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Noninvasive Molecular Imaging of MYC mRNA Expression in Human Breast Cancer Xenografts with a [99mTc]Peptide-Peptide Nucleic Acid-Peptide Chimera Xiaobing Tian,† Mohan R. Aruva,‡ Wenyi Qin,| Weizhu Zhu,| Edward R. Sauter,| Mathew L. Thakur,‡,§ and Eric Wickstrom†,§,* Departments of Biochemistry and Molecular Pharmacology, Radiology, and Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and Department of Surgery, University of Missouri, Columbia, Missouri 65212. Received August 28, 2004; Revised Manuscript Received October 28, 2004
Human estrogen receptor-positive breast cancer cells typically display elevated levels of Myc protein due to overexpression of MYC mRNA, and elevated insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. We hypothesized that scintigraphic detection of MYC peptide nucleic acid (PNA) probes with an IGF1 peptide loop on the C-terminus, and a [99mTc]chelator peptide on the N-terminus, could measure levels of MYC mRNA noninvasively in human IGF1R-overexpressing MCF7 breast cancer xenografts in nude mice. We prepared the chelator-MYC PNA-IGF1 peptide, as well as a 4-nt mismatch PNA control, by solid-phase synthesis. We imaged MCF7 xenografts scintigraphically and measured the distribution of [99mTc]probes by scintillation counting of dissected tissues. MCF7 xenografts in nude mice were visualized at 4 and 24 h after tail vein administration of the [99mTc]PNA probe specific for MYC mRNA, but not with the mismatch control. The [99mTc]probes distributed normally to the kidneys, livers, tumors, and other tissues. Molecular imaging of oncogene mRNAs in solid tumors with radiolabel-PNA-peptide chimeras might provide additional genetic characterization of preinvasive and invasive breast cancers.
INTRODUCTION
MYC was one of the first proto-oncogenes to be identified (1), and MYC was the first oncogene to be inhibited by antisense oligonucleotides, blocking the transition from G1 to S phase in normal peripheral blood mononuclear cells (2) and in promyelocytic leukemia cells (3). MYC mRNA and Myc protein are overexpressed in a variety of cancers, including breast (4), bladder (5), kidney (6), lung cancer (7), and lymphoma (8). MYC antisense oligonucleotides were able to ablate Myc protein levels in peripheral blood mononuclear cells of Eµmyc transgenic mice (9), delay the onset of congenital lymphoma in Eµ-myc transgenic mice (10), and delay the proliferation of Eµ-myc lymphoma cells transplanted into congenic parental mice (11). Similarly, in patients with advanced solid tumors, a phase I trial of our MYC antisense oligonucleotide sequence, combined with cisplatin, revealed no significant toxicity at therapeutic doses, and displayed clinical responses in ovarian and colorectal cancer (12). Myc is a nuclear phosphoprotein in the basic/helix-loophelix/leucine zipper family of transcription factors that * Corresponding author: Dr. Eric Wickstrom, Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, 219 Bluemle Life Sciences Building, 233 S. 10th St., Philadelphia PA 19107-5541, voice: 215-955-4578, fax: 215-9554580 or 215-923-9214,
[email protected], website: http:// tesla.jci.tju.edu. † Department of Biochemistry and Molecular Pharmacology, Thomas Jefferson University. ‡ Department of Radiology, Thomas Jefferson University. § Kimmel Cancer Center, Thomas Jefferson University. | University of Missouri.
associates with Max to activate proliferative genes (13). Myc protein accelerates cells into S phase by induction of cyclin D1 (14), which increases shortly after estrogen induction of MYC mRNA expression in mammary cells (15) via an atypical estrogen response element (16). As a corollary of MYC overexpression in breast cancers (4), MYC antisense oligonucleotides inhibited estrogenstimulated MCF7 breast cancer cell proliferation, as well as the estrogen-independent MDA-MB-231 breast cancer line (17). Correspondingly, induction of MYC expression in antiestrogen-arrested cells mimicked the effects of estrogen by reinitiating cell cycle progression (18), while constitutive overexpression of Myc protein conferred resistance to antiestrogen treatment in human MCF7 breast cancer cells (19). In the cell signaling pathway, antisense inhibition of MYC mRNA in MCF7 breast cancer cells inhibited cyclin D1 expression and cell proliferation (15, 20), even though MYC gene induction did not increase cyclin D1 expression. Thus, while cyclin D1 expression is dependent on Myc protein, MYC gene expression is not limited by cyclin D1. Clinical examination and mammography, the currently accepted breast cancer screening methods, miss almost half of breast cancers in women younger than 40 years, approximately one-quarter of cancers in women aged 4049 years, and one-fifth of cancers in women over 50 years old (21). The breast is the most common site of noncutaneous cancer in US women (22). Moreover, if an abnormality is found, an invasive diagnostic procedure must still be performed to determine if the breast contains atypia or cancer. Despite recent improvements in BIRADS standardization of mammography (including the use of two views of the breast rather than one, spot compression, and digital images) that have improved sensi-
10.1021/bc0497923 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/31/2004
Noninvasive MYC mRNA Imaging
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Figure 1. [99mTc]AcGlyD(Ala)GlyGlyAba-MYC PNA-AEEA-D(CysSerLysCys), WT4219, designed to bind to the receptor for IGF1, internalize, and hybridize with MYC mRNA. Scintigraphic imaging of γ-particles emitted upon decay of 99mTc might identify sites of high MYC mRNA expression.
tivity and specificity (23), 66-85% of women with suspicious mammograms who undergo surgery are found to have a benign abnormality (24). In many other patients recently determined to have a benign mammogram, clinical examination reveals a mass found to be malignant. A noninvasive method to evaluate women for the presence of atypia and/or cancer of the breast would be very beneficial. SestaMIBI has demonstrated utility in imaging lesions in a dense breast, but is limited by the fact that other cell types with high mitochondrial activity avidly take up the tracer, leading to false positive results in breasts with inflammation/infection (25), false negatives due to sensitivity limits of the technology, in which tumors
