Evaluation of Continuous Infusion Suramin in Metastatic Breast Cancer: Impact on Plasma Levels of Insulin-Like Growth Factors (Igfs) and IGF-Binding Proteins’

Vol. 3, 1713-1720, October 1997 Clinical Cancer Research 1713

Evaluation of Continuous Infusion Suramin in Metastatic Breast Cancer: Impact on Plasma Levels of Insulin-like Growth Factors (IGFs) and IGF-binding Proteins’

James B. Lawrence, Cheryl A. Conover, after suramin raises concern and underscores the importance of measuring relevant biomarkers in clinical trials. Tufia C. Haddad, James N. Ingle, Joel M. Reid, Matthew M. Ames, Vera J. Suman, INTRODUCTION Randolph S. Marks, Charles Erlichman, and Breast cancer is a leading cause of death, and its treatment Lynn C. Hartmann2 produces significant morbidity for hundreds of thousands of Endocrine Research Unit IJ. B. L., C. A. C.. 1. C. HI, Department of women annually. Innovative therapeutic approaches for breast Oncology [J. N. I., J. M. R., M. M. A., R. S. M., C. E., L. C. H.], and cancer are needed, especially for those cancers unresponsive to Section of Biostatistics IV. J. SI, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905 current hormonal and chemotherapeutic strategies. Breast car- cinogenesis has been shown to be dependent on a complex autocrine and paracrine network of growth factors, and attempts ABSTRACT to exert an antitumor effect through manipulation of peptide Suramin represents a new class of antitumor drugs that growth factors represent one new therapeutic approach. targets growth factor networks. In this Phase II trial, sura- Multiple reports over the last decade have established mm was administered by continuous infusion to 10 patients IGF-13 and IGF-II as important mitogens for transformed cells with advanced breast cancer. The target level of 280 iWml and tissues, including breast cancer ( 1-3). The IGFs exert their suramin was achieved in a median of 10 days; toxicities in mitogenic action through binding to IGF-I receptors on the this patient group were low. We monitored the insulin-like plasma membrane of cells (4). This receptor is expressed in growth factor (IGF) network in these patients because of the most breast cancer cell lines and tumor biopsies (5). Activation previously defined growth-promoting role of the IGFs in of the IGF-I receptor by IGFs initiates a mitogenic signal breast cancer. Plasma levels of total IGF-I and total IGF-II transduction cascade. This response in breast cancer cell lines showed variable responses to suramin with median de- can be inhibited by antisense RNA or antibody specific to the creases of 24 and 23%, respectively, for the 10 patients; for IGF-I receptor (6, 7). Arteaga ci a!. (8) reported that breast total IGF-I levels, this did not reach statistical significance. cancer tumor growth in nude mice could be inhibited in a On the other hand, free IGF-I plasma levels were consis- dose-dependent manner by IGF-I receptor antibody. This anti- tently and dramatically increased (over 250%) after sura- body also inhibited the growth of T6 I breast cancer xenografts mm infusion. IGF-binding proteins (IGFBPs), modulators of (9). Because the IGFs serve as potent activators of breast cancer IGF bioavailability, were also measured. Levels of IGFBP-3, cell growth in vito and in vitro, the ability to regulate these the major carrier of IGFs in the circulation, were decreased mitogens may well provide a means to moderate breast cancer 21 % after suramin treatment when measured by immuno- cell growth. radiometric assay. However, the majority of the plasma Suramin represents a new class of antitumor drugs that IGFBP-3 remaining after suramin was not the intact high- targets growth factor networks. Although suramin’s mechanism affinity IGF-binding form but rather a 30-kDa fragment of action is not completely understood, in vitro studies have with markedly reduced affinity for IGF-I. IGFBP-3 protease shown that it inhibits the binding of peptide growth factors, activity was evident in the plasma of 3 of 10 patients after including IGFs, to their receptors (10, 1 1). Suramin has shown suramin. Measurements of plasma IGFBP-1, IGFBP-2, and promising antineoplastic effects on several tumor types, includ- IGFBP-4 revealed no significant changes in response to ing prostate and adrenocortical malignancies and some lympho- suramin. The dramatic increase in active free IGF-I seen mas (12-14). In breast cancer, suramin has growth-inhibitory effects in vitro in both hormone-insensitive ER-negative breast cancer cell lines and hormone-responsive ER-positive breast cancer cells (IS). In these cultured cells, suramin inhibited Received 3/25/97; revised 6/24/97; accepted 7/7/97. IGF-I- and IGF-II-stimulated proliferation in a time- and dose- The costs of publication of this article were defrayed in part by the dependent manner. payment of page charges. This article must therefore be hereby marked In a pilot study involving eight patients with metastatic advertisement in accordance with I 8 U.S.C. Section 1 734 solely to indicate this fact. cancer resistant to standard therapy, including four patients with I Supported by NIH Grants DK-07352 (to J. B. L.), DK-43258 (to C. A. C.), MO1RROO585-24 (to the Mayo General Clinical Research Center), and P30-CA15083 (Cancer Center Support Grant), Phase Il-Ill Contract CM-07309, and the Mayo Foundation. 2 To whom requests for reprints should be addressed, at Mayo Clinic, 3 The abbreviations used are: IGF, insulin-like growth factor: IGFBP, Medical Oncology, 200 First Street SW, Rochester, MN 55905. Phone: IGF-binding proteins; ER, estrogen receptor; ALS, acid-labile subunit; (507) 284-4718; Fax: (507) 284-1803. IRMA, immunoradiometric assay; CV, coefficient of variation.

