The Telomerase Template Antagonist GRN163L Alters MDA-MB-231 Breast Cancer Cell Morphology, Inhibits Growth, and Augments the Effects of Paclitaxel

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Published OnlineFirst June 9, 2009; DOI: 10.1158/1535-7163.MCT-08-1188 Published Online First on June 9, 2009 as 10.1158/1535-7163.MCT-08-1188

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The telomerase template antagonist GRN163L alters MDA-MB-231 breast cancer cell morphology, inhibits growth, and augments the effects of paclitaxel

Erin M. Goldblatt,1 Erin R. Gentry,1,2 These data support a rationale for potentially combining Melanie J. Fox,1,2 Sergei M. Gryaznov,4 GRN163L with paclitaxel for the treatment of breast can- Changyu Shen,3 and Brittney-Shea Herbert1,2 cer in the clinical setting. [Mol Cancer Ther 2009;8(7): OF1–9] 1Department of Medical and Molecular Genetics, 2Indiana University Melvin and Bren Simon Cancer Center, and 3Division of Biostatistics, Indiana University School of Medicine, Indianapolis, Introduction 4 Indiana; and Geron Corporation, Menlo Park, California Breast cancer is one of the most common malignancies among women. Typically after surgical removal of the tu- Abstract mor mass, breast cancer patients are treated with radiation and/or chemotherapeutic drugs that lack selectivity, and re- Telomeres are repetitive (TTAGGG)n DNA sequences ∼ found at the end of chromosomes that protect the ends sistance often develops quickly. Additionally, 20% of from recombination, end to end fusions, and recognition women with a history of early breast cancer will develop as damaged DNA. Telomerase activity can be detected metastatic disease. Metastatic breast cancer, although treat- in 85% to 90% of human tumors, which stabilizes telo- able, is largely incurable with a low number of patients achieving long-term survival after standard chemotherapy meres to prevent apoptosis or cellular senescence. Previ- ∼ ous reports showed the efficacy of the novel telomerase (1). Because metastatic tumors are responsible for 90% of template antagonist, GRN163L, as a potential anticancer cancer-related deaths, the development of new treatment re- agent. The objective of the present study was to elucidate gimens that reduce metastasis is highly important in cancer the molecular effects of GRN163L in MDA-MB-231 breast therapy. Anticancer therapies aim to prolong survival, con- cancer cells and to determine whether GRN163L could be trol symptoms, and maintain the quality of life for breast used in mechanism-based combination therapy for breast cancer patients (2, 3). cancer. We observed that GRN163L reduced MDA-MB- Telomerase activity can be detected in 85% to 90% of hu- 231 growth rates without a significant effect on breast man tumors, but not in the majority of normal somatic cells, cancer cell viability within the first 14 days in vitro. In ad- making telomerase an attractive target for cancer therapy dition, GRN163L altered cell morphology, actin filament (4). Telomerase inhibition results in the erosion of telomeric DNA, genomic instability, and eventually to apoptosis or organization, and focal adhesion formation in MDA-MB- – in vitro 231 cells. Importantly, the cellular response to GRN163L cellular senescence (5 7). Previous studies have significantly augmented the effects of the microtubule shown that the telomerase template antagonist GRN163L stabilizer paclitaxel in MDA-MB-231 breast cancer cell effectively inhibits the tumorigenicity of breast cancer cells within 2 weeks. In addition, GRN163L reduces human growth in vitro and in vivo compared with paclitaxel alone in vivo or a mismatch control oligonucleotide plus paclitaxel. Fur- breast tumor volume and metastases within 30days thermore, in vitro MDA-MB-231 invasive potential was of the treatment with GRN163L (8). This reduction in metas- significantly inhibited with GRN163L and paclitaxel. tases may be explained by an inhibition of migration, adherence, or growth at distant sites by GRN163L (8, 9). Previous reports have also shown that inhibiting telomerase activity Received 12/17/08; revised 3/16/09; accepted 4/5/09; alone or in combination with current therapeutic techniques published OnlineFirst 6/9/09. can inhibit tumor cell growth in vitro without significant ad- Grant support: Indiana University School of Medicine Translational verse effects on normal cells (7, 8, 10–12). Research Predoctoral Fellowship (E.M. Goldblatt), Mary Kay Ash Microtubules are the major components of the cytoskele- Charitable Foundation, Phi Beta Psi National Sorority, American Cancer Society IRG-84-002-19, the Indiana University Simon Cancer Center, ton. Microtubules are vital for the maintenance of cell shape, Geron Corporation, and the Indiana Genomics Initiative. Indiana Genomics signaling, proliferation, and migration, making them an im- Initiative of Indiana University is supported in part by Lilly Endowment, Inc. portant target for anticancer drugs (13, 14). Paclitaxel is one of The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked the most extensively used anticancer agents, with clinical ef- advertisement in accordance with 18 U.S.C. Section 1734 solely to ficacy in a wide range of cancers (3, 15). Paclitaxel acts to sta- indicate this fact. bilize microtubules, thus inhibiting cell proliferation at the Requests for reprints: Brittney-Shea Herbert, Department of Medical and metaphase/anaphase boundary (16–18). However, the suc- Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN 46202-5251. Phone: 317-278-6147; Fax: 317-274-1069. cess of paclitaxel in the clinical setting can often be tempered E-mail: [email protected] by drug resistance, which severely limits the effectiveness Copyright © 2009 American Association for Cancer Research. of chemotherapy. Although the exact mechanisms remain doi:10.1158/1535-7163.MCT-08-1188 unclear, this phenomenon is considered to be mediated by

