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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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Related in: MedlinePlus

PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells. Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A), Tunicamycin (B), or Thapsigargin (C). Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. (D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.
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Figure 9: PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells. Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A), Tunicamycin (B), or Thapsigargin (C). Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. (D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.

Mentions: We have previously discovered that PrP can inhibit Bax-mediated cell death [2,4]. Immunoprecipitation of the pro-apoptotic 6A7-immunoreactive form of Bax indicated that BFA, TM and Thps all induce Bax activation (Figure 9A-C). However, silencing PrP before ER stress did not increase the level of Bax activation indicating that PrP cannot prevent ER stress-induced Bax activation and that PrP delays ER stress-mediated cell death by another mechanism.


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)

PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells. Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A), Tunicamycin (B), or Thapsigargin (C). Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. (D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672785&req=5

Figure 9: PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells. Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A), Tunicamycin (B), or Thapsigargin (C). Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. (D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.
Mentions: We have previously discovered that PrP can inhibit Bax-mediated cell death [2,4]. Immunoprecipitation of the pro-apoptotic 6A7-immunoreactive form of Bax indicated that BFA, TM and Thps all induce Bax activation (Figure 9A-C). However, silencing PrP before ER stress did not increase the level of Bax activation indicating that PrP cannot prevent ER stress-induced Bax activation and that PrP delays ER stress-mediated cell death by another mechanism.

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