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`Clinical Cancer Research
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`697
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`Tamoxifen-resistant Fibroblast Growth Factor-transfected MCF-7
`
`Cells Are Cross-Resistant in Vivo to the Antiestrogen
`ICI 182,780 and Two Aromatase Inhibitorsl
`
`Sandra W. McLeskey, Lurong Zhang,
`Dorraya El-Ashry, Bruce J. Trock,
`Cecilia A. Lopez, Samir Kharbanda,
`Christopher A. Tobias, Lori A. Lorant,
`Rachel S. Hannum, Robert B. Dickson, and
`Francis G. Kern2
`Lombardi Cancer Center [5. W. M.. L. 2.. B. J. T.. C. A. L.. S. K..
`C. A. T.. R. S. H.. D. E-A.. R. B. D.. F. G. K.l. Departments of
`Biochemistry and Molecular Biology lD. E—A.. F. G. K.|. Cell
`Biology [R. B. D.. L. 2.]. Medicine [8. l. T.]. and Pharmacology
`(S. W. M.]. and the School of Nursing [8. W. M.l. Georgetown
`University Medical Center. Washington. D. C. 20007
`
`ABSTRACT
`
`Although the antiestrogen tamoxifen has been the
`mainstay of therapy for estrogen receptor (ER)-positive
`breast cancer, successful treatment of responsive tumors is
`often followed by the acquisition of tamoxifen resistance.
`Subsequently, only 30-40% of patients have a positive re-
`sponse to second hormonal therapies. This lack of response
`might be explained by mechanisms for tamoxifen resistance
`that sensitize ER pathways to small amounts of estrogenic
`activity present in tamoxifen or that bypass ER pathways
`completely. To elucidate one possible mechanism of tamox-
`ifen resistance, we treated ovariectomized tumor-bearing
`mice injected with fibroblast growth factor (FGF)-trans-
`fected MCF-7 breast carcinoma cells with the steroidal an-
`
`tiestrogen 1C1 182,780 or one of two aromatase inhibitors,
`4-OHA or letrozole. These treatments did not slow estrogen-
`independent growth or prevent metastasis of tumors pro-
`duced by FGF-transfected MCF-7 cells in ovariectomized
`nude mice. FGF-transfected cells had diminished responses
`to [Cl 182,780 in vitro, suggesting that autocrine activity of
`the transfected FGF may be replacing estrogen as a mito-
`
`Received 7/3/97: revised 11/26/97: accepted l2/l()/97.
`The costs of publication of this article were defrayed in pan by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with l8 U.S.C. Section 1734 solely to
`indicate this fact.
`lThis work was supported by NIH Grants CA50376 (to F. G. K.).
`CA092|8 (to F. G. K. and S. W.M.), CA53185 (to F. G. K. and
`R. B. D.). CA66I54 (to S. W. M.). CA71465 (to D. E—A.), and Cancer
`Center Grant CA51008; American Cancer Society Grant lRG-l93 (to
`S. W. M.): US. Army Medical Research and Material Command Grants
`DAMD l7-94-4l72 (to D. E-A.)
`and DAMD l7-94-J-4l73 (to
`S. W. M.): and a Susan Komen Foundation Fellowship (to L. 2.).
`2To whom requests for reprints should be addressed. at Southem
`Research lnstitute. P. O. Box 55305. 20(X) Ninth Avenue South. Bir-
`mingham. AL 35255-5305. Phone:
`(205)581—2480: Fax: (205)58l-
`2877; E-mail: kern@sri.org.
`
`genie stimulus for tumor growth. ER levels in FGF trans-
`fectants were not down-regulated, and basal levels of tran-
`scripts for estrogen-induced genes or of ER-mediated
`transcription of estrogen response element (ERE) luciferase
`reporter constructs in the FGF expressing cells were not
`higher than parental cells, implying that altered hormonal
`responses are not due to down-regulation of ER or to FGF-
`mediated activation of ER. These studies indicate that estro-
`
`gen independence may be achieved through FGF signaling
`pathways independent of ER pathways. If so, therapies di-
`rected at the operative mechanism might produce a thera-
`peutic response or allow a response to a second course of
`antiestrogen treatment.
`
`INTRODUCTION
`
`therapy is not usually curative in
`Because conventional
`clinical breast cancer, development of tamoxifen resistance.
