B-RAF

Human B-RAF, also known as V-raf murine sarcoma viral oncogene homologue B1, belongs to a family of three human RAF genes: A-RAF1, B-RAF and RAF-1 (also known as c-RAF) (Ref. Reference de Snoo and Hayward28). RAF proteins are serine/threonine kinases that link extracellular mitogenic stimuli to transcription of cell cycle progression or progrowth genes (Ref. Reference de Snoo and Hayward28).

Since its discovery in 2002, as a gene mutated in melanoma (Ref. Reference Davies34), B-RAF has proved to have a great impact on the quest for novel melanoma therapies (Ref. Reference Wellbrock and Hurlstone35). Somatic mutations in B-RAF are the most common genetic alterations in melanoma, occurring in up to 66% of malignant melanomas (Refs Reference Davies34, Reference Pollock and Meltzer36, Reference Brose37). B-RAF mutations are thought to be induced by UV radiation or reactive oxygen species that are generated as a byproduct of melanogenesis or phaeomelanin absorption of UVA radiation (Ref. Reference Thomas26). However, UV radiation alone might not be sufficient for B-RAF mutagenesis, because the presence of the B-RAF^V600E mutation has been reported in a subset of acquired nevi from glabrous skin and genitalia (81.4%), and in congenital nevi (69%), implying that other factors are required as well (Ref. Reference Ichii-Nakato38).

There are at least 30 documented melanoma mutations of B-RAF that activate the MAPK pathway. The most common is V600E (previously mistakenly reported as position 599), which accounts for 89% of all B-RAF mutations (Refs Reference Wellbrock and Hurlstone35, Reference Davies and Samuels39). A significant novel observation is that melanomas with CDK4 amplification do not show concomitant N-RAS or B-RAF mutations (Ref. Reference Curtin40), indicating overlapping oncogenic functions in the MAPK and CDK4 pathways. B-RAF mutations are more frequent in skin without chronic sun-induced damage and without N-RAS mutations (Ref. Reference Curtin40).

A summary of the frequency of B-RAF mutations by melanoma subtypes is shown in Figure 2. B-RAF mutations are present in up to 82% of benign nevi (Ref. Reference Pollock41). Surprisingly, it has been reported that mutated B-RAF induces cell cycle arrest and senescence in nevi. The mechanism by which B-RAF induces senescence in melanocytes remains controversial and is not entirely understood (Refs Reference Michaloglou42, Reference Cotter43, Reference Houben44, Reference Wajapeyee45, Reference Kuilman46, Reference Scurr47). In nevi, it has been reported that B-RAF induces cell senescence by means other than telomerase attrition. It appears that B-RAF might directly induce p16INK4a and acidic β-galactosidase [SA-β-Gal (Ref. Reference Michaloglou42)]. A different group has shown that ectopic expression of B-RAF^V600E induces melanocyte senescence by increasing the levels of MAPK activity and by upregulating the expression of cyclin-dependent kinase (CDK) inhibitors (Refs Reference Houben44, Reference Wajapeyee45). RNA interference data have suggested pro-inflammatory cytokines, particularly IL-6 and IL-8, as key regulators of melanocyte senescence (Ref. Reference Kuilman46). However, the role of insulin growth factor-like binding protein 7 remains controversial (Refs Reference Cotter43, Reference Scurr47). Hence, B-RAF mutations should be followed or accompanied by additional molecular events, such as loss of tumour suppressor genes or upregulation of oncogenes, for melanoma transformation (Refs Reference Dankort48, Reference Zhao49).

In general, patient treatment with selective B-RAF inhibitors as single-agent targeted therapy is usually followed by relapse as a result of acquired drug resistance. Intense research has recently focused on understanding the mechanism by which melanoma develops resistance to BRAF inhibition. Several other genes appear to be involved in the acquired resistance to B-RAF inhibitors. Among other genes, COT (Ref. Reference Johannessen50), CRAF (Ref. Reference Montagut51), RTK, NRAS (Ref. Reference Nazarian52), PERK (Ref. Reference Paraiso53), PTEN (Ref. Reference Paraiso54) and CCND1 (Ref. Reference Smalley55) become either upregulated or downregulated, whereas B-RAF inhibitors become ineffective. Some authors have suggested that drug resistance to RAF inhibitors could be overruled by the use of combined RAF and MEK inhibitors (Ref. Reference Johannessen50), or cotargeting MEK and PI3K pathways (Ref. Reference Villanueva56). An excellent review on the subject has been published (Ref. Reference Solit and Rosen57).

