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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

A. Butein modulates the ALDH1A3 promoter activity by modulating DDIT3 levels. Normalized luciferase activity of MSTO-211H and HP-1 cells transfected with a ALDH1A3-luciferase expressing vector and treated with butein (18 μM) at the indicated times. B. Butein treatment reduces the occupancy of the ALDH1A3 promoter by CEBPβ. Quantitative PCR. Amplification of the CEBPβ binding region from chromatin immunoprecipitated with anti-CEBPβ and control rabbit IgG from MSTO-211H and HP-1 cells treated with vehicle or butein (18 μM), respectively, for 18 hours. Percentage of enrichment relative to the input chromatin is reported. Amplification of a DNA sequence not containing the CEBPβ binding site was used as an “off target” control to probe the anti-CEBPβ immunoprecipitated material. C-D. Butein affects DDIT3 protein levels. C. Left. Representative fluorescence micrographs of MSTO-211H cells treated with vehicle or butein (18 μM), for 24hrs and stained with anti-DDIT3 antibodies (right). Cell nuclei were stained with DAPI (left). A minimum of 8 fields (containing ≥40 nuclei) was counted in duplicate experiments. Scale bar: 20μm. Right. Histograms showing the average percentage of DDIT3 positive nuclei from duplicate experiments. D. Left. Western blotting of whole cell lysates from MSTO-211H cells treated with butein (18 μM), as indicated and stained with anti-DDIT3, anti-ALDH1A3 and anti-GAPDH antibodies (as a loading control). Right. Histograms showing the changes in intensity signal of DDIT3 and ALDH1A3 (normalized to actin with Image J software). E. Increased interaction of DDIT3 and CEBPβ in butein-treated cells. Western Blotting with anti-DDIT3 and anti CEBPβ antibodies of whole cell lysates immunoprecipitated with anti-DDIT3 antibody and isotype matched mouse IgG (as a control), respectively. F. RNAi-mediated downregulation of CEBPβ mimicks the effects of butein on ALDH1A3 expression. Left. Western blotting with anti- CEBPβ antibodies of MSTO-211H and HP-1 cells transfected with control (scrambled) and CEBPβ-targeting siRNA. (s.e: short exposure; l.e.: long exposure). GAPDH used as a loading control. Right. mRNA levels of ALDH1A3 in HP-1 and MSTO-211H cells transfected with scrambled or CEBPβ-targeting siRNAs assessed by quantitative PCR. G. Luciferase activity of HP-1 and MSTO-211H cells transfected with an ALDH1A3 luciferase expression vector and, 24hrs later, with scrambled or CEBPβ-targeting siRNAs. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were otherwise indicated. Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group).
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Figure 2: A. Butein modulates the ALDH1A3 promoter activity by modulating DDIT3 levels. Normalized luciferase activity of MSTO-211H and HP-1 cells transfected with a ALDH1A3-luciferase expressing vector and treated with butein (18 μM) at the indicated times. B. Butein treatment reduces the occupancy of the ALDH1A3 promoter by CEBPβ. Quantitative PCR. Amplification of the CEBPβ binding region from chromatin immunoprecipitated with anti-CEBPβ and control rabbit IgG from MSTO-211H and HP-1 cells treated with vehicle or butein (18 μM), respectively, for 18 hours. Percentage of enrichment relative to the input chromatin is reported. Amplification of a DNA sequence not containing the CEBPβ binding site was used as an “off target” control to probe the anti-CEBPβ immunoprecipitated material. C-D. Butein affects DDIT3 protein levels. C. Left. Representative fluorescence micrographs of MSTO-211H cells treated with vehicle or butein (18 μM), for 24hrs and stained with anti-DDIT3 antibodies (right). Cell nuclei were stained with DAPI (left). A minimum of 8 fields (containing ≥40 nuclei) was counted in duplicate experiments. Scale bar: 20μm. Right. Histograms showing the average percentage of DDIT3 positive nuclei from duplicate experiments. D. Left. Western blotting of whole cell lysates from MSTO-211H cells treated with butein (18 μM), as indicated and stained with anti-DDIT3, anti-ALDH1A3 and anti-GAPDH antibodies (as a loading control). Right. Histograms showing the changes in intensity signal of DDIT3 and ALDH1A3 (normalized to actin with Image J software). E. Increased interaction of DDIT3 and CEBPβ in butein-treated cells. Western Blotting with anti-DDIT3 and anti CEBPβ antibodies of whole cell lysates immunoprecipitated with anti-DDIT3 antibody and isotype matched mouse IgG (as a control), respectively. F. RNAi-mediated downregulation of CEBPβ mimicks the effects of butein on ALDH1A3 expression. Left. Western blotting with anti- CEBPβ antibodies of MSTO-211H and HP-1 cells transfected with control (scrambled) and CEBPβ-targeting siRNA. (s.e: short exposure; l.e.: long exposure). GAPDH used as a loading control. Right. mRNA levels of ALDH1A3 in HP-1 and MSTO-211H cells transfected with scrambled or CEBPβ-targeting siRNAs assessed by quantitative PCR. G. Luciferase activity of HP-1 and MSTO-211H cells transfected with an ALDH1A3 luciferase expression vector and, 24hrs later, with scrambled or CEBPβ-targeting siRNAs. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were otherwise indicated. Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group).

