Hee-Young Park’s research while affiliated with Boston University and other places

Dysregulation of the CXCR4/CXCL12 axis, relevant in melanoma progression, activates cell cycle progression and migration via stimulation of the MAPK-pathway. We sought to ascertain the cooperativity of the CXCR4/CXCL12 axis with established prognosticators and BRAF status in melanoma. Samples (n=107) of primary cutaneous melanoma were assessed for protein expression of CXCR4 and CXCL12 and molecular analyses were performed to ascertain BRAF status. Univariate analyses of CXCR4 protein showed that the proportion of CXCR4 positives was greater in melanomas with absence of mitoses (p<0.0001), absence of ulceration (p=0.0008) and absence of regression (p=0.02). Patients presenting at shallower stages (AJCC 1-2) exhibited a larger proportion of CXCR4 positives (76.9%, p<0.0001 and 69.0%, p=0.008), while those at deeper stages (AJCC 3-4) exhibited a larger proportion of negatives (75.0%, p=0.004 and 66.7%, p=0.22). In a multivariable analysis, lower odds of CXCR4 protein expression were associated with AJCC stage-3 (OR=0.16, p=0.01), stage-4 (OR=0.17, p=0.04), and mitoses (OR=0.21, p=0.01). Univariate analyses of CXCL12 protein showed that the proportion of CXCL12 negatives was significantly smaller in melanomas with: depth ≥1 mm, absence of ulceration and absence of vascular invasion (p<0.0001 for all). CXCR4 and CXCL12 appear to be biomarkers associated with established prognosticators of good and poor clinical outcome respectively in primary cutaneous melanoma. A BRAF mutation does not appear to be associated with CXCR4/CXCL12 axis upregulation in primary cutaneous melanoma.

Hyperpigmentation disorders are generally difficult to treat because of the limited availability of effective therapeutics with minimal side effects. In this issue, Lehraiki et al. report that metformin, an antidiabetic drug, inhibited melanogenesis, in vitro and in vivo, and they suggest that metformin may be used to treat hyperpigmentation disorders. This commentary reviews the molecular mechanisms through which metformin inhibits melanogenesis and examines metformin as a potential drug to treat hyperpigmentation.

Dysregulation of the chemokine receptor CXCR4 is relevant in melanoma progression, and the CXCR4/CXCL12 axis has been shown to activate cell cycle progression and malignant cell migration through stimulation of the mitogen-activated protein kinase pathway. Studies ascertaining the potential utility of CXCR4 mRNA as a prognosticator in melanoma have focused mainly on metastatic melanoma with conflicting results. In the light of this, we sought to explore the potential relationship between CXCR4 mRNA expression with established histopathologic prognosticators and BRAF status in melanoma. Archived consecutive samples (n=107) of primary cutaneous melanoma were retrieved and assessed for the following: CXCR4 mRNA (semiquantitative RT-PCR) and BRAF exon 15 status (DNA Sanger sequencing). Statistical analyses included correlation between CXCR4 mRNA levels and established histopathologic prognosticators as well as the BRAF status using univariate and multiple linear methods. Multivariable analyses revealed a significant correlation between elevated CXCR4 mRNA (low ΔCt value) and the presence of BRAF mutation (P=0.02). Absence of a brisk host response was associated with elevated CXCR4 mRNA expression (P=0.04). CXCR4 mRNA was significantly lower in AJCC stage 2 compared with stage 1 after controlling for significant clinical prognosticators (P=0.02). The association between elevated CXCR4 mRNA and absence of a brisk host response suggests that CXCR4 may be involved in regulation of the host immune response in melanoma and is a molecule of potential utility as a biomarker for recruiting melanoma patients for immunotherapy. Higher CXCR4 mRNA in patients with a BRAF mutation suggests its utility as a putative therapeutic target.

Cutaneous pigmentation or skin color is the body's natural protection against sun-induced damage. Skin color is determined primarily by melanin, a biopolymer that is synthesized within epidermal melanocytes, packaged in cellular organelles called melanosomes, and then dispersed to neighboring keratinocytes. The process of melanogenesis involves numerous molecules and intracellular pathways that are subject to regulation by endogenous and exogenous factors. Tyrosinase is the central and rate-limiting enzyme in melanin biosynthesis. Therefore, elucidation of the molecules and pathways that regulate tyrosinase levels and activity could identify target areas for the development of compounds to decrease excessive pigmentation on one hand or induce pigmentation on the other. The following commentary will summarize the key regulatory molecules and pathways involved in tyrosinase function.

Melanocytes are pigment-producing cells that originate from the dorsal portions of the closing neural tube in vertebrate embryos. Similar to neurons, they are derived from pluripotent neural crest cells that differentiate into numerous cell lineages. The development of melanocytes and neurons, as well as their differentiated function, within the mature tissue, is influenced by signaling molecules produced by neighboring cells. The same signaling molecules that have a role in the central and peripheral nervous tissue also have a role in cutaneous melanocytes. These include Wnt, bone morphogenetic proteins, endothelins, steel factor, hepatocyte growth factor, fibroblast growth factors, and neurotrophins. Signaling pathways including PKC- and p53/p73-dependent pathways are also common to melanocytes and neurons. The similarity between melanocytes and neurons suggests that melanocytes could provide a valuable model for studies of diseases that affect the nervous system, as well as for development of potential therapies for these diseases.

