Role of Tumor Necrosis Factor-A^Stimulatedgene6 Gretchen E

Cancer Prevention

2-Methoxyestradiol Inhibits Prostate Tumor Development in Transgenic Adenocarcinoma of Mouse Prostate: Role of Tumor Necrosis Factor-A^StimulatedGene6 Gretchen E. Garcia,7 Hans-Georg Wisniewski,3 M. Scott Lucia,4 Nicole Arevalo,8 Thomas J. Slaga,2 Susan L. Kraft,5 Robert Strange,6 and Addanki P. Kumar1

Abstract Purpose: 2-Methoxyestradiol, an estrogenic metabolite, is in clinical trials for the treatment of hormone-refractory prostate cancer. However, neither the chemopreventive role nor the mechanism of 2-methoxyestradiol ^ induced biological activities is fully understood. Experimental Design: Eight- and 24-week-old transgenic adenocarcinoma of mouse prostate (TRAMP) mice were fed a diet containing 50 mg 2-methoxyestradiol/kg body weight for 16 and 8weeks, respectively. Chemopreventive efficacy was evaluated by magnetic resonance imaging, determining the prostate-seminal vesicle complex volume and histologic analysis of prostate tumor or tissue. Tumor invasion assays were used to show the role of tumor necrosis factor-a^ stimulated gene (TSG-6),a 2-methoxyestradiol ^ up-regulated gene identified by DNA array analysis. Expression of TSG-6 was analyzed in a human tissue array containing different grades of prostate tumors. Results: Dietary administration of 2-methoxyestradiol prevented the development of preneoplastic lesions independent of progression stage. TSG-6 was low or undetectable in prostate cancer cells (LNCaP, PC-3, and DU145) and TRAMP tumors but up-regulated in response to 2-methoxyestradiol. Immunohistochemistry of the human prostate tumor array showed a de- crease inTSG-6^ positive cells with increasing grade relative to normal prostate (P =0.0001). Although overexpression of TSG-6 inhibited invasion of androgen-independent cells (P = 0.007), antisenseTSG-6 reversed this effect. Conclusions: To the best of our knowledge, this is the first report showing the potential of 2-methoxyestradiol as a chemopreventive agent. We have also identified TSG-6 as a potential marker that could be used for early diagnosis and prognosis of cancerous or precancerous lesions.

Prostate cancer is the second leading cause of cancer-related in young men in the twenties and are fairly common in men deaths in men and affects one man in nine over the age of in fifties (2). However, clinically detectable prostate cancer 65 years (1). Although it is not clear whether appearance does not generally manifest until the age of 60 or 70 years. In of prostatic intraepithelial neoplasia predicts the appearance of addition, the occurrence of precancerous lesions is more prostate cancer in men, preneoplastic lesions have been found prevalent (f1 in 3 men) than the incidence of carcinoma (f1 in 9 men; ref. 3). It is of utmost importance at this juncture to develop strategies for the prevention of early-stage prostate cancer to ensure quality of life for elderly men. 1 2 Authors’ Affiliations: Departments of Urology and Pharmacology, University of Furthermore, although prostate-specific antigen is used as a Texas Health Science Center, San Antonio,Texas; 3Department of Microbiology and Kaplan Cancer Center, NewYork University Medical Center, New York; 4Department marker to detect prostate cancer, its reliability has been found of Pathology, University of Colorado Health Sciences Center; 5Department of to be questionable in some cases (4, 5). Also presently, there is Radiation Biology, Colorado State University, Fort Collins; 6AMC Cancer Research no molecular marker that can be used to detect a precancerous 7 Center; National Jewish Research and Medical Center, Denver, Colorado; and state or identify which premalignant lesions will develop into 8Affinity Bioreagents, Golden, Colorado Received 9/21/05; revised 11/7/05; accepted 11/29/05. invasive prostate cancer. Grant support: American Cancer Society grants RSG-04-169-01 and R21 CA Recent results from different groups, including ours, have 98744 (A.P. Kumar) and San Antonio Cancer Institute/National Cancer Institute’s shown that 2-methoxyestradiol inhibits the growth of andro- Barbara Bowman Cancer Prevention award P30 CA-054174-16 (A.P. Kumar). gen-responsive and androgen-independent human prostate The costs of publication of this article were defrayed in part by the payment of page cancer cells through induction of apoptosis (6, 7). However, charges. This article must therefore be hereby marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact. the chemopreventive role of 2-methoxyestradiol has not been Note:This work was conducted in the Center for Cancer Causation and Prevention studied. We used the transgenic adenocarcinoma of mouse at AMC Cancer Research Center, Denver, Colorado. prostate (TRAMP) model of prostate cancer to test the chemo- Requests for reprints: Addanki P. Kumar, Department of Urology, University of preventive ability of 2-methoxyestradiol. This model was Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229. Phone: 210-567-5647; Fax: 210-567-6868; E-mail: [email protected]. developed by prostate-specific expression of SV40 large T F 2006 American Association for Cancer Research. antigen using the probasin (PB) promoter. TRAMP mice doi:10.1158/1078-0432.CCR-05-2068 develop progressive forms of prostate cancer with lesions