Sorafenib, originally developed as an inhibitor of RAF-1, is 10 times less potent as an inhibitor of B-RAF than as an inhibitor of RAF-1 (Ref. Reference Flaherty and McArthur58). Recent studies demonstrated that sorafenib is indeed a multikinase inhibitor that prevents tumour cell proliferation and angiogenesis by inhibiting the RAF–MEK–ERK pathway. Sorafenib also affects vascular endothelial growth factor (VEGF), KIT and other growth factor receptor tyrosine kinases in vitro and in vivo (Refs Reference Flaherty59, Reference Treisman and Garlie60). Sorafenib has already been approved by the FDA for the treatment of renal cell and hepatocellular carcinoma (Ref. Reference Treisman and Garlie60). Preclinical studies demonstrated that sorafenib inhibits the growth and survival of B-RAF mutant human melanoma cell lines, and slows the growth of melanoma xenografts in animal models (Ref. Reference Karasarides61). However, a Phase II study of sorafenib as a single agent showed little or no efficacy in the majority of advanced melanoma patients, even though 19% of 37 patients experienced stable disease (Ref. Reference Eisen62).

In vitro and in vivo studies show that sorafenib enhances the efficacy of regional chemotherapy (Refs Reference Murray63, Reference Augustine64). It was reported that sorafenib in combination with melphalan or temozolomide is more effective in slowing down tumour growth in an animal model of melanoma than either treatment alone (Ref. Reference Augustine64). Also, sorafenib plus etoposide, paclitaxel or temozolomide significantly inhibits the growth of melanoma cell lines in vitro (Ref. Reference Murray63). Similarly, a Phase I dose-escalation trial (Ref. Reference Flaherty59) reported that sorafenib in combination with carboplatin and paclitaxel (CP) had a better clinical outcome than CP alone. The combination of sorafenib and CP resulted in one complete response and nine partial responses among 24 patients with advanced melanoma. However, a subsequent randomised Phase III trial showed no difference in the clinical outcome for patients treated with CP and patients treated with sorafenib plus CP (Ref. Reference Hauschild65).

PLX4032, also known as RG7204 or RO5185426, is a newly developed and highly selective B-RAF inhibitor (Refs Reference Garber66, Reference Livingstone67, Reference Bollag68, Reference Flaherty, Puzanov and Sosman69). PLX4032 has higher affinity for mutated B-RAF^V600E (IC₅₀ = 31 nM) than for wild-type B-RAF (IC₅₀ = 100 nM) (Ref. Reference Livingstone67). Preliminary reports characterised the antiproliferative effects of PLX4032 on melanoma cell lines with B-RAF mutations (Ref. Reference Yang70). PLX4032 exhibited encouraging results in a xenograft model of melanoma harbouring B-RAF^V600E. In this model, PLX4032 induced partial or complete tumour regression and increased survival in a dose-dependent manner (Ref. Reference Yang70).

PLX4032 was the first selective B-RAF inhibitor clinically tested (Ref. Reference Flaherty71). Preliminary results of a Phase I trial showed a 79% overall response rate in patients with melanoma and other cancers (Ref. Reference Flaherty, Puzanov and Sosman69). Of 16 patients with melanoma expressing B-RAF^V600E, 14 showed significant tumour regression (Ref. Reference Flaherty, Puzanov and Sosman69). The six-month survival of these patients also improved; however, progression-free survival is still being monitored for these patients.

Another Phase I clinical trial has shown that PLX4032, administered at 960 mg PO twice daily, has a remarkably high response rate (81%) in metastatic melanoma patients harbouring B-RAF^V600E mutations (Ref. Reference Flaherty71). The trial also reported that 24 of 32 (75%) patients had partial response and 6.25% (2 of 32) had complete response. The median progression-free survival for all patients was estimated to be more than 7 months (Ref. Reference Flaherty71). Similar results were found in a Phase II trial that reported an 82% overall response rate to PLX4032. Thirty per cent of the patients experienced stable disease, and the median progression-free survival rate was reported to be 6.2 months (Ref. Reference Vultur, Villanueva and Herlyn72). This astounding response rate is unfortunately followed by relapse in most patients, caused by acquired drug resistance.

In vitro studies show that melanoma cells might acquire resistance to PLX4032 and other BRAF inhibitors, possibly by activating either the PI3K–Akt or RAF–MEK–ERK pathways (Refs Reference Villanueva56, Reference Livingstone67). One of the mechanisms of resistance to B-RAF^V600E inhibitors is the generation of BRAF–C-RAF complexes, C-RAF–C-RAF homodimers or A-RAF kinase switching that will activate the MAPK pathway (Ref. Reference Villanueva56). Also, increased PI3K signalling promotes the survival of cells resistant to BRAF inhibitors (Ref. Reference Villanueva56). In addition, when PLX4032 is administered to N-RAS-mutated melanoma cell lines, resistance to the drug is accompanied by hyperactivation of MEK–ERK1/2 signalling (Ref. Reference Kaplan73). Parallel sequencing of 138 cancer genes from a melanoma tumour in a patient who, after an initial dramatic response, developed resistance to PLX4032 detected an activating mutation in MEK1 that was not present before treatment (Ref. Reference Wagle74). It was concluded that this particular MEK1 mutation conferred resistance to RAF and MEK inhibitors in vitro (Ref. Reference Wagle74). Finally, another proposed mechanism is N-RAS upregulation through RTK or NRAS upregulation (Ref. Reference Nazarian52); and by COT activation of ERK through MEK-dependent mechanisms that do not require RAF signalling (Ref. Reference Johannessen50).

