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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 restores myogenic differentiation of embryonal RMS cells in the presence of growth medium (GM). RD cells were analyzed for the induction of muscle-like differentiation after 6 days of 5 μM DZNep (a) and MC1945 (c) treatments. Representative immunofluorescence showing de novo expression of endogenous Myosin Heavy Chain (MHC, red) in multinucleated fibers of DZNep and MC1945 treated RD cells. Untreated (UN) and control cells treated with vehicle (i.e., water or DMSO) are shown. Representative immunofluorescence of three assays. mRNA levels (real time qRT-PCR) of Myogenin and MCK in RD treated for 72 h with 5 μM DZNep (b) and 5 μM MC1954 (d) were normalized to GAPDH levels and expressed as fold increase over Untreated condition (1 arbitrary unit, not reported). Columns, means; Bars, SD. Results from two independent experiments are shown. (e) RD cells Untreated or treated for 96 h with DZNep (left) or MC1945 (right) at the indicated concentrations were stained for Annexin V and 7-AAD, and the frequency of Annexin V and 7-AAD-positive labeling (% cell death) was recorded by flow cytometry. Representative cytofluorometric plots are shown. Annexin V+/7-AAD- events (lower right quadrants) represent early stages of apoptosis, whereas Annexin V+/7-AAD + events (upper right quadrants) stand for late apoptotic cells. Representative of three independent experiments run in duplicate.
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Figure 6: Pharmacological inhibition of EZH2 restores myogenic differentiation of embryonal RMS cells in the presence of growth medium (GM). RD cells were analyzed for the induction of muscle-like differentiation after 6 days of 5 μM DZNep (a) and MC1945 (c) treatments. Representative immunofluorescence showing de novo expression of endogenous Myosin Heavy Chain (MHC, red) in multinucleated fibers of DZNep and MC1945 treated RD cells. Untreated (UN) and control cells treated with vehicle (i.e., water or DMSO) are shown. Representative immunofluorescence of three assays. mRNA levels (real time qRT-PCR) of Myogenin and MCK in RD treated for 72 h with 5 μM DZNep (b) and 5 μM MC1954 (d) were normalized to GAPDH levels and expressed as fold increase over Untreated condition (1 arbitrary unit, not reported). Columns, means; Bars, SD. Results from two independent experiments are shown. (e) RD cells Untreated or treated for 96 h with DZNep (left) or MC1945 (right) at the indicated concentrations were stained for Annexin V and 7-AAD, and the frequency of Annexin V and 7-AAD-positive labeling (% cell death) was recorded by flow cytometry. Representative cytofluorometric plots are shown. Annexin V+/7-AAD- events (lower right quadrants) represent early stages of apoptosis, whereas Annexin V+/7-AAD + events (upper right quadrants) stand for late apoptotic cells. Representative of three independent experiments run in duplicate.

Mentions: In order to evaluate whether the strong inhibitory effects on RD proliferation obtained by blocking EZH2 methyltransferase activity was associated to the triggering of myogenic-like differentiation we treated RD cells with 1 μM of MC1948 for 6 days and then we analyzed myogenic differentiation by immunocytochemistry. We noticed the appearance of multinucleated myotube-like structures expressing MHC in RD cells treated with MC1948 compared to vehicle-treated cells (Additional file 4: Figure S3c). Then we extended the study enrolling DZNep and MC1945. Treatment of RD cells for 6 days with either 5 μM of DZNep or MC1945 resulted in the formation of MHC-positive multinucleated myotube-like structures (Figure 6a and 6c) and in the induction of Myogenin and MCK gene transcription 72 h post-treatment (Figure 6b and 6d). Consistently with these results, no sign of apoptosis testified by the lack of appearance of apoptotic Annexin V-positive cells was evidenced in both DZNep- and MC1945-treated RD cells (Figure 6e). Altogether, these results suggest that the two pharmacological inhibitory approaches of EZH2 function are capable to restore myogenic differentiation of embryonal RMS cells as occurs in the case of EZH2 genetic depletion.