Mentions: To investigate the modulation of ALDH1A3 mRNA by butein (Fig. 1E), we transfected MSTO-211H and HP-1 MPM cells with a luciferase reporter vector containing the ALDH1A3 promoter (from −900 to +170bp). Treatment of the cells with vehicle or butein 24hrs later revealed that the latter strongly downregulated the luciferase expression in a time dependent way (Fig. 2A). This experiment suggested that butein may directly affect the ALDH1A3 mRNA levels by modulating its promoter activity.


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)

A. Butein modulates the ALDH1A3 promoter activity by modulating DDIT3 levels. Normalized luciferase activity of MSTO-211H and HP-1 cells transfected with a ALDH1A3-luciferase expressing vector and treated with butein (18 μM) at the indicated times. B. Butein treatment reduces the occupancy of the ALDH1A3 promoter by CEBPβ. Quantitative PCR. Amplification of the CEBPβ binding region from chromatin immunoprecipitated with anti-CEBPβ and control rabbit IgG from MSTO-211H and HP-1 cells treated with vehicle or butein (18 μM), respectively, for 18 hours. Percentage of enrichment relative to the input chromatin is reported. Amplification of a DNA sequence not containing the CEBPβ binding site was used as an “off target” control to probe the anti-CEBPβ immunoprecipitated material. C-D. Butein affects DDIT3 protein levels. C. Left. Representative fluorescence micrographs of MSTO-211H cells treated with vehicle or butein (18 μM), for 24hrs and stained with anti-DDIT3 antibodies (right). Cell nuclei were stained with DAPI (left). A minimum of 8 fields (containing ≥40 nuclei) was counted in duplicate experiments. Scale bar: 20μm. Right. Histograms showing the average percentage of DDIT3 positive nuclei from duplicate experiments. D. Left. Western blotting of whole cell lysates from MSTO-211H cells treated with butein (18 μM), as indicated and stained with anti-DDIT3, anti-ALDH1A3 and anti-GAPDH antibodies (as a loading control). Right. Histograms showing the changes in intensity signal of DDIT3 and ALDH1A3 (normalized to actin with Image J software). E. Increased interaction of DDIT3 and CEBPβ in butein-treated cells. Western Blotting with anti-DDIT3 and anti CEBPβ antibodies of whole cell lysates immunoprecipitated with anti-DDIT3 antibody and isotype matched mouse IgG (as a control), respectively. F. RNAi-mediated downregulation of CEBPβ mimicks the effects of butein on ALDH1A3 expression. Left. Western blotting with anti- CEBPβ antibodies of MSTO-211H and HP-1 cells transfected with control (scrambled) and CEBPβ-targeting siRNA. (s.e: short exposure; l.e.: long exposure). GAPDH used as a loading control. Right. mRNA levels of ALDH1A3 in HP-1 and MSTO-211H cells transfected with scrambled or CEBPβ-targeting siRNAs assessed by quantitative PCR. G. Luciferase activity of HP-1 and MSTO-211H cells transfected with an ALDH1A3 luciferase expression vector and, 24hrs later, with scrambled or CEBPβ-targeting siRNAs. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were otherwise indicated. Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group).
