ARTICLES

Reversal of Tamoxifen Resistance of Human Breast Carcinomas In Vivo by Neutralizing Antibodies to Transforming Growth Factor-ß

Carlos L. Arteaga, Katri M. Koli, Teresa C. Dugger, Robert Clarke

Affiliations of authors: C. L. Arteaga, Department of Medicine, Vanderbilt University School of Medicine, Vanderbilt Cancer Center, Department of Veteran Affairs Medical Center, Nashville, TN; K. M. Koli, T. C. Dugger, Department of Cell Biology, Vanderbilt University School of Medicine, Nashville; R. Clarke, Lombardi Cancer Center, Georgetown University, Washington, DC.

Correspondence to: Carlos L. Arteaga, M.D., Division of Medical Oncology, Vanderbilt University School of Medicine, 22nd Ave., S., 1956 TVC, Nashville, TN 37232-5536 (e-mail carlos.arteaga{at}mcmail.vanderbilt.edu).


    ABSTRACT
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BACKGROUND: Overexpression of transforming growth factor (TGF)-ß has been reported in human breast carcinomas resistant to antiestrogen tamoxifen, but the role of TGF-ß in this resistant phenotype is unclear. We investigated whether inhibition of TGF-ß2, which is overexpressed in LCC2 tamoxifen-resistant human breast cancer cells, could modify antiestrogen resistance. METHODS: TGF-ß2 expression was evaluated in LCC2 cells and tamoxifen-sensitive LCC1 cells by northern blot analysis. Secreted TGF-ß activity was quantified by use of an 125I-TGF-ß competitive radioreceptor assay. Sensitivity to tamoxifen was measured in a soft agarose colony-forming assay and in a xenograft model in nude and beige/nude mice. Natural killer (NK) cell cytotoxicity was measured by 51Cr release from LCC1 and LCC2 cell targets coincubated with human peripheral blood mononuclear cells. Decrease in TGF-ß2 expression in LCC2 cells was achieved by treatment with antisense oligodeoxynucleotides and confirmed by TGF-ß2 immunoblot analysis. RESULTS AND CONCLUSIONS: The proliferative response of LCC2 cells to tamoxifen in vitro was not altered by TGF-ß neutralizing antibodies. However, established LCC2 tumors in nude mice treated with tamoxifen plus TGF-ß antibodies failed to grow, whereas tumors treated with tamoxifen plus a control antibody continued to proliferate. This reversal of tamoxifen resistance by TGF-ß antibodies did not occur in beige/nude mice, which lack NK-cell function, suggesting that immune mechanisms may be involved in the antitumor effects of tamoxifen. Antisense TGF-ß2 oligodeoxynucleotides enhanced the NK sensitivity of LCC2 cells in the presence of tamoxifen. Finally, LCC1 tumors were markedly more sensitive to tamoxifen in NK-active than in NK-deficient mice. IMPLICATIONS: These data suggest that host NK function mediates, in part, the antitumor effect of tamoxifen and that TGF-ß2 may abrogate this mechanism, thus contributing to tamoxifen resistance.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factors (TGF)-ßs represent a large family of polypeptides involved in the regulation of cellular growth, differentiation, development, morphogenesis, and production of extracellular matrix (1,2). Three homologous mammalian isoforms have been reported: TGF-ß1, TGF-ß2, and TGF-ß3. Although these isoforms overall share similar receptor-binding properties and biologic effects in multiple experimental systems (1,2), the mouse knockout phenotypes for each isoform are different (3-5), suggesting that their effects in vivo may not fully overlap. Expression of TGF-ß1, -ß2, and -ß3 in human breast carcinoma cell lines and tumor tissues varies considerably [see(6)]. Several lines of data support the notion that mammary cell TGF-ßs are autocrine regulators of tumor and nontumor breast epithelial cells (7-9). Although growth inhibition of breast tumor cells with antiestrogens is associated with enhanced secretion of TGF-ß protein (9), transfection of a dominant negative type II TGF-ß receptor into MCF-7 breast cancer cells does not abrogate the response to tamoxifen (10), suggesting that autocrine TGF-ßs may not be universal mediators of tamoxifen action. On the other hand, several reports suggest that, in breast cancer cells, the TGF-ßs are important mediators of tumor progression by fostering critical tumor/host cell interactions like the enhancement of peritumoral angiogenesis and stroma and the inhibition of mechanisms of immune surveillance [reviewed in (6)]. In some cases, overexpression of TGF-ß1 has been temporally associated with estrogen independence and/or resistance to antiestrogens (11-15). Increased immunohistochemical staining for TGF-ß1 protein in breast tumor sections is associated with shorter postmastectomy survival independent of other prognostic factors (16). We have studied the expression and function of TGF-ßs in the tamoxifen-sensitive line LCC1 and the tamoxifen-resistant line LCC2 derived from MCF-7 human breast cancer cells. These cell lines were derived from MCF-7 mouse xenografts established in ovariectomized athymic mice. The LCC2 subline was selected in vitro in the presence of 4-hydroxytamoxifen. Both cell lines exhibit functional estrogen receptors (ERs) and progesterone receptors (PgRs) and form tumors in athymic nude mice in the presence or absence of added estradiol (17,18). This situation provides an experimental model to study mechanisms of tamoxifen in vivo resistance that do not involve loss of the ER.


