The androgen receptor is a negative regulator of eIF4E phosphorylation at S209: implications for the use of mTOR inhibitors in advanced prostate cancer.

The antiandrogen bicalutamide is widely used in the treatment of advanced prostate cancer (PCa) in many countries, but its effect on castration-resistant PCa (CRPC) is limited. We previously showed that resistance to bicalutamide results from activation of mechanistic target of rapamycin (mTOR). Interestingly, clinical trials testing combinations of the mTOR inhibitor RAD001 with bicalutamide were effective in bicalutamide-naïve CRPC patients, but not in bicalutamide-pretreated ones. Here we investigate causes for their difference in response. Evaluation of CRPC cell lines identified resistant vs sensitive in vitro models, and revealed that increased eIF4E(S209) phosphorylation is associated with resistance to the combination. We confirmed using a human-derived tumor xenograft mouse model that bicalutamide pre-treatment is associated with an increase in eIF4E(S209) phosphorylation. Thus, AR suppressed eukaryotic initiation factor 4E (eIF4E) phosphorylation, while the use of antiandrogens relieved this suppression, thereby triggering its increase. Additional investigation in human prostatectomy samples showed that increased eIF4E phosphorylation strongly correlated with the cell proliferation marker Ki67. Small interfering RNA-mediated knockdown (k/d) of eIF4E-sensitized CRPC cells to RAD001+bicalutamide, whereas eIF4E overexpression induced resistance. Inhibition of eIF4E phosphorylation by treatment with CGP57380 (an inhibitor of mitogen-activated protein kinase-interacting serine-threonine kinases MAP kinase-interacting kinase 1 (Mnk1/2), the eIF4E upstream kinase) or inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2), the upstream kinase-regulating Mnk1/2, also sensitized CRPC cells to RAD001+bicalutamide. Examination of downstream targets of eIF4E-mediated translation, including survivin, demonstrated that eIF4E(S209) phosphorylation increased cap-independent translation, whereas its inhibition restored cap-dependent translation, which could be inhibited by mTOR inhibitors. Thus, our results demonstrate that while combinations of AR and mTOR inhibitors were effective in suppressing tumor growth by inhibiting both AR-induced transcription and mTOR-induced cap-dependent translation, pre-treatment with AR antagonists including bicalutamide increased eIF4E phosphorylation that induced resistance to combinations of AR and mTOR inhibitors by inducing cap-independent translation. We conclude that this resistance can be overcome by inhibiting eIF4E phosphorylation with Mnk1/2 or ERK1/2 inhibitors.

The effect of sirolimus on prostate-specific antigen (PSA) levels in male renal transplant recipients without prostate cancer.

In kidney recipients, the immunosuppressant sirolimus has been associated with a decreased incidence of de novo posttransplant malignancies (including prostate cancer). But the effect of sirolimus on the prostate-specific antigen (PSA) blood level, an important prostate cancer screening tool, remains unknown. We studied male kidney recipients >50 years old (transplanted from January 1994 to December 2006) without clinical evidence for prostate cancer. Pre- and posttransplant PSA levels were analyzed for 97 recipients (n = 19 on sirolimus, n = 78 on tacrolimus [control group]). Pretransplant PSA was similar for sirolimus versus tacrolimus recipients (mean, 1.8 versus 1.7 ng/mL, p = 0.89), but posttransplant PSA was significantly lower for recipients on sirolimus (mean, 0.9 versus 1.9 ng/mL, respectively, p < 0.001). The mean difference between pretransplant and posttransplant PSA was -0.9 ng/mL (50.0%, p = 0.006) for the sirolimus group versus +0.2 ng/mL (+11.8%, p = 0.24) for the tacrolimus group. By multivariate analysis, only pretransplant PSA and immunosuppression with sirolimus independently impacted posttransplant PSA. Our data strongly suggest that sirolimus is associated with a significant PSA decrease in kidney recipients. Future studies must investigate the clinical implications of our findings for the use of PSA for prostate cancer screening in male kidney recipients on sirolimus.

Regulation of androgen receptor transcriptional activity by rapamycin in prostate cancer cell proliferation and survival.

