Identification of glycogen synthase kinase 3α as a therapeutic target in melanoma

Deregulated expression or activity of kinases can lead to melanomas, but often the particular kinase isoform causing the effect is not well established, making identification and validation of different isoforms regulating disease development especially important. To accomplish this objective, an siRNA screen was undertaken that which identified glycogen synthase kinase 3α (GSK3α) as an important melanoma growth regulator. Melanocytes and melanoma cell lines representing various stages of melanoma tumor progression expressed both GSK3α and GSK3β, but analysis of tumors in patients with melanoma showed elevated expression of GSK3α in 72% of samples, which was not observed for GSK3β. Furthermore, 80% of tumors in patients with melanoma expressed elevated levels of catalytically active phosphorylated GSK3α (pGSK3αY279), but not phosphorylated GSK3β (pGSK3βY216). siRNA‐mediated reduction in GSK3α protein levels reduced melanoma cell survival and proliferation, sensitized cells to apoptosis‐inducing agents and decreased xenografted tumor development by up to 56%. Mechanistically, inhibiting GSK3α expression using siRNA or the pharmacological agent AR‐A014418 arrested melanoma cells in the G0/G1 phase of the cell cycle and induced apoptotic death to retard tumorigenesis. Therefore, GSK3α is a key therapeutic target in melanoma.     

ATP-independent glucose stimulation of sphingosine kinase in rat pancreatic islets

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet β-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and α-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in β-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported β-cell growth appears to be in part mediated by SPHK activity.     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.     

Constitutive aberrant endogenous interleukin-1 facilitates inflammation and growth in human melanoma

Interleukin (IL)-1-mediated inflammation is proposed to contribute to the development and progression of some cancers. IL-1 family member proteins are known to be expressed constitutively in many melanoma tumor cells, and we hypothesize that these support molecular pathways of inflammation and facilitate tumor growth. To investigate the expression of IL-1α and IL-1β in melanoma patients, and their association with disease progression, immunohistochemical staining was carried out on tissues from 170 patients including benign nevi, primary melanomas, and metastatic melanomas. IL-1β levels were low (or zero) in benign nevi and higher in primary and metastatic melanomas (P < 0.0001). IL-1α was expressed in about 73% of nevi and 55% of metastatic melanomas, with levels significantly higher in primary tumors (P < 0.0001); most (98%) primary melanoma samples were positive for IL-1α. In vitro studies with seven human melanoma cell lines showed that five cell lines expressed IL-1α and IL-1β proteins and mRNA. We identified for the first time several important downstream signaling pathways affected by endogenous IL-1, including reactive oxygen and nitrogen species, COX-2, and phosphorylated NF-κB inhibitor (IκB) and stress-activated protein kinase/c-jun-NH(2)-kinase; all of which were decreased by siRNA to IL-1s. Downregulation of IL-1α, IL-1β, or MyD88 substantially increased p21 and p53 levels. Treatment with IL-1 receptor type I neutralizing antibody or IL-1 pathway-specific siRNAs led to growth arrest in IL-1-positive melanoma cells. Furthermore, blocking the IL-1 pathway increased autophagy in IL-1-positive melanoma cells. These results indicate that the endogenous IL-1 system is functional in most human melanoma and interrupting its signaling inhibits the growth of IL-1-positive melanoma cells.     

Involvement of vacuolar H+‐ATPase in killing of human melanoma cells by the sphingosine kinase analogue FTY720

Targeting the sphingosine 1‐phosphate (S1P)/S1P receptor (S1PR) signalling axis is emerging as a promising strategy in the treatment of cancer. However, the effect of such an approach on survival of human melanoma cells remains less understood. Here, we show that the sphingosine analogue FTY720 that functionally antagonises S1PRs kills human melanoma cells through a mechanism involving the vacuolar H⁺‐ATPase activity. Moreover, we demonstrate that FTY720‐triggered cell death is characterized by features of necrosis and is not dependent on receptor‐interacting protein kinase 1 or lysosome cathepsins, nor was it associated with the activation of protein phosphatase 2A. Instead, it is mediated by increased production of reactive oxygen species and is antagonized by activation of autophagy. Collectively, these results suggest that FTY720 and its analogues are promising candidates for further development as new therapeutic agents in the treatment of melanoma.     

An ERBB3/ERBB2 oncogenic unit plays a key role in NRG1 signaling and melanoma cell growth and survival

We recently identified neuregulin‐1 (NRG1) as a novel target of Notch1 required in Notch‐dependent melanoma growth. ERBB3 and ERBB4, tyrosine kinase receptors specifically activated by NRG1, have been shown to be either elevated in melanoma cell lines and tumors or to be mutated in 20% of melanomas, respectively. While these data support key roles of NRG1 and its receptors in the pathogenesis of melanoma, whether ERBB3 and ERBB4 display redundant or exclusive functions is not known. Here, we show that ERBB3 and ERBB4 inhibition results in distinct outcomes. ERBB3 inhibition ablates the cellular responses to NRG1, results in AKT inactivation and leads to cell growth arrest and apoptotic cell death. In contrast, ERBB4 knockdown mildly affects cell growth, has no effects on cell survival and, importantly, does not alter the responses to NRG1. Finally, we identified ERBB2 as a key coreceptor in NRG1‐dependent ERBB3 signaling. ERBB2 forms a complex with ERBB3, and its inhibition recapitulates the phenotypes observed upon ERBB3 ablation. We propose that an NRG1‐ERBB3‐ERBB2 signaling unit operates in melanoma cells where it promotes growth and survival.     

