Telomerase TemplateAntagonist GRN 163 L Disrupts Telomere Maintenance , Tumor Growth , andMetastasis of Breast Cancer

Purpose:Maintenance of telomeres by telomerase is critical for the continuing proliferation of most advanced cancer cells.Telomerase activity has been detected in the vast majority of cancer cells but not most normal cells, making the enzyme an attractive target for anticancer therapy. The aim of this study was to address the breast cancer translational potential of the novel telomerase inhibitor, GRN163L. Experimental Design: In the present study, we investigated the effects of GRN163L treatment on a panel of breast cancer cells representing different tumor subtypeswith varying genetic backgrounds, including ER+, ER , HER2+, BRCA1mutant breast tumor cells as well as doxorubicinresistant cancer cells.To investigate the in vivo effects of GRN163L, we employed a breast cancer xenograft andmetastasis model that simulates a clinical situation inwhich a patient arrives with a primary tumor that may be then treated or surgically removed. Results: GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in all breast cancer cell lines resulting in progressive telomere shortening. A mismatch control oligonucleotide showedno effect on telomerase activity andGRN163L didnot significantly affect telomere shortening in normal human mammary epithelial cells or in endothelial cells. Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases (P = 0.017) of MDA-MB-231cells in vivo after 4 weeks of treatment. Conclusions:These results show in vivo effectiveness of GRN163L inbreast cancer and support its promising clinical potential for breast cancer treatment. Breast cancer remains as the most common malignant disease in Western women. Although early detection and improved therapy of early disease has led to an overall decline in breast cancer mortality, metastatic breast cancer remains largely incurable with a median survival of 2 to 3 years. Recent genomic studies have confirmed that breast cancer is a heterogeneous disease consisting of different subtypes with potentially different survival outcomes or responses to treatment (1–3). It is therefore necessary to develop and test new molecular targets for their potential in breast cancer therapy. A hallmark of cancer is its limitless proliferative potential predominately achieved by telomere maintenance (4, 5). Telomeres are specialized structures at the end of chromosomes that protect the ends from fusion, recombination, or being recognized as uncapped DNA breaks (6–8). Human telomerase is a ribonucleoprotein complex consisting of a cellular reverse transcriptase catalytic subunit (hTERT) that uses the telomerase RNA component (hTR or hTERC) of the complex as a template for adding TTAGGG repeats to the ends of the chromosomes (reviewed in ref. 9). Telomerase may preferentially elongate the critically short telomeres, stabilize telomere lengths, and permit continued cancer cell division (10). Telomerase activity has been detected in the vast majority of human tumors cells as well as in cells of continually renewable tissues (e.g., stem cells) and germ-line cells, but not in most normal somatic cells. The observed differences of telomerase activity in tumor-derived versus normal cells, coupled with the much more rapid rate of cell division of cancer cells, resulted in the hypothesis that telomerase may represent a suitable target for specific anticancer therapies. Telomerase activity has been detected early in breast cancer progression and correlated with hTERT mRNA levels and prognosis (11–14). Furthermore, the observation Cancer Therapy: Preclinical Authors’Affiliations: Department of Medical and Molecular Genetics, Indiana University Cancer Center, Division of Hematology and Oncology, and Department of Pathology, Indiana University School of Medicine, Indianapolis, Indiana and Geron Corp., Menlo Park, California Received12/19/05; revised 2/18/06; accepted 3/9/06. Grant support: American Cancer Society grant IRG-84-002-19 and Phi Beta Psi National Sorority (B-S.Herbert), philanthropic funds to the IndianaUniversityBreast Care and Research Center, and Indiana Genomics Initiative. Indiana Genomics Initiative of Indiana University is supported in part by Lilly Endowment, Inc. The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Brittney-Shea Herbert, Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street, IB 242, Indianapolis, IN 46202-5251. Phone: 317-278-6147; Fax: 317-2741069; E-mail: brherber@iupui.edu. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2760 www.aacrjournals.org Clin Cancer Res 2006;12(10)May15, 2006 3184 Research. on May 1, 2017. © 2006 American Association for Cancer clincancerres.aacrjournals.org Downloaded from that the majority of epithelial cancers cells have significantly shorter telomeres compared with other normal cells suggests that those cancer cells represent an attractive target for antitelomerase treatments (15–20). Following this appealing scientific rationale, several classes of telomerase inhibitors targeting different sites of the telomerase complex or telomeres themselves have been evaluated (reviewed in refs. 21, 22). In particular, agents that target the 11-base template region of hTR, such as peptide nucleic acids, 2¶-O-meRNA, 2¶-O-methoxyethyl RNA, and phosphoramidate oligonucleotides, have been well studied in a variety of cancer cell lines (21–24). Furthermore, a new class of oligonucleotides targeting the hTR, N3¶!P5¶ thiophosphoramidates, have been shown to form stable duplexes with ssRNA, to be resistant to nuclease degradation, and to have both high affinity and specificity for targets (25). These N3¶!P5¶ thiophosphoramidate oligomers (such as GRN163) are effective in telomerase inhibition, telomere shortening, and inhibition of cancer cell growth (25–30). Agents that target the hTR template region act as classic, competitive enzyme inhibitors of telomerase activity and work at pharmacologic concentrations (31–33). The synthesis and properties of a new telomerase template antagonist, GRN163L, were recently reported. This lipidmodified N3¶!P5¶ thiophosphoramidate oligonucleotide is complementary to the template region of hTR and is a potent inhibitor of telomerase activity in vitro (34). GRN163L is a competitive telomerase enzyme inhibitor in that it does not target hTERT protein per se but blocks the binding of chromosomal telomere substrates to telomerase. Due to its 5¶-lipid palmitoyl domain, which increases its lipophilicity, GRN163L was shown to exhibit increased bioavailability, cellular uptake in tumors relative to nonlipidated counterparts, and it is more acid resistant than other telomerase-addressed phosphoramidate oligonucleotides (34). Recent in vivo studies in hepatoma and lung cancer suggest the translational potential for GRN163L as a novel anticancer therapeutic in these cancers (35, 36). Currently, GRN163L is in phase I/II clinical trials for chronic lymphocytic leukemia. In the present study, we investigated the effects of GRN163L as a telomerase inhibitor on telomere shortening, anchoragedependent and anchorage-independent growth, and plating efficiency of various breast cancer cell lines representing different lineages and/or genetic backgrounds. Furthermore, in the first evaluation of GRN163L using an in vivo breast xenograft tumor and metastasis model, we show that GRN163L is effective in reducing breast tumor growth and the amount of breast cancer metastases to the lung. Materials andMethods Oligonucleotides. The oligonucleotide GRN163L (5¶-Palm-TAGGGTTAGACAA-NH2-3¶) has a sequence complementary to the hTR template region. This lipid-modified N3¶!P5¶ thiophosphoramidate oligonucleotide and the 5¶-palmitoyl mismatch control oligonucleotide (5¶-Palm-TAGGTGTAAGCAA-NH2-3¶ with the mismatch bases italicized) were prepared and analyzed as described previously (34). Cell culture. A tumorigenic human mammary epithelial (HME) cell line (HME50-T) was established by infecting preimmortal HME cells from a patient with Li-Fraumeni syndrome, which contain a germ-line mutation at codon 133 in one of the two alleles of the p53 gene [Met-to-Thr (M133T)] that affects wild-type p53 protein conformation, with hTERT and H-RasV12, and then collecting clones that grew in soft agar and nude mice xenografts (34). These cells and other breast cancer cells (MCF-7, HCC1937, SKBR3, and MDA-MB231 breast carcinoma cells) were grown in DMEM (Invitrogen, Carlsbad, CA) containing 10% cosmic calf serum (HyClone, Logan, UT) and 50 Ag/mL gentamicin (Invitrogen). Nontumorigenic 21NT breast epithelial and normal HME cells were grown in MEGM (Cambrex, East Rutherford, NJ). Human endothelial progenitor cells were maintained as described (37). Population doublings were calculated as the log [(the number of cells collected) / (number of cell initially plated)] / log 2 for each passage. Treatment with GRN163L. To determine the efficacy and dose response for GRN163L in breast cancer cells, a 1:2 serial dilution series of GRN163L or its mismatch control (5.0, 2.5, 1.25, 0.625, and 0.325 Amol/L) was prepared and given to cells plated on cell culture plates. After 24 hours, 1.0 10 cells were collected for telomeric repeat amplification protocol (TRAP) assay as described below. In addition, cells were treated with GRN163L at an effective concentration (1-2.5 Amol/L, decided on by the dose-response data of the cells) for 24, 48, and 72 hours and then collected for TRAP. For long-term treatment of GRN16