p38 Protects Human Melanoma Cells from UV-Induced Apoptosis Through Down-Regulation of NF-κB Activity and Fas Expression

Identifying mechanisms that underlie the resistance of human melanoma to radiation and chemotherapy is expected to assist in developing new strategies for the treatment of this tumor type. We recently demonstrated that through up-regulation of TNFα, ATF2 increases the resistance of late stage melanoma cells to apoptosis induced by UV-irradiation. In elucidating the role of ATF2 kinases, we now demonstrate that ASK1/MKK6/p38 elicits suppression of Fas expression. ASK1/p38 downregulates the expression of a Fas via NF-κB/SP1 site on the Fas promoter. Deletion or mutation of NF-κB/SP1 within the Fas promoter abrogates p38 effect. ASK1/p38 silences the Fas promoter by inhibition of IκBα phosphorylation - thereby limiting NF-κB activity. Forced expression of a dominant negative form of p38 (p38-ASP) or treatment with p38 pharmacological inhibitor, SB203580, increases NF-κB activity, Fas expression and the levels of UVC-induced apoptosis in late stage melanoma cells. Inhibition of p38 activity also restored NF-κB activity and Fas expression in early-phase melanoma cells, suggesting that p38 elicited suppression of Fas expression is not restricted to late phase melanoma. Identifying p38-mediated down-regulation of Fas expression illustrates a novel regulatory pathway by which ASK1/MKK6/p38 alters the degree and nature of the UV-induced apoptosis of melanoma cells

Regulation of Fas Expression by STAT3 and c-Jun Is Mediated by Phosphatidylinositol 3-Kinase-AKT Signaling

Cooperation between STAT3 and c-Jun results in suppression of Fas Receptor (FasR) transcription, which is often seen in advanced human tumors. To identify requirements for STAT3-Jun cooperation, we elucidated the role of protein kinases that affect both transcription factors. The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway was found capable of down-regulating both STAT3- and c-Jun-dependent transcription, resulting in derepression of FasR transcription. Conversely, inhibition of PI3K-AKT signaling via the specific pharmacological inhibitor LY294002 up-regulated AP1/Jun- and STAT-dependent transcriptional activities, resulting in suppression of the FasR promoter activities and decreased FasR surface expression. PI3K-AKT's ability to affect FasR transcription was not observed in c-jun null fibroblasts, suggesting that c-Jun is required for PI3K/AKT-mediated regulation of FasR transcription. Interestingly, the dominant negative form of Rac1 (RacN17) was also efficient in relieving FasR expression, suggesting that the increase in FasR expression following AKT stimuli could be mediated via AKT ability to elicit suppression of Rac1, which in turn decreases JNK activities and c-Jun phosphorylation. Overall, our findings demonstrate that through its negative effects on both c-Jun and STAT3, the PI3K-AKT pathway disrupts cooperation between c-Jun and STAT3, which is required for silencing the FasR promoter, resulting in increased expression of surface FasR and concomitant sensitization to FasL-mediated programmed cell death

Expression of ring finger-deleted TRAF2 sensitizes metastatic melanoma cells to apoptosis via up-regulation of p38, TNFα and suppression of NF-κB activities

Mechanisms underlying radiation and chemotherapy resistance, the hallmark of human melanoma, are not well understood. Here we demonstrate that expression levels of signal adaptor protein TRAF2 coincide with melanoma resistance to UV-irradiation. Altered TRAF2 signaling by a form of TRAF2, which lacks the ring finger domain (TRAF2ΔN), increases activities of p38 MAPK, ATF2, and the level of TNFα expression. Forced expression of TRAF2ΔN in HHMSX highly metastatic melanoma cells that lack Fas expression and thus utilize the TNFα-TNFR1 as the major apoptotic pathway sensitized cells to UV-induced apoptosis. An over twofold increase in degree of apoptosis was observed in TRAF2ΔN expressing cells that were treated with actinomycin D, anisomycin or with the radiomimetic drug neocarzinostatin. Sensitization by TRAF2ΔN is selective since it was not observed in response to either Taxol or cis-platinum treatment. TRAF2ΔN effects are primarily mediated via p38 since inhibition of p38 reduces, whereas activation of p38 promotes the level of UV-induced apoptosis. Conversely, activation of IKK attenuates the sensitization of melanoma by TRAF2ΔN, indicating that p38-mediated suppression of NF-κB activity is among TRAF2ΔN effects. Our finding identifies p38, TNFα and NF-κB among key players that efficiently sensitizes melanoma cells to UV-, ribotoxic (anisomycin) and radiomimetic chemicals-induced programmed cell death in response to aberrant TRAF2 signaling

