Are the used ER mouse models suitable for knockout experiments?

To date, different in vivo approaches were performed to elucidate the role of ERs in PCa.

The two types of the ER, namely ER α and ER β, were described to play a role in prostate and PCa formation. Whereas ER α is described to be mainly expressed in the stromal

compartment of the prostate, ER β is mainly expressed in basal-epithelial prostate cells (Leav et al. 2001; Royuela et al. 2001). Neonatal or long term exposure to estrogens together with testosterone leads in 100 % of cases to a development of PCa in rats (Bosland et al. 1995). Studies using ER α knockout (ERKO) and ER β knockout (BERKO) mice showed that this estrogen/testosterone-induced carcinogenesis is mediated by the ER α (Ricke et al. 2008). Another study by Wang et al. (2004) described that ER α expression is increased in TRAMP mice prostate compared to wild type prostate, further supporting the evidence that ER α exhibits PCa promoting function. Furthermore, studies performed by McPherson et al. (2007), using aromatase-deficient mice that lack the capability to produce estrogens, showed that ER β but not ER α activation suppresses development of prostatic hyperplasia. Further evidence for the role of ER β in prostate cancer was described by Horvath et al. (2001). These authors analyzed the expression of ER β in five normal prostates and 159 specimens from radical prostatectomies. Horvath et al. (2001) identified high ER β expression in 100 % of normal prostate and declining ER β expression in hyperplasic prostate tissue (24.2 % ER β-positive) and carcinomas (11.3 % ER β-positive).

Furthermore, ER β expression was found to be associated with a longer relapse-free survival (Horvath et al. 2001). Taken together, these data indicate that ER α rather promotes PCa development, whereas ER β might act as a tumor suppressor in PCa.

In the present study, ERKO/TRAMP and BERKO/TRAMP mouse models previously described by Slusarz et al. (2012) were used to investigate the role of ERs in PCa. The TRAMP (transgenic adenocarcinoma of mouse prostate) mouse model expresses the simian virus 40 (SV40) T/t antigens under the control of the prostate-specific probasin promoter. T/t antigens are dominant active oncoproteins, inactivating the tumor suppressor proteins retinoblastoma (pRb) and p53 (Ahuja et al. 2005). Due to the SV40 T/t expression, PCa development is induced. In the present study one of the major aims were to investigate how the knockout of ER α or ER β could influence prostate cancer development and progression in ERKO/TRAMP and BERKO/TRAMP mice. For further investigations primary PCa cell lines should be generated from tumors of these mice in order to elucidate cellular ER signaling.

Here, it was observed that ERKO/TRAMP mice developed PIN lesions and massive SGCa.

The incidence of SGCa was not increased in ERKO/TRAMP mice compared to TRAMP and BERKO/TRAMP mice, but the SGCa growth proceeded faster and mice had to be sacrificed earlier. The data collected in this study correspond with the current knowledge concerning the growth promoting role of ER α in PCa (Ricke et al. 2008; McPherson et al. 2007).

However, no long term studies could be performed using the ERKO/TRAMP model because the accelerated growth of SGCa constrained early termination of the experiments. The development of SGCa in TRAMP mice is a well-known disadvantage of the TRAMP mouse model since SGCa do only seldom occur in humans (Tani et al. 2005; Tarjan et al. 2009).

However, most studies using TRAMP mice did not comment on SGCa growth, i.e. Slusarz et

al. (2012). It remains unclear why SGCa growth is accelerated in ERKO/TRAMP mice.