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OF2 Synergism between Telomerase Antagonists and Paclitaxel

altered drug uptake, variations in tubulin structure, and eva- Western Immunoblotting sion of apoptotic pathways (19). Cells were harvested after treatment and lysed in buffer The overall goal of the present study was to determine containing 1M Tris (pH 7.0), 2% SDS, and 5% sucrose. the cellular response to GRN163L in MDA-MB-231 breast Protein concentration was quantified using the Pierce BCA cancer cells to understand how this compound may be used Protein Assay according to manufacturer's instructions. to augment breast cancer therapy. We observed that Fifty-microgram protein lysate was subjected to SDS-PAGE GRN163L induced a rapid change in cellular architecture, followed by transfer to polyvinylidene difluoride mem- leading to the hypothesis that GRN163L can augment the brane. Blocking and antibody incubation were done in 5% effects of the microtubule stabilizer paclitaxel in reducing milk in PBS containing 0.05% Tween 20. Membranes were the cell growth of breast cancer cells. The combined treat- exposed to X-ray film using the ECL Western Blotting Sub- ment of nmol/L/sub-nmol/L concentrations of GRN163L strate (Pierce). Antibodies used in this study included the and paclitaxel resulted in a significant synergistic combina- following: anti–β-tubulin and anti–γ-tubulin (Sigma), anti- tion index (CI) in reducing the cellular proliferation and in- Pyk2 and anti-FAK (Upstate Cell Signaling Solutions), and vasive potential of MDA-MB-231 breast cancer cells. glyceraldehyde-3-phosphate dehydrogenase (Chemicon In- Furthermore, this reduction in MDA-MB-231 breast cancer ternational). Densitometry levels for each blot were deter- cell growth by combination treatment suggests a potential mined using glyceraldehyde-3-phosphate dehydrogenase use for GRN163L in adjuvant therapy for breast cancer. as a loading control; relative intensity levels for each sample were plotted and normalized to untreated samples. Cell Growth and Viability Assays to Determine the CI Materials and Methods Cells (5 × 104) were plated in triplicate with 1:2 serial Cell Culture and Reagents dilutions of GRN163L (0.005–1.25 μmol/L) or paclitaxel MDA-MB-231 breast cancer cells, MDA-087 human mam- (0.5–12.5 nmol/L) in culture media as monotherapy, or com- mary stromal cells, and BJ foreskin fibroblasts were cultured bined in which the two agents were at a 1:10ratio of each in DMEM media containing 10% cosmic calf serum (Hy- other (paclitaxel/GRN163L). Cells were collected after 5 d Clone). Nontumorigenic MCF10A breast epithelial cells to obtain the number of viable cells as measured by the try- were cultured in supplemented MEGM (Cascade Biologi- pan blue exclusion assay. CalcuSyn software (Biosoft) was cals). The lipid-modified telomerase template antagonist used to determine the combined effect of multiple drug GRN163L and its mismatch control oligonucleotide were treatment via the Chou and Talalay method (21). A CI value prepared as previously described (6). Paclitaxel