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Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1.

Gonzalez ME, Li X, Toy K, DuPrie M, Ventura AC, Banerjee M, Ljungman M, Merajver SD, Kleer CG - Oncogene (2008)

Bottom Line: EZH2 knockdown decreased proliferation and delayed the G(2)/M cell-cycle transition, although not affecting apoptosis.Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G(2)/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis.Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
Increased levels of enhancer of zeste homolog 2 (EZH2), a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER-negative breast cancer progression, EZH2 expression was inhibited in ER-negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G(2)/M cell-cycle transition, although not affecting apoptosis. In vivo, EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown upregulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G(2)/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins. Taken together, we show that EZH2 is important in ER-negative breast cancer growth in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER-negative breast cancer progression.

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The effects of EZH2 on cell proliferation and G2/M transition require BRCA1A. CAL51 cells were transfected with vector or EZH2 shRNA alone or in combination with BRCA1 shRNA. Immunoblots show strong up-regulation of BRCA1 in EZH2 shRNA CAL51 cells when compared to CAL51 cells transfected with the empty vector. BRCA1 knockdown in the setting of EZH2 down-regulation abrogates the increase in BRCA1 protein to levels similar to vector transfected cells. B. BRCA1 knockdown is sufficient to rescue the effect of EZH2 inhibition on cell proliferation. Inhibition of BRCA1 in CAL51 cells does not affect cell proliferation, as has been previously reported by Rosen and coworkers (Bae et al., 2005). C. BRCA1 knockdown rescues the effect of EZH2 inhibition on G2/M cell cycle arrest by DNA content analysis by flow cytometry. D and E. Consistently, BRCA1 knockdown reverts the effect of EZH2 down-regulation on crucial proteins that regulate G2/M transition: phosphorylated Cdc25C and total and phosphorylated Cyclin B1 in nuclear fractions of CAL51 cells.
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Figure 6: The effects of EZH2 on cell proliferation and G2/M transition require BRCA1A. CAL51 cells were transfected with vector or EZH2 shRNA alone or in combination with BRCA1 shRNA. Immunoblots show strong up-regulation of BRCA1 in EZH2 shRNA CAL51 cells when compared to CAL51 cells transfected with the empty vector. BRCA1 knockdown in the setting of EZH2 down-regulation abrogates the increase in BRCA1 protein to levels similar to vector transfected cells. B. BRCA1 knockdown is sufficient to rescue the effect of EZH2 inhibition on cell proliferation. Inhibition of BRCA1 in CAL51 cells does not affect cell proliferation, as has been previously reported by Rosen and coworkers (Bae et al., 2005). C. BRCA1 knockdown rescues the effect of EZH2 inhibition on G2/M cell cycle arrest by DNA content analysis by flow cytometry. D and E. Consistently, BRCA1 knockdown reverts the effect of EZH2 down-regulation on crucial proteins that regulate G2/M transition: phosphorylated Cdc25C and total and phosphorylated Cyclin B1 in nuclear fractions of CAL51 cells.

Mentions: To investigate whether the effects of EZH2 down-regulation on cell proliferation and transition from the G2 phase to mitosis necessitate BRCA1 protein, we employed shRNA to knockdown BRCA1 in CAL51 cells with EZH2 down-regulation and controls. As expected, BRCA1 shRNA resulted in almost complete inhibition of nuclear BRCA1 protein compared with cells transfected with the empty vector. In CAL51 shEZH2 cells, BRCA1 shRNA effectively reduced BRCA1 protein to levels similar to the empty vector transfected cells, and therefore, specifically abrogated the increase in BRCA1 caused by EZH2 knockdown (Figure 6A).


Downregulation of EZH2 decreases growth of estrogen receptor-negative invasive breast carcinoma and requires BRCA1.