Apaf‐1 expression in human cutaneous melanoma progression and in pigmented nevi

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf‐1, an important mediator of mitochondrial‐dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf‐1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf‐1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf‐1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf‐1 mRNA by fluorescence in situ hybridization and of Apaf‐1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf‐1. A significant decrease in Apaf‐1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf‐1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf‐1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf‐1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf‐1 may be an important contributory factor in the development of the disease.     

Acute effects of two melanocytolytic agents, hydroquinone and beta-mercaptoethanolamine, upon tyrosinase activity and cyclic nucleotide levels in murine melanomas

The acute in vitro actions of two potent melanocytolytic agents, hydroquinone (HQ) and beta-mercaptoethanolamine (MEA), were determined in the B-16, Cloudman S-91 and Harding-Passey (HP) murine melanomas grown in vivo. Drug treated melanoma dice (5--480 min) were analyzed for tyrosinase activity and cyclic nucleotide levels (cAMP, cGMP). HQ and MEA effects on tyrosinase activity are complex and vary with tumor type, duration of treatment and agent tested. MEA or HQ inhibited B-16 tyrosinase activity. With combined drug therapy, low concentrations of MEA plus HQ stimulate B-16 tyrosinase activity while high concentrations of the drugs have little effect on enzymatic activity. MEA depresses tyrosinase activity while HQ elevates enzymatic activity in the S-19 melanoma. Both high and low concentrations of the combined drugs (MEA plus HQ) elicit the same response, stimulation at 10 min followed by continued depression of tyrosinase activity for the remainder of the 4 h study period. MEA initially stimulates HP tyrosinase activity followed by depression of enzymic activity. In contrast, HQ initially depresses HP tyrosinase activity followed by stimulation of enzyme activity. In combination the drugs inhibit HP tyrosinase activity. The effects of MEA and/or HQ on murine melanoma cyclic nucleotide levels are equally complex. MEA or HQ elevate cAMP and cGMP levels in all three tumors with the exception of S-91 cGMP levels which are not altered. In combination the drugs increase cyclic nucleotide levels in each of the three tumor types but at different times. No correlation is present between cyclic nucleotide levels and tyrosinase activity. Thus, the action of increased cyclic nucleotide levels in melanogenesis can not be separated from the direct actions of MEA and HQ upon melanogenesis. The divergent effects of MEA and/or HQ on tyrosinase activity and cyclic nucleotide levels in these melanomas are not correlated with the known in vivo melanocytolytic activity of these drugs. Thus, these parameters appear to be inadequate indicators of melanoma cell viability in chemotherapeutic screening of drugs effective in destroying malignant melanoma.     

Regulation of NR4A nuclear receptor expression by oncogenic BRAF in melanoma cells

Activating mutations in the MAPK pathway effectors, NRAS or BRAF, are detected in over 70% of melanomas. Accordingly, the identification of downstream targets of constitutive MAPK signalling in melanoma represents a major goal in understanding the genesis of this disease. We report here the regulation of members of the NR4A family of nuclear receptors by the BRAF-MEK-ERK cascade in melanoma cells. Expression profiling of melanoma cells in which both the NR4A1 and NR4A2 family members have been down-regulated by siRNA revealed alterations in genes associated with proliferation, survival and invasiveness of tumour cells. Notably, the up-regulation of Wnt/β-catenin pathway antagonists, DACT1 and CITED1, following NR4A1/2 ablation suggests a possible link between NR4A and β-catenin activity in melanoma cells. Taken together, these data suggest that dysregulation of NR4A nuclear receptors expression and function by the MAPK pathway may contribute to melanoma tumourigenicity.     

Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like synoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro

To investigate the potential regulation of sphingosine kinase 1 (SPHK1) on the migration, invasion, and matrix metalloproteinase (MMP) expression in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). RA-FLS were transfected control siRNA or SPHK1 siRNA. The migration and invasion of unmanipulated control, control siRNA or SPHK1 siRNA- transfected RA-FLS in vitro were measured by the transwell system. The relative levels of SPHK1, PI3K, and AKT as well as AKT phosphorylation in RA-FLS were determined by Western blot. The levels of MMP-2/9 secreted by RA-FLS were detected by ELISA. Knockdown of SPHK1 significantly inhibited the spontaneous migration and invasion of RA-FLS, accompanied by significantly reduced levels of PI3K expression and AKT phosphorylation. Similarly, treatment with LY294002, an inhibitor of the PI3K/AKT pathway, inhibited the migration and invasion of RA-FLS. Knockdown of SPHK1 and treatment with the inhibitor synergistically inhibited the migration and invasion of RA-FLS, by further reducing the levels of PI3K expression and AKT phosphorylation. In addition, knockdown of SPHK1 or treatment with LY294002 inhibited the secretion of MMP-2 and MMP-9, and both synergistically reduced the production of MMP-2 and MMP-9 in RA-FLS in vitro. Knockdown of SPHK1 expression inhibits the PI3K/AKT activation, MMP-2 and MMP-9 expression, and human RA-FLS migration and invasion in vitro. Potentially, SPHK1 may be a novel therapeutic target for RA.     

