Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth

Atique U. Ahmed, Rebecca L. Schmidt, Cheol Hong Park, Nanette R. Reed, Shayla E. Hesse, Charles F. Thomas, Julian R Molina, Claude Deschamps, Ping Yang, Marie C. Aubry, Amy H. Tang

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Background: Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia - SIAH-1 and SIAH-2 - are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth. Methods: The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2PD] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase- mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 × 106 cells each at the dorsal left and right scapular areas). All statistical tests were two-sided. Results: SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P <. 001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2PD vs control, 30.1% vs 0.0%, difference = 30.1%, 95% confidence interval [CI] = 23.1% to 37.0%, P <. 001; SIAH-2-shRNA#6 vs control shRNA, 27.9% vs 0.0%, difference = 27.9%, 95% CI = 23.1% to 32.6%, P <. 001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2PD vs control, 124.7 vs 57.3, difference = 67.3, 95% CI = 49.4 to 85.3, P <. 001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95% CI = 69.8 to 115.5, P <. 001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2 PD infected vs uninfected, 191.0 vs 558.5 mm3, difference = 367.5 mm3, 95% CI = 237.6 to 497.4 mm3, P <. 001; SIAH-2PD infected vs control infected, 191.0 vs 418.3 mm3, difference = 227.5 mm3, 95% CI = 87.4 to 367.1 mm3, P =. 003; mean resected tumor weight: SIAH-2PD infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95% CI = 0.23 to 0.50 g, P <. 001; SIAH-2PD infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95% CI = 0.04 to 0.31 g, P =. 016). Conclusions: SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.

Original languageEnglish (US)
Pages (from-to)1606-1629
Number of pages24
JournalJournal of the National Cancer Institute
Volume100
Issue number22
DOIs
StatePublished - Nov 2008

Fingerprint

Lung Neoplasms
Confidence Intervals
Small Interfering RNA
Growth
Epidermal Growth Factor Receptor
Nude Mice
In Situ Nick-End Labeling
Tumor Burden
Agar
Carcinogenesis
Lentivirus Infections
Cell Proliferation
Apoptosis
Gene Knockdown Techniques
Uridine Triphosphate
Injections
Ubiquitin-Protein Ligases
DNA Nucleotidylexotransferase
Immunoblotting
Antineoplastic Agents

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Ahmed, A. U., Schmidt, R. L., Park, C. H., Reed, N. R., Hesse, S. E., Thomas, C. F., ... Tang, A. H. (2008). Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. Journal of the National Cancer Institute, 100(22), 1606-1629. https://doi.org/10.1093/jnci/djn365

Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. / Ahmed, Atique U.; Schmidt, Rebecca L.; Park, Cheol Hong; Reed, Nanette R.; Hesse, Shayla E.; Thomas, Charles F.; Molina, Julian R; Deschamps, Claude; Yang, Ping; Aubry, Marie C.; Tang, Amy H.

In: Journal of the National Cancer Institute, Vol. 100, No. 22, 11.2008, p. 1606-1629.

Research output: Contribution to journalArticle

Ahmed, AU, Schmidt, RL, Park, CH, Reed, NR, Hesse, SE, Thomas, CF, Molina, JR, Deschamps, C, Yang, P, Aubry, MC & Tang, AH 2008, 'Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth', Journal of the National Cancer Institute, vol. 100, no. 22, pp. 1606-1629. https://doi.org/10.1093/jnci/djn365
Ahmed, Atique U. ; Schmidt, Rebecca L. ; Park, Cheol Hong ; Reed, Nanette R. ; Hesse, Shayla E. ; Thomas, Charles F. ; Molina, Julian R ; Deschamps, Claude ; Yang, Ping ; Aubry, Marie C. ; Tang, Amy H. / Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth. In: Journal of the National Cancer Institute. 2008 ; Vol. 100, No. 22. pp. 1606-1629.
@article{b38f7777411e45f18dea98c60feb9ae0,
title = "Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth",
abstract = "Background: Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia - SIAH-1 and SIAH-2 - are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth. Methods: The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2PD] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase- mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 × 106 cells each at the dorsal left and right scapular areas). All statistical tests were two-sided. Results: SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P <. 001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2PD vs control, 30.1{\%} vs 0.0{\%}, difference = 30.1{\%}, 95{\%} confidence interval [CI] = 23.1{\%} to 37.0{\%}, P <. 001; SIAH-2-shRNA#6 vs control shRNA, 27.9{\%} vs 0.0{\%}, difference = 27.9{\%}, 95{\%} CI = 23.1{\%} to 32.6{\%}, P <. 001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2PD vs control, 124.7 vs 57.3, difference = 67.3, 95{\%} CI = 49.4 to 85.3, P <. 001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95{\%} CI = 69.8 to 115.5, P <. 001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2 PD infected vs uninfected, 191.0 vs 558.5 mm3, difference = 367.5 mm3, 95{\%} CI = 237.6 to 497.4 mm3, P <. 001; SIAH-2PD infected vs control infected, 191.0 vs 418.3 mm3, difference = 227.5 mm3, 95{\%} CI = 87.4 to 367.1 mm3, P =. 003; mean resected tumor weight: SIAH-2PD infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95{\%} CI = 0.23 to 0.50 g, P <. 001; SIAH-2PD infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95{\%} CI = 0.04 to 0.31 g, P =. 016). Conclusions: SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.",
author = "Ahmed, {Atique U.} and Schmidt, {Rebecca L.} and Park, {Cheol Hong} and Reed, {Nanette R.} and Hesse, {Shayla E.} and Thomas, {Charles F.} and Molina, {Julian R} and Claude Deschamps and Ping Yang and Aubry, {Marie C.} and Tang, {Amy H.}",
year = "2008",
month = "11",
doi = "10.1093/jnci/djn365",
language = "English (US)",
volume = "100",
pages = "1606--1629",
journal = "Journal of the National Cancer Institute",
issn = "0027-8874",
publisher = "Oxford University Press",
number = "22",

}

TY - JOUR

T1 - Effect of disrupting seven-in-absentia homolog 2 function on lung cancer cell growth

AU - Ahmed, Atique U.

