signal transduction pathways involved in cell proliferation, survival, behavior and fate. Wnt proteins activate at least three different intracellular signaling pathways including the Wnt/β-catenin, the Wnt/ Ca2⁺ and the Wnt/planar polarity pathways. The first pathway involves in stabilization of β-catenin. The other two involve activation of protein kinase C (PKC) and cJun N-terminal kinases (JNKs), respectively. In the canonical Wnt pathway, in the absence of a Wnt signal, cytoplasmic β-catenin is phosphorylated through the action of casein kinase Iα and GSK3β and degraded in a complex that also includes adenomas polyposis coli (APC) and axin. Upon Wnt binds to its receptor Frizzled and to a low-density lipoprotein receptor-related protein-5 or -6 (LRP5 or 6) coreceptors, the pathway can be activated. Subsequently, this ternary complex causes activation of the cytoplasmic phosphoprotein Dishevelled, which inhibits the degradation of β-catenin. Finally, β-catenin interacts with specific transcription factors T cell factor/lymphoid-enhancing factor (TCF/LEF) by the nuclear translocation to regulate target genes. Mutations of genes especially CTNNB1 and APC encoding members of the Wnt-signaling cascade are frequent in various types of human cancer such as colorectal carcinoma, hepatocellular carcinoma, hepatoblastoma and melanoma.

1.4 JNK/c-JUN signaling cascade

JNKs kinases have been studied for their importance in the regulation of mammalian physiology, including cell proliferation, cell survival, cell death, DNA repair and metabolism. JNK can be activated by a large number of extracellular stimuli, growth factors, cytokines, tumor promoters, UV radiation and hormones, which is activated by sequential protein phosphorylation through a MAP kinase module (MAP3KMAP2K-MAPK). Two MAP2Ks (JNKK1/MKK4/SEK1 and JNKK2/MKK7) have been demonstrated for JNK. Phosphorylation of JNK by these dual-specificity protein kinases is essential for its activation. Some MAP3Ks, including members of the map-erk kinase kinase (MEKK) family, activator of S-phase kinase 1 (ASK1), mixed lineage kinase (MLK), TGF-beta-activated kinase 1 (TAK1) and Tumor progression loci-2 (TPL 2), have been found to serve as MAP3Ks for JNK. JNK can elicit both positive and negative effects on tumor development based on the cellular and genetic context. JNK activation is required for Ras-mediated transformation to regulate proliferation and tumor growth.

1.5 NF-κB signaling cascade

The mammalian NF-κB family contains five members, i.e. p105/p50 (NF-κB1), p100/p52 (NF-κB2), RelA (p65), RelB and cRel. NF-κB1 and NF-κB2 are synthesized as the inactive cytosolic precursors p105 and p100, respectively. The canonical activation of NF-κB pathway relates to TNF-α stimuli to cause the activation of TNFR and association of TRAF2/MAP3K module with subsequent phosphorylation/activation of IKK. In turn, IKK-mediated phosphorylation of IκB can result in IκB ubiquitination and proteasomal degradation, releasing an active NF-κB complex. The composition of activated NF-κB complexes will determine the type of genes that will be trans-activated. Numerous mechanisms were proposed to contribute to the elevated level of NF-κB activity in malignant melanoma. For example, sustained NF-κB activation leads to induction of chemokines CXCL1 and CXCL8. CXCL1, in turn can activate IKK and NF-κB demonstrating the presence of a feed-forward mechanism that could contribute to the constitutive activation of NF-κB in melanoma cells. The CXC chemokine MGSA/GROα is constitutively expressed in melanoma that can induce NF-κB (RelA) activation in a manner dependent on Ras-MEKK1-MEK3/6-p38 pathway. By contrast, UV-induced activation of ASK1-p38 disrupts IκBα phosphorylation and decreases transcriptional activity of NF-κB, suggesting that multiple factors are cooperating, in concert, in the activation of NF-κB in melanoma.