The most commonly reported adverse effects to PLX4032 included rash, fatigue, photosensitivity, nausea, alopecia and arthralgia (Refs Reference Livingstone67, Reference Bollag68, Reference Flaherty, Puzanov and Sosman69). However, significant adverse effects were observed in up to 23% of patients. Well-differentiated cutaneous squamous cell carcinoma, kerato-acanthoma type, occurs in a significant number of patients (10 of 32 patients in an extension cohort) (Ref. Reference Flaherty71). It has been postulated that the development of squamous cell carcinoma is possibly due to PLX4032-mediated activation of ERK in keratinocytes (Ref. Reference Hatzivassiliou75).

The efficacy of PLX4032 as a melanoma therapeutic continues to be studied in ongoing clinical trials (Table 1). Emerging broad-spectrum kinase inhibitors with greater potency and improved selectivity for B-RAF are RAF-265 and XL-281, which are currently being tested in clinical trials (Table 1) (Ref. Reference Flaherty and McArthur58). They are potent RAF1 inhibitors, with relevance for targeting N-RAS mutant melanomas as well. The success rate of selective and potent inhibitors of B-RAF, such as PLX4032, indicates that targeting an early mutation is a feasible way of treating melanoma.

Table 1.

Selected drugs investigated in clinical trials for patients with melanoma caused by specific gene mutations (http://clinicaltrials.gov/) (Ref. 23)

MEK

MEK is a member of the MAP kinase pathway that is infrequently mutated in cancer cells. MEK is an important oncotherapeutic target because it is the only known direct substrate of B-RAF, and because inhibiting MEK might inhibit the growth of melanomas with mutated B-RAF and RAS (Ref. Reference Flaherty and McArthur58). PD0325901 (Ref. Reference Ciuffreda76) and AZD6244 (also known as selumetinib, ARRY-142886) (Refs Reference Banerji77, Reference Denton and Gustafson78) are selective second-generation MEK inhibitors. In vitro studies show that PD0325901 strongly inhibits the growth of melanoma cell lines independent of their B-RAF mutation status by promoting apoptosis and decreasing the production of proangiogenic factors (Ref. Reference Ciuffreda76). However, Phase I and Phase II studies were discontinued in 2007 because of ocular and neurological toxicity (NCT00147550) (Ref. 23). Experiments in nude mice inoculated with the human melanoma cell line A375 demonstrated that AZD6244 effectively inhibits cell proliferation by inhibiting ERK phosphorylation (Ref. Reference Denton and Gustafson78). AZD6244 inhibited the in vitro proliferation of melanoma cell lines with mutant B-RAF^V600E and N-RAS, but exhibited no significant effect on cells with wild-type B-RAF/N-RAS (Refs Reference Davies79, Reference Solit80). AZD6244 showed clinical efficacy in Phase I and Phase II clinical trials in patients with advanced melanoma and other advanced cancers (Refs Reference Banerji77, Reference Adjei81). Ongoing clinical trials involving AZD6244 are listed in Table 1.

KIT

KIT (also called c-KIT or CD117) is a receptor tyrosine kinase expressed on the surface of some cells. It is an upstream activator of the MAP kinase pathway and has a central role in melanocyte development and survival (Ref. Reference Berger and Garraway82). Mutation and/or amplification of KIT are present in ALM, LMM, MM and UM (Refs Reference Davies and Samuels39, Reference Berger and Garraway82, Reference Curtin83, Reference Beadling84, Reference Hofmann85, Reference Woodman and Davies86, Reference Garrido and Bastian87) (Fig. 2). KIT mutations in either the juxtamembrane domain or the kinase domain result in gain of function. KIT mutations are usually mutually exclusive with B-RAF and N-RAS mutations (Refs Reference Beadling84, Reference Garrido and Bastian87).

Cancers with KIT mutations have been successfully treated with KIT kinase inhibitors (Ref. Reference Garrido and Bastian87). As in gastrointestinal stromal tumours (GIST), most KIT mutations in melanoma occur in exon 11 (Refs Reference Berger and Garraway82, Reference Curtin83, Reference Flaherty, Hodi and Bastian88). Patients with GIST harbouring KIT mutations have been successfully treated with the KIT inhibitor imatinib mesylate or GLEEVEC. Partial responses have been reported in up to 84% of treated patients (Ref. Reference Berger and Garraway82).

