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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

Survivin plays an important role in SAHA-induced autophagy in breast cancer cells. (A, upper panels) MCF7 and MDA-MB-231 cells were transfected with either pCMV-XL (control), pCMV-XL4-survivin [overexpresses (O/E) survivin] or pCMV-XL-5-XIAP (O/E XIAP) plasmid for 72 h. Expression of different proteins was analyzed by Western blotting. (A, lower panels) MCF7 and MDA-MB-231 cells were transfected with pCMV-XL (control), pCMV-XL4-survivin (O/E survivin) or pCMV-XL-5-XIAP (O/E XIAP) for 24 h prior to 72 h SAHA treatment. Cell viability was assessed by MTT assay. Experiment was repeated three times. A statistically significant difference in the viability of cells treated with O/E survivin or O/E XIAP + SAHA vs. SAHA alone is denoted by “*” (p < 0.05). “N.S.,” denotes no significant difference between the testing groups. (B) MDA-MB-231 cells were treated with either scramble siRNA or XIAP-specific siRNA (si-XIAP) for 72 h and cell viability was analyzed by MTT assay. (C) MCF7 cells were treated with either scramble siRNA or survivin-specific siRNA (si-Survivin) for 72 h. Expression of various proteins was analyzed by Western blotting. The numbers under each survivin blot are intensity of the blot relative to that of the scramble control. Signals in the p62/SQSTM1 blots (of all repeats) were quantitated and a graph was generated to show the effect of survivin on the expression of p62/SQSTM1. (D) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Expression of p62/SQSTM1 was analyzed by Western blotting. (E) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Formation of LC3B (red fluorescent) puncta in cells was observed under a fluorescence microscope and pointed out by the arrows in the photos. Nuclei were counterstained blue with DAPI.
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Figure 2: Survivin plays an important role in SAHA-induced autophagy in breast cancer cells. (A, upper panels) MCF7 and MDA-MB-231 cells were transfected with either pCMV-XL (control), pCMV-XL4-survivin [overexpresses (O/E) survivin] or pCMV-XL-5-XIAP (O/E XIAP) plasmid for 72 h. Expression of different proteins was analyzed by Western blotting. (A, lower panels) MCF7 and MDA-MB-231 cells were transfected with pCMV-XL (control), pCMV-XL4-survivin (O/E survivin) or pCMV-XL-5-XIAP (O/E XIAP) for 24 h prior to 72 h SAHA treatment. Cell viability was assessed by MTT assay. Experiment was repeated three times. A statistically significant difference in the viability of cells treated with O/E survivin or O/E XIAP + SAHA vs. SAHA alone is denoted by “*” (p < 0.05). “N.S.,” denotes no significant difference between the testing groups. (B) MDA-MB-231 cells were treated with either scramble siRNA or XIAP-specific siRNA (si-XIAP) for 72 h and cell viability was analyzed by MTT assay. (C) MCF7 cells were treated with either scramble siRNA or survivin-specific siRNA (si-Survivin) for 72 h. Expression of various proteins was analyzed by Western blotting. The numbers under each survivin blot are intensity of the blot relative to that of the scramble control. Signals in the p62/SQSTM1 blots (of all repeats) were quantitated and a graph was generated to show the effect of survivin on the expression of p62/SQSTM1. (D) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Expression of p62/SQSTM1 was analyzed by Western blotting. (E) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Formation of LC3B (red fluorescent) puncta in cells was observed under a fluorescence microscope and pointed out by the arrows in the photos. Nuclei were counterstained blue with DAPI.

Mentions: Although survivin and its binding partner, XIAP, have been known for their anti-apoptotic functions in regulating cell survival and cell proliferation (Altieri, 2003; Potts et al., 2003), evidence that survivin and XIAP function as an endogenous repressor of autophagy has emerged (Wang et al., 2011; Huang et al., 2013; Cheng et al., 2015). SAHA is a class I HDAC (HDAC1, 2, and 3) and HDAC6 co-inhibitor and HDAC6 has been shown to regulate survivin expression through de-acetylation and cytoplasmic retention of survivin. Therefore, to determine whether SAHA-induced autophagy and/or decreased cell viability were at least partially caused by altering the expression of survivin and XIAP, the effects of SAHA on survivin and XIAP expression in both MCF7 and MDA-MB-231 cells were examined. Western blot analysis revealed that SAHA decreased the expression of survivin and XIAP in both concentration- and time-dependent manners (Figures 1A,B). Then, we evaluated the impact of the expression of survivin and XIAP on the effectiveness of SAHA in reducing cell viability and up-regulating autophagy in breast cancer cells. MTT assay revealed that ectopic over-expression of survivin significantly attenuated the inhibitory effect of SAHA (at 1x IC50 conc.) on cell viability in both MCF7 and MDA-MB-231 cells (Figure 2A). Surprisingly, ectopic over-expression of XIAP only attenuated the cell viability inhibitory effect of SAHA in MCF7 but not in MDA-MB-231 breast cancer cells (Figure 2A). In addition, down-regulation of XIAP alone by siRNA did not affect the viability of MDA-MB-231 cells in vitro (Figure 2B). These results indicated that XIAP might only play a minor role and/or a cell line-dependent role in SAHA-induced cell viability reduction, whereas, survivin might play a major role in facilitating SAHA induced cell viability reduction in human 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)