Comparing the data of the present study to the data presented by Slusarz et al. (2012), it is apparent that classification of PCa stage used by Slusarz et al. (2012) differs from the classification used in the present study. Slusarz et al. performed all experiments with 5-month old mice and differentiated between non-cancerous stages (normal tissue, hyperplasic tissue and PIN lesions) and cancerous stages (well-differentiated carcinoma (WDC), moderate-differentiated carcinoma (MDC) and poorly-differentiated carcinoma (PDC)). MDC were only described in few cases and were not further described by Slusarz et al., i.e. by a representative picture. The current study differentiated between normal prostate tissue (which was not observed in any mouse with a TRAMP genotype), PIN lesions, PCa and metastatic PCa. Furthermore, the present study analyzed the incidence of metastatic PCa or SGCa, which were both not described by Slusarz et al. (2012). However, reduced PCa incidence in ERKO/TRAMP mice observed in the present study was also shown by Slusarz et al. (2012), i.e. these authors found a reduction of PDC incidence in ERKO/TRAMP by 75 % compared to TRAMP mice. In contrast to the present study, Slusarz et al. (2012) reported a reduced incidence of PIN lesions in ERKO/TRAMP mice compared to TRAMP mice (only 5 % in ERKO/TRAMP mice compared to 23 % in TRAMP mice) and an increased incidence of WDC (85 % in ERKO/TRAMP compared to 50 % in TRAMP mice) (see Tab.

4.1). However, comparing the representative tissue sections shown by Slusarz et al. (2012) for PIN lesions, WDC and PDC to the criteria used in the present study for PIN lesions, PCa and metastatic PCa, it became clear that a comparison between the data could be misleading. The representative pictures of WDCs shown in Slusarz et al. (2012) would have been classified as PIN lesions in the present study. Therefore, a comparison between the present study and Slusarz et al (2012) is limited by the differently chosen classification of tumor sections. In the present study ERKO/TRAMP mice developed only PIN, but progression to PCa was not observed, thus, primary cell cultures could not be established to further investigate the molecular mechanisms behind the ER α knockout.

In contrast to Slusarz et al. (2012), the present study could not show a tumor suppressive function of ER β in PCa. The BERKO/TRAMP mice developed PCa, but the occurrence of PCa in BERKO/TRAMP compared to TRAMP mice was not increased. Slusarz et al. (2012) described that twice as much PDCs were found in BERKO/TRAMP compared to TRAMP mice. Furthermore Slusarz et al. (2012) described a 15 % reduced incidence of WDC compared to TRAMP mice. In the present study, an increase of tumor incidence and evidence for increased tumor aggressiveness was not observed, e.g. increased metastasis formation could also not be detected in BERKO/TRAMP PCa compared to TRAMP PCa (see Tab. 4.1).

Moreover, in vitro analysis in the present study did not reveal increased proliferation rate in BERKO/TRAMP murine primary PCa cells compared to TRAMP murine primary PCa cells.

Table 4.1: Development of PCa in TRAMP, ERKO/TRAMP and BERKO/TRAMP mice observed in the present study compared to Slusarz et al. (2012). Listed are the incidences and progression stages of PCa in TRAMP, ERKO/TRAMP and BERKO/TRAMP mice observed in the present study and by Slusarz et al. (2012). PIN: prostatic intraepithelial neoplasia, WDP: well-differentiated PCa, PDC: poorly-differentiated PCa

The present study Slusarz et al. (2012)

Mouse strain PIN PCa metastatic

PCa

PIN WDC PDC

TRAMP 56 % 17 % 21 % 23 % 50 % 20 %

ERKO/TRAMP 100 % 0 % 0 % 5 % 85 % 5 %

BERKO/TRAMP 70 % 15 % 15 % 26 % 35 % 39 %

However, in the present study it could not be definitely proven that the BERKO mice indeed did not express ER β. The genotyping PCR as well as the sequencing analysis confirmed the BERKO genotype of the used mouse model. Nonetheless, protein analysis detected ER β protein expression in kidney of BERKO/TRAMP mice, liver, testis and prostate of TRAMP-FVB mice, human VCaP cells, primary, murine PCa cell line T244 and mouse brain. The ER β expression was diminished by an ER β-specific blocking peptide. Two possible explanations for this phenomenon seem to be reasonable. First, the observed expression of ER β protein is an artifact which is attributable to a non-specific binding of the ER β antibodies used in the western blot experiments. Variable specificity of ER β antibodies was already discussed in a recent study (Skliris et al. 2002). Skliris and coworkers examined the specificity of seven ER β-specific antibodies on human breast cancer samples. They found that specificity is strongly dependent on the used method (immunohistochemistry, western blot and flow cytometry) and differs strongly between the tested antibodies. Therefore, specificity of ER β-detecting antibodies constitutes a major problem in research. Assuming that the lack of antibody specificity leads to the detection of an unspecific signal, the mouse model used in the present study exhibits indeed an ER β knockout and could therefore not support the described tumor suppressive function of ER β in PCa (Horvath et al. 2001;

