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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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In vitro and in vivo interaction between pRb and ESR1 proteins(A) RB1 kd1 and kd2 MCF7 cell lines have about 30% less mRNA levels of ESR1. y axis represents the ratio between ESR1 and GAPDH mRNAs. (B) MCF7 cells were infected with a lentivirus that expresses ESR1-V5 under the CMV promoter. RB1 kd1 cells express less endogenous and transfected ESR1 protein, demonstrating that the effect is at the posttranslational level. Quantification represents the ratio between the indicated protein and alpha-tubulin and normalized to scrambled cells. (C) In vitro interaction. ESR1 AB, CD and EF domains were pulleddown with GST-pRb N-terminal, Pocket and C-terminal domains in MCF7 cells. There is an interaction between the ESR1CD and pRb N-terminal domains. p.RED, pounceau red. (D) In vivo interaction. ESR1 was immunoprecipitated in MCF7 cells and analyzed by western blot with a pRb antibody. IgG was used as a negative control.
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Figure 2: In vitro and in vivo interaction between pRb and ESR1 proteins(A) RB1 kd1 and kd2 MCF7 cell lines have about 30% less mRNA levels of ESR1. y axis represents the ratio between ESR1 and GAPDH mRNAs. (B) MCF7 cells were infected with a lentivirus that expresses ESR1-V5 under the CMV promoter. RB1 kd1 cells express less endogenous and transfected ESR1 protein, demonstrating that the effect is at the posttranslational level. Quantification represents the ratio between the indicated protein and alpha-tubulin and normalized to scrambled cells. (C) In vitro interaction. ESR1 AB, CD and EF domains were pulleddown with GST-pRb N-terminal, Pocket and C-terminal domains in MCF7 cells. There is an interaction between the ESR1CD and pRb N-terminal domains. p.RED, pounceau red. (D) In vivo interaction. ESR1 was immunoprecipitated in MCF7 cells and analyzed by western blot with a pRb antibody. IgG was used as a negative control.

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)

In vitro and in vivo interaction between pRb and ESR1 proteins(A) RB1 kd1 and kd2 MCF7 cell lines have about 30% less mRNA levels of ESR1. y axis represents the ratio between ESR1 and GAPDH mRNAs. (B) MCF7 cells were infected with a lentivirus that expresses ESR1-V5 under the CMV promoter. RB1 kd1 cells express less endogenous and transfected ESR1 protein, demonstrating that the effect is at the posttranslational level. Quantification represents the ratio between the indicated protein and alpha-tubulin and normalized to scrambled cells. (C) In vitro interaction. ESR1 AB, CD and EF domains were pulleddown with GST-pRb N-terminal, Pocket and C-terminal domains in MCF7 cells. There is an interaction between the ESR1CD and pRb N-terminal domains. p.RED, pounceau red. (D) In vivo interaction. ESR1 was immunoprecipitated in MCF7 cells and analyzed by western blot with a pRb antibody. IgG was used as a negative control.
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Related In: Results  -  Collection

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Figure 2: In vitro and in vivo interaction between pRb and ESR1 proteins(A) RB1 kd1 and kd2 MCF7 cell lines have about 30% less mRNA levels of ESR1. y axis represents the ratio between ESR1 and GAPDH mRNAs. (B) MCF7 cells were infected with a lentivirus that expresses ESR1-V5 under the CMV promoter. RB1 kd1 cells express less endogenous and transfected ESR1 protein, demonstrating that the effect is at the posttranslational level. Quantification represents the ratio between the indicated protein and alpha-tubulin and normalized to scrambled cells. (C) In vitro interaction. ESR1 AB, CD and EF domains were pulleddown with GST-pRb N-terminal, Pocket and C-terminal domains in MCF7 cells. There is an interaction between the ESR1CD and pRb N-terminal domains. p.RED, pounceau red. (D) In vivo interaction. ESR1 was immunoprecipitated in MCF7 cells and analyzed by western blot with a pRb antibody. IgG was used as a negative control.
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