A Tmprss2-CreERT2 Knock-In Mouse Model for Cancer Genetic Studies on Prostate and Colon

Fusion between TMPRSS2 and ERG, placing ERG under the control of the TMPRSS2 promoter, is the most frequent genetic alteration in prostate cancer, present in 40–50% of cases. The fusion event is an early, if not initiating, event in prostate cancer, implicating the TMPRSS2-positive prostate epithelial cell as the cancer cell of origin in fusion-positive prostate cancer. To introduce genetic alterations into Tmprss2-positive cells in mice in a temporal-specific manner, we generated a Tmprss2-CreERT2 knock-in mouse. We found robust tamoxifen-dependent Cre activation in the prostate luminal cells but not basal epithelial cells, as well as epithelial cells of the bladder and gastrointestinal (GI) tract. The knock-in allele on the Tmprss2 locus does not noticeably impact prostate, bladder, or gastrointestinal function. Deletion of Pten in Tmprss2-positive cells of adult mice generated neoplasia only in the prostate, while deletion of Apc in these cells generated neoplasia only in the GI tract. These results suggest that this new Tmprss2-CreERT2 mouse model will be a useful resource for genetic studies on prostate and colon.


Introduction
The prostate epithelium is comprised of two distinct cell layers-an outer layer of basal cells in contact with the stroma and an inner layer of secretory luminal cells that produce constituents of the prostatic fluid. Prostate cancer shares many molecular and histologic similarities with luminal cells, including growth dependence on the androgen receptor (AR), as well as ARdependent expression of seminal fluid proteases PSA and TMPRSS2. In contrast, prostate cancer seldom express basal markers and the absence of basal markers is a pathological criteria to diagnose prostate cancer.
Although most primary prostate cancers have luminal cell histology, the etiology and cell of origin of prostate cancer remain controversial. Early studies based on sphere formation in vitro and graft formation in vivo suggested that basal cells form the stem cells of the normal prostate and are the cells origin of prostate cancer [1,2]. However, recent studies using lineage tracing in genetically engineered mouse (GEM) models support the existence of a stem cell population in both the basal and luminal epithelia of the prostate [3,4] Within the luminal compartment, castration-resistant Nkx3-1-expressing cells (CARNs) have been shown to represent a source of stem cells that can regenerate the prostate epithelium and can be transformed when Pten is deleted [5]. Using the recently developed prostate 3D organoid culture system, we have shown that both single luminal and basal cells from either human or mouse prostates can give rise to organoids, implicating the existence of bipotential stem cells in each compartment [6,7]. Recent evidence also indicated that luminal cells are favored as cells of origin of prostate cancer [8].
Genomic fusion between the membrane-bound serine protease TMPRSS2 and the ETS-family transcription factor ERG is an early genetic alteration occurring in~50% of prostate cancers [9,10]. The fusion event results in ERG overexpression under the TMPRSS2 promoter. These findings support the notion that TMPRSS2-expressing cells are important for prostate cancer initiation, and genomic alterations of these cells may trigger pathogenetic events. The identity of TMPRSS2-expressing cells is not fully elucidated. Different studies have reported that Tmprss2 is preferentially expressed in basal cells, or luminal cells, or both [11,12,13].
In order to identify Tmprss2 expressing cells, trace their lineage, and determine their tumorigenic capacity, we generated a tamoxifen-inducible knock-in mouse model carrying the CreER T2 gene under the control of the Tmprss2 promoter. We demonstrate the high efficiency of this model to selectively delete genes in the prostate luminal epithelium and colon epithelium. Furthermore, we show that conditional deletion of Pten and Apc in Tmprss2 expressing cells lead to prostate and colorectal transformation, respectively.

Materials and Methods
Generation of the Tmprss2-CreER T2 mouse All mouse studies are approved by MSKCC Institutional Animal Care and Use Committee under protocol 11-12-027. Institutional guidelines for the proper, humane use of animals in research were followed.
To generate Tmprss2-FRT-NEO-FRT-CreER T2 targeting construct, we started with pRo-sa26PAm1 (a gift from Douglas Melton, Addgene #15036) [14,15], a targeting plasmid that contains PacI and AscI cloning sites between the 5' and 3' homology arms of the Rosa26 locus, followed by a diphtheria toxin cassette (DTA). We replaced the Rosa26 5' arm with 3.15 kb fragment 5' of exon 2 of the Tmprss2 gene, generated by PCR using the 5'-TGG CTT CTG CTT CTG ATG-3' and 5'-GCG TTA ATT AAG CCT TCA GCC TTC ACT TCA C-3' primer pair on a mouse BAC clone, and cloned using PacI and SacII sites. We then replaced the Rosa26 3' arm with a 5.05 kb fragment 3' of exon 2 of the Tmprss2 gene, generated by PCR using the 5'-GGG GCG CGC CTG GCC TTT TCC TTG TTC CT-3' and 5'-GGG GGT CGA CAT GTG GCT CAG TGG TAA A-3' primer pair, and cloned using AscI and SalI sites. PCR was performed using PFU turbo (Stratagene). We named the product pTmprss2PAm1.
