Antitumor Effects of Thalidomide Analogs in Human Prostate Cancer Xenografts Implanted in Immunodeficient Mice

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4192 Vol. 10, 4192–4197, June 15, 2004 Clinical Cancer Research

Sylvia S. W. Ng,1 Gordon R. MacPherson,1 xenografts was significantly decreased by CPS45 and Michael Gu¨tschow,2 Kurt Eger,3 and CPS49. CPS49 also reduced MVD in PC3 xenografts. 1 Conclusions: Thalidomide analogs CPS11 and 49 are William D. Figg promising anti-cancer agents. PDGF signaling pathway may 1 Molecular Pharmacology Section, Cancer Therapeutics Branch, be a potential target for these thalidomide analogs. Detailed Center for Cancer Research, National Cancer Institute, NIH, microarray and functional analyses are under way with the Bethesda, Maryland; 2Pharmaceutical Institute, Poppelsdorf, University of Bonn, Bonn, Germany; and 3Institute of Pharmacy, aim of elucidating the molecular mechanism(s) of action of Pharmaceutical Chemistry, University of Leipzig, Leipzig, Germany these thalidomide analogs.

INTRODUCTION ABSTRACT Prostate carcinoma is the most common cancer and the Purpose: Thalidomide has demonstrated clinical activ- second leading cause of cancer death in American men. Current ity in various malignancies including androgen-independent standard therapy for advanced prostate cancer involves andro- prostate cancer. The development of novel thalidomide an- gen ablation using luteinizing hormone-releasing hormone ago- alogs with better activity/toxicity profiles is an ongoing re- nists with or without an androgen antagonist such as flutamide search effort. Our laboratory previously reported the in or bicalutamide. However, the disease eventually becomes an- vitro antiangiogenic activity of the N-substituted thalidomide drogen independent, at which point therapeutic options are analog CPS11 and the tetrafluorinated analogs CPS45 and limited. Novel treatment modalities are required to improve CPS49. The current study evaluated the therapeutic poten- clinical outcome. tial of these analogs in the treatment of prostate cancer in Angiogenesis, the formation of new blood vessels from vivo. preexisting vessels, plays a significant role in solid tumor Experimental Design: Severely combined immunodefi- growth and metastasis (1). In particular, using the transgenic cient mice bearing s.c. human prostate cancer (PC3 or adenocarcinoma of the mouse prostate model, Huss et al. (2) 22Rv1) xenografts were treated with the analogs at their have identified two distinct angiogenic switches in prostate maximum tolerated doses. Tumors were then excised and cancer progression. It seems logical to speculate that inhibition processed for ELISA and CD31 immunostaining to deter- of angiogenesis would represent an effective treatment strategy mine the levels of various angiogenic factors and microvessel for prostate cancer. Thalidomide has been shown to inhibit basic density (MVD), respectively. fibroblast growth factor (bFGF)- and vascular endothelial Results: CPS11, CPS45, and CPS49 induced prominent growth factor (VEGF)-induced angiogenesis (3, 4). The antitu- and modest growth inhibition in PC3 and 22Rv1 tumors, mor activity of thalidomide has also been reported in numerous respectively. Thalidomide had no effect on tumor growth in clinical trials (5–8). In androgen-independent prostate cancer, either xenograft. Vascular endothelial growth factor and thalidomide caused a decrease in serum prostate-specific anti- basic fibroblast growth factor levels were not significantly gen and an improvement of clinical symptoms in 27% of pa- altered by any of the thalidomide analogs or thalidomide in tients (9). The common side effects of thalidomide, including both PC3 and 22Rv1 tumors. CPS45, CPS49, and thalido- dose-dependent somnolence and dizziness as well as peripheral mide significantly reduced PC3 tumor platelet-derived neuropathy (10), have prompted the development of thalidomide growth factor (PDGF)-AA levels by 58–82% (P < 0.05). analogs with better pharmacological profiles. Our laboratory Interestingly, treatment with the analogs and thalidomide previously demonstrated the superior antiangiogenic activity of resulted in differential down-regulation (>1.5-fold) of genes the N-substituted analog CPS11 and the tetrafluorinated analogs encoding PDGF and PDGF receptor isoforms as determined CPS45 and CPS49 in comparison with thalidomide in multiple by DNA microarray analysis. Intratumoral MVD of 22Rv1 in vitro assays (11). The current study investigated the in vivo therapeutic efficacy of these analogs in the treatment of prostate cancer.

