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The bromodomain and extra-terminal inhibitor CPI203 enhances the antiproliferative effects of rapamycin on human neuroendocrine tumors.

Wong C, Laddha SV, Tang L, Vosburgh E, Levine AJ, Normant E, Sandy P, Harris CR, Chan CS, Xu EY - Cell Death Dis (2014)

Bottom Line: We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation.Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo.Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Foundation Laboratory, 195 Little Albany Street, New Brunswick, NJ 08901, USA.

ABSTRACT
Endogenous c-MYC (MYC) has been reported to be a potential pharmacological target to trigger ubiquitous tumor regression of pancreatic neuroendocrine tumors (PanNETs) and lung tumors. Recently inhibitors of bromodomain and extra-terminal (BET) family proteins have shown antitumor effects through the suppression of MYC in leukemia and lymphoma. In this paper, we investigated the antitumor activity of a BET protein bromodomain inhibitor (BETi) CPI203 as a single agent and in combination with rapamycin in human PanNETs. We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation. In addition, overexpression of MYC suppressed the growth inhibition caused by CPI203 and knockdown of MYC phenocopied the effects of CPI203 treatment. These findings indicate that suppression of MYC contributed to the antiproliferative effects of BETi inhibition in human PanNET cells. Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo. Furthermore, the combination treatment attenuated rapamycin-induced AKT activation, a major limitation of rapamycin therapy. Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients. This provides a novel clinical strategy for PanNETs, and possibly for other tumors as well.

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BETi enhanced the growth inhibition of rapamycin and reduced the rapamycin-induced activation of AKT in PanNET Cells. (a) Synergistic growth inhibition of CPI203 and rapamycin on BON-1 or QGP-1 cells. BON-1 or QGP-1 cells were treated with CPI203, rapamycin or in combination as indicated. Cell viability was determined at 72 h. Error bars represent S.E.M., n=3. Combination index (CI) was calculated using CompuSyn software. *P<0.01 versus single treatment; #P<0.01 versus one single treatment but P=0.01 versus the other single treatment. (b) Synergistic growth inhibition of (+)-JQ1 and rapamycin on BON-1 or QGP-1 cells, which was performed in the same way as that in (a). (c) Immunoblots of MYC, mTOR downstream targets and AKT at 24 and 48 h of BON-1 and QGP-1 cells treated with 50 nM CPI203 (BON-1) or 500 nM (QGP-1), 1 μM rapamycin or in combinations. (d) and (e) Immunoblots of MYC, mTOR downstream targets and AKT at 48 h of BON-1 cells (d) or of QGP-1 cells (e) treated with a range of doses of CPI203 or rapamycin as indicated
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fig4: BETi enhanced the growth inhibition of rapamycin and reduced the rapamycin-induced activation of AKT in PanNET Cells. (a) Synergistic growth inhibition of CPI203 and rapamycin on BON-1 or QGP-1 cells. BON-1 or QGP-1 cells were treated with CPI203, rapamycin or in combination as indicated. Cell viability was determined at 72 h. Error bars represent S.E.M., n=3. Combination index (CI) was calculated using CompuSyn software. *P<0.01 versus single treatment; #P<0.01 versus one single treatment but P=0.01 versus the other single treatment. (b) Synergistic growth inhibition of (+)-JQ1 and rapamycin on BON-1 or QGP-1 cells, which was performed in the same way as that in (a). (c) Immunoblots of MYC, mTOR downstream targets and AKT at 24 and 48 h of BON-1 and QGP-1 cells treated with 50 nM CPI203 (BON-1) or 500 nM (QGP-1), 1 μM rapamycin or in combinations. (d) and (e) Immunoblots of MYC, mTOR downstream targets and AKT at 48 h of BON-1 cells (d) or of QGP-1 cells (e) treated with a range of doses of CPI203 or rapamycin as indicated

Mentions: Rapamycin (sirolimus) and analogues are specific inhibitors of mTORC1 and demonstrated a broad-spectrum antitumor activity both in vitro and in vivo with an acceptable safety profile.22 It has also been reported that rapamycin treatment showed limited clinical efficacy, which may be due to the feedback activation of AKT triggered by mTORC1 inhibition.23,24 Combination of rapalogs with other anticancer drugs might improve efficacy. Since everolimus is an FDA-approved treatment option for NETs, we sought to explore the growth inhibitory effects of BETi in combination with rapamycin as well as its underlying mechanisms in PanNETs. BON-1 or QGP-1 cells were treated with 50 or 500 nM CPI203 alone, 100 nM or 1 μM rapamycin alone, or in combinations. For both cell lines, all the combination treatments were more effective at blocking cell growth than either agent alone (P<0.01 or P=0.01) (Figure 4a). Both cell lines were also treated with a range of doses of CPI203 and rapamycin alone at the same time (data not shown). According to the Chou–Talalay method,25 the combination index (CI) was calculated using CompuSyn software26 (ComboSyn Inc.; Paramus, NJ, USA) for each combination and CIs were less than 1 for all the combination treatments in both cell lines, indicating synergistic effects of CPI203 and rapamycin in both cell lines. The synergistic effects of (+)-JQ1 and rapamycin were observed in both cell lines as well (Figure 4b). These indicated that co-treatment of BETi and rapamycin resulted in synergistic effects on cell proliferation in PanNET cells. Consistent with the synergistic growth inhibition, co-treatment of CPI203 and rapamycin led to stronger G1 cell cycle arrest at 48 h (Supplementary Figure S1b), but not apoptosis. Annexin-V positive cells and PARP cleavage were not detected at 72 h upon co-treatment of CPI203 and rapamycin (Supplementary Figure S1c).


