The protein kinase C (PKC) family of serine/threonine protein kinases is a heterogeneous group of enzymes receiving and integrating signals involved in both normal melanocyte biology and melanoma pathology. Alterations in PKC enzyme expression and activation contribute to the malignant phenotype of melanoma in both oncogenic and tumor suppressive roles. Delineating the diverse and often context-dependent functions of PKC enzymes in melanocyte/melanoma biology is key to capitalize on these kinases as drug targets. This review summarizes several of the diverse functions of PKC in melanocyte and melanoma biology with a focus on PKC enzyme regulation and function.
Protein kinase C enzymes occupy a central node integrating signals from receptor tyrosine kinases and G protein-coupled receptors. The canonical PKC activation mechanism involves the second messengers, diacylglycerol (DAG) and calcium, generated in response to phospholipase C activation, binding to the C1 and C2 domains of PKC enzymes, respectively (Figure 1). Cofactor binding induces allosteric activation by removing the inhibitory pseudosubstrate motif from the catalytic site and facilitating membrane localization. PKC enzymes (PKCα, PKCβ, PKCγ, PKCδ, PKCθ, PKCε, PKCη, PKCι, and PKCζ) are encoded by nine genes (PRKCA, PRKCB, PRKCG, PRKCD, PRKCQ, PRKCE, PRKCH, PRKCI, and PRKCZ), which can be classified into three sub-classes based on their requirements for DAG and calcium (Figure 1). The sub-classes are classical or cPKCs (PKCα, PKCβ, PKCγ) activated by calcium and diacylglycerol, novel or nPKCs (PKCδ, PKCθ, PKCε, PKCη) activated by diacylglycerol but not calcium, and atypical or aPKCs (PKCι and PKCζ), which are not activated by either calcium or diacylglycerol. The novel PKCs contain a C2-related domain that does not bind calcium, but is involved in regulating membrane translocation and can bind phosphotyrosine in the case of PKCδ (Benes et al., 2005). The atypical PKCs lack C2 domain homology sequences and contain only a single C1 domain, which cannot bind DAG/TPA. Atypical PKCs are activated by distinct lipids (e.g., ceramides, phosphatidylserine, phosphatidylinositol 3,4,5-trisphosphate) as well as phosphorylation and protein–protein interactions (Hirai and Chida, 2003; Lozano et al., 1994; Muller et al., 1995; Nakanishi et al., 1993; Wang et al., 2005). RNA splicing generates additional heterogeneity within the PKC protein family (e.g., PKCβ-I, PKCβ-II, PKCδ-IX), although the complete extent of PKC RNA splice products has not been thoroughly described (Kim et al., 2011; Ono et al., 1986).
What is well described is that individual PKC enzymes are involved in a varied array of biological process including proliferation, migration, cell polarity, differentiation, and apoptosis. Delineating the diverse and sometimes cell-type specific functions of PKC enzymes has been a major challenge, although significant progress has been made. Differences in substrate specificity of individual PKC enzymes are not sufficient to explain the diversity of PKC function. PKC enzymes have highly conserved ATP-binding C3 and substrate-binding C4 domains, and consequently the substrate specificity of PKC enzymes is similar, but not identical, when assayed in in vitro kinase assays (Kazanietz et al., 1993; Nishikawa et al., 1997). The diversity in PKC enzyme cellular function is because of a combination of differing substrate specificity, expression, cofactor requirements, and importantly anchoring/scaffolding proteins. The anchoring proteins for PKC, including receptors for activated C kinases (RACK) and AKAP79, target-active PKC enzymes to distinct subcellular locations where they have restricted substrate access, resulting in isotype-selective functions (Hoshi et al., 2010; Klauck et al., 1996; Schechtman and Mochly-Rosen, 2001). The subcellular locations of individual PKC enzymes are highly variable and dependent on the cell type, activation mechanism, scaffolding/adaptor proteins present, and even the stage of cell cycle, making it difficult to generalize about PKC enzyme subcellular localization (Perego et al., 2002; Schechtman and Mochly-Rosen, 2001; Wang et al., 1999, 2000). Studies in melanocytes have localized PKCβ to melanosomes dependent on the scaffolding protein RACK1 and have found mitochondrial localization of PKCβ-II (Park et al., 2004b; Voris et al., 2010). Nuclear PKCδ is associated with apoptosis in melanoma, and PKC enzymes have also been localized to the cytoskeleton, consistent with their role in cell migration (Szalay et al., 2001; Zhang et al., 2005). Thus, PKC enzyme location dictates their function in melanocytes and melanoma, and defining factors specifying PKC localization is important to understand PKC enzyme heterogeneity.