    METHODS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cell lines and reagents. The LCC1 (tamoxifen-sensitive) and LCC2 (tamoxifen-resistant) breast cancer lines were maintained in phenol red-containing improved minimal essential medium (IMEM; Life Technologies, Inc. [GIBCO BRL], Gaithersburg, MD) supplemented with 10% fetal calf serum (FCS; Hazleton Laboratories, Madison, WI) and 10 nM human insulin. Tamoxifen citrate was purchased from Sigma Chemical Co. (St. Louis, MO) and stored as a 1-mM stock solution in absolute ethanol. The 2G7 and 12H5 immunoglobulin G2 (IgG2) monoclonal TGF-ß-specific antibodies (provided by B. M. Fendly; Genentech Inc., South San Francisco, CA) were generated against human recombinant TGF-ß1 and have been described previously (19). The 12H5 IgG2 is devoid of TGF-ß neutralizing activity, while the 2G7 antibody blocks growth inhibition by TGF-ß1, -ß2, and -ß3 when tested against Mv1Lu mink lung epithelial cells (19).

RNA isolation and northern blot hybridization. Poly(A)+ RNA was purified from subconfluent monolayer cultures using oligo-dT cellulose chromatography as described (20). For northern blot analysis, 3 µg of messenger RNA (mRNA) was fractionated on 1.2% agarose gels containing formaldehyde and transferred to nylon membranes (Micron Separations Inc., Westboro, MA) by capillary transfer. Prehybridization and hybridization were performed at 42 °C in 50% formamide, 250 µg/mL single-stranded DNA, 1x Denhardt's solution, 50 µg/mL poly(A), 0.1% sodium dodecyl sulfate (SDS), and 5x standard saline citrate. Complementary DNA (cDNA) probes for TGF-ß1 (21), TGF-ß2 (22), and cyclophilin (23) were labeled with [32P]deoxycytidine triphosphate (>3000 Ci/mmol; Amersham Life Science Inc., Arlington Heights, IL) using a Rediprime labeling kit (Amersham Life Science Inc.).

Preparation of cell-conditioned medium and TGF-ß radioreceptor assay. Secreted TGF-ß bioactivity was measured in serum-free IMEM conditioned for 24 hours by adherent breast cancer cells as described previously (24). To activate secreted latent TGF-ß, the conditioned medium was acidified with 1 N HCl to pH 1.5 for 1 hour at 4 °C and then neutralized with 1 N NaOH before testing in a TGF-ß radioreceptor assay (24) utilizing AKR-2B (84A) mouse fibroblasts. Binding was performed in six-well plates in 1 mL/well binding buffer (24) containing 0.25 ng/mL. 125I-TGF-ß1 (specific activity, 173 µCi/µg; Du Pont NEN, Boston, MA) competed with variable volumes of conditioned medium. Human recombinant TGF-ß1 (Genentech Inc.) was used to generate a standard curve from which the receptor binding activity of conditioned medium was calculated and then standardized to the number of cells from which the conditioned medium was collected. In this binding assay with AKR-2B (84A) cells, TGF-ß1 and TGF-ß2 are equipotent in displacing 125I-TGF-ß1 binding (25). Addition of 1 µM tamoxifen directly to the binding buffer had no effect on TGF-ß1 binding in this assay (Arteaga CL: unpublished data).

Natural killer (NK) and lymphokine-activated killer (LAK) cell cytotoxicity assays. Sparse adherent cultures of LCC1 or LCC2 target cells (104 cells per well in a 96-well plate) were labeled for 4-6 hours at 37 °C with approximately 200 µCi/mL 51Cr (specific activity, 400-1200 Ci/g; Du Pont NEN) in IMEM supplemented with 10% FCS. Unbound radioactivity was removed after two washes with growth medium before the addition of effector cells. Human peripheral blood lymphocytes (PBLs) from healthy volunteers were prepared by Ficoll-Hypaque gradient centrifugation. After two washes, variable numbers of NK effector cells were added to the 51Cr-labeled breast cancer cell targets in a final volume of 0.2 mL/well in quadruplicate. To generate LAK effector cells, PBLs were incubated for 5 days at 37 °C in 5% CO2 with 1000 U/mL human recombinant interleukin 2 (Chiron Therapeutics, Emeryville, CA) and then added in ratios ranging from 5 : 1 to 100 : 1 to freshly labeled tumor cell targets. Spontaneous 51Cr release and PBL-induced (experimental) 51Cr release were measured in both assays after an overnight coincubation, and percent cytotoxicity was calculated as described previously (26) by the formula: ([experimental release - spontaneous release]/total release) x 100.

