ZD4054) in Colorectal Cancer: a Preclinical Study

Published OnlineFirst May 30, 2013; DOI: 10.1158/1535-7163.MCT-12-0975

Molecular Cancer Cancer Therapeutics Insights Therapeutics

Efﬁcacy of the Speciﬁc Endothelin A Receptor Antagonist Zibotentan (ZD4054) in Colorectal Cancer: A Preclinical Study

Samer-ul Haque1, Michael R. Dashwood2, Mohammed Heetun1, Xu Shiwen3, Noreen Farooqui1, Bala Ramesh1, Hazel Welch1, Felicity J. Savage1, Olagunju Ogunbiyi1, David J. Abraham3, and Marilena Loizidou1

Abstract Endothelin 1 (ET-1) is overexpressed in cancer, contributing to disease progression. We previously showed

that ET-1 stimulated proliferative, migratory, and contractile tumorigenic effects via the ETA receptor. Here, for

the first time, we evaluate zibotentan, a specific ETA receptor antagonist, in the setting of colorectal cancer, in cellular models. Pharmacologic characteristics were further determined in patient tissues. Colorectal cancer lines (n ¼ 4) and fibroblast strains (n ¼ 6), isolated from uninvolved areas of colorectal cancer specimens, were

exposed to ET-1 and/or ETA/B receptor antagonists. Proliferation (methylene blue), migration (scratch

wounds), and contraction (gel lattices) were assessed. Receptor distribution and binding characteristics (Kd,

Bmax) were determined using autoradiography on tissue sections and homogenates and cytospun cells, > < supported by immunohistochemistry. Proliferation was inhibited by ETA (zibotentan BQ123; P 0.05), > migration by ETB ETA, and contraction by combined ETA and ETB antagonism. Intense ET-1 stromal binding correlated with fibroblasts and endothelial cells. Colorectal cancer lines and fibroblasts revealed high density ¼ 6 ¼ ¼ 6 and affinity ET-1 binding (Bmax 2.435 fmol/1 10 cells, Kd 367.7 pmol/L; Bmax 3.03 fmol/1 10 cells, Kd ¼ 213.6 pmol/L). In cancer tissues, ETA receptor antagonists (zibotentan; BQ123) reduced ET-1 binding more m effectively (IC50:0.1–10 mol/L) than ETB receptor antagonist BQ788 ( IC50, 1 mmol/L). ET-1 stimulated cancer-contributory processes. Its localization to tumor stroma, with greatest binding/affinity to fibro-

blasts, implicates these cells in tumor progression. ETA receptor upregulation in cancer tissues and its role in tumorigenic processes show the receptor’s importance in therapeutic targeting. Zibotentan, the most

speciﬁc ETA receptor antagonist available, showed the greatest inhibition of ET-1 binding. With its known safety proﬁle, we provide evidence for zibotentan’s potential role as adjuvant therapy in colorectal cancer. Mol Cancer Ther; 12(8); 1556–67. 2013 AACR.

Introduction ments in drug development, the success of these agents Colorectal cancer is one of the most common malig- is limited. nancies in the West and a leading cause of worldwide Endothelin-1 (ET-1) is a small vasoactive peptide morbidity and mortality. If detected at an early stage, overexpressed in plasma and tissue samples from the disease is potentially curable with surgical resection patientswithvarioussolidcancers(Fig.1A;ref.2). as the "gold standard" treatment. However, up to 60% of ET-1 effects are mediated by two distinct G protein- patients will have regional or metastatic disease at coupled receptors, ETA and ETB. Receptor binding and initial presentation (1), limiting the potential for surgi- signaling often causes opposite effects on proliferation: cal intervention. For these patients, disease manage- in the majority of cells, binding to ETA promotes ment focuses on the use of chemotherapeutic agents, growth, whereas binding to ETB leads to apoptosis. either individually or in combination. Despite improve- Receptor expression is altered in a number of solid cancers, with ETA reported as upregulated, and ETB either up- or downregulated, using various techniques. Using autoradiography, our previous results were sim- Authors' Affiliations: 1Cancer Nanotechnology Group, UCL Division of ilar to those reported in ovarian and prostate cancers Surgery and Interventional Science; 2Department of Clinical Biochemistry, Royal Free Hospital; 3Centre for Rheumatology and Connective Tissue (3, 4), in that ETA receptors were overexpressed, where- Diseases, UCL Division of Medicine, London, United Kingdom as ETB receptors were downregulated in cancer tissue Note: S. Haque and M.R. Dashwood are joint first authors. compared with normal colon. Specifically, binding to the ETA receptor was greatest in cancer-associated fibro- Corresponding Author: Marilena Loizidou, Division of Surgery and Inter- ventional Science, UCL Medical School, 9th floor, Royal Free Hospital, blasts and blood vessels and to a lesser extent in Pond Street, London, NW3 2QG, United Kingdom. Phone: 020-7794-0500, epithelial cancer cells. In contrast, ETB receptors were ext. 35575; Fax: 020-7472-6444; E-mail: [email protected] the predominant receptors in normal colon and doi: 10.1158/1535-7163.MCT-12-0975 were markedly downregulated in all cell types within 2013 American Association for Cancer Research. cancer (5).

1556 Mol Cancer Ther; 12(8) August 2013

Efﬁcacy of Zibotentan in Colorectal Cancer

A B H Ser Ser Leu Ser N Cys Cys O NH2 Met

Asp OH Lys Asp lle Glu Cys Cys lle O Leu Tr p COOH H Val Phe His O Tr p ET-1 Peptide HN N N O H N N O H H O Figure 1. Structures of ET-1 H C CH 3 3 H C CH peptide (A), ETA receptors BQ123 3 3 and ZD4054 (B), and ETB receptor C antagonist BQ788 (C). BQ123 CH3 O Receptor antagonists

ONa A N Me ET N H O HN O O CH N 3 N S N N O N H CO N N O 3 O H O H OMe Receptor antagonist Receptor