`in
`which breast tumors previously growth-inhibited by tamoxifen
`become refractory.
`represents an important
`therapeutic di-
`lemma. However, the development of tamoxifen resistance is
`not necessarily associated with progression to an ERJ-negative
`phenotype. In many cases of clinical tamoxifen resistance, ER
`expression may be retained (1—4). implying that the resistance is
`due an alteration in activity of the tamoxifen/ER complex.
`Tamoxifen resistance in such a case could result from three
`
`possible mechanisms that. according to present knowledge,
`would not preclude successful
`treatment with an alternative
`hormonal therapy. First. alterations in the ER could arise. which
`might diminish or extinguish inhibitory responses to tamoxifen.
`leaving only its partial agonist effects to predominate (5—8).
`Second. tamoxifen resistance arising in the setting of an intact
`ER could be a result of altered intratumoral tamoxifen metab-
`
`olism. which might produce more estrogenic metabolites locally
`(7. 9—l 1). Third. available tamoxifen could be sequestered by an
`increase in antiestrogen binding sites not associated with ERs
`(12). As mentioned. in each of these three instances. substitution
`of a hormonal therapy different from tamoxifen might result in
`a clinical response. Two such alternative therapies used in this
`report are steroidal estrogen antagonists. such as lCI 182.780.
`which lack the partial agonist activity of tamoxifen. and aro-
`matase inhibitors, which inhibit endogenous estrogen produc-
`tion by all tissues. depriving the ER of its ligand.
`Although the mechanisms of tamoxifen resistance de-
`
`" The abbreviations used are: ER. estrogen receptor: FGF. fibroblast
`growth factor:
`lMEM.
`improved minimal essential medium: X-gal.
`5-bromo-4-chloro-3-indoyl-B-D—galactopyranoside: FBS.
`fetal bovine
`serum: 4-OHA. 4—hydroxyandrostenedione; NK. natural killer: CCS.
`charcoal-stripped calf serum: ERE. estrogen response element: CAT.
`chloramphenicol acetyltransferase: RT. reverse transcription.
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`698 ICI 182.780 Effects on FGF-transfected MCF—7 Cells
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`scribed above should be amenable to alternative hormonal ther-
`
`apy. early results for small numbers of tamoxifen-resistant pa-
`tients have shown that only about 30—40% of such patients have
`a positive response to subsequent
`ICI 182.780 or aromatase
`inhibitor therapy (13—20). These data imply alternative mecha-
`nisms for tamoxifen resistance. Constitutive production of au-
`tocrine growth factor(s) or growth factor receptors by tumor
`cells has been proposed as a mechanism for tamoxifen resist-
`ance that may or may not
`involve ER pathways. Evidence
`supporting this hypothesis is gained from the acquisition of
`estrogen-independent growth in tumor models,
`including the
`one used in this report. in which growth factors or growth factor
`receptors have been overexpressed in estrogen-dependent breast
`carcinoma cell lines (21—26). In addition. recent clinical data
`showing decreased efficacy of tamoxifen in treating tumors
`overexpressing c-erbBZ (27) supports a role for growth factor
`signaling in Clinical tamoxifen resistance. Because some growth
`factor signaling pathways. including the ERB-B pathway. have
`been shown to interact with ER signaling pathways (25. 28-32).
`increased growth factor signaling could be one mechanism by
`which cells could become sensitive to previously ineffective
`amounts of estrogenic stimulation produced by the partial ago—
`nist activity of tamoxifen itself or its estrogenic metabolites.
`above. In cases in which such interactions have been demon-
`
`strated. the growth factor and ER pathways may act collabora-
`tively (25), making the final outcome susceptible to pharmaco-
`logical manipulations of either pathway and implying that
`second line hormonal therapies might have an effect. However.
`increased autocrine or intracrine growth factor signaling might
`also bypass the need for ER—mediated growth stimulation in
`tumor cells or affect stromal components of the tumor. such as
`endothelial or immune cells (33—36). to alter the tumor envi-
`ronment in ways conducive to tumor growth.
`In either case,
`alternative hormonal therapies might not be effective.
