*3.2. Genetically Engineered Mouse Models of Prostate Cancer*

The mouse models are beneficial resources to improve our understanding of the disease pathobiology and to establish the role of candidate oncogenes in the pathogenic processes. As discussed below, several genetically engineered mouse models of PCa have been developed that have provided insights into tumor initiation, progression, and metastasis and are being used in preclinical research.

#### 3.2.1. TRAMP

The transgenic adenocarcinoma of the mouse prostate (TRAMP) mice model was generated and characterized in 1996. The chloramphenicol acetyltransferase (CAT) gene was introduced into the germ line of mice under the control of the rat probasin (PB) promoter. In TRAMP mice, expression of both the large and small SV40 T antigens (TAG) is regulated by the prostate-specific rat PB promoter [154]. The PB-SV40 T antigen (PB-Tag) transgene is spatially restricted to the dorsolateral and ventral lobes of the prostate. The gene expression is male specific and restricted to the epithelial cells of the lateral, dorsal, and ventral prostatic lobes of the murine prostate [155]. TRAMP is a very useful model for studying the pathology of PCa as the progression occurs through PIN lesions to malignant disease, like human disease, in a predictable time. Epithelial hyperplasia develops by 10 weeks of age, PIN by 18 weeks of age, and lymphatic metastases after 28 weeks of age [154,156,157].

The TRAMP model has been used for PCa prevention and treatment studies [158,159]. It is also the first genetically engineered mouse model (GEMM) that displays castration-resistant disease progression [160]. One of the limitations of the TRAMP model, however, is that these mice often develop neuroendocrine PCa [161]. A simultaneous loss of *Rb* and *p53* could be the reason for the development of neuroendocrine cancer [161,162]. Considering the higher chances of neuroendocrine disease, the TRAMP mouse model is clinically more relevant to study PCa of neuroendocrine origin.

#### 3.2.2. LADY

The LADY PCa mouse model was developed in 1998 and is similar to the TRAMP model [163]. There are, however, a few key differences between the TRAMP and LADY. In the LADY, a larger fragment (12 kb) of the PB (a.k.a. LPB) promoter upstream of the SV40 T-antigen is used that contains additional androgen and growth factor-responsive sequences and thus allows consistently high transgene expression. Additionally, the LPB promoter is linked with a deletion mutant of the SV40 T-antigen (deleted small T-antigen) to allow the expression of large T-antigen, unlike small t-antigen in the TRAMP model. The purpose of deleting small t-antigen was to analyze the importance of neuroendocrine differences in metastatic lesions developed by LADY [164]. LADY model mice develop metastases to the liver, lymph nodes, and bones [164]. The metastases, however, primarily contain neuroendocrine cells, which is unlike the human metastasis [135,165]. Thus, the LADY mice are different from the most common type of human PCa from the perspective of rapid tumor growth and neuroendocrine tumor development. Nevertheless, the LADY model possesses the molecular changes similar to the human prostate, such as the multifocal nature of tumorigenesis, histopathologically changes from low- to high-grade dysplasia similar to PIN in humans, and the androgen-dependent growth of the primary tumors. Hence, the LADY model could be beneficial for investigating the stepwise mechanisms of PCa progression as well as therapeutic intervention [163].

#### 3.2.3. Pten Deficient Mice

Loss of the *PTEN* tumor suppressor is a critical event in PCa initiation, as discussed above. However, homozygous knockout of *Pten* in mice embryonic stem cells through the deletion of the phosphatase domain led to embryonic lethality [166,167]. To overcome this limitation, Wang et al. generated *Pten* null mice by conditional deletion of *Pten* in the murine prostatic epithelium. They generated *Pten* loxp/loxp: PB-Cre4 mice in order to attain the prostate-specific *Pten* biallelic deletion. They showed that *Pten* null PCa progressed with a short latency of PIN formation by 6 weeks of age compared to heterozygous *Pten* deletion mice, which developed PIN by 10 months. Moreover, homozygous *Pten* deletion mice developed invasive adenocarcinoma by 9 weeks of age and metastasis to the lymph node and lung by 12 weeks of age. The effect of hormone ablation therapy on *Pten* null mice was evaluated by performing the castration of mice at week 16. The response of *Pten* null tumors at day 3 and day 6 post-castration was analyzed. In response to androgen abolition, the AR-positive prostatic epithelium showed an increase in the apoptosis leading to the decrease of prostate volume. Hence, these homozygous *Pten* mutant mice recapitulate the PCa by mimicking the histopathological features of human disease [40]. In contrast, heterozygous mutant *(Pten*+/−*)* mice developed neoplasia in multiple tissues, including mammary glands, lymphoid cells, small intestines, thyroid, endometrial, and adrenal glands [166,168,169], further limiting the applicability of the heterozygous mutant over *Pten* null mice.

The *Pten* knockout model has been used to demonstrate the role of the tumor microenvironment, particularly interleukin-17 (IL-17), in the growth and progression of PCa [170,171]. To test how tumor suppressor *Rb* interacts with *Pten*, Bai et al. developed mice with double mutations in both the cyclin-dependent kinase (CDK) inhibitor *p18Ink4c* and *Pten* [172]. The double mutant mice develop a broader spectrum of prostate tumors in the anterior and dorsolateral lobes at an accelerated rate [172]. Loss of function of *Nkx3.1* is crucial for PCa progression and has been associated with the development of prostatic epithelial hyperplasia, dysplasia, and PIN [30,67,173]. *Nkx3.1* and *Pten* are shown to cooperate in prostate carcinogenesis in mice. *Nkx3.1;Pten* double mutant mice demonstrated an increased incidence of HGPIN, which resembles the early stages of human PCa [69].
