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1.  Isolation of melanoma cell subpopulations using negative selection 
Melanomas are phenotypically and functiwonally heterogeneous tumors comprising of distinct subpopulations that drive disease progression and are responsible for resistance to therapy. Identification and characterization of such subpopulations are highly important to develop novel targeted therapies. However, this can be a challenging task as there is a lack of clearly defined markers to distinguish the melanoma subpopulations from a general tumor cell population. Also, there is a lack of optimal isolation methods and functional assays that can fully recapitulate their phenotype. Here we describe a method for isolating tumor cells from fresh human tumor tissue specimens using an antibody coupled magnetic bead sorting technique that is well established in our laboratory. Thus, melanoma cells are enriched by negative cell sorting and elimination of non-tumor cell population such as erythrocytes, leukocytes, and endothelial cells. Enriched unmodified tumor cells can be further used for phenotypic and functional characterization of melanoma subpopulations.
doi:10.1007/978-1-62703-727-3_26
PMCID: PMC4251796  PMID: 24258995
Subpopulations; Tumor cell isolation; Magnetic beads; Tumorigenic potential
2.  LIMD2 Is a Small LIM-Only Protein Overexpressed in Metastatic Lesions That Regulates Cell Motility and Tumor Progression by Directly Binding to and Activating the Integrin-Linked Kinase 
Cancer research  2014;74(5):1390-1403.
Proteins that communicate signals from the cytoskeleton to the nucleus are prime targets for effectors of metastasis as they often transduce signals regulating adhesion, motility, and invasiveness. LIM domain proteins shuttle between the cytoplasm and the nucleus, and bind to partners in both compartments, often coupling changes in gene expression to extracellular cues. In this work, we characterize LIMD2, a mechanistically undefined LIM-only protein originally found to be overexpressed in metastatic lesions but absent in the matched primary tumor. LIMD2 levels in fresh and archival tumors positively correlate with cell motility, metastatic potential, and grade, including bladder, melanoma, breast, and thyroid tumors. LIMD2 directly contributes to these cellular phenotypes as shown by overexpression, knockdown, and reconstitution experiments in cell culture models. The solution structure of LIMD2 that was determined using nuclear magnetic resonance revealed a classic LIM-domain structure that was highly related to LIM1 of PINCH1, a core component of the integrin-linked kinase–parvin–pinch complex. Structural and biochemical analyses revealed that LIMD2 bound directly to the kinase domain of integrin-linked kinase (ILK) near the active site and strongly activated ILK kinase activity. Cells that were null for ILK failed to respond to the induction of invasion by LIMD2. This strongly suggests that LIMD2 potentiates its biologic effects through direct interactions with ILK, a signal transduction pathway firmly linked to cell motility and invasion. In summary, LIMD2 is a new component of the signal transduction cascade that links integrin-mediated signaling to cell motility/metastatic behavior and may be a promising target for controlling tumor spread.
doi:10.1158/0008-5472.CAN-13-1275
PMCID: PMC4183205  PMID: 24590809
3.  SECTM1 Produced by Tumor Cells Attracts Human Monocytes Via CD7-mediated Activation of the PI3K Pathway 
Tumor-associated macrophages (TAMs) play essential roles in tumor progression and metastasis. Tumor cells recruit myeloid progenitors and monocytes to the tumor site, where they differentiate into TAMs; however, this process is not well studied in humans. Here we show that human CD7, a T cell and NK cell receptor, is highly expressed by monocytes and macrophages. Expression of CD7 decreases in M-CSF differentiated macrophages and in Melanoma-conditioned Medium Induced Macrophages (MCMI/Mϕ) in comparison to monocytes. A ligand for CD7, SECTM1 (Secreted and transmembrane protein 1), is highly expressed in many tumors, including melanoma cells. We show that SECTM1 binds to CD7 and significantly increases monocyte migration by activation of the PI3K pathway. In human melanoma tissues, tumor-infiltrating macrophages expressing CD7 are present. These melanomas, with CD7-positive inflammatory cell infiltrations, frequently highly express SECTM1, including an N-terminal, soluble form, which can be detected in the sera of metastatic melanoma patients but not in normal sera. Taken together, our data demonstrate that CD7 is present on monocytes and tumor macrophages, and that its ligand, SECTM1, is frequently expressed in corresponding melanoma tissues, possibly acting as a chemoattractant for monocytes to modulate the melanoma microenvironment.
doi:10.1038/jid.2013.437
PMCID: PMC3961532  PMID: 24157461
4.  EGFR Inhibition Promotes an Aggressive Invasion Pattern Mediated by Mesenchymal-like Tumor Cells within Squamous Cell Carcinomas 
Molecular cancer therapeutics  2013;12(10):2176-2186.
