AR in CaP progression

The efficacy of many CaP treatments is often temporary, as CaP cells often become refractory to hormone ablation therapies. The current therapeutics are largely targeted towards the inhibition of AR activation, such as anti-androgens, chemical castration (treatment with gonadotropin releasing hormone (GnRH) super agonists to inhibit testosterone secretion from the testes), or surgery (orchidectomy) [7]. AI-CaP or castration resistant CaP is thought to occur due to the androgen deprivation therapies as they may induce altered protein activity and expression in the cancer cells. Despite androgen blockade in AI-CaP patients, expressions of AR target genes such as PSA remain high. Furthermore, hormone refractory CaP continues to rely on AR expression, suggesting that the AR is necessary to maintain proliferative and anti-apoptotic effects. Therefore, CaP acquires the phenotype of oncogenic addiction to the AR for its continued growth and resistance to therapy. The progression of CaP from an hormone sensitive AD to a hormone resistant AI state is likely due to mechanisms involving alterations in AR expression, amplification, mutations, and/or AR activity.

AR mutations in primary CaP are relatively low when compared to metastatic CaP where frequencies are as high as 50% [1, 55-57]. Germline or somatic mutations of the AR leads to AR overexpression and hypersensitivity due to point mutations and promiscuous mutant AR proteins. Germline mutations of the AR are rarely found. Familial inheritance of CaP with at least two first degree relatives account for 20% of cases and transmission compatible with Mendellian inheritance is described to be 50% of the cases observed [3]. Genetic susceptibility seems to be more significant in patients <55 years old [3]. Recently, a R726L mutation was reported in only Finnish patients with sporadic or familial CaP [3, 58, 59]. Genomic alterations to the AR have been found in both non-coding and coding sequences such as polymorphisms of CAG and GGC repeats, single nucleotide polymorphisms, as well as silent and missense mutations [3, 58, 60, 61]. Koochekpour et al., (2010) screened 60 CaP patients of African-American and Caucasian families with a history of familial CaP. Using exon-specific PCR, bidirectional sequencing and restriction enzyme genotyping, they found that one African-American family had a novel germline AR misssense mutation (exon 2 of DBD A1675T; T559S) in three siblings with early onset CaP. This mutation was transmitted in an X-linked pattern and located at the N-terminal region of the DBD. Koochekpour et al., (2010) reason that the location of this particular mutation likely affected AR ligand binding.

Somatic mutations are largely single base substitutions: 49% at the LBD, 37% at the NTD, and 7% at the DBD [3]. For those CaP that harbor gain of function mutations the result is primarily an increase in ligand promiscuity. The AR is activated by testosterone and DHT; however, mutations in the LBD make the AR less stringent of its partners. For example, in LNCaP cells, a Threonine (Thr; T) to Alanine (Ala;A) mutation (T877A) caused the expansion of ligand binding activity [1, 8]. This mutation permitted AR activation by androgens, estrogens, progesterones as well as the non-steroidal antagonist, flutamide. A study by Gaddipati et al., (1994) found that 25% of patient metastatic tumors had a T877A mutation. Patients that were treated with flutamide often experienced a worsening of symptoms over time. Once flutamide was withdrawn, patients tended to do better. Interestingly, some patients also experienced a rise in serum PSA levels upon flutamide treatment. Taplin et al., (1999) studied patients that were on flutamide treatment relative to those that were not given this particular treatment. Tumor cells that had the T877A mutation increased in proliferation while patients who were not treated with flutamide harboured different mutations of the AR that were not activated by flutamide. Therefore, there seems to be a strong selective pressure for AR mutants arising from flutamide treatment such that discontinuation of flutamide resulted in tumor regression before growth resumed again. Other mutations such as the H874Y (Histadine to Tyrosine) mutation in the CWR22 cell line have been found to affect co-activator interactions by altering the conformation of Helix 12 of the LBD. Helix 12 regulates co-activator binding and creates a specific groove with helices 3, 4, and 5 [63-67]. Helix 12 rotates over the ligand binding pocket and assumes favorable or unfavorable positions depending on agonist or antagonist binding, respectively. Helix 12 mutations have also been detected in CaP patients, such as Q902R (Glutamine to Arginine), and M894D (Methionine to Aspartic Acid) (an androgen insensitive mutation) [56, 67, 68]. The importance of Helix 12 and the NTD-LBD interaction for AR activity is underscored by the fact that spontaneous mutations in Helix 12, NTD, and LBD caused either complete or partial androgen insensitivity [67, 68]. Additionally, a L701H mutation was also identified in conjunction with the T877A mutation in MDA CaP 2a cell lines [1, 69]. L701H mutation alone decreased the ability of AR to bind DHT, but increased binding of other non specific adrenal corticosteroids. The presence of the T877A mutation together with L701H potentiated this interaction by more than 300% as both mutations were located within the LBD [1, 70]. Hence, the susceptibility of the AR to minimize its ligand specificity in AI-CaP makes AR dependent disease progression difficult to treat. On the other hand, other anti-androgens such as Casodex (bicalutamide) do not seem to have the same response to T877A AR [1]. Novel truncated AR mutant, mRNA splice variants and mutant AR lacking exon 3 (coding for C-terminal portion of the DBD) tandem duplication have also been found in the 22RV1 cell line (AI-CaP), derived from the CWR22R cell line [3, 71]. Furthermore, an important study by Han et al., (2001) demonstrated that prostate tumors from a genetically engineered mouse model upon androgen ablation resulted in AR gene mutations within AR NTD. Specifically, amino acid substitution A229T and E231G (Glutamic Acid to Glycine) within the AR NTD signature motif: ARNSM (Ala-Arg-Asn-Ser-Met), increased ligand independent basal activity, whereas, E231G increased responsiveness to androgen receptor co-activator ARA160 and ARA70. The ARNSM motif is unique to the AR and the most highly conserved region of the AR NTD.

