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Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer.

Déry MA, Jodoin J, Ursini-Siegel J, Aleynikova O, Ferrario C, Hassan S, Basik M, LeBlanc AC - Breast Cancer Res. (2013)

Bottom Line: Site-directed mutagenesis identified the ER stress response elements (ERSE).Higher PrP and BiP levels correlated with increasing tumor grade in TMA.Functionally, PrP delayed ER stress-induced cell death.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: High prion protein (PrP) levels are associated with breast, colon and gastric cancer resistance to treatment and with a poor prognosis for the patients. However, little is known about the underlying molecular mechanism(s) regulating human PrP gene (PRNP) expression in cancers. Because endoplasmic reticulum (ER) stress is associated with solid tumors, we investigated a possible regulation of PRNP gene expression by ER stress.

Methods: Published microarray databases of breast cancer tissues and breast carcinoma cell lines were analyzed for PrP mRNA and ER stress marker immunoglobulin heavy chain binding protein (BiP) levels. Breast cancer tissue microarrays (TMA) were immunostained for BiP and PrP. Breast carcinoma MCF-7, MDA-MB-231, HS578T and HCC1500 cells were treated with three different ER stressors - Brefeldin A, Tunicamycin, Thapsigargin - and levels of PrP mRNA or protein assessed by RT-PCR and Western blot analyses. A human PRNP promoter-luciferase reporter was used to assess transcriptional activation by ER stressors. Site-directed mutagenesis identified the ER stress response elements (ERSE). Chromatin immunoprecipitation (ChIP) analyses were done to identify the ER stress-mediated transcriptional regulators. The role of cleaved activating transcription factor 6α (ΔATF6α) and spliced X-box protein-1 (sXBP1) in PRNP gene expression was assessed with over-expression or silencing techniques. The role of PrP protection against ER stress was assessed with PrP siRNA and by using Prnp cell lines.

Results: We find that mRNA levels of BiP correlated with PrP transcript levels in breast cancer tissues and breast carcinoma cell lines. PrP mRNA levels were enriched in the basal subtype and were associated with poor prognosis in breast cancer patients. Higher PrP and BiP levels correlated with increasing tumor grade in TMA. ER stress was a positive regulator of PRNP gene transcription in MCF-7 cells and luciferase reporter assays identified one ER stress response element (ERSE) conserved among primates and rodents and three primate-specific ERSEs that regulated PRNP gene expression. Among the various transactivators of the ER stress-regulated unfolded protein response (UPR), ATF6α and XBP1 transactivated PRNP gene expression, but the ability of these varied in different cell types. Functionally, PrP delayed ER stress-induced cell death.

Conclusions: These results establish PRNP as a novel ER stress-regulated gene that could increase survival in breast cancers.