Squamous cell carcinomas (SCCs) with an infiltrative invasion pattern carry a higher risk of treatment failure. Such infiltrative invasion may be mediated by a mesenchymal-like subpopulation of malignant cells that we have previously shown to arise from epithelial to mesenchymal transition (EMT) and resist epidermal growth factor receptor (EGFR) targeting. Here we demonstrate that SCCs with infiltrative, high risk invasion patterns contain abundant mesenchymal-like cells, which are rare in tumors with low risk patterns. This cellular heterogeneity was modeled accurately in three dimensional culture using collagen-embedded SCC spheroids, which revealed distinct invasive fronts created by collective migration of E-cadherin-positive cells versus infiltrative migration of individual mesenchymal-like cells. Because EGFR expression by mesenchymal-like cells was diminished in the spheroid model and in human SCCs, we hypothesized that SCCs shift toward infiltrative invasion mediated by this subpopulation during anti-EGFR therapy. Anti-EGFR treatment of spheroids using erlotinib or cetuximab enhanced infiltrative invasion by targeting collective migration by E-cadherin-positive cells while sparing mesenchymal-like cells; by contrast, spheroid invasion in absence of mesenchymal-like cells was abrogated by erlotinib. Similarly, cetuximab treatment of xenografts containing mesenchymal-like cells created an infiltrative invasive front comprised of this subpopulation, whereas no such shift was observed upon treating xenografts lacking these cells. These results implicate mesenchymal-like SCC cells as key mediators of the infiltrative invasion seen in tumors with locally aggressive behavior. They further demonstrate that EGFR inhibition can promote an infiltrative invasion front comprised of mesenchymal-like cells preferentially in tumors where they are abundant prior to therapy.
doi:10.1158/1535-7163.MCT-12-1210
PMCID: PMC3796003  PMID: 23939378
pattern of invasion; EGFR inhibition; squamous cell carcinoma; EMT; tumor heterogeneity
5.  Concurrent MEK2 mutation and BRAF amplification confer resistance to BRAF and MEK inhibitors in melanoma 
Cell reports  2013;4(6):1090-1099.
Summary
Although BRAF and MEK inhibitors have proven clinical benefits in melanoma, most patients develop resistance. We report a de novo MEK2-Q60P mutation and BRAF gain in a melanoma from a patient who progressed on the MEK inhibitor trametinib and did not respond to the BRAF inhibitor dabrafenib. We also identified the same MEK2-Q60P mutation along with BRAF amplification in a xenograft tumor derived from a second melanoma patient resistant to the combination of dabrafenib and trametinib. Melanoma cells chronically exposed to trametinib acquired concurrent MEK2-Q60P mutation and BRAF-V600E amplification, which conferred resistance to MEK and BRAF inhibitors. The resistant cells had sustained MAPK activation and persistent phosphorylation of S6K. A triple combination of dabrafenib, trametinib, and the PI3K/mTOR inhibitor GSK2126458 led to sustained tumor growth inhibition. Hence, concurrent genetic events that sustain MAPK signaling can underlie resistance to both BRAF and MEK inhibitors, requiring novel therapeutic strategies to overcome it.
doi:10.1016/j.celrep.2013.08.023
PMCID: PMC3956616  PMID: 24055054
7.  Overcoming intrinsic multi-drug resistance in melanoma by blocking the mitochondrial respiratory chain of slow-cycling JARID1Bhigh cells 
Cancer cell  2013;23(6):10.1016/j.ccr.2013.05.003.