Another possible mechanism for the progression of AI disease is mediated by AR amplification. Overexpression of the AR causes hypersensitivity of the AR under low levels of androgens. Visakorpi et al., (1995) were the first to show that the AR was amplified in 305 hormone refractory tumors subsequent to androgen ablation therapy. Although these tumors were clinically presenting as AI-CaP, there was increased levels of the AR, and, continued proliferation of the tumor still required androgen. This suggested that some AR amplified tumors may require the presence of residual androgens that remain in the serum after monotherapy [1, 74]. Similarily, mouse models of CaP progression characterized by high expression of AR, increased AR stability, and AR nuclear localization, had hypersensitive tumor growth promoting effects upon DHT administration. DHT concentrations of 4 orders of magnitude lower were able to stimulate growth relative to DHT levels required for AD LnCaP cell proliferation [1, 75].

Although AR gene amplification and hypersensitivity serves to be a sound model for AI-CaP progression, the AR may be activated by alternative means including activation by co-regulators, increased androgen production, and/or intermediary downstream signaling pathways. Greater levels of co-activator expression such as SRC-1, ARA70, and TIF2 were demonstrated to be elevated in CaP and correlated with increased CaP grade, stage, and decreased disease free survival. For example, Cdk-activating phophatase B, an identified co-activator of the AR was overexpressed and also highly amplified in tumors with high Gleason scores [3]. Local production of androgens within the prostate can also increase AR transacti-vation by compensating for decreased serum testosterone resulting from androgen ablation therapy. Studies have shown that serum testosterone levels can decrease 95%, contrasting the DHT levels within prostate tissue which only reduce by 60% [1, 76]. Locke et al., (2008) demonstrated that there was de novo and organ synthesis of androgens in LNCaP xenograft mouse models, suggesting that CaP cells had steroidogenic properties that enable them to survive in androgen depleted environments. Moreover, this was also indicative of greater levels of intratumoral 5-alpha-reductase activity. It is likely then, that during AI-CaP disease progression, there is a switch in androgen source whereby testicular androgens are replaced by prostatic androgen. Bennett et al., (2010) have deemed this as 'androgen self-sufficient'. There is also a hypothesis that conversion of adrenal steroids can sustain the androgen signal by supplying adrenal androgens such as DHEA and androstenedione [78]. After castration, adrenal androgens could account for as much as 40% of the total DHT in the prostate [76, 78].

Hormone receptors that are activated by ligand independent mechanisms are known as 'outlaw' receptors [1]. Certain growth factors such as Insulin Growth Factor (IGF)-1, Kerati-nocyte Growth Factor (KGF), and Epidermal Growth Factor (EGF) have been demonstrated to activate AR and induce the expression of AR target genes. Culig et al., (1994) showed that there was a 5-fold increase in PSA levels in LNCaP cells upon IGF-1 stimulation. Moreover, the addition of Casodex abolished the activation of the AR by IGF-1, KGF and EGF, indicating that the LBD was necessary for this activation. Overexpression of these growth factors has been observed in CaP; however, it is unclear whether it is the AR pathway or indirect downstream effects that are mediating tumorigenesis. In fact, patients with AI-CaP can fail Casodex therapy suggesting that other mechanisms are in play for ligand independent activation of the AR.

Furthermore, patients who received androgen ablation therapy have tumor cells that overexpress growth factor receptors, the receptor tyrosine kinases. Craft et al., (1999) demonstrated that an AI-CaP cell line, generated from xenografts implanted in castrated mice, consistently overexpressed Her-2/neu (from the EGF receptor family of receptor tyrosine kinases) [1]. Interestingly, AD-CaP cell lines could also be converted to AI-CaP cells by overexpressing Her-2/neu. This pathway was not blocked by Casodex, which indicated that the LBD of the AR was not necessary to transduce the effects of Her-2/neu. Although Trastuzumab (Hercep-tin) is used primarily to treat breast cancer, Herceptin had anti-proliferative effects on AD- and AI-CaP xenografts when combined with the chemotherapeutic drug paclitaxel. Yeh et al., (1999) believe that Her-2/neu activated AR via the MAPK pathway, as inhibitors of MAPK decreased HER-2/neu mediated activation of the AR. In effect, a positive feedback loop is created where the AR can activate kinases and in turn, where kinases can activate the AR through its phosphorylation (in the presence or absence of ligand), regardless of the varying levels of androgens [1].

The AR pathway is thought to be in interplay with other signaling pathways. AR activation due to cross regulation by receptor tyrosine kinases and their downstream effectors provides alternative and sustained routes for AR activation despite androgen depletion. Currently, there has been accumulating evidence that the phosphatidylinositol 3-kinase (PI3K)-AKT pathway plays a significant role in CaP tumor progression. The cross-regulatory mechanism by which the PI3K/Akt pathway modulates the expression and activity of AR is a novel area of study. Growing evidence continues to support the increased role of the PI3K/Akt and AR signaling pathways in mediating the progression of CaP to castrate resistant disease.

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