Mouse studies. Five- to 8-week-old female athymic (nude) mice (Harlan Sprague-Dawley, Inc., Madison, WI) or NIH-III beige/nude mice (Taconic Farms, Inc., Germantown, NY) were inoculated subcutaneously just caudal to the forelimb with approximately 5 x 106 LCC1 or LCC2 tumor cells in 0.25 mL of serum-free IMEM via a 22-gauge needle. Variable times after tumor cell inoculation, 25-mg, 60-day release tamoxifen pellets (Innovative Research, Toledo, OH) were implanted subcutaneously in the back at a distant site from the tumor via a 10-gauge trocar. In some experiments, animals received daily intraperitoneal injections of 2G7 or 12H5 antibodies (100 µg each) in a 0.2-mL volume via a 26-gauge needle. Tumor diameters were measured serially with calipers, and tumor volume was calculated by the formula: volume = width2 x length/2. At the completion of the experiments, some tumors were removed, fixed in 10% formalin, and paraffin-embedded, and 5-µm-thick sections were cut, stained with hematoxylin-eosin, and subjected to light microscopy. Care of all mice used in these studies was in accord with institutional guidelines.

Effects of antisense TGF-ß2 oligonucleotides and immunoblotting of TGF-ß2 secreted in cell-conditioned medium. TGF-ß2 antisense phosphorothioate oligodeoxynucleotides as well as a nonsense oligonucleotide sequence of similar length and with the same GC content (used as control) were synthesized by and purchased from Ransom Hill Bioscience, Inc. (Ramona, CA), on the basis of a recent report (27). A search in the European Molecular Biology Laboratory (EMBL) GenBank revealed no homologies between the 14-mer antisense sequence used in this study and the 5' coding region of any known gene sequence. For inhibition of TGF-ß2 expression, LCC2 cells were treated for 48 hours at 37 °C in serum-free IMEM with 3 µM antisense or nonsense oligonucleotides. Cell medium was then collected, cleared of cell debris by centrifugation, concentrated approximately 30-fold in a Centriprep 30 MW column (Millipore Corp., Bedford, MA), boiled in Laemmli sample buffer containing the reducing agent dithiothreitol (DTT), and resolved by 15% SDS-polyacrylamide gel electrophoresis. Following transfer to nitrocellulose, membranes were subjected to an immunoblot procedure that utilizes a TGF-ß2 polyclonal antiserum R & D Systems Inc., Minneapolis, MN). Detection of TGF-ß2 reactive bands in autoradiograms was performed with horseradish peroxidase-linked antirabbit immunoglobulins and enhanced chemiluminescence.

Statistical analyses. To analyze tumor growth curves, we used a repeated-measures analysis of variance (ANOVA) model with a serial correlation structure. The analysis was based on the logarithm of the tumor volume to stabilize the variance. To avoid strict assumptions about normality, the General Estimating Equation approach was used to fit the model, as implemented in SAS/PROC GENMOD software (SAS Institute, Inc., Cary, NC). The in vitro NK and LAK cytotoxicity data were analyzed using ANOVA, as implemented in SAS/PROC GLM software. For these cytotoxicity experiments, each data point represents the mean cpm of 51Cr released from labeled target cells in four wells. If the standard deviations of the quadruplicate determinations were less than 10% of the mean value (before the experimental release was subjected to the formula for calculating % cytotoxicity [above]), they were not included in the figures.


    RESULTS
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TGF-ß Expression and Immune Sensitivity of LCC1 and LCC2 Breast Cancer Cells

We first examined steady-state mRNA levels of TGF-ß1 and TGF-ß2 in tamoxifen-sensitive and tamoxifen-resistant cells. LCC2 cells express similar low levels of TGF-ß1 mRNA (not shown) but greater than 20-fold higher levels of TGF-ß2 mRNA relative to LCC1 cells (Fig. 1, A).Go Protein levels in serum-free medium conditioned by LCC2 cells, as measured in a 125I-TGF-ß1 radioreceptor assay, were 6.8 ng of TGF-ß equivalents/106 cells per 24 hours in the absence of acid activation in vitro, whereas LCC1 cells only secreted lower levels of latent TGF-ß activity (Fig. 1, B)Go. A 24-hour incubation with 1 µM tamoxifen up-regulated TGF-ß2 mRNA levels in LCC2 cells but not in LCC1 cells (Fig 1, A)Go. By radioreceptor assay of serum-free cell medium, tamoxifen (1 µM) treatment for 24 hours increased the secretion of (active) TGF-ß activity to 13.9 ng/106 cells per 24 hours, whereas, in LCC1 cell medium, this bioactivity remained undetectable. To test the paracrine effects of TGF-ß2 overexpression in the context of a multicellular experimental system, we measured NK and LAK sensitivity of 51Cr-labeled LCC1 and LCC2 cells. NK and LAK cells are potently suppressed by exogenous TGF-ß1 and TGF-ß2 (28). Consistent with these data and the overexpression of TGF-ß2, LCC2 cells were statistically more resistant to NK cell-mediated lysis (P = .0001) and LAK cell-mediated lysis (P = .0002) than tamoxifen-sensitive LCC1 cells (Fig. 2).Go




