Activated cAMP response element binding protein is overexpressed in human mesotheliomas and inhibits apoptosis

Our plain-English summary of Activated cAMP response element binding protein is overexpressed in human mesotheliomas and inhibits apoptosis, by A. Shukla, M.W. Bosenberg, M.B. MacPherson, et al (The American Journal of Pathology, 2009).

The study

• Authors: A. Shukla, M.W. Bosenberg, M.B. MacPherson, K.J. Butnor, N.H. Heintz, H.I. Pass, M. Carbone, J.R. Testa, B.T. Mossman
• Journal: The American Journal of Pathology
• Published: 2009;175(5):2197-206
• Cited by: 43 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Little is known about the cellular mechanisms contributing to the development and chemoresistance of malignant mesothelioma (MM), an aggressive asbestos-associated tumor. A human mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells showed rapid and protracted asbestos-induced cAMP response element binding protein (CREB1) phosphorylation, which was inhibited in LP9/TERT-1 cells by small molecule inhibitors of epidermal growth factor receptor phosphorylation and protein kinase A. Asbestos increased expression of several CREB target genes (c-FOS, EGR-1, MKP1, BCL2, and MMP13) and apoptosis, which was enhanced using small interfering CREB. Human MM tissue arrays showed elevated endogenous levels of phosphorylated nuclear CREB1 as compared with reactive mesothelial hyperplasias and normal lung tissue. Significantly increased phosphorylated CREB1 and mRNA levels of BCL2, c-FOS, MMP9, and MMP13 were also observed in MM cells in vitro, which were further augmented after addition of Doxorubicin (Dox). Small interfering CREB inhibited migration of MMs, increased apoptosis by Dox, and decreased BCL2 and BCL-xL expression, suggesting a role for these molecules in CREB-induced MM survival. These data indicate that CREB1 and its target genes are up-regulated in asbestos-exposed human mesothelial cells through an epidermal growth factor receptor/protein kinase A pathway. Since activated CREB1 also is increased endogenously in human MM and modifies migration and resistance to Dox-induced apoptosis, inhibition of CREB1 may be a new strategy for MM therapy.