was pur- of <1.0denotes synergistic interaction (more than additive), chased from Sigma and dissolved as a 10mmol/L stock a CI of 1.0shows summation (additive), and a CI of >1.0 in DMSO. indicates antagonistic interaction (less than additive). Telomerase Activity Assay Colony Formation Assays Telomerase activity was measured using the PCR-based MDA-MB-231 cells were treated with 40to 80nmol/L Telomeric Repeat Amplification Protocol as previously de- GRN163L/mismatch control oligonucleotides, media only, scribed (6, 20). or combination of 40nmol/L GRN163L plus 4 nmol/L pac- Cell Growth and Viability Assays litaxel for 5 d before collection. Cells (2 × 102)fromeach To determine the effect of GRN163L on cell growth treatment group were plated in triplicate onto 6-well dishes and viability, 5 × 104 cells were plated in triplicate with and colonies were allowed to grow for 10d without chang- 2.5 μmol/L GRN163L, mismatch control oligonucleotide, ing the media or adding any drugs before staining with 20% or media only (untreated) on 12-well dishes. Cells were col- Giemsa. Colonies with >50cells were counted indepen- lected weekly and counted via a Beckman Coulter Counter dently by two individuals and averaged. Plating efficiency and the trypan blue exclusion assay to determine cell num- was calculated by dividing the average number of colonies ber and viability. Triplicate samples were combined before by the number of cells initially plated. replating to avoid selection of resistant cells. Population Xenograft mice studies doublings were calculated as the log [(the number of cells Athymicnudemice(nu/nu;HarlanSprague-Dawley, collected)/(number of cell initially plated)]/log 2. Inc.) were maintained in pathogen-free conditions within Focal Adhesion and Actin Filament Immunofluorescence the Laboratory Animal Resources Center at the Indiana Uni- Staining for focal adhesion formation and actin filament versity School of Medicine according to approved institu- organization was done using the Actin Cytoskeleton and tional protocols. MDA-MB-231 breast cancer cells (1 × 106) Focal Adhesion Staining kit from Chemicon International were injected into the left and right mammary fat pads of 6- per manufacturer's protocol. Briefly, 5 × 104 MDA-MB-231 to 8-wk-old mice. Following a recovery period of 2 d, mice cells were plated in chamber slides and grown for 72 h in began receiving treatment of 30mg/kg GRN163L ( n = 15) the presence of 2.5 μmol/L GRN163L or mismatch control or a PBS solvent control (n = 14) thrice weekly i.p., or oligonucleotides. Cells were fixed in 4% paraformaldehyde 15 mg/kg paclitaxel (n = 15) once weekly via i.p., alone or and permeabilized in 0.1% Triton-X-100. Staining was done in combination with 30mg/kg GRN163L ( n = 14). Average in 3% bovine serum albumin (Sigma) in 1 × PBS. Images weights of animals were similar among treatment groups. were captured with a digital camera attached to a Leica Tumor volume was calculated as (length × width2)/2 (in DM500 fluorescence microscope. millimeters). Mixed-effects statistical model was fitted to

