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FGF8, isoform b, is an etiological factor in prostate tumorigenesis

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FGF8, isoform b, is an etiological factor in prostate tumorigenesis

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Content FGF8, ISOFORM b, IS AN ETIOLOGICAL FACTOR IN PROSTATE TUMORIGENESIS Copyright 2002 by Zhigang Song A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PATHOLOGY) August 2002 Zhigang Song Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3094373 UMI UMI Microform 3094373 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA The Graduate School University Park LOS ANGELES, CALIFORNIA 90089 1695 This dissertation , w ritten b y S o h J G r ____ U nder th e direction o f h. D issertation C om m ittee, an d approved b y a ll its m em bers, has been p resen ted to an d a ccep ted b y The Graduate School, in p a rtia l fu lfillm en t o f requirem ents fo r th e degree o f DOCTOR OF PHILOSOPHY *v» /L3 . ~TT OrrwlutirJ1 . ' - ~ — — dS€an o f G raduate S tu d ies D ztc _Augus£.j6»_2jQ0Z__________ DISSERTATIi T T E E Chairperson 0 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION This dissertation is dedicated to my parents, Peihong Song and Dengyin Li. They founded and nurtured a most wonderful family which has been a source of my growth and inspiration leading to my current achievement. This work is also dedicated to my beloved wife, Lihua Zhang, who has selflessly contributed to this project through her full support, scientific insights and technical advice, and especially to our beautiful daughter, Helen Song. Love you all. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I am pleased to acknowledge the many people who have assisted me in achieving this goal in life. My fellow lab members: Xiantuo Wu, M.D., Ph.D., who started the PB-FGF8b transgenic animal project; William Powell, who helped me from the very beginning of my work; Jiapeng Huang, who joined in this lab at the same time as I did. I also like to thank reputed pathologists from whom I learned a lot of human and mouse prostate pathology. They were: Gary J. Miller, M.D. and Ph.D. (deceased, May 25, 2001), who was the first expert pathologist to review the histopathology of FGF8b transgenic animals; Robert D. Cardiff, M.D., Ph.D., who systemically studied the histopathology of FGF8b transgenic mice and helped me photographing many high-quality pictures for the manuscript; and Michael B. Cohen, M.D., who further reviewed the histopathology of FGF8b transgenic mice in support of our manuscript. I will remember Dr. Gary J. Miller forever for his teaching ability and help with the research that this dissertation was based on. I wish to thank my committee members, W. French Anderson, M.D., and Noriyuki Kasahara, M.D. and Ph.D., who sacrificed their valuable time to make sure I developed as a scientist. Most especially I thank my mentor, Pradip Roy-Burman, Ph.D., who so patiently guides and supports me through my whole study. Without your guidance I would not have been able to accomplish this goal. Thank you all. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Dedication......................................................................................................ii Acknowledgments........................................................................................iii List of Figures.............................................................................................. vi List of Tables.............................................................................................. vii Abstract....................................................................................................... viii Chapter 1: Introduction................................................................................. 1 Fibroblast Growth Factor 8, Isoform b ....................................... 1 Transgenic Mouse Models For Prostatic Tumorigenesis..........3 Epithelial-Stromal Cross-Talks in Prostate Tumorigenisis.......4 Thesis Hypothesis and Experimental Strategies.........................5 Chapter 2: The Effect of FGF8b on the Biology of Prostate Carcinoma cells........................................................................... 7 Abstract.....................................................................................7 Introduction.............................................................................. 8 Materials and Methods............................................................ 9 Results..................................................................................... 13 Discussion.............................................................................. 27 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapter 3: FGF8b Overexpression In Prostate Epithelium: A New Mouse Model For Prostatic Intraepithelial Neoplasia.......... 33 Abstract.............................................................................. 33 Introduction.......................................................................... 34 Materials and Methods........................................................35 Results.................................................................................40 Discussion............................................................................ 60 Chapter 4: Conclusion and Future Directions...........................................64 Conclusions..........................................................................64 Future Directions................................................................. 66 References.................................................................................................. 69 Bibliography............................................................................................... 80 V Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Fig. 1, Three-plasmid expression system and production of the lentivirus................ 14 Fig. 2. Detection of GFP and FGF8b expression in transduced LNCaP cells............16 Fig. 3. The effect of FGF8b on the growth of LNCaP cells and stromal cells...........17 Fig. 4. Stimulation of in vitro invasion by FGF8b.......................................................20 Fig. 5. Results of in vivo tumorigenesis/invasion assays..............................................21 Fig. 6. Estimation of cell population by flow cytometry..............................................24 Fig. 7. Effect of FGF8b in epithelial-stromal co-cultures........................................... 25 Fig. 8. Effect of CM from transduced LNCaP cells on proliferation of prostatic stromal cells.........................................................................................................28 Fig. 9. Detection of transgene and its expression in transgenic animals....................41 Fig. 10. In situ hybridization assays.............................................................................. 43 Fig. 11. Gross examination of prostate tissues............................................................ 48 Fig. 12. Illustrations of histopathology of PB-FGF8b transgenic animals (H&E)... 50 Fig. 13. HGPIN in PB-FGF8b transgenic animals......................................................51 Fig. 14. Histopathology of PB-FGF8b transgenic animals........................................ 54 Fig. 15. Temporal incidence of prostatic phenotypic lesions in PB-FGF8b transgenic animals, line 3.................................................................................57 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1. In vivo tumorigenesis/diaphragm invasion assay with transduced LNCaP Cells................................................................................................................. 23 Table 2. Pathology of PB-FGF8b line 3 mice.........................................................47 Table 3. Pathology of other PB-FGF8b lines..........................................................58 v ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Fibroblast growth factor 8, isoform b (FGF8b) is naturally up-regulated in human prostate cancer. We found that over-expression of FGF8b in LNCaP cells could increase their growth rate, soft agar clonogenicity, and in vitro and in vivo invasion ability. In co-culture, FGF8b produced in LNCaP cells, besides its role as an autocrine factor, could also stimulate stromal cell growth. It appeared that rather than a direct action of FGF8b on stromal cells, epithelial-stromal cell-cell contact and some unknown soluble factors secreted by LNCaP cells upon stimulation by FGF8b, were required for the maximal effect on stromal proliferation. We also found that FGF8 gene expression was naturally elevated in prostatic intraepithelial neoplasia (PIN) lesions of a SV40 T antigen-driven mouse model. To further explore its biological role in prostatic tumorigenesis, we generated transgenic mice with targeted overexpression of FGF8b in the prostatic epithelium. The transgene expression was readily demonstrated by RT-PCR, and its localization to the prostatic epithelium by in situ hybridization. In this model, prostatic hyperplasia appeared as early as 2 to 3 months, followed by the dysplasia at 5 to 6 months. The incidence of phenotypic changes during the first 14 months was determined to be 100% epithelial hyperplasia of multi-focal nature, and 35% LGPIN. This profile changed in subsequent months (15 to 24 months) to display a higher incidence of LGPIN (66%) along with high grade (HG) PIN lesions (51%). Similar to HGPIN, stromal proliferation and appearance of papillary hyperplasia with atypia, exhibited a delayed viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pattern. The affected stroma consisted primarily of the smooth muscle cell component. Taken together, results of this investigation indicate that overexpression of FGF8b is an initiating event in the development o prostatic hyperplasia which, in turn, is perhaps conducive to the manifestation of other genetic lesions, that may represent the rate-limiting factors responsible for a temporal progression from hyperplasia to HGPIN. At this time, while FGF8b is proven to be an etiological factor in prostate tumorigenesis, its role as a progression factor, along with other hitherto unidentified secondary factors, remains to be determined. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1 Introduction Fibroblast Growth Factor 8, Isoform b The fibroblast growth factor (FGF) family consists of an increasing number of structurally related polypeptide mitogens, which elicit their effects by binding to high affinity tyrosine kinase receptors on the cell surface, encoded by at least four genes (FGFR1-4) in mammals (1, 2). FGF molecules are important in cell-cell interactions during embryogenesis and tissue differentiation, as well as during tumorigenesis. FGF8, the eighth member of this family, was originally identified as the androgen-induced growth factor (AIGF) from the conditioned medium (CM) of Shionogi mouse mammary carcinoma cell line, SC-3 (3). It was demonstrated to mediate the androgen-dependent growth of SC-3 cells. Its expression was correlated with murine and chicken embryogenesis in regions of outgrowth and patterning, such as the elongating body axis, midbrain/hindbrain junction, limb and face (4-10). While knock-out of fgf& gene resulted in early embryonic lethality in mice (11), FGF8 was identified by the use of the Cre/loxP system as an epithelial signal necessary for the outgrowth and patterning of the first branchial arch primordium (12). The fg/8 was localized to mouse chromosome 19 and human FGF8 to chromosome 10q24-26 (6, 7, 13, 14). This gene is unusual in its exon 1, which in fact, consists of at least four exons in comparison to one exon in other FGF genes. 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Alternative splicing of these four exons in mouse results in potentially eight protein isoforms that vary in their amino termini (3, 6, 15, 16). The human FGF8 gene is similar to its murine counterpart in structure. However, only four protein isoforms (FGF8a, 8b, 8e and 8f) are predicted due to a blocked reading frame in the human exon IB (17, 18). The FGF8 isoforms share the same signal peptide and identical carboxy-terminal region. NIH3T3 cells transfected with fgftib cDNA, or treated with recombinant FGF8b (rFGF8b), became highly transformed compared to fgftia, or fg f8c (15, 19). Additionally, fg fi was demonstrated to cooperate with Writ-1 gene as a proto-oncogene in murine mammary tumorigenesis in Wnt-1 transgenic mice (16). Of the four possible human isoforms, three (FGFSa, FGFSb and FGFSo) were cloned in our laboratory from a human prostate tumor cell line, DU145 (20). The protein products of these cDNAs share extensive amino acid homology with mouse FGF8 isoforms in that FGF8a and FGF8b exhibit identical amino acid sequences to those of their murine counterparts. The human FGF8 isoforms, although weakly expressed in human adult tissues or cell lines, are nevertheless differentially expressed. FGF8b appears to be the primary species in prostatic epithelial cell lines (20). Consistent with previous reports, FGF8b but not FGF8a or FGF8e displays robust transforming and tumorigenic activities in NIH3T3 cells. This oncogenic activity becomes more relevant when evidence points to a significant up-regulation of FGF8b expression in high-grade prostate carcinomas (21, 22). A high frequency of FGF8 over-expression, which is associated with decreased patient survival and 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. persistent in androgen independent disease, was detected by immunohistochemical analyses in clinical specimens of prostate cancers (22). Furthermore, targeted over expression of this isoform in the mammary glands of FGF8b transgenic mice results in mammary tumorigenesis (23). In cultured human prostate cancer cells, expression of antisense FGF8b reduces their growth rate and inhibits their soft agar clonogenic activity and in vivo tumorigenicity (24). The above findings strongly suggest that FGF8b is involved in hormone-related carcinogenesis of the prostate and mammary glands. Transgenic Mouse Models For Prostatic Tumorigenesis Prostatic intraepithelial neoplasia (PIN) is generally considered as a preneoplastic lesion in humans (25-27). To understand mechanisms involved in the genesis and progression of prostatic preneoplastic lesions, the availability of suitable animal models for the disease process is critical. Although several mouse models for the study of prostate tumorigenesis have recently been described (28-32), there are, as yet, no good mouse models of prostate adenocarcinoma. As such, attempts to generate other animal models targeting specific genes for growth factors, oncoproteins or tumor suppressors known to be naturally involved in this malignancy should have a high priority. Perhaps, through a logical combination of these animals, the resultant hybrid animals may more closely resemble either early or late, if not the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. complete histopathological and clinicophysiologic characteristics of the human prostate cancer. Epithelial-Stromal Cross-Talks in Prostate Tumorigenisis It’s well known that androgen regulates the development and growth of the prostate via reciprocal mesenchymal-epithelial interactions in which the mesenchyme is the actual target and mediator of androgenic stimulation (33-43). The communication between the epithelium and stroma is in a paracrine fashion that occurs in part via growth factor pathways (44-46). Androgens appear to act on mesenchymal cells to elicit the secretion of growth factors, which regulate epithelial growth and morphogenesis. Alternatively, the epithelial cells upon androgenic stimulation release various growth factors to induce the stromal cell differentiation and patterning. The epithelial-stromal interactions continue in adulthood, and growth factors are more relevant to tissue homeostasis rather than growth in mature prostate (43, 47, 48). Dysregulated interactions (43, 47-56) and peptide growth factors (57-60) have been implicated in the initiation and progression of prostate cancer. Among them, members of FGF family are believed to be involved in the cell-cell interactions and carcinogenesis of prostate cancer (61-66). For example, FGF7, which is exclusively expressed in the stromal cells, is believed to mediate the epithelial- stromal interaction through certain FGFR isoforms, such as FGFR2IIIb, that are expressed strictly in the epithelial cells (65, 66). This directionally specific paracrine 4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. communication is suggested to maintain normal prostate homeostasis, influencing cell growth, morphogenesis, differentiation and apoptosis (1). Disruption of this directional interaction between stromal and epithelial cells occurs, through the splice switching of FGFR2IIIb to FGFR2IIIc isoform, during the tumor progression (65). Meantime, activation of other FGF members, such as FGF2 or FGF8, or other growth factors may confer autonomy and enhance evolution of the malignant cell phenotype (1, 67, 68). Thesis Hypothesis and Experimental Strategies Based on the accumulated evidence mentioned above, we were motivated to test the following hypotheses: 1) overexpression of FGF8b in a weakly tumorigenic prostate carcinoma cell line could increase its tumorigenic properties. 