Progression Of Melanoma Cells Research Articles

Protein 4.1B Suppresses Tumor Metastasis by Regulating Epithelial-mesenchymal Transition Progression in Melanoma Cells.

Epithelial-mesenchymal transition (EMT), which involves the dramatic reorganization of the cytoskeleton, is a crucial initiating step in tumor invasion and metastasis. Protein 4.1B is a membrane-cytoskeleton cross-linker that plays an important role in tumor progression and metastasis; however, the functional roles of 4.1B in melanoma remain unclear. In this study, we aimed to investigate the effect and underlying mechanism of 4.1B on melanoma cells. Our results demonstrated that 4.1B expression was downregulated in murine B16 and B16-F10 melanoma cell lines. Ectopic 4.1B expression significantly inhibited the migration of melanoma cells and pulmonary metastasis. We further investigated the possible mechanism underlying the effect of 4.1B on EMT. The results showed that ectopic 4.1B expression altered the expression of representative EMT markers (E-cadherin, vimentin and N-cadherin), and inhibited the expression of three important transcription factors (Slug, Snail, and Twist) related to EMT in melanoma cells. Moreover, the expression of integrin α5, β3 and matrix metalloproteinase 9 (MMP-9), which is known to regulate cell adhesion, migration and invasion, were suppressed. In conclusion, our data indicate that 4.1B is an important regulator during EMT progression in melanoma cells, which may present a potential target for the prevention and treatment of melanoma.

Mitochondrial dynamic alterations regulate melanoma cell progression.

Research on mitochondrial fusion and fission (mitochondrial dynamics) has gained much attention in recent years, as it is important for understanding many biological processes, including the maintenance of mitochondrial functions, apoptosis, and cancer. The rate of mitochondrial biosynthesis and degradation can affect various aspects of tumor progression. However, the role of mitochondrial dynamics in melanoma progression remains controversial and requires a mechanistic understanding to target the altered metabolism of cancer cells. Therefore, in our study, we disrupted mitochondrial fission with mdivi-1, the reported inhibitor of dynamin related protein 1 (Drp1), and knocked down Drp1 and Mfn2 to evaluate the effects of mitochondrial dynamic alterations on melanoma cell progression. Our confocal study results showed that mitochondrial fission was inhibited both in mdivi-1 and in Drp1 knockdown cells and, in parallel, mitochondrial fusion was induced. We also found that mitochondrial fission inhibition by mdivi-1 induced cell death in melanoma cells. However, silencing Drp1 and Mfn2 did not affect cell viability, but enhanced melanoma cell migration. We further show that dysregulated mitochondrial fusion by Mfn2 knockdowns suppressed the oxygen consumption rate of melanoma cells. Together, our findings suggest that mitochondrial dynamic alterations regulate melanoma cell migration and progression.

Abstract B19: Modulation of cytotoxic effects of vemurafenib by chloroquine in malignant melanoma cells G-361: Role of dermcidin

Abstract Dermcidin (DCD) is a candidate oncogene localized at 12q13.1 and co-amplified with multiple oncogenes in many tumor cells, including breast carcinomas and melanomas. DCD is one of the 100 gene signature for melanomas. To further understand the role of DCD on growth, survival and progression of melanoma cells, we selected G-361 melanoma cell clones expressing shRNA to DCD and established in vitro and in vivo models. In this study we evaluated the pharmacologic effects of vemurafenib (BRAFV600E inhibitor) and chloroquine (autophagy inhibitor) in cell viability and tumor growth of two melanoma cell lines identified as G-361 pLKO, which expresses DCD and BRAFV600E, and G-361 IBC I which express shRNA to silence DCD constitutively. Vemurafenib induces features of stress-induced senescence in addition to apoptosis. G-361 melanoma cells responded to vemurafenib (1-2 μM) alone or combined with chloroquine (50-100 μM) which increased apoptosis rates, while decreasing senescent cells expressing β-galactosidase enzyme. Vemurafenib (50 mg/kg / 21 days) inhibited melanoma growth in immunodeficient mice dependent on DCD expression. Chloroquine (30 mg/kg) in combination with vemurafenib, accelerated (given at 24-hour interval), and reduced (given at 72 hours interval), melanoma growth. Tumor cells resistant to vemurafenib and chloroquine displayed atypical cell morphology and nuclear histological patterns and diferential expression of melanocytic differentiation biomarkers S100, HMB-45, Melan-A and pancytokeratins. This work confirms the efficacy of vemurafenib and suggests potential adjuvant effects of chloroquine. It also confirms the role of dermcidin as growth factor and oncogene for melanoma cells. Citation Format: Jennifer Montoya Neyra, Jose Belizario. Modulation of cytotoxic effects of vemurafenib by chloroquine in malignant melanoma cells G-361: Role of dermcidin [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B19.

