Oncogenic B-RAFV600E Signaling Induces the T-Box3 Transcriptional Repressor to Repress E-Cadherin and Enhance Melanoma Cell Invasion

Approximately 50% of melanomas require oncogenic B-RAFV600E signaling for proliferation, survival, and metastasis, and the use of highly selective B-RAF inhibitors has yielded remarkable, although short-term, clinical responses. Reactivation of signaling downstream of B-RAF is frequently associated with acquired resistance to B-RAF inhibitors, and the identification of B-RAF targets may therefore provide new strategies for managing melanoma. In this report, we applied whole-genome expression analyses to reveal that oncogenic B-RAFV600E regulates genes associated with epithelial–mesenchymal transition in normal cutaneous human melanocytes. Most prominent was the B-RAF-mediated transcriptional repression of E-cadherin, a keratinocyte–melanoma adhesion molecule whose loss is intimately associated with melanoma invasion and metastasis. Here we identify a link between oncogenic B-RAF, the transcriptional repressor Tbx3, and E-cadherin. We show that B-RAFV600E induces the expression of Tbx3, which potently represses E-cadherin expression in melanocytes and melanoma cells. Tbx3 expression is normally restricted to developmental embryonic tissues and promoting cell motility, but it is also aberrantly increased in various cancers and has been linked to tumor cell invasion and metastasis. We propose that this B-RAF/Tbx3/E-cadherin pathway has a critical role in promoting the metastasis of B-RAF-mutant melanomas

Inducible but not constitutive expression of PD-L1 in human melanoma cells is dependent on activation of NF-κB.

Monoclonal antibodies against immune checkpoint blockade have proven to be a major success in the treatment of melanoma. The programmed death receptor-1 ligand-1 (PD-L1) expression on melanoma cells is believed to have an inhibitory effect on T cell responses and to be an important escape mechanism from immune attack. Previous studies have shown that PD-L1 can be expressed constitutively or can be induced by IFN-γ secreted by infiltrating lymphocytes. In the present study we have investigated the mechanism underlying these two modes of PD-L1 expression in melanoma cells including cells that had acquired resistance to the BRAF inhibitor vemurafenib. PD-L1 expression was examined by flow cytometry and immunoblotting. Specific inhibitors and siRNA knockdown approaches were used to examine the roles of the RAF/ MEK, PI3K, NF-κB, STAT3 and AP1/ c-Jun pathways. IFN-γ inducible expression of PD-L1 was dependent on NF-κB as shown by inhibition with BMS-345541, an inhibitor of IκB and the BET protein inhibitor I-BET151, as well as by siRNA knockdown of NF-κB subunits. We were unable to implicate the BRAF/MEK pathway as major regulators in PD-L1 expression on vemurafenib resistant cells. Similarly the PI3K/AKT pathway and the transcription factors STAT3 and c-Jun had only minor roles in IFN-γ induced expression of PD-L1. The mechanism underlying constitutive expression remains unresolved. We suggest these results have significance in selection of treatments that can be used in combination with monoclonal antibodies against PD1, to enhance their effectiveness and to reduce inhibitory effects melanoma cells have against cytotoxic T cell activity

Acquired resistance to BRAF inhibition can confer cross-resistance to combined BRAF/MEK inhibition

Aberrant activation of the BRAF kinase occurs in ~60% of melanomas, and although BRAF inhibitors have shown significant early clinical success, acquired resistance occurs in most patients. Resistance to chronic BRAF inhibition often involves reactivation of mitogen-activated protein kinase (MAPK) signaling, and the combined targeting of BRAF and its downstream target MAPK/ERK kinase (MEK) may delay or overcome resistance. To investigate the efficacy of combination BRAF and MEK inhibition, we generated melanoma cell clones resistant to the BRAF inhibitor GSK2118436. These BRAF inhibitor-resistant sublines acquired resistance through several distinct mechanisms, including the acquisition of activating N-RAS mutations and increased accumulation of COT1. These alterations uniformly promoted MAPK reactivation and most conferred resistance to MEK inhibition and to the concurrent inhibition of BRAF and MEK. These data indicate that melanoma tumors are likely to develop heterogeneous mechanisms of resistance, many of which will confer resistance to multiple MAPK inhibitory therapies. Corrigendum exists for this article and can be found in the Journal of investigative dermatology, 133(10), p. 2493, doi:10.1038/jid.2013.12510 page(s

Characterization of the Host Immune Response in Human Ganglia after Herpes Zoster▿

