- Email: [email protected]
Transgenic models for prostate cancer research
Advances in the Development of Animal and Cell Culture Mode/s
Ural Oncol /996:2:99-128
Johnston B, Dodd JG, Matusik RJ. Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia. Cancer Res 1986;46:1535-8. 27. Eagle LR, Yin X, Brothman AR, Williams BJ, Atkin NB, Pro28.
29.
30.
31
32.
chownik EV. Mutation of the MXll gene in prostate cancer. _ Nature Genetics 1995;9:249-55. Thomoson TC. Timme TL. Enawa S. Park SH. Kadmon D. Yoshida K. ‘Genetic ‘predispositio< and ‘mesenchymalepithklial interactions in ms + myc-induced carcinogenesis fn reconstituted mouse nrostate. Mol Carcinog 1993:7:165-79. Thompson Td. Growth factors and oncogenes in prostate cancer. Cancer Cells 1990;2:345-54. Merz VW, Miller GJ, Krebs TK, Timme TL, Kadmon D, Park SH, Egawa S, Scardino PT, Thompson TC. Elevated transforming growth factor-b1 and b3 mRNA levels are associated with ras + myc-induced carcinomas in reconstituted mouse prostate: Evidence for a paracrine role during progression. Mol Endocrinol 1991;5:503-13. Truong LD, Kadmon D, McCune BK, Flanders KC, Scardino PT, Thompson TC. Association of transforming growth factor-b1 with prostate cancer: An immunohistochemical study. Hum Path01 1993;24:4-9. Eastham JA, Truong LD, Rogers E, Kattan M, Flanders KC, Scardino PT, Thompson TC. Transforming growth factor-bl: Comparative immunohistochemical localization in human primary and metastatic prostate cancer. Lab lnvest 1995;5:628-35.
Transgenic
119
33. Bookstein R, MacGrogan D, Hilsenbeck SG, Sharkey F, Allred DC. p.53 is mutated in a subset of advanced-stage prostate cancers. Cancer Res 1993;53:3369-73. 34. Navone NM, Troncosco P, Pisters LL, et al. ~53 protein accumulation and gene mutation in the progression of human prostate carcinoma. J Nat1 Inst 1993;85:1657-69. 35 Eastham JA, Stapleton AMF, Gousse AE, Timme TL, et al. Association of ~53 mutations with metastatic prostate cancer. Clin Can Res 1995;1:1111-8. 36. Yang G, Stapleton AMF, Wheeler TM, Truong LD, Timme TL, Scardino PT, Thompson TC. Clustered ~53 immunostaining: A novel pattern associated with prostate cancer progression. Clin Cancer Res 1996;2:399-I01. 37 Slawin K, Kadmon D, Park SH, Scardino PT, Anzano M, Sporn MB, Thompson TC. Dietary fenretinide, a synthetic retinoid, decreases the tumor incidence and the tumor mass of ras + myc-induced carcinomas in the mouse prostate reconstitution model system. Cancer Res 1993;53:4461-3. 38 Baley PA, Yoshida K, Qian W, Sehgal I, Thompson TC. Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. J Steroid Biochem Mol Biol 1995;52:403-13. 39. Eastham JA, Chen S-H, Sehgal I, Yang G, Timme TL, Hall SJ, Woo SLC, Thompson TC. Prostate cancer gene therapy: Herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse prostatic cancer models. Human Gene Therapy 1996;7:515-23.
Models for Prostate
Cancer
Research
N.M. Greenberg Adenocarcinoma of the prostate has become the most commonly diagnosed cancer in men in the United States. Despite this fact, rapid progress toward understanding the molecular mechanisms involved in the initiation, progression, and metastasis of the disease has been slow. This is largely due to the scarcity of animal model systems that adequately reproduce the heterogeneity of human prostatic disease, owing to the fact that this is a disease rather unique to humans. Although spontaneous prostatic disease has been reported in some canine’ and rodentzV3 spe ties, there is no known naturally occurring animal model to adequately study the spectrum of human prostate cancer. However, recent developments in gene transfer technology present exciting opportunities for the establishment of new animal models that can be used to characterize the molecular mechanisms governing normal prostate development and how these mechanisms have deviated in human prostate cancer. Based on the hypothesis that cancer is a genetic disease and the observation that genetic perturbation studies in uioo have successfully been used to model a number of human cancers (see reference 4 for review), it is appropriate at this time to review the current data on the development of transgenic mouse models for prostate cancer. To do so, the various strategies that have been employed to genetically manipulate the prostate in uiuo will be examined and the particular details of the transgenic models generated will be discussed.