ClinCancerRes2006;12(3)February1,2006 980 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Loss of TSG-6 in High-gradeTumors ranging from mild prostatic intraepithelial neoplasia to large per manufacturer’s recommendations (Diagnostic System Laboratories, multinodular malignant neoplasia over their lifetime (8). Inc., Webster, TX). Values for the samples were derived by interpolating Because these animals are predisposed to develop prostate using standards available with the kit. cancer, it served as a good model to test the chemopreventive Tumor histology. Tumors were harvested and fixed in 10% neutral buffered formalin. Tumors were paraffin embedded, sectioned, placed ability of 2-methoxyestradiol (refs. 9–11 and references on polylysine slides, and stained with H&E to visualize cell nuclei and therein). cytoplasm. Images were recorded using a light microscope. Results presented here show that dietary intervention with Immunochemical analysis. We used immunochemistry to examine 2-methoxyestradiol prevents the development of prostate the levels of TSG-6 using a rabbit polyclonal antibody generated against tumors with no indication of neoplasia irrespective of the the COOH terminus in prostate cancer cell lines using standard stage of intervention. 2-Methoxyestradiol–induced cell prolif- protocols. eration inhibition involves up-regulation of tumor necrosis Tumor invasion assay. We used Cell Invasion Assay (Oncogene factor-a (TNF-a)–stimulated gene 6 (TSG-6). TSG-6 was Research Products, San Diego, CA) to determine the direct role of TSG-6 originally isolated from TNF-treated human fibroblasts and in inhibiting tumor invasion. This assay uses an invasion chamber with has been shown to be induced in response to cytokines, such an 8-Am pore size polycarbonate membrane. The upper surface of this membrane is coated with a uniform layer of basement membrane as TNF-a, interleukin-1, and growth factors (12). Although TSG- matrix solution that forms an effective extracellular matrix protein 6 was very well studied with respect to arthritis (inflammatory barrier and prevents noninvasive cells from going through the pores. In disease), its role in cancer remains largely unexplored (13). We contrast, invasive cells are able to degrade the matrix proteins that analyzed the expression of TSG-6 in different grade prostate occlude the pores and allow them to pass through. The ability to invade tumors and show lower expression of TSG-6 in high-grade can be quantified by labeling the invaded cells with calcein-AM human prostate tumors compared with normal prostate followed by fluorescence measurement. LNCaP and PC-3 cells were (P = 0.0001). Furthermore, TSG-6 inhibited the invasive ability transfected with expression vectors containing TSG-6 cDNA in sense or of androgen-independent PC-3 cells (P = 0.007). These antisense orientation or vector alone. The experiment was carried out observations suggest that 2-methoxyestradiol inhibits the according to the manufacturer’s recommendations. progression of preneoplastic lesions through up-regulation of Gene array analysis. The Micromax microarray containing 281 human cancer–related genes, including oncogenes and tumor suppres- anti-inflammatory molecule, such as TSG-6. sor genes (Perkin-Elmer Life Sciences, Inc., Boston, MA), was used in this study. Total RNA from control and 2-methoxyestradiol–treated Materials and Methods LNCaP cells was isolated using TRIZOL reagent (Ambion, Inc., Austin, TX). After assessing the integrity of the RNA by agarose gel Transgenic mouse experiments. TRAMP mice in pure C57BL/6 electrophoresis, RNA from control cells was labeled with cyanine 5 background were bred at the AMC Cancer Research Center. All mice and treated RNA with cyanine 3 as per manufacturer’s recommenda- were maintained in a