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Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells.

Jorgensen E, Stinson A, Shan L, Yang J, Gietl D, Albino AP - BMC Cancer (2008)

Bottom Line: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential.Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Public Health Division, Vector Research LLC, New York, NY, USA. ejorgensen@vectorgroupltd.com

ABSTRACT

Background: Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer.

Methods: Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.

Results: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.

Conclusion: These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

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Dependence of eIF2α phosphorylation on PERK in A549 cells treated with cigarette smoke (and thapsigargin. Panel A: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. Panel B: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.
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Figure 2: Dependence of eIF2α phosphorylation on PERK in A549 cells treated with cigarette smoke (and thapsigargin. Panel A: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. Panel B: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.

Mentions: In addition to PERK, three other kinases capable of phosphorylating eIF2α (PKR, HRI, and GCN2, reviewed in reference [52]) have been identified in mammals. Although current understanding indicates that these other kinases are regulated by different stress stimuli [52], their catalytic domains are homologous. Thus, phosphorylation of eIF2α by any of these four kinases results in similar downstream events, including translation attenuation and activation of transcriptional programs that augment the cell's ability to cope with problematic conditions such as the accumulation of unfolded proteins, amino acid deprivation, or oxidative stress [52]. However, PERK-induced activation of eIF2α is believed to occur exclusively upon the induction of ER stress [11]. Consequently, in order to show that CS treatment induces ER stress, it is important to demonstrate the dependence of CS-induced phosphorylation of eIF2α phosphorylation on PERK. Thus, A549 cells were transiently transfected with a plasmid containing siRNA designed to silence PERK, and then exposed to CS or to thapsigargin as an activation control. As shown in Figure 2, PERK siRNA transfected A549 cells show notably reduced phosphorylation of eIF2α when treated with CS (Panel A) or thapsigargin (Panel B), indicating that PERK is the major effector of CS-induced phosphorylation of eIF2α in both these systems. Total eIF2α levels remained constant in all samples regardless of treatment conditions.


Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells.

Jorgensen E, Stinson A, Shan L, Yang J, Gietl D, Albino AP - BMC Cancer (2008)

Dependence of eIF2α phosphorylation on PERK in A549 cells treated with cigarette smoke (and thapsigargin. Panel A: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. Panel B: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Dependence of eIF2α phosphorylation on PERK in A549 cells treated with cigarette smoke (and thapsigargin. Panel A: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were exposed to air (lanes 1, 3, and 5) or 2R4F cigarette smoke with 35 cc puffs diluted in 250 cc air (lanes 2,4, and 6) for 20 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified. Western blots of whole cell lysates were probed with antibodies to phosphorylated eIF2α, eIF2α, and GAPDH as a loading control. Panel B: A549 cells were transfected with control siRNA (lanes 3 and 4) or PERK siRNA (lanes 5 and 6) as described in Methods. 24 h post-tranfection the cells were treated with either 1 uM thapsigargin in DMSO (lanes 2,4, and 6) or DMSO (lanes 1, 3, and 5) for the times specified.
Mentions: In addition to PERK, three other kinases capable of phosphorylating eIF2α (PKR, HRI, and GCN2, reviewed in reference [52]) have been identified in mammals. Although current understanding indicates that these other kinases are regulated by different stress stimuli [52], their catalytic domains are homologous. Thus, phosphorylation of eIF2α by any of these four kinases results in similar downstream events, including translation attenuation and activation of transcriptional programs that augment the cell's ability to cope with problematic conditions such as the accumulation of unfolded proteins, amino acid deprivation, or oxidative stress [52]. However, PERK-induced activation of eIF2α is believed to occur exclusively upon the induction of ER stress [11]. Consequently, in order to show that CS treatment induces ER stress, it is important to demonstrate the dependence of CS-induced phosphorylation of eIF2α phosphorylation on PERK. Thus, A549 cells were transiently transfected with a plasmid containing siRNA designed to silence PERK, and then exposed to CS or to thapsigargin as an activation control. As shown in Figure 2, PERK siRNA transfected A549 cells show notably reduced phosphorylation of eIF2α when treated with CS (Panel A) or thapsigargin (Panel B), indicating that PERK is the major effector of CS-induced phosphorylation of eIF2α in both these systems. Total eIF2α levels remained constant in all samples regardless of treatment conditions.

Bottom Line: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential.Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Public Health Division, Vector Research LLC, New York, NY, USA. ejorgensen@vectorgroupltd.com

ABSTRACT

Background: Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer.

Methods: Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry.

Results: We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2 alpha) or phosphorylation (i.e., phospho-eIF2 alpha) in a majority of human lung cancers.

Conclusion: These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2 alpha and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

Show MeSH
Related in: MedlinePlus