MATERIALS AND METHODS Received 12/10/03; revised 3/19/04; accepted 3/30/04. The costs of publication of this article were defrayed in part by the Drugs. Thalidomide analogs CPS11, CPS45, and CPS49 payment of page charges. This article must therefore be hereby marked were synthesized by Dr. Kurt Eger and his group at the Univer- advertisement in accordance with 18 U.S.C. Section 1734 solely to sity of Leipzig (Leipzig, Germany). Worldwide patents of these indicate this fact. analogs have been filed by the NIH and licensed to Celgene Requests for reprints: William D. Figg, Molecular Pharmacology Section, National Cancer Institute, NIH, Building 10, Room 5A01, MSC Corp. (Warren, NJ). Their chemical structures have been pub- 1910, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 402- lished previously (11). Thalidomide was obtained from Celgene 3622; Fax: (301) 402-8606; E-mail: [email protected]. Corp.

Fig. 1 Effects of thalidomide and thalidomide analogs on PC3 (A) and 22Rv1 (B) tumor growth and on the body weight of PC3 (C) and 22Rv1 (D) tumor-bearing mice. f, vehicle control; F, CPS11; Œ, CPS45; ࡗ, CPS49; ƒ, thalidomide.

Cell Lines. Human prostate cancer cell lines PC3 and and b is the width. Before euthanasia, the mice were anesthe- 22Rv1 and Lewis lung cancer cell line LLC1 were obtained tized with 2% isoflurane. Approximately 800-1000 ␮l of blood from the American Type Culture Collection (Manassas, VA). were drawn by cardiac puncture and collected in EDTA-con- Prostate cancer cells and Lewis lung cancer cells were main- taining microtainer tubes (Becton Dickinson, Franklin Lakes, tained at 37°C and 5% CO2 in RPMI 1640 and DMEM, respec- NJ). Plasma samples were separated by centrifugation and then tively, supplemented with 10% fetal bovine serum and antibi- stored at –80°C for subsequent detection of angiogenic factors. otics (100 units/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 Harvested tumors were snap frozen in OCT (Miles Inc., Elkhart, ␮g/ml amphotericin B). IN) in liquid nitrogen and subsequently processed for immuno- Human Prostate Cancer Xenograft Model. All animal histochemistry. experiments were done in accordance with institutional guide- Measurement of Angiogenic Factors. The Quantikine lines for animal welfare. PC3 (5 ϫ 106) and 22Rv1 (3 ϫ 106) human VEGF, platelet-derived growth factor (PDGF)-AA, and cells were injected s.c. into 5–6-week-old male severely com- bFGF ELISA kits (R&D Systems, Minneapolis, MN) were used bined immunodeficient mice. When tumor volume reached to determine plasma levels of human VEGF, PDGF-AA, and ϳ150–200 mm3, animals were randomized into five groups bFGF, respectively, according to the manufacturer’s instruc- (n ϭ 5 each). Each group was treated with i.p. bolus injections tions. of either the drug vehicle (0.5% carboxymethylcellulose), Immunohistochemistry. Five-␮m-thick sections ob- CPS11 (100 mg/kg), CPS45 (100 mg/kg), CPS49 (12.5 mg/kg), tained from each frozen tumor were stained with H&E for or thalidomide (100 mg/kg) 5 days a week for 4 weeks. These histological examination. For detection of microvessels, sec- doses were the maximum tolerated doses determined in a pre- tions were stained with the polyclonal anti-CD31/PECAM-1 vious study (11). Tumors were measured with a caliper once a antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA). week, and their volumes were calculated using the formula Antigens were visualized using the streptavidin-biotin-peroxi- ␲/6 ϫ a ϫ b2, where a is the longest dimension of the tumor, dase method.

