MCAM/MUC18 REGULATES MELANOMA PROGRESSION BY MODULATING THE EXPRESSION OF ID-1

The acquisition of the metastatic melanoma phenotype is associated with increased expression of the melanoma cell adhesion molecule MCAM/MUC18 (CD146). However, the mechanism by which MUC18 contributes to melanoma metastasis remains unclear. Herein, we stably silenced MUC18 expression utilizing lentivirus-incorporated small hairpin RNA, in two metastatic melanoma cell lines, A375SM and C8161, and conducted cDNA microarray analysis. We identified and validated that the transcriptional regulator, Inhibitor of DNA Binding-1 (Id-1), previously shown to function as an oncogene in several malignancies, was downregulated by 5.6-fold following MUC18 silencing. Additionally, we found that MUC18 regulated Id-1 expression at the transcriptional level via ATF-3. Interestingly, ATF-3 was upregulated by 6.9 fold in our cDNA microarray analysis following MUC18 silencing. ChIP analysis showed increased binding of ATF-3 to the Id-1 promoter after MUC18 silencing, while mutation of the ATF-3 binding site on the Id-1 promoter increased Id-1 promoter activity in MUC18-silenced cells. These Data suggest that MUC18 silencing promotes inhibition of Id-1 expression by increasing ATF-3 expression and binding to the Id-1 promoter. Rescue of MUC18 reverted the expression of Id-1 and ATF-3, thus validating that they are not off-target effects of MUC18. To further assess the role of Id-1 in melanoma invasion and metastasis, we overexpressed Id-1 in MUC18-silenced cells. Overexpression of Id-1 in MUC18-silenced cells resulted in increased cell invasion, as well as increased expression and activity of MMP-2. Our data further reveal that Id-1 regulates MMP-2 at the transcriptional level through Sp1 and Ets-1. This is the first report to demonstrate that MUC18 does not act exclusively in cell adherence, but is also involved in cell signaling that regulates the expression of genes, such as Id-1 and ATF-3, thus contributing to the metastatic melanoma phenotype

Src activation by β-adrenoreceptors is a key switch for tumor metastasis

Norepinephrine (NE) can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here, we identify Src as a key regulator of phosphoproteomic signaling networks activated in response to beta-adrenergic signaling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumor cell migration, invasion and growth. In human ovarian cancer samples, high tumoral NE levels were correlated with high pSrcY419 levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signaling in the tumor microenvironment

CREB Inhibits AP-2α Expression to Regulate the Malignant Phenotype of Melanoma

The loss of AP-2alpha and increased activity of cAMP-responsive element binding (CREB) protein are two hallmarks of malignant progression of cutaneous melanoma. However, the molecular mechanism responsible for the loss of AP-2alpha during melanoma progression remains unknown.Herein, we demonstrate that both inhibition of PKA-dependent CREB phosphorylation, as well as silencing of CREB expression by shRNA, restored AP-2alpha protein expression in two metastatic melanoma cell lines. Moreover, rescue of CREB expression in CREB-silenced cell lines downregulates expression of AP-2alpha. Loss of AP-2alpha expression in metastatic melanoma occurs via a dual mechanism involving binding of CREB to the AP-2alpha promoter and CREB-induced overexpression of another oncogenic transcription factor, E2F-1. Upregulation of AP-2alpha expression following CREB silencing increases endogenous p21(Waf1) and decreases MCAM/MUC18, both known to be downstream target genes of AP-2alpha involved in melanoma progression.Since AP-2alpha regulates several genes associated with the metastatic potential of melanoma including c-KIT, VEGF, PAR-1, MCAM/MUC18, and p21(Waf1), our data identified CREB as a major regulator of the malignant melanoma phenotype

Targeting polyIC to EGFR over-expressing cells using a dsRNA binding protein domain tethered to EGF

<div><p>Selective delivery of drugs to tumor cells can increase potency and reduce toxicity. In this study, we describe a novel recombinant chimeric protein, dsRBEC, which can bind polyIC and deliver it selectively into EGFR over-expressing tumor cells. dsRBEC, comprises the dsRNA binding domain (dsRBD) of human PKR (hPKR), which serves as the polyIC binding moiety, fused to human EGF (hEGF), the targeting moiety. dsRBEC shows high affinity towards EGFR and triggers ligand-induced endocytosis of the receptor, thus leading to the selective internalization of polyIC into EGFR over-expressing tumor cells. The targeted delivery of polyIC by dsRBEC induced cellular apoptosis and the secretion of IFN-β and other pro-inflammatory cytokines. dsRBEC-delivered polyIC is much more potent than naked polyIC and is expected to reduce the toxicity caused by systemic delivery of polyIC.</p></div

Analysis of dsRBEC activity.

<p><b>A)</b> EMSA showing reduced mobility in 2% agarose of polyIC/dsRBEC complexes. Lane 1, polyIC (0.5 μg); Lanes 2–5, polyIC (0.5 μg) pre-incubated with dsRBEC (0.5–4 μg). <b>B)</b> Displacement of ¹²⁵-I EGF by dsRBEC (■) or unlabeled hEGF (▲) in A431 cells. The graph shows means ±SD from a representative experiment, performed using duplicate samples. The Kd was calculated as the mean from three independent experiments. <b>C)</b> Western blot analysis of EGFR phosphorylation following treatment of MDA-MB-468 cells with dsRBEC at various concentrations for 15 minutes.</p

PolyIC/dsRBEC induces the expression and secretion of pro-inflammatory cytokines in MDA-MB-468 cells.

<p><b>A)</b> qRT-PCR analysis of IFN-β, CCL5, IP10 and TNFα mRNA expression following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 2 and 4 hours. Data were normalized to GAPDH and are expressed as fold change relative to vehicle-treated samples. A representative experiment out of 3 experiments is shown. Error bars represent RQ max. <b>B)</b> Protein levels of IFN-β, CCL5, IP10 and TNFα were measured by ELISA following treatment with dsRBEC alone, polyIC alone or polyIC/dsRBEC for 24 hours. Values are averages of triplicate biological samples from one representative experiment. (***,P<0.0001 for effect of polyIC/dsRBEC vs polyIC alone, for polyIC/dsRBEC vs dsRBEC alone and for polyIC/dsRBEC vs vehicle).</p

Purification of dsRBEC.

<p><b>A)</b> dsRBEC was purified on Ni Sepharose under native conditions. Samples of total lysate (T), soluble fraction (S), unbound fraction (UB) and eluate (EL) were electrophoresed by SDS-PAGE. dsRBEC was visualized with Coomassie dye and by western blot analysis using an anti-His antibody. <b>B)</b> Samples of EL electrophoresed on 1% agarose and stained with ethidium bromide. Treatment with 10μg/ml RNase A eliminated staining. <b>C)</b> dsRBEC was purified on Ni Sepharose under denaturing conditions <b><i>(</i></b>4M urea). Samples of total lysate (T), soluble fraction (S), unbound fraction (UB) and eluate (EL) were analyzed as in (A). <b>D)</b> Equal amounts of eluted protein, isolated under native or denaturing conditions, were electrophoresed and stained with ethidium bromide. <b>E)</b> SDS-PAGE analysis of final dsRBEC purification, (15% bis-acrylamide gel stained with Coomassie dye) T, total crude lysate before purification; Ni, eluate from 4 ml Ni Sepharose column; S-75, eluate from final purification on Superdex75 (Uncropped gel can be visualized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162321#pone.0162321.s002" target="_blank">S2 Fig</a>).</p