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Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer.

Wyce A, Degenhardt Y, Bai Y, Le B, Korenchuk S, Crouthame MC, McHugh CF, Vessella R, Creasy CL, Tummino PJ, Barbash O - Oncotarget (2013)

Bottom Line: BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation.Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models.Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics DPU, Oncology R and D GlaxoSmithKline, Collegeville, PA, USA.

ABSTRACT
BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

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I-BET762 inhibits tumor growth in primary xenograft models with high MYC, AR, and TMPRSS2-ERGexpressionA, qPCR determination of basal expression of the indicated genes in the LUCaP 35CR and LUCaP 145.2 primary xenograft models. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. Data represents the mean value ± SEM from three animals. B, qPCR analysis of TMPRSS2-ERG expression as described in A. C, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 35CR xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 36 days. Samples were collected 8 hours after dosing on day 36. Data were normalized to expression of GAPDH, and are presented as fold induction compared to vehicle treated controls. Data represents the mean ± SEM from three animals. Asterisks indicate significant changes as determined by two-tailed, unpaired t test (p< 0.05). D, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 145.2 xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 30 days. Samples were collected 8 hours after dosing on day 30. Data were analyzed and presented as described in C. E, Mean absolute tumor volumes ± SEM for LUCaP 35CR xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. Asterisks indicate p < 0.05 as determined by the Mann-Whitney test. TGI for 8 mg/kg was 27% on Day 36 (n= 10; p= 0.44). TGI for 25 mg/kg was 57% on Day 36 (n=10; p =0.006). F, Mean absolute tumor volumes ± SEM for LUCaP 145.2 xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. No significant TGI was observed at either dose.
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Figure 5: I-BET762 inhibits tumor growth in primary xenograft models with high MYC, AR, and TMPRSS2-ERGexpressionA, qPCR determination of basal expression of the indicated genes in the LUCaP 35CR and LUCaP 145.2 primary xenograft models. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. Data represents the mean value ± SEM from three animals. B, qPCR analysis of TMPRSS2-ERG expression as described in A. C, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 35CR xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 36 days. Samples were collected 8 hours after dosing on day 36. Data were normalized to expression of GAPDH, and are presented as fold induction compared to vehicle treated controls. Data represents the mean ± SEM from three animals. Asterisks indicate significant changes as determined by two-tailed, unpaired t test (p< 0.05). D, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 145.2 xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 30 days. Samples were collected 8 hours after dosing on day 30. Data were analyzed and presented as described in C. E, Mean absolute tumor volumes ± SEM for LUCaP 35CR xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. Asterisks indicate p < 0.05 as determined by the Mann-Whitney test. TGI for 8 mg/kg was 27% on Day 36 (n= 10; p= 0.44). TGI for 25 mg/kg was 57% on Day 36 (n=10; p =0.006). F, Mean absolute tumor volumes ± SEM for LUCaP 145.2 xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. No significant TGI was observed at either dose.

Mentions: We next sought to determine if I-BET762 inhibits prostate tumor growth in vivo. We characterized AR, MYC, and TMRPSS2-ERG expression in two established primary prostate cancer xenograft models: the castration-resistant LuCaP 35CR model (formerly named LuCaP 35V; [21]) and the neuroendocrine prostate cancer model LuCaP 145.2. While the levels of BRD2, BRD3, and BRD4 are comparable between the two models, LuCaP 35CR expresses high levels of AR, MYC, and TMPRSS2-ERG, whereas expression of these genes are low or undetectable in LuCaP 145.2 (Figure 5A, B). Repeated dosing of mice bearing LuCaP 35CR xenografts with I-BET762 resulted in significant down-regulation of MYC as well as MLKL (Figure 5C), a gene that was down-regulated in several cell lines upon I-BET762 treatment in culture (Supplemental Table S4). In contrast, we observe no significant change in the low-level expression of MYC in the LUCaP 145.2 model; although significant changes in MLKL and the BET target gene BCL2 were detected (Figure 5D; [22]). Consistent with our in vitro data, I-BET762 produced little to no effect on ERG, or TMPRSS2-ERG expression in either xenograft model (Figure 5C, 5D). AR expression was undetectable in LUCaP 145.2, and we observed only small, variable effects on AR expression in LuCaP 35CR.


Inhibition of BET bromodomain proteins as a therapeutic approach in prostate cancer.

