Journal of the National Cancer Institute Advance Access published online on January 8, 2008
JNCI Journal of the National Cancer Institute, doi:10.1093/jnci/djm288
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Kidney-Targeted Birt-Hogg-Dubé Gene Inactivation in a Mouse Model: Erk1/2 and Akt-mTOR Activation, Cell Hyperproliferation, and Polycystic Kidneys
Affiliations of authors: Urologic Oncology Branch (MB, SBH, MBW, BZ, WML, LSS) and Mouse Cancer Genetics Program (LT), Center for Cancer Research, Pathology/Histotechnology Laboratory (DCH), Basic Research Program (ES, LSS), and Small Animal Imaging Program, Laboratory Animal Sciences Program (LI), SAIC-Frederick, Inc, and Data Management Services, Inc (WGA, RL), National Cancer Institute-Frederick, Frederick, MD; Urologic Oncology Branch (MB, SBH, MBW, BZ, WML, LSS) and Molecular Imaging Program (MB, PC), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; Department of Pathology, Kochi Medical School, Kochi, Japan (MF);Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX (VP, PI); Department of Urology and Molecular Genetics, Yokohama City University Graduate School of Medicine, Yokohama, Japan (MY)
Correspondence to: Laura S. Schmidt, PhD, Urologic Oncology Branch, National Cancer Institute-Frederick, Bldg 560, Rm 12-69, Frederick, MD 21702 (e-mail: schmidtl{at}ncifcrf.gov).
Background: Patients with Birt-Hogg-Dubé (BHD) syndrome harbor germline mutations in the BHD tumor suppressor gene that are associated with an increased risk for kidney cancer. BHD encodes folliculin, a protein that may interact with the energy- and nutrient-sensing 5'-AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathways.
Methods: We used recombineering methods to generate mice with a conditional BHD allele and introduced the cadherin 16 (KSP)–Cre transgene to target BHD inactivation to the kidney. Kidney cell proliferation was measured by BrdU incorporation and phospho-histone H3 staining. Kidney weight data were analyzed with Wilcoxon’s rank-sum, Student's t, and Welch's t tests. Hematoxylin and eosin staining and immunoblot analysis and immunohistochemistry of cell cycle and signaling proteins were performed on mouse kidney cells and tissues. BHD knockout mice and kidney cells isolated from BHD knockout and control mice were treated with the mTOR inhibitor rapamycin. Mouse survival was evaluated by Kaplan-Meier analyses. All statistical tests were two-sided.
Results: BHD knockout mice developed enlarged polycystic kidneys and died from renal failure by 3 weeks of age. Targeted BHD knockout led to the activation of Raf-extracellular signal-regulated protein kinase (Erk)1/2 and Akt-mTOR pathways in the kidneys and increased expression of cell cycle proteins and cell proliferation. Rapamycin-treated BHD knockout mice had smaller kidneys than buffer-treated BHD knockout mice had (n = 4–6 mice per group, relative kidney/body weight ratios, mean = 4.64% vs 12.2%, difference = 7.6%, 95% confidence interval = 5.2% to 10.0%; P < .001) and longer median survival time (n = 4–5 mice per group, 41.5 vs 23 days; P = .0065 ).
Conclusions: Homozygous loss of BHD may initiate renal tumorigenesis in the mouse. The conditional BHD knockout mouse may be a useful research model for dissecting multistep kidney carcinogenesis, and rapamycin may be considered as a potential treatment for Birt-Hogg-Dubé syndrome.
| CONTEXT AND CAVEATS Prior knowledge Patients with Birt-Hogg-Dubé (BHD) syndrome who carry inactivating mutations in both copies of the BHD tumor suppressor gene have an increased risk for kidney cancer. Folliculin, the protein encoded by the BHD gene, may interact with the 5’AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling pathway, a key regulator of cell growth and proliferation. Study design Kidney-targeted BHD knockout mouse model to examine how loss of BHD affects kidney function, the potential pathways involved, and therapies that target those pathways. Contribution BHD knockout mice died from renal failure and had enlarged cystic kidneys due to hyperproliferation and activation of the Erk1/2 and mTOR pathways. Treatment with the mTOR inhibitor rapamycin reduced kidney size and prolonged survival. Implications Loss of both copies of the BHD gene may lead to renal tumorigenesis in the mouse. The model used in this study may also be useful to determine the mechanisms of kidney carcinogenesis. Limitations The mouse model used in this study may not precisely recapitulate BHD syndrome in humans; additional genetic changes may be required for progression to kidney carcinogenesis.
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The content of this publication does not necessarily reflect the views or policies of the Department of Heath and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.
The NCI-Frederick is accredited by Association for Assessment and Accreditation of Laboratory Animal Care International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals (National Research Council, 1996, National Academy Press, Washington, DC).
Manuscript received July 19, 2007; revised October 24, 2007; accepted November 26, 2007.
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