climate-controlled environment with a 12-hour tions. The combined cyanine 3– and cyanine 5–labeled mRNA was light/12-hour dark cycle. Diet and water were supplied ad libitum. After purified and used in hybridization. Data was analyzed using Custom weaning at 3 to 4 weeks of age, tail DNA from males was used to Scanning and Data Processing service for Micromax system (Perkin- determine the presence of transgene by PCR as described previously Elmer Life Sciences). Results are expressed as the ratio of the signals (10). Eight-week-old transgenic males were fed AIN-96A pellets from cyanine 3 and cyanine 5 for each of the 281 cDNAs, and ratio of (a semipurified diet containing 2-methoxyestradiol, 50 mg/kg body >2 is considered significantly differentially expressed. These were weight; n = 9) or control diet (without 2-methoxyestradiol) for 16 confirmed through independent approaches, including Western blot weeks (group I). In another study, 24-week-old TRAMP mice were fed analysis or immunocytochemistry or immunohistochemistry. the same pelleted diet with or without 2-methoxyestradiol for 8 weeks Statistical analysis. Two-tailed statistical analyses were conducted at (n = 5; group II). Development and progression of prostate cancer was P = 0.05 (16). assessed using magnetic resonance imaging in the second group of animals. Animals were imaged with a small-phased array transmit- Results and Discussion receive coil of a 1.5-T General Electric LX MR instrument essentially as described in ref. (14). Each mouse was weighed once a week. Animal TRAMP mice develop prostate tumors with 100% frequency care and handling was conducted in accordance with established in progressive stages exhibiting hyperplasia (8-12 weeks), guidelines and protocols approved by the Institute’s Animal Care and neoplasia (15-18 weeks), and metastasis (24 weeks). We asked Use Committee. whether 2-methoxyestradiol could be used to prevent the Cell lines. Human prostate cancer cell lines, including androgen- responsive (LNCaP) and androgen-independent (PC-3 and DU145) cell progression of prostate tumors at various stages of develop- lines, were grown as described earlier (6, 15). TRAMP cell lines ment. To do this, one group of 8-week-old TRAMP males was were obtained from Dr. Norman Greenberg (Fred Hutchinson Cancer shifted to 2-methoxyestradiol diet for 16 weeks (group I). Research Center, Seattle, WA) and were grown as described earlier (15). Another group of 24-week-old TRAMP males was shifted to PC-3 cells overexpressing TSG-6 and vector transfected (PC3-Neo) were 2-methoxyestradiol diet for 8 weeks (group II). Necropsy was generated using standard protocols. conducted at 24 weeks of age for group I and at 32 weeks for Preparation and analysis of tissues. Necropsy was conducted on group II animals. all animals to determine if there were any gross organ abnormalities The dose of 50 mg/kg body weight was chosen from a dose in response to 2-methoxyestradiol. The prostate was dissected and escalation xenograft study (data not shown). The ability of weighed before additional processing for histopathologic evaluation. 2-methoxyestradiol to inhibit neoplastic development was Prostate lesions were scored using the established grading for TRAMP mice (8). assessed by determining the weight of the prostate gland Analysis of hormone levels in serum. The LAB-4000 testosterone and histologic evaluation of the prostate gland or tumor. assay and the LAB-4800 Ultra sensitive estradiol assay were done using 2-Methoxyestradiol–fed group I animals displayed f33% the DSL-4000–coated RIA and DSL-4800 double antibody RIA for reduction in the weight of the prostate gland compared with measuring the serum levels of testosterone and estradiol, respectively, as mice on normal diet. There was no significant change in the

