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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

Butein affects the survival of MPM chemoresistant cell subpopulations (ALDHbright cells)A. Butein reduces the number of ALDHbright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDHbright 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 ALDHbright 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 ALDHbright 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 ALDHbright and ALDHlow 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 ALDHbright 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 ALDHbright 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 ALDHbright 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).
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Figure 1: Butein affects the survival of MPM chemoresistant cell subpopulations (ALDHbright cells)A. Butein reduces the number of ALDHbright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDHbright 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 ALDHbright 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 ALDHbright 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 ALDHbright and ALDHlow 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 ALDHbright 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 ALDHbright 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 ALDHbright 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).

Mentions: Given that the ALDHbright MPM cells are the main subcellular population resistant to pemetrexed [4] and given the ability of butein to counteract the chemoresistance of MPM cells in vitro and in vivo [35], we tested the hypothesis that the latter compound may affect the survival of the ALDHbright cell subpopulations. Treatment with butein (B: 18μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) for 96hrs strongly reduced the number of ALDHbright cells in multiple unrelated MPM cell lines (n=10) and prevented their increase after pemetrexed+cisplatin (P+C) treatment (Fig. 1A-1B, p < 0.05). Since disappearance of ALDHbright cells may follow direct enzyme inhibition or downregulation of ALDH expression, we explored which of the two processes underlied the effects of butein. Short term (0-12hrs) treatment of MPM cells with butein did not affect the ALDH activity (suppl. Fig. 1A, upper and lower). To assess whether butein may modulate the expression rather than the activity of ALDHs, we first determined which ALDH isoform(s) would be enriched in the ALDHbright cells (Fig. 1C). We assessed (by quantitative PCR) the mRNA levels of the (detectable) ALDH isoforms in FACS sorted ALDHbright and ALDHlow cells from unrelated MPM cell lines (average purity of the ALDHbright cells: 92-96%, n=6). Quantitative PCR revealed that the ALDH1A3 (and, to a much lesser extent, ALDH1A1 and ALDH2) was enriched in the ALDHbright cells of all the analyzed cell lines (p < 0.05) (Fig. 1C, heat map).


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)

Butein affects the survival of MPM chemoresistant cell subpopulations (ALDHbright cells)A. Butein reduces the number of ALDHbright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDHbright 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 ALDHbright 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 ALDHbright 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 ALDHbright and ALDHlow 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 ALDHbright 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 ALDHbright 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 ALDHbright 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).
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Figure 1: Butein affects the survival of MPM chemoresistant cell subpopulations (ALDHbright cells)A. Butein reduces the number of ALDHbright cells in MPM cultures. Representative flow cytometry plots showing the percentage of ALDHbright 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 ALDHbright 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 ALDHbright 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 ALDHbright and ALDHlow 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 ALDHbright 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 ALDHbright 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 ALDHbright 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).
Mentions: Given that the ALDHbright MPM cells are the main subcellular population resistant to pemetrexed [4] and given the ability of butein to counteract the chemoresistance of MPM cells in vitro and in vivo [35], we tested the hypothesis that the latter compound may affect the survival of the ALDHbright cell subpopulations. Treatment with butein (B: 18μM), alone or in combination with pemetrexed + cisplatin (P+C: 10 μM + 5 μM, respectively) for 96hrs strongly reduced the number of ALDHbright cells in multiple unrelated MPM cell lines (n=10) and prevented their increase after pemetrexed+cisplatin (P+C) treatment (Fig. 1A-1B, p < 0.05). Since disappearance of ALDHbright cells may follow direct enzyme inhibition or downregulation of ALDH expression, we explored which of the two processes underlied the effects of butein. Short term (0-12hrs) treatment of MPM cells with butein did not affect the ALDH activity (suppl. Fig. 1A, upper and lower). To assess whether butein may modulate the expression rather than the activity of ALDHs, we first determined which ALDH isoform(s) would be enriched in the ALDHbright cells (Fig. 1C). We assessed (by quantitative PCR) the mRNA levels of the (detectable) ALDH isoforms in FACS sorted ALDHbright and ALDHlow cells from unrelated MPM cell lines (average purity of the ALDHbright cells: 92-96%, n=6). Quantitative PCR revealed that the ALDH1A3 (and, to a much lesser extent, ALDH1A1 and ALDH2) was enriched in the ALDHbright cells of all the analyzed cell lines (p < 0.05) (Fig. 1C, heat map).

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