Initial in vitro preclinical studies indicated that imatinib has potent antiproliferative and proapoptotic effects on melanoma cells by inhibiting MAPK and PI3K–AKT pathways (Ref. Reference Jiang89). Case report studies showed promising results in imatinib-treated melanoma patients with reported KIT mutations (Refs Reference Woodman and Davies86, Reference Jiang89, Reference Terheyden90, Reference Hodi91, Reference Lutzky, Bauer and Bastian92). Unfortunately, early Phase II clinical trial studies reported poor efficacy for treating metastatic melanoma (Refs Reference Wyman93, Reference Ugurel94). One possible explanation for these findings is that these studies were performed before the discovery of KIT mutations in melanoma. Early studies assessing the effect of imatinib did not set the presence of KIT mutations as a criterion for entry.

A newer Phase II clinical trial of imatinib indicated that one patient with metastatic ALM and the highest KIT expression of all enrolled patients showed significant improvement after 6 weeks of therapy and had partial response lasting 12.8 months (Ref. Reference Kim95). After 2 weeks of treatment, patients showed a marked increase in apoptosis of tumour cells (Ref. Reference Kim95). In general, imatinib was relatively well tolerated, with fatigue and oedema the most commonly reported adverse reactions. However, grade 3 or 4 toxicity occurred in >10% of patients (Ref. Reference Kim95). A clinical study of three patients exhibiting KIT mutations treated with imatinib and one with sorafenib showed that all four patients responded to treatment, but three developed brain metastasis. This is probably due to the inability of imatinib to cross the blood–brain barrier (Ref. Reference Handolias96). Other KIT inhibitors, such as dasatinib, appear to penetrate the blood–brain barrier better than imatinib (Ref. Reference Porkka97).

Multiple ongoing clinical trials target melanoma patients with KIT mutations (Table 1). Interim results from a Phase II study of imatinib in stage III or IV patients showed that two out of 12 patients had complete response, two patients had partial response, and six patients had stable disease with imatinib (Ref. Reference Carvajal98). The two patients with complete response had both amplification and mutation of KIT, whereas the two patients who showed no response had KIT mutations that confer resistance to imatinib in GIST (Ref. Reference Carvajal98). In conclusion, KIT inhibitor therapy is reasonably effective for selected patients with melanoma harbouring KIT mutations.

Bcl-2 family

The disruption of key apoptotic regulatory genes makes malignant melanoma resistant to chemotherapy (Ref. Reference Hocker, Singh and Tsao99). The Bcl-2 family of genes comprises both antiapoptotic (such as Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1) and proapoptotic proteins [multidomain proteins (Bak and Bax) and BH3-only proteins (Bid, Bim, Bad, NOXA and PUMA)] (Ref. Reference Hocker, Singh and Tsao99). The role of Bcl-2 and Bax in melanoma progression remains controversial (Ref. Reference Bush and Li100). Although some studies report that Bcl-2 expression is upregulated in advanced melanoma, other studies report the opposite effect (Ref. Reference Tron101). Similarly, Bax expression is reported to be both downregulated (Ref. Reference Bush and Li100) and upregulated during melanoma progression (Ref. Reference Tang102). By contrast, antiapoptotic family members Mcl-1 and Bcl-xl are consistently reported to be overexpressed in melanoma cells (Refs Reference Bush and Li100, Reference Tang102, Reference Zhuang103), most probably at an early stage in melanoma development (Ref. Reference Tang102). Keuling et al. demonstrated a progression-related upregulation of Mcl-1 and that inhibition of Mcl-1 using RNAi sensitised melanoma cells to the Bcl-2-selective inhibitor ABT-737 (Ref. Reference Keuling104).

The in vivo role of Bcl-2 in melanoma was shown to differ significantly from its role in vitro (Ref. Reference Benimetskaya105). In this study, downregulation of Bcl-2 protein and mRNA expression in 518A2 melanoma cells was achieved by using the novel Bcl-2 inhibitor G3139 (Ref. Reference Benimetskaya105). G3139 (oblimersen) is an antisense oligonucleotide targeted to the initiation region of the Bcl-2 mRNA (Ref. Reference Bedikian106). The results of the study indicated that in vitro downregulation of Bcl-2 expression does not affect tumour cell growth rate or sensitivity to standard cytotoxic chemotherapeutic agents (Ref. Reference Benimetskaya105). In addition, G3139-induced apoptosis was independent of Bcl-2. However, in vivo results on immunodeficient mice with xenografted melanoma cells having silenced Bcl-2 demonstrated that the tumour either failed to grow or grew to a low volume with subsequent regression (Ref. Reference Benimetskaya105). By contrast, mice with xenografted melanoma cells with normal levels of Bcl-2 exhibited large, necrotic tumours (Ref. Reference Benimetskaya105).

G3139 combined with DTIC has been tested on 771 patients with advanced melanoma enrolled in a Phase III clinical trial (Ref. Reference Bedikian106). The study reported that the drug combination produced better clinical outcomes than DTIC alone. The clinical trial reported a better median overall survival when the drugs were combined (9 months) compared with DTIC only (7.8 months). In the same manner, better outcomes were observed by the dual therapy on progression-free survival: median (2.6 versus 1.6 months), overall response (13.5% versus 7.5%), complete response (2.8% versus 0.8%) and durable response (7.3% versus 3.6%) (Ref. Reference Bedikian106).