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the data. Fixed effects include group, a linear and a qua- Results dratic component of day, and the interaction between group Treatment With GRN163L Alters the Morphology of and the linear/quadratic component. Random effects in- Breast Cancer Cells clude mouse, tumor within mouse, slope of day within To test for the cellular responses to GRN163L, MDA-MB- mouse, and slope of day within tumor. The analysis was 231 breast cancer cells were treated with 19.5 nmol/L to conducted by SAS 9.1. 2.5 μmol/L GRN163L or 10 μmol/L mismatch control oligo- Cell Invasion Assay nucleotides, and morphology was monitored daily. When Matrigel invasion assays were done using the Cell Inva- GRN163L was given to the cells at the same time as plating, sion Assay kit from Chemicon International per manufac- the morphology of MDA-MB-231 cells was altered, result- turer's protocol. MDA-MB-231 cells were treated with ing in a rounding-up effect on adherent cells as well the 0.156 to 0.3125 μmol/L GRN163L or mismatch oligonucleo- extension of fillipodia-like structures (Fig. 1). This morpho- tides, or 16 to 31 nmol/L paclitaxel for 5 d. Parallel studies logic effect by GRN163L was not observed with doses lower were done in combination with paclitaxel (16–31 nmol/L), than 0.3125 μmol/L. In contrast, normal mammary epithelial in which the two agents were at a 1:10ratio of each other. cells did not show a morphologic response to treatment with After 5 d of treatment, cells were collected and the invasive GRN163L, suggesting that this response was cancer cell spe- potential was measured. Normal human BJ fibroblasts cific. Interestingly, MDA087 immortalized breast fibroblasts, served as negative controls for the assay. Statistical analysis which use the alternative lengthening of telomeres pathway was done using a Student's t test (Microsoft Excel). and do not contain telomerase activity, also showed a

Figure 1. GRN163L induces morphologic changes in MDA-MB-231 breast cancer cells. MDA-MB-231 breast cancer cells, MDA087 telomerase– negative, immortalized breast fibroblasts, and nontumorigenic MCF10A cells were treated with 2.5 μmol/L GRN163L or mismatch (MM) control oligonucleotide for 72 h. Arrows, rounded cells; arrowheads, fillipodia-like structures (×200).

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morphologic response to treatment with GRN163L similar to that observed in MDA-MB-231 cells. This finding indicates that the effect of this telomerase template antagonist may be independent of telomerase inhibition. The mismatch control oligonucleotide did not have an effect on cell morphology. This differential response may be due to the triplet guanine sequence unique to GRN163L, which is not contained in the mismatch control oligonucleotide (9). Additionally, we observed a cell type–specific change in morphology in response to GRN163L (Supplementary Table S1; Supplementary Fig. S15). For example, luminal breast cancer cell lines displayed complete loss of adherence to culture substrates yet displayed cohesion, or adhered to each other in cell aggregates. The effects of GRN163L on cell adhesion and architecture was reversible with the addition of extracellular matrix proteins to cell culture dishes (9).6 GRN163L Significantly Inhibits Cell Proliferation in MDA-MB-231 Cells To confirm GRN163L as an effective telomerase inhibitor during the course of these studies, MDA-MB-231 breast cancer cells were treated with 2.5 μmol/LGRN163Lormis- match control oligonucleotide (MM) for 6 days. As shown in Fig. 2A, GRN163L effectively inhibited telomerase activity in MDA-MB-231 cells through 6 days after a single treatment. The mismatch control oligonucleotide had no significant effect on telomerase activity. As shown in Fig. 2B, treatment with GRN163L at the same time as plating dramatically inhibited cell growth. Treatment with mismatch control oligonucleotide had no effect on cell growth, indicating that the effects on cell growth by GRN163L are not related to the use of lipidated oligonucleotides. The total population doublings per week were calculated to deter- minetherateofgrowthinhibition.AsshowninFig.2C,

