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pRb controls estrogen receptor alpha protein stability and activity.

Caligiuri I, Toffoli G, Giordano A, Rizzolio F - Oncotarget (2013)

Bottom Line: Here we show that the N-terminal domain of pRb interacts with the CD domain of ESR1 to allow for the assembly of intermediate complex chaperone proteins HSP90 and p23.We demonstrated that a loss of pRb in human/mouse breast cells decreases the expression of the ESR1 protein through the proteasome pathway.Our work reveals a novel regulatory mechanism of ESR1 basal turnover and activity and an unanticipated relationship with the pRb tumor suppressor.

View Article: PubMed Central - PubMed

Affiliation: Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA.

ABSTRACT
A cross talk between the Estrogen Receptor (ESR1) and the Retinoblastoma (pRb) pathway has been demonstrated to influence the therapeutic response of breast cancer patients but the full mechanism remains poorly understood. Here we show that the N-terminal domain of pRb interacts with the CD domain of ESR1 to allow for the assembly of intermediate complex chaperone proteins HSP90 and p23. We demonstrated that a loss of pRb in human/mouse breast cells decreases the expression of the ESR1 protein through the proteasome pathway. Our work reveals a novel regulatory mechanism of ESR1 basal turnover and activity and an unanticipated relationship with the pRb tumor suppressor.

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Nuclear accumulation of ESR1 prevents proteasomal degradation in RB1 kd1 cells(A) MCF7 cells were treated with leptomycin B, estradiol or a combination for indicated time. Estradiol allows the translocation of ESR1 in the nucleus and Leptomycin B prevents the nuclear export. After 3 or 6 hours of treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells. (B) T47D cells were treated with CHX, estradiol, or a combination for 3 hours. CHX allows nuclear accumulation of ESR1. After CHX plus estradiol treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells.
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Figure 4: Nuclear accumulation of ESR1 prevents proteasomal degradation in RB1 kd1 cells(A) MCF7 cells were treated with leptomycin B, estradiol or a combination for indicated time. Estradiol allows the translocation of ESR1 in the nucleus and Leptomycin B prevents the nuclear export. After 3 or 6 hours of treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells. (B) T47D cells were treated with CHX, estradiol, or a combination for 3 hours. CHX allows nuclear accumulation of ESR1. After CHX plus estradiol treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells.

Mentions: ESR1, as with most of the hormonal receptors, is finely regulated at the transcriptional and posttranscriptional levels. A key role in protein half-life is played by the proteasome pathway [19–21]. Without the hormone, the ESR1 is associated with HSP70, HSP40 and the adapter HIP protein (HSP70-interacting protein) to form an early complex. Later on, HSP90 and the adapter protein HOP (HSP70/HSP90- organizing protein) displace HSP40 and bind the hydrophobic hormone-binding domain of ESR1 to form an intermediate complex [22]. After ATP binding, HSP90 interacts with p23 and Cyclophilin 40 (CYP40) to form a mature complex [22]. To test if pRb is involved in the ESR1 degradation via the proteasome pathway, we have treated the MCF7 cells with the proteasome inhibitor MG132 [23]. After 4 hours of treatment, RB1 kd cells had the same level of ESR1 as scrambled cells, thus rescuing the phenotype observed in untreated cells (Figure 3A,B). This finding was confirmed in RB1 kd2 cells (Supplemental Figure 2A) and utilizing the drug Bortezomib (Supplemental Figure 2B), another proteasome inhibitor. The same results were also observed in T47D cells (Supplemental Figure 2C). To assess if ESR1 is more ubiquitinated in RB1 kd MCF7 cells, we treated the sample with MG132, immunoprecipitated with an ESR1 antibody and analyzed the ubiquitin level. Figure 3C confirmed that ESR1 is more ubiquitinated in RB1 kd cells as compared to scrambled cells. A typical smear is observed in the stacking gel. As a consequence of the obtained results, we have analyzed the chaperone proteins involved in ESR1 protein stability [24]. In Figure 3D, we show the co-immunoprecipitaion of ESR1 with HSP90. It appears that RB1 kd1 cells have less HSP90 bound to ESR1 in untreated and MG132-treated samples compared to scrambled cells. The HSP90 co-chaperone, p23, is also reduced as expected. The level of HSP70 is unaltered, suggesting that pRb influences the intermediate complex that stabilizes the ESR1 protein [22]. Under basal conditions, the ESR1 is associated with chaperone proteins in the cytoplasm. After estradiol stimulation, ESR1 is shuttled to the nucleus to exert its genomic function on target genes [25]. The re-cycling of ESR1 on promoters of target genes is very fast [26], after which the ESR1 is exported again to the cytoplasm for proteasomal degradation [23]. Inhibitors of nuclear export such as Leptomycin B [27] or, nuclear stabilizing agents such as cycloheximide lead to ESR1 protein accumulation. When RB1 kd1 cells were treated with Leptomycin B (Figure 4A) or cycloheximide (Figure 4B) in combination with estradiol, the level of ESR1 were comparable with scrambled cells, confirming a cytoplasmic ESR1 degradation driven by the proteasome pathway.


pRb controls estrogen receptor alpha protein stability and activity.