Summary
Despite success with BRAFV600E–inhibitors, therapeutic responses in patients with metastatic melanoma are short-lived because of the acquisition of drug resistance. We identified a mechanism of intrinsic multi-drug resistance based on the survival of a tumor cell subpopulation. Treatment with various drugs, including cisplatin and vemurafenib, uniformly leads to enrichment of slow-cycling, long-term tumor-maintaining melanoma cells expressing the H3K4-demethylase JARID1B/KDM5B/PLU-1. Proteome-profiling revealed an upregulation in enzymes of mitochondrial oxidative-ATP-synthesis (OXPHOS) in this subpopulation. Inhibition of mitochondrial respiration blocked the emergence of the JARID1Bhigh subpopulation and sensitized melanoma cells to therapy, independent of their genotype. Our findings support a two-tiered approach combining anti-cancer agents that eliminate rapidly proliferating melanoma cells with inhibitors of the drug-resistant slow-cycling subpopulation.
doi:10.1016/j.ccr.2013.05.003
PMCID: PMC3810180  PMID: 23764003
8.  Rhenium Complexes with Visible-Light-Induced Anticancer Activity 
ChemMedChem  2013;8(6):924-927.
doi:10.1002/cmdc.201300060
PMCID: PMC3665699  PMID: 23568508
visible light; antiproliferation; singlet oxygen; organometallic complexes; rhenium
9.  Hypoxia induces phenotypic plasticity and therapy resistance in melanoma via the tyrosine kinase receptors ROR1 and ROR2 
Cancer discovery  2013;3(12):1378-1393.
An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the non-canonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Further, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In melanoma patients treated with the BRAF inhibitor, Vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.
doi:10.1158/2159-8290.CD-13-0005
PMCID: PMC3918498  PMID: 24104062
10.  There is a world beyond protein mutations: The role of non-coding RNAs in melanomagenesis 
Experimental dermatology  2013;22(5):303-306.
Until recently, the general perception has been that mutations in protein coding genes are responsible for tumorigenesis. With the discovery of V600EBRAF in about 50% of cutaneous melanomas there was an increased effort to find additional mutations. However, mutations characterized in melanoma to date cannot account for the development of all melanomas. With the discovery of microRNAs as important players in melanomagenesis protein mutations are no longer considered the sole drivers of tumors. Recent research findings have expanded the view for tumor initiation and progression to additional non-coding RNAs. The data suggest that tumorigenesis is likely an interplay between mutated proteins and deregulation of non-coding RNAs in the cell with an additional role of the tumor environment. With the exception of microRNAs, our knowledge of the role of non-coding RNAs in melanoma is in its infancy. Using few examples we will summarize some of the roles of non-coding RNAs in tumorigenesis. Thus, there is a whole world beyond protein coding sequences and microRNAs, which can cause melanoma.
doi:10.1111/exd.12117
PMCID: PMC3637432  PMID: 23489578
melanoma; non-coding RNA; microRNA; epigenetic modifications; alternative splicing
11.  The FBXO4 Tumor Suppressor Functions as a Barrier to BrafV600E-Dependent Metastatic Melanoma 
Molecular and Cellular Biology  2013;33(22):4422-4433.
Cyclin D1–cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16INK4A, an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16INK4A deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.
doi:10.1128/MCB.00706-13
PMCID: PMC3838190  PMID: 24019069
12.  Suppression of Nucleotide Metabolism Underlies the Establishment and Maintenance of Oncogene-Induced Senescence 
Cell reports  2013;3(4):10.1016/j.celrep.2013.03.004.
Summary
Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here we show that a decrease in deoxyribonucleotide triphosphates (dNTPs) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of RRM2, the rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Further, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.
doi:10.1016/j.celrep.2013.03.004
PMCID: PMC3840499  PMID: 23562156
13.  The novel SMAC mimetic birinapant exhibits potent activity against human melanoma cells 
Purpose
Inhibitor of apoptosis proteins (IAPs) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases (SMAC) mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as tumor necrosis factor (TNF)-α in melanomas
Experimental Design
Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in in 2D, 3D spheroid and in vivo xenograft models.
Results
When birinapant was combined with TNF-α, strong combination activity, i.e. neither compound was effective individually but the combination was highly effective, was observed in twelve out of eighteen cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line.