Death receptors and melanoma resistance to apoptosis

Impaired ability to undergo programmed cell death in response to a wide range of external stimuli acquires melanomas a selective advantage for progression and metastasis as well as their notorious resistance to therapy. Better understanding of mechanisms that govern apoptosis has enabled identification of diverse routes by which melanomas manage to escape stimuli of apoptosis. Changes at genomic, transcriptional and post-translational levels of G-proteins and protein kinases (Ras, B-Raf) and their transcription factor effectors (c-Jun, ATF2, Stat3 and NF-kappaB) affects TNF, Fas and TRAIL receptors, which play important roles in acquiring melanoma's resistance to apoptosis. Here, we summarize our current understanding of changes that alters the regulation of death receptors during melanoma development

Opposite Roles of FAP-1 and Dynamin in the Regulation of Fas (CD95) Translocation to the Cell Surface and Susceptibility to Fas Ligand-mediated Apoptosis

Human melanoma is the most aggressive form of skin cancer and is extremely resistant to radiation and chemotherapy. One of the critical parameters of this resistance is down-regulation of Fas (CD95) cell-surface expression. Using TIG3 normal human fibroblasts and human melanoma cell lines, we investigated transcriptional regulation of FAP-1, a regulator of Fas translocation in the cell. Protein-tyrosine phosphatase FAP-1 (PTPN13, PTP-BAS) interacts with human Fas protein and prevents its export from the cytoplasm to the cell surface. In contrast, dynamin-2 facilitates Fas protein translocation from the Golgi apparatus via the trans-Golgi network to the cell surface. Suppression of dynamin functions by dominant negative dynamin K44A blocks Fas export, whereas the down-regulation of FAP-1 expression by specific RNA interference restores Fas export (a phenomenon that could still be down-regulated in the presence of dominant-negative dynamin). Based on the FAP-1- and dynamin-dependent regulation of Fas translocation, we have created human melanoma lines with different levels of surface expression of Fas. Treatment of these melanoma lines with soluble Fas ligand resulted in programmed cell death that was proportional to the pre-existing levels of surface Fas. Taking into consideration the well known observations that FAP-1 expression is often up-regulated in metastatic tumors, we have established a causal connection between high basal NF-κB transcription factor activity (which is a hallmark of many types of metastatic tumors) and NF-κB-dependent transcriptional regulation of FAP-1 gene expression that finally restricts Fas protein trafficking, thereby, facilitating the survival of cancer cells

ERK and PI3K negatively regulate STAT-transcriptional activities in human melanoma cells: implications towards sensitization to apoptosis

Signal transducers and activators of transcription (STAT) proteins nuclear translocation and transcriptional activity are regulated by diverse protein kinases in response to extracellular stimuli by cytokines, growth factors and stress. Using two melanoma-derived cell lines that exhibit marked differences in basal activities of MAPKs and PI3K-AKT, we studied changes both in STAT activities and in their sensitization to apoptosis. Activating mutations of B-RAF (T1796A) and impaired expression of PTEN are detected in LU1205, but not in FEMX melanoma cells, and are reflected in high basal levels of expression and activities of MAPKs and PI3K-AKT. Treatment with either PD98059 (PD) or LY294002 (LY), the pharmacological inhibitors of MEK-ERK and PI3K, respectively, markedly increased GAS-Luc activity in LU1205, but not in FEMX cells. Tyrosine phosphorylation of STAT3/5 and of JAK2 also increased upon treatment of LU1205 cells with either PD or LY, suggesting that constitutive active MAPK and PI3K signals inhibit tyrosine phosphorylation of JAK/STATs. Treatment of FEMX and LU1205 with PD sensitized the cells to apoptosis, albeit by TNFalpha and TRAIL death cascades, respectively, indicating that additional yet distinct targets are affected by each signaling pathway. Indeed, the combination of LY and PD treatment synergistically increased the apoptosis of LU1205 and FEMX cells. Overall, whereas PI3K and MAPK downregulate JAK-STAT signaling, additional targets are affected by these kinases and sensitizes melanoma to apoptosis via distinct death cascades

Glutathione S-Transferase p Elicits Protection against H2O2-induced Cell Death via Coordinated Regulation of Stress Kinases