Caligiuri I, Toffoli G, Giordano A, Rizzolio F - Oncotarget (2013)

Nuclear accumulation of ESR1 prevents proteasomal degradation in RB1 kd1 cells(A) MCF7 cells were treated with leptomycin B, estradiol or a combination for indicated time. Estradiol allows the translocation of ESR1 in the nucleus and Leptomycin B prevents the nuclear export. After 3 or 6 hours of treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells. (B) T47D cells were treated with CHX, estradiol, or a combination for 3 hours. CHX allows nuclear accumulation of ESR1. After CHX plus estradiol treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells.
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Related In: Results  -  Collection

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Figure 4: Nuclear accumulation of ESR1 prevents proteasomal degradation in RB1 kd1 cells(A) MCF7 cells were treated with leptomycin B, estradiol or a combination for indicated time. Estradiol allows the translocation of ESR1 in the nucleus and Leptomycin B prevents the nuclear export. After 3 or 6 hours of treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells. (B) T47D cells were treated with CHX, estradiol, or a combination for 3 hours. CHX allows nuclear accumulation of ESR1. After CHX plus estradiol treatment, the RB1 kd1 cells express the same level of ESR1 as scrambled cells.
Mentions: ESR1, as with most of the hormonal receptors, is finely regulated at the transcriptional and posttranscriptional levels. A key role in protein half-life is played by the proteasome pathway [19–21]. Without the hormone, the ESR1 is associated with HSP70, HSP40 and the adapter HIP protein (HSP70-interacting protein) to form an early complex. Later on, HSP90 and the adapter protein HOP (HSP70/HSP90- organizing protein) displace HSP40 and bind the hydrophobic hormone-binding domain of ESR1 to form an intermediate complex [22]. After ATP binding, HSP90 interacts with p23 and Cyclophilin 40 (CYP40) to form a mature complex [22]. To test if pRb is involved in the ESR1 degradation via the proteasome pathway, we have treated the MCF7 cells with the proteasome inhibitor MG132 [23]. After 4 hours of treatment, RB1 kd cells had the same level of ESR1 as scrambled cells, thus rescuing the phenotype observed in untreated cells (Figure 3A,B). This finding was confirmed in RB1 kd2 cells (Supplemental Figure 2A) and utilizing the drug Bortezomib (Supplemental Figure 2B), another proteasome inhibitor. The same results were also observed in T47D cells (Supplemental Figure 2C). To assess if ESR1 is more ubiquitinated in RB1 kd MCF7 cells, we treated the sample with MG132, immunoprecipitated with an ESR1 antibody and analyzed the ubiquitin level. Figure 3C confirmed that ESR1 is more ubiquitinated in RB1 kd cells as compared to scrambled cells. A typical smear is observed in the stacking gel. As a consequence of the obtained results, we have analyzed the chaperone proteins involved in ESR1 protein stability [24]. In Figure 3D, we show the co-immunoprecipitaion of ESR1 with HSP90. It appears that RB1 kd1 cells have less HSP90 bound to ESR1 in untreated and MG132-treated samples compared to scrambled cells. The HSP90 co-chaperone, p23, is also reduced as expected. The level of HSP70 is unaltered, suggesting that pRb influences the intermediate complex that stabilizes the ESR1 protein [22]. Under basal conditions, the ESR1 is associated with chaperone proteins in the cytoplasm. After estradiol stimulation, ESR1 is shuttled to the nucleus to exert its genomic function on target genes [25]. The re-cycling of ESR1 on promoters of target genes is very fast [26], after which the ESR1 is exported again to the cytoplasm for proteasomal degradation [23]. Inhibitors of nuclear export such as Leptomycin B [27] or, nuclear stabilizing agents such as cycloheximide lead to ESR1 protein accumulation. When RB1 kd1 cells were treated with Leptomycin B (Figure 4A) or cycloheximide (Figure 4B) in combination with estradiol, the level of ESR1 were comparable with scrambled cells, confirming a cytoplasmic ESR1 degradation driven by the proteasome pathway.

Bottom Line: Here we show that the N-terminal domain of pRb interacts with the CD domain of ESR1 to allow for the assembly of intermediate complex chaperone proteins HSP90 and p23.We demonstrated that a loss of pRb in human/mouse breast cells decreases the expression of the ESR1 protein through the proteasome pathway.Our work reveals a novel regulatory mechanism of ESR1 basal turnover and activity and an unanticipated relationship with the pRb tumor suppressor.

View Article: PubMed Central - PubMed

Affiliation: Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA.

ABSTRACT
A cross talk between the Estrogen Receptor (ESR1) and the Retinoblastoma (pRb) pathway has been demonstrated to influence the therapeutic response of breast cancer patients but the full mechanism remains poorly understood. Here we show that the N-terminal domain of pRb interacts with the CD domain of ESR1 to allow for the assembly of intermediate complex chaperone proteins HSP90 and p23. We demonstrated that a loss of pRb in human/mouse breast cells decreases the expression of the ESR1 protein through the proteasome pathway. Our work reveals a novel regulatory mechanism of ESR1 basal turnover and activity and an unanticipated relationship with the pRb tumor suppressor.

Show MeSH
Related in: MedlinePlus