YM155 down-regulates survivin and XIAP, modulates autophagy and induces autophagy-dependent DNA damage in breast cancer cells

Abstract

Background and purpose: The aim of this study was to determine the potency and molecular mechanism of action of YM155, a first-in-class survivin inhibitor that is currently under phase I/II clinical investigations, in various drug-resistant breast cancers including the oestrogen receptor positive (ER(+) ) tamoxifen-resistant breast cancer and the caspase-3-deficient breast cancer.

Experimental approach: The potency of YM155 in SK-BR-3, MDA-MB-231, MCF7 and its tamoxifen-resistant sublines, TamR6, TamR7, TamR8, TamC3 and TamC6, were determined by MTT assay. Western blot analysis, flow cytometric analysis, reverse transcription-PCR, fluorescent microscopy and comet assay were used to determine the molecular mechanism of action of YM155 in different breast cancer cell lines.

Key results: YM155 was equally potent towards the parental ER(+) /caspase-3-deficient MCF7 breast cancer cells and its tamoxifen-resistant sublines in vitro. The ER(-) /HER2(+) SK-BR-3 breast cancer cells and the triple-negative/caspase-3-expressing metastatic aggressive MDA-MB-231 breast cancer cells were also sensitive to YM155 with IC50 values in the low nanomolar range. Targeting survivin by YM155 modulated autophagy, induced autophagy-dependent caspase-7 activation and autophagy-dependent DNA damage in breast cancer cells. Interestingly, YM155 also induced XIAP degradation and the degradation of XIAP might play an important role in YM155-induced autophagy in breast cancer cells.

Conclusions and implications: YM155 is a potent survivin inhibitor that has potential for the management of various breast cancer subtypes regardless of the expression of ER, HER2 and caspase-3. Importantly, this study provides new insights into YM155's molecular mechanism of action and therapeutic potential in the treatment of tamoxifen-resistant breast cancer.

Figure 1

Targeting survivin by YM155 induces LC3B conversion in breast cancer cells. (A) Caspase-3 expression was analysed in MCF7, MCF7-TamR7, MCF7-TamC3 and MDA-MB-231 by Western blotting. Equal protein loading was verified by actin. (B) MDA-MB-231 cells were treated with UV (100 J·m⁻²) for various durations before being subjected to Western blot analysis with antibodies to caspase-3. Equal protein loading was verified by actin. (C) Breast cancer cells were treated with either DMSO (-ve control) or the indicated concentrations of YM155, and the expression of survivin, LC3B, caspase-3 and caspase-7 were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (-ve control). (D) Breast cancer cells were treated with 2×IC₅₀ of YM155 at the indicated time points. Survivin, LC3B, caspase-8, caspase-9, caspase-3 and caspase-7 expression were examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (0 h post-treatment).

Figure 2

Targeting survivin by YM155 and siRNA induces caspase-7 activation and autophagic vesicle (AVO) formation in breast cancer cells. (A) MCF7-TamC3 cells were treated with either DMSO (-ve control) or YM155 co-treated with or without the pan-caspase inhibitor Z-DEVD-FMK. Caspase-7 expression was examined by Western blotting. Equal protein loading was verified by actin. (B) Breast cancer cells were treated with either DMSO (-ve control) or YM155 co-treated with or without the autophagy inhibitor 3MA. The expression of LC3B was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the untreated control. (C) MCF7-TamC3 breast cancer cells were transfected with either scrambled siRNA or survivin-specific siRNA for 72 h. Cell lysates were then prepared and subjected to Western blot analysis with antibodies to the indicated proteins. The numbers under each blot are intensity of the blot relative to that of the control (scramble siRNA). (D) MCF7 and MDA-MB-231 breast cancer cells were treated with transfection reagent alone (vehicle control) or transfected with either scrambled siRNA or survivin-specific siRNA for 72 h. Cell lysates were then prepared and subjected to Western blot analysis with antibodies to the indicated proteins. The numbers under each blot are intensity of the blot relative to that of the control (vehicle control). (E) Breast cancer cells expressing the EGFP-tagged LC3B were treated with either DMSO (-ve control) or 2×IC₅₀ YM155 for 24 h. Autophagosomes in cells were observed under a fluorescence microscope. The number of puncta present in cells was analysed by ImageJ software. (F) Breast cancer cells were treated with 2×IC₅₀ YM155, transfection reagent alone (vehicle control) or transfected with either scramble siRNA or survivin-specific siRNA for the indicated durations. Cells were processed by MDC staining. AVOs in cells were observed under a fluorescence microscope. (G) Breast cancer cells were treated with either DMSO or 2×IC₅₀ YM155 for 48 h and then processed by MDC staining. AVOs formation in cells was analyzed by flow cytometry. Red line represents cells treated with DMSO and blue line represents cells treated with 2xIC₅₀ YM155.