Historical
Perspective
Historically, efforts to establish cancer research have focused
animal models for prostate primarily on the effects of
sex hormones and other chemical carcinogens on the rodent prostate. Although many of these studies are reviewed elsewhere in this chapter, it should be mentioned that the rodent is an attractive experimental system owing to the low frequency of spontaneous prostate disease and the ease with which the animals can be manipulated chemically, hormonally, and physically in keeping with acceptable surgical interventions and other therapeutic strategies currently employed to treat the human disease. To this end, investigators have established a number of rodent models for prostate cancer as a consequence of a number of hormonal- and/or carcinogen-based strategies. For example, rats treated with sex hormones5 were shown to develop adenocarcinoma of the prostate, as did rats treated with N-methylnitrosourea,6 testosterone propionate and cyproterone acetate7 or N-methylnitrosourea, dimethylbenzanthracene, or dimethylaminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate.8,g Although these studies were essentially successful at inducing prostate cancer in the rodent prostate, the primary tumors were unfortunately highly variable in nature and extrapolation of these studies to a paradigm for the human condition proves difficult. In addition to animal based models, efforts to develop in oitro models for prostate cancer have also been quite successful and have produced a number of long term cell culture models including the Dunning,“*” PC-3,12 and PC-8213 models, and more recently LnCaP sublines that display androgen-independent cancer progression and bone metastasis.14.15 However, the question remains whether experimental systems based on long term cultured cell lines representing late stage metastatic disease are an appropri-
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ate paradigm for studying the individual molecular events associated with the initiation and progression of the disease.
Transgenic
Mouse Models
Transgenic mice are the product of a gene transfer experiment wherein recombinant DNA molecules, called transgenes, are introduced by direct microinjection into the male pronucleus of a fertilized one cell zygote and the successful integration of the transgene into a zygote chromosome (see references 16,17 for review). Mice born with at least one integrated copy of the transgene are founder animals, and subsequent passage of the transgene through the germ line establishes a line of transgenic mice. Depending on the design of the transgene, which consists of regulatory elements that establish the temporal and spatial pattern of transgene expression and the structural gene to be expressed, transgenic mice may exhibit an inheritable, reproducible phenotype such as organ specific cancer.” The severity of the observable phenotype is usually related to the capacity of the integrated transgene to be “expressed” in the tissue type or cell type of interest, the level of expression in all cells of that type (“penetrance”), and the restriction of transgene expression to any particular tissue or cell type (“tissue specificity”). By definition, transgenic mice represent a unique class of genetically engineered animals and are distinct from ex-vivo systems such as the mouse prostate reconstitution model (MPR), reviewed elsewhere in this article, that require the genetic manipulation in vitro of harvested normal mouse tissue and the subsequent grafting of cells into mice.” In both the human and the mouse the prostate gland develops in a temporally, hormonally, and spatially restricted fashion. Furthermore, adenocarcinoma of the prostate develops in the secretory epithelial cells of the prostate gland. By these criteria, a transgene expression system capable of directing expression of a heterologous gene in a manner spatially restricted to prostate epithelial cells and regulated by hormones, particularly androgens, would provide the cornerstone of studies designed to genetically manipulate the prostate epithelium in vivo. Fortunately, regulatory elements derived from various genes that exhibit prostate-restricted patterns of expression have been isolated and characterized. These include the rat C3(1) gene, *’ the mouse mammary tumor virus long terminal repeat (MMTV-LTR), the gene encoding rat seminal vesicle secreted protein(SVSll),2’*22 the human prostate specific antigen gene (PSA),“3 and the rat probasin (PB) gene.24-26 Most of the candidate prostate specific regulatory systems have now been evaluated in transgenic studies. Mice harboring a C3(1) transgene were found to express preferentially in the prostate of transgenic mice27; however, when a proximal fragment of the rat C3(1) gene was fused to a heterologous P-gal reporter gene, the spatial pattern of CS(l)-pgal fusion transgene expression was not found to be uniform. 28 Mice carrying a MMTV-LTR-int-2 cDNA construct were originally reported to exhibit a phenotype consistent with benign prostatic hyperplasia2g,30 but the pathology actually occurs in the ampulary gland (see reference 31). When a 600 bp fragment of the promoter for
/996;2:99-
I28