Targeting the intrinsic apoptosis pathway as a strategy for melanoma therapy

Melanoma drug resistance is often attributed to abrogation of the intrinsic apoptosis pathway. Targeting regulators of apoptosis is thus considered a promising approach to sensitizing melanomas to treatment. The development of small‐molecule inhibitors that mimic natural antagonists of either antiapoptotic members of the BCL‐2 family or the inhibitor of apoptosis proteins (IAPs), known as BH3‐ or SMAC‐mimetics, respectively, are helping us to understand the mechanisms behind apoptotic resistance. Studies using BH3‐mimetics indicate that the antiapoptotic BCL‐2 protein MCL‐1 and its antagonist NOXA are particularly important regulators of BCL‐2 family signaling, while SMAC‐mimetic studies show that both XIAP and the cIAPs must be targeted to effectively induce apoptosis of cancer cells. Although most solid tumors, including melanoma, are insensitive to these mimetic drugs as single agents, combinations with other therapeutics have yielded promising results, and tests combining them with BRAF‐inhibitors, which have already revolutionized melanoma treatment, are a clear priority.     

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation

Although most of pharmacological therapies for cancer utilize the apoptotic machinery of the cells, the available anti-cancer drugs are limited due to the ability of prostate cancer cells to escape from the anti-cancer drug-induced apoptosis. A human prostate cancer cell line PC3 is resistant to camptothecin (CPT). To elucidate the mechanism of this resistance, we have examined the involvement of sphingosine kinase (SPHK) and sphingosine 1-phosphate (S1P) receptor in CPT-resistant PC3 and -sensitive LNCaP cells. PC3 cells exhibited higher activity accompanied with higher expression levels of protein and mRNA of SPHK1, and also elevated expression of S1P receptors, S1P(1) and S1P(3), as compared with those of LNCaP cells. The knockdown of SPHK1 by small interfering RNA and inhibition of S1P receptor signaling by pertussis toxin in PC3 cells induced significant inhibition of cell growth, suggesting implication of SPHK1 and S1P receptors in cell proliferation in PC3 cells. Furthermore, the treatment of PC3 cells with CPT was found to induce up-regulation of the SPHK1/S1P signaling by induction of both SPHK1 enzyme and S1P(1)/S1P(3) receptors. These findings strongly suggest that high expression and up-regulation of SPHK1 and S1P receptors protect PC3 cells from the apoptosis induced by CPT.     

Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells

Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells.     

LTBP-1 blockade in dioxin receptor-null mouse embryo fibroblasts decreases TGF-beta activity: Role of extracellular proteases plasmin and elastase

In mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR), high levels of latent transforming growth factor-beta (TGF-beta)-binding protein-1 (LTBP-1) correlated with increased TGF-beta1 activity, an observation suggesting that LTBP-1 could contribute to maintain TGF-beta1 levels. Here, using small interfering RNAs (siRNA), we have first analyzed if LTBP-1 expression affected TGF-beta1 activity in MEF cells. We have then determined how LTBP-1 levels could alter the activity of extracellular proteases known to activate TGF-beta1, and finally, whether protease inhibition could reduce TGF-beta1 activation. LTBP-1 inhibition by siRNA in AhR-/- MEF decreased the amount of active TGF-beta1 and reduced plasminogen activators (PA)/plasmin and elastase activities and thrombospondin-1 (TSP-1) expression, without significantly affecting their mRNA levels. On the contrary, LTBP-1 siRNA restored matrix metalloproteinase-2 (MMP-2) activity in AhR-/- MEF. Interestingly, whereas a TGF-beta1 neutralizing antibody mimicked many of the LTBP-1 siRNA effects on extracellular proteases, addition of recombinant TGF-beta1 protein increased proteases activity over basal levels in AhR-/- MEF. These proteases contributed to TGF-beta activation since their specific inhibitors reduced active TGF-beta levels in these cells. These results suggest that LTBP-1 contributes to TGF-beta1 activation in MEF, possibly by influencing the activities of PA/plasmin, elastase, TSP-1, and MMP-2. TGF-beta1, on the other hand, could be also involved in maintaining the activity of these extracellular proteases. Thus, LTBP-1 appears to play a role in TGF-beta1 activation through a process involving extracellular protease activities, which, in turn, could be affected by TGF-beta1 levels.