The bromodomain and extra-terminal inhibitor CPI203 enhances the antiproliferative effects of rapamycin on human neuroendocrine tumors.

Wong C, Laddha SV, Tang L, Vosburgh E, Levine AJ, Normant E, Sandy P, Harris CR, Chan CS, Xu EY - Cell Death Dis (2014)

BETi enhanced the growth inhibition of rapamycin and reduced the rapamycin-induced activation of AKT in PanNET Cells. (a) Synergistic growth inhibition of CPI203 and rapamycin on BON-1 or QGP-1 cells. BON-1 or QGP-1 cells were treated with CPI203, rapamycin or in combination as indicated. Cell viability was determined at 72 h. Error bars represent S.E.M., n=3. Combination index (CI) was calculated using CompuSyn software. *P<0.01 versus single treatment; #P<0.01 versus one single treatment but P=0.01 versus the other single treatment. (b) Synergistic growth inhibition of (+)-JQ1 and rapamycin on BON-1 or QGP-1 cells, which was performed in the same way as that in (a). (c) Immunoblots of MYC, mTOR downstream targets and AKT at 24 and 48 h of BON-1 and QGP-1 cells treated with 50 nM CPI203 (BON-1) or 500 nM (QGP-1), 1 μM rapamycin or in combinations. (d) and (e) Immunoblots of MYC, mTOR downstream targets and AKT at 48 h of BON-1 cells (d) or of QGP-1 cells (e) treated with a range of doses of CPI203 or rapamycin as indicated
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: BETi enhanced the growth inhibition of rapamycin and reduced the rapamycin-induced activation of AKT in PanNET Cells. (a) Synergistic growth inhibition of CPI203 and rapamycin on BON-1 or QGP-1 cells. BON-1 or QGP-1 cells were treated with CPI203, rapamycin or in combination as indicated. Cell viability was determined at 72 h. Error bars represent S.E.M., n=3. Combination index (CI) was calculated using CompuSyn software. *P<0.01 versus single treatment; #P<0.01 versus one single treatment but P=0.01 versus the other single treatment. (b) Synergistic growth inhibition of (+)-JQ1 and rapamycin on BON-1 or QGP-1 cells, which was performed in the same way as that in (a). (c) Immunoblots of MYC, mTOR downstream targets and AKT at 24 and 48 h of BON-1 and QGP-1 cells treated with 50 nM CPI203 (BON-1) or 500 nM (QGP-1), 1 μM rapamycin or in combinations. (d) and (e) Immunoblots of MYC, mTOR downstream targets and AKT at 48 h of BON-1 cells (d) or of QGP-1 cells (e) treated with a range of doses of CPI203 or rapamycin as indicated
Mentions: Rapamycin (sirolimus) and analogues are specific inhibitors of mTORC1 and demonstrated a broad-spectrum antitumor activity both in vitro and in vivo with an acceptable safety profile.22 It has also been reported that rapamycin treatment showed limited clinical efficacy, which may be due to the feedback activation of AKT triggered by mTORC1 inhibition.23,24 Combination of rapalogs with other anticancer drugs might improve efficacy. Since everolimus is an FDA-approved treatment option for NETs, we sought to explore the growth inhibitory effects of BETi in combination with rapamycin as well as its underlying mechanisms in PanNETs. BON-1 or QGP-1 cells were treated with 50 or 500 nM CPI203 alone, 100 nM or 1 μM rapamycin alone, or in combinations. For both cell lines, all the combination treatments were more effective at blocking cell growth than either agent alone (P<0.01 or P=0.01) (Figure 4a). Both cell lines were also treated with a range of doses of CPI203 and rapamycin alone at the same time (data not shown). According to the Chou–Talalay method,25 the combination index (CI) was calculated using CompuSyn software26 (ComboSyn Inc.; Paramus, NJ, USA) for each combination and CIs were less than 1 for all the combination treatments in both cell lines, indicating synergistic effects of CPI203 and rapamycin in both cell lines. The synergistic effects of (+)-JQ1 and rapamycin were observed in both cell lines as well (Figure 4b). These indicated that co-treatment of BETi and rapamycin resulted in synergistic effects on cell proliferation in PanNET cells. Consistent with the synergistic growth inhibition, co-treatment of CPI203 and rapamycin led to stronger G1 cell cycle arrest at 48 h (Supplementary Figure S1b), but not apoptosis. Annexin-V positive cells and PARP cleavage were not detected at 72 h upon co-treatment of CPI203 and rapamycin (Supplementary Figure S1c).

Bottom Line: We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation.Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo.Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Foundation Laboratory, 195 Little Albany Street, New Brunswick, NJ 08901, USA.

ABSTRACT
Endogenous c-MYC (MYC) has been reported to be a potential pharmacological target to trigger ubiquitous tumor regression of pancreatic neuroendocrine tumors (PanNETs) and lung tumors. Recently inhibitors of bromodomain and extra-terminal (BET) family proteins have shown antitumor effects through the suppression of MYC in leukemia and lymphoma. In this paper, we investigated the antitumor activity of a BET protein bromodomain inhibitor (BETi) CPI203 as a single agent and in combination with rapamycin in human PanNETs. We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation. In addition, overexpression of MYC suppressed the growth inhibition caused by CPI203 and knockdown of MYC phenocopied the effects of CPI203 treatment. These findings indicate that suppression of MYC contributed to the antiproliferative effects of BETi inhibition in human PanNET cells. Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo. Furthermore, the combination treatment attenuated rapamycin-induced AKT activation, a major limitation of rapamycin therapy. Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients. This provides a novel clinical strategy for PanNETs, and possibly for other tumors as well.

Show MeSH
Related in: MedlinePlus