breast cancer, Miglietta ci a!. ( 16) observed a significant inverse only patients experiencing clinical benefit were to be continued relationship between the plasma levels of suramin and the levels on suramin therapy. of total IGF-I and IGF-II. These results are similar to the effects The objective response criteria were as follows: complete of treatment with the antiestrogen tamoxifen. Tamoxifen, an regression, disappearance of all evidence of tumor; partial re- effective hormonal agent in ER-positive breast cancers, has been gression, 50% or greater reduction in the product of the longest shown to decrease total IGF-I concentrations in women with perpendicular diameters of the indicator lesion(s); stable, failure advanced breast cancer (17, 18). to qualify for complete regression, partial regression, or pro- Total IGF concentrations represent IGFs in association gression; and progression, increase of 25% in the product of the with specific IGFBPs, seven of which have been identified to longest perpendicular diameters of the indicator lesion(s) or the date ( 19, 20). Greater than 90% of plasma IGFs are tightly appearance of new lesions. bound to IGFBP-3 and an ALS within a high molecular weight Suramin (NCS 3496) was provided by the Pharmaceutical

complex of - I SO kDa that prevents extravascular transit. IGFs Resources Branch, National Cancer Institute (Bethesda, MD). not captured in the ternary complex can traverse the capillary The first day’s dose was divided into an initial test dose of 200 endothelium and are available to local tissues (21). Thus, this mg given iv. over 10 mm (in SO ml of dextrose 5% in water), portion of circulating IGFs may be biologically important. which was followed after 20 mm with the initiation of a con- Given the recognized importance of growth factor net- tinuous infusion at 350 mg/m2/day. Plasma suramin concentra- works in breast cancer and suramin’s antitumor activity in vitro, tions were measured starting on day 3, and the subsequent rate we initiated a Phase II study of suramin in patients with mini- of suramin infusion was adjusted to reach a desired suramin mally pretreated advanced breast cancer. In this study, we level of 280-300 p.g/ml. One cycle was completed at this point measured IGFs and IGFBPs in the plasma of patients both of suramin infusion. Because of suramin’s tendency to cause before and after suramin infusion to assess the potential impact adrenal cortical damage, all patients were started on oral hydro- of this treatment on IGF bioavailability. Our data underscore the cortisone, 25 mg every am. and IS mg every p.m. at the time of importance of monitoring relevant biomarkers in clinical trials initiation of suramin treatment. and confirm the complexity of the IGF system in human breast Plasma Plasma samples were obtained daily from each cancer. patient during suramin infusion. EDTA was used as the antico- agulant. IGF studies were performed on sera presuramin and after plasma suramin levels reached 280 p.g/ml. Plasma was MATERIALS AND METHODS stored at -70#{176}Cuntil use. Eligibility Criteria. Eligible patients had histologically High-Performance Liquid Chromatography Assay. or cytologically confirmed advanced breast cancer; measurable Suramin plasma concentrations were determined by the reverse- disease; Eastern Cooperative Oncology Group performance phase high-performance liquid chromatography procedure of

scores of 0, 1 , or 2: life expectancy of at least 3 months; WBC Supko and Malspeis (22). Separations were achieved on a Nova-

counts 3000 mm3; platelets 150,000 mm3; plasma creatinine Pak C18 Radial-Pak cartridge column (100 X 5 mm; inside I .5 mg/dl; creatinine clearance 60 ml/min; aspartate ami- diameter, 4 p.m) installed in an RCM cartridge holder. A guard- notransferase/alanine aminotransferase <2X normal; and a di- Pak Nova-Pak C18 precolumn insert was placed before the rect plasma bilirubin O.3 mg/dl. Patients were excluded from analytical column. The mobile phase consisted of49% methanol participation if they had any of the following: rapidly progres- and S 1% of an aqueous solution of 19.6 msi ammonium acetate sive or life-threatening disease; more than one prior chemother- and 1.96 mM tetrabutylammonium phosphate (pH 6.5). UV apy regimen for metastatic disease; prior chemotherapy within absorbance of suramin was monitored at 238 nm. To 25 p.1 of the preceding 4 weeks (6 weeks for mitomycin C or nitro- patient plasma was added 500 p.1 of acetonitrile containing SO soureas); prior radiation within the preceding 2 weeks; brain mM tetrabutylammonium phosphate and S p.g of the internal metastases; previous life-threatening malignancy; plasma cal- standard 2-naphthol. After rapid mixing for 30 s and centrifu- cium > 1 2 mg/dl; or a serious intercurrent medical illness, gation (10 mm at 15,000 X g), 200 p.1 of the supernatant were including significant cardiac disease, a bleeding diathesis, a combined with 200 p.1 of 10 mrvi ammonium acetate, and JO p.1 prior history of cerebrovascular accident, concurrent use of were injected onto the column. coumadin or heparin, significant peripheral neuropathy, or cen- IRMAs. Levels of IGF-I, IGF-II, IGFBP- 1 , IGFBP-2, tral nervous system disease. Pregnant or nursing females were and IGFBP-3 were measured in plasma using specific and excluded from this study. All patients signed an informed con- highly sensitive two-site IRMAs (Diagnostic Systems Labora- sent form. Concomitant radiation therapy, chemotherapy, im- tories, Webster, TX). For determination of total IGF concentra- munotherapy, anticoagulants, or calcium channel blockers were tions, samples were acid/ethanol-extracted to separate IGF/ not allowed. IGFBP complexes and remove IGFBPs before assay. Suramin Dosing and Treatment Evaluation. The din- Unextracted plasma were used for determination of free IGF-I. ical objectives of the study were to assess the antitumor efficacy Free IGF is defined as IGF not bound by or readily dissociable and toxicity of suramin in patients with metastatic breast cancer. from IGFBPs. Because suramin inhibits the binding of IGFs to The treatment plan was as follows: all patients received an IGF receptors (10, 1 1), we needed to determine whether suramin initial loading cycle of suramin; for all patients with responding would interfere with the binding of IGFs to antibodies in the or stable disease, a second loading cycle was to be administered assays. When tested with samples in all six IRMAs, direct 2 months after the initiation of cycle I ; and after those initial addition of suramin at the maximal levels achieved in plasma two cycles of therapy, all patients were restaged. Subsequently, (300 p.g/ml) did not interfere in the assays. The free IGF-I