Different PKC enzyme expression profiles have been identified in normal melanocytes and melanoma cells (Figure 1B) (Lau et al., 2012; Oka et al., 2006; Selzer et al., 2002; Voris et al., 2010). Most cell types express at least one of each of the three sub-classes of PKCs, and melanocytes/melanoma cells are no exception. Melanocytes express PKCα, PKCβ, PKCδ, PKCε, PKCη, PKCζ, and low levels of PKCι. Melanoma cells express markedly reduced levels of PKCβ but elevated levels of PKCε and the atypical PKC enzymes PKCζ and PKCι. The reduced PKCβ expression and higher expression of aPKCs in melanoma contributes the growth and invasion of melanoma cells, as will be described in this review.
Oncogenic protein kinase C signaling
Protein kinase C enzymes are involved in transducing signals from mitogenic growth factors by virtue of their allosteric activation by second messengers generated from phospholipase C hydrolysis of phosphatidylinositols. Several growth factor/receptor pairs mitogenic for melanocytes, including HGF/c-Met and Endothelin-1/ET(B)-R, lead to PKC activation, and in fact chronic treatment with the PKC agonist 12-O-tetradecanoylphobol-13-acetate (TPA) is mitogenic for normal melanocytes (Arita et al., 1992; Eisinger et al., 1983; Eisinger and Marko, 1982; Hirobe, 2001; Imokawa et al., 1997; Pittelkow and Shipley, 1989; Sharma et al., 2005, 2007; Swope et al., 1995). The stimulation of phosphatidylinositol metabolism by growth factors mitogenic in melanoma promotes the generation of phosphatidylinositol, 4,5-bisphosphate, which can be cleaved to generate the PKC activator DAG and inositol 1,4,5-trisphosphate (Nishizuka, 1992). In addition, the phosphatidylinositol 3,4,5-trisphosphate generated induces membrane localization of 3-phosphoinositide-dependent protein kinase-1 (PDK1) (Lu et al., 2010). PDK1 is involved in the priming phosphorylation of the PKC activation loop, a necessary step for PKC maturation (Newton, 2003) (Figure 2). PDK1 also activates AKT and is required for the growth and survival of melanoma cells (Feldman et al., 2005; Lu et al., 2010). Activation of the oncogenic G protein-coupled receptor Grm1 in melanoma cells stimulates phosphatidylinositol metabolism and activation of ERK1/2 in a PKCε-dependent manner (Marin et al., 2006). Thus, multiple mechanisms exist for the activation of PKC in melanoma cells.
Protein kinase C is involved in multiple self-reinforcing mitogenic signaling loops in melanoma (Figure 3). PI3K/AKT signaling in melanoma is activated through multiple mechanisms, including the loss, mutation, or transcriptional silencing of PTEN (Guldberg et al., 1997; Mirmohammadsadegh et al., 2006; Stahl et al., 2003; Teng et al., 1997). PKC enzymes are integral to positive feedback regulation of PI3K/AKT signaling involving the MAP kinases ERK and JNK. The ERK target c-Jun promotes the transcription of PDK1, and PDK1 phosphorylates and contributes to maturation and activation PKC and AKT (Lopez-Bergami et al., 2010). PKC also phosphorylates JNK in a RACK1 dependent manner to stimulate JNK activity and increase c-Jun phosphorylation. As RACK1 is a c-Jun target gene, RACK1 is also induced by the PKC/JNK/c-Jun axis, establishing another positive feedback loop for JNK activation (Lopez-Bergami et al., 2005, 2007).
