Abstract
Whilst androgen ablation therapy is used to treat locally advanced or metastatic forms of prostate cancer, side-effects can include the emergence of an androgen-independent neuroendocrine cell population which is associated with poor prognosis. Here we have examined how cyclic AMP elevation regulates early events in the neuroendocrine differentiation process. We demonstrate that selective activation of protein kinase A is necessary and sufficient for cyclic AMP (cAMP) elevation to rapidly promote a neuroendocrine phenotype in LNCaP cells independent of de novo protein synthesis. Furthermore, the effects of cAMP could be recapitulated by inhibition of RhoA signalling or pharmacological inhibition of Rho kinase. Conversely, expression of constitutively active Gln63Leu-mutated RhoA acted as a dominant-negative inhibitor of cAMP-mediated NE phenotype formation. Consistent with these observations, cAMP elevation triggered the PKA-dependent phosphorylation of RhoA on serine 188, and a non-phosphorylatable Ser188Ala RhoA mutant functioned as a dominant-negative inhibitor of cAMP-mediated neuroendocrine phenotype formation. These results suggest that PKA-mediated inhibition of RhoA via its phosphorylation on serine 188 and the subsequent inhibition of ROCK activity plays a key role in determining initial changes in cellular morphology during LNCaP cell differentiation to a neuroendocrine phenotype. It also raises the possibility that targeted suppression of this pathway to inhibit neuroendocrine cell expansion might be a useful adjuvant to conventional prostate cancer therapy.
Highlights
Prostate cancer (PCaI) is the most frequently diagnosed malespecific malignancy and causes the greatest number of male cancerrelated deaths after lung cancer [1]
NE cells play a key role in governing differentiation and proliferation of the developing prostate gland and, whilst their role in Abbreviations: PCa, prostate cancer; cAMP, cyclic AMP; NE, neuroendocrine; prostate specific antigen (PSA), prostate-specific antigen; PKA, cAMP-dependent protein kinase; bFGF, basic fibroblast growth factor; NGF, nerve growth factor; 6-Bnz-cAMP, N6-benzoyl-cAMP; Fsk, forskolin; C3T, cell-permeable C. botulinum C3 transferase; cAMP responsive element binding protein (CREB), cAMP response element binding protein; ERK, extracellular signal-regulated kinase; RACK1, receptor of activated C kinase 1; EPAC, exchange protein directly activated by cAMP; Rho-associated protein kinase (ROCK), Rhoassociated protein kinase
Fsk + Emetine can be almost entirely recapitulated following inhibition of RhoA signalling and that RhoA itself is a PKA substrate in these cells. These results suggest that PKA-mediated inhibition of RhoA plays a key role in determining cellular morphology during initial LNCaP cell differentiation to a NE phenotype
Summary
Prostate cancer (PCaI) is the most frequently diagnosed malespecific malignancy and causes the greatest number of male cancerrelated deaths after lung cancer [1]. Prostate cell growth is reliant on the presence of androgens such as testosterone with traditional therapeutic strategies focussing on androgen ablation therapy [1]. NE cells form foci of non-dividing cells surrounded by regions of proliferating tumour cells due to the release of mitogenic compounds such as bombesin [8] and chromogranin A [9]. Due to their resistance to many conventional chemotherapeutics, it is possible that inhibition of NE cell differentiation may be a useful avenue by which to complement existing therapies and prevent expansion of this cell type in late-stage PCa
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