`Recently. cell-specific coactivators and corepressors have
`been identified for steroid hormone receptors. including the ER,
`which may influence steroid receptor—induced transcription pos-
`itively or negatively (37, 38). Thus. the activity of tamoxifen in
`inhibiting or even stimulating tumor growth might depend on
`the relative expression of various stimulatory or inhibitory co-
`factors in a particular tumor (39. 40). However. transient trans-
`fection experiments suggest that tamoxifen-resistant tumors pro-
`duced by such mechanisms should still be sensitive to pure
`antiestrogens (40).
`FGFs and their receptors have been shown to be present
`with high frequency in breast cancer specimens (41—50). Evi-
`dence for a possible role for FGF signaling in the estrogen-
`independent growth of breast tumors is gained from study of
`clonal and polyclonal FGF-transfected MCF-7 cell lines. which
`are capable of forming large. progressively growing tumors in
`ovariectomized or tamoxifen-treated nude mice. Moreover. the
`
`FGF—transfected cells are metastatic. forming micrometastases
`in lymph nodes.
`lungs. and other organs (21. 22. 51). The
`estrogen-independent and tamoxifen-resistant growth of FGF-
`transfected MCF-7 cells suggests an interaction between FGF
`signaling pathways and ER-activated pathways that could occur
`at the level of the ER itself or at the end point of both pathways.
`where they impinge on growth mechanisms. If FOE—mediated
`growth pathways bypass the ER pathway to affect growth di-
`
`rectly. we would expect that growth would be unaffected by
`hormonal treatments devoid of agonist activity. We therefore
`sought to determine the sensitivity of the estrogen-independent
`tumor growth of FGF—transfected MCF-7 cells to ICI l82.780 or
`aromatase inhibitors. In contrast to what was seen with ERB-B
`
`that FGF-mediated pathways
`signaling pathways. we report
`appear to provide an alternative growth stimulatory signal that is
`not dependent on ER activation.
`
`MATERIALS AND METHODS
`Cell Lines. FGF-transfected MCF-7 cell lines have been
`
`described previously (21. 22.51. 52). Briefly, the ML-20 clonal
`cell line is a MCF—7-derived cell line that is stably transfected
`with a lac-Z expression vector. The in vitro and in vivo growth
`characteristics of ML-20 cells are indistinguishable from wild-
`type MCF-7 cells (51). and >90% of the cells routinely stain
`positive for B-galactosidase expression by X-gal staining (52).
`MKL-F (FGF—4-transfected; Ref. 52) and FGF-l clone 18 (FGF-
`1-transfected) cells (22) resulted from the stable transfection of
`the ML-20 clonal cell line with expression vectors for FGF-4
`(also known as hst-l/K-FGF) and FGF-l (also known as acidic
`FGF or aFGF). respectively. Both cell lines continue to stably
`express B-galactosidase. allowing effects of FGF overexpres-
`sion on metastatic capability to be assessed by X—gal staining of
`organs and tissues of tumor-bearing mice. The MKL-4 cell line
`was derived by transfecting wild-type MCF-7 cells (of similar
`passage number used for the ML-20 transfection) with an exv
`pression vector for FGF-4. which produced the clonal MKS-I
`cells (21). These cells were then retransfected with an expres—
`sion vector for lm'Z. yielding MKL-4 cells (51). Cells were
`maintained in IMEM (Biofluids. Rockville. MD) supplemented
`with 5% FBS in a humidified. 37°C. 5% CO2 incubator in
`routine culture until used for tumor cell injection.
`Drugs.
`ICI 182,780 was kindly donated by Dr. Alan
`Wakeling of Zeneca Pharmaceuticals (Macclesfield, England).
`and was administered s.c. at a dose of 5 mg in 0.1 ml of vehicle
`every week. For the experiment depicted in Fig. 1. powdered
`drug was first dissolved in 100% ethanol and spiked into
`warmed peanut oil (Eastman Kodak. Rochester. NY) to give a
`final concentration of 50 mg/ml. For the experiments depicted in
`Fig. 1. B and C. 50 mg/ml preformulated drug in a vehicle of
`10% ethanol. 15% benzyl benzoate. 10% benzyl alcohol.
`brought to volume with castor oil. was supplied by B. M. Vose
`(Zeneca Pharmaceuticals). 4-OHA was donated by Angela Bro-
`die (University of Maryland. Baltimore. MD) and was admin-
`istered s.c. at a dose of l mg/mouse/day 6 days of the week in
`a vehicle of 0.3% hydroxypropylcelluose. Letrozole was do-
`nated by Dr. Ajay Bhatnagar (Novartis. Ltd.. Basil. Switzerland)
`and was administered via gavage at a dose of I mg/mouse/day
`6 days of the week in a vehicle of 0.3% hydroxypropylcellulose.