B H C 3 H3C CH3 ET H3C BQ788 ZD4054

In vitro, ET-1 production has been detected in a number gated in a panel of colorectal cancer cell lines and colonic of human cancer cell lines, such as colorectal, stomach, fibroblasts, and compared with the standard laboratory breast, and prostate (6, 7). Furthermore, exogenous ET-1 ET receptor antagonists BQ123 (ETA selective) and BQ788 addition to ovarian, prostate, and melanoma cancer cells (ETB selective; Fig. 1). Furthermore, pharmacologic char- stimulates proliferation (3, 4, 8). ET-1 may also contribute acteristics of zibotentan were determined in normal and to tumor progression by stimulating angiogenesis and cancer tissues and cells from patient specimens taken desmoplasia and promoting invasion. Previously, we during surgery for colorectal cancer. investigated ET-1 effects on colorectal cancer cells and fibroblasts isolated from the colons of patients with can- Materials and Methods cer. ET-1 via ETA provided a growth stimulus to colorectal Biologic materials cancer cells; the signal was also propagated via the trans- Cells. The human colorectal cancer cell lines used activation of the EGF receptor, when this receptor was were HT29, SW480 (moderately differentiated), SW620 present (9). Furthermore, colonic fibroblasts, when (poorly differentiated; European Collection of Cell Cul- exposed to ET-1, exhibited increased growth, migration, tures, ECACC, Sigma-Aldrich Company Ltd); cell line and contraction (10). As the fibroblasts were isolated from authentication at source includes species and identity macroscopically uninvolved areas of colorectal cancer verification by DNA barcoding and profiling, and PCR specimens, our findings are consistent with ET-1 proof short tandem repeat sequences within chromosomal duced by cancer cells activating adjacent fibroblasts in a microsatellite DNA (STR-PCR); and the human colorectal paracrine manner. cancer cell line LIM1215 isolated and immortalized at Clinically, specific ETA receptor blockade has been the Ludwig Institute for Cancer Research (HNPCC famil- proposed as a potential target for anticancer therapy; ial cancer; donated by Professor M O’Hare, Ludwig Insti- however, despite encouraging preclinical data (11, 12) tute for Cancer Research, London, United Kingdom). and early clinical signals thus far, data obtained with this Further authentication was not undertaken in our class of agent in prostate and non–small cell lung cancer laboratory. Submucosal fibroblasts were isolated from (NSCLC) have been disappointing. To date, there are no specimens of patients undergoing colorectal cancer resec- data for the ETA receptor antagonist zibotentan (ZD4054) tion and extracted from tissue adjacent to, but not in colorectal cancer, either preclinically or clinically, to macroscopically involved with, cancer. Briefly, after col- determine its usefulness either as monotherapy or in lagenase digestion, epithelial and endothelial cells were combination with cytotoxic drugs. removed using antibody-coated magnetic beads and The purpose of the present study was to determine the the remaining cells (b-actin positive) were grown and effects of the specific ETA receptor antagonist zibotentan used (passages 4–12; untransformed cell strains; ref. 10). on cellular processes integral to cancer growth and pro- Cells were discarded thereafter due to loss of the fibro- gression, namely proliferation, migration, contraction, blastoid phenotype (13). The strains were labeled CF and protein expression. These parameters were investi- (colorectal fibroblasts) and allocated numbers according

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1557

Haque et al.

to specimens received—here, we used CF35, CF42, Gel contraction. Cells were grown in three-dimen- CF56, CF65, CF75, CF78; isolated 2001–2004 with sional collagen gel lattices (18) set in 24-well plates informed patient consent. Further authentication was (100,000 cells/lattice). Gels were impregnated with not undertaken. All cell lines and cell strains were antagonists alone or in combination (zibotentan, BQ123; passaged for less than 6 months after resuscitation. Cells BQ788; zibotentan plus BQ788; 1 hour) and then "floated" were cultured in a humidified atmosphere, 37C, 5% by the addition of 1 mL serum-free ET-1. Control gels were CO2/air, in glutamine-enriched Dulbecco’s Modified floated by serum-free medium. Gels were further incu- Eagle Medium supplemented with 10% fetal calf serum bated (72 hours) and fixed in 10% formaldehyde. For and gentamycin (50 mg/mL). For cytospins, cells (2 106 imaging, gels were placed on glass slides and scanned. for gross autoradiography and 0.5 106 for microautor- Excess moisture was blotted off and gels weighed to adiography) were loaded onto polylysine-coated slides, determine weight loss proportional to contraction. centrifuged, air dried, and stored (70C; Shandon Cytos- Statistical analysis. Data were analyzed using one- pin3, WolfLabs Inc). way ANOVA [post hoc Tukey honestly significant differ- Tissues. Specimens were obtained from patients (n ¼ ence (HSD), P < 0.05]. For growth, six independent repeats 6) undergoing colorectal cancer resection; adjacent nor- were conducted for each fibroblast strain (n ¼ 5) and each mal and cancer samples were snap frozen, stored (liquid cancer line (n ¼ 2). For migration, six independent repeats m ¼ N2), and used for: (i) 5 m frozen sections on polylysine- were conducted for each fibroblast strain (n 4) and each coated slides (Leica CM3050 cryostat, Milton Keynes); (ii) cancer line (n ¼ 2). Collagen gel contraction experiments Homogenates: tissues were placed into a ball-bearing were carried out in three fibroblast strains and one cancer cage, dipped in liquid N2, and homogenized (2 minutes, line (3 independent repeats). Graphical representations Mikro-Dismembranator U, Braun Biotech). The resulting (growth, migration, contraction) combine data from all powder homogenates were diluted in molecular grade strains or cancer lines used (e.g., means of means, or water (2 mL) and stored (70C; Ethical approval, REC normalizing data/converting control datasets to 100% to No 08/H0720/162, UCL Hospitals). facilitate presentation/discussion).

in vitro Zibotentan ETA receptor antagonism— Zibotentan ETA receptor binding characteristics studies Saturation analysis: Kd/Bmax determination. Recep- Agents. For colorectal cancer line studies, ET-1 was tor binding studies were conducted as described previ- 8 used at 10 mol/L; specific ETA receptor antagonists ously (19): cytospins and tissue homogenates were incu- zibotentan (AstraZeneca) and BQ123 (Bachem Ltd) and bated in increasing 125I-ET-1 concentrations (GE, specific 12 9 specific ETB receptor antagonist BQ788 (Bachem Ltd) activity 2,200 Ci/mmol: 3 10 –10 mol/L) with were used at 10 7 mol/L. For fibroblast studies, ET-1 was nonspecific binding established in the presence of unla- used at 10 7 mol/L; zibotentan, BQ123, and BQ788 at 10 6 beled ET-1(1 mmol/L). After incubation; slides were post- mol/L (Fig. 1). Doses determined previously (9, 14). Other fixed in paraformaldehyde vapor; and homogenates fil- reagents were purchased from Sigma-Aldrich Co, unless tered and washed (3). 125I standards were prepared otherwise specified. where 50 mL aliquots of each of the serial dilutions of Growth. Cells were seeded in 96-well plates (fibro- radioligand used were spotted onto filter paper and blasts: 10,000–15,000/well; HT29, SW620: 20,000/well), attached to microscope slides that were coexposed to grown for 36 hours (60%–70% confluence) and starved radio-sensitive film alongside the cytospins. in serum-free medium (24 hours). This was replaced by Inhibition analysis. Antagonist-binding affinities serum-free medium containing ET-1 and/or ETA/ETB were assessed by inhibition studies: cytospins/tissues receptor antagonists (zibotentan, BQ123, BQ788). Plates were incubated in a fixed 125I-ET-1 concentration (150 were incubated (48 hours), fixed (10% formal saline), and pmol/L Kd) in the presence of increasing concentrations proliferation measured by the methylene blue assay (15), (3 10 9–3 10 6 mol/L) of the receptor antagonists— with absorbance 630 nm (Anthos-2010 platereader, Bio- zibotentan, BQ123, BQ788. Two fixed concentrations chrom Ltd) proportional to cell number. (high ¼ 25 mmol/L; low ¼ 5 mmol/L) for each antagonist Migration. A modified "scratch wound" assay was were used for autoradiography as described below. used (16, 17). Cells were seeded (12-well plates; Autoradiography. Low resolution. Slide-mounted tis- 100,000/well) in fully supplemented medium. At 90%– sues and cytospins were apposed to Hyperfilm MP (GE) in 100% confluence, a scratch was made through the center of X-ray cassettes and exposed for 7 to 21 days (4C), after the well. Medium was replaced with serum-free medium which films were processed following manufacturer’s (with mitomycin C to inhibit proliferation—1 mg/mL for instructions and used for densitometry; representative fibroblasts; 0.5 mg/mL for HT29, SW620) and ET-1 ET images were photographed. receptor antagonists (zibotentan, BQ123, BQ788). Photo- High resolution. Selected slides were used for micro- graphs were taken every 6 hours (0–24 hours). Means of 3 scopic localization of radioligand binding whereby tis- scratch-width measurements per well at fixed positions sues/cytospins were dipped in molten K2 emulsion were used to calculate migration as the percentage of (Ilford), stored in light-proof boxes (4C, 7–21 days), reduction in width compared with that at time ¼ 0. and the emulsion processed following manufacturer’s

