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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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Related in: MedlinePlus

Cigarette smoke inhibits XBP1 splicing. A549 cells were exposed to air (Panel A, mock treatment) or 2R4F cigarette smoke (Panel B) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh media with or without 1 uM thapsigargin or 10 μg/ml tunicamycin for the time periods specified, followed by cell lysis and RNA purification. Spliced and unspliced XBP1 mRNA was detected using PCR methodology. Spliced XBP1 is 398 base pairs and the unspliced variant is 434 base pairs. For each lane the extent of splicing was quantified as described in the Methods section and is presented in additional file 3 – Supplemental Table S3: Suppression of XBP1 splicing by cigarette smoke.
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Figure 8: Cigarette smoke inhibits XBP1 splicing. A549 cells were exposed to air (Panel A, mock treatment) or 2R4F cigarette smoke (Panel B) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh media with or without 1 uM thapsigargin or 10 μg/ml tunicamycin for the time periods specified, followed by cell lysis and RNA purification. Spliced and unspliced XBP1 mRNA was detected using PCR methodology. Spliced XBP1 is 398 base pairs and the unspliced variant is 434 base pairs. For each lane the extent of splicing was quantified as described in the Methods section and is presented in additional file 3 – Supplemental Table S3: Suppression of XBP1 splicing by cigarette smoke.

Mentions: The third UPR activation pathway is mediated by a cascade of events triggered by the dimerization and autophosphorylation of the IRE1 transmembrane protein. Under non-stressful conditions, IRE1 is inactive, however upon ER stress the conformational alteration of IRE1 via phosphorylation exposes a ribonuclease capability that removes an intron from XBP1 mRNA (denoted XBP1S), resulting in the generation of a functional protein that is a potent transcriptional regulator of genes involved in protein folding and degradation, two necessary mechanisms needed to restore ER homeostasis. The impact of CS exposure on XBP1 splicing was assessed. First, we determined the ability of A549 cells to undergo typical XBP1 splicing upon ER stress. Figure 8 (Panel A, lanes 1–4) shows that under non-stressed conditions (i.e., air exposure) only the unspliced form of XBP1 mRNA is detectable during the time-frame examined (30 min – 4 h) as expected. Figure 8 (Panel A, lanes 5–12) show that when A549 cells are exposed either to the ER stressors thapsigargin (which inhibits the ER Ca2+ pump) or tunicamycin (which blocks N-linked glycosylation), there is a significant increase in XBP1S starting at 30 minutes and continuing for up to 4 hrs post-exposure with a concomitant decrease in the unspliced form. In contrast, however, upon exposure to 2R4F CS the XBP1 mRNA remains in its unspliced form (Panel B, lanes 1–4) during the 0 – 4 h post-exposure assessment period. Indeed, when CS-exposed samples collected up to 24 h post-exposure were analyzed, no splicing of XBP1 was observed (data not shown). For the extent of splicing, see Additional File 3.


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)

Cigarette smoke inhibits XBP1 splicing. A549 cells were exposed to air (Panel A, mock treatment) or 2R4F cigarette smoke (Panel B) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh media with or without 1 uM thapsigargin or 10 μg/ml tunicamycin for the time periods specified, followed by cell lysis and RNA purification. Spliced and unspliced XBP1 mRNA was detected using PCR methodology. Spliced XBP1 is 398 base pairs and the unspliced variant is 434 base pairs. For each lane the extent of splicing was quantified as described in the Methods section and is presented in additional file 3 – Supplemental Table S3: Suppression of XBP1 splicing by cigarette smoke.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2527015&req=5

Figure 8: Cigarette smoke inhibits XBP1 splicing. A549 cells were exposed to air (Panel A, mock treatment) or 2R4F cigarette smoke (Panel B) for 20 minutes with 35 cc puffs diluted in 250 cc air. Cells were then placed in fresh media with or without 1 uM thapsigargin or 10 μg/ml tunicamycin for the time periods specified, followed by cell lysis and RNA purification. Spliced and unspliced XBP1 mRNA was detected using PCR methodology. Spliced XBP1 is 398 base pairs and the unspliced variant is 434 base pairs. For each lane the extent of splicing was quantified as described in the Methods section and is presented in additional file 3 – Supplemental Table S3: Suppression of XBP1 splicing by cigarette smoke.
Mentions: The third UPR activation pathway is mediated by a cascade of events triggered by the dimerization and autophosphorylation of the IRE1 transmembrane protein. Under non-stressful conditions, IRE1 is inactive, however upon ER stress the conformational alteration of IRE1 via phosphorylation exposes a ribonuclease capability that removes an intron from XBP1 mRNA (denoted XBP1S), resulting in the generation of a functional protein that is a potent transcriptional regulator of genes involved in protein folding and degradation, two necessary mechanisms needed to restore ER homeostasis. The impact of CS exposure on XBP1 splicing was assessed. First, we determined the ability of A549 cells to undergo typical XBP1 splicing upon ER stress. Figure 8 (Panel A, lanes 1–4) shows that under non-stressed conditions (i.e., air exposure) only the unspliced form of XBP1 mRNA is detectable during the time-frame examined (30 min – 4 h) as expected. Figure 8 (Panel A, lanes 5–12) show that when A549 cells are exposed either to the ER stressors thapsigargin (which inhibits the ER Ca2+ pump) or tunicamycin (which blocks N-linked glycosylation), there is a significant increase in XBP1S starting at 30 minutes and continuing for up to 4 hrs post-exposure with a concomitant decrease in the unspliced form. In contrast, however, upon exposure to 2R4F CS the XBP1 mRNA remains in its unspliced form (Panel B, lanes 1–4) during the 0 – 4 h post-exposure assessment period. Indeed, when CS-exposed samples collected up to 24 h post-exposure were analyzed, no splicing of XBP1 was observed (data not shown). For the extent of splicing, see Additional File 3.

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