`Sustained-release (60 day) pellets containing 5 mg of tamoxifen
`were obtained from Innovative Research of America (Sarasota,
`FL) and implanted s.c. in the interscapular area at the time of
`tumor cell injection.
`Tumor Cell Injection. The procedure for tumor cell
`injection has been described previously (2]). Briefly.
`tumor
`cells were scraped into their normal growth medium. and viable
`cells were quantified using trypan blue exclusion. The cells were
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`resuspended in their normal growth medium at a density of
`66.7 X 10“ cells/ml. and 0.15 ml (containing 10 million cells)
`were used to inject ovariectomized mice (nude or beige/nude/
`xid) into the mammary fat pad. For the experiment involving
`MKL-4 cells and nude mice (Fig. 1A). each mouse was injected
`bilaterally into the thoracic mammary fat pads (two injections
`per mouse). There were seven mice in the vehicle group and five
`mice in each treatment group. For the experiments involving
`MKL—4 cells and beige/nudelrid mice (Fig. 2). four tumor cell
`injections were given. two on each side in the thoracic mam—
`mary fat pad and two in the inguinal mammary fat pad; treat-
`ment groups consisted of four mice. For the experiments involv-
`ing MKL—F and FGF-1, clone 18 cells (Fig.
`l. B and C). each
`mouse was injected once in the right thoracic mammary fat pad.
`There were seven mice in the each vehicle group, and treatment
`groups consisted of five or six mice each. Tumors resulting from
`the injections were measured twice weekly in three dimensions
`using calipers. Tumor volume is the product of the largest
`dimension. the orthogonal measurement. and the tumor depth. as
`described previously (21). Because the FGF-l-transfected clone
`18 cell line produces tumors that in some cases are surrounded
`by a fluid-filled sac that confounds tumor measurements (22).
`these tumors were measured postmortem by weighing them.
`Determination of Metastasis. Organs were harvested
`from tumor-bearing animals. fixed briefly. and stained with
`X-gal as reported previously (51) and viewed through a dissect-
`ing microscope (Olympus SZH). Clusters of blue-staining cells
`were identified as micrometastases. In accordance with previous
`results, no macrometastases were identified (21. 22. 51. 53).
`Growth Assays. Anchorage-dependent and anchorage—
`independent growth assays were performed as described (21).
`Briefly. for anchorage-dependent growth. cells were plated in
`24-well culture dishes at a density of 10,000 cells/well for the
`time course experiments (Fig. 4) and 20,000 or 30.000 cells/well
`for the concentration-response experiments (Fig. 5). For growth
`in FBS. following overnight attachment. treatments were added
`at the indicated concentrations. and cells were counted on the
`indicated days. For growth assays under estrogen-depleted con-
`ditions. cells were stripped of estrogens during a 24-h period the
`day following plating by changing the medium four times to
`phenol red-free IMEM supplemented with 5% CCS (21). We
`have found that this stripping procedure allows complete re-
`moval of estrogens without substantial proliferation of cells
`before treatments are added. Following the stripping procedure.
`on day 0. treatments were added. and counting of cells was done
`as above.
`
`Doubling times were determined according to the follow-
`ing equation: doubling time = t2 — t,/3.3210g(N2/N,), where N2
`and NI are the number of cells at times 12 and 1,. respectively. Nl
`and N: are the means of quadruplicate determinations.
`Anchorage-independent assays in PBS-containing medium
`were done as described previously (21 ). For experiments using
`estrogen-depleted conditions. cells were stripped of estrogens
`over a 24-h period as described above before being plated in soft
`agar. Colonies greater than 60 jun were counted using an
`Omnicon 3600 Image Analysis system.
`ER Assays.
`[JHlEstradiol binding has been described
`previously (54. 55). Briefly. cells grown to 70% confluence
`were stripped with twice daily medium changes over 4 days
`
`with 5% CCS in phenol red-free IMEM. The prolonged strip-
`ping method allows ERs to become up-regulated to maximal
`levels. Cells were harvested. washed sequentially at 4°C with
`serum-free. phenol red-free IMEM followed by TEG (10 mM
`Tris. pH 7.4. 1 mM EDTA, 10% glycerol). and resuspended in 1
`ml of TEG plus 1 mM DTT. 0.5 M NaCl and a cocktail of
`protease inhibitors (1 mg/ml leupeptin. 77 jag/ml aprotinin.
