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Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

Ciarapica R, Carcarino E, Adesso L, De Salvo M, Bracaglia G, Leoncini PP, Dall'agnese A, Verginelli F, Milano GM, Boldrini R, Inserra A, Stifani S, Screpanti I, Marquez VE, Valente S, Mai A, Puri PL, Locatelli F, Palacios D, Rota R - BMC Cancer (2014)

Bottom Line: Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1.These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect.These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S, Onofrio 4, 00165 Rome, Italy. roberta.ciarapica@yahoo.com.

ABSTRACT

Background: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.

Methods: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.

Results: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.

Conclusions: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

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Pharmacological inhibition of EZH2 prevents embryonal RMS cell proliferation. (a) RD cells cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) were treated daily with either the S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (left panels) or the EZH2 catalytic inhibitor MC1945 (right panels) at the reported concentrations or with vehicle (i.e., water for DZNep or DMSO for MC1945) and harvested and counted at the indicated time points. *P < 0.05 (Student’s t-test); Bars, SD. Three independent experiments in duplicate. (b) Western blot showing EZH2 along with histone H3 trimethylation on Lys27 (H3K27me3), and on Lys9 (H3K9me3) levels in RD cells treated for 72 h with 5 μM DZNep (left panel) and 5 μM MC1945 (right panel) or with vehicle (i.e., water or DMSO). Total H3 and - tubulin amounts were shown as the loading controls. Representative of three independent experiments.
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Figure 5: Pharmacological inhibition of EZH2 prevents embryonal RMS cell proliferation. (a) RD cells cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) were treated daily with either the S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (left panels) or the EZH2 catalytic inhibitor MC1945 (right panels) at the reported concentrations or with vehicle (i.e., water for DZNep or DMSO for MC1945) and harvested and counted at the indicated time points. *P < 0.05 (Student’s t-test); Bars, SD. Three independent experiments in duplicate. (b) Western blot showing EZH2 along with histone H3 trimethylation on Lys27 (H3K27me3), and on Lys9 (H3K9me3) levels in RD cells treated for 72 h with 5 μM DZNep (left panel) and 5 μM MC1945 (right panel) or with vehicle (i.e., water or DMSO). Total H3 and - tubulin amounts were shown as the loading controls. Representative of three independent experiments.

Mentions: To translate our results toward a future potential clinical intervention for aggressive embryonal RMS, we assessed the feasibility of pharmacological inhibition of EZH2 in RD cells. We treated RD cells with a well known EZH2 inhibitor, the S-adenosyl-L-homocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep), which induces degradation of EZH2 [17,31,39]. In parallel, we used two new catalytic EZH2 inhibitors that inhibit the activity of the protein, the already validated EZH2 inhibitor MC1948 [28] and a new, more potent, derivative, MC1945 [32,40]. A significant reduction in the proliferation rate was noticed in RD cells treated for 72 h and 96 h with 1 μM of either DZNep or MC1945 compared to untreated or vehicle-treated cells (Figure 5a). Moreover, a significant greater inhibition of cell proliferation was obtained when RD cells were treated with 5 μM of each compound, suggesting a dose-dependent inhibitory effect (Figure 5a). These effects were accompanied by a down-regulation of EZH2 protein levels upon DZNep treatment (Figure 5b, left panel) whereas the levels remained constant after treatment with the catalytic inhibitors MC1945, as expected (Figure 5b, right panel) [28]. Both DZNep and MC1945 treatments resulted in a decrease in global levels of the EZH2 repressive mark H3K27me3 (Figure 5b) (28–30). On the contrary, the levels of H3K9me3, another repressive mark, remained unchanged after both treatments, demonstrating the specificity of the two compounds in targeting EZH2-containing complexes in our experimental conditions (Figure 5b). Same results were obtained in preliminary experiments with MC1948 (Additional file 5: Figure S4a and b). Similarly to what happened for EZH2-silenced cells, culture condition in differentiation medium (low serum) was unable to significantly potentiate the formation of MHC-positive multinucleated structures 4 days post-treatment as compared to growth (10% serum) medium condition (Additional file 6: Figure S5). By contrast, 5 days of treatment in DM lead to detachment of cells from the well surface, maybe due to cytotoxic effects of nutrient-deprived conditions (data not shown).


Pharmacological inhibition of EZH2 as a promising differentiation therapy in embryonal RMS.

Ciarapica R, Carcarino E, Adesso L, De Salvo M, Bracaglia G, Leoncini PP, Dall'agnese A, Verginelli F, Milano GM, Boldrini R, Inserra A, Stifani S, Screpanti I, Marquez VE, Valente S, Mai A, Puri PL, Locatelli F, Palacios D, Rota R - BMC Cancer (2014)

Pharmacological inhibition of EZH2 prevents embryonal RMS cell proliferation. (a) RD cells cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) were treated daily with either the S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (left panels) or the EZH2 catalytic inhibitor MC1945 (right panels) at the reported concentrations or with vehicle (i.e., water for DZNep or DMSO for MC1945) and harvested and counted at the indicated time points. *P < 0.05 (Student’s t-test); Bars, SD. Three independent experiments in duplicate. (b) Western blot showing EZH2 along with histone H3 trimethylation on Lys27 (H3K27me3), and on Lys9 (H3K9me3) levels in RD cells treated for 72 h with 5 μM DZNep (left panel) and 5 μM MC1945 (right panel) or with vehicle (i.e., water or DMSO). Total H3 and - tubulin amounts were shown as the loading controls. Representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016511&req=5