Elevated <i>O</i>-GlcNAcylation in the tumor microenvironment promotes B16 melanoma cell progression through the suppression of p38 MAPK

Elevated <i>O</i>-GlcNAcylation in the tumor microenvironment promotes B16 melanoma cell progression through the suppression of p38 MAPK

Emerging role of RNA binding protein UNR/CSDE1 in melanoma.

The prognosis of metastatic melanoma remains poor despite recent advances in targeted and immunotherapy. Deciphering the mechanisms of tumor invasion and metastasis, and elucidating new targets for drug development could substantially improve survival in patients with metastatic melanoma. In this regard, RNA binding proteins (RBP) play an important role in RNA metabolism and are drawing considerable attention as drivers of oncogenesis and therapeutic targets. RNA binding proteins synchronize with the target RNA to play a key role in the regulation of cellular processing and are important for gene transcription and post-translational regulation (1). Wurth et al . recently published an article in Cancer Cell exploring the role of Upstream-of-N-Ras (UNR) in invasion and progression of melanoma cells (2). The UNR gene was identified as a transcription unit located immediately upstream of N-Ras in the genome of several mammalian species (3). UNR protein consists of five cold-shock domains (CSDs). These domains bind single stranded DNA and RNA and consist of ~70 amino-acid residues (4). CSD containing proteins are involved in transcriptional and post-transcriptional control of gene expression. Experiments in Drosophila species have shown the role of UNR in regulating translation of oncogenic transcripts (5).

Varying effects of EGF, HGF and TGFβ on formation of invadopodia and invasiveness of melanoma cell lines of different origin.

The understanding of melanoma malignancy mechanisms is essential for patient survival, because melanoma is responsible for ca. 75% of deaths related to skin cancers. Enhanced formation of invadopodia and extracellular matrix (ECM) degradation are two important drivers of cell invasion, and actin dynamics facilitate protrusive activity by providing a driving force to push through the ECM. We focused on the influence of epidermal growth factor (EGF), hepatocyte growth factor (HGF) and transforming growth factor β (TGFβ) on melanoma cell invasiveness, since they are observed in the melanoma microenvironment. All three factors stimulated invasion of A375 and WM1341D cells derived from primary tumor sites. In contrast, only EGF and HGF stimulated invasion of WM9 and Hs294T cells isolated from lymph node metastasis. Enhanced formation of invadopodia and ECM degradation underlie the increased amount of invasive cells after stimulation with the tested agents. Generally, a rise in invasive potential was accompanied by a decrease in actin polymerization state (F:G ratio). The F:G ratio remained unchanged or was even increased in cell lines from a metastasis treated with TGFβ. Our findings indicate that the effects of stimulation with EGF, HGF and TGFβ on melanoma cell invasiveness could depend on melanoma cell progression stage.

MiR-26b inhibits melanoma cell proliferation and enhances apoptosis by suppressing TRAF5-mediated MAPK activation

Alterations in microRNA-26b (miR-26b) expression have been shown to participate in various malignant tumor developments. However, the possible function of miR-26b in human melanoma cells remains unclarified. In this study, quantitative polymerase chain reaction was used to explore the expression profiles of miR-26b in melanoma cells. The effect of miR-26b on cell viability was determined by using MTT assays and colony formation assay. The apoptosis levels were evaluated by using Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit and the apoptosis cells were confirmed by Transmission Electron Microscopy (TEM). Luciferase reporter plasmids were constructed to confirm direct targeting. Our study found that the expression of miR-26b was downregulated in human melanoma specimens. Overexpression of miR-26b significantly increased the anti-proliferative effects and apoptosis in A375 and B16F10 melanoma cells. In addition, luciferase gene reporter assays confirmed that TRAF5 was a direct target gene of miR-26b and the anti-tumor effect of miR-26b in melanoma cells was significantly counteracted by treatment with TRAF5 overexpression. Furthermore, the molecular mechanisms underlying the tumor suppressor of miR-26b in malignant melanomas may be due to the dephosphorylation of MAPK pathway caused by the decrease in TRAF5 expression when miR-26b is up-regulated in melanoma cells. These findings indicate that miR-26b might influence TRAF5-MAPK signaling pathways to facilitate the malignant progression of melanoma cells.