Gene targeting was performed at the Rockefeller University Gene Targeting Resource Center (Head: Chingwen Yang). The targeting plasmid was electrophoresed into albino C57BL/6J ES cells and G418 resistant clones were isolated by standard procedures. The clones were screened by Southern blotting using an external 3' probe generated by PCR primers 5'-GTC ACC CCT CAC TGC ATT TT-3' and 5'-ATG GAC ACT CCC AGG CTA GA-3' cut by Hin-dIII which gave a wild-type 7.5kb band and targeted 8.2kb band. Two positive clones were injected into C57BL/6J blastocysts by the MSKCC Mouse Genetics Core Facility (Head: Willie Mark), and chimeras were mated with albino C57BL/6J females. Germline transmission was confirmed in albino offspring using Southern blotting.
Mice tamoxifen treatment, sacrifice and tissue analysis TY, TP and TA mice were injected with tamoxifen at 4mg/40g body weight using a 27-gauge needle and injected once every other day, a total of 3 times. Mice are euthanized by carbon dioxide asphyxiation as recommended by MSKCC Institutional Animal Care and Use Committee under protocol 11-12-027.
For histology and IHC, tissue was harvested from 10-week-old TY males, 22-week-old TP males and 12-week-old TA males. Tissues were fixed in 4% paraformaldehyde (w/v) at 4 degrees Celsius for 12 hours. Tissues were washed three times with cold PBS. All immunohistochemical and histological analyses were conducted by the MSKCC Molecular Cytology Core.
For immunofluorescence, tissues were fixed in 4% paraformaldehyde (w/v) at 4 degrees Celsius for 2 hours. Tissues were washed three times with cold PBS, cryopreserved by overnight incubation in 30% sucrose (w/w), frozen in OCT (Tissue Tek, Sakura Finetek) and sectioned.

Results
In order to define Tmprss2-expressing cells in prostate, we generated an inducible CreER T2 -IRES-nlsGFP mouse model under the control of the mouse Tmprss2 gene promoter. At baseline levels, nuclear GFP expression marks Tmprss2-expressing cells and the Tmprss2-positive lineage can be traced and genetically manipulated when crossed with LoxP-lines and exposed to tamoxifen.
In human prostate cancer, the first exon of TMPRSS2 is entirely within the non-coding 5' UTR and most TMPRSS2-ERG fusions involve the first intron of TMPRSS2, with the resulting fusion transcript containing no coding sequence of TMPRSS2. We therefore replaced exon 2 with a cassette including an adenovirus splice acceptor (SA), followed by a PGK-driven neomycin selection cassette flanked by FRT recombination sites, followed by the CreER T2 -IRES-nlsGFP. After excision of the neomycin cassette, the mouse should express a chimeric transcript containing exon1 of Tmprss2 as the 5' UTR followed by CreER T2 cDNA (Fig 1A).
After homologous recombination in embryonic stem cells and germline transmission, we observed two independent lines with the correct integration verified by Southern blot analysis using both 5' and 3' probes ( Fig 1B). We next crossed F1 mice with Actb-FlpE mice and Southern blot analysis using the 5' probe confirmed excision of the neomycin cassette in all FlpE and CreER T2 double-positive F2 mice (Fig 1C). Genotyping for the Tmprss2-CreER T2 allele was performed by polymerase chain reaction (PCR), resulting in a 380-bp product for the knock-in (a) Schematic of targeting strategy. A cassette including an adenovirus splice acceptor (SA), followed by PGK-driven neomycin selection cassette flanked by FRT recombination sites, followed by the CreER T2 -IRES-nlsEGFP was used to replace exon 2 of mouse Tmprss2. The cassette is flanked by 3.5kb 5' and 5kb 3' homology arms. 5' and 3' Southern probes as was as HindIII (H) and EcoRI (E) sites and genotyping PCR primers (universal F1, wild-type specific R1, and knock-in specific R2) are depicted. The final transcript includes the non-coding exon 1 of Tmprss2 spliced into the CreER T2 -IRES-nlsEGFP gene. (b) Southern blot using 3' probe and HindIII digestion. WT mice give a 7.5 kb band and the targeted mice (regardless of neomycin cassette) give a 8.2 kb band. (c) Southern blot using 5' probe and EcoRI digestion. WT mice give a 9.5 kb band; the targeted mice with neomycin cassette (T) give a 9.9 kb band, while the mice with excised neomycin cassette (E) give a 9.5 kb band. (d) Genotype determination of wild-type and heterozygous mice by PCR. Wild-type fragment is 300-bp and mutant is 380-bp. Tmprss2-CreER T2 Knock-In Mouse allele and a 300-bp product for wild-type (Fig 1A and 1D). Homozygous Tmprss2-CreER T2 had no visible phenotype and were generated at Mendelian ratio, consistent with prior observation that Tmprss2 knockout mice had no phenotype [24].
