Kwang Hong Tay

Apoptosis of human melanoma cells induced by inhibition of B-RAFV600E involves preferential splicing of bimS

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, BimS, remains largely unappreciated. Here, we show that inhibition of the mutant B-RAFV600E triggers preferential splicing to produce BimS, which is particularly important in induction of apoptosis in B-RAFV600E melanoma cells. Although the specific B-RAFV600E inhibitor PLX4720 upregulates all three major isoforms of Bim, BimEL, BimL, and BimS, at the protein and mRNA levels in B-RAFV600E melanoma cells, the increase in the ratios of BimS mRNA to BimEL and BimL mRNA indicates that it favours BimS splicing. Consistently, enforced expression of B-RAFV600E in wild-type B-RAF melanoma cells and melanocytes inhibits BimS expression. The splicing factor SRp55 appears necessary for the increase in BimS splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of BimS and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in BimS splicing is also mirrored in freshly isolated B-RAFV600E melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAFV600E inhibitors.

2-Deoxy-D-glucose enhances TRAIL-induced apoptosis in human melanoma cells through XBP-1-mediated up-regulation of TRAIL-R2

BackgroundPast studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells.Results2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1α and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1α, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells.ConclusionThese results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1α/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

Inositol polyphosphate 5-phosphatases can terminate downstream signalling of phosphatidylinositol-3 kinase; however, their biological role in the pathogenesis of cancer is controversial. Here we report that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase, has a tumour suppressive role in melanoma. Although it is commonly downregulated in melanoma, overexpression of phosphatidylinositol 4,5-bisphosphate 5-phosphatase blocks Akt activation, inhibits proliferation and undermines survival of melanoma cells in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of phosphatidylinositol 4,5-bisphosphate 5-phosphatase results in increased proliferation and anchorage-independent growth of melanocytes. Although DNA copy number loss is responsible for downregulation of phosphatidylinositol 4,5-bisphosphate 5-phosphatase in a proportion of melanomas, histone hypoacetylation mediated by histone deacetylases HDAC2 and HDAC3 through binding to the transcription factor Sp1 at the PIB5PA gene promoter appears to be another commonly involved mechanism. Collectively, these results establish the tumour suppressive role of phosphatidylinositol 4,5-bisphosphate 5-phosphatase and reveal mechanisms involved in its downregulation in melanoma.

Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIPL from degradation by the E3 ligase itch in human melanoma cells

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIPL as the levels of FLIPL were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIPL, decreased the amount of the E3 ligase Itch associated with FLIPL, and reduced FLIPL ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIPL and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIPL and thereby TRAIL-induced apoptosis in melanoma cells.

Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.

Suppression of PP2A is critical for protection of melanoma cells upon endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells, which involves dephosphorylation of BimEL by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that ER stress reduces PP2A activity leading to increased ERK activation and subsequent phosphorylation and proteasomal degradation of BimEL. Despite sustained upregulation of Bim at the transcriptional level, the BimEL protein expression was downregulated after an initial increase in melanoma cells subjected to pharmacological ER stress. This was mediated by increased activity of ERK, whereas the phosphatase activity of PP2A was reduced by ER stress in melanoma cells. The increase in ERK activation was, at least in part, due to reduced dephosphorylation by PP2A, which was associated with downregulation of the PP2A catalytic C subunit. Notably, instead of direct dephosphorylation of BimEL, PP2A inhibited its phosphorylation indirectly through dephosphorylation of ERK in melanoma cells. Taken together, these results identify downregualtion of PP2A activity as an important protective mechanism of melanoma cells against ER stress-induced apoptosis.

Contrasting Effects of Nutlin-3 on TRAIL- and Docetaxel-Induced Apoptosis Due to Upregulation of TRAIL-R2 and Mcl-1 in Human Melanoma Cells

Wild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligase MDM2. However, we found in this study that blockade of the interaction of p53 and MDM2 by the MDM2 antagonist nutlin-3 in melanoma cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless, nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, nutlin-3 upregulated Mcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as nutlin-3 upregulated the Mcl-1 protein to a much greater extent than the Mcl-1 mRNA, and this was associated with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in fresh melanoma isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and, importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in addition to docetaxel in melanoma. Mol Cancer Ther; 9(12); 3363–74. ©2010 AACR.

