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Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

Steinert G, Oancea C, Roos J, Hagemeyer H, Maier T, Ruthardt M, Puccetti E - PLoS ONE (2011)

Bottom Line: Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific.Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling.We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Goethe-University, Frankfurt, Germany.

ABSTRACT
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

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Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation.(A) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. (B) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. (C) Effects of SSi on Wnt target genes in X-RARα-positive Sca1+/lin- HPCs. Results are represented as 2-ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.
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pone-0022540-g004: Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation.(A) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. (B) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. (C) Effects of SSi on Wnt target genes in X-RARα-positive Sca1+/lin- HPCs. Results are represented as 2-ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.

Mentions: That PML/RARα induces activation of Wnt signaling similar to Wnt ligands and increases HSC self-renewal prompted us to ask whether SSi interferes with Wnt signaling activation by PML/RARα, assessed by TCF/LEF-dependent transactivation. Therefore, we transduced 293 cells with PML/RARα and S33A as a positive control (Figure 4A and 4B) and assessed Wnt signaling activation by luciferase activity using the Topflash/Fopflash system. PML/RARα and S33A induced promoter activation, which decreased upon treatment with SSi (Figure 4A). Furthermore, we used qRT-PCR to investigate the effect of SSi on Wnt signaling in X-RARα-positive Sca1+/lin− HPCs via expression of the Wnt target genes, LEF1, CyclinD1 and Axin2, in the presence/absence of 40 µM SSi. As shown in Figure 4C, expression of LEF1, cyclin D1 and Axin2 was inhibited by SSi treatment in X-RARα-expressing Sca1+/lin− HPCs. Taken together, these data show that SSi reduce PML/RARα-mediated aberrant activation of Wnt-signaling and downregulate Wnt-specific target genes.


Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα.

Steinert G, Oancea C, Roos J, Hagemeyer H, Maier T, Ruthardt M, Puccetti E - PLoS ONE (2011)

Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation.(A) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. (B) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. (C) Effects of SSi on Wnt target genes in X-RARα-positive Sca1+/lin- HPCs. Results are represented as 2-ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139642&req=5

pone-0022540-g004: Effect of SSi on PML/RARα-mediated Wnt signaling pathway activation.(A) Transactivation assay for Wnt-signaling-related TCF/LEF-dependent transcription. Indicated expression vectors were co-transfected with either Topflash (OT) or Fopflash (OF) (the pGL3-OT promoter contains three TCF/LEF binding sites, whereas pGL3-OF contains mutated inactive binding sites and is a negative control) reporter constructs into 293 cells and exposed them to either 100 µM SSi or 0.02% DMSO. After 48 h, luciferase activity was measured and normalized to co-transfected Renilla activity. (B) Western blots for the expression of PML/RARα and S33A using the indicated antibodies, α-β-actin-loading control. (C) Effects of SSi on Wnt target genes in X-RARα-positive Sca1+/lin- HPCs. Results are represented as 2-ΔΔCT values. Each experiment was performed three times in triplicate, with similar results obtained each time. One representative experiment with SD is shown.
Mentions: That PML/RARα induces activation of Wnt signaling similar to Wnt ligands and increases HSC self-renewal prompted us to ask whether SSi interferes with Wnt signaling activation by PML/RARα, assessed by TCF/LEF-dependent transactivation. Therefore, we transduced 293 cells with PML/RARα and S33A as a positive control (Figure 4A and 4B) and assessed Wnt signaling activation by luciferase activity using the Topflash/Fopflash system. PML/RARα and S33A induced promoter activation, which decreased upon treatment with SSi (Figure 4A). Furthermore, we used qRT-PCR to investigate the effect of SSi on Wnt signaling in X-RARα-positive Sca1+/lin− HPCs via expression of the Wnt target genes, LEF1, CyclinD1 and Axin2, in the presence/absence of 40 µM SSi. As shown in Figure 4C, expression of LEF1, cyclin D1 and Axin2 was inhibited by SSi treatment in X-RARα-expressing Sca1+/lin− HPCs. Taken together, these data show that SSi reduce PML/RARα-mediated aberrant activation of Wnt-signaling and downregulate Wnt-specific target genes.

Bottom Line: Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific.Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling.We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Goethe-University, Frankfurt, Germany.

ABSTRACT
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.

Show MeSH
Related in: MedlinePlus