The mTOR (mammalian target of rapamycin) inhibitor rapamycin caused growth arrest in both androgen-dependent and androgen-independent prostate cancer cells; however, long-term treatment induced resistance to the drug. The aim of this study was to investigate methods that can overcome this resistance. Here, we show that rapamycin treatment stimulated androgen receptor (AR) transcriptional activity, whereas suppression of AR activity with the antiandrogen bicalutamide sensitized androgen-dependent, as well as AR-sensitive androgen-independent prostate cancer cells, to growth inhibition by rapamycin. Further, the combination of rapamycin and bicalutamide, but not the individual drugs, induced significant levels of apoptosis in prostate cancer cells. The net effect of rapamycin is determined by its individual effects on the mTOR complexes mTORC1 (mTOR/raptor/GbetaL) and mTORC2 (mTOR/rictor/sin1/GbetaL). Inhibition of both mTORC1 and mTORC2 by rapamycin-induced apoptosis, whereas rapamycin-stimulation of AR transcriptional activity resulted from the inhibition of mTORC1, but not mTORC2. The effect of rapamycin on AR transcriptional activity was mediated by the phosphorylation of the serine/threonine kinase Akt, which also partially mediated apoptosis induced by rapamycin and bicalutamide. These results indicate the presence of two parallel cell-survival pathways in prostate cancer cells: a strong Akt-independent, but rapamycin-sensitive pathway downstream of mTORC1, and an AR-dependent pathway downstream of mTORC2 and Akt, that is stimulated by mTORC1 inhibition. Thus, the combination of rapamycin and bicalutamide induce apoptosis in prostate cancer cells by simultaneously inhibiting both pathways and hence would be of therapeutic value in prostate cancer treatment.

A 90 kDa fragment of filamin A promotes Casodex-induced growth inhibition in Casodex-resistant androgen receptor positive C4-2 prostate cancer cells.

Prostate tumors are initially dependent on androgens for growth, but the majority of patients treated with anti-androgen therapy progress to androgen-independence characterized by resistance to such treatment. This study investigates a novel role for filamin A (FlnA), a 280 kDa cytoskeletal protein (consisting of an actin-binding domain (ABD) followed by 24 sequential repeats), in androgen-independent (AI) growth. Full-length FlnA is cleaved to 170 kDa (ABD+FlnA1-15) and 110 kDa fragments (FlnA16-24); the latter is further cleaved to a 90 kDa fragment (repeats 16-23) capable of nuclear translocation and androgen receptor (AR) binding. Here, we demonstrate that in androgen-dependent LNCaP prostate cancer cells, the cleaved 90 kDa fragment is localized to the nucleus, whereas in its AI subline C4-2, FlnA failed to cleave and remained cytoplasmic. Transfection of FlnA16-24 cDNA in C4-2 cells restored expression and nuclear localization of 90 kDa FlnA. Unlike LNCaP, C4-2 cells proliferate in androgen-reduced medium and in the presence of the AR-antagonist Casodex. They also exhibit increased Akt phosphorylation compared to LNCaP, which may contribute to their AI phenotype. Nuclear expression of 90 kDa FlnA in C4-2 cells decreased Akt phosphorylation, prevented proliferation in androgen-reduced medium and restored Casodex sensitivity. This effect was inhibited by constitutive activation of Akt indicating that FlnA restored Casodex sensitivity in C4-2 cells by decreasing Akt phosphorylation. In addition, FlnA-specific siRNA which depleted FlnA levels, but not control siRNA, induced resistance to Casodex in LNCaP cells. Our results demonstrate that expression of nuclear FlnA is necessary for androgen dependence in these cells.

Arginine vasopressin stimulates mesangial cell proliferation by activating the epidermal growth factor receptor.

