Nuclear Factor-kappa B (NF-kappa B) is an inducible transcription factor that regulates the expression of many genes involved in the immune response. Recently, NF-kappa B activity has been shown to be upregulated in many cancers, including melanoma. Data indicate that the enhanced activation of NF-kappa B may be due to deregulations in upstream signaling pathways such as Ras/Raf, PI3K/Akt, and NIK. Multiple studies have shown that NF-kappa B is involved in the regulation of apoptosis, angiogenesis, and tumor cell invasion, all of which indicate the important role of NF-kappa B in tumorigenesis. Thus, understanding the molecular mechanism of melanoma progression will aid in designing new therapeutic approaches for melanoma. In this review, the association between NF-kappa B and melanoma tumorigenesis are discussed. Additionally, the potential of emerging selective NF-kappa B inhibitors for the treatment of melanoma is reviewed.

Purpose: Constitutive activation of inhibitor of nB kinase (IKK) confers melanoma resistance to apoptosis and chemotherapy.Whether IKK is able to serve as a therapeutic target in melanoma is unknown. We explored the possibility of exploiting IKK as a therapeutic target in melanoma by using BMS-345541, a novel compound with a highly selective IKKh inhibitory activity, to trigger melanoma cell apoptosis. Experimental Design:Three human melanoma cell lines (SK-MEL-5, Hs 294T, and A375), all of which have high constitutive IKK activities, served as in vitro and in vivo melanoma models for treatment with BMS-345541. Two known antitumor drugs (temozolomide and bortezomib) were used as parallel controls for evaluation of the therapeutic efficiency and toxicity of BMS-345541. The effects of BMS-345541on nuclear factor nB (NF-nB) signaling and on the apoptosis machinery were investigated. Results: Inhibition of constitutive IKK activity by BMS-345541resulted in the reduction of NF-nB activity, CXCL1 chemokine secretion by cultured melanoma cells and melanoma cell survival in vitro and in vivo. The effect of BMS-345541on tumor cell growth was through mitochondriamediated apoptosis, based on the release of apoptosis-inducing factor, dissipation of mitochondrial membrane potential, and reduced ratio of B cell lymphoma gene-2 (Bcl-2)/Bcl-associated X protein (Bax) in mitochondria. The BMS-345541execution of apoptosis was apoptosis-inducing factor^dependent, but largely caspase-independent. Conclusion: BMS-345541 down-regulation of IKK activity results in mitochondria-mediated apoptosis of tumor cells because the programmed cell death machinery in melanoma cells is highly regulated by NF-nB signaling.Therefore, IKK may serve as a potential target for melanoma therapy.

Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.

Purpose The purpose of this preclinical study was to determine the effectiveness of RAF265, a multi-kinase inhibitor, for treatment of human metastatic melanoma and to characterize traits associated with drug response. Experimental Design Advanced metastatic melanoma tumors from 34 patients were orthotopically implanted to nude mice. Tumors that grew in mice (17 of 34) were evaluated for response to RAF265 (40 mg/kg, every day) over 30 days. The relation between patient characteristics, gene mutation profile, global gene expression profile, and RAF265 effects on tumor growth, mitogen-activated protein/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation, proliferation, and apoptosis markers was evaluated. Results Nine of the 17 tumors that successfully implanted (53%) were mutant BRAF (BRAFV600E/K), whereas eight of 17 (47%) tumors were BRAF wild type (BRAFWT). Tumor implants from 7 of 17 patients (41%) responded to RAF265 treatment with more than 50% reduction in tumor growth. Five of the 7 (71%) responders were BRAFWT, of which 1 carried c-KITL576P and another N-RASQ61R mutation, while only 2 (29%) of the responding tumors were BRAFV600E/K. Gene expression microarray data from nonimplanted tumors revealed that responders exhibited enriched expression of genes involved in cell growth, proliferation, development, cell signaling, gene expression, and cancer pathways. Although response to RAF265 did not correlate with pERK1/2 reduction, RAF265 responders did exhibit reduced pMEK1, reduced proliferation based upon reduced Ki-67, cyclin D1 and polo-like kinase1 levels, and induction of the apoptosis mediator BCL2-like 11. Conclusions Orthotopic implants of patient tumors in mice may predict prognosis and treatment response for melanoma patients. A subpopulation of human melanoma tumors responds to RAF265 and can be characterized by gene mutation and gene expression profiles.

Melanoma poses a great challenge to patients, oncologists, and biologists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor B is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor B activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor B by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1-10 nM and in combination with the chemotherapeutic agent temozolomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strikingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma.

