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A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations.

Canino C, Luo Y, Marcato P, Blandino G, Pass HI, Cioce M - Oncotarget (2015)

Bottom Line: RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH(bright) cells at steady state and, much more, after pemetrexed + cisplatin treatment.This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression.The possible broad translational relevance of the described mechanism is discussed.

View Article: PubMed Central - PubMed

Affiliation: Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University, New York, USA.

ABSTRACT
Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDH(bright) cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDH(bright) cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH(bright) cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPβ-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPβ complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDH(bright) cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo. The possible broad translational relevance of the described mechanism is discussed.

No MeSH data available.


Related in: MedlinePlus

STAT3 inhibition underlies the effect of Butein on the DDIT3 levelsA. ALDHbright cells exhibit increased activation of the STAT3 pathway. Western blotting with specific anti-STAT3 and anti-phospho-STAT3(tyr705) antibodies of whole cell lysates from purified ALDHbright and ALDHlow of three representative MPM cell lines (s.e: short exposure; l.e.: long exposure). B. mRNA levels of multiple STAT3 target genes in MSTO-211H ALDHbright vs ALDHlow cells, upon treatment with vehicle or butein (18 μM) for 24hrs. C. Upper. Combined bright field + fluorescent micrographs of MSTO-211H cells transfected with a mCherry reporter driven by the minimal DDIT3 promoter (−649/+170) and treated with butein (18 μM) for 6hrs. Scale bar: 20 μm. Lower. Percentage of mCherry positive cells in butein-treated cell cultures. A minimum of 8 fields (containing ≥30 cells) was counted in duplicate experiments. D-F. Butein affects the binding of STAT3 and NFkB to the DDIT3 promoter. D. DNA Affinity Precipitation assay (DAPA) with a biotinylated oligonucleotide containing either a STAT3 binding site in the DDIT3 promoter (STAT3) or a control sequence (CTRL), respectively. Western blotting of the DAPA-eluted from nuclear extracts of MSTO-211H cells treated with vehicle and butein (18 μM, 6hrs). Staining with antibodies against pSTAT3(Tyr705), STAT3 and NFkB(p65), respectively. E. In vivo occupancy of the DDIT3 promoter. Chromatin immunoprecipitation assays. Quantitative PCR revealing enrichment for the STAT3 containing DDIT3 promoter fragment in the eluate of STAT3, pSTAT3 and NFkB immunoprecipitates from vehicle or butein-treated MSTO-211H cells (18 μM, 20hrs). A rabbit IgG and a “off target” DNA region in the same promoter were used to control for the specificity of immunoprecipitation and of the PCR reaction, respectively. F. RE-CHIP assays. Chromatin eluted from STAT3 immunoprecipitated material of vehicle- and butein –treated MSTO-211H cells (as from 3E) was re-immunoprecipitated with a rabbit IgG, STAT3, pSTAT3 and NFKB antibodies, respectively. Quantitative PCR revealed specific amplification of the DDIT3 promoter fragment suggesting the existence of a STAT3-NFKB complex. Duplicate experiments. G. RNAi-mediated downregulation of STAT3 and NFkB mimicked the effects of butein on DDIT3 and ALDH1A3 mRNA levels. Left. Western blotting with anti-STAT3 and anti-NFkB antibodies of whole cell lysates from MSTO-211H and HP-1 cells transfected with control (scrambled), STAT3 and NFkB targeting siRNA revealed effective downregulation of the protein levels. Actin used as a loading control. Right. Quantitative PCR revealed higher levels of DDIT3 mRNA and reduced levels of ALDH1A3 mRNA in the cells with reduced expression of STAT3 and NFkB. Values expressed as folds over controls (scrambled siRNAs). Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control).
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Figure 3: STAT3 inhibition underlies the effect of Butein on the DDIT3 levelsA. ALDHbright cells exhibit increased activation of the STAT3 pathway. Western blotting with specific anti-STAT3 and anti-phospho-STAT3(tyr705) antibodies of whole cell lysates from purified ALDHbright and ALDHlow of three representative MPM cell lines (s.e: short exposure; l.e.: long exposure). B. mRNA levels of multiple STAT3 target genes in MSTO-211H ALDHbright vs ALDHlow cells, upon treatment with vehicle or butein (18 μM) for 24hrs. C. Upper. Combined bright field + fluorescent micrographs of MSTO-211H cells transfected with a mCherry reporter driven by the minimal DDIT3 promoter (−649/+170) and treated with butein (18 μM) for 6hrs. Scale bar: 20 μm. Lower. Percentage of mCherry positive cells in butein-treated cell cultures. A minimum of 8 fields (containing ≥30 cells) was counted in duplicate experiments. D-F. Butein affects the binding of STAT3 and NFkB to the DDIT3 promoter. D. DNA Affinity Precipitation assay (DAPA) with a biotinylated oligonucleotide containing either a STAT3 binding site in the DDIT3 promoter (STAT3) or a control sequence (CTRL), respectively. Western blotting of the DAPA-eluted from nuclear extracts of MSTO-211H cells treated with vehicle and butein (18 μM, 6hrs). Staining with antibodies against pSTAT3(Tyr705), STAT3 and NFkB(p65), respectively. E. In vivo occupancy of the DDIT3 promoter. Chromatin immunoprecipitation assays. Quantitative PCR revealing enrichment for the STAT3 containing DDIT3 promoter fragment in the eluate of STAT3, pSTAT3 and NFkB immunoprecipitates from vehicle or butein-treated MSTO-211H cells (18 μM, 20hrs). A rabbit IgG and a “off target” DNA region in the same promoter were used to control for the specificity of immunoprecipitation and of the PCR reaction, respectively. F. RE-CHIP assays. Chromatin eluted from STAT3 immunoprecipitated material of vehicle- and butein –treated MSTO-211H cells (as from 3E) was re-immunoprecipitated with a rabbit IgG, STAT3, pSTAT3 and NFKB antibodies, respectively. Quantitative PCR revealed specific amplification of the DDIT3 promoter fragment suggesting the existence of a STAT3-NFKB complex. Duplicate experiments. G. RNAi-mediated downregulation of STAT3 and NFkB mimicked the effects of butein on DDIT3 and ALDH1A3 mRNA levels. Left. Western blotting with anti-STAT3 and anti-NFkB antibodies of whole cell lysates from MSTO-211H and HP-1 cells transfected with control (scrambled), STAT3 and NFkB targeting siRNA revealed effective downregulation of the protein levels. Actin used as a loading control. Right. Quantitative PCR revealed higher levels of DDIT3 mRNA and reduced levels of ALDH1A3 mRNA in the cells with reduced expression of STAT3 and NFkB. Values expressed as folds over controls (scrambled siRNAs). Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control).