Varicella-zoster virus (VZV) causes varicella (chicken pox) and establishes latency in ganglia, from where it reactivates to cause herpes zoster (shingles), which is often followed by postherpetic neuralgia (PHN), causing severe neuropathic pain that can last for years after the rash. Despite the major impact of herpes zoster and PHN on quality of life, the nature and kinetics of the virus-immune cell interactions that result in ganglion damage have not been defined. We obtained rare material consisting of seven sensory ganglia from three donors who had suffered from herpes zoster between 1 and 4.5 months before death but who had not died from herpes zoster. We performed immunostaining to investigate the site of VZV infection and to phenotype immune cells in these ganglia. VZV antigen was localized almost exclusively to neurons, and in at least one case it persisted long after resolution of the rash. The large immune infiltrate consisted of noncytolytic CD8+ T cells, with lesser numbers of CD4+ T cells, B cells, NK cells, and macrophages and no dendritic cells. VZV antigen-positive neurons did not express detectable major histocompatibility complex (MHC) class I, nor did CD8+ T cells surround infected neurons, suggesting that mechanisms of immune control may not be dependent on direct contact. This is the first report defining the nature of the immune response in ganglia following herpes zoster and provides evidence for persistence of non-latency-associated viral antigen and inflammation beyond rash resolution

Productive Varicella-Zoster Virus Infection of Cultured Intact Human Ganglia▿

Varicella-zoster virus (VZV) is a species-specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and nonneuronal cells. Enveloped virions were visualized by transmission electron microscopy in neurons and nonneuronal cells and within the extracellular space. Moreover, rather than remaining highly cell associated during infection of cultured cells, such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG

Targeting NF-κB reduces PD-L1.

<p><b>A</b>. KMJR138 cells were treated with DMSO (control) or 100ng/ml IFN-γ (IFN) or indicated doses of BMS-345541 in the presence of IFN-γ for 48 hours before PD-L1 was analysed by flow cytometry. Mean Fluorescence Intensity (MFI) of PD-L1 expression is indicated; the average of two independent replicates is shown. <b>B</b>. Indicated cells were treated with either dimethyl sulfoxide (DMSO) (control) or 10μM of I-BET151 (IBET) or 5μM of BMS-345541 (BMS) for 48 hours in the absence or presence of 100ng/ml IFN-γ and PD-L1 cell surface expression was determined by flow cytometry. Mean fluorescence intensity (MFI) plotted is the average of three independent experiments. <b>C</b>. Sub-cellular fractionation of cellular lysates from a representative cell line (KMJR138) was probed for the indicated proteins. Cells were treated with DMSO (control), IFN-γ (IFN, +), I-BET151 (IBET), BMS-345541 (BMS) for 48 hours and separated into cytosolic (C) or nuclear (N) fractions. One representative blot out of two independent experiments is shown. <b>D</b>. KMJR138 cells treated with DMSO (control) or 100ng/ml IFN-γ, 5μM BMS-345541 or a combination of both for 48 hours in the absence or presence of 10μM pan-caspase inhibitor Q-VD-OPh were stained with Annexin V-APC, and PI to determine the number of apoptotic cells (left panel) and anti-PD-L1 antibody to determine the expression of PD-L1 (right panel) which is represented as the mean fluorescence intensity (MFI) over isotype controls. Average value from three independent experiments is indicated. <b>E.</b> Dual renilla-luciferase NF-κB reporter assay: KMJR138 cells were transfected with the control and NF-κB constructs for 24 hours and treated with the indicated inhibitors (10μM I-BET151 or 5μM BMS-345541) with or without 100ng/ml IFN-γ. Luciferase was measured and normalised to renilla values to determine promoter activity. Transfected cells were treated with the inhibitors for 24 hours and IFN-γ induction was carried out 1 hour prior to harvesting and recording luminescence. ***p-value = 0.0001; **p-value = 0.001; *p-value = 0.01. Average of two independent replicates experiments is indicated</p

STAT3 and c-Jun independent expression of inducible PD-L1.

<p><b>A</b>. KMJR138 cells were transduced using lentiviral constructs of either control pSIH-copGFP (-) or STAT3-copGFP (+) for 72 hours and treated with 5μM BMS-345541 (BMS) with or without 100ng/ml IFN-γ or DMSO (control) for an additional 48 hours. A representative immunoblot (out of three independent blots) for the indicated proteins is shown. <b>B</b>. KMJR138 cells were transfected with control siRNA (-) or with silencer (si) molecules against c-Jun for 48 hours and treated with DMSO (control) or IFN-γ (IFN) for an additional 48 hours and cellular lysates were immunoblotted for the indicated proteins. A representative blot out of two independent experiments is shown.</p