Ascorbic acid and ascorbate-2-phosphate decrease HIF activity and malignant properties of human melanoma cells.

BackgroundHypoxia inducible factor-1 alpha (HIF-1α) is thought to play a role in melanoma carcinogenesis. Posttranslational regulation of HIF-1α is dependent on Prolyl hydroxylase (PHD 1–3) and Factor Inhibiting HIF (FIH) hydroxylase enzymes, which require ascorbic acid as a co-factor for optimal function. Depleted intra-tumoral ascorbic acid may thus play a role in the loss of HIF-1α regulation in melanoma. These studies assess the ability of ascorbic acid to reduce HIF-1α protein and transcriptional activity in metastatic melanoma and reduce its invasive potential.MethodsHIF-1α protein was evaluated by western blot, while transcriptional activity was measured by HIF-1 HRE-luciferase reporter gene activity. Melanoma cells were treated with ascorbic acid (AA) and ascorbate 2-phosphate (A2P) to assess their ability to reduce HIF-1α accumulation and activity. siRNA was used to deplete cellular PHD2 in order to evaluate this effect on AA’s ability to lower HIF-1α levels. A2P’s effect on invasive activity was measured by the Matrigel invasion assay. Data was analyzed by One-way ANOVA with Tukey’s multiple comparisons test, or Student-T test as appropriate, with p < .05 considered significant.ResultsSupplementation with both AA and A2P antagonized normoxic as well as cobalt chloride- and PHD inhibitor ethyl 3, 4-dihydroxybenzoate induced HIF-1α protein stabilization and transcriptional activity. Knockdown of the PHD2 isoform with siRNA did not impede the ability of AA to reduce normoxic HIF-1α protein. Additionally, reducing HIF-1α levels with A2P resulted in a significant reduction in the ability of the melanoma cells to invade through Matrigel.ConclusionThese studies suggest a positive role for AA in regulating HIF-1α in melanoma by demonstrating that supplementation with either AA, or its oxidation-resistant analog A2P, effectively reduces HIF-1α protein and transcriptional activity in metastatic melanoma cells. Our data, while supporting the function of AA as a necessary cofactor for PHD and likely FIH activity, also suggests a potential non-PHD/FIH role for AA in HIF-1α regulation by its continued ability to reduce HIF-1α in the presence of PHD inhibition. The use of the oxidation-resistant AA analog, A2P, to reduce the ability of HIF-1α to promote malignant progression in melanoma cells and enhance their response to therapy warrants further investigation.

PRL-3 Promotes the Malignant Progression of Melanoma via Triggering Dephosphorylation and Cytoplasmic Localization of NHERF1

Phosphatase of regenerating liver-3 (PRL-3) has been reported to have a critical role in metastatic progression of cancers. Here, we investigate how PRL-3 increases the malignant degree of melanoma cells. The expression of PRL-3 increased gradually during the malignant progression of melanoma. The phosphorylation of Akt was elevated in highly malignant melanoma cells, which was accompanied by a decrease in nuclear phosphatase and tensin homolog (PTEN). The phosphorylation of NHERF1 in the serine site was regulated by PRL-3 and showed cytoplasmic translocation upon dephosphorylation, which resulted in a decrease in nuclear PTEN. The co-translocation of NHERF1 and PTEN from the nucleus to the cytoplasm was observed during the malignant progression of melanoma cells. Tumor growth was inhibited significantly, and the survival was prolonged upon knockdown of cytoplasmic NHERF1 in B16BL6 cells prior to the inoculation into mice. Taken together, to our knowledge previously unreported, we have identified NHERF1 as a potential substrate of PRL-3. Its phosphorylation status as well as its change in cellular localization and association with PTEN correlated with the malignant progression of melanoma. Our data provide an explanation for how PRL-3 promotes the malignant progression of melanoma, as well as a diagnostic marker or therapeutic target for malignant melanoma.

Knockdown of EIF3D suppresses proliferation of human melanoma cells through G2/M phase arrest.

