Fundamentos de la argumentación

The AR protein is encoded by the AR gene on the Xq11-12 chromosome (Hsing et al., 2002). The AR is a member of the steroid hormone receptor family of ligand-activated nuclear transcription factor and contains four functional regions, viz.: an amino terminal regulatory domain (amino terminal), a DNA-binding domain (DBD), a hinge region containing a nuclear localization signal, and a carboxy-terminal ligand-binding domain (LBD) (fig 3.9). ARs localized in the cytoplasm are unligated, but bound to heat shock proteins (HSPs), which stabilize the ARs tertiary structure in a conformation that allows androgen binding. Once ligand bound, the AR dissoc iates from the HSPs, dimerizes and subsequent to tyrosine kinase phosphorylation, is translocated into the nucleus. In the nucleus, the AR forms an active transcription complex by binding the androgen response elements (AREs) located in the promoter and enhancer regions of target genes (Taplin, 2007).

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Figure 3.9: AR structure and function, representing the binding of DHT and the formation of a transcription complex of co-regulatory proteins and transcription of androgen-regulated genes. Abbreviations are as follows: AES, amino-terminal enhancer of split; C, carboxy terminal; CBP, CREB -binding protein; HAT, histone acetylases; HDAC3, histone deacetylase 3; HDAC6, histone deacetylase 6; HEY1, hairy/enhancer - of-split related with YRPW motif 1; HSP90, heat shock protein 90; N, amino terminal; NCOR, nuclear corepressor; PCAF, p300/CBP- associated factor; PSA, prostate-specific antigen; RNA pol II, RNA polymerase II; SMRT, silencing mediator of retinoid and thyroid; SRC, nuclear receptor co-activator; TF7L2, transcription factor 7-like 2; TLE, transducin-like enhancer of split. Reproduced with permission from (Taplin, 2007).

The AR plays a central role in normal and PCa cell growth and proliferation. The AR is express ed in all stages of PCa, with somatic mutations of the AR gene involved in the progression and aggressiveness of PCa. Therefore, with the onset of PCa, ADT - castration, as well as AR blockage through usage of AR antagonists – is included in the treatment regime. When PCa recurs it is likely to be more aggressive, with AR amplification reported in one third of the cases, demonstrating an adaptation within the PCa cells (Holzbeierlein et al., 2004; Taplin et al., 1995). Furthermore, AR mutations can also occur which allow activation of the AR by weak androgens which may not have activated the AR prior to CRPC. AR mutation frequencies ranging from 0 to 44% and 0 to 50% have been reported for androgen-dependent PCa and CRPC, respectively (Mohler et al., 2004). In addition, it also possible for increased expression of transcriptional co- activator proteins, as another adaptation in PCa cells, to allow the activation of signal transduction pathways which would enhance AR responses to weak androgens (Yuan & Balk, 2009).

Together with increased AR expression, an increase in AR-regulated genes such as PSA, also occur. Transcription of PSA is positively regulated by the AR, and consistently expressed in PCa. PSA, a serum marker for PCa and treatment response, is a serine protease protein which is androgen-regulated. PSA, a member of the kallikrein family proteases, is a major part of semen (0.5 – 2 mg/mL), with the function to cleave semenogelins in the seminal coagulum. PSA is

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produced by prostate ductal and acinar epithelium and is secreted into prostatic ducts as an inactive 244-amino acid pro-enzyme (proPSA) that is activated by cleavage of seven N-terminal amino acids. PSA that enters circulation is rapidly inactivated in the lumen by proteolysis and circulates as free PSA, or is bound by protease inhibitors, including α1-antichymotrypsin (ACT) (Balk et al., 2003).

The normal physiology of the human prostate reflects a single layer of secretory epithelial cells, which are surrounded by a continuous layer of basal cells and a basement membrane. There is a concomitant disruption of the basal cell layer and basement membrane with the onset of PCa, and the loss of normal glandular architecture appears to allow direct access of PSA to peripheral circulation, as shown in Fig. 3.10 (Balk et al., 2003; Hayward & Cunha, 2000). Partial basal cell loss has been reported in prostate intraepithelial neoplasia, while complete basal cell loss is characteristic of PCa.

Figure 3.10: Model of PSA activation in normal prostate epithelium versus cancer. Reproduced with permission from (Balk et al., 2003).

Total PSA serum levels are increased in PCa, hence the screening of PSA as an indicator of PCa progression. Analyses of free and total PSA levels can increase the screening accuracy of PSA in PCa, allowing for discrimination between levels detected in the case of the healthy prostate and levels detected in PCa, as the PSA index (free PSA: total PSA) has been reported to be decreased in PCa (Catalona et al., 1998). An initial decline in PSA levels, in response to ADT, is partly due to tumour cell death and partly due to reduced AR-stimulated PSA production by surviving tumour cells. However, PSA expression continues to be maintained even after ADT. The AR is therefore still stabilized and activated by tissue androgens. Indeed, the presence of PSA in CRPC consistently correlates with the presence of an activated AR, as differential expression, subtractive

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hybridization and cDNA microarrays showed PSA expression before and after castration in tumour models of androgen-dependent PCa (Mohler et al., 2004).

In castrate tumours DHT levels, between 0.5 and 1.0 ng/g, are sufficient to activate the AR, which subsequently stimulate the expression of AR-driven genes, such as PSA, and thereby mediates tumour growth and progression of PCa to CRPC (Culig et al., 1999; Gregory et al., 1998; Gregory et al., 2001; Mohler et al., 2004). Two cellular events occur in prostate cells: cellular proliferation and PSA production – however, cellular proliferation has been shown at low DHT concentrations, and high PSA levels at high DHT concentrations. It is necessary to note, that although the secretion of PSA in prostate cells is stimulated by androgens (DHT), it does not correlate with cell proliferation. A study by Lee et al., (1995), showed that at low DHT concentrations LNCaP cells proliferate at a high rate, but at high DHT concentrations, cell proliferation decreases with a concomitant rise in PSA levels (Lee et al., 1995).

In addition ERs should also be mentioned, especially ERβ, which is anti-proliferative and proapoptotic (Imamov et al., 2004) and is expressed in both normal prostate as well as in PCa (Lai et al., 2004; Leav et al., 2001). ERβ was reported to be predominantly localized in basal cells and to a lesser extent in stromal cell nuclei (Leav et al., 2001). AR mutations occur in invasive prostate carcinomas (Haapala et al., 2001; Marcelli et al., 2000), as mentioned above, however, increased estrogen sensitiveness as a result of increased expression of ERs occurs concurrently (Lai et al., 2004). Furthermore, it is possible for ERβ to antagonize androgen-dependent prostate and PCa proliferation, as prostatic hyperplasia occurred in ERβ knock-out mice (Krege et al., 1998). Of interest, ERβ expression increases in PCa progression, although it has also been reported that ERβ expression was diminished in high-grade dysplasia and grade 4/5 carcinoma of the peripheral zone of the prostate. The down-regulation of ERβ expression occurring during the late stage of prostatic carcinogenesis, possibly contributes the loss of control over growth processes mediated by ERβ and subsequently allows the proliferative stimuli to be reactivated mediated through the AR (Leav et al., 2001).