www.aacrjournals.org 981 Clin Cancer Res 2006;12(3) February 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Prevention body weight or weight of seminal vesicles (data not shown). also exert its effects by influencing the expression of androgen- Gross appearance of prostate and seminal vesicles from a regulated genes. In view of this, we determined the expression representative 24-week-old untreated (i) and treated mouse (ii) of PB-Tag endogenous mouse PB and glyceraldehyde-3- is shown in Fig. 1A. phosphate dehydrogenase (non–androgen-regulated gene) Histologic analysis of the prostate from a control TRAMP using immunohistochemistry in prostate from age-matched mice (i) displayed major epithelial proliferation in a charac- control and treated animals. As shown in Fig. 2C, PB-Tag teristic cribriform pattern: hyperchromatic nuclei, mitotic and glyceraldehyde-3-phosphate dehydrogenase expression figures, and apoptotic bodies (Fig. 1B). In contrast, prostate was detected both in control tumor and treated tumor/tissue from TRAMP mice on 2-methoxyestradiol diet showed glands composed of columnar epithelium with round to oval nuclei with no indication of neoplasia (Fig. 1B, ii). Analysis of other tissues, including lungs, liver, and lymph nodes, showed normal morphology, indicating that 2-methoxyestradiol had no effect on any of these organs (data not shown). These data show that 2-methoxyestradiol feeding inhibits neoplastic progression in TRAMP mice. In 24-week-old TRAMP mice that were fed 2-methoxyestradiol diet (50 mg/kg body weight) for 8 weeks, the efficacy of 2-methoxyestradiol was evaluated by volumetric analysis of the prostate-seminal vesicle complex using in vivo magnetic resonance imaging. Representative images in Fig. 1C show that the prostate from control group was indistinct due to a very large tumor that infiltrated into seminal vesicles (i), whereas treatment of animals showed very small prostate with no evidence of cancer (ii). Figure 1D shows that the prostate- seminal vesicle complex was larger in the control animal (i) compared with the animal on 2-methoxyestradiol diet (ii)at the time of necropsy. Volumetric analysis at the time of magnetic resonance imaging showed that the average prostate-seminal vesicle complex volume was 3,263 F 105 mm3 in the control animals (n = 5) compared with 235 F 99 mm3 in the treated animals (n = 5; Fig. 2A). We have measured the levels of total testosterone and estradiol in serum collected from TRAMP animals that were on this study as described in Materials and Methods. Values for the sample analysis were derived by interpolation using standards available with the kit. Serum levels of estradiol were very low (<5 pg/mL). Dietary intervention with 2-methoxyestradiol did not affect the levels of estradiol. However, serum levels of total testosterone were reduced in 2-methoxyestradiol–treated animals (Fig. 2B). These data suggest that dietary intervention with 2-methoxyestradiol produced a regression of prostate tumors in TRAMP mice that is associated with a decline in serum testosterone levels with no effect on estradiol. In line with our findings, raloxifen, a selective estrogen receptor modulator, has been shown to regress prostate cancer in SV40Tag transgenic rats and male accessory sex organs in intact Sprague-Dawley rats associated with decline in serum testosterone levels (17). Studies to investigate whether Fig. 1. A, gross appearance of prostate and seminal vesicles of 24-week-old 2-methoxyestradiol reduces testosterone levels by enhancing TRAMP mice (group I). Prostate gland from 24-week-oldTRAMP mice on (i) metabolic clearance or altering hepatic function to change the normal diet and (ii) 2-methoxyestradiol diet. B, histologic analysis of prostate from untreated and treatedTRAMP mice. Histologic sections of prostate tissues excised secretion of circulating proteins capable of binding steroids are fromTRAMP mice from group I on (i) control or (ii) 2-methoxyestradiol diet. currently in progress. In both experiments, feeding animals with Tissue sections were stained with H&E. Magnification, Â200. C, magnetic 2-methoxyestradiol produced negligible body weight losses resonance imaging of 32-week-oldTRAMP mice (group II).Two groups of five 24-week-oldTRAMP mice were on a diet with or without 2-methoxyestradiol ranging from 1.2% to 2.8%. (50 mg/kg body weight) for 8weeks. At the end of this schedule, they were imaged Because TRAMP mice were generated with SV40 large T with a small phased array transmit-receive coil of a 1.5-T General Electric LX MR instrument. Representative images show that the prostate of (i) control mice was antigen coupled with PB promoter (8), it is possible that 2- unrecognizable due to a very large tumor that infiltrated into seminal vesicles; (ii) methoxyestradiol mediated effects are a consequence of down- age-matched mice on 2-methoxyestradiol fed diet showed very small prostate with regulation of the PB-Tag transgene by 2-methoxyestradiol. The no evidence of cancer as confirmed by H&E analysis after necropsy. D, gross appearance of prostate 32-week-oldTRAMP mice (group II). Prostate tumor along PB promoter is androgen regulated, and 2-methoxyestradiol with vesicles from (i) control and (ii) 2-methoxyestradiol ^ fedTRAMPmouse at the is an estrogenic metabolite; therefore, 2-methoxyestradiol may time of sacrifice (32 weeks).