HERV-K

Approximately 8% of the human genome is composed of sequences derived from HERVs (Ref. Reference Singh107). Most HERV sequences are defective as a result of mutations, deletions or termination signals in their coding regions (Ref. Reference Serafino108). However, HERV-K has been implicated in the pathogenesis of breast (Ref. Reference Golan109), ovarian (Ref. Reference Wang-Johanning110) and haematological cancer (Ref. Reference Depil111). HERV-K might also have a role in both the malignant transformation of melanocytes (Refs Reference Haqq7, Reference Singh107, Reference Serafino108, Reference Hahn112) and the ability of melanoma cells to evade immune surveillance (Ref. Reference Oricchio113). Metastatic melanoma cell lines express HERV-K-type antigens, whereas primary melanocytes do not (Ref. Reference Haqq7). Although HERVs are poorly expressed in normal somatic tissues, HERV-derived RNA transcripts can be induced in response to UV radiation (Refs Reference Hohenadl114, Reference Reiche, Pauli and Ellerbrok115), chemicals (Ref. Reference Khan, Muller and Sears116) and other endogenous factors (Ref. Reference Ono, Kawakami and Ushikubo117). For example, it was shown that UV induces activation of HERV-K in normal human epidermal melanocytes 24 h after exposure to 10 mJ/cm² UVC (Ref. Reference Reiche, Pauli and Ellerbrok115).

Recently, detectable levels of anti-HERV-K antibodies were found in 51 of 312 (16%) melanoma patients. By contrast, no retroviral antigens were found in any healthy controls (Ref. Reference Hahn112). Higher levels of anti-HERV-K antibodies were found in patients with melanoma at sun-protected sites (ALM, MM or UM) compared with patients affected by LMM, NM or SSM (Ref. Reference Hahn112). The data suggested that the presence of anti-HERV-K antibodies in patient serum at early stages of disease correlates with lower overall survival (Ref. Reference Hahn112).

In vitro studies show that cellular stress promotes the transformation of human melanoma cells from adherent to nonadherent, less to more proliferative, and less to more aggressive and malignant (Ref. Reference Serafino108). This transformation occurs simultaneously with the activation of HERV-K and the production of a large amount of viral-like particles (Ref. Reference Serafino108). The transition from adherent to nonadherent might be prevented by the downregulation of HERV-K expression by RNA interference (Ref. Reference Serafino108).

The above findings indicate a possible role of HERV-K in melanoma development and progression. Furthermore, understanding the molecular events leading to increased HERV expression and determining why ALM, MM and UM paradoxically express higher levels of HERVs might represent a fertile area of research for new melanoma therapeutics.

MicroRNA-based melanoma therapeutics

MicroRNAs (miRNAs) are small noncoding RNA molecules 21–23 nucleotides long that modulate gene expression at the post-transcriptional or translational level (Ref. Reference Wang and Lee118). miRNAs have been found in all multicellular organisms. Although they represent only 1% of the mammalian genome, there are approximately 650 human miRNAs, each of which can regulate the expression of hundreds of genes and can directly or indirectly modulate the expression of thousands of proteins (www.microRNA.org) (Refs 119, Reference Selbach120). Approximately 30% of protein-coding genes in the human genome are thought to be regulated by miRNAs (Ref. Reference Chen and Rajewsky121).

About half of all miRNA genes are located in genomic regions that are frequently amplified or deleted in cancer cells (Ref. Reference Calin122), and miRNA deregulation has been specifically associated with brain, thyroid, breast, lung, liver, gastrointestinal, gynaecological, prostate and other cancers (reviewed in Ref. Reference Wang and Lee118). Some miRNAs are deregulated in several cancers whereas others are deregulated only in specific tumour types (Ref. Reference Wang and Lee118).

There are several reasons and approaches for pursuing miRNAs as therapeutic targets: (1) miRNAs can be efficiently downregulated or upregulated using miRNA antagonising molecules (antagomiRs/antimiRs) or mimics; (2) miRNAs simultaneously regulate many target genes that might function in several oncogenic pathways; (3) miRNAs can be used as noninvasive analysis tools for detecting cancers, because of their presence in the blood of cancer patients in addition to cancer tissue (Refs Reference Mueller and Bosserhoff3, Reference Leidinger123).

miRNA-focused study of melanoma is an important emerging research area (Ref. Reference Sigalotti124). In this regard, miRNA-193b and miR-211 are of particular interest, because they are downregulated in melanoma relative to nevi. When miR-193b was overexpressed in melanoma cells in vitro, the expression of approximately 300 mRNAs decreased. Once this list was crossed with potential in silico targets from Targetscan, the list was reduced to 18, including cyclin D1 and Mcl-1 (Ref. Reference Chen125). A signature of 18 miRNAs was identified, one of which was miRNA-193b, whose overexpression is significantly correlated with longer survival (Ref. Reference Segura126). miR-211 is also markedly downregulated in melanoma relative to benign nevi. When reintroduced into melanoma cell lines, a significant reduction in melanoma invasiveness was noted. miR-211 might decrease invasiveness by directly regulating the expression of TGFBR2 and NFAT5 (Ref. Reference Levy127).

