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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 increases the expression of 26S proteasome and decreases the expression of Hsp90 in breast cancer cells. (A) Breast cancer cells were treated with the pharmacological inhibitor of proteasome, MG132, for 48 h. Expression of survivin was determined by Western blotting. (B) Breast cancer cells were treated with indicated concentrations of SAHA for 72 h and the expression of 26S proteasome was analyzed by Western blotting. (C) Breast cancer cells were treated with SAHA for 72 h and the intracellular proteasome activity in the treated cells were assessed using proteasome activity fluorometric assay kit. Experiment was repeated three times. A statistically significant difference in the proteasome activity in cells treated with SAHA vs. without SAHA (control) is denoted by “*” (p < 0.05). (D) MCF7 cells were treated with SAHA for indicated durations and expression of Hsp90 was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with SAHA vs. without SAHA (0 h) is denoted by “**” (p < 0.01). (E) Breast cancer cells were transfected with scramble, HDAC3, HDAC1, or HDAC2 siRNA for 72 h. Expression of various proteins was determined by Western blotting. (F) Breast cancer cells were treated with BML281 for 24–72 h. Expression of different proteins was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of BML281 on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with BML281 vs. without BML281 (0 h) is denoted by either “*” (p < 0.01) or “***” (p < 0.001).
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Figure 5: SAHA increases the expression of 26S proteasome and decreases the expression of Hsp90 in breast cancer cells. (A) Breast cancer cells were treated with the pharmacological inhibitor of proteasome, MG132, for 48 h. Expression of survivin was determined by Western blotting. (B) Breast cancer cells were treated with indicated concentrations of SAHA for 72 h and the expression of 26S proteasome was analyzed by Western blotting. (C) Breast cancer cells were treated with SAHA for 72 h and the intracellular proteasome activity in the treated cells were assessed using proteasome activity fluorometric assay kit. Experiment was repeated three times. A statistically significant difference in the proteasome activity in cells treated with SAHA vs. without SAHA (control) is denoted by “*” (p < 0.05). (D) MCF7 cells were treated with SAHA for indicated durations and expression of Hsp90 was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with SAHA vs. without SAHA (0 h) is denoted by “**” (p < 0.01). (E) Breast cancer cells were transfected with scramble, HDAC3, HDAC1, or HDAC2 siRNA for 72 h. Expression of various proteins was determined by Western blotting. (F) Breast cancer cells were treated with BML281 for 24–72 h. Expression of different proteins was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of BML281 on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with BML281 vs. without BML281 (0 h) is denoted by either “*” (p < 0.01) or “***” (p < 0.001).

Mentions: Survivin expression is also post-translationally regulated by the proteasomal protein degradation pathway. Binding of Hsp90 prevents survivin undergoing ubiquitination and the subsequent protein degradation by proteasome (Fortugno et al., 2003). To determine whether SAHA affects the cellular proteasomal protein degradation pathway, leading to the reduction of survivin protein stability in breast cancer cells, the expression of 26S proteasome and Hsp90 in SAHA treated breast cancer cells was determined. Western blot analysis was first performed to confirm the importance of proteasome in regulating survivin expression in breast cancer cells. Here, inhibiting proteasome by MG132 clearly increased survivin expression in both MCF7 and MDA-MB-231 cells, confirming that survivin expression is regulated post-translationally by proteasome (Figure 5A). Western blot analysis and proteasome activity assay revealed that SAHA treatment increased both the expression and activity of 26S proteasome in breast cancer cells (Figures 5B,C). In contrast, the same treatment decreased the expression of Hsp90 in MCF7 cells (Figure 5D). Interestingly, down-regulation of HDAC3, but not other HDAC isoforms, by siRNA increased the expression of 26S proteasome in breast cancer cells (Figure 5E). In addition, similar to the results of cells treated with SAHA, inhibiting HDAC6 by BML281 also down-regulated the expression of Hsp90 in both MCF7 and MDA-MB-231 cells, indicating that SAHA might down-regulate Hsp90 expression through HDAC6 inhibition (Figure 5F). Collectively, these results indicated that SAHA might promote survivin protein degradation in part through decreasing the ubiquitination protection from Hsp90 via HDAC6 inhibition and increasing the expression of 26S proteasome via HDAC3 inhibition in breast cancer cells.