The potent vasoconstrictor arginine vasopressin (AVP) is also a mitogen for mesangial cells. Treatment with AVP decreased transit time through the cell cycle. AVP-stimulated mesangial cell growth by activating both the Ras mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) cell signaling pathways. Both the selective PI3K inhibitor LY-294002 and the MAPK kinase (MEK) inhibitor PD-98059 inhibited AVP-stimulated mesangial cell proliferation. However, LY-294002 was more potent, indicating an important role for PI3K activation in AVP-stimulated mesangial cell proliferation. AVP appeared to exert its effect on MAPK and PI3K activation, as well as on cell proliferation, by activating the epidermal growth factor receptor (EGF-R). Pretreatment with the tyrphostin-derived EGF-R antagonist AG-1478 inhibited mesangial cell proliferation as well as the activation of extracellular signal-regulated kinase 1/2 (ERK1/2 or p42/p44(MAPK)), and p70S6 kinase, a downstream effector of PI3K, providing evidence that MAPK and PI3K activation, respectively, occurred downstream of EGF-R activation. Treatment with rapamycin, an inhibitor of the p70S6 kinase activator mTOR, also resulted in growth inhibition, further suggesting the importance of the PI3K signaling pathway in AVP-induced proliferation. AVP treatment appeared to transactivate EGF-R by inducing tyrosine phosphorylation of the Ca(2+)/protein kinase C (PKC)-dependent nonreceptor tyrosine kinase, Pyk2, leading to Pyk2/c-Src association and c-Src activation. This was followed by association of c-Src with EGF-R and EGF-R activation. These data suggested that AVP-stimulated Pyk2 tyrosine phosphorylation to activate c-Src, thereby leading to EGF-R transactivation.

Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells.

The biologic effects and mechanisms by which bone morphogenetic proteins (BMPs) function in breast cancer cells are not well defined. A member of this family of growth and differentiation factors, BMP-2, inhibited both basal and estradiol-induced growth of MCF-7 breast tumor cells in culture. Flow cytometric analysis showed that in the presence of BMP-2, 62% and 45% of estradiol-stimulated MCF-7 cells progressed to S-phase at 24 h and 48 h, respectively. Estradiol mediates growth of human breast cancer cells by stimulating cyclins and cyclin-dependent kinases (CDKs). BMP-2 significantly increased the level of the cyclin kinase inhibitor, p21, which in turn associated with and inactivated cyclin D1. BMP-2 inhibited estradiol-induced cyclin D1-associated kinase activity. Also estradiol-induced CDK2 activity was inhibited by BMP-2. This inhibition of CDK activity resulted in hypophosphorylation of retinoblastoma protein thus keeping it in its active form. These data provide the first evidence by which BMP-2 inhibits estradiol-induced proliferation of human breast cancer cells.

Effect of cyclin E overexpression on lovastatin-induced G1 arrest and RhoA inactivation in NIH3T3 cells.

The HMG-CoA reductase inhibitor, lovastatin, blocks targeting of the Rho and Ras families of small GTPases to their active sites by inhibiting protein prenylation. Control NIH3T3 cells, and those overexpressing human cyclin E protein were treated with lovastatin for 24 h to determine the effects of cyclin E overexpression on lovastatin-induced growth arrest and cell rounding. Lovastatin treatment (10 microM) of control 3T3 cells resulted in growth arrest at G1 accompanied by actin stress fiber disassembly, cell rounding, and decreased active RhoA from the membranous protein fraction. By contrast, in NIH3T3 cells overexpressing cyclin E, lovastatin did not cause loss of RhoA from the membrane (active) protein fraction, actin stress fiber disassembly, cell rounding or growth arrest within 24 h. Analysis of cell cycle proteins showed that 24 h of lovastatin treatment in the control cells caused an elevation in the levels of the cyclin-dependent kinase inhibitor p27(kip1), inhibition of both cyclin E- and cyclin A-dependent kinase activity, and decreased levels of hyperphosphorylated retinoblastoma protein (pRb). By contrast, lovastatin treatment of the cyclin E overexpressors did not suppress either cyclin E- or cyclin A-dependent kinase activity, nor did it alter the level of maximally phosphorylated pRb, despite increased levels of p27(kip1). However, by 72 h, the cyclin E overexpressors rounded up but remained attached to the substratum, indicating a delayed response to lovastatin. In contrast with lovastatin, inactivation of membrane-bound Rho proteins (i.e., GTP-bound RhoA, RhoB, RhoC) with botulinum C3 transferase caused cell rounding and G1 growth arrest in both cell types but did not inhibit cyclin E-dependent histone kinase activity in the cyclin E overexpressors. In addition, 24 h of cycloheximide treatment caused depletion of RhoA from the membrane (active) fraction in neo cells, but in the cells overexpressing cyclin E, RhoA remained in the active (membrane-associated) fraction. Our observations suggest that (1) RhoA activation occurs downstream of cyclin E-dependent kinase activation, and (2) overexpression of cyclin E decreased the turnover rate of active RhoA.

Role of RhoA activation in the growth and morphology of a murine prostate tumor cell line.