human PSA was used to target an activated Ha-ras oncogene to the prostate in transgenic mice, the mice developed salivary gland tumors with no apparent pathology in the prostate gland.32 It is interesting to note that the promoter for the PSA gene, a member of the kallikrein gene family, 32 directed expression in salivary gland in transgenie mice, in contrast to findings reported in previous transgenic studies using a kallikrein rKlk1 promoter Tag constructs.33 Recently, a large genomic construct carrying the entire human PSA gene including approximately 5 kbp of 5’ flanking sequence has been demonstrated to be expressed specifically in the prostate of transgenic mice.34 However, it remains to be determined whether the longer PSA promoter fragment is sufficient to target expression of a heterologous gene in a similar fashion or if other intragenie control elements are required for this spatially restricted expression. In contrast to these studies, a minimal 0.5 kb fragment of the rat probasin gene has been shown to direct developmentallyand hormonally-regulated expression of a heterologous gene reproducibly to the dorsolatera1 and ventral prostatic lobes of transgenic mice.35 The ability to manipulate prostate gene expression in vivo facilitated the establishment of a transgenic mouse model for prostate cancer. However, because no single dominant prostate cancer allele has yet been identified, it seemed logical to many groups to design a construct that would abrogate functional expression of the p53 and Rb tumor suppressor genes using SV40 T antigen because it acts as an oncoprotein through interactions wtih both Rb36 and ~53.~~~~~The rationale for this approach was based on the observations that the most frequently observed genetic lesions associated with prostate cancer appeared to be the progressive loss of wildtype ~59’~~ and Rb.44*4w8. A C3(1) promoter-SV40 T antigen construct (C3(1)TAG) has been used to develop one transgenic mouse model for prostate cancer. 4g In this model, which uses the FVB/N inbred strain, adenomas occurred in approximately onethird of the males aged between 6 and 8 months, with the majority of the male mice developing prostate cancer by 8 months. At least one adenocarcinoma was reported to metastasize to the lung in these mice. Consistent with the promiscuous expression of other C3(1) based constructs, independent lines of transgenic mice carrying the C3(1)Tag transgene exhibited a range of pathologies including prostate hyperplasias, mammary adenocarcinoma, chondrodysplasia, salivary gland proliferative disorders, nasal turbinate proliferative lesions, harderian hyperplasia, thyroid proliferative lesions, osteosarcomas, and lung carcinomas. These mice have since been used as a source for the development of new resource material, and at least two novel prostate cancer cell lines have been established.50 A probasinSV40 T antigen (PB-Tag) transgene has been used to generate an independent transgenic model for prostate cancer in the C57B1/6 inbred strain of mice.31 These mice are henceforth designated the transgenic adenocarcinoma mouse prostate (TRAMP) model. Expression of the PB-Tag transgene is restricted to the epithelial cells of the dorsolateral and ventral lobes of the male TRAMP mice, and expression of the transgene correlates with sexual maturity and is regulated by androgens. At 12
Urol Oncol /996;2:99-
I28
weeks of age, prostate structures in the TRAMP mice histologically display mild to severe hyperplasia with cribriform structures. Immunohistochemistry demonstrates Tag oncoprotein in normal epithelium as well as nests of obviously transformed cells. That oncogene expression precedes the transformed state in both the TRAMP and C3(1)TAG models supports the hypothesis that transgene expression is required but not sufficient for neoplastic transformation. Hence these animals are well suited for studies designed to identify, isolate, and characterize the gene(s) responsible to confer the neoplastic phenotype. Severe hyperplasia and adenocarcinoma is observed in the TRAMP model by 18 weeks of age. The epithelial origin of the tumors and metastatic deposits to lymph nodes, lung, and bone has been determined with antibodies specific for dorsolateral specific proteins [31] and E-cadherin [51]. By the time the mice reach 24 to 30 weeks of age, they will all display primary tumors. At this time, invasion of epithelial cells into underlying smooth muscle and stroma have been observed. By the time the mice are 24-30 weeks of age, they will all display primary tumors. As one of the hallmarks of human prostate cancer is the progression to a metastatic disease, the pattern and incidence of metastatic spread in the TRAMP model has been characterized [52]. Distant site metastases are detected in TRAMP mice as early 12 weeks of age, with the most common sites being the periaortic lymph nodes and lungs. Occasional metastases have been detected in the kidney, adrenal gland and bone. By the time the TRAMP mice reach 28 weeks of age 100% will harbor metastatic prostate cancer in the lymph
Advances in the Development of Animal and Cell Culture
2. 3. 4. 5. 6. 7. 8.
9.
10.
11.
nodes or lungs. In contrast to the CS(l)TAG mice, no other primary pathologies are observed in the TRAMP mice and
female mice are normal and fertile. It is anticipated that the establishment of the TRAMP model will facilitate many new avenues of research toward better prevention, diagnosis, and treatment of prostate cancer.
12.
13.
Summary
14.
The ability to introduce novel or specifically altered genes into the germ line of mice and directly perturb gene expression in a specific tissue can facilitate characterization of the
15.
molecular mechanisms governing transformation of differentiating tissue within the context of an intact developing animal. Transgenics provide a powerful and remarkably flexible system that can be used to study the cooperation between proto-oncogenes, tumor suppressor genes, and other epige netic factors in the development of cancer. For more information on the use and availability of transgenic models for prostate cancer research, please contact Dr. Norman M. Greenberg (Department of Cell Biology and Scott Department of Urology, Baylor College of Medicine, One Baylor Plaza, M 626, Houston, TX 77030; phone: 713-798-3819, fax: 713-798-8012, email: [email protected]).
16. 17. 18.
19. 20. 21.
References 1. Berry SJ, Coffey DS, Ewing LL. A comparison of human and canine benign prostatic hyperplasia. In: Bruchovsky N, Chap-
22.