Conclusions
Birinapant in combination with TNF-α exhibits a strong anti-melanoma effect in vitro. Birinapant as a single agent shows in vivo anti-tumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.
doi:10.1158/1078-0432.CCR-12-2518
PMCID: PMC3618495  PMID: 23403634
birinapant; IAP inhibitor; SMAC mimetic; TNF-α; melanoma
14.  Targeting ER stress–induced autophagy overcomes BRAF inhibitor resistance in melanoma 
The Journal of Clinical Investigation  2014;124(3):1406-1417.
Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAFV600E melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAFV600E melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
doi:10.1172/JCI70454
PMCID: PMC3934165  PMID: 24569374
15.  Development of a novel class of B-RafV600E-selective inhibitors through virtual screening and hierarchical hit optimization 
Organic & biomolecular chemistry  2012;10(36):7402-7417.
Oncogenic mutations in critical nodes of cellular signaling pathways have been associated with tumorigenesis and progression. The B-Raf protein kinase, a key hub in the canonical MAPK signaling cascade, is mutated in a broad range of human cancers and especially in malignant melanoma. The most prevalent B-RafV600E mutant exhibits elevated kinase activity and results in constitutive activation of the MAPK pathway, thus making it a promising drug target for cancer therapy. Herein, we described the development of novel B-RafV600E selective inhibitors via multi-step virtual screening and hierarchical hit optimization. Nine hit compounds with low micromolar IC50 values were identified as B-RafV600E inhibitors through virtual screening. Subsequent scaffold-based analogue searching and medicinal chemistry efforts significantly improved both the inhibitor potency and oncogene selectivity. In particular, compounds 22f and 22q possess nanomolar IC50 values with selectivity for B-RafV600E in vitro and exclusive cytotoxicity against B-RafV600E harboring cancer cells.
doi:10.1039/c2ob26081f
PMCID: PMC3427755  PMID: 22875039
16.  CONTROL OF TUMOR BIOENERGETICS AND SURVIVAL STRESS SIGNALING BY MITOCHONDRIAL HSP90s 
Cancer cell  2012;22(3):331-344.
SUMMARY
Tumors successfully adapt to constantly changing intra- and extra-cellular environments, but the wirings of this process are still largely elusive. Here, we show that Heat Shock Protein 90 (HSP90)-directed protein folding in mitochondria, but not cytosol, maintains energy production in tumor cells. Interference with this process activates a signaling network that involves phosphorylation of nutrient-sensing AMP-activated kinase (AMPK), inhibition of rapamycin-sensitive mTOR complex 1 (mTORC1), induction of autophagy, and expression of an endoplasmic reticulum (ER) unfolded protein response (UPR). This signaling network confers a survival and proliferative advantage to genetically disparate tumors, and correlates with worse outcome in lung cancer patients. Therefore, mitochondrial HSP90s are adaptive regulators of tumor bioenergetics, and tractable targets for cancer therapy.
doi:10.1016/j.ccr.2012.07.015
PMCID: PMC3615709  PMID: 22975376
HSP90; mitochondria; bioenergetics; ER; unfolded protein response; AMPK; autophagy
17.  Paracrine signaling between carcinoma cells and mesenchymal stem cells generates cancer stem cell niche via epithelial-mesenchymal transition 
Cancer discovery  2012;2(9):775-777.
Summary
Li and colleagues present data that cancer cell-derived intereleukin-1 induces prostaglandin E2 and cytokine secretion in mesenchymal stem cells (MSC) to activate β-catenin signaling in the cancer cell. This paracrine signaling between carcinoma cells and MSC leads to the creation of a cancer stem cell niche via epithelial-mesenchymal transition.
doi:10.1158/2159-8290.CD-12-0312
PMCID: PMC3646332  PMID: 22969117
18.  Melanoma-Derived Conditioned Media Efficiently Induce the Differentiation of Monocytes to Macrophages that Display a Highly Invasive Gene Signature 
Pigment cell & melanoma research  2012;25(4):493-505.