The nuclear GFP signal of CreER T2 -IRES-nlsGFP is dim at baseline levels and is difficult for direct lineage trace. To test the efficiency and specificity of the novel knock-in line, we crossed Tmprss2-CreER T2 mice with Rosa26-EYFP mice with a CAG driven YFP Cre-reporter [19]. We examined the YFP expression pattern in the prostate gland of 10-week-old male Tmprss2-CreER T2 ; Rosa26-EYFP /EYFP (TY) mice after 2 weeks of tamoxifen treatment. YFP immunohistochemistry (IHC) showed that labeling was highly efficient, and the majority of the prostate epithelium appeared labeled (Fig 2A). Analysis of direct YFP fluorescence combined with immunofluorescence (IF) against luminal marker Cytokeratin 8 (Ck8) or basal maker p63 showed that only the Ck8 positive luminal cells exhibited YFP signal, while the p63 positive basal cells and stroma cells were negative for YFP in the anterior, dorsolateral and ventral lobes of the prostate (Fig 2B and 2C). Quantification of YFP-expressing cells indicated that in the anterior and dorsolateral lobes, approaching 100% of luminal epithelial cells (n = 635) were positive and in the ventral lobe, approximately 80% of luminal cells (n = 476) were positive. We separated YFP-positive and YFP-negative prostate epithelia cells from TY mice anterior prostate tissue using florescence activated cell sorting (FACS). Consistent with our histological observation, the YFP-positive population had high luminal-cell-specific gene expression, Ck8 and Ck18, while the YFP-negative population had high basal-cell-specific gene expression, P63, Ck5 and Ck14 (Fig 2D).We compared the Tmprss2-CreER T2 recombinase activity with that of Nkx3-1-CreER T2 , a commonly used inducible Cre-driver for prostate luminal cells. We crossed both lines to Rosa26-mT/mG, a mouse that expresses membrane-targeted tdTomato (mT) at baseline-and membrane-targeted GFP (mG) after excision [20] and found two important differences that highlight the utility of the Tmprss2-CreER T2 mouse model. First, the efficiency of recombination within prostate luminal cells is much higher in Tmprss2-CreER T2 (99.6%, n = 234) compared to Nkx3-1-CreER T2 (23.7%, n = 223). Second, within the proximal peri-urethral prostate, which is comprised of more compact epithelial cells and is thought to have a greater number of stem cells, Tmprss2-CreER T2 is highly active (97.7%, n = 215), whereas Nkx3-1-CreER T2 lacks activity (1.04%, n = 192) (Fig 2E and 2F).
Mining of normal tissue gene expression data showed that TMPRSS2 is not prostate-specific and is expressed in multiple organs, specifically of the GI tract in both human and mouse ( Fig  3A and 3B). Notably, TMPRSS2 expression also have difference between human and mouse, such as bladder, which has relatively high expression in mouse, but it appears to be quite low in human. To determine the tissue specificity of CreER T2 activity beyond the prostate, nineteen other tissues, namely the testis, epididymis, seminal vesicle, pancreas, colon, small intestine, stomach, esophagus, bladder, bone marrow, brain, heart, kidney, liver, lung, skin, spleen, thymus and thyroid were collected from male TY mice and analyzed for YFP staining. We found that almost all the epithelial cells of colon and bladder were positive for YFP staining, while Tmprss2-CreER T2 Knock-In Mouse there were no detectable YFP positive stroma cells in these organs (Fig 3C and 3D). Recombination was also observed in epididymis, seminal vesicle, pancreas, small intestine, stomach, esophagus, kidney, liver, lung, skin and thyroid (Fig 3C).