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delaine A, Neumann F, (eds). Regulation of Androgen Action. Berlin: Congressduck, 1984; 59-68. Pollard M. Spontaneous adenocarcinomas in aged germfree Wistar rats. J Nat1 Cancer Inst 1973;51:1235-41. Shain SA, McCullough B, Segaloff A. Spontaneous adenocarcinomas of the ventral prostate of aged AX rats. J Nat1 Cancer lnst 1975;55:177-80. Adams JM, Cory S. Transgenic models of tumor development. Science 1991;254:1161-7. Noble RL. The development of prostatic adenocarcinoma in Nb rats following prolonged sex hormone administration. Cancer Res 1977;37:1927-33. Pollard M, Luckert PH. Autochthonous prostate adenocarcinomas in Lobund-Wistar rats: A model system. Prostate 1987; 11:219-27. Pollard M, Luckert PH, Snyder L. The promotional effect of testosterone on induction of prostate-cancer in MNUsensitized LW rats. Cancer Lett 1989;45:209-12. Bosland MC, Prinsen MK, Dirksen TJM, Spit BJ. Characterization of adenocarcinoma of the dorsolateral prostate induced in wistar rats by N-methyl-N-nitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2’dimethyl_4_aminobiphenyl, following sequential treatment with cyproterone acetate and testosterone proprionate. Cancer Res 1990;50:700-9. Bosland MC, Prinsen MK. Induction of dorsolateral prostate adenocarcinoma and other accessory sex gland lesions in male Wistar rats by a single administration of N-methyl-Nnitrosourea, 7,12-dimethylbenz(a)anthracene, and 3,2’-dimethyl4aminobiphenyI after sequential treatment with cyproterone acetate and testosterone propionate. Cancer Res 1990;50:691-9. Bogden AE, Taylor JE, Moreau JP, Coy DH. Treatment of R-3327 prostate tumors with a somatostatin analogue (somatuline) as adjuvant therapy following surgical castration. Cancer Res 1990;50:2646-50. Cooke DB, Quarmby VE, Mickey DD, lsaacs JT, French FS. Oncogene expression in prostate cancer: Dunning r3327 rat dorsal prostatic adenocarcinoma system. The Prostate 1988;13: 263-72. Rubin SJ, Hallahan DE, Ashman CR, Brachman DG, Beckett MA, Virudachalam S, Yandell DW, Weichselbaum RR. Two prostate carcinoma cell lines demonstrate abnormalities in tumor sup pressor genes. J Surg Oncol 1991;46:31-6. van-Steenbrugge GJ, Schroder FH. Androgendependent human prostate cancer in nude mice. The PC82 tumor model. Am J Clin Oncol 1988;2. Chung LW, Li W, Gleave ME, et al. Human prostate cancer model: Roles of growth factors and extracellular matrices. J Cell Biochem 1992;(suppl.). Thalman GN, Anezinis PE, Chang S-M, Zhau HE, Kim E, Hopwood VL, Pathak S, von Eschenbach AC, Chung LWK. Androgen-independent cancer progression and bone metastasis in the LnCaP model of human prostate cancer. Cancer Research 1994;54:2577-81. Palmiter RD, Brinster RL. Transgenic mice. Cell 1985;41:343-5. Palmiter RD, Brinster RL. Germ-line transformation of mice. Ann Rev Genetics 1986;20:465-99. Hanahan D. Heritable formation of pancreatic B-cell tumors in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 1985;315:115-22. Thompson TT, Southgate J, Kitchener G, and Land H. Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ. Cell 1989;56:917-30. Hurst HC, Parker MG. Rat prostatic steroid binding protein: DNA sequence and transcript maps of the two C3 gene. EMBO J 1983;2:769-74. Harris SE, Parker MC, Webb P, Needham M, White R, Ham J, Harris MA. Mapping cis-DNA regulatory elements in seminal vesicle secretion genes, prostate C3(1) gene, and the mouse mammary tumor virus-long terminal repeat DNA. Prog Clin Biol Res 1988;284:53-76. Harris SE, Harris MA, Johnson CM, Bean MF, Dodd JG, Matusik RJ, Carr SA, Crabb JW. Structural characterization of the rat
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seminal vesicle secretion II protein and gene. J Biol Chem 1990;265:9896-903. Riegman PH, Vlietstra RJ, van dKJA, Romijn JC, Trapman J. Characterization of the prostate-specific antigen gene: A novel human kallikrein-like gene. Biochem Biophys Res Commun 1989;159:95-102. Dodd JG, Sheppard PC, Marusik RJ. Characterization and cloning of highly abundant mRNAs of the rat dorsal prostate. J Biol Chem 1983;258:10731-7. Matusik RJ, Cattini PA, Leco KJ, Sheppard PC, Nickel BE, Neubauer Bl, Davie JR, Chang C, Liao S, Matuo Y, McKeehan WL. Regulation of gene expression in the prostate. In: Karr JP, (ed). Molecular and Cellular Biology of Prostate Cancer. New York: Plenum Press. 