2) overexpression of FGF8b specifically in the prostate epithelium could lead to pre neoplastic or neoplastic changes in the prostate in FGF8b transgenic animals. 3) overexpression of FGF8b in the epithelial cell component could influence the biological behavior of the stromal cells. First, in order to explore the multiple effects of FGF8b on the biological properties of the prostate carcinoma cells, LNCaP cells, a lentiviral transfer vector carrying the human FGF8b gene along with a GFP marker gene was constructed. The parameters used to test the various functions of this molecule included the proliferation, anchorage-independent clonogenicity, in vitro matrigel invasion abilities, and in vivo 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tumorigenesis/diaphragm invasion abilities of 8b-LNCaP cells with vector-LNCaP cells and LNCaP cells alone served as controls. Second, to examine the effect of FGF8b on the epithelial-stromal interactions in vitro, we used a co-culture system in which GFP expression in LNCaP cells served as a marker for separating the cellular components. The proliferation rates of both epithelial and stromal cells were measured. To study the effect of FGF8b on the epithelial-stromal interactions in vivo, histopathology and proliferation index of epithelial as well as stromal cell components in the prostate tissues of the FGF8b transgenic animals were extensively examined in the sequence of age groups. Third, to investigate the in vivo tumorigenic effect of FGF8b, we established four independent FGF8b transgenic mouse lines in which FGF8b expression is driven by a high-efificiency and prostate tissue-specific promoter ARR2PB. The histopathology of the prostate tissues was followed in different age groups of different lines starting from 1 month of age up to 24 months, in which line 3 was most extensively studied. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 2 The Effect of FGF8b on the Biology of Prostate Carcinoma Cells and Their Interaction with Stromal Cells Abstract Fibroblast growth factor 8, isoform b (FGF8b), has been implicated in oncogenesis of the prostate and mammary glands. We examined whether over-expression of FGF8b in a weakly tumorigenic prostate carcinoma cell line, LNCaP, could alter the growth and tumorigenic properties of these cells. The LNCaP cells were infected with a lentivirus vector carrying FGFSb cDNA and the green fluorescent protein (GFP) cDNA in the same construct, and the infected cell population was sorted based on GFP protein expression. It was demonstrated that, in comparison to the cells transduced with GFP-vector alone, LNCaP cells with FGF8b-GFP expression manifested increased growth rate, higher soft agar clonogenic efficiency, and enhanced in vitro invasion and in vivo tumorigenic properties. Most strikingly, while parental or vector-control LNCaP cells failed to grow at all in the in vivo tumorigenesis/diaphragm invasion assay in nude mice, the cells over-expressing FGF8b proliferated as deposits of tumor cells on the diaphragm, frequently with indications of potential tumor cell invasion into the diaphragm. Co-culturing of primary prostatic or non-prostatic stromal cells with the infected LNCaP cells led to 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. these observations: (i) stromal cells, irrespective of tissue origin, strongly suppressed LNCaP cell growth; (ii) FGF8b producing LNCaP cells could partially evade the stromal inhibition, perhaps from autocrine stimulatory effect of FGF8b; and (iii) production of FGF8b in the co-culture had a stimulatory effect on the proliferation of the stromal cells, prostatic or non-prostatic. This stimulation was not due to direct action of FGF8b on stroma, and it appeared that epithelial-stromal cell-cell contact and some unknown soluble factors secreted by LNCaP cells upon stimulation of FGF8b, were required for the maximal effect. Together, these results suggested that the growth rate and biological behavior of prostatic cancer cells, could be altered to more aggressive properties by up-regulation of FGF8b expression which also influenced the interaction of the affected cells with the stromal cells. The data obtained may have direct relevance to the progression of prostate tumor, recognizing that FGF8b is naturally over-expressed in advanced prostate cancers. Introduction Dysregulation of several growth factors has been implicated in prostate tumorigenesis (1, 69). One such factor, FGF8b was reported to be associated with the development or progression of this malignancy (20-22, 24, 68, 70). The increasing body of observations has prompted us to investigate the biological effects of over expression of FGF8b in prostatic cancer cells. A lentiviral transfer vector was 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. developed for transduction of FGF8b gene along with a GFP marker gene into the weakly tumorigenic LNCaP prostate carcinoma cell line. The proliferation, anchorage-independent growth ability, in vitro and in vivo invasion ability of transduced LNCaP cells were determined. To examine the epithelial-stromal interactions under the condition of FGF8b over-expression in LNCaP cells, we used a co-culture system in which GFP expression served as a marker for the separate quantitation of the cellular components. Materials and Methods Materials. Transducing vector, packaging construct (pCMVA8.71) and vesicular stomatitis virus (VSV) env-coding plasmids were originally obtained from Dr. Luigi Naldini (University of Torino, Italy) (71). The transducing vector was modified to include an internal ribosome entry site (IRES)-green fluorescent protein (GFP) marker gene cassette. Primary explant cultures of prostate and seminal vesicle stromal cells were established from residual portions of radical prostatectomy specimens or suprapubic prostatectomy specimens following fresh pathologic inspection. The tissues were freed of connective tissue elements by sharp dissection and minced into small pieces (approximately 1 mm3 ) using crossed No. 11 scalpel blades. They were first plated in a minimal volume of DMEM supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin and were incubated in a 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. humidified atmosphere of 5% CO2 at 37°C. Following primary outgrowth, the medium was changed to RPMI 1640 supplemented with 10% FBS. When the stromal cells had reached 70-80% confluency, they were detached using 0.25% trypsin/1 mM EDTA and were replated at 20% densities. Construction of Transducing Vector and Generation of Lentivirions. Human FGFSb cDNA (20), which was flanked with the CMV immediate early promoter at the 5’ end and IRES-GFP at the 3’ end, was incorporated into the poly-cloning sites of the transducing vector. Plasmids were amplified in E. coli and purified by Qiagen Maxi Prep kit. Using Superfect reagent (Stratagene Inc.), human 293T cells, at about 80% confluency, were co-transfected with the transducing vector, packaging construct and VSV e«v-encoding plasmids at a ratio of 5:5:1. The transducing vector construct containing only the CMV-driven IRES-GFP cassette, without FGF8b, was used in parallel co-transfection to produce the control vector. The medium containing virions was harvested daily starting from the third day to the fifth day after transfection. Infection of LNCaP Cells and Sorting by Fluorescence Activated Cell Sorting (FACS). The lentivirus-containing medium was concentrated 10-fold by using 300 kDa molecular weight cut-off spin columns (Gelman). One milliliter of concentrated CMV-FGF8b-GFP or CMV-GFP vector lentiviral medium was applied to 80% confluent LNCaP cells in T25 culture flasks. After 4 hrs of incubation, lentiviral medium was removed. Cells were washed with phosphate buffered saline (PBS) 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. twice and grown in complete medium for two days. The 8b- and vector-LNCaP cells were released from flasks by trypsinization and sorted by FACS based on expression of GFP. Northern Blot Analyses. Total RNAs were extracted from 8b-, vector-, and non infected LNCaP cells by using RNeasy Mini Kit (Qiagen). RNAs were separated by electrophoresis on a 1 % denaturing formaldehyde agarose gel, and transferred to Hybond N membrane (Amersham Corp.). The blots were hybridized to 3 2 P-labeled full-length FGF8b cDNA probe and exposed to X-ray film. NIH3T3 Cell Transformation Assay. To produce CM for use in the transformation assay, fresh medium without fetal bovine serum (FBS) plus 10 pg/ml heparan sulfate was applied to transduced LNCaP cells when cells were at 80% confluency. After 48 to 72 hrs of incubation, CM was collected and cell debris was removed by centrifugation. The CM, diluted 1:10 in DMEM medium, was added to NIH3T3 cells when cells grew to 70% confluency. After 2 days of incubation, cell morphology was examined microscopically as described previously (20, 24). Soft Agar Clonogenicity Assay. The 8b-, vector- and non-infected LNCaP cells were released by trysinization and suspended in 0.4% Seakem agarose at a cell density of lx l0 4 per 2 ml. The suspensions were over-layed on 4 ml of 0.8% Seakem agarose in a 6 cm-diameter dish, and incubated at 37°C in 5% CO2 for 21 days. The visible colonies were counted. The colony forming efficiency was calculated by 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dividing the number of soft agar colonies by the number of cells plated and multiplied by 100 to convert to percentages. Matrigel Invasion Chamber Assay. Inserts of 8 um pore-sized membranes for 24-well plates were prepared by coating with Matrigel basement membrane matrix (Becton Dickinson Labware, Bedford, MA) following the manufacturer’s instructions. Each chamber was separated into upper and lower portion by the insert with a thin layer of Matrigel basement membrane matrix. The 8b-, vector- or non infected LNCaP cells were placed on the upper chamber at a cell density of 1 x 105 cells/insert. The CM obtained by incubating NIH3T3 cells for 24 hours in serum-free DMEM in presence of 50 pg/ml ascorbic acid was added to the lower chamber to serve as chemoattractant. After 24 hours of incubation, the upper surface of the inserts was wiped with cotton swabs, and the inserts stained with hematoxylin and eosin (H&E). Cells that migrated through the Matrigel and the filter pores to the lower surface were counted under a light microscope with five random high-power fields per insert (72). In Vivo Tumorigenicity and Immunohistochemistry Studies. The 8b-, vector- and non-infected LNCaP cells were grown with complete medium in log phase and released from flasks by trypsinization. One million cells of each type were injected intra-peritoneally into athymic nude mice as described previously (72, 73). Each cell type was injected in triplicate. After 9 weeks of incubation, mice were sacrificed. Diaphragms were fixed in 10% formalin overnight and embedded with paraffin. 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sections were incubated with a purified goat polyclonal anti-GFP antibody (Santa Cruz) at 4ug/ml concentration. The bound antibody was detected with biotinylated anti-goat immunoglobulin. Sections incubated without primary antibody served as negative controls. Proliferation Assay of LNCaP-Stromal Co-culture. Approximately lxlO4 8b- or vector-LNCaP cells and lx l0 4 stromal cells were mixed and seeded into 6-well plates (Corning), while lxlO4 8b-, vector- non-infected LNCaP cells and stromal cells alone were also plated as controls. Each sample was seeded in triplicate. Total cell numbers of co-cultures were counted at different time points. After counting, 8b- or vector-LNCaP cell and stromal cell co-cultures were applied to flow cytometry assay based on GFP expression. The fractions of transduced LNCaP or stromal cells were measured and multiplied by total cell numbers to calculate the exact number for each cell type in co-cultures. Results Examination of Autocrine and Paracrine Mitogenic Activity of FGF8b. Chimeric lentivirions capable of a single cycle of infection were produced in a human embryonic kidney cell line, 293 T, through three-plasmid co-transfection (Fig. 1) (71). LNCaP cells, which are responsive to rFGF8b treatment and only weakly tumorigenic in vivo, were infected with these virions which carried either the 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. HIV Provirus Transducing Vector TAT, REV LTR LTR LTR LTR CMV FGF8b RRE IRES GFP GAG POL ENV SD'F Packaging Construct Poly A CMV GAG POL SDA'F Env-coding Plasmid CMV VSV.G Poly A Fig. 1. Three-plasmid expression system and production of the lentivirus. In HIV provirus, the coding regions for GAG, POL and ENV, are shown. The splice donor site (SD) and the packaging signal sequence (W) in the 5’ untranslated region are indicated. The transducing vector contained the human FGFSb gene and a linked GFP marker gene driven by the CMV promoter. An IRES sequence was placed between the genes. This expression cassette was flanked by the HIV LTRs and contained the W and rev-responsive element (RRE) of HIV at the 5’ end. The packaging construct contained the coding sequence for all necessary viral proteins, while W was deleted and the reading frame of the envelope and one accessory protein Vpu were blocked. The ewv-coding plasmid was the VSV e«v-coding sequence with the CMV promoter and SV40 polyadenylation signal sequence. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FGF'Sb-GFP or only the GFP control gene. After infection and propagation, the cells were sorted twice by FACS based on GFP expression. Thus two types of populations, cells transduced with human FGFSb gene (designated as 8b-LNCaP) and cells transduced with vector control construct (vector-LNCaP), were established. Under a fluorescent microscope, GFP was an excellent visible marker to determine if the cells were indeed transduced (Fig. 2A). The expression of FGFSb in 8b-LNCaP cells was readily detected by Northern blot assay using 3 2 P-labeled full-length FGF8b probe, while the expression in vector- or non-infected cells was too low to detect by Northern technique (Fig. 2B). To demonstrate FGFSb expression at the functional protein level, we conducted N1H3T3 cell biological transformation assay using CM from transduced LNCaP cells. In agreement with our previous work (20), CM from 8b-cells displayed a strong ability to transform the NIH3T3 cells morphologically while CM from vector-LNCaP cells did not (data not shown). To avoid the potential clonal variation, the pooled populations of sorted cells rather than single clones of transduced cells were used in all experiments. First the effect of FGF8b on growth of LNCaP cells was examined. The experiments were repeated four times with cells at different passages and representative results are illustrated in Fig. 3A. Clearly the over-expression of FGF8b in 8b-LNCaP cells increased the growth rate in comparison to that of vector-LNCaP cells. After 14 days of culture, the proliferation rate of 8b-LNCaP cells was two-fold higher than that of vector- LNCaP cells. There was no difference in the growth rate between vector- and non- 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FGF8b wmm 2 8 S - * 1 8 S - ^ Fig. 2. Detection of GFP and FGF8b expression in transduced LNCaP cells. A, GFP was detected by fluorescence microscopy. B, FGF8b mRNA was detected by Northern blot using a 3 2 P-labeled FGFSb cDNA probe. Sample loading in (B) was visualized by the 18S and 28S ribosomal RNA levels. 16 a * . ' jj a « ., T tlM<l ill rV< WC»MSct ■hHvv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LNCaP cells vector 8b S U B Days Stromal cells ■ ® - no r8b • 1 0 ng/ml r8b ■ “■100 ng/ml r8b 6 10 Days LNCaP cells no r8b 10 ng/ml r8b 100 ng/ml r8b 4 Days Fig. 3. The effect of FGF8b on the growth of LNCaP cells and stromal cells. A, proliferation assay of vector- or 8b-LNCaP cells; B, growth of prostatic stromal cells in the presence or absence of rFGF8b; C, growth of LNCaP cells treated with rFGF8b. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. infected LNCaP cells (data not shown). Next we determined whether FGF8b could function as a mitogen to stromal cells. Two different human primary prostatic stromal cell cultures and one primary stromal cell culture from human seminal vesicle were used to examine the effect of rFGF8b on stromal cell growth. Cells were treated with rFGF8b at two different concentrations, namely 10 ng/ml and 100 ng/ml. Each experiment was repeated twice. It was found that rFGF8b had no mitogenic effect on either prostatic (Fig. 3B) or non-prostatic stromal cells (data not shown). In contrast, rFGF8b, consistent with a previous study (17), was able to stimulate proliferation of LNCaP cells. The stimulation noted with 10 ng/ml of the protein factor, however, could not be further enhanced by increasing the concentration by ten-fold (Fig. 3C). Determination of FGF8b effect on tumorigenic properties of LNCaP cells. The soft agar assay was performed to investigate the effect of FGF8b on the anchorage- independent clonogenicity of transduced LNCaP cells. Each cell sample was seeded in triplicate and each assay was repeated twice. After 21 days of culture, 8b-LNCaP cell group displayed clonogenic efficiency of 332 ± 45.74 colonies (3.32%), while a reduced efficiency of 250 ± 36.62 colonies (2.5%) was observed with the vector- LNCaP cell group. By Student t-test the difference in clonogenic efficiency between these two cell groups was determined to be significant (P<0.05). Additionally the size of at least one third of colonies formed by 8b-LNCaP cells was generally larger than those formed by vector- or non-infected LNCaP cells (results not shown). The 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. latter two cell types showed no significant difference in either clonogenic efficiency or colony size. Next the in vitro invasion ability of transduced cells was measured with the Matrigel invasion chamber assay. After 24 hours of incubation, vector- (Fig. 4A) or 8b-LNCaP cells (Fig. 4B) that migrated through the Matrigel basement membrane matrix and the filter pores to the lower surface were counted under a light microscope with five random high-power fields per insert. From 1x10s seeded cells, 583 ± 37.47 8b-LNCaP cells were counted compared to 173 ± 60.70 vector-LNCaP cells (Fig. 4C). While the difference between these two cell types was significant (P=0.001), there was practically no difference between vector- or non-infected LNCaP cells. This assay was repeated three times with cells at different passages and each cell sample was done in triplicate. To assess the survival ability of transduced LNCaP cells in vivo, one million of 8b-, vector-, or non-infected LNCaP cells were injected intra-peritoneally into the athymic nude mice. Five mice were inoculated with each cell type. After 9 weeks of incubation, the mice were sacrificed and diaphragms were fixed with 10 % formalin. After paraffin embedding, sections were stained with H&E (Fig. 5A). Twelve different sections for each individual diaphragms were examined for tumor growth or tumor invasion under a microscope (72, 73). The fact that those cells were indeed tumor cells was confirmed by immunohistochemistry (IHC) assay using an anti-GFP antibody (Fig. 5B). The IHC assay was repeated twice on the sections from each diaphragm. Although the analysis involved a limited number of tissue sections, it was found that at least four 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. * ,* 5' . ' >'v . J . Vector B ■V ‘ l l : ■ - • 5 i > M W \ ■ % * * V • % ^ ‘ ‘ S ' * ^ # --<•■• ?«. J / 1M yt f *.< 40''* * *' 4 «#..* P=0.001 vector 8b Fig. 4. Stimulation of in vitro invasion by FGF8b. H&E staining was used to detect the vector- (A) or 8b-LNCaP cells (B) that migrated through the Matrigel basement membrane. The difference between migrated vector- and 8b-LNCaP cells was indicated (P=0.001) (C). The bars represent the standard deviation (SD) of means of individual experiments. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 5. Results of in vivo tumorigenesis/invasion assays. A, examples shown include detection of tumor cells on the diaphragm by H&E staining. B, the origin of tumor cells growing on the peritoneal surface of the diaphragm was confirmed by immunohistochemistry using anti-GFP antibody. 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. out of five animals in 8b-LNCaP cell-injected mice were positive for tumor growth, while all five mice in vector- or non-infected LNCaP cell injected mice were negative (Table 1). In two of these four diaphragms from 8b-LNCaP cell-injected mice, tumor cells also exhibited multi-spot attached growth on the peritoneal surface of diaphragm. The sizes of areas that tumor cells occupied in these diaphragms varied considerably. In 20% of areas examined, however, tumor cells did grow into the diaphragm and spread horizontally with evidence of vertical penetration. FGF8b-Mediated Epithelial-Stromal Cross-talk. To exploit the potential FGF8b-mediated interactions between prostatic cancer cells and stromal cells, we used a co-culture system in which GFP served as a marker to separate transduced LNCaP cells from stromal cells by the use of flow cytometry (Fig. 6A). The same two human primary prostatic stromal cell cultures stated above were also utilized in these analyses. The stromal cells in co-culture spread out on the bottom of the culture flask providing a mesenchymal bed and LNCaP cells grew on the top of stromal cells forming gland-like structure. A microscopic view of this co-culture is presented in Fig. 6B. LNCaP cells were more round-shaped and much smaller than their counterparts in cultures when they were grown alone. As illustrated in Fig. 7A the growth of prostatic stromal cells in co-culture with 8b-LNCaP cells increased significantly compared to that of stromal cells when grown with vector-LNCaP cells. The difference was approximately two-fold after 14 days of culture. The proliferation rate of stromal cells in co-culture with vector-LNCaP cells was, 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1 In vivo tumorigenesis/diaphragm invasion assay with transduced LNCaP cells Group Tumor Incidence Non-infected 0/5 Vector 0/5 8b 4/5 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LNCaP cells Stromal cells O C O 0 2 0 0 400 600 8 0 0 1000 FL1-Height B -f t 1 - * „ S , - “v I V , '.j.'f'.-: y. M sS: i ,‘ 2 * , ' v - 'Xr«- : .Q ':.-.- • 5 • - ^ u' *$ t . i* ,V ’*t 4f « * * ”’’ vtr "I" > * ' i > ' * ^ * V * * * 5 * * S r -V' ; ; w C . v - - ‘ * I ; '* V^ A " , IS ? B S ; JPf' A * Fig. 6. Estimation of cell population by flow cytometry. A, Flow cytometry assay was used to distinguish the transduced LNCaP cells from stromal cells in co-culture based on GFP expression. The axis of FLl-FIeight represents the green fluorescence intensity and “M” denotes the marker. B, A microscopic view of LNCaP-stromal co culture. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stromal cells (Prostatic) ■ stroma ■ stroma/ vector - strom a/8b 12 15 Stromal cells (SV) • stroma ■ stroma/vector j - stroma/8 b B vector/stroma 8b/stroma ~ 20 3 6 9 12 15 D ^ 20 5 - .2 E 10 Days LNCaP cells vector/stroma 8b/stroma 8 12 Days 16 Fig. 7. Effect of FGF8b in epithelial-stromal co-cultures. The rate of proliferation of prostatic stromal (A) or seminal vesicle (SV) stromal cells (C) in co-culture with transduced LNCaP cells is contrasted with the growth of transduced LNCaP cells in the presence of prostatic stromal (B) or non-prostatic stormal cells (D). 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. however, similar to that of stromal cells grown alone. The growth rate of 8b-LNCaP cells in co-culture was also higher than that of vector cells co-cultured with stromal cells. However, compared to the results with LNCaP cells cultured alone (Fig. 3A), both 8b- and vector-LNCaP cells showed inhibited growth in co-culture with stromal cells (Fig. 7B). To investigate if the stimulatory effect of stroma on 8b-LNCaP cells was prostatic tissue-specific, primary stromal cells from human seminal vesicle (SV) were grown with the transduced LNCaP cells. The cell morphology in co-culture was similar to that of prostatic stromal cell and LNCaP co-cultures. After 10 days of culture, the growth rate of these non-prostatic stromal cells in co-culture with 8b- LNCaP cells increased threefold compared to that in co-culture with vector-cells (Fig. 7C). There was again no significant difference in growth rate between SV stromal cells when cultured with vector-LNCaP or cultured alone. The growth rate of 8b- LNCaP cells in co-culture with SV stroma was slightly higher than that of vector- cells in co-culture (Fig. 7D), but both cell types showed remarkably restrained growth in presence of SV stromal cells. It was also noted that 8b-LNCaP cells and non-prostatic stromal cells developed the local confluence faster than those in co culture of prostatic stromal cells and 8b-LNCaP cells. It was quite unique that SV stromal cells became detached from the bottom of culture flask once local confluence was reached. This observation was confirmed by repeat experiments with the same primary SV stromal cells. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Since rFGF8b had no significant direct effect on proliferation of stromal cells, it was considered likely that some soluble mediators) might be secreted by 8b-LNCaP cells to stimulate stromal cells in co-culture. For that possibility, 24 hour CM from transduced LNCaP cells was applied to the primary prostatic stromal cells. As illustrated in Fig. 8, the growth rate of stromal cells treated with CM from 8b-LNCaP cells was certainly elevated compared to when vector-LNCaP cell CM was used (P=0.009). Discussion The interest in this work is three-fold. First, our results demonstrate that FGF8b produced in the LNCaP cells can function as an autocrine stimulator of the proliferation of these cells. There was a reference to this effect previously using bacterially expressed rFGF8b (17) which, unlike FGF8b produced in mammalian cells (3), lacks glycosylation. It is now documented that whether glycosylated or not, FGF8b can induce proliferation of the LNCaP cells, thus implying that the receptor activation by the growth factor is likely to be independent of glycosylation. In this regard, it was reported before that rFGF8b could efficiently activate the “c” splice form of FGFR2 or FGFR3, as well as FGFR4 (67, 74). Although these receptor forms are considered to be largely expressed in mesenchymal cells (75), there is evidence of aberrant expression of FGFR isoforms in prostate cancer cells (1, 76). It 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Stromal cells (Prostatic) 18 vector C M 8b CM 12 6 0 4 12 8 Days Fig. 8. Effect of CM from transduced LNCaP cells on proliferation of prostatic stromal cells. 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is also interesting to note that among the tissues of the male reproductive tract, only prostate appears to exhibit wide expression of the general classes of FGFRs. For example, a moderate level of expression of FGFR1 and FGFR2 is found in prostate epithelium and the microvasculature, while stromal smooth muscle cells exhibit a weak level of expression of FGFR3 (77). In another study (75), with primary cultures of human prostatic epithelial and stromal cells, FGFR3 is found to be primary product in epithelial cells with smaller amount of FGFR2, while stromal cells express primarily FGFR3 and smaller amount of FGFR1 and FGFR2. Considering these observations, and recognizing that FGF8b is naturally overexpressed in aggressive prostatic carcinoma cells (21, 22), a scenario is presented for FGF8b- FGFR signaling in regulation of the growth of prostatic epithelium. Second, besides its ability to stimulate proliferation, it is also shown here that FGF8b can also influence the various biological properties of the affected LNCaP cells. For example, in vitro parameters, like soft agar clonogenicity and matrigel invasion activity are significantly increased in 8b-LNCaP cells relative to the vector- LNCaP cells. An argument can be made that the in vitro motility and invasion may be partly related to increased proliferation, but it is unlikely to be a primary cause since the analyses were carried out only after a short period of incubation, namely 24 hours, when proliferation is deemed to be quite limited. Matrix metalloproteases (MMPs), which degrade extracellular matrix proteins, are known to be over expressed in many types of cancers (78-82). There is an accumulation of reports that 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. describe induction of MMP expression by FGF proteins in cancer cells, including prostate cancer cells, not in normal epithelial cells (76, 83-86). Thus, a possibility is considered that switch of expression patterns of FGFR isoforms that occurs in prostate cancer cells and that may be critical for abnormal proliferation by FGFBb may also be responsible for activation of MMPs, thereby facilitating tumor cell invasion. The pleiotropic effect of FGF8b signaling is clearly documented here in the study of in vivo tumorigenesis/diaphragm invasion assay. While LNCaP cells were not tumorigenic in this assay, FGF8b expression converted them to be not only tumorigenic but also to become invasive in some animals during the nine weeks period of observation. Taken together, evidence is presented that FGF8b over expression induces tumorigenic and invasive properties to the LNCaP cells. It, however, remains to be demonstrated whether results of the study using a single cell line might have broader validity when prostate cancer cells in general or prostate cancers are concerned. Finally, when we examined the effect of FGF8b on the epithelial-stromal interactions in settings of the co-culture, two important aspects were uncovered. It is clearly evident that the proliferation of the parental or transduced LNCaP cells is remarkably inhibited when co-cultured with stromal cells from prostatic or non prostatic tissues. In contrast, the growth of the stromal cells is strongly up-regulated in the presence of FGF8b-producing LNCaP cells but not the control LNCaP cells. Although the autocrine regulation of LNCaP cells by FGF8b could compensate for 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the stromal effect to a degree, the negative effect of stroma still remains quite pronounced. The stimulatory effect of 8b-LNCaP cells on stromal cells appears not to be mediated by the released FGF8b since rFGF8b is not capable of stimulating stromal cells. Considering that other soluble molecules or factors induced and secreted from the FGF8b-stimulated LNCaP cells might be responsible, we used CM on stromal cells to examine the possibility. Since there is some positive effect of the CM on stroma, although far less than what observed in the context of co-culture, this potential is still valid. Further work will be necessary to characterize the released stimulatory factors to obtain a better definition of the role of FGF8b in the environment of the prostate. However, a stronger case could be made for the importance of cell-cell contact between epithelial and stromal cells for the observed stimulatory effect on the stroma. This contact effect is explicitly dependent on the presence of FGF8b-producing LNCaP cells. Thus, FGF8b signaling, beyond the release of soluble factors, appears to be critical in stromal proliferation. It is tempting to consider alterations of cell surface molecular expression in LNCaP cells from FGF8b-FGFR interactions that may be the primary events in inducing stromal growth. In summary, the effect of over-expression of FGF8b in LNCaP cells and their interaction with stromal cells may have broad implications in the progression of human prostate cancer. The autocrine stimulatory loop of FGF8b is likely to provide advantages to the neoplastic prostatic epithelium with respect to their proliferation 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and invasive properties. Additionally, it is speculated that, on the way of invasion and metastasis, the FGF8b producing malignant cells when in physical contact with stromal cells may stimulate growth of stromal cells. The accelerated proliferation of the stromal cells has, in fact, been suggested to provide an amenable milieu in the development and progression of cancer (56). 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 3 FGF8b Overexpression In Prostate Epithelium: A New Mouse Model For Prostatic Intraepithelial Neoplasia Abstract Fibroblast growth factor 8, isoform b (FGF8b), a mitogenic and transforming polypeptide, was demonstrated to be naturally up-regulated in prostatic premalignant and malignant lesions in men. We generated four independent lines of transgenic mice with targeted overexpression of FGF8b in the prostatic epithelium using an improved rat probasin promoter, ARR2PB. Transgene expression in the prostate tissue was readily demonstrated by RT-PCR, and localized to the prostatic epithelium by in situ hybridization. The histopathology of the prostate tissues was followed in different age groups of the various lines, but most extensively in one line (line 3), starting from 1 month of age up to 24 months. Prostatic hyperplasia appeared in the lateral and ventral prostates in some animals as early as 2 to 3 months, and in other lobes between 6 to 16 months. Beginning at 5 to 7 months, dysplasia, akin to what may be considered low grade pro static intraepithelial neoplasia (LGPIN) in humans, was detected. During the first 14 months, 100% of animals exhibited multi-focal epithelial hyperplasia; 35% also had areas of LGPIN. 