1558 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Efﬁcacy of Zibotentan in Colorectal Cancer

instructions (14). Tissues/cells were stained with hema- were conjugated to BQ123 using the water soluble N- toxylin and eosin (H&E), viewed (Olympus BX50 micro- ethyl-N0-diaminopropyl-carbodiimide (EDC) method: scope), photographed (Zeiss Axiocam digital camera), Briefly, 200 mL of QDs (1 mg/mL in borate buffer (pH and images stored (KS400 system, Imaging Associates). 7.4), were mixed with EDC (1 mg/mL) for 30 minutes at Additional to densitometry, radioligand binding was also room temperature. BQ123 (10 4 mol/L) was then added assessed using a gamma counter (Perkin Elmer) where, to the above solution at approximately100 mg/mL and after film images of cytospins had been generated for mixed for 1 hour at room temperature. After this reaction densitometry, cells were digested (100 mL 4 mol/L NaOH; procedure, BQ123-QDs and unconjugated QDs were sep- 10 minutes) and radioactivity (counts per minute; CPM) arated using centrifugal filter (Millipore) with a cut-off measured. value of 10 kDa membrane. After repeated centrifuga- Calculation of binding characteristics. Densitometric tions, purified and concentrated BQ123-QDs were image analysis was conducted on a Biospectrum AC obtained. BQ 123-QDs bioconjugates were applied to Imaging System (UltraViolet Products) and analyzed slide-mounted tissues (unfixed, frozen sections) over- using VisionWorksLS Imaging software (version 6.4.3. night (at 4C), hematoxylin counterstained, and then UVP, 2007). Specific 125I-ET-1 binding was determined observed under confocal microscopy. by subtracting nonspecific from total binding at each Results concentration. Maximum receptor binding (Bmax) and in vitro affinity (Kd) were obtained using GraphPad Prism soft- Efficacy of zibotentan in models of colorectal ware (GraphPad). Radioactivity counts (gamma counter; cancer CPM) from cells removed from slides were similarly The ability of the specific ETA receptor antagonist zibo- analyzed. tentan to inhibit ET-1 stimulation of growth, migration, Immunohistochemistry. Immunohistochemistry was and contraction was tested in colorectal cancer cells and conducted using the Vectastain Alkaline Phosphatase Kit colonic fibroblasts. (Vector Labs) following manufacturer’s instructions as Proliferation. Growth effects were determined inde- described previously (5, 19). Primary antibodies used pendently in five fibroblast strains in the presence of ET-1 were AS02/Thy-1 for fibroblasts, CD31 for vascular endo- alone, ET-1, and ETA or ETB receptor antagonists (zibo- thelial cells, anti-ETA receptor and anti-ETB receptor anti- tentan, BQ123, BQ788), and all three receptor antagonists bodies (Alomone Labs), Col11A1 for collagen Type XI alone (48 hours). Exogenous ET-1 stimulated growth in all (Santa Cruz Biotech, Inc; 1:200 in PBS, 30 minutes), fol- strains, from a minimum of 28% to a maximum of 46%, lowed by universal secondary antibody (1:100 in normal compared with unstimulated controls (P < 0.05; normal- horse serum/PBS, 10 minutes). Sections were counter- ized results for all strains pooled in Fig. 2A). This effect stained (hematoxylin) and photographed as above. H&E was reduced by the specific ETA receptor antagonists, < staining was conducted on selected sections. ETA recep- zibotentan and BQ123 (P 0.05 vs. ET-1 group), but not the tors were further imaged by fluorescence of quantum dot ETB receptor antagonist BQ788. Zibotentan had similar (QD)-coupled BQ123 receptor antagonist (600 nm emis- activity to that of the laboratory standard ETA receptor sion). The water soluble QDs (manufactured in-house) antagonist BQ123. Use of the antagonists alone did not

A B ** 140 130 ** 130 * * 120 120 110 * * 110

100 100 Relative cell number Relative cell number 90 90

ET1 ET1 Control BQ123 BQ788 ZD4054 Control ZD4054 BQ123 BQ788 Fibroblast proliferation

ET-1 + BQ123ET-1 + BQ788 Cancer cell line proliferation ET1 + BQ123 ET1 + BQ788 ET-1 + ZD4054 ET-1 + ZD4054

Reagents: ET-1 (10–7 mol/L) and antagonists (10–6 mol/L) Reagents: ET-1 (10–8 mol/L) and antagonists (10–7 mol/L)

Figure 2. Growth response of fibroblast strains (A) and colorectal cancer cell lines (B) to ET-1 (10 7 mol/L for fibroblasts; 10 8 mol/L for colorectal cancer cells) 6 7 and/or receptor antagonists (anti- ETA receptor: BQ123, zibotentan, anti- ETB receptor: BQ788; 10 mol/L for fibroblasts; 10 mol/L for colorectal cancer cells). Cell number (equivalent to absorbance at 650 nm) was compared with controls (set at 100%). ETA receptor antagonists zibotentan and BQ123 significantly reduced growth in the presence ( , P < 0.05) of exogenous ET-1. ETB antagonist, BQ788, partially reduced ET-1 stimulated growth (NS). Combined results for 5 fibroblast strains (n ¼ 6 independent repeats for each strain) and 2 colorectal cancer cell lines (HT29, SW620, n ¼ 6 independent repeats for each line). Statistical analysis of original absorbance measurements using one-way ANOVA with Tukey HSD post hoc test. (, significant ET-1 growth vs. control).

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1559

Haque et al.

produce a significant effect, although ETB receptor block- 50%–51%. However, combined blockade of ETA and ETB ade (BQ788) seemed to produce a trend toward growth receptors with zibotentan and BQ788 produced an effect stimulation. A similar pattern of growth stimulation was that was less than that observed with ETB blockade, but seen for both the colorectal cancer cell lines HT29 and greater than that seen with ETA blockade (58.8% inhibi- SW620, where addition of exogenous ET-1 produced an tion, resulting in 41.2% migration). Addition of antago- increase in proliferation of approximately 22%; results nists alone did not produce a noticeable effect (results not from independent repeats are pooled for graphical shown). Colorectal cancer cells did not migrate, regardless representation (P < 0.05 vs. control, Fig. 2B). ET-1–stim- of treatment (24 hours). ulated growth was significantly blocked by ETA, (zibo- Contraction. Contraction was investigated in rat col- tentan, BQ123), but not by ETB, receptor antagonists. lagen gels containing either fibroblasts or HT29 colorectal Incubation with antagonists alone did not affect cell cancer cells. Contraction over 72 hours was recorded growth significantly. photographically and concomitant reduction in gel weight Migration. Cell migration was tested in five fibroblast measured with control gels set at 100%. ET-1 caused gel strains and both colorectal cancer cell lines. Fibroblast contraction in the three fibroblast strains investigated, migration was recorded by light microscopy up to 24 reducing the relative gel weight by approximately 40%, hours after the initial scratch was created (Fig. 3). For i.e., 61.25% of the control tissues. (Fig. 4). The addition of fibroblasts, the migration pattern was similar in four of the either ETA or ETB antagonists inhibited ET-1–induced fibroblast strains, (one strain being unresponsive contraction to varying degrees and in the following order: > > (CRF42)): By 18 to 24 hours, fibroblasts completely BQ123 BQ788 zibotentan. Combined ETA and ETB recep- obscured the scratch wound in the presence of ET-1 tor antagonism (zibotentan plus BQ788) resulted in the (100%) compared with untreated (13%). ET-1–stimulated most marked blockade of gel contraction, returning the migration was inhibited by both ETA and ETB antagonists, ET-1–induced reduced gel weights to 89.71% of untreated to varying degrees: BQ788 ¼ 71%; zibotentan and BQ123 ¼ controls. Addition of antagonists alone did not produce a