1991;299-314. Matuo Y, Adams PS, Nishi N, Yasumitsu H, Crabb JW, Matusik RJ, McKeehan WL. The androgen-dependent rat prostate protein, probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-l. In Vitro Cell Dev Biol 1989;25:581-4. Allison J, Zhang Y-L, Parker MG. Tissuespecific and hormonal regulation of the gene for rat prostatic steroid-binding protein in transgenic mice. Mol Cell Biol 1989;9:2254-7. Buttyan R, Slawin K. Rodent models for targeted oncogenesis of the prostate gland. Cancer and Metastasis Rev 1993;12:1 l-9. Muller WJ, Lee FS, Dickson C, Peters G, Pattengale P, Leder P. The int-2 gene product acts as an epithelial growth factor in transgenic mice. EMBO J 1990;9:907-13. Tutrone RFJ, Ball RA, Ornitz DM, Leder P, Richie JP. Benign prostatic hyperplasia in a transgenic mouse: A new hormonallv sensitive investieatorv model. J Urol 1993:149:633-g. Greenberg NM, DeMayo 6 Finegold MJ, Medina D, Tilley WD, Aspinall JO, Cunha GR, Donjacour AA, Matusik RJ, Rosen JM. Prostate cancer in a transgenic mouse. Proc Nat1 Acad Sci (USA) 1995;92:3439-43. Schaffner DL, Barrios R, Shaker M, Rajagopalan S, Huang S, Tindall DJ, Youn CYF, Overbeek PA, Lebovitz RM, Lieberman MW. Transgenic mice carrying a PSArasT24 hybrid gene develop salivary gland and Gl tract neoplasms. Laboratory lnvestigation 1994;72:283-9. Smith MS, Lechago J, Wines DR, MacDonald RJ, Hammer RE. Tissue-specific expression of kallikrein family transgenes in mice and rats. DNA Cell Biol 1992;11:345-58. Frelinger J, Wei C, Willis R, Storozynsky E, Tilton B, Barth R, Lord E. Targeted CTL-mediated immunity for prostate cancer: Development of human PSA-expressing transgenic mice. (Abstract). Proc Am Assoc Can Res 1996;37:444. Greenberg NM, DeMayo FJ, Sheppard PC, Barrios R, Lebovitz R, Finegold M, Angelopolou R, Dodd JG, Duckworth ML, Rosen JM, Matusik RJ. The rat probasin gene promoter directs hormonally- and developmentally-regulated expression of a heterologous gene specifically to the prostate in transgenic mice. Mol Endocrinol 1994;8:230-9. DeCaprio JA, Ludlow JW, Figge J, Skew J-Y, Huang C-M, Lee W-H, Marsilio E, Paucha E, Livingston DM. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 1988;17:275-83. Linzer DH, Levine AJ. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells. Cell 1979;17:43-52.
38. Lane DP, Crawford LV. T-antigen is bound to host proteins in SV-40 transformed cells. Nature 1979;278:261-3. 39. Yang G, Stapleton A, Wheeler TM, Truong LD, Timme TL, Scardino PT, Thompson TC. Clustered ~53 immunostaining-a novel pattern associated with prostate cancer progression. Clinical Cancer Research 1996;2:399-401. 40. Kubota Y, Shuin T, Uemura H, Fujinami K, Miyamoto H, Torigoe S, Dobashi Y, Kitamura H, lwasaki Y, Danenberg K, Danenberg PV. Tumor suppressor gene ~53 mutations in human prostate cancer. The Prostate 1995;27:18-24. 41. Hall MC, Navone NM, Troncoso P, Pollack A, Zagars GK, Voneschenbach AC, Conti CJ, Chung L. Frequency and characterization of ~53 mutations in clinically localized prostate cancer. Urology 1995;45:470-5. 42. Eastham JA, Stapleton A, Gousse AE, Timme TL, Yang G, Slawin KM, Wheeler TM, Scardino PT, Thompson TC. Association of p53 mutations with metastatic prostate cancer. Clinical Cancer Research 1995;l:lll l-8. 43. Bauer JJ, Sesterhenn lA, Mostofi KF, Mcleod DG, Srivastava S, Moul JW. ~53 nuclear protein expression is an independent prognostic marker in clinically localized prostate cancer patients undergoing radical prostatectomy. Clinical Cancer Research 1995;1:1295-300. 44. lsaacs WB, Bova GS, Morton RA, Bussemakers MJ, Brooks JD, Ewing CM. Molecular biology of prostate cancer. Semin Oncol 1994;21:514-21. 45. Heidenberg HB, Sesterhenn LA, Gaddipati JP, Weghorst CM, Buzard GS, Moul JW, Srivastava S. Alteration of the tumor suppressor gene p53 in a high fraction of hormone refractory prostate cancer. J Ural 1995;154:414-21. 46. Konishi N, Hiasa Y, Tsuzuki T, Matsuda H, Tao M, Nakamura
M, Naito H, Kitahori Y, Shiraishi T, Yatani R, Shimazaki J, Lin JC. Detection of rb,pl6/cdkn2 and p15(ink4b) gene alterations with immunohistochemical studies in human prostate carcinomas. lnt J Oncol 1996;8:107-12. 47. lttmann MM, Wieczorek R. Alterations of the retinoblastoma gene in clinically localized, stage b prostate adenocarcinomas. Human Pathology 1996;27:28-34. 48. Sarkar FH, Sakr W, Li YW, Macoska J, Ball DE, Crissman JD. Analysis of retinoblastoma (RB) gene deletion in human prostatic carcinomas. The Prostate 1992;21:145-52. 49. Maroulakou IG, Anver M, Garrett L, Green JE. Prostate and mammary adenocarcinoma in transgenic mice carrying a rat C3(1) simian virus 40 large tumor antigen fusion gene. Proc Nat1 Acad Sci USA 1994;91:11236-40. 50. Jorcyk CL, Liu M-L, Maroulakou JG, Shibata M-A, Resau JH, Green JE. Transgenic mouse prostate and mammary adenocarcinoma cell lines: Reagents for cancer vaccines. (Abstract). Proc Am Assoc Can Res 1996;37:444. 51. Morton RAJ, Gingrich JR, Foster BA, Madewell L, Greenberg NM. E-cadherin expression in a transgenic animal model of metastatic prostate cancer. Urology 1996;155:514A. 52. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ, Finegold MJ, Angelopoulou R, Rosen JM, and Greenberg NM. Metastatic prostate cancer in a transgenic mouse. Cancer Res 1996;56:4096-4102.