One therapeutic approach would be to reintroduce a deficient miRNA in a cancer, in order to reverse the oncogenic process. Indeed, miRNA replacement has recently been shown to be effective in a mouse lung cancer model. miR-34a was introduced using a lipid-based delivery system, resulting in reduced tumour growth (Ref. Reference Wiggins128). Thus, in melanoma, one potential therapeutic-based approach might be for miRNA replacement of miRNAs such as miR-193b and miRNA-211.

Targeting multiple pathways

The relative inefficiency of small-molecule inhibitors, such as rapamycin, sorafenib, U0126 or PD98059 (Refs Reference Lasithiotakis129, Reference Smalley and Herlyn130), as well as the poor outcomes of targeting single oncogenic proteins in treating patients with advanced melanoma, led to the testing of synergetic inhibition of multiple pathways (Refs Reference Lorigan, Eisen and Hauschild131, Reference Smalley and Flaherty132, Reference Smalley133). Simultaneously targeting the PI3K–Akt and MAPK pathways significantly inhibits melanoma development (Refs Reference Smalley and Flaherty132, Reference Tran134, Reference Krasilnikov135, Reference Lopez-Bergami, Fitchman and Ronai136). Simultaneous siRNA (small interfering RNA) targeting of Akt and B-RAF using liposomes inhibited in vitro or xenograft melanoma tumour growth (Ref. Reference Tran134). Synergistically inhibiting Akt with nanoliposomes containing ceramide and RAF kinase with sorafenib has been shown to decrease melanoma proliferation (Ref. Reference Tran137). Inhibiting MAPK (with sorafenib or MEK inhibitors) and mTORC1 (with rapamycin) is more effective in reducing melanoma cell growth compared with either agent alone (Refs Reference Dankort48, Reference Lasithiotakis129). Topical application of LY-294002 and U0126, inhibitors of PI3K/Akt and RAF/MAPK, respectively, inhibits the growth of TPras transgenic melanomas in mice compared with either agent alone (Ref. Reference Bedogni138). Targeting simultaneously the PI3K pathway with sorafenib, and the MEK pathway with rapamycin, was more effective than use of the single agents (Ref. Reference Lasithiotakis129). Concurrent inhibition of MEK and CDK4 kinases by PD98059 and 219476 significantly increased apoptosis compared with single agents (Ref. Reference Li139).

Another promising type of combination therapy being developed for melanoma seeks to inhibit an oncogenic target while increasing the expression of a tumour suppressor (Ref. Reference Zhao49). For example, B-RAF^V600E can be inhibited using siRNA, whereas expression of tumour suppressor CDKN2A is increased (Ref. Reference Zhao49). Unfortunately, combination therapy has yielded only partial but not complete regression of melanoma tumours, indicating that other targets or combinations of targets should be identified.

siRNA-based therapy

RNA interference (RNAi) is a biological process of post-transcriptional gene silencing in animals and plants. In 2001 it was demonstrated that siRNAs mediate RNAi in mammalian cells without generating an interferon response, thus opening the landscape for the use of RNAi in humans (Ref. Reference Elbashir140). siRNAs are ~21 base-pair double-stranded RNA molecules that are processed within the cell by the RNA-induced silencing complex (Refs Reference Elbashir140, Reference Tran, Watts and Robertson141, Reference Kawakami and Hashida142).

The administration of naked siRNA is not compatible with clinical applications because siRNAs have poor cellular internalisation, are unstable and are degraded rapidly by circulating enzymes (Refs Reference Tran, Watts and Robertson141, Reference Kawakami and Hashida142). As a result of these shortcomings, there are only a few clinical trials utilising this technology (Ref. Reference Chen and Zhaori143). The most important, and perhaps the rate-liming, factor for widespread use of in vivo RNAi is the paucity of effective siRNA carriers (Ref. Reference Fire144). Currently, the most commonly used delivery agents for siRNA include polyethylenimine (PEI) modified with stearic acid (StA), cationic liposomes, polyelectrolyte complex micelles, water-soluble lipopolymers, polycationic dendrimers and aptamers (see below) (Refs Reference Kawakami and Hashida142, Reference Alshamsan145). Nevertheless, siRNAs are promising therapeutic agents because of their high target selectivity and predicted low toxicity. In vitro and in vivo experiments indicate that siRNA targeting of various genes, such as MYC (Ref. Reference Chen146), AKT, BRAF ^V600E (Ref. Reference Tran134), CD147 (Ref. Reference Su, Chen and Kanekura147), BIRC7 (livin) (Ref. Reference Wang148) and STAT3 (Ref. Reference Alshamsan145) can inhibit melanoma tumour growth and decrease proliferation and invasion.