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Fig. 2. A, volumetric analysis of prostate seminal vesicle complex volume (PSVC) from group II TRAMP mice. Experiment was conducted essentially as described in Fig.1C, and prostate seminal vesicle complex volume was determined. Columns, average from five animals per group; bars, SD. B, serum levels of testosterone in TRAMP mice. Serum levels of total testosterone and estradiol were determined in control of 2-methoxyestradiol ^ fed (50 mg/kg body weight) TRAMP mice as described in Materials and Methods. Animals (n = 5) from each group were sacrificed at 24 weeks (group I) and 32 weeks (group II) of age. Serum was collected and analyzed for hormone levels. Values for the sample analysis were derived by interpolation using standards available with the kit. Since estradiol levels did not change in response to treatment, the data was not shown. C, immunohistochemical analysis of representative tumors or prostate tissue from 32-week-old control or treatedTRAMP mice. Paraffin-embedded tissues sections were stained with PB-Tag (PBT) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).The antibodies usedwereatadilutionof1:100inPBS and incubated overnight at 4jC. Immune complexes were revealed using a universal secondary antibody (100 ALfor30 minutes) followed by chromogen. Negative controls were included by omitting the primary antibody.

obtained from 32-week-old TRAMP mice. Glyceraldehyde- thought to contribute to the pathogenesis of many cancers, 3-phosphate dehydrogenase levels did not change between including prostate. Consistent with this regular use of control and treated prostatic tissues. These results show that our nonsteroidal anti-inflammatory drugs has been shown to be observations regarding 2-methoxyestradiol–induced biological associated with reduced risk of prostate cancer (35). Although effects are due to direct suppression of carcinogenesis and not it is not clear whether inflammation is a cause or effect of due to the down-regulation of PB-Tag. hyperplastic growth in the prostate, it appears that chronic or Although several groups, including ours, have shown recurrent prostatic inflammation may contribute to the that 2-methoxyestradiol inhibits the growth of cancer cells development of prostate cancer (36). However, TSG-6 has through induction of apoptosis, the detailed molecular not been studied in the context of cancer. mechanisms involved in mediating its growth inhibitory We used immunocytochemistry and Western blotting to detect activity is still unclear (6, 7, 18–22). To understand the the expression of TSG-6 in LNCaP, DU145, and PC-3 cells. molecular basis of 2-methoxyestradiol–mediated growth As shown in Fig. 3A, a brown 3,3V-diaminobenzidine reaction inhibition, we used cDNA array analysis of genes expression product was formed in the cytoplasm of cells treated with 2- change by 2-methoxyestradiol treatment using LNCaP cells. methoxyestradiol (3 Amol/L for 24 hours) but not in untreated Analysis of this data indicated genes involved in various controls of DU145 and PC-3 cells. Preimmune serum was cellular processes, including tumor suppression, cell cycle negative for 3,3V-diaminobenzidine product in these samples regulation, cytokine signaling, transcription control, and (data not shown). TSG-6 protein was detected in LNCaP cells but inflammation, were up-regulated. Genes showing >2-fold not in PC-3 cells. However, the TSG-6 protein levels increased in induction (up-regulated) and <0.5-fold (down-regulated) were response to 2-methoxyestradiol treatment both in LNCaP and considered significant and are shown in Table 1. We chose to PC-3 cells (Fig. 3B). Published reports show that constitutive conduct detailed studies on one such gene TSG-6 because of expression of TSG-6 is either low or undetectable but becomes its known role in inflammation. TSG-6, is a secreted detectable in response to cytokines and growth factors in many glycoprotein of 35 kDa, is associated with inflammation cell types both in vitro and in vivo (12, 13). Our data showing low and fertility (reviewed in ref. 13). TSG-6 is a hyaluronan- levels (basal expression) of TSG-6 in human prostate cancer cell binding protein, and its interaction with hyaluronan has been lines are consistent with these observations. studied in detail (23–33). TSG-6 has shown anti-inflamma- These observations prompted us to determine the clinical tory activity in several models of acute inflammation and application of TSG-6 for prostate cancer prognosis. Immuno- autoimmune arthritis (31–34). Inflammation has been histochemical staining for TSG-6 was conducted on a human

