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neu
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
77, 85–90 (1998)
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Antisense Oligonucleotides Specific for the HER2/neu Oncogene Inhibit the Growth of Human Breast Carcinoma Cells That Overexpress HER2/neu Haeri Roh, Ph.D., James Pippin, B.A., Craig Boswell, M.D.,1 and Jeffrey A. Drebin, M.D., Ph.D.2,3 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 Submitted for publication November 17, 1997
nase activity. The human homologue of the HER2/neu gene is located on chromosome 17 [2, 3]. The HER2/ neu gene was originally defined by its significant homology to the gene which encodes the epidermal growth factor receptor (EGFR), also known as erbB [1–3]. Subsequent studies have demonstrated several related genes, erbB-3 and erbB-4 [4]. It appears that erbB-2 can heterodimerize with other erbB family genes and plays a central role in signal transduction by all members of this receptor family [4, 5]. Elevated expression of HER2/neu, resulting from gene amplification or enhanced transcription, is seen in a variety of human tumors [6]. Though first identified in breast carcinoma [7], in which approximately 30% of tumors overexpress the gene, HER2/neu overexpression has also been seen in a significant fraction of ovarian, lung, and pancreatic tumors [6–10]. It is thought that overexpression of HER2/neu results in enhanced mitogenic signaling and drives the proliferation of tumor cells in which the gene is overexpressed. We initially demonstrated the ability of monoclonal antibodies specific for the rat HER2/neu protein product, p185, to downregulate p185 expression and inhibit tumor growth in rodent models in vitro [11] and in vivo [12]. Subsequent studies have demonstrated that monoclonal antibodies reactive with human p185 can inhibit the proliferation of human breast carcinoma cells [13]. Clinical trials of anti-HER2/neu monoclonal antibodies in breast cancer patients are in progress and results of a phase II trial were recently reported [14]. However, monoclonal antibodies may not be the ideal mechanism with which to downregulate HER2/neu expression. Antibodies only react with cell surface p185; increased levels of intracellular p185 may deliver mitogenic signals prior to display on the cell surface [15]. Furthermore, tumor cells in which p185 is downregulated by monoclonal antibody exposure rapidly reexpress p185 in the absence of antibody [11], suggesting that constant exposure to antibody is necessary for tumor inhibitory effects. Soluble antigenic p185 fragments shed from the tumor cell membrane may interfere with antibody binding to p185 on the tumor cell
The HER2/neu oncogene encodes a cell surface protein which plays a role in growth factor-stimulated mitogenic signaling. HER2/neu is overexpressed in 30– 40% of human breast carcinomas. This study tested the hypothesis that inhibiting HER2/neu expression using a phosphorothioate antisense (AS) oligonucleotide would inhibit the growth of breast cancer cells that overexpress this gene. A human breast carcinoma cell line, BT474, which overexpresses the HER2/neu oncogene was exposed to AS, sense (S), or scrambled antisense (SC) phosphorothioate oligonucleotides in tissue culture. Treatment with AS oligonucleotides specifically downregulated HER2/neu mRNA expression and resulted in lower levels of the HER2/neu protein product, p185; control oligonucleotides had no such effect. AS oligonucleotide treatment significantly inhibited the in vitro growth of BT474 cells, whereas S and SC controls had little effect on BT474 growth. HER2/neu AS oligonucleotide treatment had no effect on the growth of a distinct breast cancer line, MCF7, which expresses low levels of the HER2/neu oncogene. Breast carcinoma cells which overexpress the HER2/neu gene appear to be dependent on continued expression of this oncogene for cell growth. AS oligonucleotide pharmaceuticals which interfere with the expression of the HER2/neu oncogene may be of use in the therapy of some patients with breast carcinoma. q 1998 Academic Press Key Words: antisense; breast cancer; HER2; neu; oligonucleotide; oncogene.
INTRODUCTION
The HER2/neu oncogene, also known as erbB-2, was originally identified in a series of rat neuroblastoma cell lines [1]. This gene encodes a 185-kilodalton (kd) transmembrane glycoprotein with intrinsic tyrosine ki1
Supported by NIH Training Grant T32CA09621-09. Supported in part by a faculty fellowship from the American College of Surgeons. 3 To whom correspondence should be addressed at Suite 6108, One Barnes Hospital Plaza St. Louis, MO 63110. 2
0022-4804/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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surface, with resulting inhibition of antitumor effects [16]. Some p185-specific monoclonal antibodies may have growth stimulatory effects on tumors that overexpress p185 [17]. Finally, the immunogenicity of immunoglobulin molecules makes them a less than ideal pharmacologic modality. While this may be partially obviated by ‘‘humanization’’ of mouse monoclonal antibodies [14], it may not entirely eradicate the problem of anti-antibody immune responses. A different approach to downregulating gene expression is through the use of AS oligonucleotides [18–21]. AS oligonucleotides can directly bind mRNA and can catalyze degradation of the targeted mRNA by the endogenous cellular enzyme RNAse H. However, AS molecules can be degraded by nucleases and can have a variety of non-antisense effects, particularly at high concentrations. Methods to obviate such problems include the use of phosphorothioate oligonucleotides to enhance nuclease resistance and the use of liposome delivery techniques to minimize the concentration of oligonucleotides to which cells are exposed. Recently, Vaughn et al. demonstrated that treatment of SKBR3 breast carcinoma cells with a 15-nucleotidelong phosphorothioate AS oligonucleotide could inhibit HER2/neu mRNA and protein expression in a subpopulation of cell-sorted tumor cells that had received an optimal AS dose, with a resulting increase in cells in the G1 phase of the cell cycle [22]. Direct effects on tumor cell growth were not examined. In investigating approaches to downregulating HER2/neu expression we have replicated the observations of Vaughn et al. and have extended these studies to directly examine effects of HER2/neu antisense oligonucleotides on tumor cell growth in vitro.
