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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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ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines. Western blot of (A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, (B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, (C) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, (D) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.
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Figure 4: ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines. Western blot of (A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, (B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, (C) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, (D) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.

Mentions: A comparison of basal breast carcinoma cell lines, MDA-MB-231, HS578T and HCC1500, with luminal MCF-7 cells revealed a high level of PrP in the MDA-MB-231 and HS578T cell lines but not in the HCC1500 basal cell line. However, BFA, TM and Thps increased the levels of PrP and BiP in the HCC1500 cell line (Figure 4B) as observed in the luminal MCF-7 cell line (Figure 3A). Treatment of MDA-MB-231 and HS578T cells with 4-phenyl butyric acid (4-PBA), an inhibitor of ER stress [56], decreases both BiP and PrP levels suggesting that PrP is increased by intrinsic ER stress in these two basal carcinoma cell lines (Figure 4C). Nevertheless, the three pharmacological ER stressors further increased both BiP and PrP levels in MDA-MB-231 and HS578T cells (Figure 4D). Together, these results show that intrinsic or exogenous ER stress up-regulates PRNP gene expression in basal breast carcinoma cell lines.


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)

ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines. Western blot of (A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, (B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, (C) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, (D) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines. Western blot of (A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, (B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, (C) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, (D) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.
Mentions: A comparison of basal breast carcinoma cell lines, MDA-MB-231, HS578T and HCC1500, with luminal MCF-7 cells revealed a high level of PrP in the MDA-MB-231 and HS578T cell lines but not in the HCC1500 basal cell line. However, BFA, TM and Thps increased the levels of PrP and BiP in the HCC1500 cell line (Figure 4B) as observed in the luminal MCF-7 cell line (Figure 3A). Treatment of MDA-MB-231 and HS578T cells with 4-phenyl butyric acid (4-PBA), an inhibitor of ER stress [56], decreases both BiP and PrP levels suggesting that PrP is increased by intrinsic ER stress in these two basal carcinoma cell lines (Figure 4C). Nevertheless, the three pharmacological ER stressors further increased both BiP and PrP levels in MDA-MB-231 and HS578T cells (Figure 4D). Together, these results show that intrinsic or exogenous ER stress up-regulates PRNP gene expression in basal breast carcinoma cell lines.

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