`1
`ug/ml pepstatin A). A whole-cell extract was prepared by ho-
`mogenization with 40 strokes in a Teflon—glass Dounce homog—
`enizer followed by centrifugation at
`l05.000 X g for 30 min.
`Protein content of the supernatant was determined by the
`method of Bradford (56), and protein concentrations were ad-
`justed to 2 mg/ml. Extracts were incubated with 10 nM PH] 178-
`estradiol with or without a [00-fold excess of unlabeled estra—
`
`diol for 16 h at 4/C. Unbound ligand was removed by absorption
`with dextran-coated charcoal followed by centrifugation. Ali-
`quots of the supernatant were counted in a Beckman liquid
`scintillation counter.
`
`Northern Blots. Cells were grown to 50% confluence in
`IMEM supplemented with 5% FBS and then stripped of estro-
`gens as described for the growth assays. above. Treatments of
`0.1% ethanol (vehicle) or 10’“ M l7B-estradiol
`in the same
`medium were added. Cultures were harvested after 3 days of
`treatment. and RNA was extracted using RNAzol B (Tel—Test.
`lnc.) according to the manufacturer's directions. Thirty ug of
`each RNA were subjected to electrophoresis in a 1.2% formal-
`dehyde/agarose gel and transferred to nylon (Hybond-N. Am-
`ersham Corp., Arlington Heights. IL) by capillarity. JzP-labeled
`antisense riboprobes for pS—2. GAPDH. and cathepsin D were
`prepared and sequentially hybridized to the membrane overnight
`at 65°C [hybridization buffer was 50% formamide. 50 mM
`NazHPOJ. 0.8 M NaCl. 10 mM EDTA. 2.5 X Denhardt's solu—
`tion (1X Denhardt‘s = 0.02% polyvinylpyrrolidone. 0.02%
`BSA). 0.2% SDS. 400 jig/ml yeast
`tRNA. and 400 ug/ml
`sonicated salmon sperm DNA with 10" DPM/ml of the appro-
`priate probe]. The membrane was washed three times in 0.1%
`SDS/O.l X SSC at 80°C for the PS—2 and cathepsin D probes.
`and 75°C for the GAPDH probe. Autoradiograms and Phospho-
`rlmager (Molecular Dynamics Model 44551) quantitation of
`individual hybridization signals were obtained between the se-
`quential hybridizations. For the results depicted in Fig. 7. A and
`B. Phosphorlmager values obtained for PS-2 or cathepsin were
`normalized to those obtained for GAPDH.
`
`Progesterone Receptor mRNA Determination by RT-
`PCR. The primers for human progesterone receptor that pro-
`duce a 205-bp PCR product have been described previously
`(57). The human GAPDH primers that produce a 437-bp PCR
`product are as follows: 5’-AAG GTC GGT GTG AAC GGA
`TTT G-3’ (sense) and 5’—TGG TGC AGG ATG CAT TGC
`TG—3' (antisense). RT-PCR was performed with 0.1 ug of test
`RNAs. except T47D cells, where 0.02 pg was used, using the
`GeneAmp RNA PCR kit (PE Applied Biosystems. Foster City.
`CA) according to the manufacturer’s instructions with the fol-
`lowing modifications: the RT reaction was primed with 0.0625
`uM random hexamers in a volume of 40 a]. with 2 pl each of
`"SS-labeled UTP and SSS-labeled ATP (each 3000 Ci/mmol. 10
`aCi/ul. Amersham Corp.) substituted for water in the reaction.
`Then. 20 ul of each RT reaction were transferred into two tubes
`for separate GAPDH and progesterone receptor PCR reactions.
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`InnoPharma Exhibit 1008.0003
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`700 1C1 182.780 Effects on FOE-transfected MCF-7 Cells
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`Cycle analyses using RNA from ML-20, estradiol-treated cells
`(the highest expressors of progesterone receptor) revealed that
`amplification remained logarithmic at 35 cycles for the GAPDH
`reaction and 40 cycles for the progesterone receptor reaction.