A B 150 **

100

* * * Control 50 * 0 Hours 24 Hours Percentage migration (%) 0 1 T E 788 Q788 Q Control B Combined fibroblast migration ET1 + ZD4054ET1 + BQ123ET1 + 4054 + B

+ ZD 1 ET Control ET-1 ET-1 + ZD4054 Reagents: ET-1 (10–7 mol/L) and antagonists (10–6 mol/L) C Antagonist % Reduction of ET-1 induced migration * (*ET-1 induced migration set at 100%) % Decrease Significance ZD4054 46.88 P < 0.05 BQ123 49.75 P < 0.05 BQ788 70.11 P < 0.05 ET-1 + BQ123 ET-1 + BQ788 ET-1 + ZD4054/BQ788 ZD4054+BQ788 58.76 P < 0.05

Figure 3. Migration response of ﬁbroblast strains to ET-1 (10 7 mol/L) or ET-1 plus receptor antagonists (zibotentan, BQ123, BQ788, 10 6 mol/L): migration images (A) and migration as percentage of ET-1–induced migration (set at 100%; B); reduction of ET-1–induced migration (C). There is total migration of

fibroblasts after 24 hours in wells exposed to ET-1 only (100% y-axis). This was significantly reduced by both ETA receptor antagonists zibotentan and BQ123, with an average migration of 50% and 51%, respectively ( , P < 0.05 vs. ET-1). ETB receptor antagonist BQ788 produced the greatest inhibitory effect with a resultant average migration of 29% ( , P < 0.05 vs. ET-1). Combined ETA and ETB receptor blockade produced an in-between effect of that seen for ETA and ETB individually (P < 0.05). Combined results for 4 fibroblast strains (n ¼ 6 independent repeats for each strain). Statistical analysis on original migration measurements, using one-way ANOVA with Tukey HSD post hoc test. (, significant ET-1 vs. control).

1560 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Efﬁcacy of Zibotentan in Colorectal Cancer

A B

120 Control ET-1 ET-1 + ** BQ123 100 * * * 80 * **

60 ET-1 + ET-1 + ET-1 + ZD4054 + C BQ788 ZD4054 BQ788 Relative weight change (%) ET-1 Antagonist % Contraction of control size gel* Control ZD4054 (*Control gel set at 100%) Fibroblast contraction ET-1 +BQ788 % Significance ET-1 + ZD4054ET-1 + BQ123 ET-1 61.25% P < 0.05** ET-1 + ZD4054 70.87% P < 0.05* ET1 + BQ788 + ET-1 + BQ123 77.36% P < 0.05* Reagents: ET-1 (10–7 mol/L) and antagonists (10–6 mol/L) ET-1 + BQ788 76.51% P < 0.05* ET-1 + ZD4054+BQ788 89.71% P < 0.05* ** = contraction compared to control * = inhibition compared to ET-1 induced contraction

Figure 4. Contraction of fibroblast gels in response to ET-1 (10 7 mol/L) or ET-1 plus receptor antagonists (zibotentan, BQ123, BQ788; 10 6 mol/L). A, images of gel contraction. B and C, graph and table with weight change equivalent to contraction as percentage of control (set at 100%). Relative weight changes compared with controls (100% weight) were calculated for each strain and the data combined (y-axis). ET-1 stimulated a 38% reduction in gel weight (P < 0.05). This effect was blocked significantly by both ETA receptor antagonists (BQ123 and zibotentan) and ETB receptor antagonist BQ788 ( , P < 0.05). The effect is most marked with combined ETA and ETB blockade ( , P < 0.05). Combined results for 3 fibroblast strains (n ¼ 3 independent repeats for each strain). Statistical analysis on original contraction weight measurements, using one-way ANOVA with Tukey HSD post hoc test. (, significant ET-1 vs. control).

noticeable effect (results not shown). Colorectal cancer fibroblasts. In tumor tissues, both ETA and ETB receptors > 125 cells did not cause gel contraction. were present (ETA ETB) and localized to areas of I-ET- 1 binding. Intense 125I-ET-1 binding once again correlated Pharmacologic characteristics of zibotentan in with both fibroblast and endothelial cell staining. Colla- colorectal tissues, colorectal cancer cells, and colonic gen type XI (Col XI), which was used to further define fibroblasts tumor stroma, was only present in tumor sections and not Binding of 125I-ET-1, in the presence or absence of normal tissue, confirming previous reports of its associ- receptor antagonists, was shown by autoradiography in ation with colorectal pathology (20, 21). tissue sections and homogenates from patient specimens Receptor subtype distribution. To study ETA and ETB of colorectal cancer and normal bowel tissue; and also in receptor distribution within normal and tumor sections, cytospins of colorectal cancer cells or colonic fibroblasts. we inhibited 125I-ET-1 binding using specific receptor Localization and distribution of ET-1 binding. In antagonists, thereby showing indirectly the presence of high-resolution autoradiographs of frozen tissue sections, ETA and ETB receptors (Fig. 6). The ETA receptor antag- 125I-ET-1 exhibited intense binding that was evident as onist, BQ123, in both normal and tumor sections, showed white grains when viewed under dark field illumination; a concentration-dependent inhibitory effect with 25 this correlated mostly with stromal regions as defined by mmol/L reducing 125I-ET-1 binding to a greater degree H&E staining (Fig. 5A). To identify associated structures, than 5 mmol/L. Furthermore, the extent of inhibition was consecutive sections were stained immunohistochemi- different between normal and tumor sections: most 125I- cally for: ETA and ETB receptors, stromal fibroblasts ET-1 inhibition was observed in tumor sections, consistent (Thy-1), endothelial cells (CD31), and collagen type XI with a higher ETA receptor density in tumors compared (connective tissue; Fig. 5B). Normal colon tissue had with normal tissue. The ETB receptor antagonist BQ788 preserved structural architecture with a well-defined also showed a concentration-dependent inhibition in both epithelial mucosal layer, whereas that of tumors was normal and tumor (25 mmol/L > 5 mmol/L). Greater disorganized. In normal colon specimens, microautora- inhibition of ETB receptor binding in normal colonic tissue 125 diography revealed I-ET-1 binding in the epithelial suggests a higher ETB receptor expression in healthy mucosa, submucosa, and specific areas in the stroma; tissue than cancer. An interesting observation is the recep- there was high ETA and ETB receptor immunostaining at tor distribution in normal mucosa where ETA receptors 125 125 regions of I-ET-1binding. I-ET-1 bound strongly to appear localized closer to the luminal surface and ETB regions that stained positively for endothelial cells and receptors closer to the basal region. The extent of

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1561

Haque et al.