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Fig. 1. A) Transforming growth factor (TGF)-ß2 messenger RNA (mRNA) expression in LCC1 (tamoxifen-sensitive) and LCC2 (tamoxifen-resistant) human breast cancer cells. Poly(A)+ mRNA was isolated by oligo-dT-cellulose chromatography. Where indicated, cells were treated with 1 µM tamoxifen (Tam) for 24 hours prior to lysis and RNA collection. For northern blot analysis, 3 µg of mRNA per lane was fractionated on 1.2% agarose gels. After mRNA transfer, the nylon membranes were probed with [32P]deoxycytidine triphosphate-labeled TGF-ß2 and 1B15 (cyclophilin) probes as described in the "Methods" section. The 5.1-kilobase TGF-ß2 transcript was over-represented in LCC2 tumor cell RNA. B) Secretion of TGF-ß activity into conditioned medium (CM). Serum-free conditioned medium was collected at 24 hours and tested in a 1-mL 125I-TGF-ß1 radioreceptor competition assay as described in the "Methods" section. Left: Standard curve using AKR-2B (84A) mouse fibroblasts and 0.25 ng/mL 125I-TGF-ß1 in the absence or presence of 0.25-25 ng/mL unlabeled TGF-ß1. Right: Competing activity of 250 µL (25% conditioned medium of neutral [N] and 750 µL (75% conditioned medium) of transiently acidified (A) conditioned medium from LCC1 and LCC2 cells. When standardized on the basis of cell number, LCC2 cells secreted more than 10 ng/mL of TGF-ß1 equivalents per 106 cells in a 24-hour period. Each data point represents the mean counts per minute (cpm) ± standard deviation of triplicate wells.

 


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Fig. 2. Natural killer (NK) cell- and lymphokine-activated killer (LAK) cell-mediated cytotoxicity against LCC1 and LCC2 breast cancer cells. Breast cancer target cells were labeled with 51Cr in improved minimum essential medium supplemented with 10% fetal calf serum in 96-well plates and then incubated overnight with human peripheral blood lymphocytes (PBLs) at the indicated effector-to-target ratios (for assessment of NK function). For assessment of LAK function, PBLs were incubated at 37 °C in 5% CO2 with 1000 U/mL interleukin 2 (added only once) for 5 days and then coincubated with labeled target cells. Release of 51Cr was measured to calculate percentage cytotoxicity as described previously (23). Each data point represents the mean cytotoxicity calculated from four wells. NK, LCC1 versus LCC2: P<.0001; LAK, LCC1 versus LCC2: P = .0002 by analysis of variance.

 
TGF-ß Neutralizing Antibodies and Sensitivity to Tamoxifen In Vivo and In Vitro

To study the role of TGF-ß2 in tamoxifen resistance of LCC2 cells, we utilized the anti-TGF-ß 2G7 IgG2 antibody, which neutralizes all three TGF-ß mammalian isoforms (19). Established subcutaneous LCC2 tumors in nude mice bearing 60-day-release, 25-mg subcutaneously implanted tamoxifen pellets were randomized to 100 µg/day of 2G7 or the 12H5 non-neutralizing anti-TGF-ß control IgG2 by intraperitoneal injection. These systemic doses of 2G7 result in detectable and sustained plasma levels of an activity that blocks TGF-ß receptor binding when tested ex vivo (26). In animals treated with 2G7, but not with the 12H5 control antibody, tumor growth was arrested (Fig. 3, A)Go, suggesting that tumor cell TGF-ß2 was mediating tamoxifen resistance in this experimental system. A second experiment yielded similar results. By light microscopy, both groups of LCC2 tumors were poorly differentiated adenocarcinomas with no major histologic differences between them. In the absence of tamoxifen treatment, 2G7 had no effect on LCC2 xenograft growth relative to 12H5-treated tumors, suggesting that tumor cell TGF-ßs had no effect on basal LCC2 tumor growth (not shown).