Protein kinase Cε is oncogenic in melanoma and many other cell types in association with its ability to phosphorylate and activate STAT3 (Aziz et al., 2007b,a; Benavides et al., 2011; Lau et al., 2012). In melanoma, PKCε levels are elevated and associated with poor prognosis, and PKCε has been demonstrated to phosphorylate the ATF2 transcription factor, preventing ATF2 nuclear export to the mitochondria and inhibiting apoptosis (Lau et al., 2012). Thus, PKCε has multiple targets to drive melanoma cell survival and is an important PKC enzyme target for inhibitor development.
Expression of the non-canonical Wnt ligand Wnt5a is strongly correlated with melanoma aggressiveness (Bittner et al., 2000). Ectopic Wnt5a expression promotes the invasive behavior of melanoma cell lines, and PKC signaling is activated and required for these effects in metastatic melanoma (Bittner et al., 2000; Weeraratna et al., 2002). The effects of Wnt5a in melanoma cells are mediated by binding to the Frizzled-5 and/or ROR2 receptor and subsequent activation of PKC (O’Connell et al., 2010; Weeraratna et al., 2002). Wnt5a triggers an epithelial-to-mesenchymal transition in a PKC-dependent fashion (Dissanayake et al., 2007). In addition, TPA stimulated Wnt5a expression in melanoma cells with low metastatic potential and was required for increased Snail and decreased E-cadherin expression (Dissanayake et al., 2007). Wnt5a also reduced expression of melanocyte antigens MART-1, GP100, and tyrosinase in a PKC and STAT3-dependent manner, indicating that Wnt/PKC signaling may also be involved in immune evasion (Dissanayake et al., 2008). Note that the PKC enzyme required for the cellular effects of Wnt5a has not been demonstrated conclusively but is consistent with PKCα (Weeraratna et al., 2002).
Changes in integrins expression and signaling are important for melanoma invasion and metastasis, with high β3 subunit expression significantly associated with melanoma invasion and progression (Albelda et al., 1990). PKC enzymes have profound influences on actin cytoskeleton organization and PKCα and PKCδ have been linked to melanoma invasion mediated by αvβ3 integrin and Src (Lewis et al., 1996; Putnam et al., 2009). High expression of these PKC enzymes was required for enhanced invasion even in melanoma cells with αvβ3 integrin activation, suggesting that they provide a critical costimulatory signal. PKCα was associated with disruption of Rac and PAK activity and focal adhesions, while PKCδ primarily disrupted stress fibers. Thus, alterations in the expression and/or activation of PKCα or PKCδ cooperate with β3 integrin signaling to disrupt stable cell-matrix adhesion and facilitate melanoma invasion.
Tumor suppressive protein kinase C signaling
Treatment of melanoma cells with PKC agonists can induce senescence, growth inhibition and differentiation, implicating that PKC signaling in tumor suppressive functions (Arita et al., 1994; Cozzi et al., 2006; Mason et al., 2009; Staudt et al., 2008). These effects are most likely mediated by activation of DAG-responsive classical and novel PKC enzymes, although non-PKC phorbol ester receptors can be responsible for the effects of TPA and other C1 domain pharmacophores (Kazanietz, 2000). Determining which PKC enzymes are responsible for the growth inhibitory function of PKC activators on melanoma cells is a necessary step for the rationale design of drugs promoting the expression and/or activation of tumor suppressive PKC enzymes.