A Normal sections Tumor sections

B Normal sections Tumor sections

Thy-1 (fibroblast) CD31 (endothelial) Col XI (connective) ETA Receptor ETB Receptor

Figure 5. Microscopic localization of 125I-ET-1 in patient sections (A) showing dark field illumination of radiolabeled 125I-ET-1 binding (left) and H&E staining of underlying sections (right). Binding is predominantly seen on connective tissues within both normal colon and tumor stroma. B, immunohistochemistry was used to identify cell type–specific staining to regions of 125I-ET-1 bound to normal (top) and tumor (bottom sections) tissue as visualized in low-resolution autoradiography film. Thy-1 (fibroblast) had similar distribution to stromal 125I-ET-1 bound regions in both normal and tumor sections. CD31 endothelial 125 cell staining (tumor > normal) localized to I-ET-1 dense regions. ETA staining (tumor > normal) and ETB staining (normal ¼ tumor) also correlated with 125I-ET-1 binding. Tumor-specific collagen type XI was only seen in tumor stroma.

zibotentan concentration–dependent inhibition was not fmol/mg, respectively; mg of tissue protein). Overall 125I- as great as either BQ123 or BQ788. On densitometry ET-1 receptor binding to cytospins of cancer cells and analysis, there was more extensive inhibition observed fibroblasts is shown in Fig. 7C, with calculations of Kd in tumors than normal colonic tissue specimens, again and Bmax in Fig. 7D and E. The fibroblasts’ combined Kd consistent with a higher concentration of ETA receptors in value of 213.6 pmol/L (0.213 nmol/L) is regarded as high ¼ m cancer. ETA receptor overexpression in cancer tissues was affinity (near 1 nmol/L or less high affinity; 1 mol/L or corroborated by immunofluorescent detection of QD-con- more ¼ low affinity). Fibroblasts also showed a relatively 125 6 jugated BQ123 in patient specimens. Binding to ETA high maximal I-ET-1 receptor binding (3.03 fmol/1 10 receptors presented as a punctate pattern evident mostly cells). The Kd and Bmax values for all fibroblasts were in stromal areas surrounding epithelial glands (Fig. 7A similar with only one strain (CF56) showing lower values. and B). The colorectal cancer cell lines (HT29, SW480) had a 6 Binding characteristics. To examine the general bind- combined Bmax and Kd value of 2.43 fmol/1 10 cells ing characteristics of 125I-ET-1 in normal and tumor sec- and 0.367 nmol/L respectively. Both cell lines had a 125 tions, we used tissue homogenates (not shown). The Kd similar Bmax to the fibroblasts with an I-ET-1–binding shows that binding affinity of 125I-ET-1 was similar in both affinity that was marginally less (average HT29 and 6 the normal and tumor specimens (203 pmol/L and 204 SW480: Bmax, 2.435 fmol/1 10 cells; Kd, 0.367 nmol/L; 6 pmol/L). Maximum binding (Bmax) was also similar in average fibroblasts: Bmax, 3.03 fmol/1 10 cells; Kd,0.213 both normal and tumor samples (57.83 fmol/mg and 58.31 nmol/L). For receptor antagonist inhibition, the IC50 was

1562 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Efﬁcacy of Zibotentan in Colorectal Cancer

Normal tissue Tumor tissue

Figure 6. Receptor selective antagonists (BQ123, BQ788 or zibotentan; high concentration, Non-Specific Total binding 25 mmol/L; low concentration, 5 Non-Specific Total binding m 125 mol/L) ability to inhibit I-ET-1 25 µmol/L 5 µmol/L 25 µmol/L 5 µmol/L (150 pmol/L) binding to normal (left) and tumor (right) slide- mounted tissue from patients. ZD4054 125I-ET-1 total binding in the absence of antagonist. Low- resolution autoradiographs were produced by opposing the slide mounted tissues to Hyperﬁlm. Inhibition of 125I-ET-1 binding was greatest with ETA receptor BQ123 antagonists (BQ123 > zibotentan) within tumor tissue. The ETB receptor antagonist (BQ788) had a greater effect reducing 125I-ET-1 binding in normal tissue. BQ788

determined using two methods, the first measuring radio- by variations in protocols and also by the nature of the activity bound (CPM) in a gamma counter and the second specific fibroblasts; for example, ours were isolated from through exposure to radiation-sensitive film and densito- individual adult colons, whereas Kernochan and collea- metric analysis, with readings categorized into high IC50 gues used a cell line (No Co18) isolated from the gastro- (<10 mmol/L), medium (10–100 mmol/L), and low (>100 intestinal tract of a 2.5 month old (16). m mol/L). Both methods gave similar IC50 results for fibro- Fibroblast growth in the presence of exogenous ET-1 m m blasts (BQ123, 0.1–2.2 mol/L; zibotentan, 10–15.1 mol/L; was reduced significantly by ETA receptor antagonists and BQ788, 0.96–1 mmol/L). The IC50 of colorectal cancer zibotentan and BQ123, with zibotentan as effective as lines also indicated that ETA receptor antagonists more BQ123 but not as effective as the ETB receptor antagonist effectively inhibited 125I-ET-1 binding (BQ123, 4.43–10 BQ788, findings that expand on our previous work (10). m m mol/L; zibotentan, 0.1–1.01 mol/L and BQ788, 0.013– These findings are consistent with ETA as the major 1mmol/L). receptor responsible for propagating growth signals reported in a variety of cell types, including fibroblasts. Discussion Interestingly, in our series, there was some response The efficacy of the ETA receptor antagonist zibotentan variation within the fibroblast strains to BQ788 antago- against ET-1–driven proliferation, migration, and contrac- nism: for strain CF65, BQ788 antagonism resulted in a tion was investigated in a panel of colonic fibroblasts and significant decrease in ET-driven cell growth. This is not colorectal cancer cell lines. obvious in the graphical representation (Fig. 2A), as the All ET-1–treated fibroblast strains showed significant results were pooled from experiments conducted in five growth compared with controls. Previous work on colonic fibroblast strains. Conversely, in ovarian cancer-associat- fibroblasts from our group reported only one of six strains ed fibroblasts, ET-1–induced proliferation was inhibited showing ET-1–stimulated growth increase (10). This dis- by BQ123 and BQ788, suggesting that in the described crepancy is explainable by (i) the inclusion of a 24-hour model both ET receptors contribute to growth signaling starvation period before ET-1 addition and that (ii) here (22). Successful inhibition of proliferation by combined we only used two of the previously tested fibroblast ETA and ETB receptor antagonism has been previously strains. The only other study using colonic fibroblasts reported in lung myofibroblasts (23). reported small nonsignificant increases in cell numbers Treatment by receptor antagonists alone, especially ETA in response to ET-1 (16). On the other hand, Moraitis and receptor antagonists zibotentan and BQ123, did not colleagues showed that ET-1 stimulated growth signifi- result in significant differences compared with controls. cantly in three fibroblast lines isolated from patients with However, use of the ETB receptor antagonist BQ788 ovarian cancers (22). Differences may be explained both alone seemed to produce a trend towards increased

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1563

Haque et al.