Figure 5: Pharmacological inhibition of EZH2 prevents embryonal RMS cell proliferation. (a) RD cells cultured in proliferating growth medium (GM, i.e. supplemented with 10% of fetal calf serum) were treated daily with either the S-adenosyl-L-homocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) (left panels) or the EZH2 catalytic inhibitor MC1945 (right panels) at the reported concentrations or with vehicle (i.e., water for DZNep or DMSO for MC1945) and harvested and counted at the indicated time points. *P < 0.05 (Student’s t-test); Bars, SD. Three independent experiments in duplicate. (b) Western blot showing EZH2 along with histone H3 trimethylation on Lys27 (H3K27me3), and on Lys9 (H3K9me3) levels in RD cells treated for 72 h with 5 μM DZNep (left panel) and 5 μM MC1945 (right panel) or with vehicle (i.e., water or DMSO). Total H3 and - tubulin amounts were shown as the loading controls. Representative of three independent experiments.
Mentions: To translate our results toward a future potential clinical intervention for aggressive embryonal RMS, we assessed the feasibility of pharmacological inhibition of EZH2 in RD cells. We treated RD cells with a well known EZH2 inhibitor, the S-adenosyl-L-homocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep), which induces degradation of EZH2 [17,31,39]. In parallel, we used two new catalytic EZH2 inhibitors that inhibit the activity of the protein, the already validated EZH2 inhibitor MC1948 [28] and a new, more potent, derivative, MC1945 [32,40]. A significant reduction in the proliferation rate was noticed in RD cells treated for 72 h and 96 h with 1 μM of either DZNep or MC1945 compared to untreated or vehicle-treated cells (Figure 5a). Moreover, a significant greater inhibition of cell proliferation was obtained when RD cells were treated with 5 μM of each compound, suggesting a dose-dependent inhibitory effect (Figure 5a). These effects were accompanied by a down-regulation of EZH2 protein levels upon DZNep treatment (Figure 5b, left panel) whereas the levels remained constant after treatment with the catalytic inhibitors MC1945, as expected (Figure 5b, right panel) [28]. Both DZNep and MC1945 treatments resulted in a decrease in global levels of the EZH2 repressive mark H3K27me3 (Figure 5b) (28–30). On the contrary, the levels of H3K9me3, another repressive mark, remained unchanged after both treatments, demonstrating the specificity of the two compounds in targeting EZH2-containing complexes in our experimental conditions (Figure 5b). Same results were obtained in preliminary experiments with MC1948 (Additional file 5: Figure S4a and b). Similarly to what happened for EZH2-silenced cells, culture condition in differentiation medium (low serum) was unable to significantly potentiate the formation of MHC-positive multinucleated structures 4 days post-treatment as compared to growth (10% serum) medium condition (Additional file 6: Figure S5). By contrast, 5 days of treatment in DM lead to detachment of cells from the well surface, maybe due to cytotoxic effects of nutrient-deprived conditions (data not shown).

Bottom Line: Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1.These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect.These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncohematology, Laboratory of Angiogenesis, Ospedale Pediatrico Bambino Gesù, IRCCS, Piazza S, Onofrio 4, 00165 Rome, Italy. roberta.ciarapica@yahoo.com.

ABSTRACT

Background: Embryonal Rhabdomyosarcoma (RMS) is a pediatric soft-tissue sarcoma derived from myogenic precursors that is characterized by a good prognosis in patients with localized disease. Conversely, metastatic tumors often relapse, leading to a dismal outcome. The histone methyltransferase EZH2 epigenetically suppresses skeletal muscle differentiation by repressing the transcription of myogenic genes. Moreover, de-regulated EZH2 expression has been extensively implied in human cancers. We have previously shown that EZH2 is aberrantly over-expressed in RMS primary tumors and cell lines. Moreover, it has been recently reported that EZH2 silencing in RD cells, a recurrence-derived embryonal RMS cell line, favors myofiber-like structures formation in a pro-differentiation context. Here we evaluate whether similar effects can be obtained also in the presence of growth factor-supplemented medium (GM), that mimics a pro-proliferative microenvironment, and by pharmacological targeting of EZH2 in RD cells and in RD tumor xenografts.

Methods: Embryonal RMS RD cells were cultured in GM and silenced for EZH2 or treated with either the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) that induces EZH2 degradation, or with a new class of catalytic EZH2 inhibitors, MC1948 and MC1945, which block the catalytic activity of EZH2. RD cell proliferation and myogenic differentiation were evaluated both in vitro and in vivo.

Results: Here we show that EZH2 protein was abnormally expressed in 19 out of 19 (100%) embryonal RMS primary tumors and cell lines compared to their normal counterparts. Genetic down-regulation of EZH2 by silencing in GM condition reduced RD cell proliferation up-regulating p21Cip1. It also resulted in myogenic-like differentiation testified by the up-regulation of myogenic markers Myogenin, MCK and MHC. These effects were reverted by enforced over-expression of a murine Ezh2, highlighting an EZH2-specific effect. Pharmacological inhibition of EZH2 using either DZNep or MC inhibitors phenocopied the genetic knockdown of EZH2 preventing cell proliferation and restoring myogenic differentiation both in vitro and in vivo.

Conclusions: These results provide evidence that EZH2 function can be counteracted by pharmacological inhibition in embryonal RMS blocking proliferation even in a pro-proliferative context. They also suggest that this approach could be exploited as a differentiation therapy in adjuvant therapeutic intervention for embryonal RMS.

Show MeSH
Related in: MedlinePlus