Protein kinase Cβ activation is associated with melanocyte differentiation functions (melanin biosynthesis), reduced invasion and increased apoptosis, suggesting a tumor suppressive function (Oka et al., 2008; Park et al., 1993, 1999; Voris et al., 2010). In addition, PKCβ expression is reduced or undetectable in ∼90% of melanoma cell lines and ∼50% of benign nevi and melanomas (Gilhooly et al., 2001; Oka et al., 1996, 2006; Powell et al., 1993; Ryu et al., 2007; Voris et al., 2010; Yamanishi et al., 1991). The function of PKCβ in melanin biosynthesis is linked to its ability to phosphorylate and activate tyrosinase, the rate-limiting enzyme in melanin production (Park et al., 1999, 2004b). Consistent with this, PKC activators can stimulate melanocyte pigmentation and PKC inhibitors block pigment production in mouse skin (Park et al., 1993, 1996, 2004a). Selectivity for PKCβ phosphorylation of tyrosinase on melanosomes is dependent on the anchoring protein RACK1 (Park et al., 2004b; Stebbins and Mochly-Rosen, 2001). The loss of PKCβ expression and pigment production would be predicted to increase damage from UV radiation, the principle etiological agent for both melanoma and non-melanoma skin cancers. Ectopic expression of PKCβ in melanoma cells inhibited tyrosine phosphorylation of Gab1 and the hepatocyte growth factor-induced activation of PI3K, and these effects were associated with reduced melanoma cell invasion (Voris et al., 2010). PKCβ can also phosphorylate the adaptor protein p66Shc involved in increasing mitochondrial permeability and inducing apoptosis (DelCarlo and Loeser, 2006; Pinton et al., 2007). Consistent with pro-differentiation and pro-apoptotic functions of PKCβ, re-expression of PKCβ in melanoma cells suppresses growth in soft agar (Voris et al., 2010). Taken together, PKCβ participates in multiple tumor suppressive function in melanoma targeting melanoma-specific (e.g., pigmentation) and general (e.g., apoptosis, invasion) activities.
Protein kinase Cβ expression and activation are regulated by several pathways of known importance to melanoma. PKCβ is activated in response to oxidative stress, which is produced by UV radiation and is elevated in melanoma because of oncogene activation (e.g., BRAF) (de Souza et al., 2006; Fried and Arbiser, 2008; Govindarajan et al., 2007; Meyskens et al., 2001; Zaidi et al., 2008). Elevated reactive oxygen species (ROS) are also produced in response to PKCβ activation because of the phosphorylation of p66Shc and activation of the mitochondrial permeability pore, resulting in a positive feedback involving PKCβ activation and ROS generation (DelCarlo and Loeser, 2006; Giorgio et al., 2005; Pinton et al., 2007; Voris et al., 2010). PRKCB gene expression is in part regulated by the microphthalmia-associated transcription factor, MITF, a master melanocyte developmental transcription factor, consistent with the role of PKCβ in melanin biosynthesis (Park et al., 2006). However, MITF is amplified in melanoma where it functions as an oncogenic lineage determinant, making the role of MITF in PKCβ expression in the context of melanoma is unclear (Garraway et al., 2005).
Protein kinase Cδ is a pro-apoptotic PKC enzyme in many cell types, including melanoma. In addition to the classical DAG-mediated activation mechanism, PKCδ can be activated by caspase-mediated proteolytic cleavage to generate a constitutively active catalytic fragment, which is highly apoptotic (Denning et al., 1998; Emoto et al., 1995). In melanoma cells, PKCδ has been linked to activation of JNK and induction of apoptosis (Mhaidat et al., 2007, 2008). The pro-apoptotic functions of PKCδ is associated with its translocation to either the mitochondria or nucleus, with nuclear PKCδ linked to apoptosis in melanoma cells (Denning et al., 2002; DeVries et al., 2002; Li et al., 1999; Zhang et al., 2005). PKCδ functions as a tumor suppressor in some cancers, such as cutaneous squamous cell carcinoma, where its expression is lost (D’Costa et al., 2006; Yadav et al., 2010). While PKCδ mRNA and protein levels are not consistently reduced in melanoma compared to melanocytes, PKCδ may still function as a tumor suppressor in melanoma as post-translational mechanisms to inhibit its activity have been described (Denning et al., 1993; Voris et al., 2010).
Prospects for protein kinase C therapeutics in melanoma
The goals of genetic, biochemical, and cell biologic studies on altered signaling in melanoma are to improve diagnosis, prevention, and/or treatment of melanoma. The prospects for achieving these goals have been bolstered by the relatively rapid progress and recent success of therapeutics targeting melanomas with activating BRAF mutations (Flaherty et al., 2010). Kinase-targeted therapeutics such as PLX4032 (Vemurafenib) have the real potential to provide effective treatment options to patients with metastatic melanoma, historically a disease with no viable treatment options (Sosman et al., 2012). Key to the success of targeted therapeutics is target identification and validation, and work on identifying and validating PKC targets in melanoma is progressing. These studies have identified PKC enzymes involved in promoting or inhibiting melanomagenesis, and these different scenarios invoke starkly different therapeutic strategies.