Table I Patient characteristics

Age Adjuvant Patient (yr) Lag’ Rx Prior adjuvant drugs Metastatic sites Prior drugs for metastatic disease PS” ER

A 44 1.1 Yes CMF” Axillary nodes/chest wall/lung TAM’ 0 15 B 50 10.7 Yes Unknown Bones TAM 2 24 chemotherapy C 58 2.2 Yes CMF Lung/cervical node TAM ADR’ 0 17 D 56 9.9 No Kidney/adrenal/liver TAM, megace ADR + I 482 E 39 7.3 Yes CMF Supraclv N/chest wall/bone TAM. megace. DES” CMF 0 13 F 71 23.9 No Lung/bone TAM. megace. DES, HAL’ CMF 0 Unknown G 51 1.5 Yes CMF Lung TAM Taxol + ADR 0 Negative H 38 2.7 Yes CAF zoladex Pleura/lung/bone TAM. megace I 16 I 62 2.6 No Lung TAM CMF 0 Negative J 54 4.4 No Chest wall/lungs TAM, megace 6TGA 1 Negative

(4 Lag years between primary diagnosis and initiation of suramin. I, p5, Eastern Cooperative Oncology Group performance score. ‘ ER, last available ER status before initiation of suramin (fmol/mg cytosolic protein, ligand-binding assay).

.1 CMF. cyclophosphamide, methotrexate, 5-fluorouracil. ,. TAM, tamoxifen. f ADR, Adriamycin. A. IFL-RA, interferon a.

I, DES, diethylstilbestrol. ‘ HAL, halotestin.

J CAF, cyclophosphamide, Adriamycin. 5-fluoruracil. 6-TG, 6-thioguanine.

IRMA had a lower detection limit of 0.03 ng/ml and a CV of Statistical Methods. For each biomarker, the Wilcoxon 10% at 0.3 ng/mI. The total IGF-I IRMA had a lower detection signed rank test was used to assess whether the percentage limit of 0.08 ng/ml and a CV of 3.0% at 55 ng/ml. The total change in peak levels from pretreatment levels was significantly IGF-II IRMA had a lower detection limit of 0. 13 ng/ml and a different from zero. CV of 4.3% at 416 ng/ml. The IGFBP-l IRMA had a lower detection limit ofO.l I ng/ml and a CV of4.O% at 10 ng/ml. No lower detection limit or CV was available for the IGFBP-2 RESULTS IRMA. The IGFBP-3 IRMA had a lower detection limit of 0.5 Clinical End Points. Ten patients were enrolled in the

ng/ml and a CV of 2% at 83 ng/ml. trial, and their characteristics are outlined in Table 1 . The target Western Ligand Blot. Western ligand blotting was per- level of suramin (median, 284 p.g/ml; range, 239-319 p.g/ml) formed as described previously (23). Plasma ( I p.!) was sub- was achieved after a median of 10 days (range, 7-19 days) of jected to SDS-PAGE under nonreducing conditions. The pro- continuous suramin infusion. By careful monitoring during the teins were transferred to nitrocellulose using a semidry transfer loading course, there was only modest (CV < 10%) variability in apparatus. The nitrocellulose filters were blocked with 0.5% the concentration. One patient had a partial response (at least a BSA and incubated with [‘25I]-labeled IGF-I overnight at 4#{176}C. 50% reduction in the product of the longest perpendicular di- The blots were washed, dried, and exposed to either phosphor ameter) in a peripheral lymph node lasting 85 days. Another plates for Phosphorlmager analysis and quantitation (Molecular patient experienced clinical stabilization of painful, progressive Dynamics) or to film for autoradiography. bone disease for 9 months. Another was found to have brain Western Immunoblot. Plasma (0.5 p.1) was electro- metastases (persistent headaches) on day 9 of her first cycle of phoresed, transferred to nitrocellulose, and blocked as described suramin; this patient was removed from the study at that time above. The filters were incubated overnight at 4#{176}Cwith with a suramin level of 239 p.g/ml. She was not evaluable for IGFBP-3 antibody [a generous gift of Dr. R. 0. Rosenfeld (Oregon Health Science University, Portland, OR)] at a 1:1000 clinical end points but was included in the pre- and postsuramin final dilution. The filters were washed and incubated for I h biomarker analyses. The clinical outcomes of treatment effect, with horseradish peroxidase-linked goat antirabbit antibody at number of cycles of suramin administered, time to progression, room temperature. The filters were again washed and developed and time to death are provided in Table 2. Overall, the toxicities in enhanced chemiluminescence reagent (Amersham, Chicago, seen in this patient group with carefully monitored blood levels IL) and exposed to film. were tolerable (Table 3). The most common side effect was a Protease Assays. IGFBP-3 protease activity was deter- pruritic maculopapular rash. No lasting severe side effects were mined by the method of Lamson et a!. (24). Briefly, I p.1 of encountered. plasma was incubated overnight at 37#{176}Cwith [‘2511-labeled Pharmacokinetics. The pharmacokinetics of suramin IGFBP-3 in 25 p.1 of 1 ms CaCl2 and SO mrvi Tris buffer (pH were characterized for three patients from whom plasma spec- 7.4). The samples were subjected to SDS-PAGE under reducing imens were obtained during and after the continuous infusion. conditions. The gels were dried and exposed to either phosphor Modeling of suramin disposition to a two-compartment open plates or film as in the Western ligand blot analysis. model indicated slow plasma clearance (0.01 12 liter/hIm2) and