Figure 3

Targeting survivin by YM155 and siRNA induces biphasic expression of p62/SQSTM1 in breast cancer cells. (A) MCF7 cells were treated with 2×IC₅₀ rapamycin for indicated durations. Expression of p62/SQSTM1 was examined by Western blotting. Equal protein loading was verified by actin. (B) MCF7 cells were treated with either DMSO (-ve control) or CQ for 24 h. Expression of p62/SQSTM1 and LC3B was examined by Western blotting. Equal protein loading was verified by actin. (C, D, E and F) Breast cancer cells were treated with 2×IC₅₀ YM155 for indicated durations and expression of p62/SQSTM1 was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (0 h post-treatment). (G) MCF7 cells were transfected with either scramble siRNA or survivin siRNA for indicated durations. Expression of p62/SQSTM1 was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (scramble siRNA).

Figure 4

YM155 concurrently promotes p62/SQSTM1 protein degradation and induces p62/SQSTM1 gene transcription in breast cancer cells. (A) MCF7 cells were treated with DMSO (-ve control), chloroquine, bafilomycin A1, rapamycin, YM155 or YM155 combined with chloroquine for 24 h and subsequently co-treated with the de novo protein synthesis inhibitor, CHX. The rate of p62/SQSTM1 protein degradation in MCF7 cells was examined by Western blotting. Actin was used as an internal control. The numbers under each blot are intensity of the blot relative to that of the control (0 h post-CHX treatment). (B) MCF7 cells were treated either with DMSO (-ve control) or 2×IC₅₀ YM155. Expression of p62/SQSTM1 was examined by RT-PCR. Equal sample loading was verified by actin. The numbers under each band are intensity of the band relative to that of the control (-ve control). (C) MCF7 cells were treated either with DMSO (-ve control) or 2×IC₅₀ YM155. Expression of p62/SQSTM1 was examined by quantitative real-time PCR.

Figure 5

YM155 reduces the expression of XIAP in breast cancer cells. (A) Breast cancer cells were treated with either DMSO (-ve control) or the indicated concentrations of YM155 for 48 h. Expression of XIAP was examined by Western blotting. Equal protein loading was verified by actin. (B) Breast cancer cells were treated with either DMSO (-ve control) or the indicated concentrations of YM155 for 48 h. Amount of XIAP transcript present in cells was determined by RT-PCR. Equal DNA loading was verified by actin. The numbers under each band are intensity of the band relative to that of the control (-ve control). (C) MCF7 and MDA-MB-231 breast cancer cells were transfected with either scramble siRNA or survivin-specific siRNA for 72 h or treated with YM155 for 48 h. Expression of various proteins were determined by Western blot analysis. Equal protein loading was verified by actin. (D) Breast cancer cells were treated with either scramble siRNA or XIAP siRNA for 72 h. Expression of various proteins was determined by Western blot analysis. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (scramble siRNA). (E) MCF7 cells were transfected with either scramble siRNA or XIAP siRNA for 72 h or treated with 2×IC₅₀ YM155 for 48 h. Cells were stained with LysoTracker Red and formation of AVOs (red puncta) was determined by fluorescent microscopy. The nucleus was counterstained blue with Hoechst 33342. (F) MCF7 cells were transfected with scramble siRNA, survivin siRNA or XIAP siRNA for 72 h. Expression of various proteins was determined by the Western blot analysis. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (scramble siRNA). (G) MCF7 cells were treated with DMSO (-ve control), YM155 alone, embelin alone or YM155-embelin combinations for 48 h. Expression of LC3B was determined by the Western blot analysis. Equal protein loading was verified by actin.