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Fig. 3. Effect of anti-transforming growth factor (TGF)-ß antibodies on tamoxifen (Tam) resistance of LCC2 breast cancer cell-induced tumors in vivo. A) Studies in athymic nude mice. Approximately 5 x 106 LCC2 cells were inoculated subcutaneously in the flank of female nude mice. On day 14, once the tumors had reached a volume of greater than 100 mm3, all mice received a 25-mg, 60-day-release tamoxifen pellet subcutaneously at a site distant from the tumor. The following day, 100 µg/day of the 2G7 monoclonal antibody or the control immunoglobulin G2 (IgG2) 12H5 was injected intraperitoneally, and antibody injections were continued daily for the next 3 weeks. Tumor diameters were measured serially with calipers and tumor volumes in mm3 were calculated as described in the "Methods" section. Each data point represents the mean tumor volume ± standard deviation from six mice. 12H5-treated versus 2G7-treated tumor growth: P<.0001 by analysis of variance. B) Studies in NK-deficient beige/nude mice. NIH-3 beige/nude mice (NK-deficient) were inoculated subcutaneously with 5 x 106 LCC2 tumor cells. On day 10, once tumors had reached a volume of greater than 100 mm3, tamoxifen pellets were implanted subcutaneously. The following day, daily intraperitoneal injections with 100 µg of the 2G7 or 12H5 monoclonal antibodies were started and continued for the next 3 weeks as in panel A. Each data point represents the mean tumor volume ± standard deviation from six mice.

 
Previous studies (26-29) have shown that systemic administration of anti-TGF-ß antibodies can up-regulate NK function in nude mice that, in turn, exerts an antitumor effect. In addition, tamoxifen can induce and/or sensitize tumor cells to NK and LAK cells in vitro and in vivo (discussed below), thus implying that some of its antitumor effect is mediated by modulating this biologic response. These observations plus the high levels of NK activity present in athymic nude mice (30) and the relative resistance of LCC2 cells to NK activity (Fig. 2Go), a known cellular target for TGF-ß2, suggested to us a model in which tamoxifen exerts some of its antitumor effect by up-regulating host NK function in vivo. Overexpression of immunosuppressive cytokines, like TGF-ß2, can then counteract this tamoxifen-mediated host response and in part contribute to resistance to this antiestrogen. To test this hypothesis, we repeated the experiment shown in Fig. 3, A,Go in NK-deficient beige/nude mice. Neutralization of TGF-ß2 with 2G7 did not restore tamoxifen sensitivity to LCC2 tumors in NK-deficient mice (Fig. 3, BGo), suggesting that, in this resistance model, NK host function is critical for the antitumor effect of tamoxifen in vivo.

To further support the hypothesis that tumor host mechanisms were involved in the restoration of tamoxifen sensitivity by neutralizing antibodies, we examined the effect of 2G7 in tamoxifen-treated LCC2 cells in vitro. In this cell-autonomous experimental system, LCC2 colony formation in the presence of 1 or 10 µg/mL 2G7 antibody was similar in the presence or absence of 1 µM tamoxifen (Fig. 4).Go Tamoxifen stimulated LCC2 colony formation approximately 25% above ethanol (solvent for tamoxifen, used as control) in the presence of 1 or 10 µg/mL of the 12H5 control monoclonal antibody.



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Fig. 4. Effect of neutralizing anti-transforming growth factor (TGF)-ß antibodies on LCC2 breast cancer cell colony-forming ability in vitro. A single-cell suspension of 3 x 104 LCC2 tumor cells per dish were plated in soft agarose on 0.1% ethanol (EtOH) or 1 µM tamoxifen (Tam; in 0.1% ethanol) as described in the "Methods" section. For each condition and where indicated, cells were coincubated or not (control [ctl]) with 1 or 10 µg/mL 12H5 or 2G7 antibodies. In the presence of either antibody, there were no differences in clonogenicity between tamoxifen-treated and ethanol-treated control cultures (P = .35; Pearson's x2 test). Each data point represents the mean number of colonies ± standard deviation from three dishes.

 
Antisense Oligodeoxynucleotide-Mediated Inhibition of Tumor Cell TGF-ß2 Expression and Enhancement of Tamoxifen-Induced NK Toxicity Against LCC2 Cells In Vitro

Several published data [see(31)] indicate that tamoxifen can stimulate immune effector cellular mechanisms in the tumor host as well as sensitize tumor cell targets to cytotoxicity independent of ER expression. Therefore, we first examined in a multicellular experimental system whether this sensitization to NK action was different in the tamoxifen-sensitive LCC1 versus tamoxifen-resistant LCC2 cells. Consistent with their phenotype in vivo, coincubation of NK effector cells with target cells with tamoxifen in vitro resulted in sensitization of the LCC1 cells (P = .0001). On the contrary, addition of tamoxifen reduced the lower level of NK-mediated 51Cr release from LCC2 cells even further (P = .001; Fig. 5).Go



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Fig. 5. Effect of tamoxifen (Tam) on natural killer (NK) cell activity against LCC1 and LCC2 breast cancer cells. Breast tumor cells (104 per well) in 96-well plates were labeled with 51Cr in the presence or absence of 1 µM tamoxifen and then incubated overnight with peripheral blood lymphocytes at the indicated effector-to-target ratios in the presence or absence of an identical concentration of tamoxifen. Each data point represents the mean cytotoxicity derived from four identical wells. Tamoxifen-sensitive LCC1, but not tamoxifen-resistant LCC2 cells, were sensitized by tamoxifen to NK-mediated lysis. LCC1 with tamoxifen versus LCC1 without tamoxifen: P<.0001; LCC2 with tamoxifen versus LCC2 without tamoxifen: P = .001 (analysis of variance).

