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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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ATF6α and XBP1 transactivate PRNP promoter. (A) Ethidium bromide stained gel showing ΔATF6α, sXBP1 and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and pCGN-ATF6α (1-373) and/or pCGN-sXBP1. (B) Luciferase luminescence generated from HEK293 cells co-transfected as described in A. (C) Ethidium bromide stained gel showing ATF6α and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and siRNAs against ATF6α or XBP1. (D) Luciferase luminescence generated from HEK293 cells co-transfected as described in C. Statistical analyses on B and D were one-way ANOVA followed by a Tukey-Kramer multiple comparison test.
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Figure 7: ATF6α and XBP1 transactivate PRNP promoter. (A) Ethidium bromide stained gel showing ΔATF6α, sXBP1 and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and pCGN-ATF6α (1-373) and/or pCGN-sXBP1. (B) Luciferase luminescence generated from HEK293 cells co-transfected as described in A. (C) Ethidium bromide stained gel showing ATF6α and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and siRNAs against ATF6α or XBP1. (D) Luciferase luminescence generated from HEK293 cells co-transfected as described in C. Statistical analyses on B and D were one-way ANOVA followed by a Tukey-Kramer multiple comparison test.

Mentions: To more directly assess if sXBP1 and ΔATF6α transactivate PRNP gene expression from its promoter, HEK293 cells were co-transfected with pCGN constructs encoding sXBP1 or ΔATF6α and the pGL-538 PRNP promoter luciferase reporter construct. Overexpression of both ΔATF6α and sXBP1 was confirmed by RT-PCR (Figure 7A) and both of these transcription factors transactivated luciferase expression from the pGL-538 PRNP promoter luciferase reporter construct (Figure 7B). Furthermore, KD of ATF6α and XBP1 expression with two different siRNAs (Figure 7C), prevented Thps-induced luciferase expression from the pGL-538 construct (Figure 7D). The siATF6α SC siRNA induced an overall increase in luciferase activity compared to the siCtl but the levels did not differ between DMSO and Thps. This suggests that this siRNA may induce other transcription factors that up-regulate PRNP gene expression. Taken together, these results confirm that both ATF6α and sXBP1 can up-regulate PRNP gene expression in HEK293 cells.


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)

ATF6α and XBP1 transactivate PRNP promoter. (A) Ethidium bromide stained gel showing ΔATF6α, sXBP1 and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and pCGN-ATF6α (1-373) and/or pCGN-sXBP1. (B) Luciferase luminescence generated from HEK293 cells co-transfected as described in A. (C) Ethidium bromide stained gel showing ATF6α and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and siRNAs against ATF6α or XBP1. (D) Luciferase luminescence generated from HEK293 cells co-transfected as described in C. Statistical analyses on B and D were one-way ANOVA followed by a Tukey-Kramer multiple comparison test.
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Figure 7: ATF6α and XBP1 transactivate PRNP promoter. (A) Ethidium bromide stained gel showing ΔATF6α, sXBP1 and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and pCGN-ATF6α (1-373) and/or pCGN-sXBP1. (B) Luciferase luminescence generated from HEK293 cells co-transfected as described in A. (C) Ethidium bromide stained gel showing ATF6α and XBP1 amplicons from HEK293 cells co-transfected with the pGL-538 PRNP promoter luciferase reporter and siRNAs against ATF6α or XBP1. (D) Luciferase luminescence generated from HEK293 cells co-transfected as described in C. Statistical analyses on B and D were one-way ANOVA followed by a Tukey-Kramer multiple comparison test.
Mentions: To more directly assess if sXBP1 and ΔATF6α transactivate PRNP gene expression from its promoter, HEK293 cells were co-transfected with pCGN constructs encoding sXBP1 or ΔATF6α and the pGL-538 PRNP promoter luciferase reporter construct. Overexpression of both ΔATF6α and sXBP1 was confirmed by RT-PCR (Figure 7A) and both of these transcription factors transactivated luciferase expression from the pGL-538 PRNP promoter luciferase reporter construct (Figure 7B). Furthermore, KD of ATF6α and XBP1 expression with two different siRNAs (Figure 7C), prevented Thps-induced luciferase expression from the pGL-538 construct (Figure 7D). The siATF6α SC siRNA induced an overall increase in luciferase activity compared to the siCtl but the levels did not differ between DMSO and Thps. This suggests that this siRNA may induce other transcription factors that up-regulate PRNP gene expression. Taken together, these results confirm that both ATF6α and sXBP1 can up-regulate PRNP gene expression in HEK293 cells.

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