Androgen Independent PSA Secretion

Androgens are well documented as key factors regulating PSA gene expression.65 Since PCa cells initially depend on androgens for proliferation, androgen ablation therapy is the mainstay of treatment for advanced PCa, and leads to a drop in circulating PSA. Unfortunately, cancer cells eventually escape from the steroid requirement and progress to androgen independence, which is indicated by the rebound of PSA levels in circulation.66 The ability to treat these patients is limited. Interestingly, most of the hormone-refractory PCa cells express functional AR and PSA. The molecular mechanism of androgen-independent PSA secretion deserves careful analysis.

Biochemically, upon androgen binding, AR exhibits conformational changes, induced by modifications including phosphorylation and dimer-ization, for its activation.67 It is proposed that in hormone-refractory cancers, in the absence of androgen, AR is also activated by phosphoryla-tion.68 Hence, a direct cross-talk between AR and cellular kinases may be an important factor involved in the elevation of androgen-independent PSA secretion. Our understanding of the cross-talk between AR and protein kinases is discussed briefly as follows.

ErbB-2/HER-2/Neu (ErbB-2) ^ ERK/MAPK Pathway

Data from our lab strongly indicate that ErbB-2 plays a critical role in hormone-refractory proliferation of PCa cells as well as in their enhanced androgen-independent PSA secretion, and are of clinical relevance (Fig. 2). This notion of ErbB-2 regulation is shown in ErbB-2 cDNA-transfected C-33 cells in which the growth rate and PSA secretion are elevated, even in the absence of androgen.38,44,69 Conversely, in C-81 cells in which ErbB-2 is activated and PSA secretion is increased, the expression of a dominant-negative mutant of ErbB-2, or addition of ErbB-2 inhibitors, reduces the elevated androgen-independent PSA secretion.38

In androgen-deprived environments, ErbB-2, via the ERK/MAPK or Akt pathway, might activate AR by serine phosphorylation (Ser514 for ERK/MAPK and Ser791 for Akt).69-71 Our data indicate that ERK/MAPK plays a key role in the early stage of androgen-independent PSA secretion, as well as in tumor progression. For example, in androgen-depleted conditions, elevated PSA secretion is decreased by MEK1 inhibitor, PD98059, in C-81 cells, while PSA secretion is increased by MEK1 cDNA transfec-tion in C-33 cells.38 Furthermore, in ErbB-2 cDNA-transfected C-33 cells, p-Tyr ErbB-2

p-Tyr ErbB-2

Secretion Androgene

Fig. 2. Proposed model of regulation of PSA expression and secretion by ErbB-2 in hormone-refractory PCa. In androgen-independent human PCa cells, ErbB-2 is activated by hyper-tyrophosphorylation, in part, due to the loss of cPAcP expression. The activated ErbB-2 can transduce its signals via p52Shc to activate the downstream ERK/MAPK, which may lead to AR phosphorylation, resulting in an increase in androgen-independent expression and secretion of PSA, as well as cell proliferation. Activated ErbB-2 may also phosphorylate AR via Akt.

Androgen-independent PSA secretion & cell proliferation

Fig. 2. Proposed model of regulation of PSA expression and secretion by ErbB-2 in hormone-refractory PCa. In androgen-independent human PCa cells, ErbB-2 is activated by hyper-tyrophosphorylation, in part, due to the loss of cPAcP expression. The activated ErbB-2 can transduce its signals via p52Shc to activate the downstream ERK/MAPK, which may lead to AR phosphorylation, resulting in an increase in androgen-independent expression and secretion of PSA, as well as cell proliferation. Activated ErbB-2 may also phosphorylate AR via Akt.

PD98059 effectively abolishes the increased PSA secretion in steroid-reduced conditions. This mechanism (Fig. 2) thus provides an explanation of the clinical observations of hormone-refractory prostate carcinomas in which cPAcP is decreased,29,39-41 the ErbB-2 gene is not amplified, while the ErbB-2 protein could be either activated by tyrophosphorylation and/or elevated.45,72 Subsequently, ERK/MAPKs are activated by phosphorylation,73,74 leading to AR activation by phosphorylation,75 as well as the rebound of PSA levels in the circulation of most patients.1 Collectively, our data strongly indicate that the ErbB-2(^)ERK/MAPK pathway is crucial in androgen-independent PCa cell proliferation, as well as PSA secretion.