Quantification of Intratumor Microvessel Density (MVD). Tumor sections stained with anti-CD31/PECAM-1 antibody were examined by light microscopy. Clusters of stained endothelial cells distinct from adjacent microvessels, tumor cells, or other stromal cells were counted as one microvessel. The MVD for each tumor was expressed as the average count of the three most densely stained fields identified using a ϫ40 objective. Five different tumors per vehicle control or treatment group were analyzed. DNA Microarrays. RNA was extracted from PC3 and 22Rv1 tumor xenografts harvested from severely combined immunodeficient mice treated with the vehicle (0.5% carboxymethylcellulose), CPS11, CPS45, CPS49, or thalidomide using the RNeasy system (Qiagen, Valencia, CA). Messenger RNAs from each of the vehicle control reference tumors (n ϭ 5) and from each of the drug-treated tumors (n ϭ 5 tumors/treatment group) were converted to cDNA and labeled with cyanine 3-dUTP (Cy3) and cyanine 5-dUTP (Cy5), respectively, using the LabelStar system (Qiagen). Labeled cDNAs from each n ϭ 5 set were pooled, and aliquots were used to interrogate separate oligonucleotide array chips representing Ͼ20,000 human genes printed at the Laboratory of Molecular Technology (National Cancer Institute, Frederick, MD). Expression levels for each gene were determined after competitive binding between RNA from vehicle-treated PC3 or 22Rv1 tumors and those from CPS11-, CPS45-, CPS49- or thalidomide-treated PC3 or 22Rv1 tumors. Array slides were scanned with an Axon 4000 scanner (Axon Instruments, Foster City, CA). Data were filtered and normalized using GENEPIX software (Axon Instruments). Genes that were deregulated by Ն1.5-fold relative to the control were considered to be significant. Experimental Lung Metastasis Assay. LLC1 (1 ϫ 106) cells were injected into 5–6-week-old male severely combined immunodeficient mice via the tail vein. On the following day, animals were randomly assigned to five groups (n ϭ 5 each). Each group was treated with i.p. bolus injections of either the drug vehicle (0.5% carboxymethylcellulose), CPS11 (100 mg/kg), Fig. 2 Bar graphs of PC3 (f) and 22Rv1 (Ⅺ) tumor levels of vascular CPS45 (100 mg/kg), CPS49 (12.5 mg/kg), or thalidomide (100 endothelial growth factor (A), platelet-derived growth factor AA (B), mg/kg) 5 days a week for 3 weeks. All animals were euthanized at and basic fibroblast growth factor (C) in response to the vehicle control (0.5% carboxymethylcellulose), CPS11 (100 mg/kg), CPS45 (100 mg/ the end of the treatment period. Lungs were removed, and tumor kg), CPS49 (12.5 mg/kg), and thalidomide (100 mg/kg). Bars, mean Ϯ .significantly (P Ͻ 0.05) different from vehicle control ,ء .nodules on the surface of lungs were counted. SE Statistics. All results are presented as mean Ϯ SE. Com- parisons were made with one-way ANOVA followed by Dun- nett’s test, with P Ͻ 0.05 as the criterion for statistical signifi- Alterations of the Levels of Tumor Angiogenic Factors cance. by Thalidomide Analogs. PC3 and 22Rv1 tumors differentially expressed VEGF, PDGF-AA, and bFGF proteins (Fig. 2, AϪC). Neither the analogs nor thalidomide significantly altered RESULTS VEGF levels in PC3 or 22Rv1 tumors compared with the vehicle In Vivo Inhibition of Prostate Tumor Growth. CPS11, control (Fig. 2A). Interestingly, PDGF-AA levels were significantly CPS45, and CPS49 significantly inhibited PC3 tumor growth by reduced by 82%, 67%, and 58% in PC3 tumors treated with 90%, 51%, and 68%, respectively, compared with the vehicle CPS45, CPS49, and thalidomide, respectively (Fig. 2B). All of the control (Fig. 1A). Thalidomide had no effect (Fig. 1A). Growth analogs and thalidomide had no significant effects on 22Rv1 tumor of 22Rv1 tumors was slightly delayed by the analogs but not by PDGF-AA levels (Fig. 2B). PC3 and 22Rv1 tumor bFGF levels thalidomide (Fig. 1B). No significant reduction in body weight were unchanged by the analogs and thalidomide (Fig. 2C). was observed in PC3 tumor-bearing mice treated with the ana- Deregulation of Angiogenic Growth Factor Gene Ex- logs and thalidomide (Fig. 1C). In 22Rv1 tumor-bearing mice, pression in Prostate Cancer Xenografts by Thalidomide An- however, CPS45 significantly decreased body weight by ϳ15% alogs. DNA microarray data for the VEGF, PDGF, and compared with the vehicle control (Fig. 1D). fibroblast growth factor (FGF) gene families were examined.

