Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

Abstract

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.

Introduction

The traditional model of steroid hormone action involves binding to specific intracellular steroid receptors, translocation to the nucleus, DNA binding, and subsequent modulation of transcription and protein synthesis [1]. This genomic response generally requires hours to be manifested. In recent years however, a number of studies introduced the concept of non-genomic steroid hormone actions, explaining observations related to rapid steroid effects. According to the recently proposed classification for non-genomic steroid actions [2], a non-genomic effect occurs within minutes, is present in cells devoid of functional classical intracellular steroid receptors, and is insensitive to inhibitors of transcription and translation. Furthermore, activation of a non-genomic effect may be triggered by non-permeable (e.g. bovine serum albumin (BSA-covalently coupled) steroids and is, in most cases, insensitive to steroid antagonists. Non-genomic steroid actions have been reported for most steroids including glucocorticoids [3], [4], [5], [6], progesterone [7], [8], estrogens [9], [10], androgens [11], [12], [13] and neurosteroids [14], [15], [16]. For recent reviews, see [17], [18], [19].

We have recently reported the identification of functional membrane androgen receptors in the hormone-sensitive LNCaP human prostate cancer cell line, as well as in the hormone insensitive DU145 human prostate cancer cell line [13], [20]. Activation of these receptors in LNCaP cells by testosterone-BSA, a non-permeable testosterone conjugate was shown to induce rapid actin cytoskeleton reorganization and to increase the secretion of prostate-specific antigen (PSA) within minutes [13]. Furthermore, a specific non-genomic signaling cascade regulating the molecular mechanism of actin reorganization was identified, involving phosphorylation of FAK, the association of FAK with PI-3K and the subsequent activation of the latter, as well as the activation of the small guanosine triphosphatases Rac1/Cdc42 [21]. Finally, it was shown that mAR activation by BSA-coupled testosterone was able to induce: a) apoptosis of either androgen-dependent LNCaP or androgen-independent DU145 cells and b) regression of prostate cancer cells both, in vitro and in vivo [20], [22]. Similarly, stimulation of mAR by the impermeable androgen analog DHT-BSA was associated with apoptotic cell death in primary cortical astrocytes [23]. These findings collectively suggest that mAR activation may be an important target for the apoptotic regression of a given cell or tissue.

To further analyze the signaling pathways downstream of mAR and to correlate mAR-regulated events with intracellular androgen receptor (iAR) function, we have used the DU145 and LNCaP prostate cancer cell lines. While LNCaP cells express functional intracellular androgen receptors (iAR), the DU145 cell line has been shown to express either non-functional iAR [24], or to be iAR-deficient [25], [26] and which therefore fails to respond to androgen treatment. We compared cellular responses and pathways triggered by testosterone-BSA in more detail. Specifically, we sought to elucidate the role of actin in mAR-dependent apoptotic cell death and to identify key downstream effectors of mAR action regulating both actin reorganization and apoptosis. Our results provide new insights into mAR function and point to a central role of actin remodeling and Rho small GTPases in mAR signaling. It is concluded that the newly identified Rho/ROCK-dependent pathway and the observed actin reorganization operating in LNCaP and DU145 cells have a key functional role in regulating mAR-induced apoptosis in prostate cancer cells independently of iAR functional status.