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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 treatment affects the ALDHbright cell number in vivo and inhibits tumor growthA. 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 ALDHbright 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.
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Figure 5: Butein treatment affects the ALDHbright cell number in vivo and inhibits tumor growthA. 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 ALDHbright 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.

Mentions: In order to translate what we observed in vitro in an in vivo setting, we performed mouse xenograft experiments. Briefly, NOD-SCID mice were 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 (P+C: 45mpk + 7mpk, respectively) and butein + pemetrexed+ cisplatin (B+P+C 5mpk+45mpk+7mpk, respectively). Treatment (4 i.p injections at day 1, 3, 5 and 7) was started when the tumor ≥ 150mm3 in volume (day 0, n=6 mice/group). Weighting of the excised tumors (at day 24) revealed a significant effect of butein, both when administered alone and, more strongly, when combined to P+C (as compared to those excised from the vehicle-treated mice) (p<0.05) (Fig. 5A). Additionally, only 4/6 tumors were detectable in the B+P+C treated mice at the time of excision (Fig. 5A, left and right panel). No statistically significant reduction in weight of the P+C treated tumors as compared to the vehicle-treated tumors was observed (Fig. 5A, left panel). Butein treatment synergized with pemetrexed + cisplatin in reducing tumor weight, thus mirroring the chemosensitizing effects observed in vitro (Fig. 4C). We postulated that, as observed in vitro, the chemosensitizing effect observed in vivo should correlate with a change in the number of the ALDHbright cells within the butein treated tumor masses. FACS analysis of tumors disaggregated within one hour from harvesting revealed that the percentage of ALDHbright cells was significantly reduced in the butein treated tumors (p < 0.05) (Fig. 5B). Within the same experimental setting, we observed no statistically significant change in the number of ALDHbright cells within the pemetrexed + cisplatin treated tumors, while butein cotreatment caused the ALDHbright cell number to drop significantly and dramatically upon P+C treatment (as compared to the P+C treated mice, p < 0.05, Fig. 5B).


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 treatment affects the ALDHbright cell number in vivo and inhibits tumor growthA. 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 ALDHbright 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.
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Figure 5: Butein treatment affects the ALDHbright cell number in vivo and inhibits tumor growthA. 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 ALDHbright 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.
Mentions: In order to translate what we observed in vitro in an in vivo setting, we performed mouse xenograft experiments. Briefly, NOD-SCID mice were 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 (P+C: 45mpk + 7mpk, respectively) and butein + pemetrexed+ cisplatin (B+P+C 5mpk+45mpk+7mpk, respectively). Treatment (4 i.p injections at day 1, 3, 5 and 7) was started when the tumor ≥ 150mm3 in volume (day 0, n=6 mice/group). Weighting of the excised tumors (at day 24) revealed a significant effect of butein, both when administered alone and, more strongly, when combined to P+C (as compared to those excised from the vehicle-treated mice) (p<0.05) (Fig. 5A). Additionally, only 4/6 tumors were detectable in the B+P+C treated mice at the time of excision (Fig. 5A, left and right panel). No statistically significant reduction in weight of the P+C treated tumors as compared to the vehicle-treated tumors was observed (Fig. 5A, left panel). Butein treatment synergized with pemetrexed + cisplatin in reducing tumor weight, thus mirroring the chemosensitizing effects observed in vitro (Fig. 4C). We postulated that, as observed in vitro, the chemosensitizing effect observed in vivo should correlate with a change in the number of the ALDHbright cells within the butein treated tumor masses. FACS analysis of tumors disaggregated within one hour from harvesting revealed that the percentage of ALDHbright cells was significantly reduced in the butein treated tumors (p < 0.05) (Fig. 5B). Within the same experimental setting, we observed no statistically significant change in the number of ALDHbright cells within the pemetrexed + cisplatin treated tumors, while butein cotreatment caused the ALDHbright cell number to drop significantly and dramatically upon P+C treatment (as compared to the P+C treated mice, p < 0.05, Fig. 5B).

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