Table 1 Mean fold deregulation of major angiogenic factors and receptors in prostate cancer xenografts treated with thalidomide and thalidomide analogs PC3 22Rv1 Genes CPS11 CPS45 CPS49 Thala CPS11 CPS45 CPS49 Thal VEGFA 1.1 1.1 0.9 0.9 0.6 0.7 0.8 0.6 VEGFB 1.4 1.0 1.2 0.9 0.5 0.5 0.7 0.7 VEGFC n/a 1.1 n/a n/a n/a n/a 1.1 n/a PDGFA 1.6b 1.5b 1.4 1.1 1.6b 1.8b 1.3 1.8b PDGFB 0.8 1.2 0.6 0.9 1.5b 1.0 2.2b 1.4 PDGFC 1.1 0.4 0.8 n/a n/a n/a n/a n/a PDGFR␣ 0.9 0.7 1.0 1.4 1.8b 1.3 1.0 0.8 FGF1 (aFGF) 1.0 1.0 1.2 1.1 1.7b 1.3 0.8 1.1 FGF2 (bFGF) 0.5 0.9 0.8 1.0 0.8 0.5 1.5b 1.2 FGF3 0.7 1.2 0.8 1.0 n/a n/a 1.6b n/a FGF4 0.5 1.0 0.7 n/a 0.8 1.1 1.3 1.4 FGF5 0.7 0.7 0.7 n/a 1.1 1.0 6.5b 1.0 FGF6 1.1 1.0 1.0 0.9 1.2 1.3 1.2 1.7b FGF7 1.3 1.0 1.1 0.9 0.8 1.0 0.9 1.1 FGF8 1.2 0.7 1.0 0.8 0.9 0.7 1.2 1.3 FGF9 1.7b 1.6b 1.5b 1.1 1.2 1.6b 1.5b 1.6b FGF10 0.6 1.2 1.0 n/a 0.9 1.3 1.6b 1.0 FGF12 1.1 0.9 1.0 1.1 0.9 1.1 1.0 0.9 FGF13 1.1 0.7 0.7 0.7 0.8 0.4 0.5 0.6 FGF14 1.5b 1.3 1.6b 1.0 1.4 1.4 1.0 0.9 FGF16 1.0 0.9 1.6b 1.2 2.3b 1.9b 0.9 n/a FGF17 1.3 0.6 0.7 0.9 1.0 0.7 0.8 0.9 FGF18 0.8 0.3 0.5 0.8 0.6 0.7 1.2 0.9 FGF19 1.2 0.7 1.0 1.8b 1.0 1.7b 1.2 0.7 FGF20 0.9 0.9 1.0 1.3 0.9 1.0 1.0 1.1 FGF21 1.3 1.1 1.3 1.0 1.5b 1.5b 1.1 1.3 FGF22 1.1 1.0 1.3 1.3 0.8 0.7 1.1 0.7 FGF23 1.0 1.1 1.1 0.8 1.3 1.4 1.3 1.6b FGFR1 0.8 0.7 0.8 0.9 1.0 0.9 1.0 1.2 FGFR2 n/a n/a 0.6 n/a 0.7 1.0 1.9b 1.0 FGFR3 0.7 n/a n/a n/a n/a n/a 1.5b n/a FGFR4 1.2 0.8 1.0 0.9 1.0 1.2 1.0 1.0 a Thal, thalidomide; VEGF, vascular endothelial growth factor; n/a, no data were available for this gene because stringency criteria were not met; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor. b Significant (Ն1.5-fold) down-regulation.

Screening of transcriptomes sampled from PC3 tumors treated with the analogs and thalidomide revealed a 1.5-fold or greater differential down-regulation of PDGFA, FGF9, FGF14, FGF16, and FGF19 (Table 1). In 22Rv1 tumors, the analogs and thalidomide differentially caused Ն1.5-fold down-regulation of PDGFA, PDGFB, PDGF receptor ␣, and various isoforms of the FGF and FGF receptor family (Table 1). Transcription of VEGFA, VEGFB, and VEGFC in both PC3 and 22Rv1 tumors was not altered by the analogs or thalidomide (Table 1). Changes in Intratumoral MVD in Response to Thalid- omide Analogs. MVD was decreased by 64% in PC3 tumors treated with CPS49 (Fig. 3). In 22Rv1 tumors, CPS45 and CPS49 reduced MVD by 60% and 53%, respectively (Fig. 3). CPS11 and thalidomide did not affect MVD in PC3 and 22Rv1 tumors compared with the vehicle control (Fig. 3). Effects of Thalidomide Analogs on Lung Metastasis. Fig. 3 Bar graphs of PC3 (f) and 22Rv1 (Ⅺ) intratumoral microvessel As shown in Table 2, CPS11 and CPS49 significantly reduced density versus treatment with the vehicle control (0.5% carboxymethylcellulose), CPS11 (100 mg/kg), CPS45 (100 mg/kg), CPS49 (12.5 significantly ,ء .the number of lung metastases by 87% and 67%, respectively. mg/kg), and thalidomide (100 mg/kg). Bars, mean Ϯ SE CPS45 and thalidomide failed to do so. (P Ͻ 0.05) different from vehicle control.