Table 2 Clinical outcomes Table 3 Suramin toxicities among all 10 patients

No. of Time to Maximum grade (National Cancer Treatment suramin progression Time to death Institute common toxicity criteria) Total Patient effect cycles (mo) (mo) incidence Grade I Grade 2 Grade 3 Grade 4 per type A Progression I 1.3 13.6 B Stable 4 9.1 28.4 Dermatitis 2 0 1 1 4 C Progression 1 0.8 5.3 Anorexia 3 0 0 0 3 D Progression I 2.4 5.3 Proteinuria 3 0 0 0 3 E Partial response 2 4.2 15.1 Nausea 2 0 0 0 2 F Refusal” I 4.4 14.8 Neurosensory 2 0 0 0 2 G Progression 2 3.4 11.3 Stomatitis 1 1 0 0 2 H Progression 1 1.3 27.8 Hematuria-micro 2 0 0 0 2 Progression I 1.8 22.9 Ocular” 0 1 1 0 2 NeurCNS” 1 0 0 0 1 “ Patient refused additional suramin (insurance problems). Vomiting I 0 0 0 1 Hearing loss 0 1 0 0 1

Total incidence 17 (73.9’7 c) 3 (13.0%) 2 (8.7%) 1 (4.4%) 23 per grade a terminal elimination half-life of 46.2 ± 1.6 days, consistent a Protocol-specific criteria.

with previous published data on suramin pharmacokinetics (25). I) Neur CNS, central nervous system neuropathy. Biomarkers: The IGF System. Table 4 presents the plasma concentrations of total IGF-I and IGF-II, free IGF-I, and

IGFBP- I , IGFBP-2, and IGFBP-3, both pre- and postsuramin, as measured by specific IRMA. All biomarker analyses were sents IGFBP-2, and the lowest band on the blot indicates 24-kDa performed on first-cycle specimens. IGFBP-4. The intensities of these bands were quantified using Assays for total IGF measure unbound IGFs and IGFs Phosphorlmager analysis. The level of IGFBP-2 in each sample bound to IGFBPs. The median level of total IGF-II in the 10 varied but related to the value obtained by IRMA, and there was patients before drug treatment was 832 ng/ml. In 9 of 10 no consistent change with suramin treatment. By Western ligand patients, total IGF-II levels decreased after suramin treatment. blot analysis, IGFBP-4 concentrations decreased modestly in 8 The tenth patient’s IGF-II level remained essentially unchanged. of I 0 patients, and the overall change for the group was not The percentage decrease in total IGF-II levels from pretreatment remarkable. values was significant (P 0.004), ranging from -66 to 4%. Of particular interest was the finding that levels of Total IGF-I levels were highly variable among the 10 patients. IGFBP-3 detected by Western ligand blotting were markedly

The median pretreatment level was 129 ng/ml. Six patients had reduced in the plasma after suramin treatment (Fig. I ). In some a lower total IGF-I level after suramin treatment, and four patients, there was almost no detectable 38-/42-kDa IGFBP-3 patients had an increased total IGF-I level. The percentage postsuramin treatment. These results are in contrast to the IRMA change in total IGF-I levels from pretreatment values was not IGFBP-3 values for these patients, which suggested a moderate

found to be significant (P = 0.131). decrease in IGFBP-3 levels posttreatment. The addition of sur- The free IGF-I values likely reflect true unbound IGF-I as amin at a final concentration of 300 p.g/ml to control plasma well as IGF-I that readily dissociates from IGFBPs. The free samples had no effect on IGFBP assessment by Western ligand IGF-I levels of all 10 patients increased markedly after suramin blotting (data not shown). treatment. The median baseline free IGF-I concentration was 0.2 To address the apparent discrepancy between the IRMA ng/ml. The percentage increase in free IGF-I levels from pre- and Western ligand blot results, we assayed the plasma samples treatment values ranged from 70 to 1384%. This increase was by Western immunoblot using a specific IGFBP-3 antibody. highly significant (P 0.002). This technique can detect IGF-binding and -nonbinding forms IGFBP levels were also measured by specific IRMAs (Ta- of IGFBP-3. In all 10 of the study patients, the level of intact ble 4). The IGFBP- I and IGFBP-2 levels showed considerable 38-/42-kDa IGFBP-3 was greatly reduced after suramin treat- intragroup variation and no significant change in response to ment (Fig. 2). Some patients’ plasma had no detectable intact suramin. Absolute plasma IGFBP-3 concentrations were within IGFBP-3 after the drug treatment. A 30-kDa immunoreactive the range for healthy adults and decreased after suramin treat- IGFBP-3 form was evident in all of the plasma samples, how- ment in nine of the patients. The median decrease for the patient ever, both pre- and posttreatment. This form of IGFBP-3 was group was 21% (P 0.010). not detected by Western ligand blotting with [‘2511-labeled Assessment of immunoreactive IGFBP by IRMA alone IGF-I. In S of 10 patients, 30-kDa IGFBP-3 increased after gives insufficient information on the number and forms of suramin treatment. This fragment is similar to a 30-kDa IGFBP in the circulation. To further analyze the binding protein IGFBP-3 fragment generated by a IGFBP-3 protease found in levels, the plasma samples were analyzed by Western ligand the plasma of pregnant women (Fig. 2, Lanes 3 and 4). blotting (Fig. 1). Western ligand blotting separates proteins by To elucidate a possible mechanism for the decreased level apparent molecular size and measures the ability of the proteins of intact IGFBP-3, IGFBP-3 protease activity was measured in to bind [‘25I]-labeled IGF-I after transfer to nitrocellulose filters. the plasma by adding exogenous [‘2511-labeled IGFBP-3 and The bands at 42- and 38-kDa represent the expected glycosyla- monitoring the appearance of radiolabeled fragments. None of tion variants of IGFBP-3 (22, 23). The band at 34 kDa repre- the patients’ pretreatment plasma samples produced IGFBP-3

