Transcriptional switch by activating transcription factor 2-derived peptide sensitizes melanoma cells to apoptosis and inhibits their tumorigenicity

Abstract

The notorious resistance of melanoma cells to drug treatment can be overcome by expression of a 50-aa peptide derived from activating transcription factor 2 (ATF2(50-100)). Here we demonstrate that ATF2(50-100) induced apoptosis by sequestering ATF2 to the cytoplasm, thereby inhibiting its transcriptional activities. Furthermore, ATF2(50-100) binds to c-Jun N-terminal kinase (JNK) and increases its activity. Mutation within ATF2(50-100) that impairs association with JNK and the inhibition of JNK or c-Jun expression by RNA interference (RNAi) reduces the degree of ATF2(50-100)-induced apoptosis. In contrast, TAM67, a dominant negative of the Jun family of transcription factors, or JunD RNAi attenuates sensitization of melanoma cells expressing ATF2(50-100) to apoptosis after treatment with anisomycin, which is used as a model drug. Mutations within the JNK binding region of ATF2(50-100) or expression of TAM67 or JunD RNAi attenuates inhibition of melanoma's tumorigenicity by ATF2(50-100). We conclude that inhibition of ATF2 in concert with increased JNK/Jun and JunD activities is central for the sensitization of melanoma cells to apoptosis and inhibition of their tumorigenicity.

Fig. 4.

JunD is required for TRE-dependent transcription and for sensitization of melanoma cells that express ATF2^50–100 to apoptosis. (a) Infection of SW1 cells with pRS-JunD results in efficient inhibition of JunD expression. SW1 cells that constitutively express control or wild-type ATF2^50–100 were infected with control or pRS-JunD constructs. Proteins were prepared 48 h after infection, and Western blot analysis was performed with antibodies to JunD. β-actin was used as a loading control. (b and c) Inhibition of JunD expression increases Jun2-mediated transcription and inhibits TRE-dependent activities in cells expressing ATF2^50–100. SW1 cells that constitutively express control or ATF2^50–100 were infected with pRS control or pRS-JunD. Fortyeight hours after infection, the cells were transfected with Jun2-luc or TRE-Luc. Proteins were prepared 20 h later and assayed for luciferase activity. (d) Inhibition of TRE-mediated transcription is enhanced after inhibition of both JNK and JunD expression. SW1 cells that constitutively express control or ATF2^50–100 were infected with pRS-JunD or pRS-JNK or with both. Cells were transfected with TRE-Luc 48 h after infection, and proteins were prepared for luciferase assays after an additional 20 h. A portion of the same extracts was used to verify inhibition of JNK and JunD expression by Western blots. (e) Suppression of JunD transcription inhibits sensitization of the ATF2 peptide-expressing melanoma to apoptosis after anisomycin treatment. SW1 cells were infected with pRS-JunD or control pRS followed by transfection of the ATF2 peptide or control vectors 48 h later. Twenty hours later, cells were subjected to anisomycin treatment, and the degree of apoptosis was monitored by fluorescence-activated cell sorter analysis after an additional 24 h.

Fig. 1.

ATF2^50–100 promotes cytoplasmic localization of ATF2 in advanced melanomas. SW1 pcDNA and SW1-ATF2^50–100 wild-type were immunostained for endogenous ATF2 with ATF2 antibodies (green fluorescence) or for ATF2^50–100 with HA antibodies (red fluorescence). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Data shown represent multiple fields in over five experiments scoring >300 cells per experiment. Over 80% of the cells exhibited change in ATF2 localization. Shown is confocal microscopy. Phase contrast reflects cell shape.

Fig. 2.

ATF2^50–100 associates with JNK and requires its binding to sensitize melanoma to apoptosis and inhibit tumorigenicity. (a) Expression of the ATF2 peptide increases expression of c-Jun. Protein extracts prepared from SW1 cells that stably express empty vector (control) or wild-type HA-ATF2^50–100 peptide (ATF2 peptide) were subjected to immunoblot analysis by using antibodies to phosphorylated c-Jun followed by reprobing with control c-Jun antibodies. The membrane was reprobed with antibodies to β-actin. (b) The half-life of c-Jun increased in melanoma cells expressing ATF2^50–100. The half-life of c-Jun was monitored with cycloheximide, which was added to control or ATF2^50–100-expressing cells for increasing time periods. Proteins were then subjected to immunoblot analysis by using antibodies to c-Jun. Numbers on the bottom of each image indicate relative change in level of expression based on a densitometry analysis normalized to background control with the quantify one program. Values represent the absolute densitometric values for each band. (c) JNK2 associates with ATF2^50–100. In vitro binding assays were carried with bacterially expressed and purified GST-ATF2^50–100 wild-type or mutant peptide incubated with extracts of 293T cells expressing Flag-tagged JNK2 for 2 h at 4°C. The beads were washed, and eluted material was subjected to immunoblot analysis with antibodies to Flag. (Lower) Coommassie blue staining reflecting the quantity of proteins used for the reaction. Relative change was quantified and normalized per binding to ATF2^1–115, a commonly used substrate for JNK/p38, based on densitometry. (d) Mutation within either the phosphoacceptor sites (APF) or the ATP pocket (DLD) of JNK reduces JNK2 association with ATF2^50–100.An in vitro binding reaction was carried out as indicated above, except that the GST peptides were incubated with extracts from 293T cells expressing Flag-tagged JNK2 APF or HA-tagged JNK2 DLD. (Lower) Quantity of proteins used for analysis. Quantifications were carried out as outlined in c.(e) Binding of ATF2^50–100 to p38. p38 was immunoprecipitated from cells followed by incubation with wild-type or mutant forms of the ATF2^50–100 or with the N-terminal region of ATF2 (1–115) as indicated in c. Quantification reflects relative binding to the corresponding proteins. (f) ATF2^50–100 increases basal JNK activity and reduces p38 activation after stress. Immunokinase (IK) reactions were carried out with either JNK or p38 immunoprecipitated (IP) from control or anisomycin-treated SW1 cells or SW1 cells that express ATF2^50–100.(Lower) Amount of substrate used (Ponceau staining) and the amount of kinase (IB). Numbers reflect quantification of phosphorylation based on PhosphorImager analysis. (g) JNK binding and, to a lesser degree, p38 binding are required for sensitization of melanoma cells to apoptosis by ATF2^50–100. SW1 cells expressing the wild-type or mutant forms of ATF2^50–100 were analyzed to determine the basal degree of apoptosis by using fluorescence-activated cell sorter analysis. (h) JNK but not p38 is required for the ability of ATF2^50–100 to inhibit melanoma growth in vivo. SW1 cells expressing ATF2^50–100 in its wild-type or mutant forms were injected s.c. into C3H mice (groups of six mice per experimental condition), and tumors were excised and analyzed to determine size and weight after 21 d.

