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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.

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Related in: MedlinePlus

Effect of HDAC5 on protein acetylation of LSD1/USP28 and transcription of LSD1 target genes. (a) The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tagged USP28 or FLAG-tagged LSD1 were used as substrates for protein deacetylation assay. IgG was used as negative control. Active or heat inactivated recombinant human GST-tagged HDAC5 protein were mixed with immunoprecipitates and incubated at 37°C for 6 h as described in “Materials and Methods”. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody. FLAG-tagged USP28 or LSD1 proteins were probed with anti-FLAG antibody. HDAC5-GST protein was probed with anti-HDAC5 antibody. (b) Histograms represent the means of levels of acetyl-LSD1, acetyl-USP28 and acetyl-histone determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (c) MDA-MB-231 cell transfected with scramble or HDAC5 siRNAs for 48 h. LSD1 or IgG antibodies were added to cell lysate. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting with anti-acetyl lysine and anti-LSD1 antibodies, respectively. Effect of HDAC5 siRNA on Acetyl-H3K9 protein expression in MDA-MB-231 cells was examined by immunoblotting with anti-acetyl-H3K9 antibody. (d) Histograms represent the means of relative levels of acetyl-LSD1 determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (e) mRNA expression of indicated genes in MDA-MB-231 cells transfected with scramble siRNA or HDAC5 siRNA. Data are means ± s.d. of three independent experiments. (f) Quantitative ChIP analysis was used to determine the occupancy by acetyl-H3K9, H3K4me2, LSD1, and HDAC5 at promoters of p21 or CLDN7 in MDA-MB-231 cells transfected with scramble or HDAC5 siRNA. *p<0.05, **p<0.01, *** p<0.001, Student’s t-test.
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Figure 5: Effect of HDAC5 on protein acetylation of LSD1/USP28 and transcription of LSD1 target genes. (a) The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tagged USP28 or FLAG-tagged LSD1 were used as substrates for protein deacetylation assay. IgG was used as negative control. Active or heat inactivated recombinant human GST-tagged HDAC5 protein were mixed with immunoprecipitates and incubated at 37°C for 6 h as described in “Materials and Methods”. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody. FLAG-tagged USP28 or LSD1 proteins were probed with anti-FLAG antibody. HDAC5-GST protein was probed with anti-HDAC5 antibody. (b) Histograms represent the means of levels of acetyl-LSD1, acetyl-USP28 and acetyl-histone determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (c) MDA-MB-231 cell transfected with scramble or HDAC5 siRNAs for 48 h. LSD1 or IgG antibodies were added to cell lysate. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting with anti-acetyl lysine and anti-LSD1 antibodies, respectively. Effect of HDAC5 siRNA on Acetyl-H3K9 protein expression in MDA-MB-231 cells was examined by immunoblotting with anti-acetyl-H3K9 antibody. (d) Histograms represent the means of relative levels of acetyl-LSD1 determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (e) mRNA expression of indicated genes in MDA-MB-231 cells transfected with scramble siRNA or HDAC5 siRNA. Data are means ± s.d. of three independent experiments. (f) Quantitative ChIP analysis was used to determine the occupancy by acetyl-H3K9, H3K4me2, LSD1, and HDAC5 at promoters of p21 or CLDN7 in MDA-MB-231 cells transfected with scramble or HDAC5 siRNA. *p<0.05, **p<0.01, *** p<0.001, Student’s t-test.

Mentions: To examine whether interaction of HDAC5 with LSD1/USP28 complex deacetylates LSD1 or USP28, in vitro protein acetylation assays was first carried out by incubating GST-tagged recombinant HDAC5 protein with cellular pull-down of LSD1-FLAG or USP28-FLAG by IP, and immunoprecipitates of IgG was incubated with recombinant HDAC5 protein as negative control of assays (Figure 5a). Bulk histone was used as control substrate (Supplementary Figure 8). Quantitative immunoblots using antibody against pan-acetylated lysine showed that HDAC5 reduced acetylation level of LSD1 without altering the acetylation status of USP28 (Figure 5a and 5b). Next, the in vivo effect of HDAC5 depletion on LSD1 acetylation was investigated in MDA-MB-231 cells transfected with scramble or HDAC5 siRNAs. After immunoprecipitation with LSD1 antibody or IgG (negative control), immunoblotting was performed and the results showed that expression levels of both total LSD1 protein and acetylated LSD1 protein were decreased by HDAC5 depletion (Figure 5c). Quantitative immunoblots indicated that the relative acetylation level of LSD1 was not statistically altered by HDAC5 siRNA in MDA-MB-231 cells (Figure 5d). AcetylH3K9 was used as control of substrate and its expression was increased by HDAC5 siRNA (Figure 5c). These results suggest that inhibition of HDAC5 alone is not sufficient enough to increase LSD1 acetylation in breast cancer cells.


Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression
Effect of HDAC5 on protein acetylation of LSD1/USP28 and transcription of LSD1 target genes. (a) The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tagged USP28 or FLAG-tagged LSD1 were used as substrates for protein deacetylation assay. IgG was used as negative control. Active or heat inactivated recombinant human GST-tagged HDAC5 protein were mixed with immunoprecipitates and incubated at 37°C for 6 h as described in “Materials and Methods”. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody. FLAG-tagged USP28 or LSD1 proteins were probed with anti-FLAG antibody. HDAC5-GST protein was probed with anti-HDAC5 antibody. (b) Histograms represent the means of levels of acetyl-LSD1, acetyl-USP28 and acetyl-histone determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (c) MDA-MB-231 cell transfected with scramble or HDAC5 siRNAs for 48 h. LSD1 or IgG antibodies were added to cell lysate. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting with anti-acetyl lysine and anti-LSD1 antibodies, respectively. Effect of HDAC5 siRNA on Acetyl-H3K9 protein expression in MDA-MB-231 cells was examined by immunoblotting with anti-acetyl-H3K9 antibody. (d) Histograms represent the means of relative levels of acetyl-LSD1 determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (e) mRNA expression of indicated genes in MDA-MB-231 cells transfected with scramble siRNA or HDAC5 siRNA. Data are means ± s.d. of three independent experiments. (f) Quantitative ChIP analysis was used to determine the occupancy by acetyl-H3K9, H3K4me2, LSD1, and HDAC5 at promoters of p21 or CLDN7 in MDA-MB-231 cells transfected with scramble or HDAC5 siRNA. *p<0.05, **p<0.01, *** p<0.001, Student’s t-test.
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Figure 5: Effect of HDAC5 on protein acetylation of LSD1/USP28 and transcription of LSD1 target genes. (a) The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tagged USP28 or FLAG-tagged LSD1 were used as substrates for protein deacetylation assay. IgG was used as negative control. Active or heat inactivated recombinant human GST-tagged HDAC5 protein were mixed with immunoprecipitates and incubated at 37°C for 6 h as described in “Materials and Methods”. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody. FLAG-tagged USP28 or LSD1 proteins were probed with anti-FLAG antibody. HDAC5-GST protein was probed with anti-HDAC5 antibody. (b) Histograms represent the means of levels of acetyl-LSD1, acetyl-USP28 and acetyl-histone determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (c) MDA-MB-231 cell transfected with scramble or HDAC5 siRNAs for 48 h. LSD1 or IgG antibodies were added to cell lysate. Immunoprecipitation (IP) was performed with anti-LSD1 antibody followed by immunoblotting with anti-acetyl lysine and anti-LSD1 antibodies, respectively. Effect of HDAC5 siRNA on Acetyl-H3K9 protein expression in MDA-MB-231 cells was examined by immunoblotting with anti-acetyl-H3K9 antibody. (d) Histograms represent the means of relative levels of acetyl-LSD1 determined by quantitative immunoblotting using infrared immunoblotting detection and analysis. (e) mRNA expression of indicated genes in MDA-MB-231 cells transfected with scramble siRNA or HDAC5 siRNA. Data are means ± s.d. of three independent experiments. (f) Quantitative ChIP analysis was used to determine the occupancy by acetyl-H3K9, H3K4me2, LSD1, and HDAC5 at promoters of p21 or CLDN7 in MDA-MB-231 cells transfected with scramble or HDAC5 siRNA. *p<0.05, **p<0.01, *** p<0.001, Student’s t-test.
Mentions: To examine whether interaction of HDAC5 with LSD1/USP28 complex deacetylates LSD1 or USP28, in vitro protein acetylation assays was first carried out by incubating GST-tagged recombinant HDAC5 protein with cellular pull-down of LSD1-FLAG or USP28-FLAG by IP, and immunoprecipitates of IgG was incubated with recombinant HDAC5 protein as negative control of assays (Figure 5a). Bulk histone was used as control substrate (Supplementary Figure 8). Quantitative immunoblots using antibody against pan-acetylated lysine showed that HDAC5 reduced acetylation level of LSD1 without altering the acetylation status of USP28 (Figure 5a and 5b). Next, the in vivo effect of HDAC5 depletion on LSD1 acetylation was investigated in MDA-MB-231 cells transfected with scramble or HDAC5 siRNAs. After immunoprecipitation with LSD1 antibody or IgG (negative control), immunoblotting was performed and the results showed that expression levels of both total LSD1 protein and acetylated LSD1 protein were decreased by HDAC5 depletion (Figure 5c). Quantitative immunoblots indicated that the relative acetylation level of LSD1 was not statistically altered by HDAC5 siRNA in MDA-MB-231 cells (Figure 5d). AcetylH3K9 was used as control of substrate and its expression was increased by HDAC5 siRNA (Figure 5c). These results suggest that inhibition of HDAC5 alone is not sufficient enough to increase LSD1 acetylation in breast cancer cells.

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