Skin cancer is the most common malignancy with increasing incidence rates worldwide. The advanced form of skin cancer, melanoma, is resistant to conventional treatment methods, which motivated researchers to identify an alternative effective therapeutic approach. This study was designed to identify the effects of small interfering RNA (si-RNA) mediated silencing of eukaryotic translation initiation factor 3, subunit D (EIF3D) against melanoma cell survival. Briefly, a lentivirus-mediated RNA interference system was employed to knock down EIF3D expression in A375 and A431 melanoma cells. The cell proliferation was analyzed by methylthiazoletetrazolium (MTT) and colony formation assays. The cell cycle progression was investigated using flow cytometry. Results revealed that si-RNA-mediated knockdown of EIF3D significantly reduced the gene and protein expression levels of EIF3D in melanoma cells. Furthermore, knockdown of EIF3D led to a significant reduction in cell proliferation due to G2 /M phase cell cycle arrest. Apparently, the study demonstrated the critical involvement of EIF3D in the survival and progression of melanoma cells and depletion of EIF3D could be developed as a possible therapeutic option in the gene-targeted treatment of melanoma.

MacroH2A suppresses the proliferation of the B16 melanoma cell line.

MacroH2A is the most frequently altered histone, which participates in cancer progression. Increasing evidence demonstrates that cancer progression could be regulated by macroH2A by affecting the cell cycle. In the present study, it was demonstrated that macroH2A suppresses melanoma cell progression and the molecular mechanisms underlying this process were examined. The interference and overexpression vectors of macroH2A were constructed and then transferred into B16 melanoma cells and, following transfection, were analyzed by quantitative polymerase chain reaction (PCR), western blot analysis and immunofluorescence assays. Apoptosis and the cell cycle stage among all the treatment groups were detected. Then, cyclin D1, cyclin D3, cyclin-dependent protein kinase (CDK) 4, CDK6 and CDK8 expression was detected in order to elucidate the effects of macroH2A on cell cycle-related genes. The results demonstrated that the overexpression of macroH2A suppressed melanoma cell progression and arrested the cells in the G2/M stage. Furthermore, macroH2A inhibits cyclinD1, cyclin D2, CDK6 and CDK8 expression in B16 melanoma cells. In conclusion, the results demonstrated that macroH2A, a critical component of chromatin, suppresses the development of melanoma (which results from a disordered cell cycle) through regulating cyclin D1, cyclin D3 and CDK6 genes.

MC1R and cAMP signaling inhibit cdc25B activity and delay cell cycle progression in melanoma cells

The melanocortin 1 receptor (MC1R) mediates the tanning response through induction of cAMP and downstream pigmentary enzymes. Diminished function alleles of MC1R are associated with decreased tanning and increased melanoma risk, which has been attributed to increased rates of mutation. We have found that MC1R or cAMP signaling also directly decreases proliferation in melanoma cell lines. MC1R overexpression, treatment with the MC1R ligand, or treatment with small-molecule activators of cAMP signaling causes delayed progression from G2 into mitosis. This delay is caused by phosphorylation and inhibition of cdc25B, a cyclin dependent kinase 1-activating phosphatase, and is rescued by expression of a cdc25B mutant that cannot be phosphorylated at the serine 323 residue. These results show that MC1R and cAMP signaling can directly inhibit melanoma growth through regulation of the G2/M checkpoint.

Selenium for the Prevention of Cutaneous Melanoma

The role of selenium (Se) supplementation in cancer prevention is controversial; effects often depend on the nutritional status of the subject and on the chemical form in which Se is provided. We used a combination of in vitro and in vivo models to study two unique therapeutic windows for intervention in the process of cutaneous melanomagenisis, and to examine the utility of two different chemical forms of Se for prevention and treatment of melanoma. We studied the effects of Se in vitro on UV-induced oxidative stress in melanocytes, and on apoptosis and cell cycle progression in melanoma cells. In vivo, we used the HGF transgenic mouse model of UV-induced melanoma to demonstrate that topical treatment with l-selenomethionine results in a significant delay in the time required for UV-induced melanoma development, but also increases the rate of growth of those tumors once they appear. In a second mouse model, we found that oral administration of high dose methylseleninic acid significantly decreases the size of human melanoma xenografts. Our findings suggest that modestly elevation of selenium levels in the skin might risk acceleration of growth of incipient tumors. Additionally, certain Se compounds administered at very high doses could have utility for the treatment of fully-malignant tumors or prevention of recurrence.