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This profile changed in subsequent months (15 to 24 months) to a higher incidence of LGPIN (66%) along with high grade (HG) PIN lesions (51%). Similar to HGPIN, stromal proliferation and appearance of papillary hyperplasia with atypia, displayed a delayed pattern. The affected stroma consisted primarily o f the smooth muscle cell component. The incidence of chronic inflammation, mostly involving T cells was higher in the prostate of the transgenic mice relative to controls; however, the presence of a direct correlation between inflammation and hyperplasia or preneoplastic lesions was not identified. These transgenic mice represent a “natural” animal model for investigating the mechanism of development and progression of prostatic diseases, such as, prostatic hyperplasia and preneoplastic lesions. Introduction In order to study the in vivo effects of FGF8b, it was important to establish transgenic mouse lines by targeting FGF8b overexpression in the prostate epithelium. For this purpose, the small rat composite 468 bp probasin promoter ARR2 PB, was chosen. This promoter has been demonstrated to confer a high-level of reporter transgene expression specifically in the luminal prostatic epithelium and is strongly regulated by androgens (87, 88). Four independent transgenic lines, in which the FGF8b transgene expression is driven by this promoter, were generated. The specificity of expression of the transgene was confirmed by RT-PCR and in situ 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hybridization, and the transgenic animals heterozygous for the transgene were followed for study of the histopathology for up to 24 months of age. MATERIALS AND METHODS Construction of Transgene. The Xhol-Xbal fragment of ARR2PB (87) was blunted and ligated into pSV plasmid vector containing the SV40 poly A sequence and splicing signal sequence. Subsequently the full-length human FGF8b cDNA fragment (20), that harbored 100% amino acid sequence identity to the mouse FGF8b sequence (20), was inserted into the EcoRl site following the ARR2PB promoter (Fig. 9A). The sequence of FGF8b coding region and junction of each fragment was confirmed by automated DNA sequencing. The fragment containing the ARR2 PB promoter, FGF8b cDNA and SV40 poly A sequence was released by digestion with Noll and Kpnl, isolated by agarose gel, and purified by Qiagen spin column (Qiagen, Germany) and elutip column according to the manufacturer’s protocol. Generation of Transgenic Mice. Two rounds of pronuclear injection of ARR2PB promoter-FGF8b-SV40 poly A fragment were performed. The (C57BL/6 X DBA2)F1 hybrid fertilized eggs containing the transgene construct were placed into pseudo-pregnant females. Potential founder animals were screened by PCR and confirmed by Southern blot analysis using clipped tissue DNA samples. Four 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. productive transgenic lines were established by mating the founder animals with non transgenic (C57BL/6 X DBA2)F1 mice. Offspring were genotyped by PCR from tail DNA at 3 to 4 weeks of age. Tissue Preparation. An aliquot of 150-200 pi of 10 mg/ml bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) was injected intraperitoneally 1 hour before animals were sacrificed. The urogenital system was removed and the individual prostate lobes were dissected under a dissecting microscope. Tissues for histopathologic observation were fixed overnight in 10% neutral buffered formalin (Surgipath, Richmond, IL). Fixed tissues were processed and embedded in paraffin. Thin sections (5 pm) were cut and stained with H&E. Tissues for mRNA assays were frozen in liquid nitrogen at the time of dissection. PCR and Southern Blot Analysis. The tissue specimens were digested with 20 mg/ml proteinase K (Gibco, Buffalo, NY) in 500 pi of a buffer containing 50 mM Tris-HCL (pH: 8.0), 100 mM EDTA (pH: 8.0), 100 mM NaCl and 1% SDS, at 50°C for overnight. After centrifugation, the supernatant containing the genomic DNA was collected. After boiling for 5 minutes, 2 pi of the supernatant were used as the template in 30 pi reaction mixture containing 0.2 mM dNTP, 1.0 mM MgCh, 0.02% (w/v) DMSO, 6 pmol of each primer and 0.3 unit of Tag Polymerase (Gibco, Buffalo, NY). The sequences of the primers, F8b-3 and SV40-a, used for PCR and RT-PCR were 5 ’ - AACT AC AC AGCGCTGC AGAAT G-3 ’, which is complementary to the FGF8b cDNA sequence (20), and 5 ’ -GTTGAGAGTCAGCAGTAGCCTC-3 ’, which 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is complementary to the SV40 polyA signal sequence (Fig. 9A). The PCR was started at 94°C for 4 minutes, followed by 35 cycles at 94°C for 1 minute, 58°C for 90 seconds and 72°C for 90 seconds, and ended with 72°C for 5 minutes. Founder animals were further confirmed by Southern blot analysis. Briefly, 10 pg tail DNA was digested by Bam HI, run on a 1.5% agarose gel and transferred to Nylon membrane. A 3 2 P-labeled SV40 signal sequence was used to probe the Southern blot. RT-PCR. The tissue RNA was extracted using RNeasy Mini kit (Qiagen, Germany). The ThermoScript RT-PCR System (Gibco, Buffalo, NY) was used for RT-PCR assay. A solution of 1 pg RNA was mixed with 1 pi of random hexamer primers provided in 10 pi volume and denatured at 65°C for 5 minutes. After cooling on the ice, 10 pi cDNA synthesis mixture was added. The samples were incubated at 25°C for 10 minutes, followed by 60 minutes at 50°C and terminated at 85°C for 5 minutes. Aliquots of 2-4 pi of cDNA synthesis reaction mixtures were used as templates for PCR as described above. In Situ Hybridization Assays. The transgene construct was amplified by PCR using primers of F8b-T7 and F8b-T3, or primers of SV40-T7 and SV40-T3 (Fig. 9A). The sequence of F8b-T7 was 5’- GCGCT AAT ACGACTC ACT AT AGGGT AAGCTT GCT GCC ATGGGC AGC-3 ’, which contained the T7 promoter sequence and a segment that was complementary to FGF8b sequence at 5’ end (20). The sequence of F8b-T3 was 5’- GCGCAATTAACCCTCACTAAAGGGGCTTGATATCGAATTCAGGATG-3’, 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. which contained the T3 promoter sequence and segment that was complementary to FGF8b sequence at 3’ end (20). The sequence of SV40-T7 was 5’- GT AAT ACGACTC ACT AT AGGGCGC AGTGGTGGAATGCCTTT AATG-3 ’, which contained the T7 promoter sequence and a segment that was complementary to SV40 poly A signal at 5’ end. The sequence of SY40-T3 is 5’- GCGCAATTAACCCTCACTAAAGGGACCTCTACAAATGTGGTATGGCT-3’, which contained the T3 promoter sequence and segment that was complementary to SV40 poly A signal at 3’ end. PCR products were transcribed in vitro into probes that were labeled with digoxigenin using a digoxigenin labeling kit (Boehringer Mannheim, Germany). In Situ hybridization assay was performed as described by Nieto et al (89) with some modifications (90). In short, frozen tissues (5 pm) were hybridized overnight at 60°C in hybridization mixture with a probe diluted to 1:100. Sections were incubated in the blocking solution containing alkaline phosphatase labeled sheep anti-digoxigenin Fab fragments (1:1500, Boehringer Mannheim, Germany) overnight at room temperature. Signals were detected with a detection solution containing 4-nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3- indolyl-1 -phosphate (BCIP) (Boehringer Mannheim, Germany). Histological Classification. Consistent with previously described criteria (32, 91), a similar grading system for PIN-like lesions was used to evaluate PB-FGF8b animals. Generally, PIN lesions were categorized into low grade PIN (LGPIN) and high grade PIN (HGPIN) based on their degree of cytological atypia. LGPINs 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. showed mild cytological atypia. HGPINs were distinguished from LGPINs by more epithelial cell proliferation, nuclear stratification and cytological atypia. Other lesions, such as papillary hyperplasia, that can not be characterized by this grading system are described separately based on their architecture and cytology. Immunohistochemistry. BrdU staining was performed using the Zymed BrdU staining kit (Zymed , South San Francisco, CA). Sections were incubated with a biotinylated monoclonal anti-BrdU antibody. Signals were generated by the Streptavidin-peroxidase with diaminobenzidine (DAB) as a chromogen. The slides were counterstained with methylene green (KPL LABS, Gaithersburg, Maryland). The staining for androgen receptor (AR) was performed in a similar manner. After antigen retrieval by microwave heating in 1M urea, specimens were incubated with a rabbit anti-AR polyclonal antibody (Santa Cruz, CA) at 0.8 pg/ml concentration. The bound antibody was detected with the biotinylated goat anti-rabbit immumoglobulin. Sections incubated without primary antibody served as negative controls. For smooth muscle actin (SMA) staining, a mouse monoclonal antibody against a-SMA (Sigma Chemical Co., St. Louis, MO) was used at 1.1 pg/ml concentration with the DAKO ARK kit (DAKO, Carpinteria, CA) to eliminate mouse background staining. A rabbit polyclonal anti-human CD3 antibody (DAKO, Carpinteria, CA) was used at 0.8 pg/ml concentration for T cell staining. For B cell staining, a rat anti-mouse CD45R/B220 monoclonal antibody (Pharmingen, San Diego, CA) was used at 0.6 pg/ml concentration. 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Status of FGF8 Expression in PIN Lesions of the Mouse Prostate in LPB-Tag sublines. Although an association between FGF8 overexpression and human prostate cancer was described previously, little was known about this association in mice. Thus it was important to investigate the FGF8 expression status in prostatic lesions from mice. For this purpose, LPB-Tag sublines, 12T-7f(fast) and 12T-7s(slow), were used (29). As the names imply, 12T-7f animals developed PINs at a faster rate compared to those of 12T-7s (29). PIN lesions of these mice were analyzed for FGF8 expression by in situ hybridization assay in which FGF8-specific riboprobes were used. While signals were readily detected by a F8b-T3 antisense probe, the signal density was much higher in the epithelial lesions of 12T-7f line than those of 12T-7s line (Fig. 10A, C). Weak signals were also detected in prostate tissues of wild-type control animals only after prolonged exposure (data not shown). Sections incubated with a F8b-T7 sense probe served as an internal negative control (Fig. 10B, D). The major FGF8 RNA species expressed in the tumor sections was identified to be FGF8b following the same RT-PCR protocol which we reported before (20). A similar RT-PCR analysis of a tumor cell line, TRAMP-C, derived from the TRAMP model (92), also showed a single FGF8b species as the primary product (data not shown). 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 9. Detection of transgene and its expression in transgenic animals. A, graphic illustration of transgene construct. FGF8b gene is driven by a prostatic-specific promoter ARR2PB and followed by the SV40 polyA signal sequence, in which a 65 bp intron is included. Locations of the various primers are indicated by arrows. B, an example of PCR genotying using F8b-3 and SV40-a primer set. The expected size of the amplified product is 550 bp. “M” stands for DNA molecular weight marker. Numbers over the lanes represent DNA samples from various PB-FGF8b transgenic progeny. Lane 1 denotes a positive sample, while lane 2 is a negative sample. Transgene was detected in samples of lanes 3, 4, 5, 7, 8 and 10. C, Southern blot analysis of genomic DNA from potential founder animals using a 3 2 P-labeled SV40 poly A signal sequence probe. A band of 1.5 kb is expected. Each lane represents a putative PB-FGF8b transgenic founder animal. Lane 1 represents positive and lane 2 negative samples. Lanes 4, 5, 6 and 8 (founder animal #1, 3, 5 and A, respectively) were transgene positive. D, RT-PCR analysis of the tissue RNA from transgenic animals: presence or absence of reverse transcriptase is indicated by (+) and (-), respectively. Transgene expression was readily detected in AP, DLP and VP, and was also detectable in ductus deferens (DD), seminal vesicle (SV) and epididymis (Epi). However, transgene expression was not detected in other tissues tested, namely testes, thymus, liver, kidney, lung, spleen and heart. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. F8b-|7 F8b-3—► SV40-T7 a r r 2 p b F G F 8 b SV 40 Intron (65 bp) P o Iy A F8b-T3 ^-SV40-a S Y 4 0 -T 3 B M 1 2 3 4 5 6 7 8 9 10 11 1 f ^ * V " V » < »— * r -HHI * ft H 500 bp > ■ /" f* kt , M r'* * “ 4 « “ * « • tX* * y 9 * J 6 £ ? " * ‘ S J H 1 & £ m < v I » * * * e B ’I £ , f- ^ r i : ’ r £ % m m S '" tiiklfc. - , s c 1 2 3 4 5 6 7 8 9 1.5 k b D AP DLP VP Epi DP SV Testes Thymu Liver (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) (-) (+) 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission Fig. 10. In situ hybridization assays. Detection of FGF% gene expression in PIN lesions in DLP of LPB-Tag transgenic sublines 12T-7f (A, X200) and 12T-7s (C, X200) using a FGF8-specific antisense riboprobe, F8b-T3 (Fig. 1A). FGF8 expression was mostly detected in epithelial cells while there was only weak or no expression in stromal compartments. For illustration, some areas of the stroma are marked by Adjacent sections from 12T-7f (B, X200) and 12T-7s (D, X200) lesions were incubated with a FGF8-specific sense probe, F8b-T7, serving as the negative control. Detection of transgene expression in our PB-FGF8b transgenic animals is illustrated by a section of VP from a 6-month-old mouse using a SV40 poly A-specific antisense riboprobe, SV40-T3. Transgene expression was prominently detected in epithelial cells but not in the stroma; again, stromal areas are labeled with Section of VP from a littermate control animal using the same antisense probe setved as a negative control (F, X40). 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Generation of Productive Transgenic Lines. The founder animals were identified by the presence of a 550-bp PCR product amplified from tail DNA by the primers that spanned the FGF8b cDNA gene and the SV40 poly A signal sequence of the transgene (Fig. 9B). These founder animals were further confirmed by the presence of a 1.5-kb band on a Southern blot of BamHl -digested genomic DNA probed with the SV40 poly A signal sequence probe (Fig. 9C). Four founder animals were identified from the first pronuclear injection and marked as 1, 3, 5 and A. Another series of five founder mice, named 6, 8, 18, 20, 24, was obtained from the second injection. Founder A, a male animal, was determined to be infertile. Founders 5, 6 and 8 did not transmit the transgene to the offspring, as determined by PCR screening of tail DNA, after testing of 4 litters of total 25 offspring. Animal 24 contained the transgene, but failed to express it in the prostate tissues as determined by RT-PCR analysis using three different age groups of the offspring. Eventually, four productive lines, 1,3, 18 and 20, that contained the transgene and expressed the mRNA in prostate tissues were established. Since the primers for the detection of transgene expression were designed to flank an intron in SV40 poly A signal sequence, an additional band, of 485 bp long, was expected to be amplified if the transgene was transcribed and spliced (Fig. 9D). For all productive lines, the expression of the spliced version was detected in all prostate lobes, namely anterior prostate (AP), dorsolateral prostate (DLP) and ventral prostate (VP). A detectable level of expression was also seen in the ductus deferens (DD), 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. seminal vesicle (SV) and epididymis. To determine if there was spurious expression of the transgene, various other tissues (testes, thymus, liver, kidney, lung, spleen and heart) from animals of different lines were examined for transgene expression by RT-PCR. There was no detectable FGF8b mRNA in these tissues. When prostates of animals of different ages (4 to 24 months) were assayed by RT-PCR, it was found that the transgene continued to be expressed throughout the time period of this investigation (data not shown). The transgene expression was localized by in situ hybridization assay using a SV40 poly A-specific riboprobe. In prostate tissue sections, transgene mRNA was readily detected by the SV40-T3 anti-sense probe, while the SV40-T7 sense probe served as an internal negative control. As illustrated in Fig. 10E, the signals for the transgene transcripts were strikingly confined to the epithelial cells of the prostate. No positive signals were found on the prostate tissue sections of the nontransgenic littermate control animals under identical experimental conditions (Fig. 10F). Overexpression of FGF8b led to PIN lesions. Throughout the experimental period, no significant difference in gross body weight between the transgenic animals and wild-type siblings was found. Among the four productive lines, line 3 was studied most extensively (Table 2). In this line, after 2 to 3 months, the lateral prostate (LP) and VP of the transgenic animals were consistently larger in size, varying between 2 and 15 times than those of the age-matched control animals (Fig. 11). Histopathologically, hyperplasia was detected in the LP and VP of some animals 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2 Pathology of PB-FGF8b line 3 mice A ge Animal Histology of th e P ro sta te O ther (m onths) No. VP LP DP A P notable findings 1 310 normal norm al norm al norm al 2 '289 norm al Hy normal norm al 3 2 7 2 Hy norm al normal norm al 4 268 Hy Hy normal norm al 245 Hy Hy norm al norm al 5 25 7 Hy LGPIN In norm al 6 234 Hy, In, S p LG PIN , In LGPIN, In In 7 223 Hy, S p Hy norm al norm al 11 norm al LGPIN normal normal 203 LQ PIN LG PIN , In norm al normal 8 37 Hy Hy normal norm al 9 213 Hy norm al norm al normal 172 norm al norm al Hy normal 11 58 LGPIN, In norm al LGPIN norm al 91 Hy Hy, In norm al normal 12 2 A cute In A cu te In norm al norm al 13 128 LG PIN , In LGPIN LGPIN norm al 14 209 Hy Hy, In normal norm al 15 120 LGPIN LGPIN HGPIN norm al 16 202 Hy LGPIN Hy, In In 103 Papillary Hy/atypla H G PIN , Papillary Hy/atypla LGPIN Hy 17 178 Hy Hy Hy norm al 185 LGPIN, Sp LGPIN Hy Hy 190 Hy, In Hy, In, S p LGPIN, In LG PIN , In a, b, c, d 191 Hy LGPIN, In, S p LGPIN norm al 98 H G PIN , In, Sp H G PIN , In, S p HGPIN norm al 18 47 LG PIN , In H G PIN , In LGPIN norm al 88 LG PIN , in, S p H G PIN , In, S p H G PIN , S p LG PIN , Sp 152 Hy Hy Hy norm al 156 Papillary Hy/atypia LGPIN, In S p Hy, Sp 19 82 HG PIN, Papillary Hy/atypia, In, S p H G PIN HG PIN In 163 LGPIN LGPIN LGPIN norm al 157 H G PIN , Papillary Hy/atypla HG PIN normal norm al 150 LGPIN LGPIN, S p HG PIN, In, S p norm al 151 LGPIN, Sp HG PIN HGPIN norm al 20 90 A cute In HG PIN normal norm al 114 Hy, In, S p HG PIN HG PIN, S p LGPIN 135 HG PIN HG PIN HGPIN LGPIN 147 LGPIN H G PIN , In HGPIN norm al 99 LGPIN HG PIN HG PIN LGPIN 97 LGPIN HG PIN HG PIN norm al 21 101 Papillary Hy/atypla LGPIN, In LGPIN Hy, S p 113 LGPIN, In, Sp Papillary Hy/atypia, Sp LGPIN, In, S p normal 105 HG PIN HG PIN HGPIN norm al 108 Papillary Hy/atypia, S p Papillary Hy/atypia, S p Papillary Hy/atypla normal 119 LG PIN , In LG PIN , In In, S p H G PIN , In, S p 22 109 Hy Hy, In normal S p 143 Papillary Hy/atypia, In, S p Hy LGPIN norm al 146 Hy Hy norm al norm al 131 LGPIN, In, S p Papillary Hy/atypia, In, S p LGPIN, In, S p Hy a 138 Hy HG PIN , In, S p normal LGPIN 141 Hy In Hy S p 23 130 LGPIN LGPIN Hy In 133 LGPIN, In, S p LGPIN, In, S p Hy, In norm al 126 Hy, In, Sp Hy, In, S p normal norm al 129 LGPIN, in, S p HG PIN Hy normal 2 4 122 H G PIN , In, S p H G PIN , In, S p Hy, In S p Hy, hyperplasia. In, inflammation. Sp, stromal proliferation. Mild inflammation in DD (a), SV (b), epididymis (c). d, lymphocyte infiltration, mostly T cells, In stomach and small intestinal tissues. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 11. Gross examination of prostate tissues. A, One 19-month-old transgenic animal. B, the wild-type littermate control animal. “BL” stands for bladder, “V” stands for ventral prostate and “L” stands for lateral prostate. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. at time points as early as 2- to 3-months of age which was consistent with the results of the gross examination. The lesions were usually multi-focal in that epithelial hyperplasia was identified in many ducts of one lobe (Fig. 12A). Generally, multi focal hyperplasia developed later in dorsal prostate (DP) and AP, with the earliest starting at 6 months and 16 months, respectively. Amid the hyperplastic cells, LP-to- VP metaplasia was noted primarily in LP. As illustrated in Fig. 12B, epithelial cells with the basal nuclei and apical cytoplasm characteristic of the normal VP, were identified in the LP in which epithelial cells normally displayed centrally located nuclei with the surrounding cytoplasm staining pink. The significance of this phenomenon is unknown. Stromal proliferation surrounding the epithelial hyperplasia (Fig. 12A) became prominent in older transgenic animals, generally beginning at around 17 months of age, although in two cases, stromal hypercellularity in VP was noted as early as 6 to 7 months of age (Table 2). Between 5 and 7 months, LGPIN was readily detected in the DP, LP and VP. These lesions were organized in flattened, papillary, cribriform or tufting patterns. The atypical cells were generally larger than adjacent hyperplastic cells. Although the nuclei were larger and hyperchromatic, they were mildly pleomorphic (Fig. 12C, D). Abundant eosinophilic cytoplasm was also readily seen. Occasionally mitotic figures could be identified (Fig. 12C). Beginning at 15 to 17 months, HGPINs appeared in the DP, LP and VP. In these relatively advanced lesions, atypical cells filled or almost filled the lumina (Fig. 13A, B, C). Cellular atypia in HGPIN was 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 12. Illustrations of histopathology of PB-FGF8b transgenic animals (H&E). A, multi-focal hyperplasia (&), stromal proliferation (*) and mild chronic inflammation (arrow) in a VP of a 20-month-old transgenic animal are indicated. Uninvolved areas, marked by “A”, served as internal control. B, LP-to-VP metaplasia in a LP of a 23- month-old transgenic animal. The transition area is indicated by the arrow, where the cuboidal LP epithelial cells with centrally located nuclei appeared as taller, columnar VP-like epithelial cells with the basal nuclei and apical cytoplasm. C, LGPIN in a DP of a 21-month-old transgenic animal. A mitotic figure is indicated by the arrow. A portion of normal DP epithelium, marked by “A”, served as an internal control. D, LGPIN in a LP of a 5-month-old transgenic animal. The lumen containing the LGPIN lesion is indicated by the arrow. Lumina of normal LP epithelium, labeled with “A”, served as the internal control. 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 13. HGPIN in PB-FGF8b transgenic animals. A, HGPIN lesion in a DP of a 16- month-old transgenic animal (H&E, X400). A mitotic figure is shown by the arrow. B, HGPIN in a VP of the same 16-month-old transgenic animal (H&E, X400). C, HGPIN in a LP of an 18-month-old transgenic animal (H&E). The bulging duct appeared filled with atypical cells arranged in the cribriform pattern. Areas of thickened stroma (*) and an inflammatory reaction (arrow) are also indicated. Adjacent lumina (A), which appeared normal, served as internal control. D, high power examination of the HGPIN lesion in C (H&E). Highly pleomorphic atypical cells manifested as back-to-back glandular structures. Mitotic figures were indicated by arrows. Reactive inflammation was also prominent (right side of the arrows). E, anti-SMA immunostaining of the HGPIN lesion in C. A portion of the fibromuscular sheath of the affected lumen, pointed by the arrow, was missing in contrast to the intact lining seen surrounding the adjacent normal glands. F, presence of androgen receptors in these atypical cells of C is demonstrated by the anti-AR immunostaining. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. much more pronounced as compared with LGPIN. This was characterized by an increased nuclear to cytoplasmic ratio, marked nuclear atypia, hyperchromasia and prominent nucleoli. Mitotic figures were more common in HGPIN than LGPIN (Fig. 13A, D). As shown in Fig. 13C, a bulging duct filled with atypical cells arranged in cribriform pattern stood out from the adjacent abnormal glands. Apoptotic bodies and an inflammatory reaction were noted (Fig. 13D). Although the epithelium was still surrounded by a thin layer of laminin focally, a part of fibromuscular sheath appeared disrupted as revealed by anti-SMA immunostaining (Fig. 13E). Androgen receptor expression appeared to be intact in epithelial cells of HGPIN (Fig. 13F). Papillary hyperplasia, a frequent phenotype in PB-FGF8b animals, was found around 16 months of age. It was manifested as multiple nodules bulging into and filling the lumina with a stalk connected to the basement membrane (Fig. 14A). While VP, LP and DP of some, but not all animals exhibited papillary hyperplasia, these lesions were not observed in AP. Microscopically, the individual lesion presented as an exophytic papillary proliferation of epithelial cells overlying a solid fibromuscular core in which some blood vessels were readily visible (Fig. 14A, B). Atypical epithelial cells with different degree of cytological abnormalities, as described above, were evident. These nodules, shown in Fig. 14C, when examined for BrdU immunostaining, displayed a much higher proliferation index as compared to that of the tissue from age matched control animals (Fig. 14D). 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Fig. 14. Histopathology of PB-FGF8b transgenic animals. A, papillary hyperplasia in a LP of a 23-month-old transgenic animal (H&E). B, stromal cells occupied the core of papillary hyperplasia shown in A. Highly pleomorphic stromal cells were present with occasional mitotic figures, one indicated by the arrow. C, increased rate of cell proliferation in papillary hyperplasia, as assessed by BrdU immunostaining, is illustrated by a section of LP from a 16-month-old transgenic animal as compared to its littermate control (D). Proliferation, which was pronounced in the epithelium, was also involved in the stroma, as indicated by arrows. E, inflammation in AP of a 23- month-old transgenic animal is indicated by the arrow (H&E, X200). F, anti-CD3 immunostaining of the lymphocytes in E (X200). G, anti-CD45R/B22Q immunohistochemistry of the lymphocytes in E (X200). H, anti-SMA immunostaining of the stromal cells in a section adjacent to B. Areas of immunoreactive cells are labeled by arrows. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. In general, as summarized in Table 2 and Fig. 15, between 2 to 14 months, 100% (17/17) of line 3 transgenic animals developed multi-focal hyperplasia in, at least, one lobe of the prostate, and 35% (6/17) LGPIN. No papillary hyperplasia and HGPIN were detected in animals up to the age of 14 months. After 15 months, 100% (39/39) transgenic animals continued to display hyperplasia in increased number of lobes, 23% (9/39) developed papillary hyperplasia with atypia, the incidence of LGPIN increased to 66% (26/39) and 51% (20/39) developed HGPIN. Similar lesions were also identified in the transgenic animals of other lines (Table 3). Throughout the investigation period, none of the nontransgenic control animals developed PIN lesions while a mild hyperplasia was noted in about 20% aging controls. Overexpression of FGF8b in the epithelial cells also led to stromal hypercellularity and increased inflammation in prostate. In line 3, chronic inflammation was frequently noticed starting at 5 months and became more common in older animals (Table 2). Generally the inflammation was found in the LP and VP, although AP and DP were also involved but to a lesser extent. Similar changes were also present in animals of line 1, occasionally with a higher intensity when compared to animals of other lines (Table 3). Although the earliest time points of their appearances were similar, a direct correlation between the inflammation and PIN lesions could not be made, as there were specimens with PINs which lacked overt evidence of inflammation and vice versa. Microscopically, clusters of lymphocytes 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0 O c 0 ■O BOTH) o c 10(h 90 80 70 60 50 40 30 20 10 0 ^ * * r « : 15-24 months 2 to 14 months Age Fig. 15. Temporal incidence of prostatic phenotypic lesions in PB-FGF8b transgenic animals, line 3. Between 2 to 14 months, 100% (17/17) of line 3 transgenic animals developed multi-focal hyperplasia in one or more lobes of the prostate, and 35% (6/17) LGPIN. No papillary hyperplasia and HGPIN were detected in animals up to the age of 14 months. After 15 months, 100% (39/39) transgenic animals developed hyperplasia encompassing more lobes, 23% (9/39) papillary hyperplasia with atypia, 66% (26/39) LGPIN and 51% (20/39) HGPIN. 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3 Pathology of other PB-FGF8b lines Line Age Animal Histology of th e P ro sta te O ther No. (m onths) Ho,________________ V P ________ LP _____________ DP AP_________notable findings 4 ' 33 normal norm al norma! norm al 6 16 normal norm al normal norm al 9 3 Hy norm al norm al norm al 10 45 LG PIN , S p norm al norm al Hy 12 2 normal Hy normal norm al 13 18 Hy norm al norm al normal 14 59 In LG PIN , In Hy Hy 16 76 e e e e 17 118 Hy Hy, In In norm al 121 LGPIN, S p Hy, In e normal 18 47 ln,e a e e 29 LGPIN, In H G PIN , In In In 19 37 Hy, In LG PIN , In e In 56 In In In In 20 53 LGPIN, In, S p Papillary Hy/atypia e In, S p 5 4 In, S p Hy, In norm al normal 44 LG PIN , In, S p LG PIN , S p norm al normal 5 5 Hy, In norm al norm al S p 60 Hy LG PIN , S p Hy Hy 21 7 4 In, S p Hy, In, S p S p In 62 LGPIN Hy, In norm al normal 22 9 2 Papillary Hy/alypla, In, S p LG PIN , In, S p e In, Sp 87 Hy Hy, S p S p Hy, In 9 5 Hy, In, Sp LG PIN . In In S p 91 In, S p LG PIN , in, S p In, S p In, Sp 23 7 2 Hy, In, Sp In In, Sp In, Sp 75 In, S p, e In, S p .e In, S p , e In , S p , e 7 8 LGPIN, In, S p Papillary Hy/atypia, In , S p In, S p In, S p 79 In, Sp, e In, Sp, e In, S p , e In, Sp 24 83 Hy, In, S p In, S p In In 14 13 H G PIN , S p H G PIN , S p H G PIN normal 16 16 In H G PIN , S p LGPIN Hy 20 # i a Papillary Hy/atypia, In, S p Hy, S p In, Sp In, S p 14 5 Hy, S p LGPIN H G PIN , In In 17 8 Hy Hy, In Hy Hy 18 10 LGPIN, In Hy, S p S p norm al 7 #A Hy Hy norm al normal Hy, hyperplasia. In, inflammation. Sp, stromal proliferation. Mild inflammation in DD (a), SV (b), epididymis (c). d, lymphocyte infiltration, mostly T cell, in stomach and small intestinal tissues, e, dilated glands. 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were sparsely located in the stroma (Fig. 12, 13, 14E). While in close continuity with the ducts, these lymphocytes did not seem to invade the epithelial cells in most animals. Immunohistochemically, most of these lymphocytes were CD3 positive (Fig. 14F), while a small percentage was of B cell lineage as determined by immunoreactivity to an anti-CD45R/B220 antibody (Fig. 14G). In rare cases (2 of 56 animals in line 3), a mixed acute and chronic inflammation was found where the glandular profile was severely disrupted. In one animal of line 3 and one of line 1 (Tables 2 and 3), the CD3-positive inflammatory cells appeared in a pattern suggestive of lymphoma within the prostate tissues, a matter which remained to be further investigated. In these animals, the inflammatory infiltration was also found in some extraprostatic tissues, such as small intestine and stomach. The stromal proliferation, which was relatively more prominent in the AP than other lobes, was another remarkable change in PB-FGF8b transgenic animals. It was characterized by significantly thick stroma with hypercellularity (12A, 14A, B). Smooth muscle cells were identified by immunohistochemistry as the major component of the stroma (Fig. 14H). These stromal cells also exhibited a high proliferation index (Fig. 14C). In some animals, proliferation of stromal cells apparently led to stromal papilloma (Fig. 14A, B), which manifested the phyllodes-like pattern. These cells, having increased nuclear-cytoplasmic ratio, displayed pleomorphic, hyperchromatic nuclei with prominent nucleoli. A few scattered mitotic figures could also be noted (Fig. 14B). 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The mild chronic inflammation was also found in the stroma o f the DD, SV and epididymis in some of the transgenic animals. Interestingly, these were also the non prostatic tissues that manifested a low level of transgene expression as detected by RT-PCR assay. However, these abnormalities did not affect the fertility of the transgenic animals since all established lines could be successfully maintained. Chronic inflammation, much less severe than those identified in transgenics, was also noted in about 10% of old control animals. However, none of those control animals exhibited significant stromal hypercellularity. DISCUSSION While there is ample evidence for the up-regulation of FGF8 gene expression in human prostatic premalignant and malignant lesions (21, 22, 70, 93), until now it was not known whether this observation is unique to the human disease or more general in nature in terms of prostate carcinogenesis. Here, we demonstrate that mouse fgf& transcription is also elevated in PIN lesions of SV40 Tag-driven mouse models (29). Similar to the observations in human prostate cancers, we find that fg f8, isoform b mRNA, whose amino acid coding sequence is 100% identical between the human and mouse species (20), is specifically up-regulated in mouse PINs, with fast growing lesions exhibiting much higher level of expression relative to the slow 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. growing lesions. Thus, it was considered logical to develop mouse models in which the /g/8b is targeted for overexpression in the prostatic epithelium. We describe four productive lines of transgenic mice, in each of which the expression of FGF8b transgene is under the control of the androgen-regulated, prostate epithelium-specific ARR2PB promoter (87). These independent lines display a similar sequence of development of phenotypic changes in prostatic tissues. In general, the results point to an increased expression of FGF8b which is sufficient to drive proliferation in the prostatic epithelium preceding the development of histopathologically identifiable lesions, many of which resemble human preneoplastic prostatic lesions. A stochastic pattern of disease progression in these transgenic mice is noteworthy (Fig. 15). The prostatic abnormalities, beginning with multi-focal epithelial hyperplasia, are followed by appearance of LGPIN and subsequently, HGPIN lesions. If all the prostatic pathology is combined, irrespective of whether one of more lobes are involved, 100% of the transgenic animals manifest prostatic hyperplasia. While this high incidence of hyperplasia is followed by the development of LGPIN at a rate of 35% within the first 14 months, no HGPINs could be detected up to this time point. However, during further aging (15 to 24 months), as the incidence of LGPIN increases from 35% to 66%, there is also the first appearance of HGPIN at a remarkable high frequency of 51%. Thus, the overexpression of FGF8b appears to be a distinct initiating event in the development of hyperplasia which, in turn, is perhaps conducive to the manifestation of other 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. genetic lesions, that may represent the rate-limiting factors responsible for a potentially temporal progression from hyperplastic changes to HGPIN lesions. These properties of the transgenic mice, which are markedly prostate-restricted, also closely reflect the usual slow progression of prostatic disease in humans. While the specific mechanisms by which FGF8b drives tumorigenesis are not known, some of the observations made in vivo with this transgenic model are concordant to those previously reported in other systems. For example, in vitro mitogenic and transforming activity of FGF8b (15, 19, 20, 23) or tumorigenicity in MMTV promoter-driven mammary or ovarian epithelium (23) are consistent with the current findings. Moreover, a delayed but fairly common development of stromal hypercellularity in the prostate of the FGF8b transgenic mice, mimics the in vitro coculture experiments described before (68), implicating an indirect effect of FGF8b signaling in epithelial cells on the stromal cells. Thus, FGF8b is likely to act not only as an initiation factor but also, possibly, as a progression factor. Since transgene expression in our model is found to be continuous throughout the two-year period of life investigated, and considering that FGF8b overexpression in weakly tumorigenic human prostatic tumor LNCaP cells could significantly enhance their tumorigenicity and invasiveness (68), this potential should not be overlooked. However, because the model has yet to yield invasive cancer, and as described above, there are likely to be other rate limiting factors in the progression of the lesions, FGF8b is only proven to be an etiological factor in prostate tumorigenesis in this model. Its role as a 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. progression factor, along with other hitherto unidentified secondary factors, remains to be investigated. In summary, transgenic mice overexpressing FGF8b in prostate epithelial cells are found to develop progressively epithelial hyperplasia, LGPIN and ultimately HGPIN. Interestingly, abnormalities of stroma including hyperplasia and chronic inflammation are also observed in these animals. These findings are biologically significant since FGF8b expression is associated with progression of human prostate cancer, beginning with PIN lesions. The model is likely to be valuable in examining how FGF8b may be involved in influencing the autocrine and paracrine pathways in the prostate tissue. It is also important to note that the potential of the model could be greatly enhanced by seeking genetic synergy between FGF8b transgenics with mutated mice known to reflect other “natural” changes in prostate cancer, such as Nkx 3.1(30, 94), p27 (95), and PTEN (96). With the development of an efficient prostate-epithelium-specific Cre-loxP model (88), it is also now possible to generate animals with conditional alleles of genes whose disruption is otherwise embryonically lethal. One such allele, RXRa retinoid receptor (97, 98), has already been combined in the background of FGF8b overproduction, and there is indication of acceleration of the phenotypic changes in the prostate from this inter-crossing (our unpublished data). 