Journal of the National Cancer Institute Advance Access published online on November 11, 2008
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djn365
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© The Author 2008. Published by Oxford University Press.
ARTICLES |
Effect of Disrupting Seven-in-Absentia Homolog 2 Function on Lung Cancer Cell Growth
Affiliations of authors: Department of Surgery (AUA, CHP, NRR, SEH, CD, AHT), Department of Biochemistry and Molecular Biology (RLS, AHT), Division of Pulmonary and Critical Care Medicine, Department of Medicine (CFT), Department of Oncology (JRM), Division of Epidemiology, Department of Health Sciences Research (PY), Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology (MCA), Mayo Clinic Cancer Center, Mayo Clinic College of Medicine, Rochester, MN
Correspondence to: Amy H. Tang, PhD, Mayo, Departments of Surgery and Biochemistry and Molecular Biology, Clinic College of Medicine, 200 First Street SW MS-2-85, Rochester, MN 55905 (e-mail: tang.amy{at}mayo.edu).
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 x 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-2PD 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.
| CONTEXT AND CAVEATS Prior knowledge Activating mutations in the epidermal growth factor receptor (EGFR) gene and oncogenic mutations in the K-RAS gene are among the most common genetic lesions found in non–small-cell lung cancers. A human homolog of Drosophila seven-in-absentia—SIAH-2—is a conserved downstream component of the EGFR/RAS pathway that is required for mammalian RAS signal transduction. Targeting SIAH-2 might be an effective strategy for blocking lung tumor growth and cell proliferation. Study design The effects of lentiviral expression of a dominant-negative protease-deficient mutant of SIAH-2 and of a short hairpin RNA–mediated gene knockdown were assayed in normal human lung epithelial cells, in human lung cancer cells, and in athymic nude mice. Contribution SIAH-2 deficiency in human lung cancer cell lines reduced MAPK signaling, inhibited cell proliferation, and increased apoptosis compared with SIAH-proficient cells. SIAH-2 deficiency also reduced anchorage-independent growth of lung cancer cells in soft agar, and blocked the growth of lung cancer cell–derived tumors in nude mice. Implications SIAH-2 may be a viable target for novel anti-RAS and anticancer agents aimed at inhibiting EGFR and/or RAS-mediated tumorigenesis. Limitations The lung cancer cell lines and the nude mouse cancer models may not reflect the heterogeneity and complexity of human tumors. The lentiviral vector used to deliver the anti-SIAH-2 agents into the tumor cells may induce toxicity in cancer patients. From the Editors
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Manuscript received December 7, 2007; revised August 15, 2008; accepted September 12, 2008.
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J Natl Cancer Inst 2008 100: 1561.