Constitutive activation of the transcription factor nuclear factor-κB (NF-κB) plays a major role in inflammatory diseases as well as cancer by inducing the endogenous expression of many proinflammatory proteins such as chemokines, and facilitating escape from apoptosis. The constitutive expression of chemokines such as CXCL1 has been correlated with growth, angiogenesis, and metastasis of cancers such as melanoma. The transcription of CXCL1 is regulated through interactions of NF-κB with other transcriptional regulatory molecules such as poly(ADP-ribose) polymerase-1 (PARP-1) and cAMP response element binding protein (CREB)-binding protein (CBP). It has been proposed that these two proteins interact with NF-κB and other enhancers to form an enhanceosome at the promoter region of CXCL1 and modulate CXCL1 transcription. In addition to these positive cofactors, a negative regulator, CAAT displacement protein (CDP), may also be involved in the transcriptional regulation of CXCL1. It has been postulated that the elevated expression of CXCL1 in melanomas is due to altered interaction between these molecules. CDP interaction with the promoter down-regulates transcription, whereas PARP and/or CBP interactions enhance transcription. Thus, elucidation of the interplay between components of the enhanceosome of this gene is important in finding more efficient and new therapies for conditions such as cancer as well as acute and chronic inflammatory diseases. I. Chemotactic CytokinesChemokines are small, proinflammatory, inducible, secreted cytokines that are involved in trafficking, activation, and proliferation of many cell types such as myeloid, lymphoid, pigment epidermal, and endothelial cells (1). Chemokine proteins are encoded by 70-130 amino acids, which also include a signal peptide sequence of 20-25 amino acids. It is interesting to note that although chemokines share little homology in their primary sequence, their overall tertiary structure is similar (2). Chemokines have been observed to form dimers in concentrated solutions and on crystallization, however, these concentrations are much higher than the biological concentrations. It is now commonly accepted that chemokines act as monomers in biological systems (2-4). To date, over 50 chemokines have been identified and assigned to four classes according to their arrangement of the first two of four conserved cysteine residues: C, CC, CXC, and CX 3 C chemokines (Table I). The C chemokines such as lymphotactin (XCL1) lack two of the four conserved cysteine residues, whereas CC chemokines have the first two cysteines adjacent to each other; examples are chemoattractant protein-1 (CCL2), macrophage inflammatory protein-1α (CCL3), and Copyright 2003, Elsevier (USA). All rights reserved. NIH Public Access Author ManuscriptProg Nucleic Acid Res Mol Biol. Author manuscript; available in PMC 2011 July 20. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript regulated upon activation of normal T cells expressed and secreted (CCL5). In CX 3 C ch...

The continuous production of the CXC ligand 1 (CXCL1) chemokine by melanoma cells is a major effector of tumor growth. We have previously shown that the constitutive expression of this chemokine is dependent upon transcription factors nuclear factor-kappa B (NF-jB), stimulating protein-1 (SP1), high-mobility group-I/Y (HMGI/Y), CAAT displacement protein (CDP) and poly(ADPribose) polymerase-1 (PARP-1). In this study, we demonstrate for the first time the mechanism of transcriptional regulation of CXCL1 through PARP-1 in melanoma cells. In its inactive state, PARP-1 binds to the CXCL1 promoter in a sequence-specific manner and prevents binding of NF-jB (p65/p50) to its element. However, activation of the PARP-1 enzymatic activity enhances CXCL1 expression, owing to the loss of PARP-1 binding to the CXCL1 promoter, accompanied by enhanced binding of p65 to the promoter. The delineation of the role of NF-jB-interacting factors in the putative CXCL1 enhanceosome will provide key information in developing strategies to block constitutive expression of this and other chemokines in cancer and to develop targeted therapy.

Purpose Preclinical studies show that bortezomib, a proteasome inhibitor, blocks NF-κB activation and, combined with temozolomide, enhances activity against human melanoma xenografts and modulates other critical tumor targets. We initiated a phase I trial of temozolomide plus bortezomib in advanced melanoma. Objectives included defining a maximum tolerated dose for the combination, characterizing biomarker changes reflecting inhibition of both proteasome and NF-κB activity in blood (if possible tumor), and characterizing antitumor activity. Experimental Design Cohorts were enrolled onto escalating dose levels of temozolomide (50-75 mg/m2) daily, orally, for 6 of 9 weeks and bortezomib (0.75-1.5 mg/m2) by i.v. push on days 1, 4, 8, and 11 every 21 days. Peripheral blood mononuclear cells were assayed at specified time points for proteasome inhibition and NF-κB biomarker activity. Results Bortezomib (1.3 mg/m2) and temozolomide (75 mg/m2) proved to be the maximum tolerated dose. Dose-limiting toxicities included neurotoxicity, fatigue, diarrhea, and rash. Nineteen melanoma patients were enrolled onto four dose levels. This melanoma population (17 M1c, 10 elevated lactate dehydrogenase, 12 performance status 1-2) showed only one partial response (8 months) and three with stable disease ≥4 months. A significant reduction in proteasome-specific activity was observed 1 hour after infusion at all bortezomib doses. Changes in NF-κB electrophoretic mobility shift assay and circulating chemokines in blood failed to correlate with the schedule/dose of bortezomib, inhibition of proteasome activity, or clinical outcome. Conclusions We have defined phase II doses for this schedule of temozolomide with bortezomib. Although proteasome activity was inhibited for a limited time in peripheral blood mononuclear cells, we were unable to show consistent effects on NF-κB activation.