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

Amino terminal hydrophobic import signals target the p14ARF tumor suppressor to the mitochondria

The p14ARF tumor suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. this allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.11 page(s

Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma

Acquired resistance to BRAF inhibitors often involves MAPK re-activation, yet the MEK inhibitor trametinib showed minimal clinical activity in melanoma patients that had progressed on BRAF-inhibitor therapy. Selective ERK inhibitors have been proposed as alternative salvage therapies. We show that ERK inhibition is more potent than MEK inhibition at suppressing MAPK activity and inhibiting the proliferation of multiple BRAF inhibitor resistant melanoma cell models. Nevertheless, melanoma cells often failed to undergo apoptosis in response to ERK inhibition, because the relief of ERK-dependent negative feedback activated RAS and PI3K signalling. Consequently, the combination of ERK and PI3K/mTOR inhibition was effective at promoting cell death in all resistant melanoma cell models, and was substantially more potent than the MEK/PI3K/mTOR inhibitor combination. Our data indicate that a broader targeting strategy concurrently inhibiting ERK, rather than MEK, and PI3K/mTOR may circumvent BRAF inhibitor resistance, and should be considered during the clinical development of ERK inhibitors.11 page(s

Control of NF-kB activity in human melanoma by bromodomain and extra-terminal protein inhibitor I-BET151

The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role.12 page(s

The epigenetic regulator I-BET151 induces BIM-dependent apoptosis and cell cycle arrest of human melanoma cells

Epigenetic changes are widespread in melanoma and contribute to the pathogenic biology of this disease. In the present study, we show that I-BET151, which belongs to a new class of drugs that target the BET family of epigenetic "reader" proteins, inhibits melanoma growth in vivo and induced variable degrees of apoptosis in a panel of melanoma cells. Apoptosis was caspase dependent and associated with G1 cell cycle arrest. All melanoma cells tested had increased levels of the BH3 proapoptotic protein BIM, which appeared to be regulated by the BRD2 BET protein and to some extent by BRD3. In contrast, knockdown experiments indicated that inhibition of BRD4 was associated with decreased levels of BIM. Apoptosis was dependent on BIM in some but not all cell lines, indicating that other factors were determinants of apoptosis, such as downregulation of antiapoptotic proteins revealed in gene expression arrays. G1 cell cycle arrest appeared to be mediated by p21 and resulted from inhibition of the BRD4 protein. The activity of BET protein inhibitors appears independent of the BRAF and NRAS mutational status of melanoma, and further studies to assess their therapeutic role in melanoma are warranted.11 page(s

Identification of a single amino acid residue which is critical for the interaction between HSV-1 inner tegument proteins pUL36 and pUL37

The herpes simplex virus type 1 (HSV-1) structural tegument proteins pUL36 and pUL37 are essential for secondary envelopment during the egress of viral particles. For this study, scanning alanine mutagenesis of HSV-1 pUL37, in combination with yeast two-hybrid, identified pUL37 residue D631 as a major determinant for binding of pUL36. Further analysis of the binding of this pUL37 mutant to pUL36 by coimmunoprecipitation assay confirmed the role of pUL37 D631 in mediating binding of pUL36. A trans-complementation assay using pUL37 deletion virus FRΔUL37 was then carried out, where pUL37 wild type or D631A were provided in trans. For pUL37 D631A, a significant reduction in virus titer was observed compared to that seen when pUL37 wild type was present. The results presented here underline the crucial role of the pUL36/pUL37 interaction in replication of HSV-1 and indicate a critical role for pUL37 D631 in mediating this interaction

BRAF inhibitor resistance mechanisms in metastatic melanoma : spectrum and clinical impact

Purpose: Multiple BRAF inhibitor resistance mechanisms have been described, however, their relative frequency, clinical correlates, and effect on subsequent therapy have not been assessed in patients with metastatic melanoma. Experimental Design: Fifty-nine BRAFV600-mutant melanoma metastases from patients treated with dabrafenib or vemurafenib were analyzed. The genetic profile of resistance mechanisms and tumor signaling pathway activity was correlated with clinicopathologic features and therapeutic outcomes. Results: Resistance mechanisms were identified in 58% progressing tumors and BRAF alterations were common. Gene expression analysis revealed that mitogen-activated protein kinase (MAPK) activity remained inhibited in 21% of resistant tumors, and the outcomes of patients with these tumors were poor. Resistance mechanisms also occurred in pretreatment biopsies and heterogeneity of resistance mechanisms occurred within patients and within tumors. There were no responses to subsequent targeted therapy, even when a progressing tumor had a resistance mechanism predicted to be responsive. Conclusions: Selecting sequential drugs based on the molecular characteristics of a single progressing biopsy is unlikely to provide improved responses, and first-line therapies targeting multiple pathways will be required.13 page(s