Mentions: DDIT3 is a target gene of STAT3 and its levels are upregulated in cells where binding of STAT3 to its promoter is diminished [28], implying active repression. Since we and others have shown that butein inhibits STAT3 [35, 37], we tested whether butein increased the levels of DDIT3 by inhibiting STAT3 activation. First, we evaluated the status of the STAT3 pathway in purified ALDHbright cells. Western blotting of whole cell lysates from both ALDHbright and ALDHlow cells of three representative cell lines revealed strong enrichment for the pSTAT3(tyr705) signal (with slight changes in the levels of the total STAT3 protein) in the ALDHbright cell fraction (Fig. 3A). Accordingly, quantitative PCR of 30 representative, literature selected STAT3 target genes [38] revealed that most of the targets exhibited higher levels in the ALDHbright cells as opposed to the ALDHlow cells (suppl. Fig. 3). Butein treatment modulated the levels of most of the targets in both ALDHbright and ALDHlow cells (Suppl. Fig. 3), and, to a much higher extent, the levels of a subset of those genes in the ALDHbright cells, including DDIT3 (as compared to the ALDHlow cells) (Fig. 3B). Thus, the MPM ALDHbright cells exhibited higher activation of the STAT3 pathway and responsivity to butein treatment.


A STAT3-NFkB/DDIT3/CEBPβ axis modulates ALDH1A3 expression in chemoresistant cell subpopulations.

