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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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Expression of BiP, XBP1, and grp94 in CS-treated human lung cells. NHBE cells were exposed to air (Mock), 2R4F cigarette smoke with 35 cc puffs diluted in 500 cc air (2R4F), or Brand B cigarette smoke with 35 cc puffs diluted in 500 cc air for 15 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified prior to RNA extraction. Four separate microarray representing four separate samples were used to analyze each condition. Mean intensity values are shown. * = p < 0.01 when compared to Mock.
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Figure 5: Expression of BiP, XBP1, and grp94 in CS-treated human lung cells. NHBE cells were exposed to air (Mock), 2R4F cigarette smoke with 35 cc puffs diluted in 500 cc air (2R4F), or Brand B cigarette smoke with 35 cc puffs diluted in 500 cc air for 15 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified prior to RNA extraction. Four separate microarray representing four separate samples were used to analyze each condition. Mean intensity values are shown. * = p < 0.01 when compared to Mock.

Mentions: In addition to PERK, a second UPR effector arm is activated by proteolytic cleavage of the 90 kDa ATF6 protein and release of a functional 50 kDa transcription factor. Our initial analysis indicated that the basal level of ATF6 expression in A549 cells was below detectable levels (data not shown). Thus, in order to assess the effect of CS on ATF6 cleavage we constructed a full length ATF6/pCMV6 expression plasmid and transfected it into A549 cells which were then exposed to CS. Since robust detection of the 50 kDa end product is difficult as it is unstable and rapidly degraded [59], we monitored the disappearance of the 90 kDa ATF6 protein to assess its activation. Figure 4 (Panel A) shows that treatment of ATF6-transfected A549 cells with DTT, a potent inducer of ER-stress and the UPR due to its disruption of intramolecular disulfide bonds, results in the disappearance of the 90 kDa protein by 1 hr. Similarly, Figure 4 (Panel B) shows that treatment of ATF6-transfected A549 cells with CS also results in disappearance of ATF6 starting at 2 h post-CS exposure and continuing to at least 4 h post-exposure. The reduction in the 90 kDa ATF6 protein could also be due to increased degradation or reduced synthesis. However, when the array data was assessed to determine changes in the levels of three genes whose transcription is dependent on activated ATF6 (i.e., BiP, XBP1, and grp94) [60], we found that both XBP1 and BiP transcript levels are elevated in response to CS treatment (Figure 5). Thus, these data support the conclusion that CS-treatment activates the ATF6-signaling pathway.


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)

Expression of BiP, XBP1, and grp94 in CS-treated human lung cells. NHBE cells were exposed to air (Mock), 2R4F cigarette smoke with 35 cc puffs diluted in 500 cc air (2R4F), or Brand B cigarette smoke with 35 cc puffs diluted in 500 cc air for 15 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified prior to RNA extraction. Four separate microarray representing four separate samples were used to analyze each condition. Mean intensity values are shown. * = p < 0.01 when compared to Mock.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Expression of BiP, XBP1, and grp94 in CS-treated human lung cells. NHBE cells were exposed to air (Mock), 2R4F cigarette smoke with 35 cc puffs diluted in 500 cc air (2R4F), or Brand B cigarette smoke with 35 cc puffs diluted in 500 cc air for 15 minutes. Cells were then placed in fresh medium and returned to the incubator for the time periods specified prior to RNA extraction. Four separate microarray representing four separate samples were used to analyze each condition. Mean intensity values are shown. * = p < 0.01 when compared to Mock.
Mentions: In addition to PERK, a second UPR effector arm is activated by proteolytic cleavage of the 90 kDa ATF6 protein and release of a functional 50 kDa transcription factor. Our initial analysis indicated that the basal level of ATF6 expression in A549 cells was below detectable levels (data not shown). Thus, in order to assess the effect of CS on ATF6 cleavage we constructed a full length ATF6/pCMV6 expression plasmid and transfected it into A549 cells which were then exposed to CS. Since robust detection of the 50 kDa end product is difficult as it is unstable and rapidly degraded [59], we monitored the disappearance of the 90 kDa ATF6 protein to assess its activation. Figure 4 (Panel A) shows that treatment of ATF6-transfected A549 cells with DTT, a potent inducer of ER-stress and the UPR due to its disruption of intramolecular disulfide bonds, results in the disappearance of the 90 kDa protein by 1 hr. Similarly, Figure 4 (Panel B) shows that treatment of ATF6-transfected A549 cells with CS also results in disappearance of ATF6 starting at 2 h post-CS exposure and continuing to at least 4 h post-exposure. The reduction in the 90 kDa ATF6 protein could also be due to increased degradation or reduced synthesis. However, when the array data was assessed to determine changes in the levels of three genes whose transcription is dependent on activated ATF6 (i.e., BiP, XBP1, and grp94) [60], we found that both XBP1 and BiP transcript levels are elevated in response to CS treatment (Figure 5). Thus, these data support the conclusion that CS-treatment activates the ATF6-signaling pathway.

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