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4 Conclusions and Future Directions Conclusions The biological significance of the studies described in this dissertation is three-fold. First, we explored the multiple effects of FGF8b on the biological properties of the prostate carcinoma cells, LNCaP cells. Our results demonstrate that FGF8b is a strong mitogen that might function through the autocrine as well paracrine pathways. Overexpression of FGF8b can also increase the soft agar clonogenicity and matrigel invasion abilities of LNCaP cells. The in vivo tumorigenesis and diaphragm invasion activity of these cells are greatly enhanced by the condition of FGF8b overexpression. These data may indicate that upregulation of FGF8b contributes to the progression of prostate cancer considering that FGF8b is naturally overexpressed in this malignancy. Second, we examined the effect of FGF8b on the epithelial- stromal interactions in the settings of in vitro coculture described in the chapter 2, and of in vivo prostate tissues of FGF8b transgenic animals described in the chapter 3. In coculture of FGF8b-expressing LNCaP cells and prostate stromal cells, overexpression of FGF8b can not only stimulate the growth of epithelial cells but also increase the proliferation rate of stromal cells. This stimulation is not relevant to the direct mitogenic action of FGF8b on the stromal cells. It seems to require, at least 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in the in vitro reconstructed coculture systems, the intimate epithelial-stromal cell cell contact and some unknown soluble factors released by LNCaP cells upon stimulation of FGF8b. These in vitro findings are further supported by the following observations obtained from the studies of the FGF8b transgenic animals. In addition to the convincing epithelial abnormalities, stromal proliferation is determined to be another prominent phenomenon of these transgenic mice. It is manifested as significantly thickened stroma with hypercellularity, in which smooth muscle cells are identified to be the major component. These stromal cells generally possess a high proliferation index. In some extreme cases, proliferation of stromal cells apparently led to stromal papilloma. Third, we documented that overexpression of FGF8b in the prostatic epithelium is sufficient to lead to the preneoplastic PIN lesions. The development of these epithelial phenotypes follows a stochastic pattern. The starting abnormality, multi-focal hyperplasia, is followed by LGPDM and HGPIN. With aging of the animals, the incidences of these lesions increase dramatically. These properties of the transgenic mice, which are markedly prostate-restricted, closely mimic the usual slow progression of the prostatic disease in humans. They represent a novel animal model to study the mechanisms of development and progression of prostatic diseases, such as, prostatic hyperplasia and preneoplastic lesions. In addition, these transgenic mice also provide a unique animal model to investigate mechanisms of FGF8b in vivo signaling and unveiling of its potential downstream effects. 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Future Directions The utility of the FGF8b transgenic animals developed could be in multiple areas. First, this model provides a useful tool to study the mechanisms of prostate tumorigenesis of FGF8b in vivo. The downstream effectors of FGF8b signaling pathway could be identified with some advanced techniques, such as the microarray assay of mRNA extracted from the prostate tissues of these transgenic mice. The detection of these downstream signaling pathways would help us understand more about the various genes involved in the development of this malignancy and their interactions. The knowledge gained would also provide some meaningful clues for uncovering the mediators that carry the cross-talk between the epithelial and stromal cells. Second, these animals could be used to study the effect of androgen on the development of PIN lesions and its mechanisms. One approach for this study is to castrate the transgenic animals at different ages and follow the effect on the development of the phenotypic changes. Cellular components and their mRNA specimens of prostate tissues of these animals will be isolated and used for investigating the differential gene expressions that would show the resultant changes following androgen ablation. These observations are essential to understand the biological function of androgen in the development of prostate preneoplastic lesions. They could also provide some information about the functional role of FGF8b in the development and progression of PIN lesions recognizing the fact that expression of FGF8b is under the regulation of androgen supply. Third, this novel animal model 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. could be used to study the effects of some potential therapeutic interventions. Some of them, such as antisense L-plastin (72, 99), Endostatin (100, 101) (102, 103) and P2O2 gene (M-C, Hung, personal communication), are already being planned. With the idea to derive an accelerated transgenic mouse model for prostate tumorigenesis, intercrossed models, in which overexpression of FGF8b is combined with prostate-specific knockout of a negative regulator of prostate or a tumor suppressor gene that is known to be involved in the human disease, could be attempted. Taking advantage of the development of an efficient prostate-epithelium- specific Cre-loxP model (88), we have already developed a bitransgenic mouse line in which knock of RXRa retinoid receptor (97, 98) is combined in the background of FGF8b overproduction. There are some indications of acceleration of the phenotypic changes in the prostate from this inter-crossing (our unpublished data). Since disruption of PTEN (96) is embryonically lethal, prostate-specific knockout of this strong tumor suppressor gene using this Cre-loxP animal (88) becomes necessary in term of studying its function in prostate tumorigenesis. Subsequently the combination of this knockout with FGF8b overexpression will perhaps produce an enhanced phenotype. Meantime, one bitransgenic mouse line, the combination of FGF8b overexpression with Nkx 3.1(30, 94) has been established. Currently the characterization of its phenotype is being undertaken. Other models, such as combination of FGF8b transgenic with tissue-selective knockout of p27 (95) or other 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. negative cell cycle regulators, known to be affected in human prostate cancers, are being contemplated in collaboration with other laboratories. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1 . McKeehan, W. L., Wang, F., and Kan, M. The heparan sulfate-fibroblast growth factor family: diversity of structure and function, Progress in Nucleic Acid Research & Molecular Biology. 59: 135-76, 1998. 2. Szebenyi, G. and Fallon, J. F. Fibroblast growth factors as multifunctional signaling factors, International Review of Cytology. 185: 45-106, 1999. 3. Tanaka, A., Miyamoto, K., Minamino, N., Takeda, M., Sato, B., Matsuo, H., and Matsumoto, K. Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells, Proceedings of the National Academy of Sciences of the United States of America. 89: 8928-32, 1992. 4. Heikinheimo, M., Lawshe, A., Shackleford, G. M., Wilson, D. B., and MacArthur, C. A. Fgf-8 expression in the post-gastrulation mouse suggests roles in the development of the face, limbs and central nervous system, Mechanisms of Development. 48: 129-38, 1994. 5. Ohuchi, H., Yoshioka, H., Tanaka, A., Kawakami, Y., Nohno, T., and Noji, S. Involvement of androgen-induced growth factor (FGF-8) gene in mouse embryogenesis and morphogenesis, Biochemical & Biophysical Research Communications. 204: 882-8, 1994. 6. Crossley, P. H. and Martin, G. R. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo, Development. 121: 439-51, 1995. 7. Lorenzi, M. V., Long, J. E., Miki, T., and Aaronson, S. A. Expression cloning, developmental expression and chromosomal localization of fibroblast growth factor-8, Oncogene. 10: 2051-5, 1995. 8. Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Colquhoun, K., Martin, P., Lumsden, A., Dickson, C., and Mason, I. A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth, Current Biology. 5: 797-806, 1995. 9. Crossley, P. H., Minowada, G., MacArthur, C. A., and Martin, G. R. Roles for FGF8 in the induction, initiation, and maintenance of chick limb development, Cell. 84: 127-36, 1996. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10. Crossley, P. H., Martinez, S., and Martin, G. R. Midbrain development induced by FGF8 in the chick embryo, Nature. 380: 66-8, 1996. 11. Meyers, E. N., Lewandoski, M., and Martin, G. R. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination, Nature Genetics. 18: 136-41, 1998. 12. Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M., and Martin, G. R. Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch, Genes & Development. 13: 3136-48, 1999. 13. White, R. A., Dowler, L. L., Angeloni, S. V., Pasztor, L. M., and MacArthur, C. A. Assignment of FGF8 to human chromosome 10q25-q26: mutations in FGF8 may be responsible for some types of acrocephalosyndactyly linked to this region, Genomics. 30: 109-11, 1995. 14. Payson, R. A., Wu, J., Liu, Y., and Chiu, I. M. The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells, Oncogene. 13: 47-53, 1996. 15. MacArthur, C. A., Lawshe, A., Shankar, D. B., Heikinheimo, M., and Shackleford, G. M. FGF-8 isoforms differ in NIH3T3 cell transforming potential, Cell Growth & Differentiation. 6: 817-25, 1995. 16. MacArthur, C. A., Shankar, D. B., and Shackleford, G. M. Fgf-8, activated by proviral insertion, cooperates with the Wnt-1 transgene in murine mammary tumorigenesis, Journal of Virology. 69: 2501-7, 1995. 17. Tanaka, A., Miyamoto, K., Matsuo, H., Matsumoto, K., and Yoshida, H. Human androgen-induced growth factor in prostate and breast cancer cells: its molecular cloning and growth properties, FEBS Letters. 363: 226-30, 1995. 18. Gemel, J., Gorry, M., Ehrlich, G. D., and MacArthur, C. A. Structure and sequence of human FGF8, Genomics. 35: 253-7, 1996. 19. Kouhara, H., Koga, M., Kasayama, S., Tanaka, A., Kishimoto, T., and Sato, B. Transforming activity of a newly cloned androgen-induced growth factor, Oncogene. 9: 455-62, 1994. 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20. Ghosh, A. K., Shankar, D. B„ Shackleford, G. M., Wu, K„ A, T. A„ Miller, G. J., Zheng, J., and Roy-Burman, P. Molecular cloning and characterization of human FGF8 alternative messenger RNA forms, Cell Growth & Differentiation. 7: 1425-34, 1996. 21. Tanaka, A., Furuya, A., Yamasaki, M., Hanai, N., Kuriki, K., Kamiakito, T., Kobayashi, Y., Yoshida, H., Koike, M., and Fukayama, M. High frequency of fibroblast growth factor (FGF) 8 expression in clinical prostate cancers and breast tissues, immunohistochemically demonstrated by a newly established neutralizing monoclonal antibody against FGF 8, Cancer Research. 58: 2053- 6, 1998. 22. Dorkin, T. J., Robinson, M. C., Marsh, C., Bjartell, A., Neal, D. E., and Leung, H. Y. FGF8 over-expression in prostate cancer is associated with decreased patient survival and persists in androgen independent disease, Oncogene. 18: 2755-61, 1999. 23. Daphna-Iken, D., Shankar, D. B., Lawshe, A., Ornitz, D. M., Shackleford, G. M., and MacArthur, C. A. MMTV-FgfB transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia, Oncogene. 17: 2711-7, 1998. 24. Rudra-Ganguly, N., Zheng, J., Hoang, A. T., and Roy-Burman, P. Downregulation of human FGF8 activity by antisense constructs in murine fibroblastic and human prostatic carcinoma cell systems, Oncogene. 16: 1487-92, 1998. 25. McNeal, J. E. and Bostwick, D. G. Intraductal dysplasia: a premalignant lesion of the prostate, Human Pathology. 17: 64-71, 1986. 26. Bostwick, D. G. and Brawer, M. K. Prostatic intra-epithelial neoplasia and early invasion in prostate cancer, Cancer. 59: 788-94, 1987. 27. Bostwick, D. G. Prostatic intraepithelial neoplasia is a risk factor for cancer. [Review] [118 refs], Seminars in Urologic Oncology. 17: 187-98, 1999. 28. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D., Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J., and Rosen, J. M. Prostate cancer in a transgenic mouse, Proceedings of the National Academy of Sciences of the United States of America. 92: 3439-43, 1995. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29. Kasper, S., Sheppard, P. C., Yan, Y., Pettigrew, N., Borowsky, A. D., Prins, G. S., Dodd, J. G., Duckworth, M. L., and Matusik, R. J. Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer, Laboratory Investigation. 78: i-xv, 1998. 30. Bhatia-Gaur, R , Donjacour, A. A., Sciavolino, P. J., Kim, M., Desai, N., Young, P., Norton, C. R., Gridley, T., Cardiff, R. D., Cunha, G. R , Abate- Shen, C., and Shen, M. M. Roles for Nkx3.1 in prostate development and cancer, Genes & Development. 13: 966-77, 1999. 31. DiGiovanni, J., Kiguchi, K., Frijhoff, A., Wilker, E., Bol, D. K., Beltran, L., Moats, S., Ramirez, A., Jorcano, J., and Conti, C. Deregulated expression of insulin-like growth factor 1 in prostate epithelium leads to neoplasia in transgenic mice, Proceedings of the National Academy of Sciences of the United States of America. 97: 3455-60, 2000. 32. Masumori, N., Thomas, T. Z., Chaurand, P., Case, T., Paul, M., Kasper, S., Caprioli, R M., Tsukamoto, T., Shappell, S. B., and Matusik, R. J. A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential, Cancer Research. 61: 2239-49, 2001. 33. Chung, L. W. and Cunha, G. R. Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme, Prostate. 4: 503-11, 1983. 34. Shannon, J. M. and Cunha, G. R. Autoradiographic localization of androgen binding in the developing mouse prostate, Prostate. 4: 367-73, 1983. 35. Takeda, H., Mizuno, T., and Lasnitzki, I. Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate, Journal of Endocrinology. 104: 87-92, 1985. 36. Sugimura, Y., Cunha, G. R., and Bigsby, R. M. Androgenic induction of DNA synthesis in prostatic glands induced in the urothelium of testicular feminized (Tfm/Y) mice, Prostate. 9: 217-25, 1986. 37. Cunha, G. R., Donjacour, A. A., Cooke, P. S., Mee, S., Bigsby, R. M., Higgins, S. J., and Sugimura, Y. The endocrinology and developmental biology of the prostate. [Review] [291 refs], Endocrine Reviews. 8: 338-62, 1987. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38. Takeda, H., Suematsu, N., and Mizuno, T. Transcription of prostatic steroid binding protein (PSBP) gene is induced by epithelial-mesenchymal interaction, Development - Supplement. 110: 273-81, 1990. 39. Takeda, H. and Chang, C. Immunohistochemical and in-situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland, Journal of Endocrinology. 129: 83-9, 1991. 40. Husmann, D. A , McPhaul, M. J., and Wilson, J. D. Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis, Endocrinology. 128: 1902-6, 1991. 41. Hayashi, N., Cunha, G. R., and Parker, M. Permissive and instructive induction of adult rodent prostatic epithelium by heterotypic urogenital sinus mesenchyme, Epithelial Cell Biology. 2: 66-78, 1993. 42. Cunha, G. R. Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate, Cancer. 74: 1030-44, 1994. 43. Cunha, G. R., Hayward, S. W., Dahiya, R., and Foster, B. A. Smooth muscle- epithelial interactions in normal and neoplastic prostatic development. [Review] [57 refs], Acta Anatomica. 155: 63-72, 1996. 44. Aaronson, S. A., Bottaro, D. P., Miki, T., Ron, D., Finch, P. W., Fleming, T. P., Ahn, J., Taylor, W. G., and Rubin, J. S. Keratinocyte growth factor. A fibroblast growth factor family member with unusual target cell specificity, Annals of the New York Academy of Sciences. 638: 62-77, 1991. 45. Nilsen-Hamilton, M. Transforming growth factor-beta and its actions on cellular growth and differentiation, Current Topics in Developmental Biology. 24: 95-136, 1990. 46. Partanen, A. M. Epidermal growth factor and transforming growth factor- alpha in the development of epithelial-mesenchymal organs of the mouse, Current Topics in Developmental Biology. 24: 31-55, 1990. 47. Hayward, S. W., Cunha, G. R., and Dahiya, R. Normal development and carcinogenesis of the prostate. A unifying hypothesis, Annals of the New York Academy of Sciences. 784: 50-62, 1996. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48. Hayward, S. W., Rosen, M. A., and Cunha, G. R. Stromal-epithelial interactions in the normal and neoplastic prostate, British Journal of Urology. 79: 18-26, 1997. 49. Thompson, T. C., Southgate, J., Kitchener, G., and Land, H. Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ, Cell. 56: 917-30, 1989. 50. Thompson, T. C. Growth factors and oncogenes in prostate cancer. [Review] [103 refs], Cancer Cells. 2: 345-54, 1990. 51. Camps, J. L., Chang, S. M., Hsu, T. C., Freeman, M. R., Hong, S. J., Zhau, H. E., von Eschenbach, A. C., and Chung, L. W. Fibroblast-mediated acceleration of human epithelial tumor growth in vivo, Proceedings of the National Academy of Sciences of the United States of America. 87: 75-9, 1990. 52. Chung, L. W., Li, W., Gleave, M. E., Hsieh, J. T„ Wu, H. C., Sikes, R. A., Zhau, H. E., Bandyk, M. G., Logothetis, C. J., Rubin, J. S., and et al. Human prostate cancer model: roles of growth factors and extracellular matrices, Journal of Cellular Biochemistry - Supplement. 16H: 99-105, 1992. 53. Thompson, T. C., Truong, L. D., Timme, T. L., Kadmon, D., McCune, B. K., Flanders, K. C., Scardino, P. T., and Park, S. H. Transgenic models for the study of prostate cancer. [Review] [45 refs], Cancer. 71: 1165-71, 1993. 54. Hayward, S. W., Dahiya, R., Cunha, G. R., Bartek, J., Deshpande, N., and Narayan, P. Establishment and characterization of an immortalized but non transformed human prostate epithelial cell line: BPH-1, In Vitro Cellular & Developmental Biology. Animal. 31: 14-24, 1995. 55. Chung, L. W. The role of stromal-epithelial interaction in normal and malignant growth. [Review] [29 refs], Cancer Surveys. 23: 33-42, 1995. 56. Rowley, D. R. What might a stromal response mean to prostate cancer progression?, Cancer & Metastasis Reviews. 17: 411-9, 1998. 57. Imagawa, W., Bandyopadhyay, G. K., and Nandi, S. Regulation of mammary epithelial cell growth in mice and rats, Endocrine Reviews. 11: 494-523, 1990. 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58. Story, M. T. Polypeptide modulators of prostatic growth and development, Cancer Surveys. 11: 123-46, 1991. 59. McKeehan, W. L. Growth factor receptors and prostate cell growth, Cancer Surveys. 11: 165-75, 1991. 60. Thompson, T. C. Growth factors and oncogenes in prostate cancer, Cancer Cells. 2: 345-54, 1990. 61. Boyer, B., Valles, A. M., and Thiery, J. P. Model systems of epithelium- mesenchyme transitions, Acta Anatomica. 156: 227-39, 1996. 62. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C,, Hittmair, A., Zhang, J., Thumher, M., Bartsch, G., and Klocker, H. Regulation of prostatic growth and function by peptide growth factors, Prostate. 28: 392-405, 1996. 63. Thomson, A. A., Foster, B. A., and Cunha, G. R. Analysis of growth factor and receptor mRN A levels during development of the rat seminal vesicle and prostate, Development. 124: 2431-9, 1997. 64. Rubin, J. S., Bottaro, D. P., Chedid, M., Miki, T., Ron, D., Cheon, G., Taylor, W. G., Fortney, E., Sakata, H., Finch, P. W., and et al. Keratinocyte growth factor, Cell Biology International. 19: 399-411, 1995. 65. Yan, G., Fukabori, Y., McBride, G., Nikolaropolous, S., and McKeehan, W. L. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy, Molecular & Cellular Biology. 13: 4513-22, 1993. 66. Yan, G., Fukabori, Y., Nikolaropoulos, S., Wang, F., and McKeehan, W. L. Heparin-binding keratinocyte growth factor is a candidate stromal-to- epithelial-cell andromedin, Molecular Endocrinology. 6: 2123-8, 1992. 67. MacArthur, C. A., Lawshe, A., Xu, J., Santos-Ocampo, S., Heikinheimo, M., Chellaiah, A. T., and Ornitz, D. M. FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development, Development. 121: 3603-13, 1995. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 68. Song, Z., Powell, W. C., Kasahara, N., van Bokhoven, A., Miller, G. J., and Roy-Burman, P. The effect of fibroblast growth factor 8, isoform b, on the biology of prostate carcinoma cells and their interaction with stromal cells, Cancer Research. 60: 6730-6, 2000. 69. Djakiew, D. Dysregulated expression of growth factors and their receptors in the development of prostate cancer. [Review] [153 refs], Prostate. 42: 150-60, 2000. 70. Leung, H. Y., Dickson, C., Robson, C. N., and Neal, D. E. Over-expression of fibroblast growth factor-8 in human prostate cancer, Oncogene. 12: 1833-5, 1996. 71. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector, Science. 272: 263-7, 1996. 72. Zheng, J., Rudra-Ganguly, N., Powell, W. C., and Roy-Burman, P. Suppression of prostate carcinoma cell invasion by expression of antisense L- plastingene, American Journal of Pathology. 155: 115-22, 1999. 73. Powell, W. C., Knox, J. D., Navre, M., Grogan, T. M., Kittelson, J., Nagle, R. B., and Bowden, G. T. Expression of the metalloproteinase matrilysin in DU- 145 cells increases their invasive potential in severe combined immunodeficient mice, Cancer Research. 53: 417-22, 1993. 74. Blunt, A. G., Lawshe, A., Cunningham, M. L., Seto, M. L., Ornitz, D. M., and MacArthur, C. A. Overlapping expression and redundant activation of mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands, Journal of Biological Chemistry. 272: 3733-8, 1997. 75. Ittman, M. and Mansukhani, A. Expression of fibroblast growth factors (FGFs) and FGF receptors in human prostate, Journal of Urology. 157: 351-6, 1997. 76. Klein, R. D., Maliner-Jongewaard, M. S., Udayakumar, T. S., Boyd, J. L., Nagle, R. B., and Bowden, G. T. Promatrilysin expression is induced by fibroblast growth factors in the prostatic carcinoma cell line LNCaP but not in normal primary prostate epithelial cells, Prostate. 41: 215-23, 1999. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 77. Hughes, S. E. Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues, Journal of Histochemistry & Cytochemistry. 45: 1005-19, 1997. 78. Basset, P., Bellocq, J. P., Wolf, C., Stoll, I., Hutin, P., Limacher, J. M., Podhajcer, 0. L., Chenard, M. P., Rio, M. C., and Chambon, P. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas, Nature. 348: 699-704, 1990. 79. Newell, K. J., Witty, J. P., Rodgers, W. H., and Matrisian, L. M. Expression and localization of matrix-degrading metalloproteinases during colorectal tumorigenesis, Molecular Carcinogenesis. 10: 199-206, 1994. 80. McDonnell, S., Navre, M., Coffey, R. J., Jr., and Matrisian, L. M. Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas, Molecular Carcinogenesis. 4: 527-33, 1991. 81. Karelina, T. V., Goldberg, G. I., and Eisen, A. Z. Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia, Journal of Investigative Dermatology. 103: 482-7, 1994. 82. Pajouh, M. S., Nagle, R. B., Breathnach, R , Finch, J. S., Brawer, M. K., and Bowden, G. T. Expression of metalloproteinase genes in human prostate cancer, Journal of Cancer Research & Clinical Oncology. 117: 144-50, 1991. 83. Miyagi, N., Kato, S., Terasaki, M., Shigemori, M., and Morimatsu, M. Fibroblast growth factor-2 and -9 regulate proliferation and production of matrix metalloproteinases in human gliomas, International Journal of Oncology. 12: 1085-90, 1998. 84. Miyake, H., Yoshimura, K., Hara, I., Eto, H., Arakawa, S., and Kamidono, S. Basic fibroblast growth factor regulates matrix metalloproteinases production and in vitro invasiveness in human bladder cancer cell lines, Journal of Urology. 157: 2351-5, 1997. 85. Kurogi, T., Nabeshima, K., Kataoka, H., Okada, Y., and Koono, M. Stimulation of gelatinase B and tissue inhibitors of metalloproteinase (TEMP) production in co-culture of human osteosarcoma cells and human fibroblasts: gelatinase B production was stimulated via up-regulation of fibroblast growth factor (FGF) receptor, International Journal of Cancer. 66: 82-90, 1996. 77 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 86. Uria, J. A., Balbin, M., Lopez, J. M., Alvarez, J., Vizoso, F., Takigawa, M., and Lopez-Otin, C. Collagenase-3 (MMP-13) expression in chondrosarcoma cells and its regulation by basic fibroblast growth factor, American Journal of Pathology. 153: 91-101, 1998. 87. Zhang, J., Thomas, T. Z., Kasper, S., and Matusik, R. J. A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo, Endocrinology. 141: 4698-710, 2000. 88. Wu, X., Wu, J., Huang, J., Powell, W. C., Zhang, J., Matusik, R. J., Sangiorgi, F. O., Maxson, R. E., Sucov, H. M., and Roy-Burman, P. Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue- specific gene ablation, Mechanisms of Development. 101: 61-9, 2001. 89. Nieto, M. A., Patel, K., and Wilkinson, D. G. In situ hybridization analysis of chick embryos in whole mount and tissue sections. [Review] [9 refs], Methods in Cell Biology. 51: 219-35, 1996. 90. Ting-Xin Jiang, N. S. S., Randall B. Widelitz and Cheng-Ming Chuong Current Methods in the Study of Avian Skin Appendages. In: C.-M. Chuong (ed.) Molecular Basis of Epithelial Appendage Morphogenesis, pp. 395-408: R. G. Landes Company, 1998. 91. Shibata, M. A., Ward, J. M., Devor, D. E., Liu, M. L., and Green, J. E. Progression of prostatic intraepithelial neoplasia to invasive carcinoma in C3(1)/SV40 large T antigen transgenic mice: histopathological and molecular biological alterations, Cancer Research. 56: 4894-903, 1996. 92. Gingrich, J. R., Barrios, R. J., Kattan, M. W., Nahm, H. S., Finegold, M. J., and Greenberg, N. M. Androgen-independent prostate cancer progression in the TRAMP model, Cancer Research. 57: 4687-91, 1997. 93. Valve, E. M., Nevalainen, M. T., Nurmi, M. J., Laato, M. K., Martikainen, P. M., and Harkonen, P. L. Increased expression of FGF-8 isoforms and FGF receptors in human premalignant prostatic intraepithelial neoplasia lesions and prostate cancer, Laboratory Investigation. 81: 815-26, 2001. 94. Tanaka, M., Komuro, I., Inagaki, H., Jenkins, N. A., Copeland, N. G., and Izumo, S. Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands, Developmental Dynamics. 219: 248-60, 2000. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 95. Di Cristofano, A., De Acetis, M., Koff, A., Cordon-Cardo, C., and Pandolfi, P. P. Pten and p27KIPl cooperate in prostate cancer tumor suppression in the mouse, [see comments], Nature Genetics. 27: 222-4, 2001. 96. Podsypanina, K., Ellenson, L. H., Nemes, A., Gu, J., Tamura, M., Yamada, K. M., Cordon-Cardo, C., Catoretti, G., Fisher, P. E., and Parsons, R. Mutation of Pten/Mmacl in mice causes neoplasia in multiple organ systems, Proceedings of the National Academy of Sciences of the United States of America. 96: 1563-8, 1999. 97. Lohnes, D., Kastner, P., Dierich, A., Mark, M., LeMeur, M., and Chambon, P. Function of retinoic acid receptor gamma in the mouse, Cell. 73: 643-58, 1993. 98. Krezel, W., Dupe, V., Mark, M., Dierich, A., Kastner, P., and Chambon, P. RXR gamma null mice are apparently normal and compound RXR alpha +/- /RXR beta -/-/RXR gamma -/- mutant mice are viable, Proceedings of the National Academy of Sciences of the United States of America. 93: 9010-4, 1996. 99. Zheng, J., Rudra-Ganguly, N., Miller, G. J., Moffatt, K. A., Cote, R. J., and Roy-Burman, P. Steroid hormone induction and expression patterns of L- plastin in normal and carcinomatous prostate tissues, American Journal of Pathology. 150: 2009-18, 1997. 100. Zogakis, T. G. and Libutti, S. K. General aspects of anti-angiogenesis and cancer therapy. [Review] [210 refs], Expert Opinion on Biological Therapy. 1: 253-75, 2001. 101. Sim, B. K., MacDonald, N. J., and Gubish, E. R. Angiostatin and endostatin: endogenous inhibitors of tumor growth. [Review] [68 refs], Cancer & Metastasis Reviews. 19: 181-90, 2000. 102. Cirri, L., Donnini, S., Morbidelli, L., Chiarugi, P., Ziche, M., and Ledda, F. Endostatin: a promising drug for antiangiogenic therapy. [Review] [33 refs], International Journal of Biological Markers. 14: 263-7, 1999. 103. Cao, Y. Endogenous angiogenesis inhibitors: angiostatin, endostatin, and other proteolytic fragments. [Review] [67 refs], Progress in Molecular & Subcellular Biology. 20: 161-76, 1998. 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLIOGRAPHY Aaronson, S. A., D. P. Bottaro, T. Miki, D. Ron, P. W. Finch, T. P. Fleming, J. Ahn, W. G. Taylor, and J. S. Rubin 1991. Keratinocyte growth factor. A fibroblast growth factor family member with unusual target cell specificity Armais of the New York Academy of Sciences. 638:62-77. Basset, P., J. P. Bellocq, C. Wolf, I. Stoll, P. Flutin, J. M. Limacher, 0. L. Podhajcer, M. P. Chenard, M. C. Rio, and P. Chambon 1990. A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas Nature. 348:699-704. Bhatia-Gaur, R., A. A. Donjacour, P. J. Sciavolino, M. Kim, N. Desai, P. Young, C. R. Norton, T. Gridley, R. D. Cardiff, G. R. Cunha, C. Abate-Shen, and M. M. Shen 1999. Roles for Nkx3.1 in prostate development and cancer Genes & Development. 13:966-77. Blunt, A. G., A. Lawshe, M. L. Cunningham, M. L. Seto, D. M. Omitz, and C. A. MacArthur 1997. Overlapping expression and redundant activation of mesenchymal fibroblast growth factor (FGF) receptors by alternatively spliced FGF-8 ligands Journal of Biological Chemistry. 272:3733-8. Bostwick, D. G. 1999. Prostatic intraepithelial neoplasia is a risk factor for cancer. [Review] [118 refs] Seminars in Urologic Oncology. 17:187-98. Bostwick, D. G., and M. K. Brawer 1987. Prostatic intra-epithelial neoplasia and early invasion in prostate cancer Cancer. 59:788-94. Boyer, B., A. M. Valles, and J. P. Thiery 1996. Model systems of epithelium- mesenchyme transitions Acta Anatomica. 156:227-39. Camps, J. L., S. M. Chang, T. C. Hsu, M. R. Freeman, S. J. Hong, H. E. Zhau, A. C. von Eschenbach, and L. W. Chung 1990. Fibroblast-mediated acceleration of human epithelial tumor growth in vivo Proceedings of the National Academy of Sciences of the United States of America. 87:75-9. Cao, Y. 1998. Endogenous angiogenesis inhibitors: angiostatin, endostatin, and other proteolytic fragments. [Review] [67 refs] Progress in Molecular & Subcellular Biology. 20:161 -76. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chung, L. W. 1995. The role of stromal-epithelial interaction in normal and malignant growth. [Review] [29 refs] Cancer Surveys. 23:33-42. Chung, L. W., and G. R. Cunha 1983. Stromal-epithelial interactions: II. Regulation of prostatic growth by embryonic urogenital sinus mesenchyme Prostate. 4:503-11. Chung, L. W., W. Li, M. E. Gleave, J. T. Hsieh, H. C. Wu, R. A. Sikes, H. E. Zhau, M. G. Bandyk, C. J. Logothetis, J. S. Rubin, and et al. 1992. Human prostate cancer model: roles of growth factors and extracellular matrices Journal of Cellular Biochemistry - Supplement. 16H:99-105. Cirri, L., S. Donnini, L. Morbidelli, P. Chiarugi, M. Ziche, and F. Ledda 1999. Endostatin: a promising drug for antiangiogenic therapy. [Review] [33 refs] International Journal of Biological Markers. 14:263-7. Crossley, P. H., and G. R. Martin 1995. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo Development. 121:439-51. Crossley, P. H., S. Martinez, and G. R. Martin 1996. Midbrain development induced by FGF8 in the chick embryo Nature. 380:66-8. Crossley, P. H., G. Minowada, C. A. MacArthur, and G. R. Martin 1996. Roles for FGF8 in the induction, initiation, and maintenance of chick limb development Cell. 84:127-36. Culig, Z., A. Hobisch, M. V. Cronauer, C. Radmayr, A. Hittmair, J. Zhang, M. Thumher, G. Bartsch, and H. Klocker 1996. Regulation of prostatic growth and function by peptide growth factors Prostate. 28:392-405. Cunha, G. R. 1994. Role of mesenchymal-epithelial interactions in normal and abnormal development of the mammary gland and prostate Cancer. 74:1030-44. Cunha, G. R., A. A. Donjacour, P. S. Cooke, S. Mee, R. M. Bigsby, S. J. Higgins, and Y. Sugimura 1987. The endocrinology and developmental biology of the prostate. [Review] [291 refs] Endocrine Reviews. 8:338-62. Cunha, G. R., S. W. Hayward, R. Dahiya, and B. A. Foster 1996. Smooth muscle-epithelial interactions in normal and neoplastic prostatic development. [Review] [57 refs] Acta Anatomica. 155:63-72. 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Daphna-Iken, D., D. B. Shankar, A. Lawshe, D. M. Ornitz, G. M. Shackleford, and C. A. MacArthur 1998. MMTV-FgfB transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia Oncogene. 17:2711-7. Di Cristofano, A., M. De Acetis, A. Koff, C. Cordon-Cardo, and P. P. Pandolfi 2001. Ren and p27KIPl cooperate in prostate cancer tumor suppression in the mouse, [see comments] Nature Genetics. 