Abstract 3804: Molecular changes induced by the curcumin biphenyl analogue D6 in melanoma cells

Abstract Malignant melanoma (MM) is one of the most aggressive cancers that affect humans. It is the most common and fatal skin cancer, with an incidence that is critically increasing in Western populations (15 times in the last 40 years) and for which, in the advanced stages, there is no effective therapy. The search for new compounds able to control it is therefore essential. The hydroxylated biphenyl compound (3E,3′E)-4,4′-(5,5′,6,6′-tetramethoxy-[1,1′-biphenyl]-3,3′-diyl)bis(but-3-en-2-one), namely D6, a structural analogue of curcumin, showed a strong antitumor activity on MM cells both in vitro and in vivo, as we have recently described [1]. The mechanisms of action of this compound are not clear yet, but it inhibits cancer cells growth by inducing apoptosis without affecting normal fibroblasts growth. Firstly we investigated the ability of D6 to enter cells by Liquid Chromatography-Mass Spectrometry (LC-MS) analysis. The LC-MS data showed a quick cellular uptake of D6 with a maximum peak after about 2 hours. To investigate on the effects of D6 treatment on the cell cycle progression in MM cells, we performed flow cytometry analysis, which evidenced a cell cycle arrest in the G2/M phase. Gene expression profiles analysis using Illumina® high-density microarrays, was carried out on D6 treated MM cells and normal fibroblasts allowing the identification of 599 transcripts whose expression was modulated by D6 treatment in MM cells but not in normal fibroblast. The analysis of results, performed by Ingenuity Pathway Analysis software, showed the centrality of CDKN1A gene, encoding p21, a major negative cell cycle regulator, which was strongly over expressed, while cyclin B and cdc25 phosphatase, showed both to be down modulated. Other genes which expression interestingly appeared to be decreased by D6 activity were the oncogenes c-kit and PI3K, suggesting a down regulation of both c-kit mediated and PI3K/AKT signal transduction pathways driving to cell proliferation. Modulation of expression of these specific genes has been confirmed at a protein level by western blotting analysis. Moreover our results pointed out an upregulation of several heat shock family genes, which is probably due to a curcumin related anti inflammatory effect of D6. The preliminary results of these analysis show that D6 induces molecular changes in MM cells at both gene expression and protein levels, involving major cell proliferation regulatory pathways. [1] Marina Pisano et al. (2010).Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells. Molecular Cancer, 9:137. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3804. doi:1538-7445.AM2012-3804

MAPK and PI3K/AKT mediated YB‐1 activation promotes melanoma cell proliferation which is counteracted by an autoregulatory loop

Y-box binding protein 1 (YB-1) is an oncogenic transcription and translation factor and is overexpressed in several types of cancer. Our previous data showed that YB-1 is upregulated and translocated to the nucleus during melanoma progression and that YB-1 is an important transcription factor regulating proliferation, survival, migration, invasion and chemosensitivity of melanoma cells. It has been suggested that YB-1 is activated and translocated to the nucleus after S102-phosphorylation in the DNA binding domain. In this study, we show that activation of YB-1 by S102-phosphorylation and nuclear translocation is increased during melanoma progression using a human tissue microarray with 100 melanocytic lesions. Furthermore, we analysed the mechanisms governing the expression and activity of YB-1 in melanoma cells. We show that the PI3K/AKT and p53 signalling, growth factors and chemotherapeutic agents increase YB-1 promoter activity. This, however, resulted in no or only modest increase in YB-1 protein expression. We show that the MAPK and PI3K/AKT signalling pathways, both activated in melanoma cells, as well as p53 overexpression increase YB-1 S102-phosphorylation, whereas NFκB signalling inhibits phosphorylation. Overexpression of YB-1 in melanoma cells inhibits translation efficiency and by this proliferation and survival of melanoma cells indicating that there is an autoregulatory loop restricting YB-1 protein expression. These data suggest that there is a tightly regulated feedback mechanism regulating YB-1 expression and activation, necessary for proper cell cycle progression of melanoma cells.

Abstract 1067: FOXD3 is a B-RAFV600E-regulated inhibitor of G1/S progression in melanoma cells

Abstract The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Here we demonstrate that FOXD3 levels are up-regulated following attenuation of B-RAF and MEK signaling in mutant B-RAF harboring human melanoma cells. This effect was selective since FOXD3 was not up-regulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G1 cell cycle arrest that was largely dependent on p53 and, to a lesser extent, its downstream target p21Cip1. These studies demonstrate that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator and have implications for the repression of melanocytic lineage cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1067.

