Special Lecture

How the TRAMP Model Revolutionized the Studyof Prostate Cancer ProgressionIrwin H. Gelman

See related article by Gingrich et al., Cancer Res. 1996;56:4096–102.

FDA approval for prostate-specific antigen (PSA) testing in1994 as a screening aid led to a dramatic increase in the diagnosisof prostate cancer cases. Yet, researchers had few tools to study thisdisease beyond a handful of human cell lines (mainly LNCaP,PC-3, and DU145), pedigrees of rat carcinogen–enhanced celllines developed from autochthonous prostate cancer tumors(including the Dunning, Noble, and Pollard series), in vivogrowths of prostate cancer/mesenchyme tissue recombinants, andhuman prostate cancer xenografts (such as CWR22). None ofthese models, however, recapitulated all the parameters of pros-tate cancer progression in human disease, including formation ofinitial hyperplasia, intraepithelial neoplasia (PIN), adenocarci-noma, metastasis to peripheral organs including local lymphnodes and bone, and recurrence from androgen deprivationtherapy (1).

The few attempts to produce transgenic (Tg) mouse modelsof prostate cancer in the early 1990s suffered from a lack ofprostate specificity (2, 3) or from use of oncogenes irrelevant totypical human prostate cancer oncogenesis (4). An earlyattempt to increase prostate specificity, by using the PSA pro-moter to drive expression of a Ras oncogene, paradoxically didnot cause prostate cancer, but rather cancer of the salivaryglands and gastrointestinal tract (5). This changed, however,in 1994 when Maroulakou and colleagues (6) described Tgmice expressing the SV40 T-antigen (Tag) driven by the ratprostatic steroid binding protein C3 gene promoter. These micedevelop prostate hyperplasia that progress to PIN or adeno-carcinoma lesions after about 8 months of age. Only one case ofmetastasis (to the lung) was reported. Interestingly, females ofthis stock had more aggressive disease, succumbing to mam-mary adenocarcinomas, and in a few cases, lung metastasesafter 6 months of age. The slow prostate cancer progression andlack of metastatic potential, however, made this Tg modelsuboptimal.

Nature abhors a vacuum, and into this void came what isnow referred to as the transgenic adenocarcinoma mouseprostate (TRAMP) model, whose ability to form prostate cancerwas first described in a 1995 Proceedings of the NationalAcademy of Sciences article (7) and then characterized moreextensively for its metastatic potential in Gingrich and collea-gues (8). Unlike the model of Maroulakou and colleagues,TRAMP drives SV40-Tag oncogene expression by the rat pro-

basin (rPB) gene promoter. Specifically, the notion that rPBexpression was specific to the prostate epithelium was strength-ened by a decade of research from Robert Matusik's laboratory,culminating with the observation that the �426 to þ28 rPBpromoter region was sufficient to drive androgen-responsiveexpression (9). The clinical relevance for the use of SV40-Tag,which induces oncogenic progression by binding to and inac-tivating the Trp53 and Rb1 tumor suppressors (10), was basedon previous data showing loss of p53 and Rb in human prostatecancer (11, 12).

Because transgene chromosomal insertion sites as well as strainbackground were known to influence transgene expression levelsand phenotypic penetrance (13), initial production of TRAMPmice included eight independent founder lines in both C57BL/6and FVB backgrounds. Line 5666, which exhibited low Tagimmunohistochemical staining levels in the prostate epitheliumas well as restricted expression to the dorsal and ventral lobes ofthe prostate (compared with other prostate lobes and bodyorgans, assessed by RT-PCR), developed only epithelial hyper-plasia and low-grade PIN after 33 weeks. In contrast, line 8247exhibited PIN lesions by 10 weeks of age, hyperplasias in allprostate lobes by 22 weeks, and invasive adenocarcinomas by 20weeks of age (14). The Cancer Research article by Gingrich andcolleagues (15), where this model was first named TRAMP,followed mice over roughly 30 weeks for pathologic and immu-noreactive changes. Several common featureswereobserved. First,in the initial group of 17, all mice exhibited prostate neoplasiabetween 18 and 24 weeks, characterized by cribriform struc-tures and frequent apoptotic bodies. Second, neoplastic pros-tates were marked by stromal remodeling, specifically, hyper-proliferation of the fibromuscular stroma. This observation,now described as reactive stroma, is appreciated for the roleplayed by the prostate tumor microenvironment in prostatecancer progression (16). Third, although epithelial Tag expres-sion always preceded neoplastic transformation, individualepithelial cells exhibited variations in expression levels, espe-cially when comparing adjacent acini in the same mouse. Thissuggested cell-to-cell transgene heterogeneity, which mightaffect which cells (and acini) might progress oncogenicallywithin a lobe. Whether a similar phenomenon of variation inoncogene expression underlies the fact that a large portion ofhuman prostate-confined disease is multifocal yet marked byshared potential driver mutations (17) is not yet clear.

The most profound breakthrough of the TRAMP article was inthe frequency and breadth of metastases formed. Although thisarticle studied small cohorts, high frequencies ofmacrometastaseswere identified in lymphnodes and lungs (roughly a third of casesat 24weeks of age, rising to between two-thirds to 100%of cases at28 weeks), and lower frequencies in the kidney, adrenal glands,and bone (spine). Bone metastasis, marked by characteristicosteolytic histology, was only found in a single [TRAMP� FVB]F1mouse at 22 weeks of age, whereas no bone lesions were detectedin a 34-week-old [TRAMP � C57BL/6]F1 mouse. A subsequent

Department of Cancer Genetics, Roswell Park Cancer Institute,Buffalo, New York.