A B C Figure 7. Localization of QD-BQ- Nonspecific 123 conjugates in patient sections Total binding binding from normal (A) and tumor (B) 1,000 pmol/L specimens. Frozen sections were incubated with QD-BQ123 overnight (4C) and visualized by 300 pmol/L confocal microscopy. Punctate yellow-orange QD-BQ123 binding 100 pmol/L was more abundant in tumor versus normal sections. Green tissue autofluorescence is present. 30 pmol/L Bar, 50 mm. C, images generated by exposure of cytospins to film. Cytospun cells were incubated 10 pmol/L with increasing concentrations of 125I-ET-1 (3 1012 to 1 109 mol/L) to determine total binding 3 pmol/L and in the presence of unlabeled ET-1 (1 mmol/L) to establish nonspecific binding. D, cells were Specific binding (combined) removed with NaOH and total and 25,000 nonspecific 125I-ET-1 activity fi 20,000 quanti ed by gamma counter. Graphs of specific binding were 15,000 produced by subtracting 125 10,000 nonspecific from total I-ET-1 binding. Prism was used to Fibroblasts 5,000

Radiocount CPM calculate fibroblast and cancer cell 6 0 line Bmax (mean, 3.03 fmol/1 10 0 200 400 600 800 1,000 Specific Nonspecific cells; 2.43 fmol/1 106 cells, binding binding nmol/L Radioligand (ET-1) respectively) and Kd [mean, D E 213.6 pmol/L (0.214 nmol/L); Specific binding (combined) 367.0 pmol/L (0.367 nmol/L), respectively]. E, specific 8,000 radiolabeled 125I-ET-1 6,000 (100 pmol/L) localization to colorectal fibroblasts (top) and 4,000 cancer cell lines (bottom). Total 125I-ET-1 binding (left) and 2,000 nonspecific (right) shows specific Cancer cell lines Radiocount CPM 0 binding to cells. 0 200 400 600 800 1,000 nmol/L Radioligand (ET-1)

proliferation in the fibroblast strains. The response of the original amounts of endogenous ET-1 produced by each five strains to BQ788 growth stimulation was mixed: in individual strain. Although not measured in the fibroblast strain CF35, BQ788-stimulated increase in growth reached strains studied here, we have previously reported varied significance at P < 0.05. In another strain, it just missed endogenous ET-1 production by different cell lines (19). significance, whereas in the remaining three fibroblast The present findings, considered in the wider context of strains there was minimal response to BQ788 stimulation. fibroblast studies, show both strong overall patterns of Explanations for the observed response center around the colonic fibroblast response but also the importance of availability of endogenous ET-1 after ETB receptor block- individual-specific differences. In the future, such indi- ade. ET-1 produced by the fibroblasts themselves does not vidual-specific data could inform personalized treatment. generally seem to constitute a major growth signal, as ETA ET-1–stimulated proliferation of colorectal cancer lines receptor antagonism alone does not result in growth (HT29, SW620) was consistent with previous reports that reduction. However, the ETB receptor is part of the ET- also include similar results from LIM1215 (9, 14). Growth 1 clearance pathway and therefore blockade by BQ788 rates were reduced significantly by zibotentan and BQ123, would result in more endogenous ET-1 accumulating in but not BQ788, with zibotentan more effective than the cell environment. The peptide would bind to ETA BQ123. Generally, the majority of cancer cell types, for receptor and therefore contribute in a minor way to the example, prostate, ovarian, and colorectal cancers show a proliferation signal. Differences in responses by individ- reduction of ET-1–stimulated growth in response to ETA ual fibroblast strains would be further determined by the antagonism; the proliferative signal may be promoted

1564 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Efﬁcacy of Zibotentan in Colorectal Cancer

further through EGF receptor transactivation (4, 9, 24). previously published data regarding the characteristics of The striking exception is melanoma, where ET-1 drives ET-1 binding in other tissues (30, 31, 32). growth via ETB receptor (25). Zibotentan inhibition of ET- We previously showed a change in the ratio of receptor 1–induced growth was reported in ovarian cancer lines, subtypes, with upregulation of ETA receptors and down- with a reduction in ETA-mediated angiogenesis and inva- regulation of ETB receptors in colorectal cancer compared sive mediators (VEGF, matrix metalloproteinases; with normal colon (5, 19). In the present series, we refs. 24, 26). Zibotentan also enhanced the apoptotic activ- inhibited 125I- ET-1 binding with unlabeled receptor ity of the chemotherapeutic agent, paclitaxel, in an in vitro antagonists. BQ123 showed a concentration-dependent and in vivo model of ovarian cancer (24), indicating a inhibition of 125I-ET-1 binding in both normal and tumor possible role for combination therapy. specimens (25 mmol/L > 5 mmol/L). The most striking ET-1–stimulated migration was clearly shown in fibro- inhibition was observed in the tumor sections, consistent blasts, but not cancer cells. It was inhibited by all three with a higher ETA receptor expression in cancer compared antagonists and most markedly by BQ788, consistent with with normal colon, supporting previous findings: Specif- previous findings (10, 16). Comparing ETA receptor ically, Hoosein and colleagues (5) used the selective radi- 125 antagonists, zibotentan produced a similar inhibitory olabeled ETA receptor antagonist [ I]PD-151242 and effect on migration to BQ123. Combined ETA and ETB showed that binding on cancer tissues was increased by receptor blockade (zibotentan plus BQ788) produced a 55.5% when compared with normal colon. The ETB recep- migratory effect in between that produced by ETB block- tor antagonist BQ788 also inhibited binding in a concen- m > m ade and ETA blockade. Fibroblast migration and contrac- tration-dependent manner (25 mol/L 5 mol/L). The tion due to various stimuli are well documented. For greater inhibition of binding to ETB receptors observed in example, Shi-Wen and colleagues showed that on ET-1 normal colon than within tumor is consistent with higher exposure, normal lung fibroblasts upregulated contractile ETB receptor expression in normal colon and a down- proteins, which promoted myofibroblast contraction and regulation in colorectal cancer (5, 19). In the latter study, 125 migration (27). Fibroblasts from patients with scleroder- the specific ETB receptor agonist, [ I]BQ3020, showed a ma express high ET-1 levels suggesting a contributory role 45% decrease in ETB receptor expression in cancer com- in connective tissue diseases. pared with normal. However, this is contrary to the Gel contraction was similarly a fibroblast-specific findings elucidated by Egidy and colleagues (29), who response and both ET receptors needed to be blocked for described a quantitative increase in mRNA expression of maximum inhibition, although the contractile response both ETA and ETB receptors in colorectal cancer speci- was not completely abolished. ET-1–stimulated contrac- mens. However, increases at the mRNA level are not tion seems to be associated with an increase in cytosolic necessarily followed by translation at the protein level. calcium and myosin phosphorylation and BQ123 and Zibotentan did not display a concentration-dependent BQ788 were previously shown to inhibit this effect to inhibition to the extent observed with BQ123 and BQ788. various degrees (10, 16). In other tissues, ET-1 caused There was overall greater inhibition observed in tumor primary lung fibroblast contraction, although this specimens than normal tissues, in keeping with the over- response was mediated by ETA predominantly, rather expression of ETA receptors in cancer. The extent of than both receptors (27). Although zibotentan was able inhibition of 125I- ET-1 binding may not have been as to inhibit ET-1–mediated fibroblast contraction, it did so to expected as zibotentan is a specific ETA receptor antago- a lesser degree than either BQ123 or BQ788. nist while BQ123 and BQ788 at higher concentrations are To investigate zibotentan efficacy against 125I-ET-1 known to act on both receptor subtypes (lose selectivity; binding, we carried out studies using specimens from ref. 30). Therefore, use of these laboratory compounds patients with colorectal cancer. Autoradiography showed may not be truly subtype-selective at high concentrations. 125 125 I-ET-1–binding sites (ETA and ETB receptors) within I-ET-1 binding to specific structures was further normal colon and cancer tissues. Maximal binding was clarified by immunohistochemical localization of ETA and seen in stromal regions, which are densely populated by ETB receptors, in addition to fibroblast, endothelial cell, fibroblasts and blood vessels/endothelial cells, as shown and collagen XI mapping. ETA and ETB immunostaining by immunohistochemistry (AS02, CD31). Positive colla- closely correlated to 125I-ET-1 binding in parallel tissue gen XI staining confirmed the pathologic state of the sections. Cancer tissues showed increased ETA receptor stroma associated with cancer lesions (20). This correlates staining localized to epithelial cells and tumor stroma, and with our previous work on human colon specimens, reduced ETB receptor staining on the epithelial cell sur- which showed that radiolabeled ET-1, ETA receptor face, compared with normal tissues. Similar alterations in antagonist, and ETB receptor agonist bound in a similar receptor expression have been reported in autoradio- pattern within cancer and normal tissue (5, 14). Other graphic studies on prostate and ovarian cancer (3, 4). groups have also reported strong stromal binding of 125I 125I-ET-1 binding to vascular regions, shown here by ET-1 in intestinal tissues (28, 29). Tissue homogenates colocalization with ETB-expressing endothelial cells and were used to determine binding characteristics. The Kd its known association with ETA-expressing vascular 125 and Bmax of I-ET-1 were similar in both normal and smooth muscle cells (30, 31), highlights ET-1 angiogenic tumor homogenates. These figures are closely matched to actions, such as VEGF production (33). 125I-ET-1 also