Table 4 Plasma levels of IGFs and IGFBPs before and after suramin treatment Data represent the median and range values (in parentheses) for the 10 patients before and after suramin. Normal range values for adults, where available, are: total IGF-II, 462-1042 ng/ml; total IGF-I, 100-494 ng/ml; and IGFBP-3, 2080-4310 ng/ml. Before treatment After treatment Percentage change Protein (ng/ml) (ng/ml) from baseline P Total IGF-II 832(244, 1176) 604 (252, 825) -23% (-66%, 4%) 0.004 Total IGF-I 129 (36, 301) 90 (5, 208) -24% (-96%, 31%) 0.131 Free IGF-I 0.2 (0.1, 0.7) 1.3 (0.2, 2.8) 256% (70%, 1384%) 0.002 IGFBP- 1 69(17, 150) 58 (24, 94) -30% (-81%, 354%) 0.770 IGFBP-2 987 (644, 1419) 1203 (463, 1914) 18% (-48%, 95%) 0.275 IGFBP-3 3014 (1315, 3876) 2250 (1228, 3693) -21% (-58%, 8%) 0.010

B C DE FGHI

.. BP-.3 43- Rfs :r,,1F, 1tI.IIt. J1I :‘

31-

.. BP4 - 21-

- + - + - + - + - + + - + - + - + - +

Fig. I Western ligand blot of IGFBPs in the plasma of women with advanced breast cancer (A-f) before ( - ) and after (+ ) treatment with suramin. Plasma samples were subjected to SDS-PAGE under nonreducing conditions, and separated proteins were transferred to nitrocellulose filters. After incubation with [‘25I]-labeled IGF-I, filter-immobilized IGFBPs were identified by autoradiography as described in “Materials and Methods.” The migration positions of molecular size markers (in kilodaltons) are indicated on the left. Arrows identify IGFBP.2, IGFBP-3, and IGFBP-4.

proteolysis, whereas the postsuramin treatment samples of pa- by 70-1384% after suramin treatment. An increase in free IGF-I tients A, B, and F did exhibit low levels of an IGFBP-3 protease was seen in all 10 patients. The decrease in the total IGF-II activity similar to that seen in plasma from women in late plasma levels agrees with a previous suramin trial ( I 6) and was pregnancy. Suramin alone did not induce exogenous or endog- not due to direct effects of suramin in the assays. It is interesting enous IGFBP-3 proteolysis (data not shown). to note that levels of the IGFs and IGFBPs measured in patients B and E, who had a stabilized condition and a partial response DISCUSSION after suramin treatment, did not show any significant change Over the past decade, numerous lines of evidence have from the median values. Also, the magnitude of the suramin- shown the central importance of peptide growth factor networks induced decrease in total IGFs is similar to the previously in human breast cancer. Overexpression of specific growth reported effect of tamoxifen on IGF levels. This antiestrogen factor receptors and/or the relevant ligands has been shown in reduced total systemic IGF-I concentrations in breast cancer vitro, in vivo, and in clinical models to enhance tumor growth or patients by about 35% in two clinical trials ( 17, 18). However, unlike suramin, tamoxifen treatment resulted in a parallel de- correlate with worse outcome ( I , 2). In an attempt to target these growth factor pathways in breast cancer, we conducted a clinical crease in levels of free IGF-I.4 Although the absolute levels of trial of suramin, a prototype agent within the class of growth free IGF-I represent only 1 % of total circulating IGF-I, it is the factor inhibitors that is thought to act generally to inhibit the free form that is readily available to extravascular tissues and, at binding of growth factors to their receptors (10). We monitored these concentrations, bioactive at the cellular level (26). IGFs in the IGF network in these patients because of the previously the picomolar to nanomolar concentration range have been defined growth-promoting role of the IGFs in human solid reported to be potent mitogens in a variety of breast cancer cells tumors, especially breast cancer (1-3). Recognizing the com- (27). plexity of the IGF family, we were concerned that the measure- In an effort to elucidate the mechanism responsible for the ment of total IGF levels alone was insufficient to characterize the predominant activity within the IGF network. Hence, we examined several components of the IGF system in our patients with advanced breast cancer, pre- and postexposure to suramin. 4 J. N. Ingle, V. J. Suman, C. G. Kardinal, J. E. Krook, J. A. Mailliard, J. W. Kugler, C. L. Loprinzi, R. J. Dalton, L. C. Hartmann, C. A. The most striking result obtained in this study indicated Conover, and M. N. Pollak. Randomized trial of tamoxifen alone or that whereas total levels of plasma IGF-I decreased, but not combined with octreotide in women with metastatic breast cancer, significantly, and IGF-II decreased 23%, free IGF-I increased manuscript in preparation.