was replaced with standard culture medium. After incubating an additional 2 h the cells were taken up with trypsin–EDTA solution and extensively washed with Hanks’ balanced salt solution at 47C, and cellular fluorescence was analyzed using a FACSscan flow cytometer. Northern and Western blotting. Breast cancer cells (5 1 105) were plated into 60-mm dishes under standard culture conditions. After incubating overnight the medium was replaced with serum-free Optimem and oligonucleotides at 1 mM were added in the presence of 10 mg/ml lipofectin. Cultures were incubated for 4 h and then the medium was replaced with standard culture medium. Twenty-four hours later cell lysates were prepared using Trizol (Gibco), and RNA and protein were extracted. Northern blots were performed by electrophoresing total cellular RNA samples (10 mg/lane) through formaldehyde–agarose gels, transferring to nylon membranes, and hybridizing with a 1.9-kilobase HER2/neu human c-DNA probe (nucleotides 1025 to 2914 of the intact c-DNA). A glyceraldehyde phosphate dehydrogenase (GAPDH) probe (Clontech, Palo Alto, CA) was used to demonstrate equal RNA loading. Western blots were performed by electrophoresing protein samples through polyacrylamide gels, transferring to nylon membranes, and probing with a monoclonal antibody specific for the human HER2/neu protein product, p185 (Oncogene Research, Cambridge, MA). An actin-specific monoclonal antibody (Amersham Corp., Arlington Heights, IL) was used to control for equal protein loading. Cell growth assays. Replicate samples of 2 1 104 breast tumor cells were plated in individual wells of a 24-well plate in standard culture medium. After allowing 24–48 h for cells to become adherent, the medium was changed to Opti-mem and oligonucleotides at a final concentration of 1 mM in the presence of 10 mg/ml lipofectin were added for 4 h. The medium was then changed to standard medium containing serum, and the cells were cultured for 5 days. Cells were then taken up in trypsin–EDTA and viable cells counted using a hemocytometer in the presence of trypan blue. Each treatment group was performed in triplicate. Control growth cultures were treated with 10 mg/ml lipofectin without the addition of oligonucleotide. Statistical analysis. The possibility that differences in cell growth between treated groups were due to chance was evaluated using Student’s t test.
RESULTS METHODS Oligonucleotides. Phosphorothioate oligonucleotides were obtained from Oligos, Etc. (Wilsonville, OR). HER2/neu target sequences corresponding to the 5* portion of the HER2/neu mRNA used were based on those of Vaughn et al. [22]: AS, CTCCATGGTGCTCAC; S, GTGAGCACCATGGAG; SC, CGCCTTATCCGTAGC (underlined nucleotides indicate positions at which AS and SC differ). For determination of oligonucleotide uptake using flow cytometry a SC phosphorothioate oligonucleotide modified by coupling fluorescein isothiocyanate (FITC) to the 5* end was used as a tracer (Oligos, Etc.). Oligonucleotide stock solutions were diluted to a concentration of 10 mM in distilled water, sterilized by filtration, and maintained at 0707C. Stock solutions were thawed on ice and diluted appropriately for application to cell cultures. Cell lines. The breast carcinoma cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbecco’s modified Eagles medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin mixture. Cultures were maintained in a 5% CO2 humidified incubator, at 377C. Flow cytometric analysis of oligonucleotide uptake. BT474 cells (5 1 105) were plated into 60-mm dishes under standard culture conditions. After incubating overnight the medium was replaced with serum-free Opti-mem (Gibco, Gaithersberg, MD) and AS oligonucleotides at 1 mM plus FITC-coupled SC oligonucleotide tracer at a final concentration of 10 nM were added in the presence of 10 mg/ml lipofectin (Gibco). Cultures were incubated for 4 h and then the medium
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Oligonucleotide uptake by BT474 carcinoma cells. Initial studies to determine the efficacy of oligonucleotide delivery to tumor cells were performed using FITCcoupled phosphorothioate molecules. As shown in Fig. 1, cells treated with AS oligonucleotides, to which are added 1% of a FITC SC oligonucleotide tracer, take up a significant amount of the oligonucleotides as determined by flow cytometry 2 h posttreatment. Virtually all tumor cells take up some FITC oligonucleotide with a one- to two-log difference in fluorescence intensity, suggesting variability in cellular uptake within the treated cell population. There is still significant retention of the FITC signal when assayed at 48 h, demonstrating long-term retention of the oligonucleotides (data not shown). Downregulation of HER2/mRNA expression. While it is clear that cells exposed to HER2/neu AS oligonucleotides take up significant amounts of these molecules, the ability of HER2/neu AS treatment to downregulate HER2/neu mRNA is critical in demonstrating a specific AS effect. As shown in Fig. 2, AS oligonucleotide treatment of BT474 tumor cells results in significant downregulation of HER2/neu mRNA expression
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FIG. 1. Cellular uptake of FITC-conjugated oligonucleotides. BT474 cells were treated with FITC-conjugated oligonucleotides (treated) or with lipofectin alone (untreated) for 4 h as described under Methods. After incubating an additional 2 h the cells were washed and processed for flow cytometry. Histograms depict the fluorescence intensity of a total of 10,000 cells in each group. Greater than 97% of treated cells display fluorescence above the upper 1% limit of that seen in untreated cells. The variation in fluorescence intensity in the treated group reflects variable uptake of FITC-conjugated oligonucleotide.
compared to cells treated with lipofectin alone, as determined by Northern blotting. Treatment with control S or SC oligonucleotides has little effect on HER2/neu mRNA expression. Downregulation of p185 expression. Having demonstrated a specific AS effect on HER2/neu mRNA expression, we next examined the effect of AS oligonucleotide treatment on expression of the HER2/neu oncogene product, p185. As shown in Fig. 3, BT474 cells treated with HER2/neu AS oligonucleotides express significantly less p185 protein than do cells treated with either lipofectin alone or S or SC oligonucleotides, as determined by Western blotting. This effect is somewhat less striking than the effect on mRNA due to the significantly longer half-life of protein compared to mRNA; antisense treatment has no effect on protein
FIG. 2. Downregulation of HER2/neu mRNA by AS oligonucleotides. BT474 cells were treated with 10 mg/ml lipofectin alone or with 1 mM AS, S, or SC oligonucleotides in the presence of 10 mg/ml lipofectin for 4 h. Cells were then cultured in standard medium for 24 h and total cellular RNA was extracted and Northern blotting performed as described under Methods. Treatment with HER/neu AS oligonucleotides results in significant downregulation of HER2/ neu mRNA expression. GAPDH probe hybridization confirms equal loading of RNA in each lane.