Wyce A, Degenhardt Y, Bai Y, Le B, Korenchuk S, Crouthame MC, McHugh CF, Vessella R, Creasy CL, Tummino PJ, Barbash O - Oncotarget (2013)

I-BET762 inhibits tumor growth in primary xenograft models with high MYC, AR, and TMPRSS2-ERGexpressionA, qPCR determination of basal expression of the indicated genes in the LUCaP 35CR and LUCaP 145.2 primary xenograft models. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. Data represents the mean value ± SEM from three animals. B, qPCR analysis of TMPRSS2-ERG expression as described in A. C, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 35CR xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 36 days. Samples were collected 8 hours after dosing on day 36. Data were normalized to expression of GAPDH, and are presented as fold induction compared to vehicle treated controls. Data represents the mean ± SEM from three animals. Asterisks indicate significant changes as determined by two-tailed, unpaired t test (p< 0.05). D, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 145.2 xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 30 days. Samples were collected 8 hours after dosing on day 30. Data were analyzed and presented as described in C. E, Mean absolute tumor volumes ± SEM for LUCaP 35CR xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. Asterisks indicate p < 0.05 as determined by the Mann-Whitney test. TGI for 8 mg/kg was 27% on Day 36 (n= 10; p= 0.44). TGI for 25 mg/kg was 57% on Day 36 (n=10; p =0.006). F, Mean absolute tumor volumes ± SEM for LUCaP 145.2 xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. No significant TGI was observed at either dose.
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Figure 5: I-BET762 inhibits tumor growth in primary xenograft models with high MYC, AR, and TMPRSS2-ERGexpressionA, qPCR determination of basal expression of the indicated genes in the LUCaP 35CR and LUCaP 145.2 primary xenograft models. Data were normalized to expression of GAPDH, and are presented as relative expression compared to normal prostate RNA. Data represents the mean value ± SEM from three animals. B, qPCR analysis of TMPRSS2-ERG expression as described in A. C, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 35CR xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 36 days. Samples were collected 8 hours after dosing on day 36. Data were normalized to expression of GAPDH, and are presented as fold induction compared to vehicle treated controls. Data represents the mean ± SEM from three animals. Asterisks indicate significant changes as determined by two-tailed, unpaired t test (p< 0.05). D, qPCR analysis of gene expression changes induced by I-BET762 treatment in the LuCaP 145.2 xenograft model. Expression was analyzed in mice treated daily with vehicle, 8 mg/kg I-BET762, or 25 mg/kg I-BET762 for 30 days. Samples were collected 8 hours after dosing on day 30. Data were analyzed and presented as described in C. E, Mean absolute tumor volumes ± SEM for LUCaP 35CR xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. Asterisks indicate p < 0.05 as determined by the Mann-Whitney test. TGI for 8 mg/kg was 27% on Day 36 (n= 10; p= 0.44). TGI for 25 mg/kg was 57% on Day 36 (n=10; p =0.006). F, Mean absolute tumor volumes ± SEM for LUCaP 145.2 xenografts following treatment with 8 mg/kg or 25 mg/kg I-BET762. No significant TGI was observed at either dose.
Mentions: We next sought to determine if I-BET762 inhibits prostate tumor growth in vivo. We characterized AR, MYC, and TMRPSS2-ERG expression in two established primary prostate cancer xenograft models: the castration-resistant LuCaP 35CR model (formerly named LuCaP 35V; [21]) and the neuroendocrine prostate cancer model LuCaP 145.2. While the levels of BRD2, BRD3, and BRD4 are comparable between the two models, LuCaP 35CR expresses high levels of AR, MYC, and TMPRSS2-ERG, whereas expression of these genes are low or undetectable in LuCaP 145.2 (Figure 5A, B). Repeated dosing of mice bearing LuCaP 35CR xenografts with I-BET762 resulted in significant down-regulation of MYC as well as MLKL (Figure 5C), a gene that was down-regulated in several cell lines upon I-BET762 treatment in culture (Supplemental Table S4). In contrast, we observe no significant change in the low-level expression of MYC in the LUCaP 145.2 model; although significant changes in MLKL and the BET target gene BCL2 were detected (Figure 5D; [22]). Consistent with our in vitro data, I-BET762 produced little to no effect on ERG, or TMPRSS2-ERG expression in either xenograft model (Figure 5C, 5D). AR expression was undetectable in LUCaP 145.2, and we observed only small, variable effects on AR expression in LuCaP 35CR.

Bottom Line: BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation.Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models.Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics DPU, Oncology R and D GlaxoSmithKline, Collegeville, PA, USA.

ABSTRACT
BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

Show MeSH
Related in: MedlinePlus