One overarching theme in the development of kinase inhibitors for cancer therapeutics is specificity. The vast majority of inhibitors targeting intracellular, non-receptor kinases are analogues of ATP. As the ATP-binding sites displays significant sequence and structural homology among protein kinases, the competitive ATP inhibitors are somewhat limited in the degree of specificity possible. Given the often opposing functions of different PKC enzymes, it is theoretically preferable to have a high level of specificity for the targeted kinase (e.g., PKCε) to maximize the desired effects (Mackay and Twelves, 2007). Thus, the PKC enzyme functional heterogeneity favors a highly selective enzyme inhibitor for melanoma therapy. As PKC enzyme substrate targeting is profoundly influenced by scaffolding proteins, approaches to interfere with PKC scaffolding proteins interactions may be a promising approach (Churchill et al., 2009).
Protein kinase C inhibitors as therapeutics for melanoma
One of the most promising PKC inhibitors for melanoma is Enzastaurin (LY317615.HCl), a PKCβ-selective, orally available ATP analog that has shown some activity against melanoma in pre-clinical studies and in other advanced cancers in phase I and II clinical trials (Carducci et al., 2006; Faul et al., 2003; Hanauske et al., 2007; Wolff et al., 2011). Ironically, Enzastaurin was developed as a selective inhibitor of PKCβ, one of the PKC enzymes down-regulated in melanomas and thus would not be predicted to be effective in melanoma (Faul et al., 2003; Gilhooly et al., 2001; Voris et al., 2010). However, multiple mechanisms of action and cellular targets have been described that may account for the anti-melanoma activity of Enzastaurin. For example, Enzastaurin is able to inhibit tumor angiogenesis by interfering with VEGF function and production (Keyes et al., 2004; Teicher et al., 2002). Thus, Enzastaurin may inhibit tumor growth in part by targeting endothelial cells, which express PKCβ (Xia et al., 1996).
Enzastaurin also has direct effects on tumor cell growth, survival and invasion (Graff et al., 2005; Rizvi et al., 2006). Enzastaurin inhibited the growth of primary melanoma cultures and had a preferential growth inhibitory effect on uveal melanoma cell lines with GNAQ mutations (Hanauske et al., 2007; Wu et al., 2012). GNAQ is a phospholipase C-coupled Gαq subunit commonly mutated in uveal melanomas where it functions as a dominant oncogene (Van Raamsdonk et al., 2009, 2010). Enzastaurin was able to reduce the protein levels and phosphorylation of multiple PKC enzymes (PKCβ, PKCε, PKCθ) important for cell viability in uveal melanoma cells (Wu et al., 2012). Enzastaurin was also able to reduce viability of cell such as Mel285 lacking detectible expression of PKCβ, indicating that it has targets in addition to PKCβ (Wu et al., 2012).
Other more general PKC inhibitors are able to inhibit the growth or invasion/migration of melanoma, most likely by targeting the oncogenic PKC enzyme signaling. The PKC regulatory domain inhibitor calphostin C inhibited mouse melanoma cell migration in association with reduced MARKS phosphorylation likely due to the inhibition of PKCα (Chen and Rotenberg, 2010). PKCα knockdown or inhibition of PKC with calphostin c or the cPKC inhibitor Gö6976 also inhibited the migration of human melanoma cells (Byers et al., 2010).
Protein kinase C activators as therapeutics for melanoma
While the concept of using a PKC activator, such as a phorbol ester (e.g., the tumor promoter TPA) to treat cancer may at first alarm most cancer biologists familiar with the mouse skin chemical carcinogenesis model, the idea may have some merit for treating melanoma. The general observation that PKC activation inhibits the growth of melanoma cells while stimulating proliferation of normal melanocytes suggests an inherent therapeutic window for selectively targeting transformed melanocytes with PKC agonists (Arita et al., 1994; Cozzi et al., 2006; Eisinger et al., 1983; Eisinger and Marko, 1982; Mason et al., 2009; Pittelkow and Shipley, 1989; Powell et al., 1993; Staudt et al., 2008). The growth inhibitory effects of PKC activation are likely due to some PKC enzymes having tumor suppressive functions in melanoma.