Hurtado et al. 2008; Dey et al. 2014). Indeed, the study by Skliris et al. did not describe the ER β antibodies used in the present experiments and ER β antibody specificity might have improved since 2002. In the present work detection of ER β expression in different tissues was reproducible with different ER β antibodies and diminished by co-incubation with an ER β-specific peptide. These results indicate that the ER β antibody is specific. Interestingly, Slusarz et al. (2012) did not provide results using western blot analysis in their study to validate the ER β knockout in BERKO/TRAMP mice, but immunohistochemical staining was performed on tissue sections to prove the ER β knockout.

A second explanation for the discrepancies observed in the present BERKO/TRAMP mouse model could be that the knockout of ER β in the used mouse model was not quite complete.

Although sequencing analysis confirmed that sequence aberrations did not occur in the respective knockout cassette, the possibility of alternative splicing cannot be ruled out entirely. The knockout cassette is located in exon 3 of the mouse Esr2 (ER β) gene (Krege et al. 1998), however, an ER β splice variant with a deletion of exon 3 was already described (ER β ∆ex3) (Fig. 4.1). Exon 3 of the mouse Esr2 gene encodes for the DNA binding domain of the ER β and therefore is essential for the transcriptional activity of the ER β (Price, JR et al. 2001). Only recently Maneix et al. (2015) described that mice with expression of ER β ∆ex3 showed only mild impairments compared to a full ER β knockout. They conclude that several functions of ER β are independent of the transcriptional activity of the receptor.

Thus, it can be assumed that the expression of ER β ∆ex3 is responsible for the observed ER β signal in the present western blot experiments which in turn leads to the lack of evidence of ER β-mediated tumor suppressive function in the present study.

Figure 4.1: Structure of the wild type and ∆ex3 Esr2 gene and protein. Depicted is a schematic illustration of the wild type Esr2 gene and protein (upper figure) and the ∆ex3 Esr2 gene and protein described by Maneix et al (2015) (lower figure). The deletion of exon 3 results in the generation of an ER β protein lacking the DNA binding domain. We hypothesized that the BERKO mouse model used in the present study expresses a comparable ER β splice variant, lacking exon 3 and therefore also lacking the knockout cassette. Modified according to Maneix et al. (2015) and Smart et al. (2013).

The role of human ER β splice variants was described by different studies and it was suggested that in contrast to full length ER β, ER β splice variants exhibit rather tumor promoting than tumor suppressive functions in LNCaP and PC3 PCa cell lines (Leung et al.

2010; Dey et al. 2012; Hurtado et al. 2008). Especially the splice variants ER β 2 and ER β 5 are suspected to exhibit tumor promoting functions. Both ER β splice variants lack the AF-2 domain and differ in the LBD from full length ER β (Christoforou et al. 2014). In contrast to

human ER β, only little is known about the role of murine ER β splice variants in PCa. One study by Lu et al. (1998) suggested that human and murine ER β splice variants might not equal each other. However, to date, tumor promoting function could not be associated with the murine ER β splice variant ER β ∆ex3 (Maneix et al. 2015).

Taken together, the in vivo and in vitro results of the present study indicate that the chosen mouse models were not quite suitable for the molecular analysis of the role of ER α or ER β in PCa development and progression. On the one hand, ERKO/TRAMP mice only developed PIN lesions in the prostate, but these premalignant lesions did not further progress to PCa, and therefore primary cell cultures could not be established. On the other hand, BERKO/TRAMP mice did not show the expected phenotype with increased aggressive PCa development and moreover, could not be clearly identified to be completely ER β-negative.