Abstract 1064: Numb, a progenitor cell fate determinant and intrinsic inhibitor of Notch signaling, is cell cycle regulated and required for proper mitotic entry and progression in melanoma cells

Abstract Proper regulation of the cell cycle, particularly DNA replication and cell division, are crucial for correct cell ploidy and genomic integrity. Loss of various regulators, often tumor suppressors, during these processes leads to either cell cycle arrest or unregulated progression through mitosis. This may result in aneuploidy and neoplastic transformation. Therefore, defining the mechanisms by which various tumor suppressors exert their regulatory functions is essential for understanding cancer development as well as developing novel target-based therapies to combat cancer progression. We found that Numb expression is cell cycle regulated and that its expression is required for proper mitotic entry and exit in melanoma cells. Using a double thymidine arrest and release protocol, we evaluated the expression of Numb during the cell cycle. Numb mRNA and protein expression was found to be maintained at a low steady state level in asynchronous A375 and HS294T melanoma cells. However, as cells entered S phase Numb levels were found to increase, peak during mitosis and then rapidly decline at mitotic exit when the cells reentered G1 Phase. This cycling of Numb mirrored the mitotic specific expression of both Plk1 and CyclinB1, however Numb showed an expression pattern with an increase beginning slightly earlier (as early as S phase). Plk1 inhibition (via shRNA as well as a small molecule inhibitor) and nocodazole-mediated mitotic arrest was also found to be accompanied with an increase in Numb expression. Additionally, we found that Plk1 and Numb co-immunoprecipitated and Plk1 was capable of phosphorylating Numb indicating a possible regulation of Numb by Plk1. Further, surprisingly, we found that lentiviral shRNA mediated knockdown of Numb resulted in a G2/M cell cycle arrest (using FACS analysis) and a significant reduction in cell growth (using Trypan blue exclusion) in both melanoma cell lines. Overall, our observations suggests that Numb is a regulator of proper cell cycle progression and may possess tumor suppressor functions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1064.

FOXD3 Is a Mutant B-RAF–Regulated Inhibitor of G1-S Progression in Melanoma Cells

The forkhead box transcription factor FOXD3 is a stemness factor that prevents the production of melanocyte progenitors from the developing neural crest; however, its role in human cancers is not known. Transformation of melanocytes gives rise to melanoma. In two thirds of melanomas, the serine/threonine kinase B-RAF is mutated to a constitutively active form. Here, we show that FOXD3 levels are upregulated following attenuation of B-RAF and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) signaling in mutant B-RAF harboring human melanoma cells. This effect was selective because FOXD3 was not upregulated following MEK inhibition in wild-type B-RAF melanoma cells and mutant B-RAF thyroid carcinoma cells. Ectopic FOXD3 expression potently inhibited melanoma cell growth without altering mutant B-RAF activation of ERK1/2. Inhibition of cell growth was due to a potent G(1) cell cycle arrest and was associated with p53-dependent upregulation of p21(Cip1). FOXD3-induced cell cycle arrest was prevented by p53 depletion and, to a lesser extent, p21(Cip1) depletion. These studies show that FOXD3 is suppressed by B-RAF, uncover a novel role and mechanism for FOXD3 as a negative cell cycle regulator, and have implications for the repression of melanocytic lineage cells.

Phosphoglucose Isomerase/Autocrine Motility Factor Promotes Melanoma Cell Migration through ERK Activation Dependent on Autocrine Production of Interleukin-8

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Nuclear PTEN levels and G2 progression in melanoma cells

The phosphatase and tensin homolog (PTEN) exerts its function, in part, by negatively regulating the well-known phosphatidylinositol-3-kinase/AKT signaling pathway. Previous histological work has suggested that alterations in the nuclear/cytoplasmic compartmentalization of PTEN may play a role in the development and progression of melanoma. In this study, we examined the nuclear/cytoplasmic compartmentalization of PTEN in melanoma cell lines and its correlation with the cell cycle. Studies were performed in melanoma cells lines using classic cell biological techniques. In contrast to breast cancer cell lines, we found that increased levels of nuclear PTEN levels correlate with G2 rather than with G1 arrest. In WM164 and SKmel28 cells, overexpression of PTEN protein did not significantly increase the number of cells in the G2 phase. Differential CDC2 phosphorylation levels in cells that overexpressed PTEN compared with those where PTEN was downregulated suggest some involvement of PTEN in G2 checkpoint regulation. The data suggest that although nuclear PTEN levels correlate with the G2 phase, the role of PTEN in modulating G2/M arrest is not limiting. Further, the specific cell cycle phase regulated by nuclear PTEN is cell-type dependent. Taken together, our observations suggest that in melanoma, nuclear PTEN is involved in G2 progression possibly through the modulation of CDC2, opening up a new arena for investigation.