 
To test the contribution of TGF-ß2 to NK resistance in the presence of tamoxifen, we used antisense phosphorothioate oligodeoxynucleotides (27). Treatment of LCC2 cells with 3 µM antisense TGF-ß2 oligodeoxynucleotides for 48 hours abrogated the secretion of TGF-ß2, compared with nonsense oligodeoxynucleotides as measured by immunoblot of concentrated serum-free conditioned medium (Fig. 6).Go Proliferation of LCC2 cells was not affected during the 48-hour incubation with antisense oligonucleotides compared with nonsense oligonucleotide-treated cultures. We then examined the effect of antisense oligonucleotide-mediated inhibition of TGF-ß2 expression in LCC2 cells on NK sensitivity in the absence and presence of tamoxifen. Treatment of LCC2 cells with TGF-ß2 antisense oligonucleotides enhanced their sensitivity to NK effector cells in the presence of tamoxifen at all effector-to-target ratios tested, but not in the absence of tamoxifen (Fig. 7),Go suggesting that, in these tamoxifen-resistant cells, TGF-ß2 mediates, in part, this relative resistance to NK activity and that down-regulation of TGF-ß2 expression can unmask the NK-sensitizing effect of tamoxifen.



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Fig. 6. Antisense-mediated inhibition of transforming growth factor (TGF)-ß2 secretion. LCC2 breast cancer cells were treated for 48 hours in serum-free improved minimum essential medium in the presence of 3 µM nonsense or antisense TGF-ß2 phosphorothioate oligodeoxynucleotides. Cell medium was collected, concentrated as described in the "Methods" section, and then subjected to an immunoblot procedure utilizing a TGF-ß2 specific polyclonal antibody. Denaturation by sodium dodecyl sulfate and boiling in the presence of dithiothreitol result in reduction of the TGF-ß2 active dimer to a monomeric TGF-ß2 species of approximately 12.5 kD detectable in the medium from cells treated with nonsense oligodeoxynucleotides, but not antisense oligodeoxynucleotides.

 


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Fig. 7. Effect of transforming growth factor (TGF)-ß2 antisense oligodeoxynucleotides on basal (absence of tamoxifen) and tamoxifen-stimulated natural killer (NK) cell function. LCC2 breast cancer cells were preincubated in serum-free improved minimum essential medium for 48 hours with 3 µM nonsense or antisense TGF-ß2 oligodeoxynucleotides, and the cells were labeled with 51Cr for the last 6 hours of this incubation period. After three washes, peripheral blood lymphocytes were added at the indicated ratios of effector-to-target cells, and isotope release from the target cells was measured after an overnight coincubation in the absence (A) or presence (B) of 1 µM tamoxifen (Tam) in RPMI-1640 medium supplemented with 10% fetal calf serum. Each data point represents the mean cytotoxicity calculated from four wells. All standard deviations from these quadruplicate determinations were less than 10%. Antisense oligodeoxynucleotide treatment of NK targets markedly enhanced LCC2 sensitivity to NK effector cells compared with nonsense oligonucleotide-treated cells when coincubated in the presence of tamoxifen (P<.0001 by analysis of variance).

 
Antitumor Effect of Tamoxifen in Nude Mice Versus Beige/Nude Mice Against Tamoxifen-Sensitive LCC1 Xenografts

To test whether both the growth-inhibitory effect and the sensitizing effect of tamoxifen on tumor targets to NK effector cells were related phenomena, we concurrently examined the inhibitory effect of tamoxifen on LCC1 tumors in nude mice, which exhibit elevated NK cell function (30), as well as in beige/nude mice. In these animals, the beige mutation markedly reduces NK function (30). Tamoxifen pellets were implanted 2 days after subcutaneous tumor cell inoculation. Within 4 weeks, 100% of LCC1 tumors formed in both nude and beige/nude mice without the need for estrogen supplementation and in the absence of tamoxifen treatment (Table 1).Go Four weeks after tumor inoculation, there were no detectable LCC1 xenografts in tamoxifen-treated nude mice. Conversely, five of eight NK-deficient beige/nude animals treated with tamoxifen exhibited subcutaneous tumors that measured more than 4 mm in diameter (>100 mm3). The formation of LCC1 tumors suggested that, in this tumor model, host NK function was, in part, mediating the growth-inhibitory effect of tamoxifen (Table 1Go). At 8 weeks, there were still no detectable LCC1 tumors in nude mice treated with tamoxifen.