Figure 6

YM155 induces autophagy-dependent DNA damage. (A) Breast cancer cells were treated with 2×IC₅₀ YM155 for the indicated durations. Expression of survivin and γH2AX was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (0 h post-treatment). (B) MCF7 and MDA-MB-231 breast cancer cells were treated with transfection reagent alone (vehicle control) or transfected with either scrambled siRNA or survivin siRNA for 72 h. Cell lysates were then prepared and subjected to Western blot analysis with antibodies to the indicated proteins. The numbers under each blot are intensity of the blot relative to that of the control (vehicle control). (C) YM155-treated MDA-MB-231 cells were transfected with either the control plasmid (pCMV6 empty vector) or pCMV6-survivin that overexpresses survivin (O/E survivin) for 72 h. Expression of γH2AX and survivin was examined by Western blotting. Equal protein loading was verified by actin. (D) Breast cancer cells were treated with either DMSO (control) or 2×IC₅₀ YM155 with or without 3MA (8 mM) for 48 h. Expression of γH2AX was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the cells treated with YM155 alone. (E) Cells were treated with either DMSO (control) or 2xIC₅₀ YM155 with or without 3MA (8 mM) for 48 h. DNA damage was detected using comet assay. A statistically significant difference in the relative tail moment of cells treated with YM155 versus YM155 + 3MA is denoted by an asterisk; *P < 0.05. (F) Cells were treated with DMSO (control) or CQ for 48 h. Expression of LC3B-II was examined by Western blotting. Equal protein loading was verified by actin. DNA damage was detected using comet assay. (G) Breast cancer cells were treated with either DMSO (control) or 2×IC₅₀ YM155 with or without BAF for 48 h. Expression of survivin and γH2AX was examined by Western blotting. Equal protein loading was verified by actin. (H) MDA-MB-231 cells were treated with either scramble siRNA or LC3B siRNA for 72 h or pretransfected with scrambled siRNA, LC3B siRNA for 24 h and subsequently co-treated with 2×IC₅₀ YM155 for 48 h. Cell lysates were then prepared and subjected to Western blot analysis with antibodies to the indicated proteins. Equal protein loading was verified by actin.

Figure 7

Targeting XIAP induces DNA damage in breast cancer cells. (A) MDA-MB-231 cells were treated with DMSO (-ve control) or 2×IC₅₀ embelin for 48 h. Expression of γH2AX was examined by Western blotting. Equal protein loading was verified by actin. (B) MDA-MB-231 cells were transfected with either scramble siRNA or XIAP siRNA for 72 h. Expression of XIAP and γH2AX was examined by Western blotting. Equal protein loading was verified by actin. (C) MCF7 cells were treated with DMSO (-ve control), embelin or YM155 with or without embelin for 48 h. Expression of γH2AX was examined by Western blotting. Equal protein loading was verified by actin.

Figure 8

YM155 induces autophagic cell death in breast cancer cells. (A) MDA-MB-231 cells were cultured in RPMI containing either 10 or 1% FBS for 48 h. Expression of LC3B was examined by Western blotting. Equal protein loading was verified by actin. (B) MDA-MB-231 cells were cultured in RPMI containing 1% FBS and treated with either 3MA or CQ for 72 h. Cell viability was determined by the MTT assay. A statistically significant difference in the viability of cells treated with 1% FBS versus 1%FBS + 3MA/CQ is denoted by an asterisk; *P < 0.05. (C) Breast cancer cells were treated with the indicated concentrations of YM155 with or without 3MA or Z-DEVD-FMK for 72 h. Cell viability was determined by the MTT assay. (D) Breast cancer cells were treated with either DMSO (control) or 2×IC₅₀ YM155 with or without CQ for 72 h. Cell viability was assessed by MTT assay. A statistically significant difference in the viability of cells treated with YM155 versus YM155 + CQ is denoted, *P < 0.05. (E) MDA-MB-231 cells were treated with DMSO (control), rapamycin or YM155 with or without rapamycin for 72 h. Cell viability was assessed by MTT assay. A statistically significant difference in the viability of cells treated with YM155 + rapamycin versus YM155 or rapamycin alone is denoted, *P < 0.05. (F) MDA-MB-231 cells were treated with either DMSO (-ve control) or 2×IC₅₀ YM155 with or without CQ or BAF. Cytotoxicity was determined by LDH-cytotoxicity assay.

Figure 9

YM155 induces autophagy-dependent caspase-7 activation in MCF7-TamC3 cells. (A) MCF7-TamC3 cells were treated with either DMSO (-ve control) or 2×IC₅₀ YM155 with or without 3MA, CQ, BAF or rapamycin for 48 h. Expression of caspase-7 was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (-ve control). (B) MCF7-TamC3 cells were treated with YM155 with or without LC3B siRNA co-transfection. Expression of caspase-7 was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of the blot relative to that of the control (YM155 alone). (C) MCF7-TamC3 cells were treated with either DMSO (-ve control), 1×IC₅₀ YM155, rapamycin, or YM155 with rapamycin for 48 h. Expression of LC3B and caspase-7 was examined by Western blotting. Equal protein loading was verified by actin. The numbers under each blot are intensity of blot relative to that of the control (-ve control).

Figure 10

Proposed mechanism of action of YM155 in human breast cancer cells.