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FIG. 3. Inhibition of expression of the HER2/neu protein product, p185, by AS oligonucleotides. BT474 and MCF7 breast carcinoma cells were treated with 10 mg/ml lipofectin alone or with 1 mM AS, S, or SC oligonucleotides in the presence of 10 mg/ml lipofectin for 4 h. Cells were then cultured in standard medium for 24 h; total cellular protein was extracted and Western blotting performed as described under Methods. Total protein loaded was 2 mg/lane for BT474 and 20 mg/lane for MCF7. HER/neu AS oligonucleotide treatment results in significant inhibition of p185 expression. Western blotting using an anti-actin antibody confirms equal loading of protein in each lane.
synthesized prior to antisense treatment, but blocks de novo synthesis of p185 as the result of downregulating HER2/neu mRNA. Also demonstrated in Fig. 3 is the effect of oligonucleotide treatment on p185 expression in the MCF7 tumor line. This line expresses 20- to 50-fold less p185 than does BT474, but is equally sensitive to the effects of HER2/neu antisense oligonucleotides on p185 expression. Thus, antisense effects on p185 expression appear to be independent of the intrinsic level of p185 expressed in a particular tumor cell line. Antisense effects on cell growth. It has been demonstrated that HER2/neu AS oligonucleotides specifically downregulate HER2/neu mRNA expression, resulting in lower levels of p185 within treated tumor cells. To determine whether such antisense-mediated downregulation of p185 resulted in changes in cell proliferation, the effects of such treatment on in vitro tumor cell growth were examined. As shown in Fig. 4, AS oligonucleotide treatment results in significant inhibition of BT474 growth when compared to cells treated with S and SC control oligonucleotides. The effect of oligonucleotide treatment on MCF7 breast carcinoma cells, which express lower levels of p185, has also been examined. As shown in Fig. 4, AS oligonucleotide treatment has little effect on the growth of MCF7 tumor cells compared to S or SC controls. It is worth noting that MCF7 cells are more sensitive to the nonspecific toxic effects of phosphorothioate oligonucleotides than are BT474 cells and that both AS and SC groups achieved borderline statistical significance (P õ 0.06) when compared to the growth of cells treated with lipofectin alone. However, it is apparent from the data shown that there is no specific effect of HER2/neu AS oligonucleotides beyond the nonspecific effects caused by control oligonucleotides. This is obviously quite different than the response of BT474 cells, which also show a modest nonspecific effect of S and SC oligonucleotides, but a much more dramatic inhibition of proliferation by HER2/neu AS oligonucleotides. We have examined HER2/neu antisense effects on
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FIG. 4. Effects of oligonucleotide treatment on tumor cell growth. BT474 (elevated HER2/neu expression) and MCF7 (low HER2/neu expression) breast cancer cells were treated with AS, S, or SC oligonucleotides at a final concentration of 1 mM in the presence of 10 mg/ ml lipofectin for 4 h on day 0. Cells were then cultured for 5 days and triplicate samples were counted. Growth of the oligonucleotidetreated groups is expressed as a percentage of the growth of lipofectin-treated controls. It is apparent that AS oligonucleotide treatment inhibits the growth of BT474 cells but has little effect on the growth of MCF7 cells. Error bars indicate standard errors of the mean.
the growth of several other breast cancer lines which express varying levels of p185. As shown in Table 1, the proliferation of several other tumor lines that express low levels of p185 is minimally if at all inhibited by HER2/neu antisense oligonucleotides. Thus, HER2/ neu AS oligonucleotide-mediated downregulation of HER2/neu appears to selectively inhibit the growth of cells that overexpress the HER2/neu oncogene. DISCUSSION
We have examined the effects of HER2/neu-specific phosphorothioate AS oligonucleotides on HER2/neu expression and in vitro growth in human breast carcinoma cell lines. These studies have demonstrated that AS treatment can selectively downregulate HER2/neu mRNA and protein expression; S and SC oligonucleotides have no such effects. Furthermore, HER2/neu AS oligonucleotide treatment selectively inhibits the growth of BT474 cells, which overexpress HER2/neu, but has little effect on the growth of breast carcinoma cells which express lower levels of the HER2/neu oncogene. The effects of HER2/neu AS oligonucleotides on tumor cell proliferation are sequence-specific and targetspecific, affecting only tumor cells which express high levels of p185. It is important to note that the levels of p185 in BT474 cells after AS downregulation are still significantly higher than those of untreated MCF7 cells, and yet the growth rate of cells in MCF7 cultures is greater than that of AS-treated BT474 cultures. Thus, there is not a single threshold level of p185 re-
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quired for proliferation of breast cancer cells. Instead, cells which overexpress HER2/neu appear to be dependent on continued high levels of p185; downregulation of p185 interrupts the excessive mitogenic signaling which results from HER2/neu overexpression. It can be concluded that elevated expression of the HER2/ neu gene is a critical genetic event in the process of multistep carcinogenesis in cells which overexpress HER2/neu. In contrast, breast cancer cells which express low levels of HER2/neu may have abnormalities of a distinct (parallel) growth regulatory pathway responsible for driving neoplastic proliferation, which is independent of HER2/neu, and thus are not affected by downregulation of p185. These results are consistent with the effects of p185-specific monoclonal antibodies on the proliferation of breast carcinoma cells, which are also selectively inhibitory for tumor cells that overexpress p185 [23]. The studies presented here extend the work of Vaughn et al. [22]. The AS effects described by Vaughn et al. required cell sorting and enrichment for an optimally treated subset of tumor cells in order to demonstrate AS-mediated downregulation of HER2/neu mRNA. Effects on cell surface p185 were demonstrated by immunofluorescence, but AS effects on total cellular p185 protein were not examined. Furthermore, effects on cell growth were not directly investigated. In contrast, we have demonstrated effects of HER2/neu AS oligonucleotides on mRNA and protein expression, as well as effects on tumor cell growth, using bulk cell culture populations. Some of the differences between the studies of Vaughn et al. and the results presented here may reflect differences in the breast tumor cell lines studied— they used the SKBR3 line whereas we used BT474. While both lines overexpress HER2/neu to a comparable degree, we have found SKBR3 to be particularly sensitive to non-antisense effects resulting from phos-
TABLE 1 Effects of HER2/neu Antisense Oligonucleotides on Breast Cancer Cell Lines Breast carcinoma cell line
Baseline p185 expression
Specific inhibition of growth by AS oligonucleotidesa (%)
BT474 SKBR3 MCF7 BT20 MDA468
//// //// / / 0
60–80 30–50 õ20 õ10 õ10
Note. Summary of multiple experiments examining effects of HER2/neu AS oligonucleotides on the growth of human breast carcinoma cell lines. a Specific inhibition calculated as inhibition of growth by AS oligonucleotides compared to effects of SC control oligonucleotides. All experiments performed at 1 mM oligonucleotide concentrations, except SKBR3 carried out at 300 nM due to the greater sensitivity of this cell line to nonspecific effects of phosphorothioate oligonucleotides.
