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HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Kang Y, Nian H, Rajendran P, Kim E, Dashwood WM, Pinto JT, Boardman LA, Thibodeau SN, Limburg PJ, Löhr CV, Bisson WH, Williams DE, Ho E, Dashwood RH - Cell Death Dis (2014)

Bottom Line: On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300.Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis.These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

View Article: PubMed Central - PubMed

Affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR, USA.

ABSTRACT
Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

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Bmf has a pivotal role in the apoptotic mechanism triggered by MSP. (a) Quantitative real-time PCR (qRT-PCR) assays of survival genes normalized to β-actin (ACTB), 12 h after treatment of colon cancer cells with MSP (10 μM) or vehicle. Data indicate mean±S.D., n=3, *P<0.05 using Students t-test to compare MSP-treated cells with vehicle controls. (b, c) Time-course for induction of BMF mRNA (mean±S.D., n=3) and Bmf protein expression. (d, e) In HCT116 cells, MSP was significantly less effective at inducing BMF mRNA (mean±S.D., n=3, *P<0.05) and Bmf protein expression following siRNA-mediated knockdown of Bmf. (f) Reduced cleavage of caspases and (open arrow) PARP in MSP-treated HCT116 cells following Bmf knockdown. Results in a–f are representative findings from two or more experiments
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fig2: Bmf has a pivotal role in the apoptotic mechanism triggered by MSP. (a) Quantitative real-time PCR (qRT-PCR) assays of survival genes normalized to β-actin (ACTB), 12 h after treatment of colon cancer cells with MSP (10 μM) or vehicle. Data indicate mean±S.D., n=3, *P<0.05 using Students t-test to compare MSP-treated cells with vehicle controls. (b, c) Time-course for induction of BMF mRNA (mean±S.D., n=3) and Bmf protein expression. (d, e) In HCT116 cells, MSP was significantly less effective at inducing BMF mRNA (mean±S.D., n=3, *P<0.05) and Bmf protein expression following siRNA-mediated knockdown of Bmf. (f) Reduced cleavage of caspases and (open arrow) PARP in MSP-treated HCT116 cells following Bmf knockdown. Results in a–f are representative findings from two or more experiments

Mentions: To investigate the apoptotic mechanism in more detail, several survival factors were profiled. Induction of pro-apoptotic APAF1, BAK, BIM, and BMF, and loss of anti-apoptotic BCLXL occurred within 12 h of MSP treatment, in both HCT116 and HT29 colon cancer cell lines (Figure 2a). Induction of BMF was particularly striking, and was confirmed both at the mRNA and protein level in time-course studies (Figures 2b and c). Knockdown of Bmf using RNAi attenuated the increase in BMF mRNA levels following MSP treatment (Figure 2d), and there was a reduction in Bmf protein expression (Figure 2e). These changes were associated with reduced PARP cleavage and attenuated levels of cleaved caspases (Figure 2f). Notably, in cells treated with MSP, activated caspases 3, 6, and 9 were reduced to near background levels by Bmf knockdown. Cleaved caspase-8 also was detected after MSP treatment and this caspase apparently was unaffected by Bmf knockdown (Figure 2f). Further experiments should seek to optimize Bmf knockdown beyond the ~50–60% reduction achieved here (Figures 2d and e), and clarify the relative contributions of internal and external apoptotic pathways. Nonetheless, we conclude that Bmf was a key mediator of MSP-induced apoptosis in colon cancer cells.


HDAC8 and STAT3 repress BMF gene activity in colon cancer cells.

Kang Y, Nian H, Rajendran P, Kim E, Dashwood WM, Pinto JT, Boardman LA, Thibodeau SN, Limburg PJ, Löhr CV, Bisson WH, Williams DE, Ho E, Dashwood RH - Cell Death Dis (2014)