Summary
The presence of tumor-associated macrophages (TAMs) in melanomas is correlated with a poor clinical prognosis. However, there is limited information on the characteristics and biological activities of human TAMs in melanomas. In this study, we developed an in vitro method to differentiate human monocytes to macrophages using modified melanoma-conditioned medium (MCM). We demonstrate that factors from MCM-induced macrophages (MCMI-Mϕ) express both M1-Mϕ and M2-Mϕ markers, and inhibit melanoma-specific T cell proliferation. Furthermore, microarray analyses reveal that the majority of genes up-regulated in MCMI-Mϕ are associated with tumor invasion. The most strikingly up-regulated genes are CCL2 and MMP-9. Consistent with this, blockade of both CCL-2 and MMPs diminish MCMI-Mϕ-induced melanoma invasion. Finally, we demonstrate that both MCMI-Mϕ and in vivo TAMs express the pro-invasive, melanoma-associated gene, GPMNB. Our study provides a framework for understanding the mechanisms of crosstalk between TAMs and melanoma cells within the tumor microenvironment.
doi:10.1111/j.1755-148X.2012.01005.x
PMCID: PMC3615702  PMID: 22498258
Melanoma; macrophages; invasion; tumor microenvironment; GPMNB
19.  Identification of a novel family of BRAFV600E inhibitors 
Journal of Medicinal Chemistry  2012;55(11):5220-5230.
The BRAF oncoprotein is mutated in about half of malignant melanomas and other cancers, and a kinase activating single valine to glutamate substitution at residue 600 (BRAFV600E) accounts for over 90% of BRAF-mediated cancers. Several BRAFV600E inhibitors have been developed, although they harbor some liabilities, thus motivating the development of other BRAFV600E inhibitor options. We report here the use of an ELISA based high-throughput screen to identify a family of related quinolol/naphthol compounds that preferentially inhibit BRAFV600E over BRAFWT and other kinases. We also report the X-ray crystal structure of a BRAF/quinolol complex revealing the mode of inhibition, employ structure-based medicinal chemistry efforts to prepare naphthol analogs that inhibit BRAFV600E in vitro with IC50 values in the 80–200 nM under saturating ATP concentrations, and demonstrate that these compounds inhibit MAPK signaling in melanoma cells. Prospects for improving the potency and selectivity of these inhibitors are discussed.
doi:10.1021/jm3004416
PMCID: PMC3383862  PMID: 22537109
20.  Intratumoral Heterogeneity as a Therapy Resistance Mechanism: Role of Melanoma Subpopulations 
Malignant melanoma is an aggressive form of skin cancer whose incidence continues to increase worldwide. Increased exposure to sun, ultraviolet radiation and the use of tanning beds can increase the risk of melanoma. Early detection of melanomas is the key to successful treatment mainly through surgical excision of the primary tumor lesion. But in advanced stage melanomas, once the disease has spread beyond the primary site to distant organs, the tumors are difficult to treat and quickly develop resistance to most available forms of therapy. The advent of molecular and cellular techniques has led to a better characterization of tumor cells revealing the presence of heterogeneous melanoma subpopulations. The discovery of gene mutations and alterations of cell-signaling pathways in melanomas has led to the development of new targeted drugs that show dramatic response rates in patients. Single agent therapies generally target one subpopulation of tumor cells while leaving others unharmed. The surviving subpopulations will have the ability to repopulate the original tumors that can continue to progress. Thus, a rational approach to target multiple subpopulations of tumor cells with a combination of drugs instead of single agent therapy will be necessary for long-lasting inhibition of melanoma lesions. In this context, the recent development of immune checkpoint reagents provides an additional armor that can be used in combination with targeted drugs to expand the presence of melanoma reactive T-cells in circulation to prevent tumor recurrence.
doi:10.1016/B978-0-12-397927-8.00011-7
PMCID: PMC3677516  PMID: 22959031
Melanoma; tumor; heterogeneity; subpopulations; therapy; resistance
21.  GSK3β inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation 
This study addresses the role of glycogen synthase kinase (GSK)-3β signaling in the tumorigenic behavior of melanoma. Immunohistochemical staining revealed GSK3β to be focally expressed in the invasive portions of 12% and 33% of primary and metastatic melanomas, respectively. GSK3 inhibitors and siRNA knockdown of GSK3β were found to inhibit the motile behavior of melanoma cells in scratch wound, 3D collagen implanted spheroid and modified Boyden chamber assays. Functionally, inhibition of GSK3β signaling was found to suppress N-cadherin expression at the mRNA and protein levels and was associated with decreased expression of the transcription factor Slug. Pharmacological and genetic ablation of GSK3β signaling inhibited the adhesion of melanoma cells to both endothelial cells and fibroblasts and prevented transendothelial migration, an effect rescued by the forced overexpression of N-cadherin. A further role for GSK3β signaling in invasion was suggested by the ability of GSK3β inhibitors and siRNA knockdown to block phosphorylation of FAK and increase the size of focal adhesions. In summary, we have demonstrated a previously unreported role for GSK3β in modulating the motile and invasive behavior of melanoma cells through N-cadherin and FAK. These studies suggest the potential therapeutic utility of inhibiting GSK3β in defined subsets of melanoma.