Fig. 3.

JNK and c-Jun are required for sensitization of melanoma cells to basal apoptosis by ATF2^50–100. (a) Infection with pRS-JNK virus of SW1 cells reduces JNK expression. SW1 cells were infected with pRS control or pRS-JNK viruses. Proteins were prepared 48 h after infection and subjected to Western blot analysis with antibodies to JNK. (Lower) Depiction of the same immunoblot but with β-actin antibodies to assure equal loading. The multiple lanes truly represent the degree of inhibition of JNK expression achieved by this RNAi. (b) Inhibition of JNK expression inhibits basal but not inducible apoptosis. SW1 cells were infected with pRS-JNK and control virus followed (24 h later) by transfection of the ATF2 peptide and (20 h later) treatment with anisomycin. Twenty-four hours after treatment, the degree of apoptosis was monitored by fluorescence-activated cell sorter analysis. (c) RNAi by pRS-JNK inhibits TRE-dependent transcription. SW1 cells were infected with pRS control or pRS JNK viruses, and 48 h later the cells were transfected with the corresponding control or ATF2^50–100 constructs and TRE-Luc. Proteins were assayed for luciferase activity after an additional 20 h. Values depict absolute luciferase activity. (d) Infection of SW1 cells with pRS-c-Jun inhibits c-Jun expression. SW1 cells were infected with control construct; the pRS-c-Jun construct and proteins were prepared 48 h after infection and subjected to Western blot analysis with antibodies to c-Jun. (Middle) Control for lack of pRS-Jun effect on JunD expression. β-Actin was used as a loading control. (e) Suppression of c-Jun expression affects the degree of basal apoptosis induced in ATF2^50–100-expressing cells. SW1 cells were infected with pRS-c-Jun or control pRS followed by transfection of ATF2^50–100 or control vectors 48 h later. Twenty hours later, cells were subjected to anisomycin treatment, and the degree of apoptosis was monitored by fluorescence-activated cell sorter analysis after an additional 24 h. (f) pRS-c-Jun does not affect TRE-dependent transcription in ATF2^50–100-expressing cells. SW1 cells were infected with the pRS control pRS-c-Jun, followed by cotransfection of ATF2^50–100 or control vectors with TRE-Luc 48 h later. Twenty hours later, proteins were prepared and assayed for luciferase activity. Values depict absolute luciferase activity.

Fig. 5.

JunD is the primary factor mediating inhibition of melanoma tumorigenicity during expression of ATF2^50–100.(a) Expression of TAM67 attenuates the inhibition of melanoma tumorigenicity after expression of ATF2^50–100. SW1 cells that stably express control or ATF2^50–100 were infected with TAM67, and clones expressing TAM67 clones were selected on the basis of resistance to puromycin. Cells expressing TAM67 in combination with control or ATF2^50–100 were injected (10⁴) s.c. into C3H mice, and tumor size was monitored for 25 d. Data represent mean values (P < 0.003; t test) based on analysis of eight mice per experimental group. (b) Expression of pRS-JunD but not pRS-c-Jun abolishes ATF2^50–100 ability to inhibit tumorigenicity of SW1 melanoma. The experiment was carried out as indicated in a, except that SW1 cells were infected with indicated pRS-constructs in vitro followed by their s.c. injection into C3H mice. The analysis was performed in groups of eight mice per experimental condition (P < 0.001; t test). (Right) Pictures provide representative data on tumor size at the end of the experiment. (c) Proposed model. The expression of the ATF2^50–100 results in cytoplasmic accumulation of ATF2 with concomitant inhibition of ATF2 transcription. Consequently, the Jun2 promoter sequences are occupied by c-Jun/JunD, which sensitizes melanoma to apoptosis after treatment (Left). Consistent with this model is the finding that expression of a 10-aa ATF2 peptide, which no longer inhibits ATF2 or Jun2-dependent transcription, suffices to sensitize melanoma cells to basal apoptosis by up-regulating c-Jun/JunD (unpublished data). ATF2^50–100 association with JNK results in increased activity of c-Jun, which sensitizes melanoma cells to spontaneous (basal) apoptosis, and suffices to reduce tumorigenicity of this otherwise aggressive tumor (Right). ERK, extracellular response kinase.