1.6 JAK/STAT signaling cascade

STAT proteins consist of a family of transcription factors involved in the activation of target genes in response to cytokines and growth factors. Tyrosine-phosphorylated STATs undergo homodimerization or heterodimerization, followed by translocation to the nucleus for gene transcription. Four mammalian JAKs and seven STAT members render distinct patterns of gene transcription upon specific stimulation. Various mechanisms regulating the level and duration of STAT activation contributes to the complexity of the pathway, including dephosphorylation of the receptor complex or nuclear STAT dimers by PTPases, interaction of activated STATs with inhibitory molecules from the protein inhibitor of activated STAT family, and feedback inhibition of the pathway by suppressor of cytokine signaling (SOCS) proteins through inhibition and/or degradation of JAKs. In addition, different kinases can mediate STAT activity. There are two mechanisms to regulate STAT’s effect on carcinogenesis such as melanoma, i.e. immune surveillance and control of growth factor signaling, apoptosis and angiogenesis. Among the different STATs, STAT3 play a vital role in melanoma development. For example, expression of a STAT3 dominant-negative variant, STAT3β can induce cell death in B16 melanoma cells and cause inhibition of tumor growth and tumor regression by increased apoptosis. STAT3 can control IL-6-induced growth inhibition of normal melanocytes and early-stage melanoma cells, and facilitate growth of advanced melanomas.

1.7 TGF-β signaling cascade

The TGF-β family consists of multiple factors possessing dual tumor suppressor and oncogenic ability. TGF-β binding to membrane receptors can cause the assembly of a receptor complex where phosphorylates proteins of the SMAD family bind to DNA and regulate transcription of several genes to induce diverse effects. The overexpression of TGF-β1 in human melanoma cells can stimulate the neighboring stroma cells through increased production and deposition of extracellular matrix proteins. The activation of stroma causes a tumor cell survival advantage and increased metastasis.

1.8 Notch signaling cascade

Notch is an evolutionarily conserved signaling mechanism involved in many cellular processes including cell differentiation, proliferation, apoptosis, adhesion, EMT, migration and angiogenesis. Notch and its ligands Delta and Jagged, are transmembrane proteins expressed on an adjacent cell and can activate Notch signaling through a direct cell-cell interaction. Upon ligand binding, Notch intracellular domain is cleaved and translocated to the nucleus leading to the activation of target gene transcription. Notch signaling functions in several cancers including the melanoma. Expression of Notch-1/2 and Notch ligands are upregulated in “dysplastic nevi” and melanomas compared with common melanocytic nevi. Notch signaling is essential for melanoma survival.

Testis Cancer Diagnosis

2.1 Molecular Markers for Melanoma

For melanoma, detecting molecular markers or genetic alterations has emerged as an innovative form of testing that guides therapeutic decisions and aids the diagnosis of histologically challenging cases. Analysis methods including comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), and quantitative gene expression profiling contribute to the detection of genetic mutations and determination of expression levels. A large number of prognostic or diagnostic markers have been found and detected in melanoma. GNAQ and GNA11 mutations result in overamplification of signaling through the MAPK and PI3K pathways via inhibiting GTPase activity. With GNAQ and GNA11 mutations, GTP is persistently bound to the G protein and leads to constitutive downstream signaling. These mutations can be detected in most of uveal melanoma. CDKN2A gene mutation is the most common alteration in hereditary melanoma, which can result in hyperphosphorylation of retinoblastoma protein (RB1), releasing the E2F1 transcription factor to promote cell cycle progression from G1 to S, loss of p14ARF function to promote the ubiquitination of p53, and reducing cell cycle arrest and apoptosis. BAP1 is a tumor suppressor gene and its mutations are associated with melanoma development. SF3B1 encodes a subunit of splicing factor 3b, and its mutations are a marker of good prognosis for uveal melanoma. EIF1AX is a gene involved in regulating protein translation. EIF1AX mutations are found in uveal melanoma to be used as diagnosis marker. Other molecular markers include vitamin D receptor (VDR), melanocortin 1 receptor (MC1R), Microphthalmia transcription factor (MITF) and hyaluronan and proteoglycan link protein 1 (HAPLN1).