In plain English

This was a laboratory study using human cells and tumor tissue. The authors exposed a human peritoneal mesothelial cell line (LP9/TERT-1) and isolated human pleural mesothelial cells to crocidolite asbestos. Asbestos increased activation (phosphorylation) of a protein called CREB1, lasting 24 hours. In LP9 cells, the increase depended on two signaling pathways, called EGFR and PKA. Asbestos also raised expression of several genes controlled by CREB. After 24 hours of asbestos exposure, 44% of LP9 cells given small interfering RNA against CREB were apoptotic, compared with 28% of asbestos-exposed cells given non-targeting control RNA. Five mesothelioma cell lines showed more activated CREB than non-cancerous mesothelial cells. The chemotherapy drug doxorubicin raised it further in two of these lines. Reducing CREB increased doxorubicin-induced cell death in two mesothelioma lines and cut migration of one line (Mont) by 35%. In tissue from patients with pleural mesothelioma, 24 of 33 sections showed nuclear activated CREB. Three reactive mesothelial hyperplasias stained weakly. The authors concluded that up-regulation of activated CREB by doxorubicin is a probable mechanism of resistance to chemotherapy drugs.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory (in vitro) study of human mesothelial and mesothelioma cell lines, plus immunohistochemical staining of human mesothelioma tissue arrays
• Who was studied: Cells: LP9/TERT-1 human peritoneal mesothelial cell line; NYU474 isolated human pleural mesothelial cells; five human malignant mesothelioma cell lines, including sarcomatous Mont, epithelioid Me26, Gard and Hmeso. Tissue: three arrays containing 40 pleural mesotheliomas (18 epithelioid, 13 biphasic, 9 sarcomatoid), of which 33 could be graded, plus 7 normal lung sections and 3 reactive mesothelial hyperplasias.
• Exposure: National Institute on Environmental Health Sciences reference sample of crocidolite asbestos, added to cells at a final concentration printed as "5 g/cm2" of dish area (a micro symbol appears to have been lost in the scanned text). Mesothelioma cells were also treated with doxorubicin (Dox).
• Main results:
  • In LP9 cells and NYU474 isolated human mesothelial cells, asbestos increased CREB1 phosphorylation (reported as statistically significant), and the increase persisted for 24 hours.
  • In LP9 cells, the PKA inhibitor H89 completely abolished asbestos-induced CREB1 phosphorylation, and the EGFR inhibitor AG1478 blocked it. The authors state that asbestos activates CREB via EGFR- and PKA-dependent pathways.
  • In asbestos-exposed LP9 cells, expression of cFOS, EGR-1 and MKP-1 rose significantly at 4, 8 and 24 hours. BCL2 and MMP13 rose significantly at 24 hours.
  • All LP9 cells in this comparison were exposed to asbestos for 24 hours. Among cells given non-targeting control siRNA, 28% were apoptotic. Among cells given siRNA against CREB, 44% were apoptotic.
  • All five mesothelioma cell lines showed higher endogenous CREB activation than the untransformed human mesothelial cells (NYU474 and LP9).
  • In the Mont and Me26 mesothelioma lines, doxorubicin (0.1 to 10, units with micro symbol lost; 24 and 48 hours) increased CREB phosphorylation in a dose- and time-related way.
  • In Mont and Me26 cells, siRNA reduced CREB levels by 56% and 80% respectively. Baseline apoptosis was not affected, but siRNA against CREB significantly increased the percentage of cells undergoing doxorubicin-induced apoptosis in both lines.
  • siRNA against CREB decreased migration of Mont cells by 35% (reported as statistically significant). Similar trends were seen in Hmeso cells.
  • In Mont and Me26 cells, reducing CREB lowered expression of the prosurvival gene BCL2 over time. BCL-xL decreased only transiently, at 24 hours.
  • In human mesothelioma tissue sections, 24 of 33 (72.72%) were positive for nuclear phosphorylated CREB and 7 of 33 (21.21%) for cytoplasmic phosphorylated CREB.
  • Reactive mesothelial hyperplasias showed weak phosphorylated CREB staining. Normal lung showed only occasional staining, in alveolar type II cells.
• The authors' conclusion: The authors concluded that up-regulation of activated CREB in mesothelioma cells by doxorubicin is a probable mechanism of resistance to chemotherapy drugs. They also suggested that inhibiting CREB may be a treatment strategy.
• Limitations the authors state:
  • The antibody used to detect phosphorylated CREB also reacted with phosphorylated ATF1, another CREB family member.
  • The roles of other CREB-regulated genes in the increased apoptosis were not explored.
  • The endogenous CREB activation in mesothelioma cell lines could not be blocked by the inhibitors tested, so its mechanism was not identified.

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma, Apoptosis, Malignant Mesothelioma, Mesothelial Cells, Reactive Mesothelial Hyperplasia, Doxorubicin.

Related studies

• Extracellular signal-regulated kinase 5 and cyclic AMP response element binding protein are novel pathways inhibited by vandetanib (ZD6474) and doxorubicin in mesotheliomas — M. Sayan, A. Shukla, et al. (2014)
• Asbestos-mediated CREB phosphorylation is regulated by protein kinase A and extracellular signal-regulated kinases 1/2 — C.A. Barlow, T.F. Barrett, et al. (2007)
• Deficiency of Fyn protein is prerequisite for apoptosis induced by Src family kinase inhibitors in human mesothelioma cells — R. Eguchi, S. Kubo, et al. (2012)
• Fibroblast growth factor-10 prevents asbestos-induced alveolar epithelial cell apoptosis by a mitogen-activated protein kinase-dependent mechanism — D. Upadhyay, V. Panduri, et al. (2005)
• ERK2 is essential for the growth of human epithelioid malignant mesotheliomas — A. Shukla, J.M. Hillegass, et al. (2011)
• Distinct autophagy-apoptosis related pathways activated by Multi-walled (NM 400) and Single-walled carbon nanotubes (NIST-SRM2483) in human bronchial epithelial (16HBE14o-) cells — M. Ghosh, S. Murugadoss, et al. (2020)

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