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phorothioate oligonucleotides. Lower doses of AS oligonucleotides, 300 nM, were used in the studies of Vaughn et al., presumably to avoid confounding toxic effects, which may have compromised their ability to maximize AS-mediated HER2/neu downregulation. In contrast, we have found minimal non-antisense effects at AS concentrations of 1 mM using the BT474 cell line, as shown here. Since AS effects are clearly dose-dependent (data not shown), our ability to use higher HER2/ neu AS concentrations without encountering nonspecific effects may have allowed us to identify AS effects on HER2/neu expression and cell growth in total (nonsorted) cell cultures. Liu and Pogo, using a different AS sequence targeted to the 5* region of HER2/neu mRNA, have found effects on BT474 growth similar to those reported here [24]. AS oligonucleotides represent a potent approach at downregulating the expression of specific genes [18– 22, 24]. AS studies targeting a number of distinct growth regulatory genes in tumor cell culture systems, including Bcl-2 [25], myb [26], myc [27], protein kinase A [28], protein kinase C [29], and raf [30], have been published, and several of these AS compounds appear to have antitumor activity on human tumors implanted into nude mice. Some of these AS molecules are being tested in phase I and II clinical trials. The HER2/neu oncogene appears to be another useful genetic target for AS oligonucleotide-based therapy. We are currently investigating the in vivo activity of HER2/neu AS, alone and in conjunction with HER2/neu-specific monoclonal antibodies, in tumor xenograft models. REFERENCES
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ney, R., Soper, J. T., Dodge, R., Clarke-Pearson, D. L., Marks, P., McKinzie, S., Yin, S., and Bast, R. C., Jr. Overexpression of HER-2/neu is associated with poor survival in advanced epithelial ovarian cancer. Cancer Res. 50: 4087, 1990. 9. Kern, J. A., Robinson, R. A., Gazdar, A., Torney, L., and Weiner, D. B. Mechanisms of p185HER2 expression in human non-small cell lung cancer cell lines. Am. J. Respir. Cell Mol. Biol. 6: 359, 1992. 10. Williams, T. M., Weiner, D. B., Greene, M. I., and McGuire, H. C. Expression of c-erbB-2 in human pancreatic adenocarcinomas. Pathobiology 59: 46, 1991. 11. Drebin, J. A., Link, V. C., Stern, D. F., Weinberg, R. A., and Greene, M. I. Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies. Cell 41: 695, 1985. 12. Drebin, J. A., Link, V. C., Weinberg, R. A., and Greene, M. I. Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen. Proc. Natl. Acad. Sci. USA 83: 9129, 1986. 13. Hudziak, R. M., Lewis, G. D., Winget, E., Fendly, B. M., Shepard, H. M., and Ullrich, A. p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol. Cell. Biol. 9: 1165, 1989. 14. Baselga, J., Tripathy, D., Mendelsohn, J., Baughman, S., Benz, C. C., Dantis, L., Sklarin, N. T., Seidman, A. D., Hudis, C. A., Moore, J., Rosen, P. P., Twaddell, T., Henderson, I. C., and Norton, L. Phase II study of weekly intravenous recombinant humanized anti-p185HER2 monoclonal antibody in patients with HER2/neu-overexpressing metastatic breast cancer. J. Clin. Oncol. 14: 737, 1996. 15. Graus-Porta, D., Beerli, R. R., and Hynes, N. E. Single chain antibody-mediated intracellular retention of erbB-2 impairs neu differentiation factor and epidermal growth factor signaling. Mol. Cell. Biol. 15: 1182, 1995. 16. Brodowicz, T., Wiltschke, C., Budinsky, A. C., Krainer, M., Steger, G. G., and Zielinski, C. C. Soluble HER2/neu neutralizes biologic effects of anti-HER2/neu antibody on breast cancer cell in vitro. Int. J. Cancer 73: 875, 1997.
1. Schechter, A. L., Stern, D. F., Vaidyanathan, L., Decker, S. J., Drebin, J. A., Greene, M. I., and Weinberg, R. A. The neu oncogene: An erbB related gene encoding a 185,000 Mr tumor antigen. Nature 312: 513, 1984. 2. Schechter, A. L., Hung, M-C., Vaidyanathan, L., Weinberg, R. A., Yang-Feng, T. L., Francke, U. L., Ullrich, A., and Coussens, L. The neu gene: An erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science 229: 976, 1985. 3. Coussens, L., Yang-Feng, T. L., Liao, Y-C., Chen, E., Gray, A., McGrath, J., Seeburg, P. H., Libermann, T. A., Schlessinger, J., Francke, U., Levinson, A., and Ullrich, A. Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230: 1132, 1985. 4. Carraway, K. L., and Cantley, L. C. A neu acquaintance for erbB3 and erbB4: A role for receptor heterodimerization in growth signaling. Cell 78: 5, 1994.
