Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk

doi:10.1073/pnas.0710359105

Enhanced sensitivity to IGF-II signaling links loss of imprinting of IGF2 to increased cell proliferation and tumor risk

*Center for Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205;
^†Department of Biomedical Engineering, Johns Hopkins University School of Engineering, Clark 208C, 3400 North Charles, Street, Baltimore, MD 21218;
^‡Department of Pathology, Johns Hopkins University School of Medicine, CRB II/343, 1550 Orleans Street, Baltimore, MD 21231;
^§Department of Development and Genetics, Uppsala University, Norbyvagen 18A, Uppsala SE-752 36, Sweden;
^¶Novartis Institute for Biomedical Research, Klybeckstrasse 141, Basel CH-4002, Switzerland; and
^‖National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, MD 21224

Communicated by Bert Vogelstein, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, November 1, 2007 (received for review October 25, 2007)

Abstract

Loss of imprinting (LOI) of the insulin-like growth factor-II gene (IGF2), leading to abnormal activation of the normally silent maternal allele, is a common human epigenetic population variant associated with a 5-fold increased frequency of colorectal neoplasia. Here, we show first that LOI leads specifically to increased expression of proliferation-related genes in mouse intestinal crypts. Surprisingly, LOI(+) mice also have enhanced sensitivity to IGF-II signaling, not simply increased IGF-II levels, because in vivo blockade with NVP-AEW541, a specific inhibitor of the IGF-II signaling receptor, showed reduction of proliferation-related gene expression to levels half that seen in LOI(−) mice. Signal transduction assays in microfluidic chips confirmed this enhanced sensitivity with marked augmentation of Akt/PKB signaling in LOI(+) cells at low doses of IGF-II, which was reduced in the presence of the inhibitor to levels below those found in LOI(−) cells, and was associated with increased expression of the IGF1 and insulin receptor genes. We exploited this increased IGF-II sensitivity to develop an in vivo chemopreventive strategy using the azoxymethane (AOM) mutagenesis model. LOI(+) mice treated with AOM showed a 60% increase in premalignant aberrant crypt foci (ACF) formation over LOI(−) mice. In vivo IGF-II blockade with NVP-AEW541 abrogated this effect, reducing ACF to a level 30% lower even than found in exposed LOI(−) mice. Thus, LOI increases cancer risk in a counterintuitive way, by increasing the sensitivity of the IGF-II signaling pathway itself, providing a previously undescribed epigenetic chemoprevention strategy in which cells with LOI are “IGF-II addicted” and undergo reduced tumorigenesis in the colon upon IGF-II pathway blockade.

Footnotes

**To whom correspondence may be addressed. E-mail: afeinberg{at}jhu.edu or alev{at}jhu.edu
Author contributions: A.K. and C.J.W. contributed equally to this work; D.L.L., M.S.H.K., A.L., and A.P.F. designed research; A.K., C.J.W., W.T., P.O., B.W., C.A.I.-D., R.A., and A.A.S. performed research; R.C., R.O., M.A.P., D.L.L., and M.S.H.K. contributed new reagents/analytic tools; A.K., C.J.W., W.T., P.O., B.W., C.A.I.-D., M.A.P., A.A.S., M.S.H.K., A.L., and A.P.F. analyzed data; and A.K., A.L., and A.P.F. wrote the paper
Conflict of interest statement: R.A. and M.A.P. (Novartis, Inc.) have a proprietary interest in NVP-AEW541. The Johns Hopkins University and the National Institutes of Health have filed a patent on data presented in this manuscript.
Data deposition: The microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) Database, www.ncbi.nlm.nih.gov/geo (accession no. GSE8583).
This article contains supporting information online at www.pnas.org/cgi/content/full/0710359105/DC1.