the average time for one population doubling increased Figure 2. Effect of GRN163L on MDA-MB-231 cell growth. A, MDA- from 1.5 days in untreated cells, to 4.25 days in cells treated MB-231 breast cancer cells were treated with 2.5 μmol/L GRN163L or with GRN163L (P < 0.001). As expected, the mismatch con- mismatch (MM) control oligonucleotides and collected after 1 to 6 d to determine the duration of telomerase inhibition after a single treatment. trol oligonucleotide had no effect on population doubling Relative telomerase activity was calculated as the ratio of telomerase pro- rate compared with untreated cells (P = 0.22). Treatment ducts (ladder) intensity to the intensity of the internal control (IC) band. A with GRN163L did not have a significant effect on normal positive control cell extract (+) and NP40 lysis buffer (LB) were used as a positive and negative control, respectively. UT, untreated sample; HI, human mammary epithelial cells or BJ fibroblasts, as previ- heat-inactivated control samples. B, population doubling levels (±SD; ously reported (data not shown; refs. 7, 8). n = 3) of MDA-MB-231 cells treated with 2.5 μmol/L GRN163L, mismatch oligonucleotide, or media only (UT). C, average time for one population One possible explanation for the morphologic changes, doubling was determined for MDA-MB-231 cells in response to GRN163L reduction in cell growth, and longer population doubling or mismatch oligonucleotides. The doubling time at each weekly passage time in GRN163L-treated MDA-MB-231 cells is the induc- (of the seven passages) was averaged and plotted (±SD; ***, P < 0.001). tion of cell death, particularly apoptosis. Treatment with GRN163L did not induce apoptotic cell death in MDA- maining cells did not show significantly decreased viability MB-231 cells within 72 hours (Supplementary Fig. S2).5 (Supplementary Fig. S2).5 These results taken together indi- Alternatively to cell death, the marked decrease in MDA- cate that the growth inhibition seen in MDA-MB-231 breast MB-231 cell growth may be due to a reduction in cell viabil- cancer cells for the first 2 weeks of treatment with GRN163L ity. MDA-MB-231 cells were treated with 2.5 μmol/L is independent of a reduction in viability or an induction of GRN163L or mismatch oligonculeotides, and collected and apoptosis. counted via the trypan blue exclusion assay daily for 14 GRN163L Disrupts Focal Adhesion Formation and days. Although there was a decrease in cell number, the re- Actin Filament Organization The altered morphology and adhesion capacity observed in response to treatment with GRN163L suggests a change in the cell membrane. To test the effect of GRN163L on the 5 Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). cell structure, actin filaments and focal adhesions were 6 Goldblatt EM, Ositelu O, Herbert B-S, unpublished observations. examined to determine whether this telomerase template

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antagonist can disrupt structural organization of breast can- tle staining throughout the remaining cell membrane. This cer cells. As shown in Fig. 3, untreated cells and mismatch disruption of regular adhesion proteins/elements could control–treated cells displayed well-organized actin fila- help to explain the rounding-up/detachment or disruption ments (phalloidin staining, in red) in long, parallel projec- of cell spreading effects observed in breast cancer cells in tions equally distributed throughout the cell (22). response to GRN163L. However, in GRN163L-treated cells, the actin fiber organi- GRN163L Alters the Expression of Proteins Involved zation was observed primarily along the edges of the cell in Cellular Structure and Function rather than in filaments throughout the cell. This dense ac- The morphologic and structural changes induced by cumulation around the perimeter of the cell indicates a re- GRN163L indicate that the expression of proteins involved duction in stress fiber contractility (22). Furthermore, under in cellular attachment and intracellular organization may be normal physiologic conditions, cells form focal adhesions in altered. The protein levels of β-andγ-tubulin were mea- multiple places along the cell membrane, primarily located sured to determine changes in two of the main components at the ends of actin filaments (22). As shown in Fig. 3, un- of microtubules. Additionally, expression of FAK and Pyk2 treated and mismatch control treated cells showed this pat- were measured, as these proteins play a major role in micro- tern of focal adhesion formation (vinculin staining, in tubule organization, cell spreading, growth, and prolifera- green). However, GRN163L-treated cells displayed punctate tion (23–27). As shown in Fig. 4, the expression of Pyk2 staining concentrated around the nuclei of the cells with lit- increased in a cyclical fashion after 12- and 72-hour treatment

Figure 3. Effects of GRN163L on focal adhesion formation and actin filament organization. MDA-MB-231 cells were plated on chamber slides with 2.5 μmol/L GRN163L, mismatch control oligonucleotide, or media only (untreated) for 72 h. Cells were fixed and stained with phalloidin (actin filaments; red) or vinculin (focal adhesions; green). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). GRN163L disrupts cell adhesion as evidenced by punctate vinculin staining (arrows) rather than diffuse staining seen in untreated and mismatch control treated samples. GRN163L also disrupts cell structure, as seen by altered actin filament organization (arrows). Results are representative of three independent experiments (scale bar, 50 μm).