Corresponding Author: Irwin H. Gelman, Roswell Park Cancer Institute, Elmand Carlton Streets, Buffalo, NY 14263. Phone: 716-845-7681; Fax: 716-845-1698;E-mail: irwin.gelman@RoswellPark.org

doi: 10.1158/0008-5472.CAN-16-2636

�2016 American Association for Cancer Research.

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study by Gingrich and colleagues (18) with larger cohorts con-firmed that TRAMP mice in the FVB background suffered moreaggressive pathobiologies compared with those in the C57BL/6background. The initial prostate tumors andmetastases arising inthe 18- to 24-week old mice were identified as either HG-PIN oradenocarcinomas, marked by reduced or absent E-cadherin stain-ing, a hallmark of prostate cancer progression in humans (19).Greater than 40% of TRAMP mice castrated at 12 weeks, whichis either prior to overt neoplastic onset or at a PIN stage, developedrecurrent tumors that were muchmore poorly differentiated thanthe tumors arising in non-castrates (20), and castration increasedthe incidence of metastasis formation in the liver, salivary glands,and calvaria (21). Of note was the persistence of androgenreceptor (AR) expression during disease progression to adenocar-cinoma (18).

A provocative finding regarding the pathobiology of TRAMPmice is that poorly differentiated tumors arising in >28-week-old mice, or at a higher frequency in castrates, expressedneuroendocrine markers such as synaptophysin (21). Neuro-endocrine (NE) prostate cancer is rare, comprising less than 2%of all cases, and likely arises from rare, AR-negative NE cellsnormally found within the basal epithelial layer in acini (22).However, at least 50% of the synaptophysin-positive TRAMPtumors also expressed AR, suggesting a potential transdiffer-entiation phenomenon. This phenomenon has been describedin patient-derived xenograft models forced into castration-resistant (CR) growth (23), or in CR prostate cancer (PC)patients after prolonged therapy with AR antagonists (24). Thisalso may be influenced by the TRAMP strain background,because CR tumors in the FVB background seemed to beformed by NE lineage cells independent of those formingepithelial cell lesions (25, 26).

In the decade after the introduction of TRAMP mice, severalgroups addressed how the SV40-Tag and the nature of the PBpromoter affected the incidence of prostate adenocarcinomas,NEPC, and metastases. For example, Masumori and colleaguesproduced a Tg model expressing only the so-called "large Tag"(versus the "small t-ag" transcript included in the TRAMP con-struct) whose expression is driven by a larger, 12 Kb PB promoterthat contained additional androgen and growth factor–respon-sive sequences. This LPB (a.k.a. LADY) model produced PINlesions, adenocarcinomas andmetastases that expressed NEmar-kers starting after 24weeks, but at amuch lower frequency than inTRAMP mice (27). Zhang and colleagues (28) sought to addressthe fact that the rPB promoter in the TRAMP model had weakprostate epithelial expression, and thus, they produced a com-posite PB promoter (Arr2pb), which removed the inhibitorysequences from �426 to �287, and then introduced a secondcopy of the androgen response region (�244 to �96). Tg micewith Cre recombinase driven by the Arr2pb promoter showedstrong expression in the lateral lobe, with lesser expression in theventral, dorsal, and anterior lobes, and very low, but discernableexpression in the seminal vesicles (29). This Tg model has beenused extensively to conditionally knock out many genes in theprostate, although a caveat is that the expression of the transgene

in oocytes means that the Cre allele must be transferred throughmales only. One such use of the Arr2pb-Cre was to produce aconditional prostate-specific knockout of Tp53 and Rb1 (30)model, which exhibited both adenocarcinomas and NEPC, con-firming that the oncogenic transformation induced by Tag in theTRAMP model was likely through the inactivation of these twotumor suppressors.

TRAMP mice have been used extensively to dissect the rolesplayed by specific pathway mediators, transcription factors, ormetabolic pathways in prostate cancer progression. An earlyexample was the cross of TRAMP tomice lacking the transcriptionfactor, Egr-1 (early growth response protein-1; ref. 31), such thatthe loss of Egr-1 had no effect on the rate or time to neoplastictransformation, but blocked progression from PIN to adenocar-cinoma. Ablation of PKCe severely decreased adenocarcinomaand metastasis formation in TRAMP mice (32). Our groupaddressed the role of the Src family kinases, whose activity isknown to increase in prostate cancer progression (33), bycrossing TRAMP mice with Src-, Lyn-, or Fyn-null mice (34).Whereas the loss of Fyn or Lyn had little or no effect onadenocarcinoma or NEPC formation, the loss of Src severelyimpaired the frequency and time-to-onset of adenocarcinomas.A side note of this study is that in C57BL/6 TRAMP mice agedpast 30 weeks, a small percent (3%) of mice present as "tumor-free with metastases," that is, mice with PIN lesions yetparadoxically, exhibit extensive pulmonary metastases that arepredominantly adenocarcinomas. This suggests that, at least insome cases, prostate metastases derive from rare, aggressivecells that disseminate early. We found that the loss of Src, Fyn,or Lyn increased the frequency of this population >5-fold,strongly suggesting that these kinases might play a secondary,suppressive role in specific pathways of metastatic progression.

ConclusionsIn the two decades since the publication describing the TRAMP

model, many groups have produced multiple Tg models ofprostate cancer, incorporating other well-documented changesfound in human prostate cancer, such as Myc upregulation orPTEN loss. However, the TRAMP model has exhibited amazingstaying power in the field, even surviving its association withNEPC,whichwas originally thought to relegate it to a rare prostatecancer pathology, but which is now gaining appreciation as asignificant mechanism of disease progression arising from con-tinued AR antagonist therapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsI wish to thank David Goodrich and Barbara Foster for constructive

comments.

Received September 27, 2016; accepted September 27, 2016; publishedonline November 1, 2016.

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