www.aacrjournals.org 983 Clin Cancer Res 2006;12(3) February 1, 2006 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Cancer Prevention prostate cancer tissue array containing 72 specimens of different Gleason grades with paired normal prostate. The array was prepared by the histology core Laboratory (Univer- sity of Colorado Health Science Center, Denver, CO), the Prostate Diagnostic Laboratory, using a Beecher Instruments (Silver Springs, MD) tissue arrayer and 1.5-mm core diameter. Each specimen was analyzed for immunoreactivity using a 1 to 3+ scoring system for stain intensity and percentage of positive cells. Grading scale for intensity ranged from undetectable signal (1+) to strong signal (3+). ANOVA and bootstrap methods were used to determine the statistical significance of these observations. TSG-6 staining was pre- dominantly localized in the cytoplasm of epithelial (basal and luminal) but not stromal cells. Membrane staining was Fig. 3. A, TSG-6 in prostate cancer cells. Human prostate cancer cell lines (PC-3 and DU145) were either treated with 2-methoxyestradiol (2-ME ;3Amol/L) for 24 observed in some specimens. There was a progressive loss of hours or left untreated (solvent control). Following treatment, cells were harvested staining with increasing Gleason grade (P < 0.0001; Fig. 4); and fixed in 10% buffered formalin, and immunocytochemical analysis was done as described in Materials and Methods. Staining indicative ofTSG-6 expression was 80% to 100% of cells from normal prostate showed TSG-6 detected only in cells treated with 2-methoxyestradiol. Untreated cells showed very expression. A representative picture from normal and Gleason little or no staining. B, Western blot analysis ofTSG-6.Whole-cell extracts prepared 5 tumors is shown in Fig. 4. Although these observations are from LNCaP cells treated with 2-methoxyestradiol (3 Amol/L) for different time points (2, 4, and 6 hours) or PC-3 cells treated with different concentrations of based on a small sample size, they suggest that the loss of 2-methoxyestradiol for 24 hours (0.5, 1, and 3 Amol/L) was used in immunoblot TSG-6 expression may be a significant predictor of prostate analysis. Equal loading of protein was confirmed with h-actin antibody. cancer progression. Based on the observed inverse correlation between TSG-6 expression and prostate cancer malignancy, we hypothesized transfected with TSG-6 cDNA in the sense or antisense that TSG-6 may have a role in tumor invasion. PC-3 cells were orientation or with vector alone and used in tumor invasion assays. These three comparisons (each condition to control) were regarded as a family. To control the family-wise error rate, Ps were multiplicity adjusted and derived from a bootstrap Table 1. Genes showing >2-fold (up-regulation) resampling of the residuals from a standard additive model for or <0.5-fold (down-regulation) in response to the estimates of the group means (16). As shown in Fig. 5, 2-methoxyestradiol treatment in LNCaP cells overexpression of TSG-6 inhibited the invasive ability of PC-3 cells significantly (P = 0.007 between control and TSG-6) when TNF-inducible (TSG-6) mRNA fragment, 5.877182 compared with vector-transfected control cells. This effect was adhesion receptor CD44 putative CDS also seen in LNCaP cells, but the data were not statistically Transcriptional activator 5.211346 significant (data not shown). It has been reported that LNCaP fra-2 mRNA 4.275474 cells do not secrete matrix metalloproteinase-9 and are poorly Connective tissue growth factor ^ related 4.195513 invasive in Matrigel assays (37). proteinWISP-1 (WISP1) To confirm that increased level of TSG-6 was required for C-mpl ligand (ML) 4.142802 2-methoxyestradiol–mediated inhibition of tumor invasion, TSG-6 cDNA was stably transfected into PC-3 cells. Both PC- IMP dehydrogenase type1mRNA complete cds 3.693961 9 T-lymphoma invasion and metastasis-inducing 3.684486 3 Neo and PC-3TSG-6 clones grew equally well. PC-3TSG-6 TIAM1protein (TIAM1) clones were less invasive than vector-transfected PC-3 Neo Thrombopoietin receptor (MPL) gene 3.609131 clones that were consistent with the transient transfection Mas proto-oncogene 3.602571 data discussed above (Fig. 5B). Although these data mRNA for GLI protein 3.382681 collectively indicate that TSG-6 inhibits tumor invasion in Humos gene homologous to transforming 3.358187 prostate cancer cells, the relationship between TSG-6 and gene of mmsv prostate cancer development is unclear. We evaluated VAV2,VAVoncogene homologue 3.277642 prostate tumor/tissue from control and 2-methoxyestradiol– C-myb 3.107726 fed TRAMP mice for TSG-6 expression to determine if the mRNA for lymphotoxin (TNF-h)3.079749levels correlate with tumor development. A representative Epithelial membrane protein (CL-20). 3.054039 Western blot is shown in Fig. 6. The levels of TSG-6 was h CDK inhibitory protein (clone p15INK4B/HA5) 3.052287 quantified and normalized to -actin as a loading control. A Stem cell protein (SCL) 2.990963 representative Western blot of tumor samples from a control Cyclophilin-related protein 2.958744 animal and tissue from an animal on 2-methoxyestradiol diet Rel proto-oncogene mRNA 2.932135 is shown in Fig. 6 (inset). As shown in this figure, prostate Transcription factor SUPT3H (SUPT3H) 2.908878 from 2-methoxyestradiol–fed animals showed 1.5- to 2-fold Type-2 phosphatidic acid phosphatase 0.458625 induction of TSG-6. Results presented here show a strong a-2 (PAP2-a2 ) link between the expression of TSG-6 and prostate cancer a-Tubulin 0.457914 mRNA for h-actin 0.352991 9 A.P. Kumar and G.E. Garcia, unpublished data.