We asked whether Tmprss2-CreER T2 is active in the tumor initiating cells and if Tmprss2-CreER T2 -mediated deletion of tumor suppressor genes can generate tissue specific tumorigenesis. First, we analyzed deletion of Pten, a tumor suppressor implicated in prostate cancer initiation. To achieve Pten deletion, we crossed Pten LoxP/LoxP mice [18] to the Tmprss2-CreER T2 knock-in line. We treated the Tmprss2-CreER T2/ T2 ; Pten LoxP/LoxP (TP) mice with tamoxifen at 8 weeks of age and analyzed the prostate and colon 12 weeks later. Hematoxylin and eosin (H&E) staining of the prostate showed prevalent prostatic intraepithelial neoplasia (mPin), with cribriform growth and enlarged nuclei in tamoxifen but not vehicle injected mice (Fig 4A). IHC and IF staining of PTEN and P63 showed that Pten loss is specific to the prostate luminal epithelial cells, while Pten is expressed in some prostate basal epithelial cells and stromal cells surrounding the prostate acini (Fig 4A and 4B). The loss of Pten corresponds with an increase of AKT phosphorylation in tamoxifen treated prostate luminal cells. We next analyzed colonic epithelium in these TP mice and found no visible gross or histological abnormalities. IHC analysis showed robust loss of Pten throughout the colonic epithelium. However, AKT phosphorylation was not detectable using the same staining method as the prostate pAKT staining, suggesting very low baseline PI-3 kinase activity that cannot induce AKT phosphorylation despite loss of Pten (Fig 4C).
We next analyzed deletion of Apc, a gatekeeper in colorectal cancer tumorigenesis. We crossed Apc LoxP/LoxP mice to the Tmprss2-CreER T2 mice and treated the Tmprss2-CreER T2/T2 ; Apc LoxP/LoxP (TA) mice with tamoxifen at 8 weeks of age and analyzed the mice colon and prostate 12 weeks post tamoxifen treatment. PCR of genomic DNA isolated from prostate and colon showed the presence of PCR product specific for Apc-deletion only in tamoxifen-treated mice ( Fig 5A). The colonic epithelium of tamoxifen treated TA mice showed features characteristic of Apc-loss adenomas (Fig 5B). The loss of Apc in these mice corresponds to an increase in β-catenin staining and marked hyperproliferation with increased Ki67 and Pcna staining outside the crypts, characteristic of differentiation arrest by the identification of lysozyme-positive Paneth cells outside the base of crypt (Fig 5C). Although the prostate had loss of Apc (Fig 5A), the prostate exhibited normal histology (Fig 5D). IHC showed a subtle increase in membrane-localized β-catenin levels in cells and no change in Ki67 index (Fig 5D).

Discussion
GEM models of cancer have provided important insights into cancer initiation and progression, and have important implications for clinical studies and clinical trials. Various mouse lines were generated over recent years by selectively introducing targeted mutations in prostatic epithelium through Cre recombinase under the control of the mouse mammary tumor virus promoter MMTV-Cre [25], a modified probasin promoter PB-Cre4 [26], or the prostate-specific antigen (PSA) promoter PSA-Cre [27,28]. More recently, several prostate-specific CreER T2 mouse models, including knock-in to the Nkx3-1 locus [5], transgenic under the human PSA promoter [29] and transgenic under a modified probasin promoter (ARR2Pb) [30] have been generated, each able to induce mPin when crossed to Pten LoxP/LoxP mice. This  Tmprss2-CreER T2 knock-in strain is highly efficient and allows precise control over the timing of introducing gene alterations in the mouse prostate and colon, and accurately mimics the late onset of human prostate cancer and colon cancer. Here, we show that Tmprss2-CreER T2 is expressed in several organs and rapidly induces prostate neoplasia after induction of Pten deletion and colorectal neoplasia after induction of Apc deletion. We further find that the effects of oncogenic activation are surprisingly tissuespecific and recapitulate that of human cancer. In human prostate cancer, PTEN loss is an early tumorigenic lesion, while Wnt-pathway activation through aberrations in APC, CTNNB1, RNF43, and RSPO2 are found in metastatic castration-resistant cancer, suggesting they are associated with tumor progression [31,32]. In human colorectal cancer, Wnt-pathway activation is a gatekeeper event while mutations of loss of PTEN rarely occur.
In conclusion, we have established a new CreER T2 knock-in mouse line, and these knock-in mice can be used to investigate Tmprss2-expressing cells and their descendant cells at various stages in vivo. We believe that the generated knock-in mice in this article could be useful for studying the initiation and progression of prostate and colon cancers.