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

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.

Keywords: ALDH; CEBPβ; DDIT3; NFkB; STAT3.

Figure 1. Butein affects the survival of MPM chemoresistant cell subpopulations (ALDH^bright cells)

A. Butein reduces the number of ALDH^bright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDH^bright cells (red, gated) in MPM cell cultures treated for 24hrs with vehicle (V: DMSO 0.05%) and butein (B: 18μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) and stained for ALDH activity at 96hrs. The percentage of ALDH^bright cells was determined over the same cells treated with a specific ALDH inhibitor (DEAB) immediately after adding the ALDH substrate (BAA). B. Graph showing the average ALDH^bright cell number from the grouped MPM cell lines (n=10) treated as indicated in 1A. C-D. The ALDH1A3 is responsible for the aldehyde dehydrogenase (ALDH) activity of the ALDHbright cells. C. Heat map: mRNA levels of the detectable ALDH isoforms in purified ALDH^bright and ALDH^low cells from 6 MPM cell lines. D. Left. Representative western blotting of MSTO-211H cells infected with a pool of ALDH1A1, ALDH2 and ALDH1A3 targeting shRNAs and control (scrambled) shRNAs, selected with puromycin and stained as indicated. Right. ALDH activity in 4 representative MPM cell lines infected as indicated in the left panel. Percentage of ALDH activity is relative to cells infected with the scrambled shRNA (control). Duplicate experiments. E. Butein modulates the expression of ALDH1A3. Upper. ALDH1A3 mRNA levels of purified MSTO-211H and HP-1 ALDH^bright cells treated with butein for the indicated times, as assessed by quantitative PCR. Lower. Western Blotting with anti-ALDH1A3 specific antibodies and anti-actin (as a loading control) of whole cell lysates from purified MSTO-211H and HP-1 ALDH^bright cells treated with butein for 36hrs F-G. Butein treatment affects the viability of purified ALDHbright cells. Percentage of SYTOX red negative cells from MSTO-211H and HP-1 ALDH^bright cells infected with a vector expressing scrambled shRNA or ALDH1A3-shRNAs, respectively) and treated as indicated for 24hrs and harvested at 72hrs. G. Protein levels of stress response genes and apoptotic effectors in the indicated MPM cell lines treated as in fig. 1F and harvested at 48hrs. Duplicate experiments. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were indicated otherwise. Statistics: * p < 0.05; ns=not significant: (p > 0.05). One-way analysis of variance with Tukey's post hoc corrections-comparing the mean of each group with the mean of every other group (B) or Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group) (D, E. F. G).

Figure 1. Butein affects the survival of MPM chemoresistant cell subpopulations (ALDH^bright cells)

A. Butein reduces the number of ALDH^bright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDH^bright cells (red, gated) in MPM cell cultures treated for 24hrs with vehicle (V: DMSO 0.05%) and butein (B: 18μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) and stained for ALDH activity at 96hrs. The percentage of ALDH^bright cells was determined over the same cells treated with a specific ALDH inhibitor (DEAB) immediately after adding the ALDH substrate (BAA). B. Graph showing the average ALDH^bright cell number from the grouped MPM cell lines (n=10) treated as indicated in 1A. C-D. The ALDH1A3 is responsible for the aldehyde dehydrogenase (ALDH) activity of the ALDHbright cells. C. Heat map: mRNA levels of the detectable ALDH isoforms in purified ALDH^bright and ALDH^low cells from 6 MPM cell lines. D. Left. Representative western blotting of MSTO-211H cells infected with a pool of ALDH1A1, ALDH2 and ALDH1A3 targeting shRNAs and control (scrambled) shRNAs, selected with puromycin and stained as indicated. Right. ALDH activity in 4 representative MPM cell lines infected as indicated in the left panel. Percentage of ALDH activity is relative to cells infected with the scrambled shRNA (control). Duplicate experiments. E. Butein modulates the expression of ALDH1A3. Upper. ALDH1A3 mRNA levels of purified MSTO-211H and HP-1 ALDH^bright cells treated with butein for the indicated times, as assessed by quantitative PCR. Lower. Western Blotting with anti-ALDH1A3 specific antibodies and anti-actin (as a loading control) of whole cell lysates from purified MSTO-211H and HP-1 ALDH^bright cells treated with butein for 36hrs F-G. Butein treatment affects the viability of purified ALDHbright cells. Percentage of SYTOX red negative cells from MSTO-211H and HP-1 ALDH^bright cells infected with a vector expressing scrambled shRNA or ALDH1A3-shRNAs, respectively) and treated as indicated for 24hrs and harvested at 72hrs. G. Protein levels of stress response genes and apoptotic effectors in the indicated MPM cell lines treated as in fig. 1F and harvested at 48hrs. Duplicate experiments. Histogram bars represent the mean ± s.e.m of ≥ three experiments, except were indicated otherwise. Statistics: * p < 0.05; ns=not significant: (p > 0.05). One-way analysis of variance with Tukey's post hoc corrections-comparing the mean of each group with the mean of every other group (B) or Student's t-test (comparing each sample to its control or, when indicated, to other samples within the same group) (D, E. F. G).