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1565

Haque et al.

bound strongly to fibroblasts, which in cancer tissues ETA receptor affinity may once again be due to a loss of express predominantly ETA receptors and often produce BQ123 receptor selectivity when used at higher concen- collagen type XI. Interestingly, ET-1 affects the APC/ trations. Colorectal cancer cells exhibited IC50 values b-catenin and Wnt signaling, which is the pathway similar to that of fibroblasts (zibotentan: high; BQ123: involved in regulating COLXI and CTFG expression, medium; BQ788: low range) with ETA receptor antago- 125 molecules with tumorigenic associations (34). nists more effective at inhibiting I-ET-1 than the ETB > Within normal mucosa, ETA receptors appear closer to receptor antagonist (BQ123 1,000-fold BQ788). BQ788 the luminal surface with ETB receptors towards the mus- IC50 results were the same as that seen in fibroblasts, cularis mucosa and lamina propria (Fig. 6), a distribution suggesting they may have similar universal functions pattern not previously reported. This may reflect trophic such as an involvement in the ET-1 "clearance pathway". signaling roles for ET receptors along the basal to apical When comparing the two ETA receptor antagonists, zibo- direction of cell turnover and differentiation. Similar tentan was the most effective at inhibiting 125I-ET-1 bind- functions have been postulated for retinoic acid receptors ing in cancer cells. and vitamin D receptors, which are denser at the apex, In summary, we showed that ET-1 stimulates behavior and ERa found predominantly in basal regions (35). This consistent with tumorigenesis in colorectal cancer cells pattern is consistent with ETB associated with growth and in colonic fibroblasts. Many of the actions are via the arrest signaling and ETA associated with differentiation ETA receptor with various levels of contribution of the ETB signaling. receptor and this strengthens the evidence for the poten- The combined fibroblasts Kd value (0.213 nmol/L) is tial therapeutic use of zibotentan. Importantly, ET-1 regarded as high affinity (1 nmol/L) with the cells appears to drive colorectal tumorigenesis primarily via showing a relatively high maximal 125I-ET-1 binding the stroma and relevant anti-ET treatment would provide (3.03 fmol/1 106 cells). The colorectal cancer lines a shift in treatment paradigm. 6 exhibited a similar Bmax (2.43 fmol/1 10 cells) and affinity that was marginally less than the fibroblasts (Kd: Disclosure of Potential Conflicts of Interest 0.367 nmol/L), suggesting that fibroblasts have more ET No potential conflicts of interest were disclosed. surface receptors than cancer cells; this is supported by our high-resolution autoradiography (Fig. 7E). Fibro- Authors' Contributions Conception and design: S. Haque, M.R. Dashwood, M. Heetun, D.J. blasts also had a higher binding affinity than cancer cells. Abraham, M. Loizidou These figures were similar to previous studies using Development of methodology: S. Haque, M.R. Dashwood, M. Heetun, human colonic mucosa and human skin fibroblasts with X. Shiwen, N. Farooqui, B. Ramesh, D.J. Abraham, M. Loizidou Acquisition of data (provided animals, acquired and managed patients, Kd values of 0.41 and 0.4 nmol/L, respectively (28, 36, 37). provided facilities, etc.): S. Haque, M.R. Dashwood, M. Heetun, This is the first time that individual cellular components of F.J. Savage, O. Ogunbiyi, D.J. Abraham, M. Loizidou Analysis and interpretation of data (e.g., statistical analysis, biostatis- colonic tissue have been evaluated for ET-1 binding char- tics, computational analysis): S. Haque, M.R. Dashwood, X. Shiwen, acteristics with our overall results suggesting a higher N. Farooqui, H. Welch, D.J. Abraham, M. Loizidou Writing, review, and/or revision of the manuscript: S. Haque, M.R. binding density/Bmax within fibroblasts. Dashwood, M. Heetun, B. Ramesh, H. Welch, D.J. Abraham, M. Loizidou Competition studies were conducted using both radio- Administrative, technical, or material support (i.e., reporting or orga- activity measurements and film densitometry. Both meth- nizing data, constructing databases): S. Haque, X. Shiwen, N. Farooqui, ods gave similar IC results for fibroblasts (BQ123: high; B. Ramesh, M. Loizidou 50 Study supervision: M.R. Dashwood, N. Farooqui, O. Ogunbiyi, D.J. zibotentan: medium; BQ788: low range). Only small sam- Abraham, M. Loizidou ple numbers were used and, overall, the ETA receptor antagonists were much more effective at inhibiting 125I- Grant Support ET-1 binding than ETB receptor antagonists. The ETA This study was partially funded by AstraZeneca and awarded to M.R. Dashwood and M. Loizidou, grant code BSDD. receptor antagonist BQ123 was approximately 10,000 fold The costs of publication of this article were defrayed in part by the more effective than the ETB receptor antagonist BQ788, payment of page charges. This article must therefore be hereby marked whereas the orally active drug, zibotentan, was more advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. effective at inhibiting 125I-ET-1 binding than BQ788 by approximately 1,000-fold although approximately 1,000- Received October 14, 2012; revised May 21, 2013; accepted May 21, 2013; fold less effective than BQ123. This difference between published OnlineFirst May 30, 2013.

References 1. Colon Cancer Alliance, 2011, http://www.ccalliance.org/index.html 4. Nelson JB, Chan-Tack K, Hedican SP, Magnuson SR, Opgenorth TJ, 2. Grant K, Loizidou M, Taylor I. Endothelin-1: a multifunctional molecule Bova GS, et al. Endothelin-1 production and decreased endothelin B in cancer. Br J Cancer 2003;88:163–6. receptor expression in advanced prostate cancer. Cancer Res 3. Bagnato A, Salani D, Di Castro V, Wu-Wong JR, Tecce R, Nicotra MR, 1996;56:663–8. et al. Expression of endothelin 1 and endothelin A receptor in ovarian 5. Hoosein MM, Dashwood MR, Dawas K, Ali HM, Grant K, Savage F, carcinoma: evidence for an autocrine role in tumor growth. Cancer Res et al. Altered endothelin receptor subtypes in colorectal cancer. Eur J 1999;59:720–7. Gastroenterol Hepatol 2007;19:775–82.

