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Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells.

Lee JY, Kuo CW, Tsai SL, Cheng SM, Chen SH, Chan HH, Lin CH, Lin KY, Li CF, Kanwar JR, Leung EY, Cheung CC, Huang WJ, Wang YC, Cheung CH - Front Pharmacol (2016)

Bottom Line: In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity.It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90.Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University Tainan, Taiwan.

ABSTRACT
SAHA is a class I HDAC/HDAC6 co-inhibitor and an autophagy inducer currently undergoing clinical investigations in breast cancer patients. However, the molecular mechanism of action of SAHA in breast cancer cells remains unclear. In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity. Importantly, we found that down-regulation of survivin plays an important role in SAHA-induced autophagy and cell viability reduction in human breast cancer cells. SAHA decreased survivin and XIAP gene transcription, induced survivin protein acetylation and early nuclear translocation in MCF7 and MDA-MB-231 breast cancer cells. It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90. Interestingly, targeting HDAC3 and HDAC6, but not other HDAC isoforms, by siRNA/pharmacological inhibitors mimicked the effects of SAHA in modulating the acetylation, expression, and nuclear translocation of survivin and induced autophagy in MCF7 and MDA-MB-231 cancer cells. Targeting HDAC3 also mimicked the effect of SAHA in up-regulating the expression and activity of proteasome, which might lead to the reduced protein stability of survivin in breast cancer cells. In conclusion, this study provides new insights into SAHA's molecular mechanism of actions in breast cancer cells. Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

No MeSH data available.


Related in: MedlinePlus

SAHA affects the expression of suvivin and XIAP at the transcriptional level. (A) MCF7 and (B) MDA-MB-231 cells were treated with various concentrations of SAHA for 24 h. The relative amount of survivin and XIAP mRNA transcripts present in cells was analyzed by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with SAHA vs. without SAHA (negative control) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 cells were treated with SAHA for 24 h and expression of p53 was determined by Western blotting. (D) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 48 h. Expression of different proteins was analyzed by Western blotting. Signals in the survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDACs on the expression of survivin. (E) MCF7 cells were transfected with scramble, HDAC2, 3, or 6 siRNA for either 24 or 48 h. Relative amount of survivin mRNA transcripts present in cells was determined by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with HDAC2, 3, or 6 siRNA vs. scramble siRNA is denoted by either “*” (p < 0.05) or “**” (p < 0.01).
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Figure 3: SAHA affects the expression of suvivin and XIAP at the transcriptional level. (A) MCF7 and (B) MDA-MB-231 cells were treated with various concentrations of SAHA for 24 h. The relative amount of survivin and XIAP mRNA transcripts present in cells was analyzed by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with SAHA vs. without SAHA (negative control) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 cells were treated with SAHA for 24 h and expression of p53 was determined by Western blotting. (D) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 48 h. Expression of different proteins was analyzed by Western blotting. Signals in the survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDACs on the expression of survivin. (E) MCF7 cells were transfected with scramble, HDAC2, 3, or 6 siRNA for either 24 or 48 h. Relative amount of survivin mRNA transcripts present in cells was determined by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with HDAC2, 3, or 6 siRNA vs. scramble siRNA is denoted by either “*” (p < 0.05) or “**” (p < 0.01).

Mentions: Next, we sought to investigate which pathway could SAHA be involved in suppressing the expression of survivin and XIAP; we examined the effects of SAHA on survivin and XIAP at the transcriptional level. Quantitative real-time PCR analysis revealed that SAHA (24 h post-treatment, at 1–2x IC50 conc.) decreased the amount of survivin and XIAP mRNA transcripts present by approximately 50% in both MCF7 and MDA-MB-231 cells (Figures 3A,B), indicating that SAHA, at least at the tested concentrations, can reduce the expression of survivin and XIAP at the transcriptional level. In addition, Western blot analysis revealed that SAHA did not affect the expression of p53, which is a survivin gene transcription negative regulator, in the wild-type p53 expressing MCF7 cells, further indicating that SAHA exhibits its anti-breast cancer effect, at least at the transcriptional level, through a p53-independent mechanism (Figure 3C; Mirza et al., 2002).


Inhibition of HDAC3- and HDAC6-Promoted Survivin Expression Plays an Important Role in SAHA-Induced Autophagy and Viability Reduction in Breast Cancer Cells.