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OF6 Synergism between Telomerase Antagonists and Paclitaxel

Figure 4. GRN163L effects on the expression of signaling pathway proteins involved in cellular structure and function. MDA-MB-231 cells were treated with 2.5 μmol/L GRN163L or mismatch oligonucleotides for the depicted times and Western immunoblotting for the indicated adhesion and morphology proteins was done (A). Densitometry levels were determined by comparing intensity of FAK, Pyk2, γ-tubulin, and β-tubulin to glyceraldehyde-3-phosphate dehydrogenase and relative intensity levels for each sample were plotted and normalized to untreated samples (B). Untreated and mismatch control– treated cells were analyzed after 72 h. Results are representative of at least two independent experiments (±SE).

with GRN163L, whereas the other protein levels did not lipid-modified oligonucleotide. MDA-MB-231 cells dis- change. Although the exact role of Pyk2 in cancer remains un- played a dose-dependent growth inhibition in response to clear, it has been shown that Pyk2 is involved in maintaining both paclitaxel and GRN163L (Fig. 5A). The combination cell structure and inducing proliferation (28, 29). Specula- of GRN163L and paclitaxel resulted in an increase in the tively, the up-regulation of Pyk2 may be induced to aid the growth inhibition compared with either agent alone (Sup- cell in reshaping its actin filaments to allow the cell to con- plementary Fig. S3; Fig. 5A).5 Importantly, the mismatch tinue replicating in a normal fashion. control alone did not inhibit cell growth and did not alter In a separate attempt to understand the mechanisms un- the effects of paclitaxel. The combination of GRN163L or derlying these alterations in focal adhesion and actin orga- mismatch oligonucleotide plus paclitaxel, however, had no nization, global proteomics and Ingenuity Pathway significant effect on nontumorigenic MCF10A cell growth Analyses were done on MDA-MB-231 breast cancer cells inhibition induced by paclitaxel alone (Supplementary treated with GRN163L compared with mismatch control Fig. S3; ref. 8).5 and untreated cells (Supplementary Table S2).5 Although As shown in Fig. 5B, GRN163L acted synergistically (CI, the analyses suggested that there were not alterations in a <1.0) with paclitaxel to inhibit growth of MDA-MB-231 singular pathway that explain the phenotypic change in- cells. To address the effects of low dose combination treat- duced by GRN163L, there were changes in pathways in- ment on plating efficiency/survival capacity, MDA-MB-231 volved in structural functions as reported by others (30). cells were treated with 40nmol/L GRN163L in combination These results support the hypothesis that the phenotypic with 4 nmol/L paclitaxel for 5 days before collection and change can be explained by alterations in a structural func- replating at low density. After 10days, colonies were tion of the cells. counted and compared with controls (media only- and mis- GRN163L Synergistically Enhances the Effects of match oligonucleotide–treated cells). GRN163L in combina- Paclitaxel on Inhibiting Cell Proliferation in MDA-MB- tion with paclitaxel at low nmol/L doses significantly 231 Breast Cancer Cells reduced the plating efficiency of MDA-MB-231 breast can- The disruption of actin structures in MDA-MB-231 cells cer cells compared with either drug alone and mismatch by GRN163L suggests that GRN163L may work synergis- controls (Fig. 5C). tically with paclitaxel to inhibit breast cancer cell growth. The Combination of GRN163L and Paclitaxel To test the effect of combination therapy on cell growth, Significantly Inhibit Tumor Growth In vivo and Cell cells were plated with 0.005 to 1.25 μmol/L GRN163L ei- Invasion In vitro ther alone or with 0.5 to 12.5 nmol/L paclitaxel for 5 days. To test that the effects of combining GRN163L and pacli- This range of doses encompasses treatments that do not taxel can be seen in vivo as well as in vitro, the combination induce morphologic responses, as well as those that are treatment was examined on the growth of orthotopic MDA- known to affect cell morphology and growth (in addition MB-231 tumors. As shown in Fig. 6A, the combination of to telomerase inhibition). Low doses of GRN163L were GRN163L plus paclitaxel significantly decreased tumor used to rule out any possible cytotoxicity induced by the volume progression over PBS control–treated animals