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Figure 2: A. Butein modulates the ALDH1A3 promoter activity by modulating DDIT3 levels. Normalized luciferase activity of MSTO-211H and HP-1 cells transfected with a ALDH1A3-luciferase expressing vector and treated with butein (18 μM) at the indicated times. B. Butein treatment reduces the occupancy of the ALDH1A3 promoter by CEBPβ. Quantitative PCR. Amplification of the CEBPβ binding region from chromatin immunoprecipitated with anti-CEBPβ and control rabbit IgG from MSTO-211H and HP-1 cells treated with vehicle or butein (18 μM), respectively, for 18 hours. Percentage of enrichment relative to the input chromatin is reported. Amplification of a DNA sequence not containing the CEBPβ binding site was used as an “off target” control to probe the anti-CEBPβ immunoprecipitated material. C-D. Butein affects DDIT3 protein levels. C. Left. Representative fluorescence micrographs of MSTO-211H cells treated with vehicle or butein (18 μM), for 24hrs and stained with anti-DDIT3 antibodies (right). Cell nuclei were stained with DAPI (left). A minimum of 8 fields (containing ≥40 nuclei) was counted in duplicate experiments. Scale bar: 20μm. Right. Histograms showing the average percentage of DDIT3 positive nuclei from duplicate experiments. D. Left. Western blotting of whole cell lysates from MSTO-211H cells treated with butein (18 μM), as indicated and stained with anti-DDIT3, anti-ALDH1A3 and anti-GAPDH antibodies (as a loading control). Right. Histograms showing the changes in intensity signal of DDIT3 and ALDH1A3 (normalized to actin with Image J software). E. Increased interaction of DDIT3 and CEBPβ in butein-treated cells. Western Blotting with anti-DDIT3 and anti CEBPβ antibodies of whole cell lysates immunoprecipitated with anti-DDIT3 antibody and isotype matched mouse IgG (as a control), respectively. F. RNAi-mediated downregulation of CEBPβ mimicks the effects of butein on ALDH1A3 expression. Left. Western blotting with anti- CEBPβ antibodies of MSTO-211H and HP-1 cells transfected with control (scrambled) and CEBPβ-targeting siRNA. (s.e: short exposure; l.e.: long exposure). GAPDH used as a loading control. Right. mRNA levels of ALDH1A3 in HP-1 and MSTO-211H cells transfected with scrambled or CEBPβ-targeting siRNAs assessed by quantitative PCR. G. Luciferase activity of HP-1 and MSTO-211H cells transfected with an ALDH1A3 luciferase expression vector and, 24hrs later, with scrambled or CEBPβ-targeting siRNAs. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were otherwise indicated. Statistics: * p < 0.05; ns=not significant: (p > 0.05). Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group).
Mentions: To investigate the modulation of ALDH1A3 mRNA by butein (Fig. 1E), we transfected MSTO-211H and HP-1 MPM cells with a luciferase reporter vector containing the ALDH1A3 promoter (from −900 to +170bp). Treatment of the cells with vehicle or butein 24hrs later revealed that the latter strongly downregulated the luciferase expression in a time dependent way (Fig. 2A). This experiment suggested that butein may directly affect the ALDH1A3 mRNA levels by modulating its promoter activity.

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