It has been demonstrated that siRNA can be packed effectively into complexes suitable for cell internalisation by using a lipid-substituted PEI (PEI-StA) (Ref. Reference Alshamsan145). PEI-StA complexes were used to deliver siRNA efficiently to B16 melanoma cells, resulting in potent silencing of the gene encoding signal transducer and activator of transcription 3 (STAT3) (Ref. Reference Alshamsan145). STAT3 upregulates VEGF and antiapoptotic proteins, such as Bcl-2, Bcl-xL and Mcl-1. Melanoma cell viability was significantly reduced to 16% after the third daily dose with STAT3-siRNA–PEI–StA complexes, and in vivo experiments demonstrated regression of tumours, higher levels of IL-6, and lower levels of VEGF and STAT3 (Ref. Reference Alshamsan145).

The first-in-human (recruiting) Phase I clinical trial has been initiated to assess the safety of CALAA-01, an siRNA, using a targeted nanoparticle delivery system for treating solid tumours, including melanoma (NCT00689065) (Refs 23, Reference Davis149). Another recruiting Phase I clinical trial is assessing the safety and efficacy of an antimelanoma vaccine generated using siRNA techniques (NCT00672542) (Ref. 23).

Nanoparticles

Nanotechnology has advanced tremendously during the last two decades. A variety of delivery systems now available for tumour therapeutics use nonorganic polymers (Refs Reference Zhang150, Reference Hughes151), silica (Ref. Reference Zhao152), silicon, gold, iron (Refs Reference Zhang150, Reference Timko, Dvir and Kohane153, Reference Kim, Ghosh and Rotello154, Reference Cobley155) and carbon nanoparticles (Ref. Reference Timko, Dvir and Kohane153). The most commonly used nanoparticle delivery agents are liposomes (Refs Reference Hughes151, Reference Timko, Dvir and Kohane153, Reference Chen156, Reference Villares157), lipid-like materials (Ref. Reference Love158), quantum dots (Ref. Reference Tran, Watts and Robertson141) and viral particles (Ref. Reference Zhang150). Nanoparticle delivery systems can be loaded with several pharmacological agents or molecular agents. The advantages of this approach include protection of particle contents from degradation and interaction with the biological environment, enhanced drug uptake by tumours and reduced toxicity (Refs Reference Tran, Watts and Robertson141, Reference Peer159). Excellent reviews on the general topic of nanoparticle-based drug delivery (Refs Reference Kawakami and Hashida142, Reference Hughes151, Reference Peer159, Reference Xu and Anchordoquy160, Reference Bawa161) and more specifically on liposome-based delivery applications for melanoma (Ref. Reference Tran, Watts and Robertson141) have been published.

Nanoparticles made up of materials that react to changes in their environment, and nanoparticles controlled by a remote device are novel modalities that are being explored in cancer therapy. Light, pH, ultrasound and magnetic-field-sensitive nanoparticles are of therapeutic importance as a result of their targeting abilities (Refs Reference Timko, Dvir and Kohane153, Reference Cobley155, Reference Gao, Chan and Farokhzad162). Metallic and ceramic nanoparticles, such as silica, titanium oxide and aluminium oxide, can be fabricated to assume a porous structure, suitable for the delivery of antineoplastic agents (Ref. Reference Zhang150). Cellular uptake will be dependent on their size, functionalisation and crystalline structure, among other characteristics (Ref. Reference Buzea, Pacheco and Robbie163).

Efficient targeting of neoplastic cells can be achieved by carefully designing delivery systems conjugated to antibodies, aptamers or other cancer-specific ligands (peptides, vitamins) (Refs Reference Tran, Watts and Robertson141, Reference Zhang150, Reference Peer159). It is expected that to maximise specificity, cancer cells should overexpress a surface marker relative to normal cells (Ref. Reference Peer159). Using antibodies as targeting agents leads to the binding of immune cells to the antibody and the initiation of a signalling cascade that kills the tumour cells (Ref. Reference Peer159). Aptamers are short single-stranded DNA or RNA oligonucleotides selected to bind to a wide variety of targets, such as intracellular proteins, transmembrane proteins and, most importantly, tumour receptors (Refs Reference Barbas and White164, Reference White, Sullenger and Rusconi165).

Iron oxide, coated with phospholipids, surfactants or other compounds that improve their stability, can be used in thermotherapy (Ref. Reference Zhang150). A rodent model of melanoma has proved that thermotherapy, based on the magnetic-field-induced excitation of iron oxide nanoparticles, effectively prolonged the survival of the treated group (a 4.5-fold increase) when compared with controls (Ref. Reference Jordan166). Gold nanoparticles are of interest because of their optical properties, suitable for high infrared phototherapy (Ref. Reference Zhang150).

Liposomes can be used to encapsulate chemotherapeutic agents, siRNA, miRNA or radioactive particles, and are being explored as tools for delivery of antimelanoma agents, as discussed above (Refs Reference Tran, Watts and Robertson141, Reference Fink167, Reference Hwang168).