Human Prostate Tumor Xenografts as Representative Models for Clinical Prostate Cancer Wytske M. van Weerden
and Gert J. van Steenbrugge
Introduction Experimental models are required to investigate concepts of basic functions and properties of human prostate cancer that are most difficult to examine through clinical stud-
ies. Representative models of human prostate cancer have always been scarce due to the fact that human prostate tumor tissue is very difficult to grow both in vitro and in vivo. Therefore, many attempts have been made to estab-
Ural Oncol /996:2:99-128
Johnston B, Dodd JG, Matusik RJ. Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia. Cancer Res 1986;46:1535-8. 27. Eagle LR, Yin X, Brothman AR, Williams BJ, Atkin NB, Pro28.
29.
30.
31
32.
chownik EV. Mutation of the MXll gene in prostate cancer. _ Nature Genetics 1995;9:249-55. Thomoson TC. Timme TL. Enawa S. Park SH. Kadmon D. Yoshida K. ‘Genetic ‘predispositio< and ‘mesenchymalepithklial interactions in ms + myc-induced carcinogenesis fn reconstituted mouse nrostate. Mol Carcinog 1993:7:165-79. Thompson Td. Growth factors and oncogenes in prostate cancer. Cancer Cells 1990;2:345-54. Merz VW, Miller GJ, Krebs TK, Timme TL, Kadmon D, Park SH, Egawa S, Scardino PT, Thompson TC. Elevated transforming growth factor-b1 and b3 mRNA levels are associated with ras + myc-induced carcinomas in reconstituted mouse prostate: Evidence for a paracrine role during progression. Mol Endocrinol 1991;5:503-13. Truong LD, Kadmon D, McCune BK, Flanders KC, Scardino PT, Thompson TC. Association of transforming growth factor-b1 with prostate cancer: An immunohistochemical study. Hum Path01 1993;24:4-9. Eastham JA, Truong LD, Rogers E, Kattan M, Flanders KC, Scardino PT, Thompson TC. Transforming growth factor-bl: Comparative immunohistochemical localization in human primary and metastatic prostate cancer. Lab lnvest 1995;5:628-35.
Transgenic
119
33. Bookstein R, MacGrogan D, Hilsenbeck SG, Sharkey F, Allred DC. p.53 is mutated in a subset of advanced-stage prostate cancers. Cancer Res 1993;53:3369-73. 34. Navone NM, Troncosco P, Pisters LL, et al. ~53 protein accumulation and gene mutation in the progression of human prostate carcinoma. J Nat1 Inst 1993;85:1657-69. 35 Eastham JA, Stapleton AMF, Gousse AE, Timme TL, et al. Association of ~53 mutations with metastatic prostate cancer. Clin Can Res 1995;1:1111-8. 36. Yang G, Stapleton AMF, Wheeler TM, Truong LD, Timme TL, Scardino PT, Thompson TC. Clustered ~53 immunostaining: A novel pattern associated with prostate cancer progression. Clin Cancer Res 1996;2:399-I01. 37 Slawin K, Kadmon D, Park SH, Scardino PT, Anzano M, Sporn MB, Thompson TC. Dietary fenretinide, a synthetic retinoid, decreases the tumor incidence and the tumor mass of ras + myc-induced carcinomas in the mouse prostate reconstitution model system. Cancer Res 1993;53:4461-3. 38 Baley PA, Yoshida K, Qian W, Sehgal I, Thompson TC. Progression to androgen insensitivity in a novel in vitro mouse model for prostate cancer. J Steroid Biochem Mol Biol 1995;52:403-13. 39. Eastham JA, Chen S-H, Sehgal I, Yang G, Timme TL, Hall SJ, Woo SLC, Thompson TC. Prostate cancer gene therapy: Herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse prostatic cancer models. Human Gene Therapy 1996;7:515-23.
Models for Prostate
Cancer
Research
N.M. Greenberg Adenocarcinoma of the prostate has become the most commonly diagnosed cancer in men in the United States. Despite this fact, rapid progress toward understanding the molecular mechanisms involved in the initiation, progression, and metastasis of the disease has been slow. This is largely due to the scarcity of animal model systems that adequately reproduce the heterogeneity of human prostatic disease, owing to the fact that this is a disease rather unique to humans. Although spontaneous prostatic disease has been reported in some canine’ and rodentzV3 spe ties, there is no known naturally occurring animal model to adequately study the spectrum of human prostate cancer. However, recent developments in gene transfer technology present exciting opportunities for the establishment of new animal models that can be used to characterize the molecular mechanisms governing normal prostate development and how these mechanisms have deviated in human prostate cancer. Based on the hypothesis that cancer is a genetic disease and the observation that genetic perturbation studies in uioo have successfully been used to model a number of human cancers (see reference 4 for review), it is appropriate at this time to review the current data on the development of transgenic mouse models for prostate cancer. To do so, the various strategies that have been employed to genetically manipulate the prostate in uiuo will be examined and the particular details of the transgenic models generated will be discussed.