`making these assays semiquantitative. The GAPDH PCR reac-
`tion was performed using standard reagent conditions recom-
`mended by the manufacturer and cycles of 95°C for 45 s and
`50°C for 45 s for 35 cycles. For the progesterone receptor PCR
`reaction. final MgCl2 concentrations were adjusted to 1.25 mM,
`and 0.25 M acetamide was included. Cycles were of 95°C for
`45 s and 50°C for 45 s for 40 cycles. GAPDH and progesterone
`receptor reaction products were first visualized by ethidium
`bromide staining following electrophoresis in a 2% agarose gel.
`Products were then electrophoresed on a 4—20% acrylamide gel
`that was subjected to both autoradiography and Phosphorlmager
`quantitation as described above.
`Transient Transfection, Luciferase, and CAT Reporter
`Assays. ML-20 and clone 18 cells were plated in 6-well
`plates. allowed to attach overnight. and stripped of estrogens in
`a procedure similar to that for the growth assays (see above).
`Following stripping, cells were transfected by the calcium phos-
`phate. low-CO2 method (58). The luciferase plasmids pGLB-
`MERE or pGLB-MNON were obtained by inserting an approx-
`imately 1.48—kb fragment containing a glucocorticoid response
`element-deleted mouse mammary tumor virus promoter with
`either a substituted double consensus ERE (MERE) or the same
`sequence with the ERE palindromes scrambled (MNON) (59)
`into the HindIII site of pGLB (Promega, Madison, WI). Each
`dish received 2.5 pg of either pGLB-MERE or pGLB-MNON
`and 1.0 pg pCMV-CAT. which directs constitutive expression
`of CAT, cotransfected as a control for transfection efficiency.
`Following transfection. each well was washed twice with PBS
`and incubated for 48 h in medium containing vehicle (0.01%
`ethanol). 10’" M estradiol, 10"7 M ICI 182,780, a combination
`of E: and 1C1. 10 ng/ml FGF-l plus 10 ug/ml heparin. or a
`combination of FGF. heparin. and ICI 182.780. (Duplicate sam-
`ples of each treatment were used.) Cells were lysed and assayed
`for luciferase activity using the Luciferase Reporter Gene Assay
`(Boehringer Mannheim, Indianapolis, IN) according to the man—
`ufacturer's instructions. Luciferase values. expressed as relative
`light units. for each sample were corrected for background by
`subtracting the value of lysates of untransfected cells prepared
`in parallel. CAT expression was assayed using the CAT ELISA
`(Boehringer Mannheim. Indianapolis. IN) according to the man-
`ufacturer‘s instructions. Protein content of the lysates was de-
`termined using the BCA Protein Assay Reagent (Pierce, Rock-
`ford. 1L). Luciferase and CAT values. normalized for protein.
`were used to calculate mean specific relative light units/ng CAT.
`Statistical Analyses. Statistical methods used for tumor
`growth have been described previously (53. 60). For Figs.
`1 and
`2. only mice surviving at
`the end of the experiment were
`included in the analysis. When no tumor developed from a
`particular injection, tumor volume was recorded as zero. The
`repeated measures ANOVA (60) was used to compare tumor
`volumes among the treatment groups using measurements taken
`over the entire time course of the experiment. In addition, final
`tumor volumes (or weights in the case of clone 18) were
`compared among treatment groups at the end of each experi-
`ment using ANOVA. For analysis of metastasis in Table l. for
`
`each transfectant, analysis of covariance was used to compare
`the effects of treatment on total metastases. total distant metas—
`tases (lung metastases plus other metastases), lymph node me-
`tastases.
`lung metastases. and other metastases. The analyses
`were all conducted with final tumor volume (or weight for the
`clone 18 cells)
`included in the model as a covariate. The
`analyses considered the effects of all treatments simultaneously,
`as well as the effects of individual
`treatment comparisons
`(which were adjusted for multiple comparisons using Dunnett’s
`method). For each transfectant. the effect of final tumor volume
`(or weight for clone 18) on the number of metastases was
`evaluated using linear regression (for each of the categories of
`metastasis described above). In Fig. 3. paired t tests were per-
`formed comparing control and transfected cells under different
`conditions of treatment. For the anchorage-dependent growth
`assays depicted in Fig. 4, we examined the effect of treatment on
`the rate of cell growth, using linear regression with an interac-
`tion between time and treatment. To compare cell growth rates
`and doubling times among the cell lines under specific treatment
`conditions, nested linear regression models were used. For Fig,
`6. ANOVA was used to determine significant differences in ER
`binding among cell lines.