27:222-4. DiGiovanni, J., K. Kiguchi, A. FrijhofF, E. Wilker, D. K. Bol, L. Beltran, S. Moats, A. Ramirez, J. Jorcano, and C. Conti 2000. Deregulated expression of insulin-like growth factor 1 in prostate epithelium leads to neoplasia in transgenic mice Proceedings of the National Academy of Sciences of the United States of America. 97:3455-60. Djakiew, D. 2000. Dysregulated expression of growth factors and their receptors in the development of prostate cancer. [Review] [153 refs] Prostate. 42:150-60. Dorkin, T. J., M. C. Robinson, C. Marsh, A. Bjartell, D. E. Neal, and H. Y. Leung 1999. FGF8 over-expression in prostate cancer is associated with decreased patient survival and persists in androgen independent disease Oncogene. 18:2755-61. Gemel, J., M. Gorry, G. D. Ehrlich, and C. A. MacArthur 1996. Structure and sequence of human FGF8 Genomics. 35:253-7. Ghosh, A. K., D. B. Shankar, G. M. Shackleford, K. Wu, T. A. A, G. J. Miller, J. Zheng, and P. Roy-Burman 1996. Molecular cloning and characterization of human FGF8 alternative messenger RNA forms Cell Growth & Differentiation. 7:1425-34. Gingrich, J. R , R. J. Barrios, M. W. Kattan, H. S. Nahm, M. J. Finegold, and N. M. Greenberg 1997. Androgen-independent prostate cancer progression in the TRAMP model Cancer Research. 57:4687-91. Greenberg, N. M., F. DeMayo, M. J. Finegold, D. Medina, W. D. Tilley, J. O. Aspinall, G. R. Cunha, A. A. Donjacour, R. J. Matusik, and J. M. Rosen 1995. Prostate cancer in a transgenic mouse Proceedings of the National Academy of Sciences of the United States of America. 92:3439-43. Hayashi, N., G. R. Cunha, and M. Parker 1993. Permissive and instructive induction of adult rodent prostatic epithelium by heterotypic urogenital sinus mesenchyme Epithelial Cell Biology. 2:66-78. 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hayward, S. W., G. R. Cunha, and R. Dahiya 1996. Normal development and carcinogenesis of the prostate. A unifying hypothesis Annals of the New York Academy of Sciences. 784:50-62. Hayward, S. W., R. Dahiya, G. R. Cunha, J. Bartek, N. Deshpande, and P. Narayan 1995. Establishment and characterization of an immortalized but non transformed human prostate epithelial cell line: BPH-1 In Vitro Cellular & Developmental Biology. Animal. 31:14-24. Hayward, S. W., M. A. Rosen, and G. R. Cunha 1997. Stromal-epithelial interactions in the normal and neoplastic prostate British Journal of Urology. 79:18-26. Heikinheimo, M., A. Lawshe, G. M. Shackleford, D. B. Wilson, and C. A. MacArthur 1994. Fgf-8 expression in the post-gastrulation mouse suggests roles in the development of the face, limbs and central nervous system Mechanisms of Development. 48:129-38. Hughes, S. E. 1997. Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues Journal of Histochemistry & Cytochemistry. 45:1005-19. Husmann, D. A., M. J. McPhaul, and J. D. Wilson 1991. Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis Endocrinology. 128:1902-6. Imagawa, W., G. K. Bandyopadhyay, and S. Nandi 1990. Regulation of mammary epithelial cell growth in mice and rats Endocrine Reviews. 11:494-523. Ittman, M., and A. Mansukhani 1997. Expression of fibroblast growth factors (FGFs) and FGF receptors in human prostate Journal of Urology. 157:351-6. Karelina, T. V., G. I. Goldberg, and A. Z. Eisen 1994. Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia Journal of Investigative Dermatology. 103:482-7. Kasper, S., P. C. Sheppard, Y. Yan, N. Pettigrew, A. D. Borowsky, G. S. Prins, J. G. Dodd, M. L. Duckworth, and R. J. Matusik 1998. Development, progression, and androgen-dependence of prostate tumors in probasin-large T antigen transgenic mice: a model for prostate cancer Laboratory Investigation. 78:i-xv. 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Klein, R. D., M. S. Maliner-Jongewaard, T. S. Udayakumar, J. L. Boyd, R. B. Nagle, and G. T. Bowden 1999. Promatrilysin expression is induced by fibroblast growth factors in the prostatic carcinoma cell line LNCaP but not in normal primary prostate epithelial cells Prostate. 41:215-23. Kouhara, H., M. Koga, S. Kasayama, A. Tanaka, T. Kishimoto, and B. Sato 1994. Transforming activity of a newly cloned androgen-induced growth factor Oncogene. 9:455-62. Krezel, W., V. Dupe, M. Mark, A. Dierich, P. Kastner, and P. Chambon 1996. RXR gamma null mice are apparently normal and compound RXR alpha +/- /RXR beta -/-/RXR gamma -/- mutant mice are viable Proceedings of the National Academy of Sciences of the United States of America. 93:9010-4. Kurogi, T., K. Nabeshima, H. Kataoka, Y. Okada, and M. Koono 1996. Stimulation of gelatinase B and tissue inhibitors of metalloproteinase (TIMP) production in co-culture of human osteosarcoma cells and human fibroblasts: gelatinase B production was stimulated via up-regulation of fibroblast growth factor (FGF) receptor International Journal of Cancer. 66:82-90. Leung, H. Y., C. Dickson, C. N. Robson, and D. E. Neal 1996. Over-expression of fibroblast growth factor-8 in human prostate cancer Oncogene. 12:1833-5. Lohnes, D,, P. Kastner, A. Dierich, M. Mark, M. LeMeur, and P. Chambon 1993. Function of retinoic acid receptor gamma in the mouse Cell. 73:643-58. Lorenzi, M. V., J. E. Long, T. Miki, and S. A. Aaronson 1995. Expression cloning, developmental expression and chromosomal localization of fibroblast growth factor-8 Oncogene. 10:2051-5. MacArthur, C. A., A. Lawshe, D. B. Shankar, M. Heikinheimo, and G. M. Shackleford 1995. FGF-8 isoforms differ in NIH3T3 cell transforming potential Cell Growth & Differentiation. 6:817-25. MacArthur, C. A., A. Lawshe, J. Xu, S. Santos-Ocampo, M. Heikinheimo, A. T. Chellaiah, and D. M. Omitz 1995. FGF-8 isoforms activate receptor splice forms that are expressed in mesenchymal regions of mouse development Development. 121:3603-13. MacArthur, C. A., D. B. Shankar, and G. M. Shackleford 1995. Fgf-8, activated by proviral insertion, cooperates with the Wnt-1 transgene in murine mammary tumorigenesis Journal of Virology. 69:2501-7. 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mahmood, R., J. Bresnick, A. Hombruch, C. Mahony, N. Morton, K. Colquhoun, P. Martin, A. Lumsden, C. Dickson, and I. Mason 1995. A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth Current Biology. 5:797-806. Masumori, N., T. Z. Thomas, P. Chaurand, T. Case, M. Paul, S. Kasper, R. M. Caprioli, T. Tsukamoto, S. B. Shappell, andR. J. Matusik 2001. A probasin-large T antigen transgenic mouse line develops prostate adenocarcinoma and neuroendocrine carcinoma with metastatic potential Cancer Research. 61:2239- 49. McDonnell, S., M. Navre, R. J. Coffey, Jr., and L. M. Matrisian 1991. Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas Molecular Carcinogenesis. 4:527-33. McKeehan, W. L. 1991. Growth factor receptors and prostate cell growth Cancer Surveys. 11:165-75. McKeehan, W. L., F. Wang, and M. Kan 1998. The heparan sulfate-fibroblast growth factor family: diversity of structure and function Progress in Nucleic Acid Research & Molecular Biology. 59:135-76. McNeal, J. E., and D. G. Bostwick 1986. Intraductal dysplasia: a premalignant lesion of the prostate Human Pathology. 17:64-71. Meyers, E. N., M. Lewandoski, and G. R. Martin 1998. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination Nature Genetics. 18:136-41. Miyagi, N., S. Kato, M. Terasaki, M. Shigemori, and M. Morimatsu 1998. Fibroblast growth factor-2 and -9 regulate proliferation and production of matrix metalloproteinases in human gliomas International Journal of Oncology. 12:1085-90. Miyake, H., K. Yoshimura, I. Hara, H. Eto, S. Arakawa, and S. Kamidono 1997. Basic fibroblast growth factor regulates matrix metalloproteinases production and in vitro invasiveness in human bladder cancer cell lines Journal of Urology. 157:2351-5. 85 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Naldini, L., U. Blomer, P. Gallay, D. Ory, R. Mulligan, F. H. Gage, I. M. Verma, and D. Trono 1996. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector Science. 272:263-7. Newell, K. J., J. P. Witty, W. H. Rodgers, and L. M. Matrisian 1994. Expression and localization of matrix-degrading metalloproteinases during colorectal tumorigenesis Molecular Carcinogenesis. 10:199-206. Nieto, M. A., K. Patel, and D. G. Wilkinson 1996. In situ hybridization analysis of chick embryos in whole mount and tissue sections. [Review] [9 refs] Methods in Cell Biology. 51:219-35. Nilsen-Hamilton, M. 1990. Transforming growth factor-beta and its actions on cellular growth and differentiation Current Topics in Developmental Biology. 24:95-136. Ohuchi, H., H. Yoshioka, A. Tanaka, Y. Kawakami, T. Nohno, and S. Noji 1994. Involvement of androgen-induced growth factor (FGF-8) gene in mouse embryogenesis and morphogenesis Biochemical & Biophysical Research Communications. 204:882-8. Pajouh, M. S., R. B. Nagle, R. Breathnach, J. S. Finch, M. K. Brawer, and G. T. Bowden 1991. Expression of metalloproteinase genes in human prostate cancer Journal of Cancer Research & Clinical Oncology. 117:144-50. Partanen, A. M. 1990. Epidermal growth factor and transforming growth factor- alpha in the development of epithelial-mesenchymal organs of the mouse Current Topics in Developmental Biology. 24:31-55. Payson, R. A., J. Wu, Y. Liu, and I. M. Chiu 1996. The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells Oncogene. 13:47-53. Podsypanina, K., L. H. Ellenson, A. Nemes, J. Gu, M. Tamura, K. M. Yamada, C. Cordon-Cardo, G. Catoretti, P. E. Fisher, and R. Parsons 1999. Mutation of Pten/Mmacl in mice causes neoplasia in multiple organ systems Proceedings of the National Academy of Sciences of the United States of America. 96:1563-8. Powell, W. C., J. D. Knox, M. Navre, T. M. Grogan, J. Kittelson, R. B. Nagle, and G. T. Bowden 1993. Expression of the metalloproteinase matrilysin in DU- 145 cells increases their invasive potential in severe combined immunodeficient mice Cancer Research. 53:417-22. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Rowley, D. R. 1998. What might a stromal response mean to prostate cancer progression? Cancer & Metastasis Reviews. 17:411-9. Rubin, J. S., D. P. Bottaro, M. Chedid, T. Miki, D. Ron, G. Cheon, W. G. Taylor, E. Fortney, H. Sakata, P. W. Finch, and et al. 1995. Keratinocyte growth factor Cell Biology International. 19:399-411. Rudra-Ganguly, N., J. Zheng, A. T. Hoang, and P. Roy-Burman 1998. Downregulation of human FGF8 activity by antisense constructs in murine fibroblastic and human prostatic carcinoma cell systems Oncogene. 16:1487-92. Shannon, J. M., and G. R. Cunha 1983. Autoradiographic localization of androgen binding in the developing mouse prostate Prostate. 4:367-73. Shibata, M. A., J. M. Ward, D. E. Devor, M. L. Liu, and J. E. Green 1996. Progression of prostatic intraepithelial neoplasia to invasive carcinoma in C3(1)/SV40 large T antigen transgenic mice: histopathological and molecular biological alterations Cancer Research. 56:4894-903. Sim, B. K., N. J. MacDonald, and E. R. Gubish 2000. Angiostatin and endostatin: endogenous inhibitors of tumor growth. [Review] [68 refs] Cancer & Metastasis Reviews. 19:181-90. Song, Z., W. C. Powell, N. Kasahara, A. van Bokhoven, G. J. Miller, and P. Roy- Burman 2000. The effect of fibroblast growth factor 8, isoform b, on the biology of prostate carcinoma cells and their interaction with stromal cells Cancer Research. 60:6730-6. Story, M. T. 1991. Polypeptide modulators of prostatic growth and development Cancer Surveys. 11:123-46. Sugimura, Y., G. R. Cunha, and R. M. Bigsby 1986. Androgenic induction of DNA synthesis in prostatic glands induced in the urothelium of testicular feminized (Tfm/Y) mice Prostate. 9:217-25. Szebenyi, G., and J. F. Fallon 1999. Fibroblast growth factors as multifunctional signaling factors International Review of Cytology. 185:45-106. Takeda, H., and C. Chang 1991. Immunohistochemical and in-situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland Journal of Endocrinology. 129:83-9. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Takeda, H., T. Mizuno, and I. Lasnitzki 1985. Autoradiographic studies of androgen-binding sites in the rat urogenital sinus and postnatal prostate Journal of Endocrinology. 104:87-92. Takeda, H., N. Suematsu, and T. Mizuno 1990. Transcription of prostatic steroid binding protein (PSBP) gene is induced by epithelial-mesenchymal interaction Development - Supplement. 110:273-81. Tanaka, A., A. Furuya, M. Yamasaki, N. Hanai, K. Kuriki, T. Kamiakito, Y. Kobayashi, H. Yoshida, M. Koike, and M. Fukayama 1998. High frequency of fibroblast growth factor (FGF) 8 expression in clinical prostate cancers and breast tissues, immunohistochemically demonstrated by a newly established neutralizing monoclonal antibody against FGF 8 Cancer Research. 58:2053-6. Tanaka, A., K. Miyamoto, H. Matsuo, K. Matsumoto, and H. Yoshida 1995. Human androgen-induced growth factor in prostate and breast cancer cells: its molecular cloning and growth properties FEBS Letters. 363:226-30. Tanaka, A., K. Miyamoto, N. Minamino, M. Takeda, B. Sato, H. Matsuo, and K. Matsumoto 1992. Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells Proceedings of the National Academy of Sciences of the United States of America. 89:8928-32. Tanaka, M., I. Komuro, H. Inagaki, N. A. Jenkins, N. G. Copeland, and S. Izumo 2000. Nkx3.1, a murine homolog of Ddrosophila bagpipe, regulates epithelial ductal branching and proliferation of the prostate and palatine glands Developmental Dynamics. 219:248-60. Thompson, T. C. 1990. Growth factors and oncogenes in prostate cancer Cancer Cells. 2:345-54. Thompson, T. C. 1990. Growth factors and oncogenes in prostate cancer. [Review] [103 refs] Cancer Cells. 2:345-54. Thompson, T. C., J. Southgate, G. Kitchener, and H. Land 1989. Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ Cell. 56:917-30. 88 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thompson, T. C., L. D. Truong, T. L. Timme, D. Kadmon, B. K. McCune, K. C. Flanders, P. T. Scardino, and S. H. Park 1993. Transgenic models for the study of prostate cancer. [Review] [45 refs] Cancer. 71:1165-71. Thomson, A. A., B. A. Foster, and G. R. Cunha 1997. Analysis of growth factor and receptor mRNA levels during development of the rat seminal vesicle and prostate Development. 124:2431-9. Ting-Xin Jiang, N. S. S., Randall B. Widelitz and Cheng-Ming Chuong 1998. Current Methods in the Study of Avian Skin Appendages, p. 395-408. In C.-M. Chuong (ed.), Molecular Basis of Epithelial Appendage Morphogenesis. R. G. Landes Company. Trumpp, A., M. J. Depew, J. L. Rubenstein, J. M. Bishop, and G. R. Martin 1999. Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch Genes & Development. 13:3136-48. Uria, J. A., M. Balbin, J. M. Lopez, J. Alvarez, F. Vizoso, M. Takigawa, and C. Lopez-Otin 1998. Collagenase-3 (MMP-13) expression in chondrosarcoma cells and its regulation by basic fibroblast growth factor American Journal of Pathology. 153:91-101. Valve, E. M., M. T. Nevalainen, M. J. Nurmi, M. K. Laato, P. M. Martikainen, and P. L. Harkonen 2001. Increased expression of FGF-8 isoforms and FGF receptors in human premalignant prostatic intraepithelial neoplasia lesions and prostate cancer Laboratory Investigation. 81:815-26. White, R. A., L. L. Dowler, S. V. Angeloni, L. M. Pasztor, and C. A. MacArthur 1995. Assignment of FGF8 to human chromosome 10q25-q26: mutations in FGF8 may be responsible for some types of acrocephalosyndactyly linked to this region Genomics. 30:109-11. Wu, X., J. Wu, J. Huang, W. C. Powell, J. Zhang, R. J. Matusik, F. O. Sangiorgi, R. E. Maxson, H. M. Sucov, and P. Roy-Burman 2001. Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation Mechanisms of Development. 101:61-9. Yan, G., Y. Fukabori, G. McBride, S. Nikolaropolous, and W. L. McKeehan 1993. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostate epithelial cells accompany stromal independence and malignancy Molecular & Cellular Biology. 13:4513-22. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Yan, G., Y. Fukabori, S. Nikolaropoulos, F. Wang, and W. L. McKeehan 1992. Heparin-binding keratinocyte growth factor is a candidate stromal-to-epithelial- cell andromedin Molecular Endocrinology. 6:2123-8. Zhang, J., T. Z. Thomas, S. Kasper, and R. J. Matusik 2000. A small composite probasin promoter confers high levels of prostate-specific gene expression through regulation by androgens and glucocorticoids in vitro and in vivo Endocrinology. 141:4698-710. Zheng, J., N. Rudra-Ganguly, G. J. Miller, K. A. Moffatt, R. J. Cote, and P. Roy- Burman 1997. Steroid hormone induction and expression patterns of L-plastin in normal and carcinomatous prostate tissues American Journal of Pathology. 150:2009-18. Zheng, J., N. Rudra-Ganguly, W. C. Powell, and P. Roy-Burman 1999. Suppression of prostate carcinoma cell invasion by expression of antisense L- plastin gene American Journal of Pathology. 155:115-22. Zogakis, T. G., and S. K. Libutti 2001. General aspects of anti-angiogenesis and cancer therapy. [Review] [210 refs] Expert Opinion on Biological Therapy. 1:253-75. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Song, Zhigang (author)

Core Title FGF8, isoform b, is an etiological factor in prostate tumorigenesis

School Graduate School

Degree Doctor of Philosophy

Degree Program Pathology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, oncology,OAI-PMH Harvest

Language English

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Advisor Roy-Burman, Pradip (committee chair), Anderson, W. French (committee member), Kasahara, Noriyuki (committee member)

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