Table 2 Effects of thalidomide and thalidomide analogs on express PDGF and cognate receptors, suggesting an autocrine lung metastasis pathway for stimulation of tumor cell growth (13). In particular, Values are the mean Ϯ SE. PDGF-AA, PDGF receptor ␣, and PDGF receptor ␤ proteins Treatment No. of tumor nodules in lungs were detected in prostatic intraepithelial neoplasias and adeno- Vehicle control 15 Ϯ 1 carcinomas (14, 15). Furthermore, PDGF was shown to have CPS11 2 Ϯ 2a proangiogenic effects (16–18). It was thus hypothesized that CPS45 9 Ϯ 2 these thalidomide analogs inhibit prostate cancer growth in part a CPS49 5 Ϯ 3 via modulation of PDGF signaling. In accordance with this Ϯ Thalidomide 11 1 hypothesis, intratumoral MVD was significantly decreased in a Significantly (P Ͻ 0.05) different from vehicle control. response to CPS45 and CPS49 treatment, suggesting that the antitumor effects of these tetrafluorinated analogs may be associated with the inhibition of PDGF-mediated angiogenesis. The observation that CPS11 exhibited a potent antitumor effect DISCUSSION without concurrently decreasing the intratumoral levels of major Our results showed that the N-substituted thalidomide an- angiogenesis factors and MVD is intriguing. It is possible that alog CPS11 and the tetrafluorinated analogs CPS45 and CPS49, the N-substituted analog affects non-angiogenesis-related mo- but not thalidomide, suppress the growth of androgen-indepen- lecular pathways in addition to the angiogenic pathways tar- dent prostate cancer xenografts. It was demonstrated previously geted by the tetrafluorinated analogs, perhaps indicating a (11) that CPS45 and CPS49 inhibit rat aortic microvessel out- broader range of anticancer profile of the former. The significant growth as well as human umbilical vein endothelial cell prolif- reduction in the number of tumor nodules induced by CPS11 eration and tube formation more potently than CPS11. However, and CPS49 in the Lewis lung model indicated that these analogs CPS11 was observed to suppress prostate tumor growth to a also possess antimetastatic activity. Other thalidomide analogs greater extent than CPS45 and CPS49 and even to induce PC3 reported in the literature have also demonstrated antiangiogenic tumor regression in the current study. It was speculated that and/or antitumor properties (19, 20). One study (20) showed that CPS11 may have a better pharmacokinetic profile or multiple certain analogs promote cell cycle arrest and increase the ex- pharmacological targets. The minimal antitumor effects of these pression of proapoptotic proteins and decrease that of antiapop- analogs observed in the 22Rv1 xenografts remain elusive. totic proteins. However, it should be noted that different thalid- To understand the mechanism(s) of action underlying the omide analogs and thalidomide itself may bind to distinct target in vivo antitumor effects of these analogs, tumor levels of the protein(s). angiogenic factors VEGF, PDGF-AA, and bFGF were deter- In summary, the present data demonstrated the antitumor mined. It is well documented that VEGF and bFGF are potent effects of thalidomide analogs in human prostate cancer xe- stimulators of endothelial cell proliferation and play a signifi- nografts. One possible mechanism of action of these analogs cant role in tumor angiogenesis. All of the thalidomide analogs may involve inhibition of the PDGF signaling pathway. Detailed and thalidomide failed to reduce tumor VEGF and bFGF protein transcriptome-wide microarray analyses are under way with the levels, consistent with microarray analyses that indicated their aim of deciphering the precise mechanisms of action of these lack of effect on VEGFA and FGF2 (bFGF) gene transcription. analogs and explaining the differential effects of these analogs The exception was CPS49, which caused a 1.5-fold down- on PC3 and 22Rv1 prostate tumors. Full toxicology and terato- regulation of the FGF2 gene but did not reduce bFGF protein genicity testings are in progress before clinical development. levels in the 22Rv1 tumors. Interestingly, PC3 tumor PDGF-AA levels were decreased by CPS45, CPS49, and thalidomide, but ACKNOWLEDGMENTS not by CPS11, although microarray experiments revealed that We are grateful to Dr. Miriam R. 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Sylvia S. W. Ng, Gordon R. MacPherson, Michael Gütschow, et al.

Clin Cancer Res 2004;10:4192-4197.

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