RIPK1 regulates survival of human melanoma cells upon endoplasmic reticulum stress through autophagy

Although RIPK1 (receptor [TNFRSF]-interacting protein kinase 1) is emerging as a critical determinant of cell fate in response to cellular stress resulting from activation of death receptors and DNA damage, its potential role in cell response to endoplasmic reticulum (ER) stress remains undefined. Here we report that RIPK1 functions as an important prosurvival mechanism in melanoma cells undergoing pharmacological ER stress induced by tunicamycin (TM) or thapsigargin (TG) through activation of autophagy. While treatment with TM or TG upregulated RIPK1 and triggered autophagy in melanoma cells, knockdown of RIPK1 inhibited autophagy and rendered the cells sensitive to killing by TM or TG, recapitulating the effect of inhibition of autophagy. Consistently, overexpression of RIPK1 enhanced induction of autophagy and conferred resistance of melanoma cells to TM- or TG-induced cell death. Activation of MAPK8/JNK1 or MAPK9/JNK2, which phosphorylated BCL2L11/BIM leading to its dissociation from BECN1/Beclin 1, was involved in TM- or TG-induced, RIPK1-mediated activation of autophagy; whereas, activation of the transcription factor HSF1 (heat shock factor protein 1) downstream of the ERN1/IRE1-XBP1 axis of the unfolded protein response was responsible for the increase in RIPK1 in melanoma cells undergoing pharmacological ER stress. Collectively, these results identify upregulation of RIPK1 as an important resistance mechanism of melanoma cells to TM- or TG-induced ER stress by protecting against cell death through activation of autophagy, and suggest that targeting the autophagy-activating mechanism of RIPK1 may be a useful strategy to enhance sensitivity of melanoma cells to therapeutic agents that induce ER stress.

The melanoma-associated antigen MAGE-D2 suppresses TRAIL receptor 2 and protects against TRAIL-induced apoptosis in human melanoma cells

Emerging evidence has pointed to biological roles of melanoma-associated antigens (MAGEs) in cancer development, progression and resistance to treatment. However, the mechanisms involved remain to be fully elucidated. In this report, we show that one of the MAGE proteins, MAGE-D2, suppresses the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 2 (TRAIL-R2) and plays an important role in protecting melanoma cells from apoptosis induced by TRAIL. MAGE-D2 was commonly expressed at increased levels in melanoma cells compared with melanocytes. Although its inhibition by small interfering RNA (siRNA) did not cause cell death, it rendered melanoma cells more sensitive to TRAIL-induced apoptosis. This was associated with enhanced formation of TRAIL death-inducing signaling complex and up-regulation of TRAIL-R2, and was blocked by a recombinant TRAIL-R2/Fc chimeric protein or siRNA knockdown of TRAIL-R2. Regulation of TRAIL-R2 by MAGE-D2 appeared to be mediated by p53, in that knockdown MAGE-D2 did not up-regulate TRAIL-R2 in p53-null or mutant p53 melanoma cells. In addition, inhibition of MAGE-D2 did not result in up-regulation of TRAIL-R2 in wild-type p53 cell lines with p53 inhibited by short hairpin RNA. Indeed, knockdown of MAGE-D2 led to up-regulation of p53 due to a transcriptional increase. The regulatory effect of MAGE-D2 on TRAIL-R2 expression and TRAIL-induced apoptosis was recapitulated in studies on fresh melanoma isolates. Taken together, these results identify the expression of MAGE-D2 as an important mechanism that inhibit TRAIL-induced apoptosis and suggest that targeting MAGE-D2 may be a useful strategy in improving the therapeutic efficacy of TRAIL in melanoma.

Adipocytes Contribute to Resistance of Human Melanoma Cells to Chemotherapy and Targeted Therapy

Epidemiological evidence has linked the development and progression of several cancers including melanoma with obesity. However, whether obesity impinges on responses of cancer cells to treatment remains less understood. Here we report that human adipocytes contribute to resistance of melanoma cells to various therapeutic agents. Exposure to media from adipocyte cultures (adipocyte media) increased cell proliferation and reduced sensitivity of melanoma cells to apoptosis induced by diverse chemotherapeutic drugs, including the DNA-damaging drug cisplatin, the microtubuletargeting agent docetaxel, and the histone deacetylase inhibitor SAHA. This was associated with increased activation of PI3K/Akt and MEK/ERK signaling, and was attenuated by a PI3K or MEK inhibitor. The effect of adipocyte media on melanoma cells was, at least in part, due to the interaction between the adipokine leptin and its long form receptor OB-Rb, in that immunodepletion of leptin in adipocyte media or siRNA knockdown of OB-Rb in melanoma cells reversed the increase in Akt and ERK activation, enhancement in cell proliferation, and importantly, protection of melanoma cells against the drugs. In support, recombinant leptin partially recapitulated the effect of adipocyte media on melanoma cells. Of note, OB-Rb was increased on the surface of melanoma cells compared to melanocytes, whereas leptin short form receptors appeared to be suppressed post-transcriptionally, suggesting that OB-Rb was selectively upregulated in melanoma cells. Collectively, these results indicate that adipocytes contribute to the resistance of melanoma cells to chemotherapeutic drugs and agents targeting the PI3K/Akt and MEK/ERK pathways, and suggest that inhibition of the leptin/ OB-Rb system may be useful to improve the efficacy of multiple therapeutic approaches in the treatment of melanoma.