AU - Schmidt, Rebecca L.

AU - Park, Cheol Hong

AU - Reed, Nanette R.

AU - Hesse, Shayla E.

AU - Thomas, Charles F.

AU - Molina, Julian R

AU - Deschamps, Claude

AU - Yang, Ping

AU - Aubry, Marie C.

AU - Tang, Amy H.

PY - 2008/11

Y1 - 2008/11

N2 - Background: Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia - SIAH-1 and SIAH-2 - are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth. Methods: The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2PD] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase- mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 × 106 cells each at the dorsal left and right scapular areas). All statistical tests were two-sided. Results: SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P <. 001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2PD vs control, 30.1% vs 0.0%, difference = 30.1%, 95% confidence interval [CI] = 23.1% to 37.0%, P <. 001; SIAH-2-shRNA#6 vs control shRNA, 27.9% vs 0.0%, difference = 27.9%, 95% CI = 23.1% to 32.6%, P <. 001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2PD vs control, 124.7 vs 57.3, difference = 67.3, 95% CI = 49.4 to 85.3, P <. 001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95% CI = 69.8 to 115.5, P <. 001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2 PD infected vs uninfected, 191.0 vs 558.5 mm3, difference = 367.5 mm3, 95% CI = 237.6 to 497.4 mm3, P <. 001; SIAH-2PD infected vs control infected, 191.0 vs 418.3 mm3, difference = 227.5 mm3, 95% CI = 87.4 to 367.1 mm3, P =. 003; mean resected tumor weight: SIAH-2PD infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95% CI = 0.23 to 0.50 g, P <. 001; SIAH-2PD infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95% CI = 0.04 to 0.31 g, P =. 016). Conclusions: SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.

AB - Background: Hyperactivated epidermal growth factor receptor (EGFR) and/or RAS signaling drives cellular transformation and tumorigenesis in human lung cancers, but agents that block activated EGFR and RAS signaling have not yet been demonstrated to substantially extend patients' lives. The human homolog of Drosophila seven-in-absentia - SIAH-1 and SIAH-2 - are ubiquitin E3 ligases and conserved downstream components of the RAS pathway that are required for mammalian RAS signal transduction. We examined whether inhibiting SIAH-2 function blocks lung cancer growth. Methods: The antiproliferative and antitumorigenic effects of lentiviral expression of anti-SIAH-2 molecules (ie, a dominant-negative protease-deficient mutant of SIAH-2 [SIAH-2PD] and short hairpin RNA [shRNA]-mediated gene knockdown against SIAH-2) were assayed in normal human lung epithelial BEAS-2B cells and in human lung cancer BZR, A549, H727, and UMC11 cells by measuring cell proliferation rates, by assessing MAPK and other activated downstream components of the RAS pathway by immunoblotting, assessing apoptosis by terminal deoxynucleotidyltransferase- mediated UTP end-labeling (TUNEL) assay, quantifying anchorage-independent cell growth in soft agar, and assessing A549 cell-derived tumor growth in athymic nude mice (groups of 10 mice, with two injections of 1 × 106 cells each at the dorsal left and right scapular areas). All statistical tests were two-sided. Results: SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling and statistically significantly inhibited cell proliferation compared with those in SIAH-proficient cells (P <. 001) and increased apoptosis (TUNEL-positive A549 cells 3 days after lentivirus infection: SIAH-2PD vs control, 30.1% vs 0.0%, difference = 30.1%, 95% confidence interval [CI] = 23.1% to 37.0%, P <. 001; SIAH-2-shRNA#6 vs control shRNA, 27.9% vs 0.0%, difference = 27.9%, 95% CI = 23.1% to 32.6%, P <. 001). SIAH-2 deficiency also reduced anchorage-independent growth of A549 cells in soft agar (mean number of colonies: SIAH-2PD vs control, 124.7 vs 57.3, difference = 67.3, 95% CI = 49.4 to 85.3, P <. 001; shRNA-SIAH-2#6 vs shRNA control: 27.0 vs 119.7, difference = 92.7, 95% CI = 69.8 to 115.5, P <. 001), and blocked the growth of A549 cell-derived tumors in nude mice (mean tumor volume on day 36 after A549 cell injection: SIAH-2 PD infected vs uninfected, 191.0 vs 558.5 mm3, difference = 367.5 mm3, 95% CI = 237.6 to 497.4 mm3, P <. 001; SIAH-2PD infected vs control infected, 191.0 vs 418.3 mm3, difference = 227.5 mm3, 95% CI = 87.4 to 367.1 mm3, P =. 003; mean resected tumor weight: SIAH-2PD infected vs uninfected, 0.12 vs 0.48 g, difference = 0.36 g, 95% CI = 0.23 to 0.50 g, P <. 001; SIAH-2PD infected vs control infected, 0.12 vs 0.29 g, difference = 0.17 g, 95% CI = 0.04 to 0.31 g, P =. 016). Conclusions: SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis.

UR - http://www.scopus.com/inward/record.url?scp=56749097004&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=56749097004&partnerID=8YFLogxK

U2 - 10.1093/jnci/djn365

DO - 10.1093/jnci/djn365

M3 - Article

C2 - 19001609

AN - SCOPUS:56749097004

VL - 100

SP - 1606

EP - 1629

JO - Journal of the National Cancer Institute

JF - Journal of the National Cancer Institute

SN - 0027-8874

IS - 22

ER -