Prostate cancer cells derived from transgenic mice with adenocarcinoma of the prostate (TRAMP cells) were treated with the HMG-CoA reductase inhibitor, lovastatin. This caused inactivation of the small GTPase RhoA, actin stress fiber disassembly, cell rounding, growth arrest in the G1 phase of the cell cycle, cell detachment and apoptosis. Addition of geranylgeraniol (GGOL) in the presence of lovastatin, to stimulate protein geranylgeranylation, prevented lovastatin's effects. That is, RhoA was activated, actin stress fibers were assembled, the cells assumed a flat morphology and cell growth resumed. The following observations support an essential role for RhoA in TRAMP cell growth: (1) TRAMP cells expressing dominant-negative RhoA (T19N) mutant protein displayed few actin stress fibers and grew at a slower rate than controls (35 h doubling time for cells expressing RhoA (T19N) vs 20 h for untransfected cells); (2) TRAMP cells expressing constitutively active RhoA (Q63L) mutant protein displayed a contractile phenotype and grew faster than controls (13 h doubling time). Interestingly, addition of farnesol (FOL) with lovastatin, to stimulate protein farnesylation, prevented lovastatin-induced cell rounding, cell detachment and apoptosis, and stimulated cell spreading to a spindle shaped morphology. However, RhoA remained inactive and growth arrest persisted. The morphological effects of FOL addition were prevented in TRAMP cells expressing dominant-negative H-Ras (T17N) mutant protein. Thus, it appears that H-Ras is capable of inducing cell spreading, but incapable of supporting cell proliferation, in the absence of geranylgeranylated proteins like RhoA.

Lovastatin induces apoptosis by inhibiting mitotic and post-mitotic events in cultured mesangial cells.

Lovastatin, an inhibitor of protein prenylation, was reported to inhibit DNA synthesis and induce apoptosis in cultured cells. This report describes the morphological consequences of lovastatin treatment. Lovastatin (50 microM) induced mesangial cell rounding and disassembly of actin stress fibers within 24 to 48 h. After 48 to 72 h of lovastatin treatment, the cells detached from the substratum and underwent apoptotic cell death as evidenced by condensed nuclear chromatin, nuclear fragmentation, cell blebbing and decrease in cell size. Time lapse cinematography revealed that lovastatin caused cell rounding by either inhibiting cytokinesis or cell spreading following cytokinesis. Lovastatin-induced cell rounding, detachment, and apoptosis were dependent upon cell proliferation. These effects were prevented by serum deprivation to inhibit cell proliferation or by plating cells at densities which resulted in contact inhibition of cell growth. Lovastatin-induced mesangial cell rounding and apoptosis were also prevented by the inclusion of the isoprenoids all-trans-farnesol or all-trans-geranylgeraniol in the incubation medium. These results indicate that the effects of lovastatin were mediated by inhibition of protein isoprenylation because exogenous all-trans-geranylgeraniol can be used only in protein prenylation. The small GTP-binding protein RhoA, which may be important for cell spreading and cytokinesis, accumulated in the cytosol following treatment with lovastatin, suggestive of its inactivation. This effect was also prevented by the inclusion of either farnesol or geranylgeraniol in the incubation medium. Thus, lovastatin-induced apoptosis in mesangial cells occurs by interfering with prenylation dependent mitotic and post-mitotic events.

Monitoring electropermeabilization in the plasma membrane of adherent mammalian cells.

When an electrical potential of order one volt is induced across a cell membrane for a fraction of a second, temporary breakdown of ordinary membrane functions may occur. One result of such a breakdown is that molecules normally excluded by the membrane can now enter the cells. This phenomenon, generally referred to as electropermeabilization, is known as electroporation when actual pores form in the membrane. This paper presents a unique approach to the measurement of pore formation and closure in anchored mammalian cells. The cells are cultured on small gold electrodes, and by constantly monitoring the impedance of the electrode with a low-amplitude AC signal, small changes in cell morphology, cell motion, and membrane resistance can be detected. Because the active electrode is small, the application of a few volts across the cell-covered electrode causes pore formation in the cell membrane. In addition, the heat transfer is very efficient, and the cells can be porated in their regular growth medium. By this method, the formation and resealing of pores due to applied electric fields can be followed in real time for anchorage-dependent cells.