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).

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).

Figure 3. STAT3 inhibition underlies the effect of Butein on the DDIT3 levels

A. 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 ALDH^bright and ALDH^low 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 ALDH^bright vs ALDH^low 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).

Figure 3. STAT3 inhibition underlies the effect of Butein on the DDIT3 levels

A. 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 ALDH^bright and ALDH^low 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 ALDH^bright vs ALDH^low 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).

Figure 4. Butein unlocks the repression of DDIT3mRNA in the chemoresistant ALDH^bright cells

A. Heat map. DDIT3 mRNA levels in ALDH^bright and ALDH^low cells purified from the 6M PM cell lines and treated with vehicle (V: DMSO 0.05%) and butein (B: 18 μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) for 16hrs. B. Upper. Western blotting with DDIT3 antibodies of whole cell lysates from purified ALDH^bright and ALDH^low MSTO-211H cells treated as in 4A. Coomassie staining used as a loading control. Lower. Histograms showing the changes in intensity signal of DDIT3 from duplicate experiments (normalized to two reference protein bands in the coomassie stained gel with Image J software). C. Clonogenic assays. Number of formed colonies from purified cell subpopulations of MSTO-211H and HP-1 cells treated with butein for 16hrs before seeding at clonal density. Histogram bars represent the mean ± s.e.m of triplicate experiments. 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).

Figure 5. Butein treatment affects the ALDH^bright cell number in vivo and inhibits tumor growth

A. Average weight of tumors excised from NOD-SCID mice (n=6/group) injected subcutaneously with 3*10^6 MSTO-211H cells and treated intraperitoneally with vehicle (V, 20 %DMSO/80%corn oil), butein (B, 5mpk), pemetrexed + cisplatin (V+P+C: 45mpk + 7mpk, respectively) and butein + pemetrexed+ cisplatin (B+P+C 5mpk+45mpk+7mpk, respectively) after tumor formation. Duplicate experiments. Inset. Representative micrographs of tumors excised from P+C and B+P+C treated mice, respectively, at day 24 p.i. Scale bar: 0.5 inches. B. FACS plots show the average percentage of ALDH^bright cells from freshly excised and disaggregated tumors. C-D. Butein-treated tumors exhibit inverse regulation of DDIT3 and ALDH1A3 mRNAs. The levels of DDIT3 and ALDH1A3 mRNAs were assessed by quantitative PCR in freshly excised tumors. Asterisks indicate outliers. E. Upper panel. Representative micrographs of 3D clonogenic assays performed with cells derived from the disaggregated tumors of mice treated with V+P+C or B+P+C. Scale bar: 100 μm. Lower panel. Average number of 3D spheroids formed from the disaggregated tumors. Duplicate experiments. Histogram bars represent the mean ± s.e.m. Statistics: * p < 0.05; ns=not significant: (p > 0.05). One-way analysis of variance with Tukey's post hoc corrections-comparing the mean of each group with the mean of every other group.

Figure 6. Proposed working model

The ALDH^bright cells exhibit constitutive activation of the STAT3 pathway which triggers downregulation of DDIT3 mRNA levels both at steady state and upon pemetrexed+cisplatin treatment. Interference with STAT3-NFkB function unlocks expression of DDIT3 in the ALDH^bright cells and this reduces the occupancy of ALDH1A3 promoter by CEBPβ thereby lowering ALDH1A3 expression and the intracellular ALDH activity and strongly affecting the survival of the ALDH^bright cells to chemotherapy-induced stress.