17. Xu, F-J., Stack, S., Boyer, C., O’Briant, K., Whitaker, R., Mills, G. B., Yu, Y. H., and Bast, R. C., Jr. Heregulin and agonistic anti-p185c-erbB2 antibodies inhibit proliferation but increase invasiveness of breast cancer cells that overexpress p185c-erbB2: Increased invasiveness may contribute to poor prognosis. Clin. Cancer Res. 3: 1629, 1997.
5. Graus-Porta, D., Beerly, R. R., Daly, J. M., and Hynes, N. E. ErbB-2, the preferred heterodimerization partner of all erbB receptors, is the mediator of lateral signaling. EMBO J. 16: 1647, 1997. 6. Hynes, N. E., and Stern, D. F. The biology of erbB-2/neu/HER2 and its role in cancer. Biochem. Biophys. Acta 1198: 165, 1994.
22. Vaughn, J. P., Iglehart, J. D., Demirdji, S., Davis, P., Babiss, L. E., Caruthers, M. H., and Marks, J. R. Antisense DNA downregulation of the ErbB2 oncogene measured by a flow cytometric assay. Proc. Natl. Acad. Sci. USA 92: 8338, 1995.
7. Slamon, D. J., Godolphin, W., Jones, L. A., Holt, J. A., Wong, S. C., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ullrich, A., and Press, M. F. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707, 1989. 8. Berchuck, A., Kamel, A., Whitaker, R., Kerns, B., Olt, G., Kin-
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18. Zamecnik, P. C., and Stephenson, M. L. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 75: 280, 1978. 19. Stein, C. A., and Krieg, A. M. Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides. Antisense Res. Dev. 4: 67, 1994. 20. Wagner, R. W. The state of the art in antisense research. Nature Med. 1: 1116, 1995. 21. Crooke, S. T., and Bennett, C. F. Progress in antisense oligonucleotide therapeutics. Annu. Rev. Pharmacol. Toxicol. 36: 107, 1996.
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25. Ziegler, A., Luedke, G. H., Fabbro, D., Altmann, K-H., Stahel, R. A., and Zangemeister-Wittke, U. Induction of apoptosis in small-cell lung cancer cells by an antisense oligodeoxynucleotide targeting the bcl-2 coding sequence. J. Natl. Cancer Inst. USA 89: 1027, 1997. 26. Anfossi, G., Gewirtz, A. M., and Calebretta, B. An oligomer complementary to c-myb encoded mRNA inhibits proliferation of human myeloid leukemia cell lines. Proc. Natl. Acad. Sci. USA 86: 3379, 1989. 27. Leonetti, D., D’Agnano, I., Lozupone, F., Valentini, A., Geiser, T., Zon, G., Calebretta, B., Citro, G., and Zupi, G. Antitumor
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Antisense Oligonucleotides Specific for the HER2/neu Oncogene Inhibit the Growth of Human Breast Carcinoma Cells That Overexpress HER2/neu Haeri Roh, Ph.D., James Pippin, B.A., Craig Boswell, M.D.,1 and Jeffrey A. Drebin, M.D., Ph.D.2,3 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 Submitted for publication November 17, 1997
nase activity. The human homologue of the HER2/neu gene is located on chromosome 17 [2, 3]. The HER2/ neu gene was originally defined by its significant homology to the gene which encodes the epidermal growth factor receptor (EGFR), also known as erbB [1–3]. Subsequent studies have demonstrated several related genes, erbB-3 and erbB-4 [4]. It appears that erbB-2 can heterodimerize with other erbB family genes and plays a central role in signal transduction by all members of this receptor family [4, 5]. Elevated expression of HER2/neu, resulting from gene amplification or enhanced transcription, is seen in a variety of human tumors [6]. Though first identified in breast carcinoma [7], in which approximately 30% of tumors overexpress the gene, HER2/neu overexpression has also been seen in a significant fraction of ovarian, lung, and pancreatic tumors [6–10]. It is thought that overexpression of HER2/neu results in enhanced mitogenic signaling and drives the proliferation of tumor cells in which the gene is overexpressed. We initially demonstrated the ability of monoclonal antibodies specific for the rat HER2/neu protein product, p185, to downregulate p185 expression and inhibit tumor growth in rodent models in vitro [11] and in vivo [12]. Subsequent studies have demonstrated that monoclonal antibodies reactive with human p185 can inhibit the proliferation of human breast carcinoma cells [13]. Clinical trials of anti-HER2/neu monoclonal antibodies in breast cancer patients are in progress and results of a phase II trial were recently reported [14]. However, monoclonal antibodies may not be the ideal mechanism with which to downregulate HER2/neu expression. Antibodies only react with cell surface p185; increased levels of intracellular p185 may deliver mitogenic signals prior to display on the cell surface [15]. Furthermore, tumor cells in which p185 is downregulated by monoclonal antibody exposure rapidly reexpress p185 in the absence of antibody [11], suggesting that constant exposure to antibody is necessary for tumor inhibitory effects. Soluble antigenic p185 fragments shed from the tumor cell membrane may interfere with antibody binding to p185 on the tumor cell
The HER2/neu oncogene encodes a cell surface protein which plays a role in growth factor-stimulated mitogenic signaling. HER2/neu is overexpressed in 30– 40% of human breast carcinomas. This study tested the hypothesis that inhibiting HER2/neu expression using a phosphorothioate antisense (AS) oligonucleotide would inhibit the growth of breast cancer cells that overexpress this gene. A human breast carcinoma cell line, BT474, which overexpresses the HER2/neu oncogene was exposed to AS, sense (S), or scrambled antisense (SC) phosphorothioate oligonucleotides in tissue culture. Treatment with AS oligonucleotides specifically downregulated HER2/neu mRNA expression and resulted in lower levels of the HER2/neu protein product, p185; control oligonucleotides had no such effect. AS oligonucleotide treatment significantly inhibited the in vitro growth of BT474 cells, whereas S and SC controls had little effect on BT474 growth. HER2/neu AS oligonucleotide treatment had no effect on the growth of a distinct breast cancer line, MCF7, which expresses low levels of the HER2/neu oncogene. Breast carcinoma cells which overexpress the HER2/neu gene appear to be dependent on continued expression of this oncogene for cell growth. AS oligonucleotide pharmaceuticals which interfere with the expression of the HER2/neu oncogene may be of use in the therapy of some patients with breast carcinoma. q 1998 Academic Press Key Words: antisense; breast cancer; HER2; neu; oligonucleotide; oncogene.