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Identification of ER stress response elements (ERSE) in the PRNP promoter. (A) Schematic diagram of two ERSE (ERSEa and ERSEb), one ERSE-like, and one ERSE-II in the human PRNP promoter. (B) Luciferase activity in HEK293T cells transfected with pGL2 (empty vector), pGL-214 (vector containing the first 214 nucleotides of human PRNP promoter), or pGL-538 (first 538 nucleotides of human PRNP promoter) and treated six hours with ER stressors. Data are expressed as the mean ± SD of two experiments done in triplicate. * Indicates P ≤0.05 compared to the control (Ctl). (C) Schematic diagram of the PRNP promoter mutants showing the mutated nucleotides in bold. Putative transcription factor binding sites predicted by TRANSFAC and the conserved motif 4 affected by the mutations are shown. (D) Luciferase activity measured in HEK293T cells transfected with wild type PRNP promoter (pGL-538) or ERSE mutants of the PRNP promoter. Data represent the mean ± SEM of three experiments done in triplicate. * Indicates P ≤0.05 compared to wild type. (E) Luciferase activity in HEK293T cells transfected with wild type or ERSE mutant PRNP promoters and treated with DMSO (control) or ER stressors. The fold increase of luciferase activity calculated for each PRNP promoter construct corresponds to the ratio of the RLU in presence of ER stress over the RLU in presence of DMSO (Control). Data represent the mean ± SEM of three independent experiments done in triplicate. * Indicates P ≤0.05 compared to the control (DMSO) and # indicates P ≤0.05 between the mutants and the wild type PRNP promoter. (F) Schematic diagram showing conservation of human ERSE-like, ERSEa, ERSE-II and ERSEb. Nucleotide sequence alignment of PRNP promoters is from a previous study [48] and from an alignment done with Ensembl databases. N indicates the number of nucleotide. Compared to human PRNP promoter, * indicates a non-conserved and non-complementary nucleotide while X denotes an absent nucleotide.
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Figure 5: Identification of ER stress response elements (ERSE) in the PRNP promoter. (A) Schematic diagram of two ERSE (ERSEa and ERSEb), one ERSE-like, and one ERSE-II in the human PRNP promoter. (B) Luciferase activity in HEK293T cells transfected with pGL2 (empty vector), pGL-214 (vector containing the first 214 nucleotides of human PRNP promoter), or pGL-538 (first 538 nucleotides of human PRNP promoter) and treated six hours with ER stressors. Data are expressed as the mean ± SD of two experiments done in triplicate. * Indicates P ≤0.05 compared to the control (Ctl). (C) Schematic diagram of the PRNP promoter mutants showing the mutated nucleotides in bold. Putative transcription factor binding sites predicted by TRANSFAC and the conserved motif 4 affected by the mutations are shown. (D) Luciferase activity measured in HEK293T cells transfected with wild type PRNP promoter (pGL-538) or ERSE mutants of the PRNP promoter. Data represent the mean ± SEM of three experiments done in triplicate. * Indicates P ≤0.05 compared to wild type. (E) Luciferase activity in HEK293T cells transfected with wild type or ERSE mutant PRNP promoters and treated with DMSO (control) or ER stressors. The fold increase of luciferase activity calculated for each PRNP promoter construct corresponds to the ratio of the RLU in presence of ER stress over the RLU in presence of DMSO (Control). Data represent the mean ± SEM of three independent experiments done in triplicate. * Indicates P ≤0.05 compared to the control (DMSO) and # indicates P ≤0.05 between the mutants and the wild type PRNP promoter. (F) Schematic diagram showing conservation of human ERSE-like, ERSEa, ERSE-II and ERSEb. Nucleotide sequence alignment of PRNP promoters is from a previous study [48] and from an alignment done with Ensembl databases. N indicates the number of nucleotide. Compared to human PRNP promoter, * indicates a non-conserved and non-complementary nucleotide while X denotes an absent nucleotide.

Mentions: We identified three potential ER stress response elements (ERSE) and one ERSE-II motif in the human PRNP promoter using the transcription factor database TRANSFAC and by manually scanning the PRNP promoter (EMBL accession no. AJ289875) [48] (Figure 5A). No consensus UPR element (UPRE) or amino-acid-regulatory element (AARE) was found in the PRNP promoter. Two ERSE motifs, named here ERSEa and ERSEb to easily discriminate between them, were located at nucleotides -89 to -71 and at -20 to -2, respectively. ERSEa was in the opposite orientation and had a T to A substitution (CCAAT to CCAAa) compared to the ERSE consensus sequence. The ERSEb nucleotide sequence also contained two CG substitutions (CCACG to CgACc). One non-conventional ERSE (ERSE-like) motif, located from -231 to -196 and predicted by TRANSFAC, contained N26 rather than the canonical N9 of the glucose-regulated proteins CCAAT-N9-CCACG ERSE consensus sequence [35]. One canonical ERSE-II motif was contained within ERSEb (-20 to -10). Compared to the ERSE-II consensus sequence (ATTGG-N-CCACG), the ERSE-II motif in the PRNP gene promoter had an A to C substitution (CCACG to CCcCG).


Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer.