Clin Cancer Res 2006;12(3) February 1, 2006 984 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Loss of TSG-6 in High-gradeTumors progression and therefore its potential use as a prognostic Work from many laboratories and our own shows that growth marker in 2-methoxyestradiol–induced prevention of prostate inhibitory effect of 2-methoxyestradiol is specific for tumor carcinogenesis. cells and does not affect normal cells. Therefore, our novel 2-Methoxyestradiol is an endogenous nontoxic metabolic finding presented here showing inhibition of precancerous byproduct of estrogens that is present in human urine and cells is noteworthy. blood. 2-Methoxyestradiol has been shown to (a) inhibit Although the role of TSG-6 in prostate carcinogenesis endothelial cell proliferation implicating it in angiogenesis; (b) is unexplored, TSG-6 has been shown to be up-regulated inhibit the growth of different cancer cells, including lung, by various cytokines and growth factors and activated in breast, pancreatic, hepatocellular carcinoma, neuroblastoma, pathologic conditions, such as rheumatoid arthritis and medulloblastoma, melanoma, and gastric cancer (6, 7, 18– osteoarthritis. It has also been shown that TSG-6 forms a stable 22). Oral administration of 2-methoxyestradiol (75 mg/kg complex with inter-a-inhibitor, a Kunitz-type serine protease body weight) for 4 weeks inhibited tumor growth by about inhibitor in plasma. This combination of TSG-6 and inter-a- 60% with no evidence of toxicity in a breast cancer model, inhibitor inhibits plasmin, a serine protease that activates reduced the number of metastases in a lung tumor model by matrix metalloproteinases that degrade extracellular matrix 59%, and reduced tumor size in mice with angiosarcoma by during inflammatory reactions. Based on these observations, 68%. Furthermore, efficacy of 2-methoxyestradiol was evaluat- we speculate that treatment of prostate cancer cells with 2- ed by administering it orally to hormone-refractory prostate methoxyestradiol leads to activation of TSG-6 and complex cancer patients who had failed other treatments, including formation with inter-a-inhibitor that in turn inhibits activation hormonal therapy. This study concluded that (a) 2-methox- of serine proteases and consequently inhibits prostate cancer yestradiol was safe and well tolerated; (b) prostate-specific progression. It is also plausible that TSG-6 exert its anti- antigen levels declined or stabilized in some patients (37). inflammatory effects in an inter-a-inhibitor–independent

Fig. 4. A, TSG-6 staining in human prostate. Human prostate tissue array containing different grade tumors with paired normal prostate was stained withTSG-6.TSG-6 was used at a dilution of 1:100 in PBS and incubated overnight at 4jC. Immune complexes were revealed using a universal secondary antibody (100 AL for 30 minutes) followed by chromogen as described in Materials and Methods. Left, little or noTSG-6 staining in the cancerous tissue; right,TSG-6 expression in normal prostate (P = 0.0001). Statistical significance of the data was determined using ANOVA and bootstrap methods as described in Materials and Methods. B, variability ofTSG-6 expression in human prostate tumors.Tissue array containing human prostate tumors from different Gleason grade were incubated with a protein blocking serum-free reagent for 60 minutes to block nonspecific binding. TSG-6 was used at a dilution of 1:100 in PBS and incubated overnight at 4jC. Immune complexes were revealed using a universal secondary antibody (100 AL for 30 minutes) followed by chromogen. Negative controls were included by omitting the primary antibody (data not shown).

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for the induction of TSG-6 by TNF-a and interleukin-1h (39, 40). In a different line of research, 2-methoxyestradiol has been found to activate the transcription factor AP-1 in LNCaP cells (7). We found Fra-1 and Fra-2 (Fos-related antigen) mRNA was up-regulated by 2-methoxyestradiol treatment in LNCaP cells (Table 1). Fra-1 and Fra-2 are members of transcription factor AP-1 complex. These data implicate AP-1 in the induction of TSG-6 transcription in LNCaP cells. This could lead to the up-regulation of TSG-6 mRNA steady-state levels that we observed. Furthermore, it is known that binding of TNF-a to its receptor causes activation of transcription factor AP-1 and nuclear factor- nB that in turn induce genes involved in inflammatory response (41). It is noteworthy to mention that 2-methoxyestradiol has been shown to activate both AP-1 and nuclear factor-nB (7). Based on this, we speculate that 2-methoxyestradiol may exert its biological effects through TNF-a