INTRODUCTION
The HER2/neu oncogene, also known as erbB-2, was originally identified in a series of rat neuroblastoma cell lines [1]. This gene encodes a 185-kilodalton (kd) transmembrane glycoprotein with intrinsic tyrosine ki1
Supported by NIH Training Grant T32CA09621-09. Supported in part by a faculty fellowship from the American College of Surgeons. 3 To whom correspondence should be addressed at Suite 6108, One Barnes Hospital Plaza St. Louis, MO 63110. 2
0022-4804/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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surface, with resulting inhibition of antitumor effects [16]. Some p185-specific monoclonal antibodies may have growth stimulatory effects on tumors that overexpress p185 [17]. Finally, the immunogenicity of immunoglobulin molecules makes them a less than ideal pharmacologic modality. While this may be partially obviated by ‘‘humanization’’ of mouse monoclonal antibodies [14], it may not entirely eradicate the problem of anti-antibody immune responses. A different approach to downregulating gene expression is through the use of AS oligonucleotides [18–21]. AS oligonucleotides can directly bind mRNA and can catalyze degradation of the targeted mRNA by the endogenous cellular enzyme RNAse H. However, AS molecules can be degraded by nucleases and can have a variety of non-antisense effects, particularly at high concentrations. Methods to obviate such problems include the use of phosphorothioate oligonucleotides to enhance nuclease resistance and the use of liposome delivery techniques to minimize the concentration of oligonucleotides to which cells are exposed. Recently, Vaughn et al. demonstrated that treatment of SKBR3 breast carcinoma cells with a 15-nucleotidelong phosphorothioate AS oligonucleotide could inhibit HER2/neu mRNA and protein expression in a subpopulation of cell-sorted tumor cells that had received an optimal AS dose, with a resulting increase in cells in the G1 phase of the cell cycle [22]. Direct effects on tumor cell growth were not examined. In investigating approaches to downregulating HER2/neu expression we have replicated the observations of Vaughn et al. and have extended these studies to directly examine effects of HER2/neu antisense oligonucleotides on tumor cell growth in vitro.
was replaced with standard culture medium. After incubating an additional 2 h the cells were taken up with trypsin–EDTA solution and extensively washed with Hanks’ balanced salt solution at 47C, and cellular fluorescence was analyzed using a FACSscan flow cytometer. Northern and Western blotting. Breast cancer cells (5 1 105) were plated into 60-mm dishes under standard culture conditions. After incubating overnight the medium was replaced with serum-free Optimem and oligonucleotides at 1 mM were added in the presence of 10 mg/ml lipofectin. Cultures were incubated for 4 h and then the medium was replaced with standard culture medium. Twenty-four hours later cell lysates were prepared using Trizol (Gibco), and RNA and protein were extracted. Northern blots were performed by electrophoresing total cellular RNA samples (10 mg/lane) through formaldehyde–agarose gels, transferring to nylon membranes, and hybridizing with a 1.9-kilobase HER2/neu human c-DNA probe (nucleotides 1025 to 2914 of the intact c-DNA). A glyceraldehyde phosphate dehydrogenase (GAPDH) probe (Clontech, Palo Alto, CA) was used to demonstrate equal RNA loading. Western blots were performed by electrophoresing protein samples through polyacrylamide gels, transferring to nylon membranes, and probing with a monoclonal antibody specific for the human HER2/neu protein product, p185 (Oncogene Research, Cambridge, MA). An actin-specific monoclonal antibody (Amersham Corp., Arlington Heights, IL) was used to control for equal protein loading. Cell growth assays. Replicate samples of 2 1 104 breast tumor cells were plated in individual wells of a 24-well plate in standard culture medium. After allowing 24–48 h for cells to become adherent, the medium was changed to Opti-mem and oligonucleotides at a final concentration of 1 mM in the presence of 10 mg/ml lipofectin were added for 4 h. The medium was then changed to standard medium containing serum, and the cells were cultured for 5 days. Cells were then taken up in trypsin–EDTA and viable cells counted using a hemocytometer in the presence of trypan blue. Each treatment group was performed in triplicate. Control growth cultures were treated with 10 mg/ml lipofectin without the addition of oligonucleotide. Statistical analysis. The possibility that differences in cell growth between treated groups were due to chance was evaluated using Student’s t test.