Cont Preg A BCDEFGHIJ I I IIIIIIIII .;‘---:T1 1S P-*-42 ‘ *

lSi

- + - + + - + - + + -+ -+-+ -+

Fig. 2 Western immunoblot of IGFBP-3 in the plasma of women with advanced breast cancer (A-f) before ( -) and after (+) suramin treatment. Proteins in plasma were electrophoretically separated and transferred to nitrocellulose, as described in Fig. 1 and in “Materials and Methods,” and then probed with a specific IGFBP-3 antibody. Lanes I and 2, plasma from healthy, nonpregnant adults. Lanes 3 and 4, plasma from healthy women in their third trimester of pregnancy. Arrows identify 38-/42-kDa intact IGFBP-3 and an immunoreactive 30-kDa IGFBP-3 fragment.

dramatic rise in free IGF-I after suramin exposure, we deter- amin to decrease the high-affinity form of IGFBP-3 in the mined the plasma levels of the circulating IGFBPs. After sura- circulation might have an adverse effect in breast cancer pa- mm treatment, plasma levels of IGFBP-3, the major carrier of tients, allowing increased free IGF availability and potential the IGFs in plasma, decreased a modest 2 1% when measured by stimulation of tumor cells. Suramin has been reported to have IRMA. However, when assessed by Western ligand blot, the stimulatory effects on tumor growth in rabbits (32), although levels of intact 38-/42-kDa IGFBP-3 were dramatically de- effects on the IGF system were not examined in that study. We creased, in some instances, to almost undetectable levels. Im- saw no evidence of a tumor-stimulating effect in our study, but munoblotting with a specific IGFBP-3 antibody indicated the the sample size was small. presence of a 3O-kDa IGFBP-3 form, which did not bind [i25I. Our data do not provide any information on the effect of labeled IGF-I by Western ligand blotting. Both the IGFBP-3 suramin on the IGF system at the tissue level. However, in vitro fragment with reduced affinity for IGF-I as well as intact studies have shown that, at high concentrations, suramin inhibits IGFBP-3 with high affinity for IGF-I are measured in the IGF/receptor interactions but that suramin’s effects are revers- IGFBP-3 IRMA. Suramin treatment produced no significant ible. Wilms’ tumor growth is inhibited by suramin via interfer-

changes in IGFBP- 1 , IGFBP-2, or IGFBP-4 when assessed by ing with IGF-II binding, but this inhibitory effect of suramin is IRMA or by Western ligand blotting. These IGFBPs are acutely overcome with excess IGF-II (1 1). Moreover, Pommier et al. regulated by nutritional factors not controlled in this study (28), (33) showed that at low concentrations that do not inhibit and the resultant variability might obscure any effect. Alter- receptor binding, suramin induces release of IGF-II from local ations in the levels and saturation of these IGFBPs could affect IGFBPs and actually facilitates IGF-I binding to receptors on IGF delivery and bioactivity (see below) and will need to be colon cancer cells. addressed in future studies. The mechanism by which suramin administration disrupts What are the possible implications of a decrease in intact the ternary IGFBP-3IIGF/ALS complex is unclear. Suramin IGFBP-3 and an increase in free IGF-I in patients with breast could inhibit IGF and IGFBP interaction in vivo as has been cancer? One concern is that this could reflect an alteration in shown in vitro, leading to a general increase in free IGF. IGF availability to target tissues, including tumor. The majority However, its particular effect to decrease intact IGFBP-3 sug- of the circulating IGFs are bound with high affinity to IGFBP-3 gests a more specific mode of action. IGFBP-3 protease activity in a lSO-kDa ternary complex that includes an ALS. IGFs has been widely studied and is especially active in the plasma of tightly bound in this ternary complex are generally latent and pregnant women. This protease clips intact IGFBP-3 into a cannot cross the capillary boundary (21). Destabilization of the 30-kDa fragment that has a much lower affinity for IGFs, ternary complex results in redistribution of IGFs into the un- resulting in increased IGF bioavailability (26, 3 1). Suramin did bound form as well as into binary IGFBP complexes, which are not directly act as an IGFBP-3 protease when tested in our cleared more rapidly from the circulation (29, 30). This in- cell-free assay (data not shown). Suramin treatment was asso- creased transcapillary transfer of free, active IGF to target ciated with circulating IGFBP-3 protease activity in 3 of 10 tissues could conceivably promote tumor cell growth. Indeed, patient plasma samples. This activity produced an IGFBP-3 alterations in IGFBP-3 may be more important for IGF delivery fragment similar in size to that seen in pregnancy plasma. In to tumor cells than alterations in total plasma IGF concentra- addition, there may be periphera] proteolysis of IGFBP-3 with tions. In fact, it has been proposed that free IGF is increased to subsequent appearance of the fragment in the circulation. Lalou the human fetus by such a mechanism. IGFBP-3 modified by a et a!. (34) have reported on a cell surface-associated plasmino- specific protease induced in the maternal circulation during gen/plasminogen activator system involved in IGFBP-3 prote- pregnancy has decreased affinity for IGF-I, thus potentially olysis. This type of a cell-associated IGFBP-3 proteolysis would increasing bioavailability of IGF-I to the fetus (30, 31). not be measured by our assay but would be consistent with the Suramin’s long half-life could conceivably alter growth factor increase in immunoreactive 30-kDa IGFBP-3 in the plasma. equilibria for protracted periods. Thus, systemic effects of sur- Although not directly measured in this study, low ALS