1566 Mol Cancer Ther; 12(8) August 2013 Molecular Cancer Therapeutics

Efﬁcacy of Zibotentan in Colorectal Cancer

6. Levin ER. Endothelins. N Engl J Med 1995;333:356–63. 22. Moraitis S, Miller WR, Smyth JF, Langdon SP. Paracrine regulation of 7. Nelson J, Bagnato A, Battistini B, Nisen P. The endothelin axis: ovarian cancer by endothelin. Eur J Cancer 1999;35:1381–7. emerging role in cancer. Nat Rev Cancer 2003;3:110–6. 23. Prefontaine A, Calderone A, Dupuis J. Role of endothelin receptors on 8. Rosano L, Varmi M, Salani D, Di Castro V, Spinella F, Natali PG, et al. basal and endothelin-1-stimulated lung myofibroblast proliferation. Endothelin-1 induces tumor proteinase activation and invasiveness of Can J Physiol Pharmacol 2008;86:337–42. ovarian carcinoma cells. Cancer Res 2001;61:8340–6. 24. Rosano L, Di Castro V, Spinella F, Tortora G, Nicotra MR, Natali PG, 9. Grant K, Knowles J, Dawas K, Burnstock G, Taylor I, Loizidou M. et al. Combined targeting of endothelin A receptor and epidermal Mechanisms of endothelin 1-stimulated proliferation in colorectal growth factor receptor in ovarian cancer shows enhanced antitumor cancer cell lines. Br J Surg 2007;94:106–12. activity. Cancer Res 2007;67:6351–9. 10. Knowles JP, Shiwen X, Haque S, Bhalla A, Dashwood MR, Yang S, 25. Lahav R, Suva ML, Rimoldi D, Patterson PH, Stamenkovic I. Endothelin et al. M. Endothelin-1 stimulates colon cancer adjacent fibroblasts. Int receptor B inhibition triggers apoptosis and enhances angiogenesis in J Cancer 2012;130:1264–72. melanomas. Cancer Res 2004;64:8945–53. 11. Carducci MA, Saad F, Abrahamsson PA, Dearnaley DP, Schulman CC, 26. Rosano L, Di Castro V, Spinella F, Decandia S, Natali PG, Bagnato North SA, et al. Atrasentan Phase III Study Group Institutions. A phase A. ZD4054, a potent endothelin receptor A antagonist, inhibits 3 randomized controlled trial of the efficacy and safety of atrasentan in ovarian carcinoma cell proliferation. Exp Biol Med 2006;231: men with metastatic hormone-refractory prostate cancer. Cancer 1132–5. 2007;110:1959–66. 27. Shi-WenX,ChenY,DentonCP,EastwoodM,RenzoniEA,Bou- 12. Nelson JB, Love W, Chin JL, Saad F, Schulman CC, Sleep DJ, et al. Gharios G, et al. Endothelin-1 promotes myofibroblast induction Atrasentan Phase 3 Study Group. Phase 3, randomized, controlled trial through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt- of atrasentan in patients with nonmetastatic, hormone-refractory dependent pathway and is essential for the enhanced contractile prostate cancer. Cancer 2008;113:2478–87. phenotype of fibrotic fibroblasts. Mol Biol Cell 2004;15: 13. Valentich JD, Popov V, Saada JI, Powel DW. Phenotypic character- 2707–19. ization of an intestinal subepithelial myofibroblast cell line. Am J 28. Inagaki H, Bishop AE, Eimoto T, Polak JM. Autoradiographic locali- Physiol 1997;272:C1513–24. zation of endothelin-1 binding sites in human colonic cancer tissue. 14. Ali H, Loizidou M, Dashwood M, Savage F, Sheard C, Taylor I. J Pathol 1992;168:263–7. Stimulation of colorectal cancer cell line growth by ET-1 and its 29. Egidy G, Juillerat-Jeanneret L, Korth P, Bosman FT, Pinet F. The inhibition by ET(A) antagonists. Gut 2000;47:685–8. endothelin system in normal human colon. Am J Physiol Gastrointest 15. Oliver MH, Harrison NK, Bishop JE, Cole PJ, Laurent GJ. A rapid and Liver Physiol 2000;279:G211–22. convenient assay for counting cells cultured in microwell plates: appli- 30. Alexander SPH, Mathie A, Peters JA. Guide to Receptors and Channels cation for assessment of growth factors. J Cell Sci 1989;92:513–8. (GRAC), 4th edn. Br J Pharmacol 2009;158:S1–S254. 16. Kernochan LE, Tran BN, Tangkijvanich P, Melton AC, Tam SP, Yee HF 31. Sullivan ME, Mumtaz FH, Khan MA, Dashwood MR, Thompson CS, Jr. Endothelin-1 stimulates human colonic myofibroblast contraction Mikhailidis DP, et al. Endothelins in the urinary tract. BJU Int and migration. Gut 2002;50:65–70. 2000;86:97–106. 17. Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat 32. Hansen-Schwartz J, Szok D, Edvinsson L. Expression of ETA and ETB hepatic stellate cells is modulated by endothelin-1 through rho-kinase- receptor mRNA in human cerebral arteries. Br J Neurosurg 2002;16: mediated alterations in the acto-myosin cytoskeleton. Hepatology 149–53. 2001;33:74–80. 33. Alanen K, Deng DX, Chakrabarti S. Augmented expression of endothe- 18. Ivarsson M, McWhirter A, Black CM, Rubin K. Impaired regulation of lin-1, endothelin-3 and the endothelin-B receptor in breast carcinoma. collagen pro-alpha 1(I) mRNA and change in pattern of collagen- Histopathology 2000;36:161–7. binding integrins on scleroderma fibroblasts. J Invest Dermatol 34. Ladwa R, Pringle H, Kuvmar R, West K. Expression of CTGF and Cyr61 1993;101:216–21. in colorectal cancer. J Clin Pathol 2011;64:58–64. 19. Ali H, Dashwood M, Dawas K, Loizidou M, Savage F, Taylor I. 35. D'Errico I, Moschetta A. Nuclear receptors, intestinal architecture Endothelin receptor expression in colorectal cancer. J Cardiovasc and colon cancer: an intriguing link. Cell Mol Life Sci 2008;65: Pharmacol 2000;36:S69–71 1523–43. 20. Fischer H, Stenling R, Rubio C, Lindblom A. Colorectal carcinogenesis 36. Inagaki H, Bishop AE, Escrig C, Wharton J, Allen-Mersh TG, Polak JM. is associated with stromal expression of COL11A1 and COL5A2. Localization of Endothelin-like immunoreactivity and endothelin bind- Carcinogenesis 2001;22:875–8. ing sites in human colon. Gastroenterology 1991;101:47–54. 21. Fischer H, Salahshor S, Stenling R, Bjork€ J, Lindmark G, Iselius L, et al. 37. Kusuhara M, Yamaguchi K, Nagasaki K, Hayashi C, Suzaki A, Hori S, COL11A1 in FAP polyps and in sporadic colorectal tumors. BMC et al. Production of endothelin in human cancer cell lines. Cancer Res Cancer 2001;1:17. 1990;50:3257–61.

www.aacrjournals.org Mol Cancer Ther; 12(8) August 2013 1567

Efficacy of the Specific Endothelin A Receptor Antagonist Zibotentan (ZD4054) in Colorectal Cancer: A Preclinical Study

Samer-ul Haque, Michael R. Dashwood, Mohammed Heetun, et al.

Mol Cancer Ther 2013;12:1556-1567. Published OnlineFirst May 30, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-12-0975

Cited articles This article cites 36 articles, 11 of which you can access for free at: http://mct.aacrjournals.org/content/12/8/1556.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/12/8/1556.full#related-urls

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 Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/12/8/1556. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.