Survivin plays an important role in SAHA-induced autophagy in breast cancer cells. (A, upper panels) MCF7 and MDA-MB-231 cells were transfected with either pCMV-XL (control), pCMV-XL4-survivin [overexpresses (O/E) survivin] or pCMV-XL-5-XIAP (O/E XIAP) plasmid for 72 h. Expression of different proteins was analyzed by Western blotting. (A, lower panels) MCF7 and MDA-MB-231 cells were transfected with pCMV-XL (control), pCMV-XL4-survivin (O/E survivin) or pCMV-XL-5-XIAP (O/E XIAP) for 24 h prior to 72 h SAHA treatment. Cell viability was assessed by MTT assay. Experiment was repeated three times. A statistically significant difference in the viability of cells treated with O/E survivin or O/E XIAP + SAHA vs. SAHA alone is denoted by “*” (p < 0.05). “N.S.,” denotes no significant difference between the testing groups. (B) MDA-MB-231 cells were treated with either scramble siRNA or XIAP-specific siRNA (si-XIAP) for 72 h and cell viability was analyzed by MTT assay. (C) MCF7 cells were treated with either scramble siRNA or survivin-specific siRNA (si-Survivin) for 72 h. Expression of various proteins was analyzed by Western blotting. The numbers under each survivin blot are intensity of the blot relative to that of the scramble control. Signals in the p62/SQSTM1 blots (of all repeats) were quantitated and a graph was generated to show the effect of survivin on the expression of p62/SQSTM1. (D) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Expression of p62/SQSTM1 was analyzed by Western blotting. (E) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Formation of LC3B (red fluorescent) puncta in cells was observed under a fluorescence microscope and pointed out by the arrows in the photos. Nuclei were counterstained blue with DAPI.
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Figure 2: Survivin plays an important role in SAHA-induced autophagy in breast cancer cells. (A, upper panels) MCF7 and MDA-MB-231 cells were transfected with either pCMV-XL (control), pCMV-XL4-survivin [overexpresses (O/E) survivin] or pCMV-XL-5-XIAP (O/E XIAP) plasmid for 72 h. Expression of different proteins was analyzed by Western blotting. (A, lower panels) MCF7 and MDA-MB-231 cells were transfected with pCMV-XL (control), pCMV-XL4-survivin (O/E survivin) or pCMV-XL-5-XIAP (O/E XIAP) for 24 h prior to 72 h SAHA treatment. Cell viability was assessed by MTT assay. Experiment was repeated three times. A statistically significant difference in the viability of cells treated with O/E survivin or O/E XIAP + SAHA vs. SAHA alone is denoted by “*” (p < 0.05). “N.S.,” denotes no significant difference between the testing groups. (B) MDA-MB-231 cells were treated with either scramble siRNA or XIAP-specific siRNA (si-XIAP) for 72 h and cell viability was analyzed by MTT assay. (C) MCF7 cells were treated with either scramble siRNA or survivin-specific siRNA (si-Survivin) for 72 h. Expression of various proteins was analyzed by Western blotting. The numbers under each survivin blot are intensity of the blot relative to that of the scramble control. Signals in the p62/SQSTM1 blots (of all repeats) were quantitated and a graph was generated to show the effect of survivin on the expression of p62/SQSTM1. (D) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Expression of p62/SQSTM1 was analyzed by Western blotting. (E) Cells were transfected with pCMV-XL (control) for 72 h, pCMV-XL (control) for 24 h followed up with 48 h SAHA co-treatment, or pCMV-XL4-survivin (O/E survivin) for 24 h followed up with 48 h SAHA co-treatment. Formation of LC3B (red fluorescent) puncta in cells was observed under a fluorescence microscope and pointed out by the arrows in the photos. Nuclei were counterstained blue with DAPI.
Mentions: Although survivin and its binding partner, XIAP, have been known for their anti-apoptotic functions in regulating cell survival and cell proliferation (Altieri, 2003; Potts et al., 2003), evidence that survivin and XIAP function as an endogenous repressor of autophagy has emerged (Wang et al., 2011; Huang et al., 2013; Cheng et al., 2015). SAHA is a class I HDAC (HDAC1, 2, and 3) and HDAC6 co-inhibitor and HDAC6 has been shown to regulate survivin expression through de-acetylation and cytoplasmic retention of survivin. Therefore, to determine whether SAHA-induced autophagy and/or decreased cell viability were at least partially caused by altering the expression of survivin and XIAP, the effects of SAHA on survivin and XIAP expression in both MCF7 and MDA-MB-231 cells were examined. Western blot analysis revealed that SAHA decreased the expression of survivin and XIAP in both concentration- and time-dependent manners (Figures 1A,B). Then, we evaluated the impact of the expression of survivin and XIAP on the effectiveness of SAHA in reducing cell viability and up-regulating autophagy in breast cancer cells. MTT assay revealed that ectopic over-expression of survivin significantly attenuated the inhibitory effect of SAHA (at 1x IC50 conc.) on cell viability in both MCF7 and MDA-MB-231 cells (Figure 2A). Surprisingly, ectopic over-expression of XIAP only attenuated the cell viability inhibitory effect of SAHA in MCF7 but not in MDA-MB-231 breast cancer cells (Figure 2A). In addition, down-regulation of XIAP alone by siRNA did not affect the viability of MDA-MB-231 cells in vitro (Figure 2B). These results indicated that XIAP might only play a minor role and/or a cell line-dependent role in SAHA-induced cell viability reduction, whereas, survivin might play a major role in facilitating SAHA induced cell viability reduction in human 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