ErbB-2 can up-regulate PSA secretion at two levels: the transcriptional level and the post-translational level in the secretory pathway. In C-81 cells in which ErbB-2 is activated by hyper-tyrophosphorylation, the level of PSA mRNA is elevated in the steroid-reduced condition, compared to C-33 cells,27'43 which may be due in part to the activation of the PSA promoter by ErbB-2 via ERK/MAPK and AR.69 Alternatively, ErbB-2 may activate MEK1, leading to the up-regulation of a transcription factor(s) that subsequently activates the PSA promoter.76 ErbB-2 may also activate the secretory pathway of PSA in those cells (M. S. Lee and M. F. Lin, unpublished observations). Nevertheless, further experiments are needed.

Akt Pathway

AR phosphorylation might also occur through the PI3K ^ Akt pathway in hormone-refractory PCa cells.71 Androgen-responsive, low passage LNCaP cells have a lower Akt activity than the androgen-independent high passage cells,77 which implies that this pathway could be important in later stages of advanced metastatic cancer, where PTEN mutation leads to constant Akt activation. Nevertheless, it should be noted that pathways other than PTEN could be involved in activating Akt in higher passage LNCaP cells because PTEN is also mutated in the lower passage LNCaP cells.78,79 Interestingly, the anti-androgen flutamide blocks Akt-induced PSA secretion in IL-4-treated LNCaP cells,80 while it stimulates the growth of LNCaP cells,81 indicating the differential roles of Akt. Nevertheless, IGFBP-5 cDNA-transfected LNCaP cells express hyperac-tivated Akt and exhibit higher growth rates and PSA secretion levels in castrated mice than do control cells.82 Further experiments are required to clarify the role of Akt in androgen-independent PSA secretion vs. hormone-refractory PCa cell proliferation and its clinical relevance.

Cytokine Pathway

Interleukin-6 (IL-6) has been proposed to play a role in the development of androgen-independence in PCa cells because IL-6 enhances the growth of LNCaP cells in androgen-depleted environment and serum IL-6 levels are found to be elevated in some patients with advanced PCa. Since androgen-independent LNCaP-IL-6 + cells, which were selected by continuous passage in media containing 5 ng/ml IL-6, are resistant to the cell cycle arrest mediated by IL-6,83 it is inferred that IL-6 may contribute to PCa progression, and via the ErbB-2(^)ERK/MAPK pathway, stimulate androgen-independent PSA secretion.84'85 IL-4 might also have a role in the androgen-independent, AR-mediated PSA secretion,80 because IL-4 level is elevated in some PCa patients with hormone-refractory cancer.86

PKA Pathway

Forskolin, a PKA activator, can increase the activity of the PSA promoter in the absence of androgen.87 The activation of PKA by forskolin also results in increased PSA expression in androgen-depleted LNCaP cells.88 Butyrate, a differentiation agent, increased circulating PSA levels in castrated animals. Electromobility shift assay showed an increased AR-ARE complex formation in butyrate-treated LNCaP cells that is partially inhibited by a PKA blocker, indicating that butyrate action on LNCaP cells might, in part, involve the PKA pathway.88

Other Downstream Mechanisms

Protein phosphorylation may activate other regulatory factors, e.g., AR cofactors, leading to increased PSA secretion. Alternatively, aberrantly regulated transcriptional factors may play a role in this mode of regulation. For example, nuclear factor-Kappa B (NF-kB), which is increased in androgen-independent PCa cells, can activate PSA expression,89 while the growth of those cells is inhibited by NF-kB inhibitors.90 A 45-kDa cell-specific transcription factor (p45) has also been implicated in androgen-independent expression of the PSA gene.91

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