`
`RESULTS
`
`Estrogen-independent Growth of Tumors Produced by
`FGF-transfected MCF-7 Cells Is Not Inhibited by Treat-
`ment with a Pure Antiestrogen or with Aromatase Inhibi-
`tors. We have previously shown that both FGF-1- and FGF—
`4-transfected MCF-7 cells form progressively growing tumors
`in ovariectomized nude mice. as well as in similar mice
`treated with tamoxifen (21, 22. 53). Although ovariectomized
`mice could be expected to have substantially lower levels of
`estrogenic compounds than reproductively intact mice, some
`estrogens are synthesized at extraovarian sites. such as adre-
`nal gland, liver, fat. or possibly the tumor itself. The trans-
`fected cells evidently still possess ERs, because they respond
`to estrogen and tamoxifen administered to the mice, as well
`as to these compounds used in tissue culture (21. 22). To test
`the hypothesis that growth of the FGF-transfecled cells in
`ovariectomized or tamoxifen-treated nude mice is due to
`
`increased sensitivity to the small amounts of estrogens still
`present in ovariectomized nude mice, we tested the ability of
`a pure antiestrogen. ICI 182.780, and two aromatase inhibi-
`tors, 4-OHA and letrozole. to inhibit the estrogen-indepen-
`dent tumor growth produced by these FGF—transfected cell
`lines.
`
`In a first experiment to test the above hypothesis, FGF-4-
`transfected MKL-4 cells were injected as before. and the mice
`were treated with vehicle.
`tamoxifen. or ICI 182780. There
`
`were no significant differences in tumor volume among the
`treatment groups considered over the entire time course of the
`experiment (P = 0.72) or at the final time point (Fig. M; P =
`0.72). Treatment with ICI 182.780 did not inhibit tumor growth
`below that achieved in vehicle-treated mice (P = 0.675). Thus.
`the failure of ICI 182.780 to inhibit the estrogen-independent
`growth exhibited by this cell line supports the hypothesis that
`such growth does not result from small amounts of estrogenic
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`0
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`B MKL-F (FGF»4)
`
`
`
`
`
`TumorWeight(mg)
`
`&8
`
`C FGF-l.clone18 [Ill
`
`DJ00
`
`—N88
`
`Days after Injection
`
`Days after Injection
`
`Treatment
`
`E VEH s\\\\\‘ 4-OHA
`- TAM m LET
`
`ICI
`
`Fig. 1 Growth of FGF-transfected MCF-7 cells in ovariectomized nude mice is not inhibited by treatment with [Cl 182.780, 4-OHA. or Ietrozole.
`Ten million cells from the indicated cell lines were injected into the mammary fat pads of ovariectomized nude mice treated with vehicle (VEH); a
`5-mg, 60-day-release tamoxifen pellet (TAM); ICI 182,780, 5 mg s.c. every week (ICI):
`I mg of 4-OHA s.c. per day 6 days of the week (4-0HA);
`or 1 mg of Ietrozole per day via gavage 6 days of the week (LET). Columns, group mean; bars SE. Numbers above each column are the percentages
`of injections resulting in measurable tumors at that time point. A. volumes of tumors produced by one clonal FGF-4—transfected MCF-7 cell line.
`MKL-4, at the indicated number of days following tumor cell injection. B, volumes of tumors produced by a second clonal FGF—4-transfected MCF-7
`cell line. MKL-F, at the indicated number of days following tumor cell injection. C. weights of tumors produced by a clonal FGF- 1 -transfected MCF-7
`cell line. FGF—l, clone 18. weighed after sacrifice of the animals 28 days after tumor cell injection. (Because the FGF-l producing MCF-7 cells may
`form fluid-filled sacs around the tumor. confounding tumor measurements before sacrifice. only postmonem weights are presented here.)
`
`growth stimulation achieved by extraovarian estrogen produc-
`tion.
`
`We wished to assess the effect of ICI 182,780 on metastasis
`as well as on tumor growth.
