BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma

B-RAF is mutated to a constitutively active form in 8% of human cancers including 50% of melanomas. In clinical trials, the RAF inhibitor, PLX4032 (vemurafenib), caused partial or complete responses in 48–81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 6–7 months before tumor regrowth, indicating the acquisition of resistance to PLX4032. To understand the mechanisms of resistance, we developed mutant B-RAF melanoma cells that displayed resistance to RAF inhibition through continuous culture with PLX4720 (the tool compound for PLX4032). Resistance was associated with a partial reactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, recovery of G1/S cell-cycle events, and suppression of the pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins, Bcl-2-interacting mediator of cell death-extra large (Bim-EL) and Bcl-2 modifying factor (Bmf). Preventing ERK1/2 reactivation with MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase) inhibitors blocked G1-S cell-cycle progression but failed to induce apoptosis or upregulate Bim-EL and Bmf. Treatment with the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid, led to de-repression of Bim-EL and enhanced cell death in the presence of PLX4720 or AZD6244 in resistant cells. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway reactivation as well as ERK1/2-independent silencing of BH3-only proteins. Furthermore, combined treatment of HDAC inhibitors and MEK inhibitors may contribute to overcoming PLX4032 resistance.     

Down-Regulation of Deacetylase HDAC6 Inhibits the Melanoma Cell Line A375.S2 Growth through ROS-Dependent Mitochondrial Pathway

Previous studies have shown that histone deacetylase 6 (HDAC6) plays critical roles in many cellular processes related to cancer. However, its biological roles in the development of melanoma remain unexplored. Our aim was to investigate whether HDAC6 has a biological role in human melanoma development and to understand its underlying mechanism. In the present study, HDAC6 expression was up-regulated in melanoma tissues and cell lines. Knockdown of HDAC6 significantly inhibited the proliferation and colony formation ability of A375.S2 cells, promoted cell arrest at G0/G1 phase and apoptosis. Additionally, western blotting assay showed that HDAC6 silencing suppressed Bcl-2 level and enhanced Bax level, then activated caspase-9 and caspase-3, and further activated the release of cytochrome c from mitochondria to cytoplasm, finally induced cell apoptosis involving the mitochondrial pathway. Knockdown of HDAC6 triggered a significant generation of ROS and disruption of mitochondrial membrane potential (MMP). Furthermore, ROS inhibitor, NAC reduced HDAC6 siRNA-induced ROS production, and blocked HDAC6 siRNA-induced loss of MMP and apoptosis. NAC also significantly blocked HDAC6 siRNA-induced mtDNA copy number decrease and mitochondrial biogenesis and degradation imbalance. In conclusion, the results showed that knockdown of HDAC6 induced apoptosis in human melanoma A375.S2 cells through a ROS-dependent mitochondrial pathway.     

Impact of IFNα2b upon pSTAT3 and the MEK/ERK MAPK Pathway in Melanoma

PURPOSE: High-dose IFNalpha2b (HDI) was established as the first effective adjuvant therapy for patients with high-risk resected melanoma more than a decade ago, but its fundamental molecular mechanism of action remains unclear. STAT3 and the mitogen activated protein kinases (MAPKs), especially ERK (extracellular signal-regulating kinase) and MEK (MAPK/ERK kinase), play roles in melanoma progression and host immunity. We have therefore evaluated STAT3 and MEK/ERK MAP kinases in patients with regional lymph node metastasis (stage IIIB) of melanoma in the context of a prospective neoadjuvant trial of HDI (UPCI 00-008). PATIENTS AND METHODS: In the context of this trial, HDI was administered daily for 20 doses following diagnostic biopsy, and prior to definitive surgery. Immunohistochemistry for pSTAT3, phospho-MEK1/2, phospho-ERK1/2, and EGFR was performed on paired fixed (nine patients) biopsies. RESULTS: HDI was found to down-regulate pSTAT3 (P = 0.008) and phospho-MEK1/2 (P = 0.008) levels significantly in tumor cells. Phospho-ERK1/2 was down-regulated by HDI in tumor cells (P = 0.015), but not in lymphoid cells. HDI down-regulated EGFR (P = 0.013), but pSTAT3 activation appeared not to be associated with EGFR expression and the MEK/ERK MAPK pathway. CONCLUSION: We conclude that HDI regulates MAPK signaling differentially in melanoma tumor cells and host lymphoid cells in vivo. STAT3 activation is independent of the EGFR/MEK/ERK signaling pathway.     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

FGF9/FGFR1 promotes cell proliferation,epithelial-mesenchymal transition,M2 macrophage infiltration and liver metastasis of lung cancer

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.     