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(difference in quadratic effect of day: P < 0.0001) and dis- In addition to testing the effects of combination therapy played reduced tumor growth compared with either drug on primary tumor growth, in vitro invasion assays were alone (difference in quadratic effect of day: P < 0.0005 for done to determine the metastatic potential of MDA-MB- paclitaxel and P < 0.005 for GRN163L). 231 cells after treatment with GRN163L (or MM) and paclitaxel. As shown in Fig. 6B, GRN163L and paclitaxel alone inhibited cell invasion compared with untreated control cells (P = 0.046 and 0.006, respectively), whereas the mismatch oligonucleotide had no effect (P = 0.47). The combination of GRN163L and paclitaxel significantly decreased invasion compared with either drug alone (P = 0.002 versus GRN163L alone; P = 0.017 versus paclitaxel alone). The combination of mismatch oligonucleotide and paclitaxel had no significant effect on the inhibition of invasion induced by paclitaxel alone (P = 0.23). Invasion was complete- ly inhibited with 31 nmol/L paclitaxel, and the addition of GRN163L or mismatch oligonucleotides had no further effect (data not shown).

Discussion GRN163L is an effective telomerase inhibitor; however, the mechanisms underlying all cellular responses are not com- pletely understood. In addition to its effects as a telomerase inhibitor, GRN163L likely disrupts focal adhesion formation and actin filament organization, thus impairing cancer cell adhesion and morphology. This response significantly de- lays the growth of MDA-MB-231 cells without a reduction in cell viability or the induction of apoptosis in the short term. Importantly, this effect of GRN163L-sensitized breast cancer cells to paclitaxel, an important chemotherapeutic agent used in the treatment of breast cancer. Although the effect of combination therapy on the rate or incidence of metastasis was not tested in vivo,ourin vitro invasion assay results suggest that nanomolar amounts of GRN163L plus paclitaxel significantly reduces the invasive potential of MDA-MB-231 cells. Under normal growth conditions, cells require attachment to a solid surface before they can grow. The formation of focal adhesions helps in the organization of the cytoskeleton and activates intracellular signaling pathways, including signals for proliferation, survival, and migration (23–25). Migration occurs through a continuing cycle of dis- rupting focal adhesions, extension of membrane protrusions at the advancing edge and contraction of the trailing ends of Figure 5. GRN163L synergistically augments the effects of paclitaxel in the cell, and the formation of new focal adhesions (31). The MDA-MB-231 breast cancer cells. A, MDA-MB-231 cells were treated for 5 d with 1:2 serial dilutions of GRN163L or a mismatch oligonucleotide response to GRN163L may disrupt this process through its alone (MM), paclitaxel alone, or the combination of GRN163L/MM with triplet-G motif (9), thus inhibiting the migratory potential of paclitaxel at a 1:10 ratio. Percent growth inhibition (compared with media cancer cells and reducing metastatic disease. In addition to only, UT) was plotted versus concentration for each treatment group ± SD (n = 3 independent experiments). B, CIvalues and percent growth inhibi- the effect on cell growth, we observed that GRN163L has an tion of MDA-MB-231 breast cancer cells treated with GRN163L in combi- effect on cell architecture, resulting in altered actin filament nation with paclitaxel at a constant ratio (1:10). Data are expressed as organization and focal adhesion formation. A reduction in mean CIvalue ± SE ( ♦;n = 3 independent experiments) and mean percent growth inhibition ± SD (▪;n = 3 independent experiments). Dotted line, a migration after treatment with GRN163L has been shown in CIvalue of 1.0. C, GRN163L in combination with paclitaxel at low nmol/L lung cancer cells (9), although the mechanisms underlying doses reduces the plating efficiency of MDA-MB-231 breast cancer cells compared with either drug alone. Cells were treated with 40 nmol/L this effect remain to be elucidated. The results shown in GRN163L in combination with 4 nmol/L paclitaxel for 5 d before collection Figs. 5 and 6 indicate that the combination of GRN163L and plating a low density. After 10 d, colonies were counted and the plat- and paclitaxel, a microtubule stabilizer and mitotic inhibi- ing efficiency of each treatment group was plotted (±SD; n = 3). Control group represents the average of media only– and mismatch oligonucleo- tor, allows for an effective growth inhibition at low nanomo- tide–treated cells. lar doses of both drugs.