To elucidate mechanisms underlying glutathione S-transferase p (GSTp)-mediated cellular protection against oxidative stress-induced cell death, the effect of GSTp on stress signaling pathways was investigated before and after H2O2 treatment. Under nonstressed conditions, increased expression of GSTp via a tet-off-inducible GSTp in NIH 3T3 cells increased the phosphorylation of mitogen-activated protein (MAP) kinase kinase 4, p38, extracellular receptor kinase (ERK), and inhibitor of κ-kinase (IKK), and reduced phosphorylation of MAP kinase kinase 7 and Jun NH2-terminal kinase (JNK). Whereas H2O2 treatment of cells induced JNK, p38, and IKK activities, in the presence of H2O2 and elevated GSTp expression there was an additional increase in ERK, p38, and IKK activities and a decrease in JNK activity. GSTp-mediated protection from H2O2-induced death was attenuated upon inhibition of p38, nuclear factor κB, or MAP kinase by dominant negative or pharmacological inhibitors. Conversely, expression of a dominant negative JNK protected cells from H2O2-mediated death. These data suggest that the coordinated regulation of stress kinases by GSTp, as reflected by increased p38, ERK, and nuclear factor κB activities together with suppression of JNK signaling, contributes to protection of cells against reactive oxygen species-mediated death

The LIM domain protein UNC-95 is required for the assembly of muscle attachment structures and is regulated by the RING finger protein RNF-5 in C. elegans

Here, we describe a new muscle LIM domain protein, UNC-95, and identify it as a novel target for the RING finger protein RNF-5 in the Caenorhabditis elegans body wall muscle. unc-95(su33) animals have disorganized muscle actin and myosin-containing filaments as a result of a failure to assemble normal muscle adhesion structures. UNC-95 is active downstream of PAT-3/β-integrin in the assembly pathways of the muscle dense body and M-line attachments, and upstream of DEB-1/vinculin in the dense body assembly pathway. The translational UNC-95::GFP fusion construct is expressed in dense bodies, M-lines, and muscle–muscle cell boundaries as well as in muscle cell bodies. UNC-95 is partially colocalized with RNF-5 in muscle dense bodies and its expression and localization are regulated by RNF-5. rnf-5(RNAi) or a RING domain deleted mutant, rnf-5(tm794), exhibit structural defects of the muscle attachment sites. Together, our data demonstrate that UNC-95 constitutes an essential component of muscle adhesion sites that is regulated by RNF-5

TIP49b, a Regulator of Activating Transcription Factor 2 Response to Stress and DNA Damage

Activating transcription factor 2 (ATF2/CRE-BP1) is implicated in transcriptional control of stress-responsive genes. A yeast two-hybrid screen identified TBP-interacting protein 49b (TIP49b), a component of the INO80 chromatin-remodeling complex, as a novel ATF2-interacting protein. TIP49b's association with ATF2 is phosphorylation dependent and requires amino acids 150 to 248 of ATF2 (ATF2150–248), which are implicated in intramolecular inhibition of ATF2 transcriptional activities. Forced expression of TIP49b efficiently attenuated ATF2 transcriptional activities under normal growth conditions as well as after UV treatment, ionizing irradiation, or activation of p38 kinase, all of which induced ATF2 phosphorylation and increased TIP49b-ATF2 association. Constitutive expression of ATF2150–248 peptide outcompeted TIP49b interaction with ATF2 and alleviated the suppression of ATF2 transcriptional activities. Expression of ATF2150–248 in fibroblasts or melanoma but not in ATF2-null cells caused a profound G2M arrest and increased degree of apoptosis following irradiation. The interaction between ATF2 and TIP49b constitutes a novel mechanism that serves to limit ATF2 transcriptional activities and highlights the central role of ATF2 in the control of the cell cycle and apoptosis in response to stress and DNA damage

FAP-1 Association with Fas (Apo-1) Inhibits Fas Expression on the Cell Surface

As revealed by intracellular pools of nonactive Fas (Apo-1), export of Fas to the cell surface is often impaired in human tumors, thereby inactivating Fas ligand-mediated apoptosis. Here, we demonstrate that association with Fas-associated phosphatase 1 (FAP-1) attenuates Fas export to the cell surface. Forced expression of FAP-1 reduces cell surface Fas levels and increases the intracellular pool of Fas within the cytoskeleton network. Conversely, expression of dominant-negative forms of FAP-1, or inhibition of FAP-1 expression by short interfering RNA, efficiently up-regulates surface expression of Fas. Inhibition of Fas surface expression by FAP-1 depends on its association with the C terminus of Fas. Mutation within amino acid 275 results in decreased association with FAP-1 and greater export of Fas to the cell surface in melanomas, normal fibroblasts, or Fas null cells. Identifying the role of FAP-1 in binding to, and consequently inhibition of, Fas export to the cell surface provides novel insight into the mechanism underlying the regulation of Fas trafficking, which is commonly impaired in advanced tumors with FAP-1 overexpression