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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.


HDAC5 regulates LSD1 by altering USP28 stability. (a) MDA-MB-231 cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-HDAC5 or pcDNA3-HA-ubiquitin plasmids were treated with or without proteasome inhibitor 10µM MG132 for 10 h followed by immunoprecipitation (IP) using LSD1 antibody and immunoblots with anti-HA, LSD1 or HDAC5 antibodies. (b) Effect of siRNA of Jade-2, USP28 and HDAC5 on LSD1 protein expression in MDA-MB-231 cells. Results represent the mean of three independent experiments ± s.d. *** p<0.001, Student’s t-test. (c) MDA-MB-231 cells were transfected with scramble siRNA, HDAC5-siRNA, control vector pcDNA3.1, or pcDNA3.1-HDAC5 plasmids for 48 h. mRNA expression of Jade-2 and USP28 was measured by quantitative PCR. β-actin was used as an internal control. (d) MDA-MB-231 or MCF10A–CA1a cells were simultaneously transfected with pcDNA3.1-FLAG-Jade-2 and HDAC5 siRNA for 48 h and subjected to immunoblots with anti-HDAC5 or Jade-2 antibodies. β-actin was used as loading control to normalize target protein levels. (e) After MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h, immunoblotting was performed for expression of HDAC5 and USP28. (f) MDA-MB-231 or MCF10A–CA1a cells were transfected with scramble or HDAC5 siRNA alone, or in combination with pDZ-USP28 for 48 h. Whole cell lysates were analyzed for protein levels of HDAC5, USP28 and LSD1. β-actin was used as loading control to normalize target protein levels. The experiments were performed three times with similar results.
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Figure 4: HDAC5 regulates LSD1 by altering USP28 stability. (a) MDA-MB-231 cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-HDAC5 or pcDNA3-HA-ubiquitin plasmids were treated with or without proteasome inhibitor 10µM MG132 for 10 h followed by immunoprecipitation (IP) using LSD1 antibody and immunoblots with anti-HA, LSD1 or HDAC5 antibodies. (b) Effect of siRNA of Jade-2, USP28 and HDAC5 on LSD1 protein expression in MDA-MB-231 cells. Results represent the mean of three independent experiments ± s.d. *** p<0.001, Student’s t-test. (c) MDA-MB-231 cells were transfected with scramble siRNA, HDAC5-siRNA, control vector pcDNA3.1, or pcDNA3.1-HDAC5 plasmids for 48 h. mRNA expression of Jade-2 and USP28 was measured by quantitative PCR. β-actin was used as an internal control. (d) MDA-MB-231 or MCF10A–CA1a cells were simultaneously transfected with pcDNA3.1-FLAG-Jade-2 and HDAC5 siRNA for 48 h and subjected to immunoblots with anti-HDAC5 or Jade-2 antibodies. β-actin was used as loading control to normalize target protein levels. (e) After MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h, immunoblotting was performed for expression of HDAC5 and USP28. (f) MDA-MB-231 or MCF10A–CA1a cells were transfected with scramble or HDAC5 siRNA alone, or in combination with pDZ-USP28 for 48 h. Whole cell lysates were analyzed for protein levels of HDAC5, USP28 and LSD1. β-actin was used as loading control to normalize target protein levels. The experiments were performed three times with similar results.