Canino C, Luo Y, Marcato P, Blandino G, Pass HI, Cioce M - Oncotarget (2015)

STAT3 inhibition underlies the effect of Butein on the DDIT3 levelsA. ALDHbright cells exhibit increased activation of the STAT3 pathway. Western blotting with specific anti-STAT3 and anti-phospho-STAT3(tyr705) antibodies of whole cell lysates from purified ALDHbright and ALDHlow of three representative MPM cell lines (s.e: short exposure; l.e.: long exposure). B. mRNA levels of multiple STAT3 target genes in MSTO-211H ALDHbright vs ALDHlow cells, upon treatment with vehicle or butein (18 μM) for 24hrs. C. Upper. Combined bright field + fluorescent micrographs of MSTO-211H cells transfected with a mCherry reporter driven by the minimal DDIT3 promoter (−649/+170) and treated with butein (18 μM) for 6hrs. Scale bar: 20 μm. Lower. Percentage of mCherry positive cells in butein-treated cell cultures. A minimum of 8 fields (containing ≥30 cells) was counted in duplicate experiments. D-F. Butein affects the binding of STAT3 and NFkB to the DDIT3 promoter. D. DNA Affinity Precipitation assay (DAPA) with a biotinylated oligonucleotide containing either a STAT3 binding site in the DDIT3 promoter (STAT3) or a control sequence (CTRL), respectively. Western blotting of the DAPA-eluted from nuclear extracts of MSTO-211H cells treated with vehicle and butein (18 μM, 6hrs). Staining with antibodies against pSTAT3(Tyr705), STAT3 and NFkB(p65), respectively. E. In vivo occupancy of the DDIT3 promoter. Chromatin immunoprecipitation assays. Quantitative PCR revealing enrichment for the STAT3 containing DDIT3 promoter fragment in the eluate of STAT3, pSTAT3 and NFkB immunoprecipitates from vehicle or butein-treated MSTO-211H cells (18 μM, 20hrs). A rabbit IgG and a “off target” DNA region in the same promoter were used to control for the specificity of immunoprecipitation and of the PCR reaction, respectively. F. RE-CHIP assays. Chromatin eluted from STAT3 immunoprecipitated material of vehicle- and butein –treated MSTO-211H cells (as from 3E) was re-immunoprecipitated with a rabbit IgG, STAT3, pSTAT3 and NFKB antibodies, respectively. Quantitative PCR revealed specific amplification of the DDIT3 promoter fragment suggesting the existence of a STAT3-NFKB complex. Duplicate experiments. G. RNAi-mediated downregulation of STAT3 and NFkB mimicked the effects of butein on DDIT3 and ALDH1A3 mRNA levels. Left. Western blotting with anti-STAT3 and anti-NFkB antibodies of whole cell lysates from MSTO-211H and HP-1 cells transfected with control (scrambled), STAT3 and NFkB targeting siRNA revealed effective downregulation of the protein levels. Actin used as a loading control. Right. Quantitative PCR revealed higher levels of DDIT3 mRNA and reduced levels of ALDH1A3 mRNA in the cells with reduced expression of STAT3 and NFkB. Values expressed as folds over controls (scrambled siRNAs). Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control).
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Figure 3: STAT3 inhibition underlies the effect of Butein on the DDIT3 levelsA. ALDHbright cells exhibit increased activation of the STAT3 pathway. Western blotting with specific anti-STAT3 and anti-phospho-STAT3(tyr705) antibodies of whole cell lysates from purified ALDHbright and ALDHlow of three representative MPM cell lines (s.e: short exposure; l.e.: long exposure). B. mRNA levels of multiple STAT3 target genes in MSTO-211H ALDHbright vs ALDHlow cells, upon treatment with vehicle or butein (18 μM) for 24hrs. C. Upper. Combined bright field + fluorescent micrographs of MSTO-211H cells transfected with a mCherry reporter driven by the minimal DDIT3 promoter (−649/+170) and treated with butein (18 μM) for 6hrs. Scale bar: 20 μm. Lower. Percentage of mCherry positive cells in butein-treated cell cultures. A minimum of 8 fields (containing ≥30 cells) was counted in duplicate experiments. D-F. Butein affects the binding of STAT3 and NFkB to the DDIT3 promoter. D. DNA Affinity Precipitation assay (DAPA) with a biotinylated oligonucleotide containing either a STAT3 binding site in the DDIT3 promoter (STAT3) or a control sequence (CTRL), respectively. Western blotting of the DAPA-eluted from nuclear extracts of MSTO-211H cells treated with vehicle and butein (18 μM, 6hrs). Staining with antibodies against pSTAT3(Tyr705), STAT3 and NFkB(p65), respectively. E. In vivo occupancy of the DDIT3 promoter. Chromatin immunoprecipitation assays. Quantitative PCR revealing enrichment for the STAT3 containing DDIT3 promoter fragment in the eluate of STAT3, pSTAT3 and NFkB immunoprecipitates from vehicle or butein-treated MSTO-211H cells (18 μM, 20hrs). A rabbit IgG and a “off target” DNA region in the same promoter were used to control for the specificity of immunoprecipitation and of the PCR reaction, respectively. F. RE-CHIP assays. Chromatin eluted from STAT3 immunoprecipitated material of vehicle- and butein –treated MSTO-211H cells (as from 3E) was re-immunoprecipitated with a rabbit IgG, STAT3, pSTAT3 and NFKB antibodies, respectively. Quantitative PCR revealed specific amplification of the DDIT3 promoter fragment suggesting the existence of a STAT3-NFKB complex. Duplicate experiments. G. RNAi-mediated downregulation of STAT3 and NFkB mimicked the effects of butein on DDIT3 and ALDH1A3 mRNA levels. Left. Western blotting with anti-STAT3 and anti-NFkB antibodies of whole cell lysates from MSTO-211H and HP-1 cells transfected with control (scrambled), STAT3 and NFkB targeting siRNA revealed effective downregulation of the protein levels. Actin used as a loading control. Right. Quantitative PCR revealed higher levels of DDIT3 mRNA and reduced levels of ALDH1A3 mRNA in the cells with reduced expression of STAT3 and NFkB. Values expressed as folds over controls (scrambled siRNAs). Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control).
Mentions: DDIT3 is a target gene of STAT3 and its levels are upregulated in cells where binding of STAT3 to its promoter is diminished [28], implying active repression. Since we and others have shown that butein inhibits STAT3 [35, 37], we tested whether butein increased the levels of DDIT3 by inhibiting STAT3 activation. First, we evaluated the status of the STAT3 pathway in purified ALDHbright cells. Western blotting of whole cell lysates from both ALDHbright and ALDHlow cells of three representative cell lines revealed strong enrichment for the pSTAT3(tyr705) signal (with slight changes in the levels of the total STAT3 protein) in the ALDHbright cell fraction (Fig. 3A). Accordingly, quantitative PCR of 30 representative, literature selected STAT3 target genes [38] revealed that most of the targets exhibited higher levels in the ALDHbright cells as opposed to the ALDHlow cells (suppl. Fig. 3). Butein treatment modulated the levels of most of the targets in both ALDHbright and ALDHlow cells (Suppl. Fig. 3), and, to a much higher extent, the levels of a subset of those genes in the ALDHbright cells, including DDIT3 (as compared to the ALDHlow cells) (Fig. 3B). Thus, the MPM ALDHbright cells exhibited higher activation of the STAT3 pathway and responsivity to butein treatment.