Bmf has a pivotal role in the apoptotic mechanism triggered by MSP. (a) Quantitative real-time PCR (qRT-PCR) assays of survival genes normalized to β-actin (ACTB), 12 h after treatment of colon cancer cells with MSP (10 μM) or vehicle. Data indicate mean±S.D., n=3, *P<0.05 using Students t-test to compare MSP-treated cells with vehicle controls. (b, c) Time-course for induction of BMF mRNA (mean±S.D., n=3) and Bmf protein expression. (d, e) In HCT116 cells, MSP was significantly less effective at inducing BMF mRNA (mean±S.D., n=3, *P<0.05) and Bmf protein expression following siRNA-mediated knockdown of Bmf. (f) Reduced cleavage of caspases and (open arrow) PARP in MSP-treated HCT116 cells following Bmf knockdown. Results in a–f are representative findings from two or more experiments
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237248&req=5

fig2: Bmf has a pivotal role in the apoptotic mechanism triggered by MSP. (a) Quantitative real-time PCR (qRT-PCR) assays of survival genes normalized to β-actin (ACTB), 12 h after treatment of colon cancer cells with MSP (10 μM) or vehicle. Data indicate mean±S.D., n=3, *P<0.05 using Students t-test to compare MSP-treated cells with vehicle controls. (b, c) Time-course for induction of BMF mRNA (mean±S.D., n=3) and Bmf protein expression. (d, e) In HCT116 cells, MSP was significantly less effective at inducing BMF mRNA (mean±S.D., n=3, *P<0.05) and Bmf protein expression following siRNA-mediated knockdown of Bmf. (f) Reduced cleavage of caspases and (open arrow) PARP in MSP-treated HCT116 cells following Bmf knockdown. Results in a–f are representative findings from two or more experiments
Mentions: To investigate the apoptotic mechanism in more detail, several survival factors were profiled. Induction of pro-apoptotic APAF1, BAK, BIM, and BMF, and loss of anti-apoptotic BCLXL occurred within 12 h of MSP treatment, in both HCT116 and HT29 colon cancer cell lines (Figure 2a). Induction of BMF was particularly striking, and was confirmed both at the mRNA and protein level in time-course studies (Figures 2b and c). Knockdown of Bmf using RNAi attenuated the increase in BMF mRNA levels following MSP treatment (Figure 2d), and there was a reduction in Bmf protein expression (Figure 2e). These changes were associated with reduced PARP cleavage and attenuated levels of cleaved caspases (Figure 2f). Notably, in cells treated with MSP, activated caspases 3, 6, and 9 were reduced to near background levels by Bmf knockdown. Cleaved caspase-8 also was detected after MSP treatment and this caspase apparently was unaffected by Bmf knockdown (Figure 2f). Further experiments should seek to optimize Bmf knockdown beyond the ~50–60% reduction achieved here (Figures 2d and e), and clarify the relative contributions of internal and external apoptotic pathways. Nonetheless, we conclude that Bmf was a key mediator of MSP-induced apoptosis in colon cancer cells.

Bottom Line: On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300.Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis.These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

View Article: PubMed Central - PubMed

Affiliation: Linus Pauling Institute, Oregon State University, Corvallis, OR, USA.

ABSTRACT
Histone deacetylase (HDAC) inhibitors are undergoing clinical trials as anticancer agents, but some exhibit resistance mechanisms linked to anti-apoptotic Bcl-2 functions, such as BH3-only protein silencing. HDAC inhibitors that reactivate BH3-only family members might offer an improved therapeutic approach. We show here that a novel seleno-α-keto acid triggers global histone acetylation in human colon cancer cells and activates apoptosis in a p21-independent manner. Profiling of multiple survival factors identified a critical role for the BH3-only member Bcl-2-modifying factor (Bmf). On the corresponding BMF gene promoter, loss of HDAC8 was associated with signal transducer and activator of transcription 3 (STAT3)/specificity protein 3 (Sp3) transcription factor exchange and recruitment of p300. Treatment with a p300 inhibitor or transient overexpression of exogenous HDAC8 interfered with BMF induction, whereas RNAi-mediated silencing of STAT3 activated the target gene. This is the first report to identify a direct target gene of HDAC8 repression, namely, BMF. Interestingly, the repressive role of HDAC8 could be uncoupled from HDAC1 to trigger Bmf-mediated apoptosis. These findings have implications for the development of HDAC8-selective inhibitors as therapeutic agents, beyond the reported involvement of HDAC8 in childhood malignancy.

Show MeSH
Related in: MedlinePlus