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Table 1. Antitumor effect of tamoxifen in nude versus beige/nude mice*

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We have studied the role of TGF-ßs in the tamoxifen-resistant phenotype of LCC2 human breast cancer cells. Hormone-dependent breast carcinoma cells follow a predictable pattern from antiestrogen sensitivity to the acquisition of antiestrogen resistance. A number of molecular and cellular mechanisms are involved in the emergence of this phenotype, including the (infrequent) loss of ER expression, the selection of ER mutants with altered transcriptional responses, alterations in the intracellular pharmacology and/or binding of tamoxifen to breast cancer cells, and other non-ER-related mechanisms (see below). Several reviews (32,33) cover discussions on these mechanisms in more detail. The experimental models employing LCC1 and LCC2 cells were used in this study. These cells maintain high ER levels and up-regulate PgR levels in response to exogenous estradiol (17,18), thus providing a good system to study mechanisms of tamoxifen resistance that do not involve ER loss. Compared with other tamoxifen-resistant tumor models, LCC2 tumor growth is not stimulated by tamoxifen (18).

LCC2 tamoxifen-resistant cells markedly overexpress TGF-ß2 compared with LCC1 tamoxifen-sensitive cells. Tamoxifen resistance in LCC2 tumors was abrogated by the coadministration of the antiestrogen with neutralizing TGF-ß antibodies when tested in nude mice. This response was not seen in beige/nude mice that lack NK activity. Furthermore, tamoxifen sensitized the tamoxifen-sensitive LCC1 cells, but not the resistant LCC2 cells, to NK effector cells in vitro. Antisense oligonucleotide-mediated inhibition of TGF-ß2 secretion by LCC2 cells restored the ability of tamoxifen to sensitize LCC2 cells to NK cell-induced cytolysis. Finally, tamoxifen exhibited a greater antitumor effect against LCC1 tumor cells in NK-plus nude mice compared with that seen in NK-deficient beige/nude mice. Taken together, these data suggest that overexpression of TGF-ß2 by breast tumor cells can mediate tamoxifen resistance in vivo. Our results also suggest that host NK function is involved in the antitumor effect of the antiestrogen tamoxifen and that overexpression of potently immunosuppressive cytokines, like TGF-ß2, can counteract this antiestrogenic effect in the host and thus contribute to tamoxifen resistance. Of note, however, tamoxifen reduced the low level of NK cell-mediated toxicity against LCC2 cells (Fig. 5Go). This particular result may reflect the tamoxifen-mediated increase in TGF-ß2 mRNA and the activation of secreted TGF-ß activity in LCC2 cells (Fig. 1Go), which in turn can block NK cell function.

Our results would seem at odds with the dogma that proposes antiestrogen-induced up-regulation of autocrine TGF-ßs in breast carcinoma cells and tumor tissues as a mediator of tamoxifen's antitumor action (8,9), and that loss of this autocrine pathway can contribute to tamoxifen resistance. The LCC1/LCC2 model we used argues against this hypothesis in that tamoxifen-sensitive LCC1 cells bind TGF-ß1 poorly and do not respond to exogenous TGF-ß1. On the other hand, the LCC2 resistant cells express all three TGF-ß receptors in high levels (data not shown) and exhibit responses to exogenous TGF-ß1 in culture (34). Furthermore, a more recent study by Koli et al. (10) also challenges this dogma, in that transfection of a suppressible dominant-negative truncated type II TGF-ß receptor into antiestrogen- and TGF-ß-sensitive MCF-7 cells abrogated TGF-ß responses but not tamoxifen-mediated growth inhibition.

A rise in either the plasma levels of TGF-ß2 (35) or the tumor levels of TGF-ß2 mRNA (36) has been observed in patients with metastatic breast tumors receiving tamoxifen therapy. In one of these studies, TGF-ß1 and TGF-ß3 expression in tumors was not altered by tamoxifen therapy (36), suggesting a TGF-ß isoform-specific effect of tamoxifen. It was proposed in these studies that up-regulation of TGF-ß2 expression is a marker of clinical response to tamoxifen. However, the great majority of tamoxifen-sensitive mammary tumors will progress to a tamoxifen-resistant state, suggesting the possibility of a subsequent temporal association between breast tumor TGF-ß2 overexpression and tamoxifen resistance. This is consistent with our findings in the LCC2 cells. TGF-ß1 overexpression has also been temporally associated with antiestrogen resistance in human breast cancer cell lines (11,12) and mammary tumor tissues (13,15).

