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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 transcriptionally increases PrP levels in MCF-7 cells. One representative Western blot from at least three independent experiments is shown in A-B. (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. (D) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).
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Figure 3: ER stress transcriptionally increases PrP levels in MCF-7 cells. One representative Western blot from at least three independent experiments is shown in A-B. (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. (D) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).

Mentions: To experimentally assess if ER stressors up-regulate PRNP gene expression in breast cancer cells, we assessed the luminal subtype breast carcinoma MCF-7 cell line, which lacks endogenous PrP expression under steady state conditions. MCF-7 cells were exposed to three different ER stressors: Golgi-disaggregating BFA, N-linked glycosylation inhibitor Tunicamycin (TM), and ER Ca-ATPase family (SERCA) inhibitor Thapsigargin (Thps). Each ER stressor increased PrP levels in a dose-dependent manner within 18 hrs of treatment (Figure 3A). PrP accumulated as immature glycosylated proteins (28 to 33 kDa) with BFA, as unglycosylated (25 kDa) protein with TM, and as unglycosylated, immature, and mature (34 to 36 kDa) glycosylated proteins with Thps. Increased BiP protein levels confirmed induction of the ER stress response with the BFA, TM and Thps treatments. The ER stress-mediated up-regulation of PrP appeared specific since serum-deprivation of MCF-7 cells for 18 hrs did not increase PrP levels (Figure 3B). ER stressors increased PrP mRNA levels within six hours of treatment in MCF-7 cells with concomitant splicing of XBP1 (sXBP1), although sXBP1 was more prominent in the BFA-treated MCF-7 cells (Figure 3C). ER stress was further confirmed by Western blots showing an increase of ERP44, GRP94, ERP72 and BiP, proteins known to be up-regulated in ER stress (Figure 3D). Furthermore, the transcriptional inhibitor actinomycin D (Act D) or the translational inhibitor, cycloheximide (CHX) strongly inhibited the ER stress-mediated increase of PrP levels and the increase of the ER stress-related protein CHOP (Figure 3E). Together, these results show that ER stress of the luminal breast carcinoma MCF-7 cell line increases PRNP gene transcription.


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 transcriptionally increases PrP levels in MCF-7 cells. One representative Western blot from at least three independent experiments is shown in A-B. (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. (D) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: ER stress transcriptionally increases PrP levels in MCF-7 cells. One representative Western blot from at least three independent experiments is shown in A-B. (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. (D) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).
Mentions: To experimentally assess if ER stressors up-regulate PRNP gene expression in breast cancer cells, we assessed the luminal subtype breast carcinoma MCF-7 cell line, which lacks endogenous PrP expression under steady state conditions. MCF-7 cells were exposed to three different ER stressors: Golgi-disaggregating BFA, N-linked glycosylation inhibitor Tunicamycin (TM), and ER Ca-ATPase family (SERCA) inhibitor Thapsigargin (Thps). Each ER stressor increased PrP levels in a dose-dependent manner within 18 hrs of treatment (Figure 3A). PrP accumulated as immature glycosylated proteins (28 to 33 kDa) with BFA, as unglycosylated (25 kDa) protein with TM, and as unglycosylated, immature, and mature (34 to 36 kDa) glycosylated proteins with Thps. Increased BiP protein levels confirmed induction of the ER stress response with the BFA, TM and Thps treatments. The ER stress-mediated up-regulation of PrP appeared specific since serum-deprivation of MCF-7 cells for 18 hrs did not increase PrP levels (Figure 3B). ER stressors increased PrP mRNA levels within six hours of treatment in MCF-7 cells with concomitant splicing of XBP1 (sXBP1), although sXBP1 was more prominent in the BFA-treated MCF-7 cells (Figure 3C). ER stress was further confirmed by Western blots showing an increase of ERP44, GRP94, ERP72 and BiP, proteins known to be up-regulated in ER stress (Figure 3D). Furthermore, the transcriptional inhibitor actinomycin D (Act D) or the translational inhibitor, cycloheximide (CHX) strongly inhibited the ER stress-mediated increase of PrP levels and the increase of the ER stress-related protein CHOP (Figure 3E). Together, these results show that ER stress of the luminal breast carcinoma MCF-7 cell line increases PRNP gene transcription.

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