RESULTS METHODS Oligonucleotides. Phosphorothioate oligonucleotides were obtained from Oligos, Etc. (Wilsonville, OR). HER2/neu target sequences corresponding to the 5* portion of the HER2/neu mRNA used were based on those of Vaughn et al. [22]: AS, CTCCATGGTGCTCAC; S, GTGAGCACCATGGAG; SC, CGCCTTATCCGTAGC (underlined nucleotides indicate positions at which AS and SC differ). For determination of oligonucleotide uptake using flow cytometry a SC phosphorothioate oligonucleotide modified by coupling fluorescein isothiocyanate (FITC) to the 5* end was used as a tracer (Oligos, Etc.). Oligonucleotide stock solutions were diluted to a concentration of 10 mM in distilled water, sterilized by filtration, and maintained at 0707C. Stock solutions were thawed on ice and diluted appropriately for application to cell cultures. Cell lines. The breast carcinoma cell lines were obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbecco’s modified Eagles medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, and penicillin/streptomycin mixture. Cultures were maintained in a 5% CO2 humidified incubator, at 377C. Flow cytometric analysis of oligonucleotide uptake. BT474 cells (5 1 105) were plated into 60-mm dishes under standard culture conditions. After incubating overnight the medium was replaced with serum-free Opti-mem (Gibco, Gaithersberg, MD) and AS oligonucleotides at 1 mM plus FITC-coupled SC oligonucleotide tracer at a final concentration of 10 nM were added in the presence of 10 mg/ml lipofectin (Gibco). Cultures were incubated for 4 h and then the medium
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Oligonucleotide uptake by BT474 carcinoma cells. Initial studies to determine the efficacy of oligonucleotide delivery to tumor cells were performed using FITCcoupled phosphorothioate molecules. As shown in Fig. 1, cells treated with AS oligonucleotides, to which are added 1% of a FITC SC oligonucleotide tracer, take up a significant amount of the oligonucleotides as determined by flow cytometry 2 h posttreatment. Virtually all tumor cells take up some FITC oligonucleotide with a one- to two-log difference in fluorescence intensity, suggesting variability in cellular uptake within the treated cell population. There is still significant retention of the FITC signal when assayed at 48 h, demonstrating long-term retention of the oligonucleotides (data not shown). Downregulation of HER2/mRNA expression. While it is clear that cells exposed to HER2/neu AS oligonucleotides take up significant amounts of these molecules, the ability of HER2/neu AS treatment to downregulate HER2/neu mRNA is critical in demonstrating a specific AS effect. As shown in Fig. 2, AS oligonucleotide treatment of BT474 tumor cells results in significant downregulation of HER2/neu mRNA expression
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FIG. 1. Cellular uptake of FITC-conjugated oligonucleotides. BT474 cells were treated with FITC-conjugated oligonucleotides (treated) or with lipofectin alone (untreated) for 4 h as described under Methods. After incubating an additional 2 h the cells were washed and processed for flow cytometry. Histograms depict the fluorescence intensity of a total of 10,000 cells in each group. Greater than 97% of treated cells display fluorescence above the upper 1% limit of that seen in untreated cells. The variation in fluorescence intensity in the treated group reflects variable uptake of FITC-conjugated oligonucleotide.
compared to cells treated with lipofectin alone, as determined by Northern blotting. Treatment with control S or SC oligonucleotides has little effect on HER2/neu mRNA expression. Downregulation of p185 expression. Having demonstrated a specific AS effect on HER2/neu mRNA expression, we next examined the effect of AS oligonucleotide treatment on expression of the HER2/neu oncogene product, p185. As shown in Fig. 3, BT474 cells treated with HER2/neu AS oligonucleotides express significantly less p185 protein than do cells treated with either lipofectin alone or S or SC oligonucleotides, as determined by Western blotting. This effect is somewhat less striking than the effect on mRNA due to the significantly longer half-life of protein compared to mRNA; antisense treatment has no effect on protein
FIG. 2. Downregulation of HER2/neu mRNA by AS oligonucleotides. BT474 cells were treated with 10 mg/ml lipofectin alone or with 1 mM AS, S, or SC oligonucleotides in the presence of 10 mg/ml lipofectin for 4 h. Cells were then cultured in standard medium for 24 h and total cellular RNA was extracted and Northern blotting performed as described under Methods. Treatment with HER/neu AS oligonucleotides results in significant downregulation of HER2/ neu mRNA expression. GAPDH probe hybridization confirms equal loading of RNA in each lane.
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FIG. 3. Inhibition of expression of the HER2/neu protein product, p185, by AS oligonucleotides. BT474 and MCF7 breast carcinoma cells were treated with 10 mg/ml lipofectin alone or with 1 mM AS, S, or SC oligonucleotides in the presence of 10 mg/ml lipofectin for 4 h. Cells were then cultured in standard medium for 24 h; total cellular protein was extracted and Western blotting performed as described under Methods. Total protein loaded was 2 mg/lane for BT474 and 20 mg/lane for MCF7. HER/neu AS oligonucleotide treatment results in significant inhibition of p185 expression. Western blotting using an anti-actin antibody confirms equal loading of protein in each lane.
synthesized prior to antisense treatment, but blocks de novo synthesis of p185 as the result of downregulating HER2/neu mRNA. Also demonstrated in Fig. 3 is the effect of oligonucleotide treatment on p185 expression in the MCF7 tumor line. This line expresses 20- to 50-fold less p185 than does BT474, but is equally sensitive to the effects of HER2/neu antisense oligonucleotides on p185 expression. Thus, antisense effects on p185 expression appear to be independent of the intrinsic level of p185 expressed in a particular tumor cell line. Antisense effects on cell growth. It has been demonstrated that HER2/neu AS oligonucleotides specifically downregulate HER2/neu mRNA expression, resulting in lower levels of p185 within treated tumor cells. To determine whether such antisense-mediated downregulation of p185 resulted in changes in cell proliferation, the effects of such treatment on in vitro tumor cell growth were examined. As shown in Fig. 4, AS oligonucleotide treatment results in significant inhibition of BT474 growth when compared to cells treated with S and SC control oligonucleotides. The effect of oligonucleotide treatment on MCF7 breast carcinoma cells, which express lower levels of p185, has also been examined. As shown in Fig. 4, AS oligonucleotide treatment has little effect on the growth of MCF7 tumor cells compared to S or SC controls. It is worth noting that MCF7 cells are more sensitive to the nonspecific toxic effects of phosphorothioate oligonucleotides than are BT474 cells and that both AS and SC groups achieved borderline statistical significance (P õ 0.06) when compared to the growth of cells treated with lipofectin alone. However, it is apparent from the data shown that there is no specific effect of HER2/neu AS oligonucleotides beyond the nonspecific effects caused by control oligonucleotides. This is obviously quite different than the response of BT474 cells, which also show a modest nonspecific effect of S and SC oligonucleotides, but a much more dramatic inhibition of proliferation by HER2/neu AS oligonucleotides. We have examined HER2/neu antisense effects on
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FIG. 4. Effects of oligonucleotide treatment on tumor cell growth. BT474 (elevated HER2/neu expression) and MCF7 (low HER2/neu expression) breast cancer cells were treated with AS, S, or SC oligonucleotides at a final concentration of 1 mM in the presence of 10 mg/ ml lipofectin for 4 h on day 0. Cells were then cultured for 5 days and triplicate samples were counted. Growth of the oligonucleotidetreated groups is expressed as a percentage of the growth of lipofectin-treated controls. It is apparent that AS oligonucleotide treatment inhibits the growth of BT474 cells but has little effect on the growth of MCF7 cells. Error bars indicate standard errors of the mean.