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 increases the expression of 26S proteasome and decreases the expression of Hsp90 in breast cancer cells. (A) Breast cancer cells were treated with the pharmacological inhibitor of proteasome, MG132, for 48 h. Expression of survivin was determined by Western blotting. (B) Breast cancer cells were treated with indicated concentrations of SAHA for 72 h and the expression of 26S proteasome was analyzed by Western blotting. (C) Breast cancer cells were treated with SAHA for 72 h and the intracellular proteasome activity in the treated cells were assessed using proteasome activity fluorometric assay kit. Experiment was repeated three times. A statistically significant difference in the proteasome activity in cells treated with SAHA vs. without SAHA (control) is denoted by “*” (p < 0.05). (D) MCF7 cells were treated with SAHA for indicated durations and expression of Hsp90 was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with SAHA vs. without SAHA (0 h) is denoted by “**” (p < 0.01). (E) Breast cancer cells were transfected with scramble, HDAC3, HDAC1, or HDAC2 siRNA for 72 h. Expression of various proteins was determined by Western blotting. (F) Breast cancer cells were treated with BML281 for 24–72 h. Expression of different proteins was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of BML281 on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with BML281 vs. without BML281 (0 h) is denoted by either “*” (p < 0.01) or “***” (p < 0.001).
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Figure 5: SAHA increases the expression of 26S proteasome and decreases the expression of Hsp90 in breast cancer cells. (A) Breast cancer cells were treated with the pharmacological inhibitor of proteasome, MG132, for 48 h. Expression of survivin was determined by Western blotting. (B) Breast cancer cells were treated with indicated concentrations of SAHA for 72 h and the expression of 26S proteasome was analyzed by Western blotting. (C) Breast cancer cells were treated with SAHA for 72 h and the intracellular proteasome activity in the treated cells were assessed using proteasome activity fluorometric assay kit. Experiment was repeated three times. A statistically significant difference in the proteasome activity in cells treated with SAHA vs. without SAHA (control) is denoted by “*” (p < 0.05). (D) MCF7 cells were treated with SAHA for indicated durations and expression of Hsp90 was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of SAHA on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with SAHA vs. without SAHA (0 h) is denoted by “**” (p < 0.01). (E) Breast cancer cells were transfected with scramble, HDAC3, HDAC1, or HDAC2 siRNA for 72 h. Expression of various proteins was determined by Western blotting. (F) Breast cancer cells were treated with BML281 for 24–72 h. Expression of different proteins was determined by Western blotting. Signals in the Hsp90 blots (of all repeats) were quantitated and a graph was generated to show the effect of BML281 on the expression of Hsp90. A statistically significant difference in the expression of Hsp90 in cells treated with BML281 vs. without BML281 (0 h) is denoted by either “*” (p < 0.01) or “***” (p < 0.001).
Mentions: Survivin expression is also post-translationally regulated by the proteasomal protein degradation pathway. Binding of Hsp90 prevents survivin undergoing ubiquitination and the subsequent protein degradation by proteasome (Fortugno et al., 2003). To determine whether SAHA affects the cellular proteasomal protein degradation pathway, leading to the reduction of survivin protein stability in breast cancer cells, the expression of 26S proteasome and Hsp90 in SAHA treated breast cancer cells was determined. Western blot analysis was first performed to confirm the importance of proteasome in regulating survivin expression in breast cancer cells. Here, inhibiting proteasome by MG132 clearly increased survivin expression in both MCF7 and MDA-MB-231 cells, confirming that survivin expression is regulated post-translationally by proteasome (Figure 5A). Western blot analysis and proteasome activity assay revealed that SAHA treatment increased both the expression and activity of 26S proteasome in breast cancer cells (Figures 5B,C). In contrast, the same treatment decreased the expression of Hsp90 in MCF7 cells (Figure 5D). Interestingly, down-regulation of HDAC3, but not other HDAC isoforms, by siRNA increased the expression of 26S proteasome in breast cancer cells (Figure 5E). In addition, similar to the results of cells treated with SAHA, inhibiting HDAC6 by BML281 also down-regulated the expression of Hsp90 in both MCF7 and MDA-MB-231 cells, indicating that SAHA might down-regulate Hsp90 expression through HDAC6 inhibition (Figure 5F). Collectively, these results indicated that SAHA might promote survivin protein degradation in part through decreasing the ubiquitination protection from Hsp90 via HDAC6 inhibition and increasing the expression of 26S proteasome via HDAC3 inhibition in breast cancer cells.

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