Lee JY, Kuo CW, Tsai SL, Cheng SM, Chen SH, Chan HH, Lin CH, Lin KY, Li CF, Kanwar JR, Leung EY, Cheung CC, Huang WJ, Wang YC, Cheung CH - Front Pharmacol (2016)

SAHA affects the expression of suvivin and XIAP at the transcriptional level. (A) MCF7 and (B) MDA-MB-231 cells were treated with various concentrations of SAHA for 24 h. The relative amount of survivin and XIAP mRNA transcripts present in cells was analyzed by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with SAHA vs. without SAHA (negative control) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 cells were treated with SAHA for 24 h and expression of p53 was determined by Western blotting. (D) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 48 h. Expression of different proteins was analyzed by Western blotting. Signals in the survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDACs on the expression of survivin. (E) MCF7 cells were transfected with scramble, HDAC2, 3, or 6 siRNA for either 24 or 48 h. Relative amount of survivin mRNA transcripts present in cells was determined by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with HDAC2, 3, or 6 siRNA vs. scramble siRNA is denoted by either “*” (p < 0.05) or “**” (p < 0.01).
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Figure 3: SAHA affects the expression of suvivin and XIAP at the transcriptional level. (A) MCF7 and (B) MDA-MB-231 cells were treated with various concentrations of SAHA for 24 h. The relative amount of survivin and XIAP mRNA transcripts present in cells was analyzed by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with SAHA vs. without SAHA (negative control) is denoted by “*” (p < 0.05) and “**” (p < 0.01). (C) MCF7 cells were treated with SAHA for 24 h and expression of p53 was determined by Western blotting. (D) MCF7 cells were transfected with HDAC1, 2, 3, or 6 siRNA for 48 h. Expression of different proteins was analyzed by Western blotting. Signals in the survivin blots (of all repeats) were quantitated and a graph was generated to show the effect of different HDACs on the expression of survivin. (E) MCF7 cells were transfected with scramble, HDAC2, 3, or 6 siRNA for either 24 or 48 h. Relative amount of survivin mRNA transcripts present in cells was determined by qPCR. Experiment was repeated three times. A statistically significant difference in the amount of mRNA transcripts present in cells treated with HDAC2, 3, or 6 siRNA vs. scramble siRNA is denoted by either “*” (p < 0.05) or “**” (p < 0.01).
Mentions: Next, we sought to investigate which pathway could SAHA be involved in suppressing the expression of survivin and XIAP; we examined the effects of SAHA on survivin and XIAP at the transcriptional level. Quantitative real-time PCR analysis revealed that SAHA (24 h post-treatment, at 1–2x IC50 conc.) decreased the amount of survivin and XIAP mRNA transcripts present by approximately 50% in both MCF7 and MDA-MB-231 cells (Figures 3A,B), indicating that SAHA, at least at the tested concentrations, can reduce the expression of survivin and XIAP at the transcriptional level. In addition, Western blot analysis revealed that SAHA did not affect the expression of p53, which is a survivin gene transcription negative regulator, in the wild-type p53 expressing MCF7 cells, further indicating that SAHA exhibits its anti-breast cancer effect, at least at the transcriptional level, through a p53-independent mechanism (Figure 3C; Mirza et al., 2002).

Bottom Line: In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity.It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90.Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University Tainan, Taiwan.

ABSTRACT
SAHA is a class I HDAC/HDAC6 co-inhibitor and an autophagy inducer currently undergoing clinical investigations in breast cancer patients. However, the molecular mechanism of action of SAHA in breast cancer cells remains unclear. In this study, we found that SAHA is equally effective in targeting cells of different breast cancer subtypes and tamoxifen sensitivity. Importantly, we found that down-regulation of survivin plays an important role in SAHA-induced autophagy and cell viability reduction in human breast cancer cells. SAHA decreased survivin and XIAP gene transcription, induced survivin protein acetylation and early nuclear translocation in MCF7 and MDA-MB-231 breast cancer cells. It also reduced survivin and XIAP protein stability in part through modulating the expression and activation of the 26S proteasome and heat-shock protein 90. Interestingly, targeting HDAC3 and HDAC6, but not other HDAC isoforms, by siRNA/pharmacological inhibitors mimicked the effects of SAHA in modulating the acetylation, expression, and nuclear translocation of survivin and induced autophagy in MCF7 and MDA-MB-231 cancer cells. Targeting HDAC3 also mimicked the effect of SAHA in up-regulating the expression and activity of proteasome, which might lead to the reduced protein stability of survivin in breast cancer cells. In conclusion, this study provides new insights into SAHA's molecular mechanism of actions in breast cancer cells. Our findings emphasize the complexity of the regulatory roles in different HDAC isoforms and potentially assist in predicting the mechanism of novel HDAC inhibitors in targeted or combinational therapies in the future.

No MeSH data available.


Related in: MedlinePlus