Ingenol-3-angelate (ingenol mebutate, PEP005) is a PKC activator effective clinically against actinic keratosis, squamous cell carcinoma, and basal cell carcinomas (Anderson et al., 2009; Hampson et al., 2005; Ramsay et al., 2011; Siller et al., 2009, 2010). In melanoma cells, ingenol-3-angelate can induce senescence, apoptosis, or necrosis in a PKC-dependent manner depending on the drug concentration (Cozzi et al., 2006; Gillespie et al., 2004). Ingenol-3-angelate activates the pro-apoptotic PKCδ, and its inhibition of melanoma cell growth is associated with induction of p21 and overstimulation of the ERK MAP kinase signaling (Cozzi et al., 2006; Hampson et al., 2005; Mason et al., 2009). In vivo, ingenol-3-angelate is able to penetrate deeply into the skin to cause vascular damage and hemorrhaging owing to its transport by the drug efflux pump P-glycoprotein (Li et al., 2010). Thus, ingenol-3-angelate is a PKC agonist with unique tissue distribution properties, which can activate PKC enzymes to inhibit the growth of melanoma cells.
Other PKC agonists with reduced skin tumor promoting activity may also be promising for melanoma treatment. Mezerein is an incomplete or weak mouse skin tumor promoter able to inhibit the growth and induce differentiation of melanoma cells (Mufson et al., 1979; Staudt et al., 2008). The Bryostatins are ultrapotent PKC agonists capable of inducing only a subset of TPA responses while inhibiting the phorbol ester responses not induced (Hennings et al., 1987; Sako et al., 1987). Brysotatins have substantially different PKC enzyme down-regulation specificity and kinetics than TPA, which may explain their selective effects on cells (Szallasi et al., 1994, 1995). Bryostatin-1 has been evaluated in advanced melanoma patients in both phase I and phase II trials, but had little activity (Bedikian et al., 2001; Gonzalez et al., 1999; Propper et al., 1998; Tozer et al., 2002). PKC agonists with differential desensitization/activation specificities may still hold promise as melanoma therapeutics, but clearly defined mechanisms of action and dose/response relationships need to be established. Indeed, it is not clear that the anti-proliferative and anti-metastatic effects of Byostatin-1 on melanoma cell lines are mediated via PKC enzymes (Schuchter et al., 1991; Szallasi et al., 1996).
As the expression of the tumor suppressor PKCβ is lost in melanoma, therapies with agonists to specifically activate the enzyme will not be effective unless expression of PKCβ can be restored. However, the PKCβ gene is transcriptionally upregulated by phorbol esters, and thus phorbol esters could be effective at inducing both PKCβ expression and activation (Obeid et al., 1992). PKC agonists with multiple mechanisms of action, such as disrupting tumor vasculature or inducing expression of their target PKC enzyme, may provide the needed pharmacological advantage for the development of PKC-based therapeutics for melanoma. The integration of PKC enzymes into AKT and MAP kinase pathways important to melanoma resistance to BRAF inhibitors suggests that targeting PKC may also hold promise for melanomas with acquired or de novo resistant to other targeted therapies. In fact, an expression library screen of almost 600 protein kinases to identify alternative kinases capable of imparting resistance to BRAF inhibition in melanoma identified PKCε and PKCη as two of only nine highly significant kinase hits (Johannessen et al., 2010). Thus, PKC activation by alternative signaling cascades or other mechanisms is likely sufficient to bypass BRAF(V600E) inhibition and making PKCε and PKCη highly attractive targets for overcoming resistance to BRAF inhibition.
In summary, the diverse functions of individual PKC enzymes and their close association with key signaling pathways activated in melanoma is both a challenge and opportunity for targeting them in melanoma. The PKC signaling landscape appears complex, and considering the successes and surprises of targeting BRAF(V600E) in melanoma, the more we understand PKC signaling mechanisms at the biochemical level, the better we will be poised to target this class of enzymes clinically.
I would like to acknowledge Drs. Brian Nickoloff, Caroline Le Poole, and Meenhard Herlyn for sparking and encouraging my interest in PKC and melanoma. This review is admittedly limited in scope, and I apologize for the many valuable publications not discussed here.