Polymer nanoparticles chemically conjugated to PD98059 have been used to target the MAPK signal transduction cascade in melanoma cells and melanoma-bearing mice (Ref. Reference Basu169). These experiments showed that this system effectively inhibits in vitro proliferation and induces apoptosis of melanoma cells (Ref. Reference Basu169). The nanoparticle–PD98059 complex also enhanced the antitumour activity of cisplatin chemotherapy (Ref. Reference Basu169). In one recent report, miRNA and siRNA were codelivered using a liposome–polycation–hyaluronic-acid nanoparticle-based formulation modified with a single-chain antibody fragment. This novel system proved to have an enhanced therapeutic effect (Ref. Reference Chen156).

Hundreds of clinical trials are already using encapsulated drugs on patients with various diseases, including melanoma (Ref. 23). A clinical Phase II study evaluated the efficacy of second-line treatment with liposomal doxorubicin in 30 patients with metastatic melanoma, previously unsuccessfully treated with chemotherapy (Ref. Reference Fink167). Liposomal doxorubin was well tolerated; the median survival was 214 days, with seven patients surviving for more than 300 days and five patients for more than 400 days (Ref. Reference Fink167).

Stem cells

Preclinical studies suggest that genetically modified neural stem cells (NSCs) can be targeted to solid tumours with high selectivity (Refs Reference Aboody, Najbauer and Danks170, Reference Aboody171, Reference Aboody172). Some researchers have used NSCs that express prodrug-activating enzymes, interleukins, interferon (IFN)-β and apoptosis-promoting factors (Ref. Reference Aboody, Najbauer and Danks170). Systemically administered progenitor cells migrate to and infiltrate solid tumours. Thus, this antineoplastic delivery system is quite promising (Ref. Reference Aboody, Najbauer and Danks170). Stem cells selectively migrate not only to solid tumours, but also to metastases at sites far away from the primary site of the tumour (Ref. Reference Porada and Almeida-Porada173). The tropism of NSCs has been used to deliver therapeutic genes selectively to tumours of various origin (brain tumours, metastatic melanoma to brain, metastatic breast carcinoma to lung and neuroblastoma) (Ref. Reference Aboody, Najbauer and Danks170). The exact mechanism that drives stem cell migration to tumours is still unknown; however, it is assumed that tumour cells produce chemotactic factors (Ref. Reference Brown174).

The extraordinary tropism of NSCs has been show to effectively target melanoma metastases in the brain (Ref. Reference Aboody171). In mice, NSCs injected into the peripheral circulation migrate to tumours such as glioma, neuroblastoma, prostate cancer and melanoma (Ref. Reference Brown174). When injected into the brain, NSCs migrate through normal brain tissue to specifically target tumour cells (Ref. Reference Aboody172). NSCs can be genetically engineered to express cytosine deaminase (CD) enzyme which is able to convert the nontoxic compound 5-fluorocytosine into a chemotherapeutic drug 5-fluorouracil (5-FU) (Ref. Reference Aboody172). In animal models, intracranial implantation of genetically engineered NSCs expressing CD resulted in a 71% reduction in tumour mass (Refs Reference Aboody171, Reference Aboody172).

A recent study evaluated the effect of NSCs secreting carboxylesterase, which converts CPT-11 into SN-38, a potent topoisomerase-1 inhibitor, on the growth of melanoma cells (Ref. Reference Gutova175). Exposure of melanoma cells to conditioned medium from the enzyme-secreting NSCs and CPT-11 enhanced cell killing 100-fold compared with the use of CPT-11 alone (Ref. Reference Gutova175). High concentrations of IFN-β inhibit malignant cell growth in vitro. Mesenchymal stem cells (MSCs) have been used to target the delivery of IFN-β to tumour cells in a mouse xenograft model with pulmonary metastases derived from breast carcinoma and melanoma cells (Ref. Reference Studeny176). In vitro coculture of MSCs and breast carcinoma or melanoma cells inhibited tumuor cell growth (Ref. Reference Studeny176). Intravenous injection of MSCs into mice with pulmonary metastases suppressed the growth of metastases and prolonged mouse survival compared with control or with mice injected with recombinant IFN-β (Ref. Reference Studeny176).

A recent study indicated that human metastatic melanoma cells can be reprogrammed in the neural-crest-rich microenvironment of the chick embryo, adopting a neural-crest-cell-like phenotype (Ref. Reference Kulesa177). This suggests that it will be important in future studies to determine the molecular events that allow melanoma cells to modulate or be modulated by constituents of the immediate microenvironment.

Emerging melanoma therapies that deserve to be mentioned, but are not covered in this review, include melanoma immunotherapy, with a focus on vaccines, cytotoxic T-lymphocyte antigen-4 antibodies and adoptive cell transfer (Refs Reference Weber178, Reference Homsi, Grimm and Hwu179).