Targeted therapy for melanoma has primarily centered on the RAS/MEK/ERK pathway

Targeted therapy for melanoma has primarily centered on the RAS/MEK/ERK pathway because of the prevalence of B-RAF and N-RAS mutations which are located in more than 50% of melanomas for B-RAF and 15-30% for N-Ras (Sekulic et al. tumors to PLX4032 was also seen in some patients as well as in V600E BRAF-bearing melanoma cell lines (Bollag et al. 2010 Flaherty et al. 2010 Lee et al. 2010 These observations underscore the plasticity of melanoma in acquiring resistance to chemotherapeutic brokers and highlight the importance of targeting multiple signaling pathways in order to achieve a sustainable therapeutic response. Two additional signaling pathways known to be dysregulated in melanoma are the AKT and NF-κB pathways. These pathways have been intensively studied over the past two decades for target discovery and small molecule inhibitor development (Madhunapantula and Robertson 2009 The protein kinase AKT is usually activated downstream of class I phosphatidylinositol-3-kinase (PI3K) which generates the lipid second messenger phosphatidylinositol-3 4 5 (PIP3) from its precursor phosphatidylinositol 4 5 (PIP2). Activation of PI3K by growth factor receptor tyrosine kinases or heterotrimeric G-protein coupled receptors results in the generation of PIP3 which subsequently recruits AKT to the plasma membrane and results in the dual phosphorylation of Rabbit Polyclonal to ZAK. AKT at T308 and S473 by the protein kinases PDK1 and mTOR respectively (Alessi et al. 1997 Sarbassov et al. 2005 Following these activating phosphorylation events AKT in turn phosphorylates a large number of downstream substrates involved in apoptosis cell-cycle regulation protein translation and metabolism (Bhaskar and Hay 2007 646502-53-6 Plas and Thompson 2005 AKT activation is usually antagonized by the tumor suppressor PTEN a lipid phosphatase that dephosphorylates PIP3 to PIP2. In melanoma activation of AKT occurs in approximately 50% of human melanomas with inactivation of PTEN by mutation or epigenetic silencing observed in approximately 60% of these cases (Dhawan et al. 2002 Robertson 2005 The NF-κB family of transcription factors is certainly involved with a diverse spectral range of mobile processes including irritation cell cycle legislation cell migration/invasion and apoptosis (Ghosh and Karin 2002 Within the canonical NF-κB pathway the transcriptional activity of NF-κB is certainly tightly governed by IκB binding which sequesters NF-κB within the cytoplasm. Activation from the canonical NF-κB pathway by ligands such as for example TNF-α results in the activation from the IκB kinase (IKK) complicated which includes the kinase subunits IKKα IKKβ as well as the regulatory subunit IKKγ (NEMO). The turned on 646502-53-6 IKK complicated phosphorylates IκB triggering its proteasomal degradation and enabling the nuclear translocation of NF-κB and transcription of focus on genes such as for example IAP Turn Bcl-XL and cyclin D (Deveraux et al. 1998 Guttridge et al. 1999 Micheau et al. 2001 Ravi et al. 2001 In melanoma constitutive activation of the NF-κB pathway is frequently observed due to upregulation of IKK activity either by increased expression of IKK activating kinases or by increased expression of cytokines and chemokines arising from B-RAF or N-RAS mutation (Dhawan and Richmond 2002 Norris and Baldwin 1999 646502-53-6 Ueda and Richmond 2006 Yang and Richmond 2001 Furthermore the requirement for IKK activity in melanoma is usually underscored by a recent publication whereby deletion of IKKβ abrogates H-Ras driven melanoma development in Ink4a/Arf ?/? mice (Yang et al. 2010 We have been studying BI-69A11 a small molecule inhibitor of AKT originally recognized through an in silico approach (Forino et al. 2005 BI-69A11 binds to the AKT catalytic site and inhibits the kinase activity in an ATP-competitive manner with an IC50 of 2.3μM in vitro (Forino et al. 2005 In cell-based assays using melanoma prostate and breast cell lines BI-69A11 reduced AKT S473 phosphorylation and inhibited cell proliferation via increased cell death. In particular cells that exhibited elevated AKT activity such as UACC 903 cells harboring both B-RAF mutation and PTEN inactivation were more sensitive to cell killing by BI-69A11 (Gaitonde et al. 2009 Furthermore BI-69A11 effectively inhibited melanoma growth as tumor 646502-53-6 xenografts in vivo (Gaitonde et al. 2009 In this study we further characterize the AKT inhibitor BI-69A11 and find that in addition to its AKT inhibitory activity BI-69A11 also targets the NF-κB pathway through a mechanism that is consistent with sphingosine-1-kinase inhibition. Importantly the dual targeting of both the AKT and NF-κB pathways is essential for the inhibition of.