Mentions: Protein ubiquitination assays indicated that HDAC5 overexpression significantly attenuated LSD1 polyubiquitination (Figure 4a), whereas depletion of HDAC5 by siRNA facilitated LSD1 polyubiquitination (Supplementary Figure 7a). Recently, Jade-2 and USP28 were identified as specific E3 ubiquitin ligase and deubiquitinase for LSD1 respectively (20, 21). Our study showing that increase of LSD1 protein expression by Jade-2 siRNA and decrease of LSD1 protein expression by USP28 siRNA in MDA-MB-231 cells confirmed the roles of Jade-2/USP28 as LSD1 ubiquitin ligase/deubiquitinase in breast cancer cells (Figure 4b; Supplementary Figure 7b). qPCR studies demonstrated that mRNA level of either Jade-2 or USP28 was not altered by HDAC5 knockdown or overexpression (Figure 4c). The regulation of HDAC5 on protein expression of Jade-2 or USP28 was subsequently assessed. Due to the lack of highly specific antibody against Jade-2, plasmids expressing Jade-2-FLAG fusion protein were transfected into cells as an alternative approach. MDA-MB-231 and MCF10A–CA1a cells expressing Jade-2-FLAG protein were simultaneously treated with HDAC5 siRNA to evaluate the effect of HDAC5 on Jade-2 protein expression. Immunoblot showed that depletion of HDAC5 did not change the protein level of Jade-2 (Figure 4d). However, overexpression of HDAC5 led to significant increase of USP28 protein expression in both cell lines (Figure 4e). In vitro pull-down assay using His-tag recombinant LSD1 protein incubated with USP28-FLAG protein indicated a direct interaction of HDAC5 and USP28 (Supplementary Figure 4), and HDAC5 overexpression significantly attenuated USP28 polyubiquitination (Supplementary Figure 7c). To understand whether HDAC5 may stabilize LSD1 protein through upregulation of USP28 protein stability, a rescue study was carried out in MDA-MB-231 and MCF10A–CA1a cells using concurrent transfection of HDAC5 siRNA and USP28 expression plasmids, and showed that overexpression of USP28 completely blocked the destabilization of LSD1 by HDAC5 depletion (Figure 4f, Supplementary Figure 7d). In an additional rescue experiment, overexpression of HDAC5 failed to promote LSD1 protein expression when cells were simultaneously treated with USP28 by siRNA (Supplementary Figure 7e). All these data support the notion that HDAC5 stabilizes LSD1 protein by enhancing protein expression of its deubiquitinase.


Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression
HDAC5 regulates LSD1 by altering USP28 stability. (a) MDA-MB-231 cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-HDAC5 or pcDNA3-HA-ubiquitin plasmids were treated with or without proteasome inhibitor 10µM MG132 for 10 h followed by immunoprecipitation (IP) using LSD1 antibody and immunoblots with anti-HA, LSD1 or HDAC5 antibodies. (b) Effect of siRNA of Jade-2, USP28 and HDAC5 on LSD1 protein expression in MDA-MB-231 cells. Results represent the mean of three independent experiments ± s.d. *** p<0.001, Student’s t-test. (c) MDA-MB-231 cells were transfected with scramble siRNA, HDAC5-siRNA, control vector pcDNA3.1, or pcDNA3.1-HDAC5 plasmids for 48 h. mRNA expression of Jade-2 and USP28 was measured by quantitative PCR. β-actin was used as an internal control. (d) MDA-MB-231 or MCF10A–CA1a cells were simultaneously transfected with pcDNA3.1-FLAG-Jade-2 and HDAC5 siRNA for 48 h and subjected to immunoblots with anti-HDAC5 or Jade-2 antibodies. β-actin was used as loading control to normalize target protein levels. (e) After MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h, immunoblotting was performed for expression of HDAC5 and USP28. (f) MDA-MB-231 or MCF10A–CA1a cells were transfected with scramble or HDAC5 siRNA alone, or in combination with pDZ-USP28 for 48 h. Whole cell lysates were analyzed for protein levels of HDAC5, USP28 and LSD1. β-actin was used as loading control to normalize target protein levels. The experiments were performed three times with similar results.