Bottom Line: RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH(bright) cells at steady state and, much more, after pemetrexed + cisplatin treatment.This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression.The possible broad translational relevance of the described mechanism is discussed.

View Article: PubMed Central - PubMed

Affiliation: Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Langone Medical Center, New York University, New York, USA.

ABSTRACT
Here we studied the relevance and modulation of aldehyde dehydrogenase (ALDH) expression in malignant pleural mesothelioma (MPM) chemoresistant cell subpopulations (ALDH(bright) cells), which survive pemetrexed + cisplatin treatment in vitro and in vivo. Expression of the ALDH1A3 isoform was invariably enriched in purified ALDH(bright) cells from multiple MPM cell lines and accounted for the enzymatic activity of those cells. RNAi mediated downregulation of ALDH1A3 reduced the survival of the ALDH(bright) cells at steady state and, much more, after pemetrexed + cisplatin treatment. We demonstrated, for the first time, that a pSTAT3(tyr705)-NFkB(p65) complex is required for the repression of DDIT3 mRNA and this ensures high levels of CEBPβ-dependent ALDH1A3 promoter activity. Inhibition of STAT3-NFkB activity allowed high levels of DDIT3 expression with increased formation of a DDIT3-CEBPβ complex. This reduced the occupancy of the ALDH1A3 promoter by CEBPβ, thus largely reducing the ALDH1A3 expression. Consequently, survival of ALDH(bright) cells in pemetrexed + cisplatin-treated cultures was impaired, following increased apoptosis. We show that such a mechanism is relevant in vivo and underlies the action of butein, a dual STAT3-NFkB inhibitor capable of abating the chemoresistance of mesothelioma cells in vivo. The possible broad translational relevance of the described mechanism is discussed.

No MeSH data available.


Related in: MedlinePlus