`In spite of its retention of the
`transfected lat-Z expression plasmid, the MKL—4 cell line be-
`comes heterogeneous over time with respect to B-galactosidase
`expression, such that a few cells have high expression. but most
`are negative (52). We therefore used a second clonal FGF-4-
`transfected MCF-7 cell
`line, MKL—F, the B-galactosidase ex-
`pression of which is stable. for a subsequent experiment involv-
`ing FGF-4-transfected MCF-7 cells. Because FGF-l has also
`been shown to produce estrogen—independent
`in vivo growth
`when transfected into MCF—7 cells (22), we also included a
`clone of FGF—I-transfected cells designated clone 18. the B-ga-
`lactosidase expression of which is also stable. For these exper-
`iments, two aromatase inhibitors, 4-OHA (61. 62) and Ietrozole
`(63), were also used to inhibit extraovarian synthesis of estro-
`gens.
`
`In agreement with the experiment using MKL—4 cells de-
`picted in Fig.
`IA, when the FGF-4-transfected MKL-F cells
`were used, there were no differences in tumor volume among
`treatment groups over all
`time points (P = 0.382), and ICI
`182,780 did not decrease tumor growth below that obtained in
`vehicle-treated animals (Fig. 18; P = 0.837 for the last time
`point). In addition, neither 4-OHA nor Ietrozole decreased tu-
`mor growth below vehicle-treated levels (P = 0.571 and 0.931
`for the last time point, respectively).
`FGF-l-transfected clone 18 cells form tumors that are
`
`accurate tumor volume measurements during the course of the
`experiment. Consequently. when these cells were used (Fig.
`1C). only terminal tumor weights were analyzed with ANOVA.
`As with the MKL-4 and MKL-F cells, ICI 182.780 did not
`
`inhibit estrogen-independent tumor growth in the clone 18 cells
`(P = 0.977). Administration of 1C1 182,780 to animals injected
`with ML-20 cells. a clonal line of B-galactosidase-transfected
`wild-type MCF-7 cells (51), also produced no effect when
`compared with vehicle-treated animals [i.e., no progressively
`growing tumors were obtained in either case (data not shown”.
`In other, separate experiments. a polyclonal population of con-
`trol vector-transfected ML-20 cells that forms progressively
`growing tumors in estrogen-supplemented mice (22) did not
`form tumors in either untreated or lCl 182,780-treated animals"
`Thus. the continued progressive in vivo growth of FGF~trans-
`fected cells in ovariectomized animals treated with either a pure
`antiestrogen or aromatase inhibitors demonstrates that the estro-
`gen-independent growth of these cells in untreated ovariecto-
`mized nude mice is not due to estrogenic activity produced at
`extraovarian sites.
`Because ICI 182,780, 4-OHA, and Ietrozole were without
`effect in the experiments described above. we injected repro-
`ductively intact female mice for 2 weeks with these compounds
`at the same doses used in the above experiments to observe for
`activity in preventing effects of endogenous estrogens on the
`
`sometimes surrounded by a fluid-filled sac (22, 53). preventing
`
`‘ Unpublished results.
`
`InnoPharma Exhibit 1008.0005
`
`
`
`702 1C] 182.780 Effects on FGF-transfected MCF-7 Cells
`
`Table I Metastasis of FGF—transfected MCF-7 cells is not inhibited
`by treatment with ICI 182.780 or aromatase inhibitors
`Mice were sacrificed and tumors and organs were subjected to
`X-gal staining as described previously (51). Mice bearing tumors pro-
`duced by injection of MKL—4 cells were sacrificed at 61 days; for
`MKL~F tumors. mice were sacrificed after 64 days: and for FGF-1 clone
`18 tumors. mice were sacrificed after 28 days.
`Metastatic site
`
`and incidence of metastasis were indeed significantly correlated
`(P = 0.014).
`Effects of FGF and/or Estrogen on the Residual Im-
`mune System of Nude Mice Is Not Responsible for the
`Estrogen-independent Growth of FGF-transfected MCF-7
`Cells. Although nude mice have a T-cell defect. they retain
`NK cell activity. It has been postulated that the residual NK
`activity in nude mice is responsible for some xenograft rejection
`and poor metastatic ability of xenografts (35). Estrogen and
`tamoxifen have been shown to decrease NK cell activity in nude
`mice (36), but estrogen increases the ability of NK cells to kil