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 restores myogenic differentiation of embryonal RMS cells in the presence of growth medium (GM). RD cells were analyzed for the induction of muscle-like differentiation after 6 days of 5 μM DZNep (a) and MC1945 (c) treatments. Representative immunofluorescence showing de novo expression of endogenous Myosin Heavy Chain (MHC, red) in multinucleated fibers of DZNep and MC1945 treated RD cells. Untreated (UN) and control cells treated with vehicle (i.e., water or DMSO) are shown. Representative immunofluorescence of three assays. mRNA levels (real time qRT-PCR) of Myogenin and MCK in RD treated for 72 h with 5 μM DZNep (b) and 5 μM MC1954 (d) were normalized to GAPDH levels and expressed as fold increase over Untreated condition (1 arbitrary unit, not reported). Columns, means; Bars, SD. Results from two independent experiments are shown. (e) RD cells Untreated or treated for 96 h with DZNep (left) or MC1945 (right) at the indicated concentrations were stained for Annexin V and 7-AAD, and the frequency of Annexin V and 7-AAD-positive labeling (% cell death) was recorded by flow cytometry. Representative cytofluorometric plots are shown. Annexin V+/7-AAD- events (lower right quadrants) represent early stages of apoptosis, whereas Annexin V+/7-AAD + events (upper right quadrants) stand for late apoptotic cells. Representative of three independent experiments run in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Pharmacological inhibition of EZH2 restores myogenic differentiation of embryonal RMS cells in the presence of growth medium (GM). RD cells were analyzed for the induction of muscle-like differentiation after 6 days of 5 μM DZNep (a) and MC1945 (c) treatments. Representative immunofluorescence showing de novo expression of endogenous Myosin Heavy Chain (MHC, red) in multinucleated fibers of DZNep and MC1945 treated RD cells. Untreated (UN) and control cells treated with vehicle (i.e., water or DMSO) are shown. Representative immunofluorescence of three assays. mRNA levels (real time qRT-PCR) of Myogenin and MCK in RD treated for 72 h with 5 μM DZNep (b) and 5 μM MC1954 (d) were normalized to GAPDH levels and expressed as fold increase over Untreated condition (1 arbitrary unit, not reported). Columns, means; Bars, SD. Results from two independent experiments are shown. (e) RD cells Untreated or treated for 96 h with DZNep (left) or MC1945 (right) at the indicated concentrations were stained for Annexin V and 7-AAD, and the frequency of Annexin V and 7-AAD-positive labeling (% cell death) was recorded by flow cytometry. Representative cytofluorometric plots are shown. Annexin V+/7-AAD- events (lower right quadrants) represent early stages of apoptosis, whereas Annexin V+/7-AAD + events (upper right quadrants) stand for late apoptotic cells. Representative of three independent experiments run in duplicate.
Mentions: In order to evaluate whether the strong inhibitory effects on RD proliferation obtained by blocking EZH2 methyltransferase activity was associated to the triggering of myogenic-like differentiation we treated RD cells with 1 μM of MC1948 for 6 days and then we analyzed myogenic differentiation by immunocytochemistry. We noticed the appearance of multinucleated myotube-like structures expressing MHC in RD cells treated with MC1948 compared to vehicle-treated cells (Additional file 4: Figure S3c). Then we extended the study enrolling DZNep and MC1945. Treatment of RD cells for 6 days with either 5 μM of DZNep or MC1945 resulted in the formation of MHC-positive multinucleated myotube-like structures (Figure 6a and 6c) and in the induction of Myogenin and MCK gene transcription 72 h post-treatment (Figure 6b and 6d). Consistently with these results, no sign of apoptosis testified by the lack of appearance of apoptotic Annexin V-positive cells was evidenced in both DZNep- and MC1945-treated RD cells (Figure 6e). Altogether, these results suggest that the two pharmacological inhibitory approaches of EZH2 function are capable to restore myogenic differentiation of embryonal RMS cells as occurs in the case of EZH2 genetic depletion.

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