Historical
Perspective
Historically, efforts to establish cancer research have focused
animal models for prostate primarily on the effects of
sex hormones and other chemical carcinogens on the rodent prostate. Although many of these studies are reviewed elsewhere in this chapter, it should be mentioned that the rodent is an attractive experimental system owing to the low frequency of spontaneous prostate disease and the ease with which the animals can be manipulated chemically, hormonally, and physically in keeping with acceptable surgical interventions and other therapeutic strategies currently employed to treat the human disease. To this end, investigators have established a number of rodent models for prostate cancer as a consequence of a number of hormonal- and/or carcinogen-based strategies. For example, rats treated with sex hormones5 were shown to develop adenocarcinoma of the prostate, as did rats treated with N-methylnitrosourea,6 testosterone propionate and cyproterone acetate7 or N-methylnitrosourea, dimethylbenzanthracene, or dimethylaminobiphenyl after sequential treatment with cyproterone acetate and testosterone propionate.8,g Although these studies were essentially successful at inducing prostate cancer in the rodent prostate, the primary tumors were unfortunately highly variable in nature and extrapolation of these studies to a paradigm for the human condition proves difficult. In addition to animal based models, efforts to develop in oitro models for prostate cancer have also been quite successful and have produced a number of long term cell culture models including the Dunning,“*” PC-3,12 and PC-8213 models, and more recently LnCaP sublines that display androgen-independent cancer progression and bone metastasis.14.15 However, the question remains whether experimental systems based on long term cultured cell lines representing late stage metastatic disease are an appropri-
120
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ate paradigm for studying the individual molecular events associated with the initiation and progression of the disease.
Transgenic
Mouse Models
Transgenic mice are the product of a gene transfer experiment wherein recombinant DNA molecules, called transgenes, are introduced by direct microinjection into the male pronucleus of a fertilized one cell zygote and the successful integration of the transgene into a zygote chromosome (see references 16,17 for review). Mice born with at least one integrated copy of the transgene are founder animals, and subsequent passage of the transgene through the germ line establishes a line of transgenic mice. Depending on the design of the transgene, which consists of regulatory elements that establish the temporal and spatial pattern of transgene expression and the structural gene to be expressed, transgenic mice may exhibit an inheritable, reproducible phenotype such as organ specific cancer.” The severity of the observable phenotype is usually related to the capacity of the integrated transgene to be “expressed” in the tissue type or cell type of interest, the level of expression in all cells of that type (“penetrance”), and the restriction of transgene expression to any particular tissue or cell type (“tissue specificity”). By definition, transgenic mice represent a unique class of genetically engineered animals and are distinct from ex-vivo systems such as the mouse prostate reconstitution model (MPR), reviewed elsewhere in this article, that require the genetic manipulation in vitro of harvested normal mouse tissue and the subsequent grafting of cells into mice.” In both the human and the mouse the prostate gland develops in a temporally, hormonally, and spatially restricted fashion. Furthermore, adenocarcinoma of the prostate develops in the secretory epithelial cells of the prostate gland. By these criteria, a transgene expression system capable of directing expression of a heterologous gene in a manner spatially restricted to prostate epithelial cells and regulated by hormones, particularly androgens, would provide the cornerstone of studies designed to genetically manipulate the prostate epithelium in vivo. Fortunately, regulatory elements derived from various genes that exhibit prostate-restricted patterns of expression have been isolated and characterized. These include the rat C3(1) gene, *’ the mouse mammary tumor virus long terminal repeat (MMTV-LTR), the gene encoding rat seminal vesicle secreted protein(SVSll),2’*22 the human prostate specific antigen gene (PSA),“3 and the rat probasin (PB) gene.24-26 Most of the candidate prostate specific regulatory systems have now been evaluated in transgenic studies. Mice harboring a C3(1) transgene were found to express preferentially in the prostate of transgenic mice27; however, when a proximal fragment of the rat C3(1) gene was fused to a heterologous P-gal reporter gene, the spatial pattern of CS(l)-pgal fusion transgene expression was not found to be uniform. 28 Mice carrying a MMTV-LTR-int-2 cDNA construct were originally reported to exhibit a phenotype consistent with benign prostatic hyperplasia2g,30 but the pathology actually occurs in the ampulary gland (see reference 31). When a 600 bp fragment of the promoter for
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human PSA was used to target an activated Ha-ras oncogene to the prostate in transgenic mice, the mice developed salivary gland tumors with no apparent pathology in the prostate gland.32 It is interesting to note that the promoter for the PSA gene, a member of the kallikrein gene family, 32 directed expression in salivary gland in transgenie mice, in contrast to findings reported in previous transgenic studies using a kallikrein rKlk1 promoter Tag constructs.33 Recently, a large genomic construct carrying the entire human PSA gene including approximately 5 kbp of 5’ flanking sequence has been demonstrated to be expressed specifically in the prostate of transgenic mice.34 However, it remains to be determined whether the longer PSA promoter fragment is sufficient to target expression of a heterologous gene in a similar fashion or if other intragenie control elements are required for this spatially restricted expression. In contrast to these studies, a minimal 0.5 kb fragment of the rat probasin gene has been shown to direct developmentallyand hormonally-regulated expression of a heterologous gene reproducibly to the dorsolatera1 and ventral prostatic lobes of transgenic mice.35 The ability to manipulate prostate gene expression in vivo facilitated the establishment of a transgenic mouse model for prostate cancer. However, because no single dominant prostate cancer allele has yet been identified, it seemed logical to many groups to design a construct that would abrogate functional expression of the p53 and Rb tumor suppressor genes using SV40 T antigen because it acts as an oncoprotein through interactions wtih both Rb36 and ~53.