Gonzalez ME, Li X, Toy K, DuPrie M, Ventura AC, Banerjee M, Ljungman M, Merajver SD, Kleer CG - Oncogene (2008)

The effects of EZH2 on cell proliferation and G2/M transition require BRCA1A. CAL51 cells were transfected with vector or EZH2 shRNA alone or in combination with BRCA1 shRNA. Immunoblots show strong up-regulation of BRCA1 in EZH2 shRNA CAL51 cells when compared to CAL51 cells transfected with the empty vector. BRCA1 knockdown in the setting of EZH2 down-regulation abrogates the increase in BRCA1 protein to levels similar to vector transfected cells. B. BRCA1 knockdown is sufficient to rescue the effect of EZH2 inhibition on cell proliferation. Inhibition of BRCA1 in CAL51 cells does not affect cell proliferation, as has been previously reported by Rosen and coworkers (Bae et al., 2005). C. BRCA1 knockdown rescues the effect of EZH2 inhibition on G2/M cell cycle arrest by DNA content analysis by flow cytometry. D and E. Consistently, BRCA1 knockdown reverts the effect of EZH2 down-regulation on crucial proteins that regulate G2/M transition: phosphorylated Cdc25C and total and phosphorylated Cyclin B1 in nuclear fractions of CAL51 cells.
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Related In: Results  -  Collection

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Figure 6: The effects of EZH2 on cell proliferation and G2/M transition require BRCA1A. CAL51 cells were transfected with vector or EZH2 shRNA alone or in combination with BRCA1 shRNA. Immunoblots show strong up-regulation of BRCA1 in EZH2 shRNA CAL51 cells when compared to CAL51 cells transfected with the empty vector. BRCA1 knockdown in the setting of EZH2 down-regulation abrogates the increase in BRCA1 protein to levels similar to vector transfected cells. B. BRCA1 knockdown is sufficient to rescue the effect of EZH2 inhibition on cell proliferation. Inhibition of BRCA1 in CAL51 cells does not affect cell proliferation, as has been previously reported by Rosen and coworkers (Bae et al., 2005). C. BRCA1 knockdown rescues the effect of EZH2 inhibition on G2/M cell cycle arrest by DNA content analysis by flow cytometry. D and E. Consistently, BRCA1 knockdown reverts the effect of EZH2 down-regulation on crucial proteins that regulate G2/M transition: phosphorylated Cdc25C and total and phosphorylated Cyclin B1 in nuclear fractions of CAL51 cells.
Mentions: To investigate whether the effects of EZH2 down-regulation on cell proliferation and transition from the G2 phase to mitosis necessitate BRCA1 protein, we employed shRNA to knockdown BRCA1 in CAL51 cells with EZH2 down-regulation and controls. As expected, BRCA1 shRNA resulted in almost complete inhibition of nuclear BRCA1 protein compared with cells transfected with the empty vector. In CAL51 shEZH2 cells, BRCA1 shRNA effectively reduced BRCA1 protein to levels similar to the empty vector transfected cells, and therefore, specifically abrogated the increase in BRCA1 caused by EZH2 knockdown (Figure 6A).

Bottom Line: EZH2 knockdown decreased proliferation and delayed the G(2)/M cell-cycle transition, although not affecting apoptosis.Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G(2)/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis.Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
Increased levels of enhancer of zeste homolog 2 (EZH2), a critical regulator of cellular memory, are associated with negative estrogen receptor (ER) expression and disease progression in breast cancer. High levels of EZH2 signal the presence of metastasis and poor outcome in breast cancer patients. To test the hypothesis that deregulation of EZH2 contributes to ER-negative breast cancer progression, EZH2 expression was inhibited in ER-negative breast cancer cells MDA-MB-231 and CAL51 using a lentivirus system. EZH2 knockdown decreased proliferation and delayed the G(2)/M cell-cycle transition, although not affecting apoptosis. In vivo, EZH2 downregulation significantly decreased breast xenograft growth and improved survival. EZH2 knockdown upregulated BRCA1 protein. Of note, BRCA1 knockdown was sufficient to rescue the effects of EZH2 downregulation on proliferation, G(2)/M arrest, and on the levels of hyperphosphorylated mitotic Cdc25C and Cyclin B1 proteins, crucial for entry into mitosis. Invasive ER-negative breast carcinomas show significant overexpression of EZH2 and downregulation of BRCA1 proteins. Taken together, we show that EZH2 is important in ER-negative breast cancer growth in vivo and in vitro, and that BRCA1 is required for the proliferative effects of EZH2. Blockade of EZH2 may provide a prime target to prevent and/or halt ER-negative breast cancer progression.

Show MeSH
Related in: MedlinePlus