Déry MA, Jodoin J, Ursini-Siegel J, Aleynikova O, Ferrario C, Hassan S, Basik M, LeBlanc AC - Breast Cancer Res. (2013)

Identification of ER stress response elements (ERSE) in the PRNP promoter. (A) Schematic diagram of two ERSE (ERSEa and ERSEb), one ERSE-like, and one ERSE-II in the human PRNP promoter. (B) Luciferase activity in HEK293T cells transfected with pGL2 (empty vector), pGL-214 (vector containing the first 214 nucleotides of human PRNP promoter), or pGL-538 (first 538 nucleotides of human PRNP promoter) and treated six hours with ER stressors. Data are expressed as the mean ± SD of two experiments done in triplicate. * Indicates P ≤0.05 compared to the control (Ctl). (C) Schematic diagram of the PRNP promoter mutants showing the mutated nucleotides in bold. Putative transcription factor binding sites predicted by TRANSFAC and the conserved motif 4 affected by the mutations are shown. (D) Luciferase activity measured in HEK293T cells transfected with wild type PRNP promoter (pGL-538) or ERSE mutants of the PRNP promoter. Data represent the mean ± SEM of three experiments done in triplicate. * Indicates P ≤0.05 compared to wild type. (E) Luciferase activity in HEK293T cells transfected with wild type or ERSE mutant PRNP promoters and treated with DMSO (control) or ER stressors. The fold increase of luciferase activity calculated for each PRNP promoter construct corresponds to the ratio of the RLU in presence of ER stress over the RLU in presence of DMSO (Control). Data represent the mean ± SEM of three independent experiments done in triplicate. * Indicates P ≤0.05 compared to the control (DMSO) and # indicates P ≤0.05 between the mutants and the wild type PRNP promoter. (F) Schematic diagram showing conservation of human ERSE-like, ERSEa, ERSE-II and ERSEb. Nucleotide sequence alignment of PRNP promoters is from a previous study [48] and from an alignment done with Ensembl databases. N indicates the number of nucleotide. Compared to human PRNP promoter, * indicates a non-conserved and non-complementary nucleotide while X denotes an absent nucleotide.
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Figure 5: Identification of ER stress response elements (ERSE) in the PRNP promoter. (A) Schematic diagram of two ERSE (ERSEa and ERSEb), one ERSE-like, and one ERSE-II in the human PRNP promoter. (B) Luciferase activity in HEK293T cells transfected with pGL2 (empty vector), pGL-214 (vector containing the first 214 nucleotides of human PRNP promoter), or pGL-538 (first 538 nucleotides of human PRNP promoter) and treated six hours with ER stressors. Data are expressed as the mean ± SD of two experiments done in triplicate. * Indicates P ≤0.05 compared to the control (Ctl). (C) Schematic diagram of the PRNP promoter mutants showing the mutated nucleotides in bold. Putative transcription factor binding sites predicted by TRANSFAC and the conserved motif 4 affected by the mutations are shown. (D) Luciferase activity measured in HEK293T cells transfected with wild type PRNP promoter (pGL-538) or ERSE mutants of the PRNP promoter. Data represent the mean ± SEM of three experiments done in triplicate. * Indicates P ≤0.05 compared to wild type. (E) Luciferase activity in HEK293T cells transfected with wild type or ERSE mutant PRNP promoters and treated with DMSO (control) or ER stressors. The fold increase of luciferase activity calculated for each PRNP promoter construct corresponds to the ratio of the RLU in presence of ER stress over the RLU in presence of DMSO (Control). Data represent the mean ± SEM of three independent experiments done in triplicate. * Indicates P ≤0.05 compared to the control (DMSO) and # indicates P ≤0.05 between the mutants and the wild type PRNP promoter. (F) Schematic diagram showing conservation of human ERSE-like, ERSEa, ERSE-II and ERSEb. Nucleotide sequence alignment of PRNP promoters is from a previous study [48] and from an alignment done with Ensembl databases. N indicates the number of nucleotide. Compared to human PRNP promoter, * indicates a non-conserved and non-complementary nucleotide while X denotes an absent nucleotide.
Mentions: We identified three potential ER stress response elements (ERSE) and one ERSE-II motif in the human PRNP promoter using the transcription factor database TRANSFAC and by manually scanning the PRNP promoter (EMBL accession no. AJ289875) [48] (Figure 5A). No consensus UPR element (UPRE) or amino-acid-regulatory element (AARE) was found in the PRNP promoter. Two ERSE motifs, named here ERSEa and ERSEb to easily discriminate between them, were located at nucleotides -89 to -71 and at -20 to -2, respectively. ERSEa was in the opposite orientation and had a T to A substitution (CCAAT to CCAAa) compared to the ERSE consensus sequence. The ERSEb nucleotide sequence also contained two CG substitutions (CCACG to CgACc). One non-conventional ERSE (ERSE-like) motif, located from -231 to -196 and predicted by TRANSFAC, contained N26 rather than the canonical N9 of the glucose-regulated proteins CCAAT-N9-CCACG ERSE consensus sequence [35]. One canonical ERSE-II motif was contained within ERSEb (-20 to -10). Compared to the ERSE-II consensus sequence (ATTGG-N-CCACG), the ERSE-II motif in the PRNP gene promoter had an A to C substitution (CCACG to CCcCG).