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Figure 4: HDAC5 regulates LSD1 by altering USP28 stability. (a) MDA-MB-231 cells transfected with pcDNA3.1-FLAG, pcDNA3.1-FLAG-HDAC5 or pcDNA3-HA-ubiquitin plasmids were treated with or without proteasome inhibitor 10µM MG132 for 10 h followed by immunoprecipitation (IP) using LSD1 antibody and immunoblots with anti-HA, LSD1 or HDAC5 antibodies. (b) Effect of siRNA of Jade-2, USP28 and HDAC5 on LSD1 protein expression in MDA-MB-231 cells. Results represent the mean of three independent experiments ± s.d. *** p<0.001, Student’s t-test. (c) MDA-MB-231 cells were transfected with scramble siRNA, HDAC5-siRNA, control vector pcDNA3.1, or pcDNA3.1-HDAC5 plasmids for 48 h. mRNA expression of Jade-2 and USP28 was measured by quantitative PCR. β-actin was used as an internal control. (d) MDA-MB-231 or MCF10A–CA1a cells were simultaneously transfected with pcDNA3.1-FLAG-Jade-2 and HDAC5 siRNA for 48 h and subjected to immunoblots with anti-HDAC5 or Jade-2 antibodies. β-actin was used as loading control to normalize target protein levels. (e) After MDA-MB-231 or MCF10A–CA1a cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 plasmids for 48 h, immunoblotting was performed for expression of HDAC5 and USP28. (f) MDA-MB-231 or MCF10A–CA1a cells were transfected with scramble or HDAC5 siRNA alone, or in combination with pDZ-USP28 for 48 h. Whole cell lysates were analyzed for protein levels of HDAC5, USP28 and LSD1. β-actin was used as loading control to normalize target protein levels. The experiments were performed three times with similar results.
Mentions: Protein ubiquitination assays indicated that HDAC5 overexpression significantly attenuated LSD1 polyubiquitination (Figure 4a), whereas depletion of HDAC5 by siRNA facilitated LSD1 polyubiquitination (Supplementary Figure 7a). Recently, Jade-2 and USP28 were identified as specific E3 ubiquitin ligase and deubiquitinase for LSD1 respectively (20, 21). Our study showing that increase of LSD1 protein expression by Jade-2 siRNA and decrease of LSD1 protein expression by USP28 siRNA in MDA-MB-231 cells confirmed the roles of Jade-2/USP28 as LSD1 ubiquitin ligase/deubiquitinase in breast cancer cells (Figure 4b; Supplementary Figure 7b). qPCR studies demonstrated that mRNA level of either Jade-2 or USP28 was not altered by HDAC5 knockdown or overexpression (Figure 4c). The regulation of HDAC5 on protein expression of Jade-2 or USP28 was subsequently assessed. Due to the lack of highly specific antibody against Jade-2, plasmids expressing Jade-2-FLAG fusion protein were transfected into cells as an alternative approach. MDA-MB-231 and MCF10A–CA1a cells expressing Jade-2-FLAG protein were simultaneously treated with HDAC5 siRNA to evaluate the effect of HDAC5 on Jade-2 protein expression. Immunoblot showed that depletion of HDAC5 did not change the protein level of Jade-2 (Figure 4d). However, overexpression of HDAC5 led to significant increase of USP28 protein expression in both cell lines (Figure 4e). In vitro pull-down assay using His-tag recombinant LSD1 protein incubated with USP28-FLAG protein indicated a direct interaction of HDAC5 and USP28 (Supplementary Figure 4), and HDAC5 overexpression significantly attenuated USP28 polyubiquitination (Supplementary Figure 7c). To understand whether HDAC5 may stabilize LSD1 protein through upregulation of USP28 protein stability, a rescue study was carried out in MDA-MB-231 and MCF10A–CA1a cells using concurrent transfection of HDAC5 siRNA and USP28 expression plasmids, and showed that overexpression of USP28 completely blocked the destabilization of LSD1 by HDAC5 depletion (Figure 4f, Supplementary Figure 7d). In an additional rescue experiment, overexpression of HDAC5 failed to promote LSD1 protein expression when cells were simultaneously treated with USP28 by siRNA (Supplementary Figure 7e). All these data support the notion that HDAC5 stabilizes LSD1 protein by enhancing protein expression of its deubiquitinase.

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.