Fig. 5. A, overexpression ofTSG-6 inhibits tumor invasion. Effect ofTSG-6 on tumor cell invasion was determined using Cell Invasion Assay (Oncogene Research Products) as described in Materials and Methods. Briefly, PC-3 cells were plated in 24-well plate coated with basement membrane matrix or transfected with sense TSG-6 cDNA either in the sense (TSG-6) or in antisense orientation (ASTSG-6). Cells that migrated to the lower chamber (invading cells) through the Matrigel were quantified by labeling with calcein-AM and measuring the fluorescence. Columns, mean % invasion with respect to untreated control set at 100% in three independent experiments; bars, SD. Statistical significance (P = 0.007 between control and TSG-6) was determined using bootstrap resampling of the data as described in Materials and Methods. B, tumor invasion ability in PC-3 cells stably expressing TSG-6. PC-3 cells stably expressingTSG-6 or control (PC-3Neo) cells were plated in 24-well plate coated with basement membrane matrix. Cells that migrated to the lower chamber (invading cells) through the Matrigel were quantified by labeling with calcein-AM and measuring the fluorescence as described above for (A). Columns, mean % invasion with respect to untreated control set at 100%; bars, SD. manner (38). Alternatively, 2-methoxyestradiol could exert its effects through TSG-6 involving other matrix degrading enzymes, such as cysteine proteases or matrix metalloproteinases. No detailed studies of the effects of 2-methoxyestradiol on Fig. 6. A, TSG-6 staining inTRAMP tissue andTRAMP cell lines. Paraffin- embedded tissues sections were deparaffinized with xylene and rehydrated using the regulation of TSG-6 transcription or TSG-6 mRNA stability graded ethanol and used in immunohistochemistry as described in Materials and have been conducted. However, some conclusions can be Methods. i, noTSG-6 staining in the prostate from animal on normal diet. drawn from studies of the transcriptional regulation of TSG-6 Magnification, Â40. ii,TSG-6 expression in prostate from a representative animal on 2-methoxyestradiol diet that showed positive staining. B, TSG-6 inTRAMP mouse expression in fibroblasts and studies of the signaling pathways prostate cancer cells.TRAMP C2, mouse prostate cells were either treated with affected by 2-methoxyestradiol in the induction of apoptosis 2-methoxyestradiol (3 Amol/L) for 24 hours or left untreated (solvent control). Following treatment, cells were harvested and fixed in 10% buffered formalin, and in LNCaP cells. TSG-6 regulation in response to inflammatory immunocytochemical analysis was done as described in Materials and Methods. cytokines has been studied in primary human fibroblasts. Staining indicative ofTSG-6 expression was detected only in cells treated with Three closely arranged cis-acting elements located in the 2-methoxyestradiol. Untreated cells showed very little or no staining. C, Western blot analysis ofTSG-6.TSG-6 expression was determined inTRAMP tumors (control) or proximal TSG-6 promoter [one activator protein (AP-1) site prostate tissue (2-methoxyestradiol ^ fed animals) depending on the case using and two nuclear factor/interleukin-6 sites] are responsible Western blot analysis. Data was quantified using Scion image analysis program.

Clin Cancer Res 2006;12(3) February 1, 2006 986 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Loss of TSG-6 in High-gradeTumors

signaling pathway. TSG-6 expression was not detected in immunoreactivity in high-grade human prostate tumors p53-deficient (WS1-E6) cells, indicating that it is under the compared with normal prostate strongly support the notion control of p53 (42). The observed low level expression of that TSG-6 could potentially be developed as a molecular TSG-6 in LNCaP cells (containing wild-type p53) is marker to detect a precancerous state or identify which consistent with this finding. However, the importance of premalignant lesions will develop into invasive prostate cancer. p53 status for biological functions of TSG-6 in prostate Loss of expression of TSG-6 in high-grade prostate tumors cancer will be the subject of future studies. is somewhat similar to the published data showing loss of One potential mechanism for the indicated effect of TSG-6 expression of homeobox gene Nkx 3.1 and transcription factor on prostate cancer is suggested by the recent determination AP-2 in high-grade prostate tumors (47, 48). Because TSG-6 that TSG-6 interacts with thrombospondin-1 (TSP-1) and is a secretory protein, it has an additional advantage of TSP-2 (43). TSP-1 and TSP-2 have antitumor effects that are being developed as a marker in urine or serum samples. At mediated by several mechanisms (reviewed in ref. 44). TSP-1 this point, it is not known whether TSG-6 is specific to prostate has a prominent antiangiogenic effect that entails inhibition or expressed in other tissues. A large number of samples of endothelial cell migration, induction of endothelial cell from different grade prostate tumors as well as other tissues apoptosis, tumor cell apoptosis, and the inhibition of growth need to be analyzed to elucidate the precise role of TSG-6 in factor mobilization (44, 45). It has been shown that TSP-1 carcinogenesis. binding to TSG-6 does not interfere with the binding of TSG-6 to hyaluronan (43). This opens the possibility that TSG-6 acts as a docking protein that mediates binding of TSP Acknowledgments to hyaluronan. Because TSG-6 has been shown to form stable We thank Dr. Norman Greenberg for providing us with the TRAMP colony and complexes with hyaluronan (27), this may result in the cell lines and for his comments on the article; Drs. Ghosh, Rao, andThompson (Uni- virtual immobilization of TSP in the extracellular matrix versity ofTexas Health Science Center, San Antonio) for their comments on the arti- of tumors, and in particular in the tumor stroma, where cle; Pamela Wolfe for statistical analysis; Ashima Gupta for technical assistance; high concentrations of accumulated hyaluronan have been Laura Chubb (Colorado State University) and Elizabeth Genova (Prostate Diagnos- tic Laboratory, University of Colorado Health Sciences Center) for their assistance detected (46). with magnetic resonance imaging andTSG-6 staining, respectively; and the Univer- 2-Methoxyestradiol may act through modulation of expres- sity of Colorado Comprehensive Cancer Center Histology core facility for assistance sion of androgen-regulated genes. Reduced levels of TSG-6 with tissue sections.

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Gretchen E. Garcia, Hans-Georg Wisniewski, M. Scott Lucia, et al.

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