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Figure 6. GRN163L and paclitaxel significantly reduce the tumor growth and invasive potential of breast cancer cells. A, efficacy of GRN163L in combination with paclitaxel in vivo. Tumor growth rate in mice treated with PBS control (n = 14), GRN163L alone (30 mg/kg; n = 15), paclitaxel alone (15 mg/kg; n = 15), or the combination of GRN163L and paclitaxel (n = 14) is shown. Points, mean of the tumor volume averages per mouse; bars, SE. B, GRN163L in combination with paclitaxel significantly inhibits invasive potential in vitro. MDAMB-231 cells were treated with 156 nmol/L GRN163L or mismatch control oligonucleotides, or media only (untreated, UT) with or without 15.6 nmol/L paclitaxel for 5 d before measuring invasive ability as described in Materials and Methods. Results are presented as the percent invasive potential relative to untreated MDA- MB-231 cells, and are the average of two independent experiments with four replicates each (±SE).

Telomerase inhibition has often shown a long lag phase omerase inhibition therapy (39–41). The effect of GRN163L before critical telomere shortening and cell growth inhibi- on cell adhesion and morphology is reversible (data not tion, which may effect the fitness of normal cells that use shown; ref. 9), with the return of the pretreatment telomer- telomerase for survival (32–35). The research presented ase activity levels, telomere elongation and maintenance, here, however, shows a rapid response to GRN163L in can- and normal cell architecture. Taking advantage of this rapid cer cells, with significant effects on MDA-MB-231 breast response by treating cells with GRN163L and paclitaxel in- cancer cell growth and morphology within 5 days. Al- dicates that shorter exposure times may be possible to though the response to GRN163L did not induce rapid se- reduce secondary effects of treatment with other therapeu- nescence, apoptotic cell death, or decreased cell viability tics (IR, chemotherapy, or molecular-targeted therapy). over 2 weeks, this does not rule out the fact that loss of a Taken together with previously published reports demon- sentinel, critically short telomere(s) may affect cell survival strating that GRN163L inhibits primary breast cancer tumor and trigger alternate forms of growth arrest or cell death, growth in vivo (8, 12), these results provide a rationale for such as autophagy, as shown by the recent report on telo- the continued investigation of this drug in breast cancer pre- mere-targeting, G-rich oligonucleotides (36). It is also pos- clinical/clinical trials. Therefore, treatment with telomerase sible that early-stage telomere dysfunction induced by template antagonists could have beneficial outcomes not GRN163L induces mitotic catastrophe, which would result only on primary tumor growth, due to the effects of telo- in failure of cell cycle arrest before or at mitosis due to ab- mere shortening, but also in therapeutic regimens for sensi- normal spindle formation (37, 38). One concern with the use tization to therapy and reduction in metastasis. of telomerase inhibitors in the clinical setting is the effect on stem and progenitor cells found in highly replicative tissues. The rapid response to GRN163L and paclitaxel shown here Disclosure of Potential Conflicts of Interest indicates that it is reasonable to expect that quiescent stem S.M. Gryaznov: employee, Geron Corporation; inventor, GRN163L. B-S. cells will not be significantly affected by short courses of tel- Herbert, research support, Geron Corporation.

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Mol Cancer Ther 2009;8(7). July 2009

The telomerase template antagonist GRN163L alters MDA-MB-231 breast cancer cell morphology, inhibits growth, and augments the effects of paclitaxel

Erin M. Goldblatt, Erin R. Gentry, Melanie J. Fox, et al.

Mol Cancer Ther Published OnlineFirst June 9, 2009.

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