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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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RB1 kd MCF7 and T47D cells down regulate the ESR1 protein(A) ESR1 expression in RB1 knocked down MCF7 cells. kd1 and kd2 represent two different shRNAs. Alpha-tubulin was used as a loading control. (B) Quantification of three independent experiments as in (A). y axis represents the ratio between the ESR1 and alpha-tubulin proteins and normalized to scrambled cells. The RB1 knocked down MCF7 cells express about 50% less of the ESR1 protein. (C) ESR1 expression in RB1 knock down T47D cells. (D) Quantification of three independent experiments as in (b). The RB1 knock down T47D cells express about 56% less of the ESR1 protein. (E) MCF7 cells were grown in CSS medium for 3 days (NT) and treated with 10−8 estradiol for 45 minutes (E2). Cells were probed with an ESR1 antibody (green) and stained with DAPI (blue). RB1 kd1 cells express less ESR1 protein.
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Figure 1: RB1 kd MCF7 and T47D cells down regulate the ESR1 protein(A) ESR1 expression in RB1 knocked down MCF7 cells. kd1 and kd2 represent two different shRNAs. Alpha-tubulin was used as a loading control. (B) Quantification of three independent experiments as in (A). y axis represents the ratio between the ESR1 and alpha-tubulin proteins and normalized to scrambled cells. The RB1 knocked down MCF7 cells express about 50% less of the ESR1 protein. (C) ESR1 expression in RB1 knock down T47D cells. (D) Quantification of three independent experiments as in (b). The RB1 knock down T47D cells express about 56% less of the ESR1 protein. (E) MCF7 cells were grown in CSS medium for 3 days (NT) and treated with 10−8 estradiol for 45 minutes (E2). Cells were probed with an ESR1 antibody (green) and stained with DAPI (blue). RB1 kd1 cells express less ESR1 protein.

Mentions: To test our hypothesis, we have generated MCF7 (ESR1 positive) cell lines knocked-down for the three members of the Retinoblastoma family, pRb, pRb2/p130, and p107 (Supplemental Figure 1A) [14,15]. Supplemental Figure 1B shows that the loss of pRb family members decreased the expression of ESR1 when compared to scrambled cells. Among the three members, only pRb is involved in this mechanism (Supplemental Figure 1B, Figure 1A). The data were obtained in basal conditions in the absence of hormones (Charcoal Stripped Serum, CSS). We decided to perform all the experiments under these conditions unless otherwise indicated. To exclude that the mechanism is a characteristic of a single cell line, we have down regulated pRb in the T47D ESR1 positive breast cancer cell line. The results in T47D cells are comparable to those in the MCF7 cells (Figure 1C). In both cell lines, the downregulation of ESR1 in RB1 kd cells is statistically significant (Figure 1B,D). To confirm the data, we have carried out immunofluorescence experiments. We observed a reduction in signal intensity of the ESR1 in MCF7 RB1 kd cells in basal and estradiol-stimulated conditions (Figure 1E). To definitively demonstrate that the activity of ESR1 was compromised, we have assessed the expression of some classical ESR1 target genes [16]. We observed that the expression of TFF1 and CTSD are down regulated in RB1 kd cells (Supplemental Figure 1C). Analysis of ESR1 mRNA also showed a reduction in RB1 kd cells (Figure 2A). Since the pRb family members could bind the ESR1 promoter [17] and the ESR1 protein itself regulates its expression [16], we have cloned the ESR1 downstream a non-endogenous promoter. A western blot analysis indicates that the reduction of ESR1 relative expression under the non-endogenous promoter is comparable with that of endogenous promoter indicating a control at the posttranscriptional level (Figure 2B). These data indicate that pRb could be a new cofactor of ESR1, regulating its protein expression level.


pRb controls estrogen receptor alpha protein stability and activity.

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

RB1 kd MCF7 and T47D cells down regulate the ESR1 protein(A) ESR1 expression in RB1 knocked down MCF7 cells. kd1 and kd2 represent two different shRNAs. Alpha-tubulin was used as a loading control. (B) Quantification of three independent experiments as in (A). y axis represents the ratio between the ESR1 and alpha-tubulin proteins and normalized to scrambled cells. The RB1 knocked down MCF7 cells express about 50% less of the ESR1 protein. (C) ESR1 expression in RB1 knock down T47D cells. (D) Quantification of three independent experiments as in (b). The RB1 knock down T47D cells express about 56% less of the ESR1 protein. (E) MCF7 cells were grown in CSS medium for 3 days (NT) and treated with 10−8 estradiol for 45 minutes (E2). Cells were probed with an ESR1 antibody (green) and stained with DAPI (blue). RB1 kd1 cells express less ESR1 protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757244&req=5

Figure 1: RB1 kd MCF7 and T47D cells down regulate the ESR1 protein(A) ESR1 expression in RB1 knocked down MCF7 cells. kd1 and kd2 represent two different shRNAs. Alpha-tubulin was used as a loading control. (B) Quantification of three independent experiments as in (A). y axis represents the ratio between the ESR1 and alpha-tubulin proteins and normalized to scrambled cells. The RB1 knocked down MCF7 cells express about 50% less of the ESR1 protein. (C) ESR1 expression in RB1 knock down T47D cells. (D) Quantification of three independent experiments as in (b). The RB1 knock down T47D cells express about 56% less of the ESR1 protein. (E) MCF7 cells were grown in CSS medium for 3 days (NT) and treated with 10−8 estradiol for 45 minutes (E2). Cells were probed with an ESR1 antibody (green) and stained with DAPI (blue). RB1 kd1 cells express less ESR1 protein.
Mentions: To test our hypothesis, we have generated MCF7 (ESR1 positive) cell lines knocked-down for the three members of the Retinoblastoma family, pRb, pRb2/p130, and p107 (Supplemental Figure 1A) [14,15]. Supplemental Figure 1B shows that the loss of pRb family members decreased the expression of ESR1 when compared to scrambled cells. Among the three members, only pRb is involved in this mechanism (Supplemental Figure 1B, Figure 1A). The data were obtained in basal conditions in the absence of hormones (Charcoal Stripped Serum, CSS). We decided to perform all the experiments under these conditions unless otherwise indicated. To exclude that the mechanism is a characteristic of a single cell line, we have down regulated pRb in the T47D ESR1 positive breast cancer cell line. The results in T47D cells are comparable to those in the MCF7 cells (Figure 1C). In both cell lines, the downregulation of ESR1 in RB1 kd cells is statistically significant (Figure 1B,D). To confirm the data, we have carried out immunofluorescence experiments. We observed a reduction in signal intensity of the ESR1 in MCF7 RB1 kd cells in basal and estradiol-stimulated conditions (Figure 1E). To definitively demonstrate that the activity of ESR1 was compromised, we have assessed the expression of some classical ESR1 target genes [16]. We observed that the expression of TFF1 and CTSD are down regulated in RB1 kd cells (Supplemental Figure 1C). Analysis of ESR1 mRNA also showed a reduction in RB1 kd cells (Figure 2A). Since the pRb family members could bind the ESR1 promoter [17] and the ESR1 protein itself regulates its expression [16], we have cloned the ESR1 downstream a non-endogenous promoter. A western blot analysis indicates that the reduction of ESR1 relative expression under the non-endogenous promoter is comparable with that of endogenous promoter indicating a control at the posttranscriptional level (Figure 2B). These data indicate that pRb could be a new cofactor of ESR1, regulating its protein expression level.

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