expression could also lead to a loss of intact IGFBP-3 and an 9. Brunner, N., Yee, D., Kern, F. G., Spang-Thomsen, M., Lippman, increase in free IGF. ALS, when part of the ternary complex, M. E., and Cullen, K. Effect of endocrine therapy on growth of T61 human breast cancer xenografts is directly correlated to a specific stabilizes both IGFBP-3 and the bound IGF. Bang Ct a!. (31) down-regulation of insulin-like growth factor II (IGF-II). Eur. J. Cancer, found that despite the prominence of the 30-kDa IGFBP-3 29A: 562-569, 1993. fragment, IGFBP-3 protease activity was not increased in fetal 10. Coffey, R. J., Leof, E. B., Shipley, G. D., and Moses, H. L. Suramin serum. Instead, they found a lower level of ALS in fetal serum inhibition of growth factor receptor binding and mitogenicity in in conjunction with an increased level of the 30-kDa IGFBP-3 AKR-2B cells. J. Cell. Physiol., 132: 143-148, 1987. fragment. More studies will be necessary to determine the I I. Vincent, T. S., Hazen-Martin, D. J., and Garvin, A. J. Inhibition of underlying mechanism for the altered IGF system with suramin insulin like growth factor II autocrine growth of Wilms’ tumor by suramin in vitro and in vivo. Cancer Lett., 103: 49-56, 1996. treatment. In addition, we must consider a possible effect of hydro- 12. Eisenberger, M. A.. Sinibaldi, V. J., Reyno, L. M., Sridhara, R., Jodrell, D. I., Zuhowski, E. G., Tkaczuk, K. H., Lowit, M. H., Hemady, cortisone on the IGF system. Because of suramin’s known R. K., Jacobs, S. C., Van Echo, D., and Egorin, M. J. Phase I and clinical adrenal toxicity, all patients receiving suramin were also treated evaluation of a pharmacologically guided regimen of suramin in patients with oral hydrocortisone. Glucocorticoid could independently with hormone-refractory prostate cancer. J. Clin. Oncol., /3: 2174- affect the levels of IGFs and IGFBPs. However, previous re- 2186, 1995. ports have shown that serum levels of IGFBP-3 increase after 13. Kobayashi, K., Vokes, E. E., Vogelzang, N. J., Janisch, L., Soliven, glucocorticoid treatment (35). Moreover, glucocorticoids have B., and Ratain, M. J. Phase I study of suramin given by intermittent infusion without adaptive control in patients with advanced cancer. been shown either to have no effect on total IGF-I levels or to J. Clin. Oncol., 13: 2196-2207, 1995. slightly elevate total IGF-I levels (36, 37). 14. Scher, H., and Kelly, W. K. Suramin: defining the role in the clinic. Exploitation of the dependence of breast cancer cells on Princ. Pract. Oncol., 7: 1-16, 1993. IGFs is a new and potentially important therapeutic approach. 15. Vignon, F., Prebois, C., and Rochefort, H. Inhibition of breast However, it is essential to consider the complexity and dynam- cancer growth by suramin. J. Natl. Cancer Inst., 84: 38-42, 1992. ics of the IGF system in designing such therapeutic strategies. 16. Miglietta, L., Barreca, A., Repetto, L., Costantini, M., Rosso, R., Our experience with suramin in a group of women with mini- and Boccardo, F. Suramin and plasma insulin-like growth factor levels mally pretreated advanced breast cancer illustrates several chal- in metastatic cancer patients. Anticancer Res., 13: 2473-2476, 1993. lenges and provides motivation for further research regarding 17. Pollock, M., Huynh, H., and Lefebvre, S. Tamoxifen reduces the biological significance of plasma IGFIIGFBP measure- plasma insulin-like growth factor-I (IGF-I). Breast Cancer Res. Treat., 22: 91-100, 1992. ments. We would recommend thorough study of relevant growth I 8. Colletti, R., Roberts, J., Devlin, J., and Copeland, K. Effect of factor intermediates as suramin’s role in the clinic continues to tamoxifen on plasma insulin-like growth factor I in patients with breast be defined. cancer. Cancer Res., 49: 1882-1889, 1989. 19. Shimasaki, S., and Ling, N. Identification and molecular character- ACKNOWLEDGMENTS ization of insulin-like growth factor-binding proteins. Prog. Growth Factor Res., 3: 243-266, 1991. We thank Jill Piens and the Mayo General Clinical Research Center 20. Oh, Y., Nagalla, S., Yamanaka, Y., Kim, H., and Wilson, E. staff for assistance in specimen collection and patient care, Pamela Synthesis and characterization of insulin-like growth factor-binding Bagniewski for analysis of patient samples, and Cheryl Collins and Julie protein (IGFBP)-7. J. Biol. Chem., 271: 30322-30325, 1996. Tienter for help in the preparation of this manuscript. 2 1. Baxter, R. C. Insulin-like growth factor-binding proteins in the human circulation: a review. Horm. Res. (Basel), 42: 140-144, 1994. REFERENCES 22. Supko, J. G., and Malspeis, L. A. A rapid isocratic HPLC assay of suramin (NCS 34936) in human plasma. J. Liq. Chromatogr., 13: 1. Dickson, R. B., and Lippman, M. E. Growth factors in breast cancer. Endocr. Rev., 16: 559-589, 1995. 727-741, 1990. 2. Daughaday, W. H. The possible autocrine/paracrine and endocrine 23. Conover, C. A., Kiefer, M. C., and Zapf, J. Posttranslational regu- roles of insulin-like growth factors of human tumors. Endocrinology, lation of insulin-like growth factor (IGF)-binding protein 4 in normal 127: 1-4, 1990. and transformed human fibroblasts: IGF dependence and biological 3. Lee, A. V., and Yee, D. Insulin-like growth factors and breast cancer. studies. J. Clin. Invest., 91: 1 129-1 137, 1993. Biomed. Pharmacother., 49: 415-421, 1995. 24. Lamson, G., Giudice, L. C., and Rosenfeld, R. G. A simple assay 4. LeRoith, D., Werner, H., Beitner-Johnson, D., and Roberts, C. T., Jr. for proteolysis ofIGFBP-3. J. Clin. Endocrinol. Metab., 72: 1391-1393, Molecular and cellular aspects of the insulin-like growth factor I recep- 1991. tor. Endocr. Rev., 16: 143-163, 1995. 25. Eisenberger, M. A., and Reyno, L. M. Suramin. Cancer Treat. Rev., 5. Peyrat, J. P., and Bonneterre, J. Type 1 IGF receptor in human breast 20: 259-273, 1994. diseases. Breast Cancer Res. Treat., 22: 59-67, 1992. 26. Lassarre, C., and Binoux, M. Insulin-like growth factor-binding 6. Neuenschwander, S., Roberts, C. T., Jr., and LeRoith, D. Growth protein 3 is functionally altered in pregnancy plasma. Endocrinology, inhibition of MCF-7 breast cancer cells by stable expression of an 134: 1254-1262, 1994. insulin-like growth factor I receptor antisense ribonucleic acid. Endo- 27. Karey, K., and Sirbaska, D. Differential responsiveness of human crinology, 136: 4298-4303, 1995. breast cancer cell lines MCF-7 and T47D to growth factors and l73- 7. Arteaga, C. L., and Osborne, C. K. Growth inhibition of human estradiol. Cancer Res., 48: 4083-4092, 1988. breast cancer cells in vitro with an antibody against the type I somato- 28. Ketelslegers, J-M., Maiter, D., Macs. M., Underwood, L. E., and medin receptor. Cancer Res., 49: 6237-6241, 1989. Thissen, J-P. Nutritional regulation of the growth hormone and insulin- 8. Arteaga, C. L., Kitten, L. J., Coronado, E. B., Jacobs, S., Kull, F. C., like growth factor-binding proteins. Horm. Res. (Basel), 45: 252-257, Jr., Allred, D. C., and Osborne, C. K. Blockade of the type I somato- 1996. medin receptor inhibits growth of human breast cancer cells in athymic 29. Prosser, C. G., and Schwander, J. Influence of insulin-like growth mice. J. Clin. Invest., 84: 1418-1423, 1989. factor-binding protein 2 on plasma clearance and transfer of insulin-like