Antiproliferative effect of panaxynol on RASMCs via inhibition of ERK1/2 and CREB

Panaxynol (PNN) occurs in many foods such as carrot, celery, and several reports have shown that it has neuritogenic and neuroprotective properties. In this study, we have investigated the antiproliferative effect and the mechanism of PNN on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (RASMCs). PNN significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of RASMCs in a concentration-dependent manner. Flow cytometry analysis showed that PNN blocked the cell cycle progression at the G(1)/S phase. Preincubation of RASMCs with 9 microM PNN resulted in a significant inhibition of PDGF-BB-induced extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylation expression and PDGF-BB-induced CREB phosphorylation expression. The results indicated that the inhibitory effect of PNN on the PDGF-BB-induced proliferation of RASMCs might be mediated by blocking phosphorylation of ERK1/2 and that of CREB.     

Hdac6 knock-out increases tubulin acetylation but does not modify disease progression in the R6/2 mouse model of Huntington's disease

Huntington's disease (HD) is a progressive neurodegenerative disorder for which there is no effective disease modifying treatment. Following-on from studies in HD animal models, histone deacetylase (HDAC) inhibition has emerged as an attractive therapeutic option. In parallel, several reports have demonstrated a role for histone deacetylase 6 (HDAC6) in the modulation of the toxicity caused by the accumulation of misfolded proteins, including that of expanded polyglutamine in an N-terminal huntingtin fragment. An important role for HDAC6 in kinesin-1 dependent transport of brain-derived neurotrophic factor (BDNF) from the cortex to the striatum has also been demonstrated. To elucidate the role that HDAC6 plays in HD progression, we evaluated the effects of the genetic depletion of HDAC6 in the R6/2 mouse model of HD. Loss of HDAC6 resulted in a marked increase in tubulin acetylation throughout the brain. Despite this, there was no effect on the onset and progression of a wide range of behavioural, physiological, molecular and pathological HD-related phenotypes. We observed no change in the aggregate load or in the levels of soluble mutant exon 1 transprotein. HDAC6 genetic depletion did not affect the efficiency of BDNF transport from the cortex to the striatum. Therefore, we conclude that HDAC6 inhibition does not modify disease progression in R6/2 mice and HDAC6 should not be prioritized as a therapeutic target for HD.     

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities of matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKCα and PKCδ phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.     

Involvement of histone deacetylation in ras-induced down-regulation of the metastasis suppressor RECK

RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Previous study demonstrated that oncogenic ras inhibited RECK expression via an Sp1 binding site in the RECK promoter. In this study, we investigated the molecular mechanism by which ras inhibited RECK expression. Co-transfection assay showed that Sp1 and Sp3 are transactivators, rather than repressors, for RECK gene. So, we tested whether ras activation induced the binding of histone deacetylases (HDACs) to Sp1 to repress RECK expression. Our data showed Sp1-associated HDAC1 in cells was increased after ras induction. By using DNA affinity precipitation assay, we found that induction of oncogenic ras enhanced the binding of HDAC1 to the DNA probe corresponding to the Sp1 site in the RECK promoter. Additionally, a HDAC inhibitor trichostatin A (TSA) potently antagonized the inhibitory action of ras on RECK. The signaling pathway by which ras suppresses RECK was also addressed. Induction of oncogenic ras activated extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK) and p38(HOG) kinase in 2-12 cells. Addition of PD98059 or overexpression of dominant-negative mutant of ERK2 indeed reversed ras-mediated inhibition of RECK promoter activity. Taken together, our results suggest that oncogenic ras represses RECK expression via a histone deacetylation mechanism.