2.2 Protein Markers for Melanoma

Melanin is produced in melanocytes through a series of enzymatic reactions resulting in production of variety of intermediates of melanogenesis. Tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TRP1, TRP2) participate in the melanin synthesis. TYR has been used in the diagnosis of melanoma for its high sensitivity and specificity. B-raf is one of the signaling kinases in the MAPK pathway, and its mutations are the most common genetic alteration in cutaneous melanoma. The N-ras GTPase is essential in the transduction of extracellular growth signals. When the oncogene is mutated, GTPase activity is reduced, leading to a constitutively active GTP-bound G protein to propagate downstream signals. In contrast to BRAF mutations, NRAS-mutant melanomas are associated with the nodular subtype and found in cumulative solar damage (CSD) skin. The contrast between this observation and the association of NRAS mutation with skin with CSD indicates that detecting the UV signature mutations can help diagnose melanoma. C-KIT is a receptor tyrosine kinase (RTK) involved in binding to growth factors as the first signal down the MAPK and PI3K pathways. The majority of c-KIT mutations are identified in mucosal and acral melanomas, and these mutations relate to worse survival as compared with wild-type melanomas. Immune checkpoint proteins cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) have been used as targets in the targeted melanoma therapy.

3 Targeted Therapy for Melanoma

Numerous molecular mechanisms involved in the pathogenesis of melanoma render effective ways for targeted therapy. Major components of cell signaling pathways, such as the RAS-RAF-MEK-ERK, PI3K/AKT, WNT, JNK/c-JUN, NF-κB, JAK/STAT, TGF-β and Notch signaling pathways, are altered in melanoma cells by oncogenes through overexpression or mutation, leading to dysregulated cell signaling and cell proliferation. Here, we summarize the potential targets and new drugs developed that have been used in recent, ongoing and future clinical trials to try to improve the clinical outcomes of this disease (Table1-12).

3.1 Melanoma therapy for RAS-RAF-MEK-ERK pathway

Inhibitors targeting BRAF have now been developed and are highly clinically effective. Several selective BRAF inhibitors have been tested in phase III studies against chemotherapy (DTIC), and showing an improvement in overall survival (OS). Vemurafenib (PLX4032) is an oral, highly selective and competitive inhibitor of mutant BRAF, which is investigated in melanoma (BRIM-3) phase III study as first-line treatment in patients with BRAF V600E-mutant metastatic melanoma. Dabrafenib (GSK2118436) is a reversible selective inhibitor against mutant BRAF. Phase III study (BREAK-3) results in unresectable stage III or IV BRAF V600E-mutated melanoma showed that Dabrafenib has a significant improvement in progression free survival (PFS) and response rate (RR) over DTIC with an acceptable safety Profile. MEK162 is the first inhibitor targeting NRAS-mutant and demonstrate clinical activity in patients with melanoma. Sorafenib (BAY43-9006) is a potent multi-kinase inhibitor that targets also the receptor tyrosine kinase-associated angiogenesis (VEGFR-2, VEGFR-3, PDGF-β) and tumor progression, which is effective in the treatment of a small percentage of melanomas. MEK1/2 inhibitors include Trametinib (GSK1120212) and Selumetinib (AZD6244).

Table 1 Clinical trials of BRAF inhibitor Vemurafenib

According to statistics, a total of 20 Vemurafenib projects targeting melanoma BRAF are currently in clinical stage, of which 11 are recruiting and 9 are not recruiting.

Table 2 Clinical trials of BRAF inhibitor Dabrafenib

According to statistics, a total of 43 Dabrafenib projects targeting melanoma BRAF are currently in clinical stage, of which 29 are recruiting and 14 are not recruiting.

Table 3 Clinical trials of NRAS inhibitor MEK162

According to statistics, a total of 19 MEK162 projects targeting melanoma NRAS are currently in clinical stage, of which 11 are recruiting and 8 are not recruiting.