doi:10.1038/jid.2012.237
PMCID: PMC3479306  PMID: 22810307
22.  Cancer Testis Antigens in human melanoma stem cells: expression, distribution and methylation status 
Journal of cellular physiology  2008;215(2):287-291.
Neoplastic populations with stem cell potential have been most recently identified in human cutaneous melanoma, and initially characterized for their phenotypic profile. Being melanoma stem cells (MSC) the most desirable target of therapeutic intervention, we asked whether they express the epigenetically-regulated Cancer Testis Antigens (CTA) on which melanoma immunotherapy is increasingly focusing. Reverse transcription-PCR analyses identified the presence of the large majority of investigated CTA (i.e., MAGE, GAGE, NY-ESO and SSX families) in different MSC populations. MSC expressed MAGE-A proteins as detected by western blot; noteworthy, the distribution of MAGE-A proteins was highly homogeneous within given MSC populations as shown by confocal immunofluorescence. Promoter methylation studies unveiled a homogeneously-demethylated MAGE-A3 promoter that paired MAGE-A3 expression in MSC. Altogether these findings demonstrate that MSC can be efficiently targeted by CTA-directed immunotherapeutic approaches, and suggest that epigenetic patterns most likely drive the expression of CTA in MSC as previously shown for melanoma cells.
doi:10.1002/jcp.21380
PMCID: PMC3624762  PMID: 18205182
cancer stem cells; melanoma; immunotherapy; DNA methylation; cancer testis antigens
23.  PLX4032, a Potent Inhibitor of the B-Raf V600E Oncogene, Selectively Inhibits V600E-positive Melanomas 
Pigment cell & melanoma research  2010;23(6):820-827.
SUMMARY
Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for melanoma patients is now underway in advanced human (Phase III) clinical trials.
doi:10.1111/j.1755-148X.2010.00763.x
PMCID: PMC3623294  PMID: 20973932
24.  Structure-based design of an organoruthenium Phosphatidyl-Inositol-3-Kinase inhibitor reveals a switch governing lipid kinase potency and selectivity 
ACS chemical biology  2008;3(5):305-316.
Mutations that constitutively activate the PI3K signaling pathway, including alterations in PI3K, PTEN and AKT are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3Kγ/E5E2 complex reveals the molecular features that give rise to this potency and selectivity towards lipid kinases with implications for the design of a subsequent generation of PI3K-isoform specific organometallic inhibitors.
doi:10.1021/cb800039y
PMCID: PMC3618672  PMID: 18484710
25.  Direct reprogramming of melanocytes to neural crest stem-like cells by one defined factor 
Stem cells (Dayton, Ohio)  2011;29(11):1752-1762.
Mouse and human somatic cells can either be reprogrammed to a pluripotent state or converted to another lineage with a combination of transcription factors suggesting that lineage commitment is a reversible process. Here we show that only one factor, the active intracellular form of Notch1, is sufficient to convert mature pigmented epidermal-derived melanocytes into functional multipotent neural crest stem-like cells. These induced neural crest stem cells (iNCSCs) proliferate as spheres under stem cell media conditions, re-express neural crest-related genes and differentiate into multiple neural crest derived mesenchymal and neuronal lineages. Moreover, iNCSCs are highly migratory and functional in ovo. These results demonstrate that mature melanocytes can be reprogrammed toward their primitive neural crest cell precursors through the activation of a single stem cell-related pathway. Reprogramming of melanocytes to iNCSCs may provide an alternate source of NCSCs for neuroregenerative applications.
doi:10.1002/stem.740
PMCID: PMC3615703  PMID: 21948558
Notch; melanocytes; neural crest stem cells; reprogramming; dedifferentiation

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