the growth of several other breast cancer lines which express varying levels of p185. As shown in Table 1, the proliferation of several other tumor lines that express low levels of p185 is minimally if at all inhibited by HER2/neu antisense oligonucleotides. Thus, HER2/ neu AS oligonucleotide-mediated downregulation of HER2/neu appears to selectively inhibit the growth of cells that overexpress the HER2/neu oncogene. DISCUSSION
We have examined the effects of HER2/neu-specific phosphorothioate AS oligonucleotides on HER2/neu expression and in vitro growth in human breast carcinoma cell lines. These studies have demonstrated that AS treatment can selectively downregulate HER2/neu mRNA and protein expression; S and SC oligonucleotides have no such effects. Furthermore, HER2/neu AS oligonucleotide treatment selectively inhibits the growth of BT474 cells, which overexpress HER2/neu, but has little effect on the growth of breast carcinoma cells which express lower levels of the HER2/neu oncogene. The effects of HER2/neu AS oligonucleotides on tumor cell proliferation are sequence-specific and targetspecific, affecting only tumor cells which express high levels of p185. It is important to note that the levels of p185 in BT474 cells after AS downregulation are still significantly higher than those of untreated MCF7 cells, and yet the growth rate of cells in MCF7 cultures is greater than that of AS-treated BT474 cultures. Thus, there is not a single threshold level of p185 re-
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quired for proliferation of breast cancer cells. Instead, cells which overexpress HER2/neu appear to be dependent on continued high levels of p185; downregulation of p185 interrupts the excessive mitogenic signaling which results from HER2/neu overexpression. It can be concluded that elevated expression of the HER2/ neu gene is a critical genetic event in the process of multistep carcinogenesis in cells which overexpress HER2/neu. In contrast, breast cancer cells which express low levels of HER2/neu may have abnormalities of a distinct (parallel) growth regulatory pathway responsible for driving neoplastic proliferation, which is independent of HER2/neu, and thus are not affected by downregulation of p185. These results are consistent with the effects of p185-specific monoclonal antibodies on the proliferation of breast carcinoma cells, which are also selectively inhibitory for tumor cells that overexpress p185 [23]. The studies presented here extend the work of Vaughn et al. [22]. The AS effects described by Vaughn et al. required cell sorting and enrichment for an optimally treated subset of tumor cells in order to demonstrate AS-mediated downregulation of HER2/neu mRNA. Effects on cell surface p185 were demonstrated by immunofluorescence, but AS effects on total cellular p185 protein were not examined. Furthermore, effects on cell growth were not directly investigated. In contrast, we have demonstrated effects of HER2/neu AS oligonucleotides on mRNA and protein expression, as well as effects on tumor cell growth, using bulk cell culture populations. Some of the differences between the studies of Vaughn et al. and the results presented here may reflect differences in the breast tumor cell lines studied— they used the SKBR3 line whereas we used BT474. While both lines overexpress HER2/neu to a comparable degree, we have found SKBR3 to be particularly sensitive to non-antisense effects resulting from phos-
TABLE 1 Effects of HER2/neu Antisense Oligonucleotides on Breast Cancer Cell Lines Breast carcinoma cell line
Baseline p185 expression
Specific inhibition of growth by AS oligonucleotidesa (%)
BT474 SKBR3 MCF7 BT20 MDA468
//// //// / / 0
60–80 30–50 õ20 õ10 õ10
Note. Summary of multiple experiments examining effects of HER2/neu AS oligonucleotides on the growth of human breast carcinoma cell lines. a Specific inhibition calculated as inhibition of growth by AS oligonucleotides compared to effects of SC control oligonucleotides. All experiments performed at 1 mM oligonucleotide concentrations, except SKBR3 carried out at 300 nM due to the greater sensitivity of this cell line to nonspecific effects of phosphorothioate oligonucleotides.
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phorothioate oligonucleotides. Lower doses of AS oligonucleotides, 300 nM, were used in the studies of Vaughn et al., presumably to avoid confounding toxic effects, which may have compromised their ability to maximize AS-mediated HER2/neu downregulation. In contrast, we have found minimal non-antisense effects at AS concentrations of 1 mM using the BT474 cell line, as shown here. Since AS effects are clearly dose-dependent (data not shown), our ability to use higher HER2/ neu AS concentrations without encountering nonspecific effects may have allowed us to identify AS effects on HER2/neu expression and cell growth in total (nonsorted) cell cultures. Liu and Pogo, using a different AS sequence targeted to the 5* region of HER2/neu mRNA, have found effects on BT474 growth similar to those reported here [24]. AS oligonucleotides represent a potent approach at downregulating the expression of specific genes [18– 22, 24]. AS studies targeting a number of distinct growth regulatory genes in tumor cell culture systems, including Bcl-2 [25], myb [26], myc [27], protein kinase A [28], protein kinase C [29], and raf [30], have been published, and several of these AS compounds appear to have antitumor activity on human tumors implanted into nude mice. Some of these AS molecules are being tested in phase I and II clinical trials. The HER2/neu oncogene appears to be another useful genetic target for AS oligonucleotide-based therapy. We are currently investigating the in vivo activity of HER2/neu AS, alone and in conjunction with HER2/neu-specific monoclonal antibodies, in tumor xenograft models. REFERENCES
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