~~~~~The rationale for this approach was based on the observations that the most frequently observed genetic lesions associated with prostate cancer appeared to be the progressive loss of wildtype ~59’~~ and Rb.44*4w8. A C3(1) promoter-SV40 T antigen construct (C3(1)TAG) has been used to develop one transgenic mouse model for prostate cancer. 4g In this model, which uses the FVB/N inbred strain, adenomas occurred in approximately onethird of the males aged between 6 and 8 months, with the majority of the male mice developing prostate cancer by 8 months. At least one adenocarcinoma was reported to metastasize to the lung in these mice. Consistent with the promiscuous expression of other C3(1) based constructs, independent lines of transgenic mice carrying the C3(1)Tag transgene exhibited a range of pathologies including prostate hyperplasias, mammary adenocarcinoma, chondrodysplasia, salivary gland proliferative disorders, nasal turbinate proliferative lesions, harderian hyperplasia, thyroid proliferative lesions, osteosarcomas, and lung carcinomas. These mice have since been used as a source for the development of new resource material, and at least two novel prostate cancer cell lines have been established.50 A probasinSV40 T antigen (PB-Tag) transgene has been used to generate an independent transgenic model for prostate cancer in the C57B1/6 inbred strain of mice.31 These mice are henceforth designated the transgenic adenocarcinoma mouse prostate (TRAMP) model. Expression of the PB-Tag transgene is restricted to the epithelial cells of the dorsolateral and ventral lobes of the male TRAMP mice, and expression of the transgene correlates with sexual maturity and is regulated by androgens. At 12
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weeks of age, prostate structures in the TRAMP mice histologically display mild to severe hyperplasia with cribriform structures. Immunohistochemistry demonstrates Tag oncoprotein in normal epithelium as well as nests of obviously transformed cells. That oncogene expression precedes the transformed state in both the TRAMP and C3(1)TAG models supports the hypothesis that transgene expression is required but not sufficient for neoplastic transformation. Hence these animals are well suited for studies designed to identify, isolate, and characterize the gene(s) responsible to confer the neoplastic phenotype. Severe hyperplasia and adenocarcinoma is observed in the TRAMP model by 18 weeks of age. The epithelial origin of the tumors and metastatic deposits to lymph nodes, lung, and bone has been determined with antibodies specific for dorsolateral specific proteins [31] and E-cadherin [51]. By the time the mice reach 24 to 30 weeks of age, they will all display primary tumors. At this time, invasion of epithelial cells into underlying smooth muscle and stroma have been observed. By the time the mice are 24-30 weeks of age, they will all display primary tumors. As one of the hallmarks of human prostate cancer is the progression to a metastatic disease, the pattern and incidence of metastatic spread in the TRAMP model has been characterized [52]. Distant site metastases are detected in TRAMP mice as early 12 weeks of age, with the most common sites being the periaortic lymph nodes and lungs. Occasional metastases have been detected in the kidney, adrenal gland and bone. By the time the TRAMP mice reach 28 weeks of age 100% will harbor metastatic prostate cancer in the lymph
Advances in the Development of Animal and Cell Culture
2. 3. 4. 5. 6. 7. 8.
9.
10.
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nodes or lungs. In contrast to the CS(l)TAG mice, no other primary pathologies are observed in the TRAMP mice and
female mice are normal and fertile. It is anticipated that the establishment of the TRAMP model will facilitate many new avenues of research toward better prevention, diagnosis, and treatment of prostate cancer.
12.
13.
Summary
14.
The ability to introduce novel or specifically altered genes into the germ line of mice and directly perturb gene expression in a specific tissue can facilitate characterization of the
15.
molecular mechanisms governing transformation of differentiating tissue within the context of an intact developing animal. Transgenics provide a powerful and remarkably flexible system that can be used to study the cooperation between proto-oncogenes, tumor suppressor genes, and other epige netic factors in the development of cancer. For more information on the use and availability of transgenic models for prostate cancer research, please contact Dr. Norman M. Greenberg (Department of Cell Biology and Scott Department of Urology, Baylor College of Medicine, One Baylor Plaza, M 626, Houston, TX 77030; phone: 713-798-3819, fax: 713-798-8012, email: [email protected]).
16. 17. 18.
19. 20. 21.
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Human Prostate Tumor Xenografts as Representative Models for Clinical Prostate Cancer Wytske M. van Weerden
and Gert J. van Steenbrugge
Introduction Experimental models are required to investigate concepts of basic functions and properties of human prostate cancer that are most difficult to examine through clinical stud-
ies. Representative models of human prostate cancer have always been scarce due to the fact that human prostate tumor tissue is very difficult to grow both in vitro and in vivo. Therefore, many attempts have been made to estab-