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Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression

View Article: PubMed Central - PubMed

ABSTRACT

We have previously demonstrated that crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) facilitates breast cancer proliferation. However, the underlying mechanisms are largely unknown. Here we report that expression of HDAC5 and LSD1 proteins were positively correlated in human breast cancer cell lines and tissue specimens of primary breast tumors. Protein expression of HDAC5 and LSD1 was significantly increased in primary breast cancer specimens in comparison with matched normal adjacent tissues. Using HDAC5 deletion mutants and co-immunoprecipitation studies, we showed that HDAC5 physically interacted with LSD1 complex through its domain containing nuclear localization sequence and phosphorylation sites. While the in vitro acetylation assays revealed that HDAC5 decreased LSD1 protein acetylation, siRNA-mediated HDAC5 knockdown did not alter the acetylation level of LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 stabilized LSD1 protein and decreased the nuclear level of H3K4me1/me2 in MDA-MB-231 cells, whereas loss of HDAC5 by siRNA diminished LSD1 protein stability and demethylation activity. We further demonstrated that HDAC5 promoted the protein stability of USP28, a bona fide deubiquitinase of LSD1. Overexpression of USP28 largely reversed HDAC5-KD induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein. Depletion of HDAC5 by shRNA hindered cellular proliferation, induced G1 cell cycle arrest, and attenuated migration and colony formation of breast cancer cells. A rescue study showed that increased growth of MDA-MB-231 cells by HDAC5 overexpression was reversed by concurrent LSD1 depletion, indicating that tumor-promoting activity of HDAC5 is an LSD1 dependent function. Moreover, overexpression of HDAC5 accelerated cellular proliferation and promoted acridine mutagen ICR191 induced transformation of MCF10A cells. Taken together, these results suggest that HDAC5 is critical in regulating LSD1 protein stability through posttranslational modification, and the HDAC5-LSD1 axis plays an important role in promoting breast cancer development and progression.

No MeSH data available.


Related in: MedlinePlus

HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.
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Figure 2: HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.

Mentions: To address whether LSD1 and HDAC5 physically interact, a co-immunoprecipitation study was carried out in MDA-MB-231 and MCF10A–CA1a cells transiently transfected with pcDNA3.1 or pcDNA3.1-FLAG-HDAC5 plasmids. After immunoprecipitation (IP) with LSD1 antibody, we found that both endogenous and exogenous HDAC5 proteins were co-immunoprecipitated with LSD1 protein (Figure 2a). The interaction between native LSD1 and HDAC5 was further validated in additional breast cancer cell lines (Figure 2b). A similar result was obtained in the reciprocal immunoprecipitation using anti-FLAG antibody to confirm that LSD1 was co-immunoprecipitated with FLAG-HDAC5 (Figure 2c). To precisely map the HDAC5 domain(s) responsible for interaction with LSD1, we expressed a series of HDAC5 deletion mutants engineered in pcDNA3.1-FLAG plasmids in MDA-MB-231 cells (Figure 2d). Immunoprecipitation assays of cells transfected with full length HDAC5 cDNA confirmed the HDAC5-LSD1 interaction. Deletion of an N-terminal myocyte enhancer factor-2 (MEF2) binding domain (HDAC5-Δ1) alone had no impact on HDAC5-LSD1 interaction. However, removal of both the MEF2 domain and nuclear localization sequence (NLS) (HDAC5-Δ2) completely abolished HDAC5-LSD1 interaction. Further deletion of an N-terminal HDAC and nuclear export sequence (NES) (HDAC5-Δ3) and MEF2 domain (HDAC5-Δ4) did not adversely alter LSD1 binding with HDAC5 fragments (Figure 2e). Immunofluorescence studies showed nuclear localization of full length HDAC5, HDAC5-Δ1, HDAC5-Δ3 and HDAC5-Δ4. Depletion of the NLS-containing domain (HDAC5-Δ2) completely blocked HDAC5 nuclear translocation (Figure 2f). In vitro pull-down assays by using His-tag recombinant LSD1 protein incubating with HDAC5 full length or deletion mutants validated that HDAC5 domain containing NLS element is essential for interaction with LSD1 (Supplementary Figure 4).


Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression
HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.
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Figure 2: HDAC5 and LSD1 physically interact in breast cancer cells. (a) MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting (IB) with anti-LSD1, anti-FLAG or anti-HDAC5 antibodies, respectively. (b) Whole cell lysates were immunoprecipitated with anti-LSD1 antibody followed by IB with anti-HDAC5 and LSD1 antibodies in indicated breast cancer cell lines. IgG was used as negative control. (c) MDA-MB-231 cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5-FLAG plasmids, and IP was performed with anti-FLAG followed by IB with anti-LSD1 and anti-FLAG antibodies, respectively. (d) Schematic representation of full length and deletion mutants of HDAC5-FLAG constructs. (e) FLAG-tagged full-length or deletion mutants of HDAC5 were expressed in MDA-MB-231 cells. Extracts were immunoprecipitated with anti-FLAG antibody, and bound LSD1 was examined by IB using anti-LSD1 antibody. IB with anti-FLAG was used to detect the levels of FLAG-tagged HDAC5 full-length or deletion mutants in IP and input samples (10%). (f) MDA-MB-231 cells were transfected with plasmids expressing FLAG-tagged full-length or deletion mutants of HDAC5 proteins. Immunofluorescence study was performed using anti-FLAG antibody. DAPI was used as a control for nuclear staining. All the experiments were performed three times with similar results.
Mentions: To address whether LSD1 and HDAC5 physically interact, a co-immunoprecipitation study was carried out in MDA-MB-231 and MCF10A–CA1a cells transiently transfected with pcDNA3.1 or pcDNA3.1-FLAG-HDAC5 plasmids. After immunoprecipitation (IP) with LSD1 antibody, we found that both endogenous and exogenous HDAC5 proteins were co-immunoprecipitated with LSD1 protein (Figure 2a). The interaction between native LSD1 and HDAC5 was further validated in additional breast cancer cell lines (Figure 2b). A similar result was obtained in the reciprocal immunoprecipitation using anti-FLAG antibody to confirm that LSD1 was co-immunoprecipitated with FLAG-HDAC5 (Figure 2c). To precisely map the HDAC5 domain(s) responsible for interaction with LSD1, we expressed a series of HDAC5 deletion mutants engineered in pcDNA3.1-FLAG plasmids in MDA-MB-231 cells (Figure 2d). Immunoprecipitation assays of cells transfected with full length HDAC5 cDNA confirmed the HDAC5-LSD1 interaction. Deletion of an N-terminal myocyte enhancer factor-2 (MEF2) binding domain (HDAC5-Δ1) alone had no impact on HDAC5-LSD1 interaction. However, removal of both the MEF2 domain and nuclear localization sequence (NLS) (HDAC5-Δ2) completely abolished HDAC5-LSD1 interaction. Further deletion of an N-terminal HDAC and nuclear export sequence (NES) (HDAC5-Δ3) and MEF2 domain (HDAC5-Δ4) did not adversely alter LSD1 binding with HDAC5 fragments (Figure 2e). Immunofluorescence studies showed nuclear localization of full length HDAC5, HDAC5-Δ1, HDAC5-Δ3 and HDAC5-Δ4. Depletion of the NLS-containing domain (HDAC5-Δ2) completely blocked HDAC5 nuclear translocation (Figure 2f). In vitro pull-down assays by using His-tag recombinant LSD1 protein incubating with HDAC5 full length or deletion mutants validated that HDAC5 domain containing NLS element is essential for interaction with LSD1 (Supplementary Figure 4).

View Article: PubMed Central - PubMed

ABSTRACT

We have previously demonstrated that crosstalk between lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) facilitates breast cancer proliferation. However, the underlying mechanisms are largely unknown. Here we report that expression of HDAC5 and LSD1 proteins were positively correlated in human breast cancer cell lines and tissue specimens of primary breast tumors. Protein expression of HDAC5 and LSD1 was significantly increased in primary breast cancer specimens in comparison with matched normal adjacent tissues. Using HDAC5 deletion mutants and co-immunoprecipitation studies, we showed that HDAC5 physically interacted with LSD1 complex through its domain containing nuclear localization sequence and phosphorylation sites. While the in vitro acetylation assays revealed that HDAC5 decreased LSD1 protein acetylation, siRNA-mediated HDAC5 knockdown did not alter the acetylation level of LSD1 in MDA-MB-231 cells. Overexpression of HDAC5 stabilized LSD1 protein and decreased the nuclear level of H3K4me1/me2 in MDA-MB-231 cells, whereas loss of HDAC5 by siRNA diminished LSD1 protein stability and demethylation activity. We further demonstrated that HDAC5 promoted the protein stability of USP28, a bona fide deubiquitinase of LSD1. Overexpression of USP28 largely reversed HDAC5-KD induced LSD1 protein degradation, suggesting a role of HDAC5 as a positive regulator of LSD1 through upregulation of USP28 protein. Depletion of HDAC5 by shRNA hindered cellular proliferation, induced G1 cell cycle arrest, and attenuated migration and colony formation of breast cancer cells. A rescue study showed that increased growth of MDA-MB-231 cells by HDAC5 overexpression was reversed by concurrent LSD1 depletion, indicating that tumor-promoting activity of HDAC5 is an LSD1 dependent function. Moreover, overexpression of HDAC5 accelerated cellular proliferation and promoted acridine mutagen ICR191 induced transformation of MCF10A cells. Taken together, these results suggest that HDAC5 is critical in regulating LSD1 protein stability through posttranslational modification, and the HDAC5-LSD1 axis plays an important role in promoting breast cancer development and progression.

No MeSH data available.


Related in: MedlinePlus