Protein kinase C (PKC) is a multigene family of serine/threonine protein kinases involved in cell signaling pathways of proliferation and motility. PKC interacts with Rho GTPases in the regulation of the actin cytoskeleton. The PKC-alpha isozyme binds the Rho GTPase cdc42, and both are coordinated with the Rac-phosphatidylinositol-3 kinase (PI3K) signaling pathway in melanoma cell invasion and migration on extracellular matrix proteins. To further define the role of PKC-alpha in melanoma cell migration, we tested the effect of PDBu and Ca dependent activation of PKC-alpha as well as treatment with the PKC-alpha inhibitors calphostin C and Go6976. Furthermore, we transfected siRNA targeted against PKC-alpha into human melanoma cells and performed time-lapse analysis of cell migration followed by western immunoblotting. We found that significant enhancement of cell migration at 0.5 h after PDBu treatment directly correlated with Ca dependent activation of PKC-alpha and was inhibited by the PKC-alpha inhibitor calphostin C. PKC-alpha siRNA transfection nearly abrogated PKC-alpha expression and significantly reduced melanoma cell migration compared with siRNA controls. These findings provide further evidence that PKC-alpha plays an important role in melanoma cell migration and may have implications in therapies designed to disrupt melanoma cell motility by alteration of PKC-alpha signaling.

Bone Morphogenetic Protein (BMP-4) was shown to down-regulate melanogenesis, in part, by decreasing the level of tyrosinase [Yaar et al. (2006) JBC:281]. Results presented here show that BMP-4 down-regulated the protein levels of TRP-1, PKC-beta, and MCI-R. When paired cultures of human melanocytes were treated with vehicle or BMP-4 (25 ng/ml), MAPK/ERK were phosphorylated within one hour of BMP-4 treatment. Then the activated MAPK/ERK caused an acute phosphorylation of MITF, followed by proteosome-mediated degradation of MITF, the key transcription factor for melanogenic proteins [Wu et al. (2000) Gene & Development:14]. However, prolonged exposure of melanocytes to BMP-4 (up to 48 hours) caused a decrease in the level of MITF-M transcript. In addition, BMP-4 decreased the intracellular level of cAMP, the key regulator of MITF expression. These results demonstrate that BMP-4 activates MAPK/ERK signaling pathway to transiently activate MITF; however, chronic treatment of BMP-4 to melanocytes causes a down-regulation of the expression of MITF, possibly in a cAMP-dependent pathway.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta family, signal in many cells including neural precursors. Two receptors, types 1 and 2, coordinately mediate BMP signaling, and type 1 receptor has two forms: A and B. Using RT-PCR we found that neural crest-derived human melanocytes express BMP receptor-1A, -1B, and -2. Furthermore, melanocytes and the surrounding keratinocytes express BMP-4, suggesting both autocrine and paracrine effects of this molecule. Moreover, BMP-4 supplementation of cultured human melanocytes decreases melanin synthesis, tyrosinase mRNA, and protein. The mechanism of this BMP-4 effect on tyrosinase and ultimately on melanogenesis involves modest decreases of tyrosinase transcription rate and mRNA stability. Moreover, ultraviolet irradiation, the best recognized environmental stimulator of melanogenesis, down-regulated the mRNA of BMP receptor-1B in melanocytes. Our data provide evidence of a novel regulatory pathway for melanogenesis in human skin.

The cAMP-dependent pathway up-regulates MITF (microphthalmia-associated transcription factor), important for key melanogenic proteins such as tyrosinase, TRP-1 (tyrosinase-related protein 1) and TRP-2. We asked whether MITF is also a key transcription factor for PKC-beta (protein kinase C-beta), required to phosphorylate otherwise inactive tyrosinase. When paired cultures of human melanocytes were treated with isobutylmethylxanthine, known to increase intracellular cAMP, both protein and mRNA levels of PKC-beta were induced by 24 h. To determine whether MITF modulates PKC-beta expression, paired cultures of human melanocytes were transfected with dn-MITF (dominant-negative MITF) or empty control vector. By immunoblotting, PKC-beta protein was reduced by 63+/-3.7% within 48 h. Co-transfection of an expression vector for MITF-M, the MITF isoform specific for pigment cells, or empty control vector with a full-length PKC-beta promoter-CAT (chloramphenicol acetyltransferase) reporter construct (PKC-beta/CAT) into Cos-7 cells showed >60-fold increase in CAT activity. Melanocytes abundantly also expressed MITF-A, as well as the MITF-B and MITF-H isoforms. However, in contrast with MITF-M, MITF-A failed to transactivate co-expressed PKC-beta/CAT or CAT constructs under the control of a full-length tyrosinase promoter. Together, these results demonstrate that MITF, specifically MITF-M, is a key transcription factor for PKC-beta, linking the PKC- and cAMP-dependent pathways in regulation of melanogenesis.

Vascular endothelial growth factor (VEGF) is constitutively produced by keratinocytes, but has no known epidermal target cell. We now report that normal human melanocytes (Mc) maintained in serum-free, hormone-, and growth factor-supplemented medium lacking phorbol ester and choleragen constitutively express VEGF receptor-1 (VEGFR-1), VEGFR-2, and neuropilin-1. Furthermore, stimulation of Mc with VEGF165 isoform leads to phosphorylation of VEGFR-2, the receptor responsible for most of the VEGF-mediated effects in endothelial cells, suggesting that the receptor is functional. Interestingly, in Mc, VEGFR-2 expression is induced by ultraviolet irradiation and is downregulated by VEGF and tumor necrosis factor-alpha. Prolonged culture (>8 weeks) in the presence of phorbol ester abrogates VEGFR-2 expression, explaining previous reports that Mc do not express VEGFR-1 and VEGFR-2. These data suggest that VEGF may play a role in Mc behavior in skin.