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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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HDAC5-LSD1 axis is implicated in breast cancer progression. (a) Depletion of HDAC5 by shRNA lentivirus infection downregulated LSD1 protein expression in MDA-MB-231 and MCF10A–CA1a cells. (b) Scramble shRNA and HDAC5-KD cells were analyzed for growth and viability by crystal violet assays. (c) Soft agar colony formation for HDAC5-KD and scramble control of MDA-MB-231 and MCF10A–CA1a cells. (d) Scramble shRNA and HDAC5-KD cells were harvested and stained for DNA with propidium iodide for flow cytometric analysis. The fractions corresponding to G1, S and G2/M phases of the cell cycle are indicated. (e) Boyden Chamber transwell migration assays for cell invasion for MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD-1 cells. (f) MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 5 days and crystal violet assays for growth were carried out. (g) MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD cells were transfected with empty or pReceiver-LSD1 expression plasmids for 5 days and crystal violet assays for growth were carried out. Bars represent the means of three independent experiments ± s.d. * p<0.05, ** p<0.01, *** p<0.001, Student’s t-test.
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Figure 6: HDAC5-LSD1 axis is implicated in breast cancer progression. (a) Depletion of HDAC5 by shRNA lentivirus infection downregulated LSD1 protein expression in MDA-MB-231 and MCF10A–CA1a cells. (b) Scramble shRNA and HDAC5-KD cells were analyzed for growth and viability by crystal violet assays. (c) Soft agar colony formation for HDAC5-KD and scramble control of MDA-MB-231 and MCF10A–CA1a cells. (d) Scramble shRNA and HDAC5-KD cells were harvested and stained for DNA with propidium iodide for flow cytometric analysis. The fractions corresponding to G1, S and G2/M phases of the cell cycle are indicated. (e) Boyden Chamber transwell migration assays for cell invasion for MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD-1 cells. (f) MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 5 days and crystal violet assays for growth were carried out. (g) MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD cells were transfected with empty or pReceiver-LSD1 expression plasmids for 5 days and crystal violet assays for growth were carried out. Bars represent the means of three independent experiments ± s.d. * p<0.05, ** p<0.01, *** p<0.001, Student’s t-test.

Mentions: To explore the functional role of the HDAC5-LSD1 axis in regulating breast cancer development, stable knockdown of HDAC5 mRNA (HDAC5-KD) was generated in MDA-MB-231 and MCF10A–CA1a cells by infection with shRNA lentiviral particles. Similar to the effect of transient inhibition of HDAC5 by siRNA, stable knockdown of HDAC5 expression significantly reduced LSD1 protein expression in two independent HDAC5-KD clones (Figure 6a). Loss of HDAC5 in both clones hindered cell proliferation and colony formation in soft agar (Figure 6b and 6c). Flow cytometry analysis showed that inhibition of HDAC5 resulted in a greater fraction of cells accumulated at G1 phase and reduction of the S-phase cell fraction (Figure 6d; Supplementary Figure 9). Moreover, loss of HDAC5 attenuated motility and invasion of MDA-MB-231 cells in a Boyden chamber assay (Figure 6e). A rescue experiment indicated that HDAC5 overexpression promoted growth of MDA-MB-231-Scramble cells, but failed to alter the growth of MDA-MB-231-LSD1-KD cells (Figure 6f). An additional rescue study revealed that LSD1 overexpression rescued growth inhibition by HDAC5 depletion in MDA-MB-231-HDAC5-KD cells (Figure 6g). Taken together, these results demonstrate that tumor promoting activity of HDAC5 is dependent on LSD1 activity in breast cancer cells.


Functional Interaction of Histone Deacetylase 5 (HDAC5) and Lysine-specific Demethylase 1 (LSD1) Promotes Breast Cancer Progression
HDAC5-LSD1 axis is implicated in breast cancer progression. (a) Depletion of HDAC5 by shRNA lentivirus infection downregulated LSD1 protein expression in MDA-MB-231 and MCF10A–CA1a cells. (b) Scramble shRNA and HDAC5-KD cells were analyzed for growth and viability by crystal violet assays. (c) Soft agar colony formation for HDAC5-KD and scramble control of MDA-MB-231 and MCF10A–CA1a cells. (d) Scramble shRNA and HDAC5-KD cells were harvested and stained for DNA with propidium iodide for flow cytometric analysis. The fractions corresponding to G1, S and G2/M phases of the cell cycle are indicated. (e) Boyden Chamber transwell migration assays for cell invasion for MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD-1 cells. (f) MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 5 days and crystal violet assays for growth were carried out. (g) MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD cells were transfected with empty or pReceiver-LSD1 expression plasmids for 5 days and crystal violet assays for growth were carried out. Bars represent the means of three independent experiments ± s.d. * p<0.05, ** p<0.01, *** p<0.001, Student’s t-test.
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Figure 6: HDAC5-LSD1 axis is implicated in breast cancer progression. (a) Depletion of HDAC5 by shRNA lentivirus infection downregulated LSD1 protein expression in MDA-MB-231 and MCF10A–CA1a cells. (b) Scramble shRNA and HDAC5-KD cells were analyzed for growth and viability by crystal violet assays. (c) Soft agar colony formation for HDAC5-KD and scramble control of MDA-MB-231 and MCF10A–CA1a cells. (d) Scramble shRNA and HDAC5-KD cells were harvested and stained for DNA with propidium iodide for flow cytometric analysis. The fractions corresponding to G1, S and G2/M phases of the cell cycle are indicated. (e) Boyden Chamber transwell migration assays for cell invasion for MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD-1 cells. (f) MDA-MB-231-Scramble or MDA-MB-231-LSD1-KD cells were transfected with control vector pcDNA3.1 or pcDNA3.1-HDAC5 for 5 days and crystal violet assays for growth were carried out. (g) MDA-MB-231-Scramble or MDA-MB-231-HDAC5-KD cells were transfected with empty or pReceiver-LSD1 expression plasmids for 5 days and crystal violet assays for growth were carried out. Bars represent the means of three independent experiments ± s.d. * p<0.05, ** p<0.01, *** p<0.001, Student’s t-test.
Mentions: To explore the functional role of the HDAC5-LSD1 axis in regulating breast cancer development, stable knockdown of HDAC5 mRNA (HDAC5-KD) was generated in MDA-MB-231 and MCF10A–CA1a cells by infection with shRNA lentiviral particles. Similar to the effect of transient inhibition of HDAC5 by siRNA, stable knockdown of HDAC5 expression significantly reduced LSD1 protein expression in two independent HDAC5-KD clones (Figure 6a). Loss of HDAC5 in both clones hindered cell proliferation and colony formation in soft agar (Figure 6b and 6c). Flow cytometry analysis showed that inhibition of HDAC5 resulted in a greater fraction of cells accumulated at G1 phase and reduction of the S-phase cell fraction (Figure 6d; Supplementary Figure 9). Moreover, loss of HDAC5 attenuated motility and invasion of MDA-MB-231 cells in a Boyden chamber assay (Figure 6e). A rescue experiment indicated that HDAC5 overexpression promoted growth of MDA-MB-231-Scramble cells, but failed to alter the growth of MDA-MB-231-LSD1-KD cells (Figure 6f). An additional rescue study revealed that LSD1 overexpression rescued growth inhibition by HDAC5 depletion in MDA-MB-231-HDAC5-KD cells (Figure 6g). Taken together, these results demonstrate that tumor promoting activity of HDAC5 is dependent on LSD1 activity 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