Table 4 Clinical trials of BRAF inhibitor Sorafenib

According to statistics, a total of 1 Sorafenib project targeting melanoma BRAF is currently in clinical stage and is not recruiting.

Table 5 Clinical trials of MEK1/2 inhibitor Trametinib

According to statistics, a total of 46 Trametinib projects targeting melanoma MEK1/2 are currently in clinical stage, of which 30 are recruiting and 16 are not recruiting.

Table 6 Clinical trials of MEK1/2 inhibitor Selumetinib

According to statistics, a total of 3 Selumetinib projects targeting melanoma MEK1/2 are currently in clinical stage, of which 1 is recruiting and 2 are not recruiting.

3.2 Melanoma therapy for PI3K/AKT pathway

A variety of oral tyrosine kinase inhibitors (TKIs) are currently under evaluation in advanced melanoma, such as imatinib, dasatinib, nilotinib and sunitinib. Imatinib has beeen evaluated in three phase II trials in patients with c-KIT-mutated metastatic melanoma. A phase II study has been evaluated the predictive role of KIT activation for response to treatment with sunitinib. A phase II study of dasatinib in a molecularly unselected population of patients with advanced melanoma demonstrated two partial responses lasting 24 weeks or most of patients evaluable for response. Nilotinib is a second-generation TKI with a similar potency as imatinib against c-KIT. It is currently being evaluated in multiple phase II studies. Other PI3K-mTOR inhibitors include BEZ235, GSK2126458, BYL719, CCI-779 (Temsirolimus) and RAD001 (Everolimus), etc.

Table 7 Clinical trials of tyrosine kinase inhibitor imatinib

According to statistics, a total of 1 imatinib project targeting melanoma tyrosine kinase is currently in clinical stage and is recruiting.

Table 8 Clinical trials of tyrosine kinase inhibitor dasatinib

According to statistics, a total of 2 dasatinib projects targeting melanoma tyrosine kinase are currently in clinical stage, of which 1 is recruiting and 1 is not recruiting.

Table 9 Clinical trials of tyrosine kinase inhibitor sunitinib

According to statistics, a total of 2 sunitinib projects targeting melanoma tyrosine kinase are currently in clinical stage, and not recruiting.

Table 10 Clinical trials of PI3K-mTOR inhibitor CCI-779

According to statistics, a total of 4 CCI-779 projects targeting melanoma PI3K-mTOR are currently in clinical stage, of which 2 is recruiting and 2 are not recruiting.

3.3 Immunotherapy for melanoma

CTLA4 is a member of the CD28:B7 immunoglobulin superfamily. It is normally expressed at low levels on the surface of effector and regulatory T cells. T cells autoregulate their activation through expression of CTLA4, which functions as a negative costimulatory molecule for the T cell. Two anti-CTLA4 antibodies ipilimumab and tremelimumab have been tested in phase III clinical trials. Ipilimumab is a fully human immunoglobulin G1 monoclonal antibody that targets CTLA4 and thereby leads to T-cell hyper responsiveness with disinhibition of antitumour immunity. Ipilimumab was the first drug in the management of metastatic melanoma to demonstrate a survival benefit. Tremelimumab is a fully human IgG2 monoclonal antibody that also targets CTLA4. The half-life of tremelimumab is longer than ipilimumab, so its dosing schedule is less frequent. It is assessed in phase II study for the melanoma treatment.

Table 11 Clinical trials of CTLA4 inhibitor Ipilimumab

According to statistics, a total of 137 Ipilimumab projects targeting melanoma CTLA4 are currently in clinical stage, of which 10 are recruiting and 7 are not recruiting.

Table 12 Clinical trials of CTLA4 inhibitor tremelimumab

According to statistics, a total of 8 tremelimumab projects targeting melanoma CTLA4 are currently in clinical stage, of which 4 are recruiting and 4 are not recruiting.