The greater inhibition of tamoxifen-sensitive LCC1 tumors in nude mice, which have elevated NK and normal LAK activities, compared with that seen in beige/nude NK-deficient mice, suggests that the antiproliferative effect of tamoxifen and the sensitization to host NK (and possibly LAK) function are related phenomena. Natural cytotoxicity, mediated by NK cells, is believed to play an important role in host antitumor surveillance mechanisms. NK cells are predominantly large granular lymphocytes, the majority of which express CD16 and CD56 cell surface antigens and represent overall 5%-8% of PBLs (37). NK cells can mediate cytolysis in the absence of major histocompatibility complex class 1 and class 2 antigen expression in target cells (37,38). In patients with breast cancer and in experimental systems, tamoxifen has been shown to enhance NK function and/or increase the sensitivity of tumor cell targets in an ER-independent fashion (31,39-42). However, in one report by Gottardis et al. (43), prolonged treatment with tamoxifen inhibited NK function in nude mice and stimulated growth of tamoxifen-resistant MCF-7 tumors. Nonetheless, NK activity is inversely related to the clinical stage of disease and/or the presence of axillary lymph node metastases in patients with breast cancer (44-46), implicating host natural cytotoxicity in the control of cancer progression. Accumulation of NK cells in tumors was temporally associated with a clinical spontaneous remission in a patient with metastatic breast carcinoma (47). Other reports (48-50) indicate that pharmacologically achievable concentrations of tamoxifen enhance immune cytolysis of tumor targets mediated by LAK cells and cytotoxic T cells. Overall and except for the observation by Gottardis et al. (43), these reports and our results are consistent with the hypothesis that host immune function may play a role in the antitumor effect of triphenylethylene antiestrogens, independent of the ER status of the tumor cell target. These reports also suggest the possibility of synergistic antitumor effects of tamoxifen with anti-TGF-ß antibodies in other ER-negative neoplasms. This possibility remains to be tested.

In vitro, anti-TGF-ß antibodies failed to reverse tamoxifen resistance in LCC2 cells. This result suggests that the observed reversal in vivo may not involve ER signaling in LCC2 cells. Other non-ER mechanisms of antiestrogen-mediated tumor inhibition that involve paracrine mechanisms have been reported. For example, tamoxifen and pure ER antagonists inhibit endothelial cell proliferation within mammary tumors and promote the apoptosis of angiogenesis-dependent breast tumors (51,52). Moreover, MCF-7 tumors transfected with fibroblast growth factor (FGF)-4 and FGF-1 develop hematogenous metastases in vivo and are unresponsive to tamoxifen (53-55), suggesting a role for angiogenic factors in ER-independent resistance to tamoxifen. Of note, TGF-ßs are potent inductors of angiogenesis (2,56), thus leading to a possible causal association between overexpression of TGF-ßs, high intratumoral microvessel density, and a tamoxifen-resistant phenotype. Whether enhanced TGF-ß2-mediated angiogenesis is involved in the antiestrogen resistance of TGF-ß2-overexpressing LCC2 tumors will require further experiments.

In summary, we have described a novel mechanism of antiestrogen resistance, as well as a potentially important immunologic component in tamoxifen response and acquired resistance in human breast carcinoma cells. It is interesting that the in vitro selection of LCC2 cells against tamoxifen produced a resistance mechanism that functions through an effect on cells or mechanisms (NK function) not involved in the initial selection. However, the greater effect of tamoxifen against LCC1 tumors in NK-competent versus NK-deficient mice, as well as the TGF-ß2 antisense oligonucleotide-mediated sensitization of LCC2 cells to NK effectors in vitro in the presence of tamoxifen, strongly argues in favor of this immunologic component. In addition, the published effects of tamoxifen therapy on the expression or content of TGF-ßs in patients' tumors and tumor models (13,15,36) suggest that tamoxifen-induced overexpression of TGF-ßs can also occur in vivo. Several practical implications could be derived from these data. First, the effects of tamoxifen on tumor host (endothelial, immune) cells will be missed in cell-autonomous in vitro screens and potentially lead to false-negative preclinical models. Second, ER-positive tumors that overexpress TGF-ßs may be clinically unresponsive de novo to tamoxifen and/or escape antiestrogens early and would, therefore, be candidates to alternative molecular therapies aimed at down-regulating the overexpressed immunosuppressive (or angiogenic) cytokines. Future prospective clinicoepidemiologic studies in patients should clarify the practical implications of these data and address how predictably the overexpression of TGF-ßs by ER-positive breast tumors may determine clinical resistance to tamoxifen and other antiestrogens in general.


    NOTES
 
Supported by Public Health Service grants R01CA58022 (R. Clarke), R01CA62212 (C. L. Arteaga), and CA65485 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; Merit Review and Clinical Investigator grants from the Department of Veteran Affairs (C. L. Arteaga); and the Susan G. Komen Foundation (C. L. Arteaga).

We thank Dr. John Hanfelt (Lombardi Cancer Center) for his helpful comments and consultation.


    REFERENCES
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Manuscript received February 5, 1998; revised October 28, 1998; accepted November 2, 1998.


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