Bottom Line: Site-directed mutagenesis identified the ER stress response elements (ERSE).Higher PrP and BiP levels correlated with increasing tumor grade in TMA.Functionally, PrP delayed ER stress-induced cell death.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: High prion protein (PrP) levels are associated with breast, colon and gastric cancer resistance to treatment and with a poor prognosis for the patients. However, little is known about the underlying molecular mechanism(s) regulating human PrP gene (PRNP) expression in cancers. Because endoplasmic reticulum (ER) stress is associated with solid tumors, we investigated a possible regulation of PRNP gene expression by ER stress.

Methods: Published microarray databases of breast cancer tissues and breast carcinoma cell lines were analyzed for PrP mRNA and ER stress marker immunoglobulin heavy chain binding protein (BiP) levels. Breast cancer tissue microarrays (TMA) were immunostained for BiP and PrP. Breast carcinoma MCF-7, MDA-MB-231, HS578T and HCC1500 cells were treated with three different ER stressors - Brefeldin A, Tunicamycin, Thapsigargin - and levels of PrP mRNA or protein assessed by RT-PCR and Western blot analyses. A human PRNP promoter-luciferase reporter was used to assess transcriptional activation by ER stressors. Site-directed mutagenesis identified the ER stress response elements (ERSE). Chromatin immunoprecipitation (ChIP) analyses were done to identify the ER stress-mediated transcriptional regulators. The role of cleaved activating transcription factor 6α (ΔATF6α) and spliced X-box protein-1 (sXBP1) in PRNP gene expression was assessed with over-expression or silencing techniques. The role of PrP protection against ER stress was assessed with PrP siRNA and by using Prnp cell lines.

Results: We find that mRNA levels of BiP correlated with PrP transcript levels in breast cancer tissues and breast carcinoma cell lines. PrP mRNA levels were enriched in the basal subtype and were associated with poor prognosis in breast cancer patients. Higher PrP and BiP levels correlated with increasing tumor grade in TMA. ER stress was a positive regulator of PRNP gene transcription in MCF-7 cells and luciferase reporter assays identified one ER stress response element (ERSE) conserved among primates and rodents and three primate-specific ERSEs that regulated PRNP gene expression. Among the various transactivators of the ER stress-regulated unfolded protein response (UPR), ATF6α and XBP1 transactivated PRNP gene expression, but the ability of these varied in different cell types. Functionally, PrP delayed ER stress-induced cell death.

Conclusions: These results establish PRNP as a novel ER stress-regulated gene that could increase survival in breast cancers.

Show MeSH
Related in: MedlinePlus