growth factors I and II from plasma into mammary-derived lymph and 34. Lalou, C., Silve, C., Rosato, R., Segovia, B., and Bioux, M. Inter- milk of goats. J. Endocrinol., 150: 121-127, 1996. actions between insulin-like growth factor I (IGF-I) and the system of 30. Blat, C., Villaudy, J., and Binoux, M. In vivo proteolysis of plasma plasminogen activators and their inhibitors in the control of IGF-binding insulin-like growth factor (IGF)-binding protein 3 results in increased protein 3 production and proteolysis in human osteosarcoma cells. availability of IGF to target cells. J. Clin. Invest., 93: 2286-2290, 1994. Enodcrinology, 135: 2318-2326, 1994. 31. Bang, P., Stangenberg, M., Westgren, M., and Rosenfeld, R. De- 35. Baxter, R., Holman, S., Corbould, A., Stranks, S., Ho, P., and creased ternary complex formation and predominance of a 29-kDa Braund, W. Regulation of the insulin-like growth factors and their IGFBP-3 fragment in human fetal plasma. Growth Regul., 4: 68-76, binding proteins by glucocorticoid and growth hormone in nonislet cell 1994. tumor hypoglycemia. J. Clin. Endocrinol. Metab., 80: 2700-2708, 1995. 32. Ramirez, L. H., Julieron, M., Bonnay, M., Koscielny, S., Thao, Z., 36. Price, W., Stiles, A., Moats-Staats, B., and D’Erchole, J. Gene Gouyette, A., and Munck, J-N. Stimulation of tumor growth in vitro and in expression of insulin-like growth factors (IGFs), the type I IGF receptor, vivo by suramin on the VX2 model. Invest. New Drugs, 13: 51-53, 1995. and IGF-binding proteins in dexamethasone-induced fetal growth retar- 33. Pommier, G., Garrouste, F., El Atiq, F., Roccabianca, M., Marvaldi, J., dation. Endocrinology, 130: 1424-1432, 1992. and Remade-Bonnet, M. Potential autocrine role of insulin-like growth 37. Allen, D., and Goldberg, B. Stimulation of collagen synthesis and factor II during suramin-induced differentiation of HT29-D4 human co- linear growth by growth hormone in glucocorticoid-treated children. lonic adenocarcinoma cell line. Cancer Res., 52: 3182-3188, 1992. Pediatrics, 89: 416-421, 1992.

Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 1997 American Association for Cancer Research. Evaluation of continuous infusion suramin in metastatic breast cancer: impact on plasma levels of insulin-like growth factors (IGFs) and IGF-binding proteins.

J B Lawrence, C A Conover, T C Haddad, et al.

Clin Cancer Res 1997;3:1713-1720.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/3/10/1713

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/3/10/1713. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.