Mecanismos de señalización que intervienen en la diferenciación neuroendocrina del cáncer de próstatapapel de la autofagia y del cannabinoide WIN 55-212-2

Abstract

INTRODUCTION: Most prostate tumours appear as adenocarcinoma form whose development and progression are androgen-dependent. Therefore, conventional therapy focuses on blocking the synthesis of endogenous androgens and/or inhibiting the function of the androgen receptor. Androgen deprivation therapy rarely eliminates all the tumour cells and, with time, patients develop resistance to this treatment and, consequently prostate cancer resistant to castration (CRPC) occurs. In some cases, these types of cancer evolve into a very aggressive subtype with neuroendocrine differentiation (NED) whose survival rate might reach approximately one year. The comprehensive knowledge of the molecular mechanisms that underly the appearance of neuroendocrine cells and overall the determination of the processes that allow their survival and their biology, is of utmost importance in order to develop new therapeutic strategies that enable to treatment of this highly lethal cancer. It has been demonstrated that prostate cancer cells express high levels of the cannabinoid receptors CB1 and CB2. Activation of the CB2 receptor by JWH-015 cannabinoid induces cell cycle arrest and apoptosis (Ruiz-Llorente et al. 2003; Nithipatikom et al. 2004; Olea-Herrero et al. 2009a). However, it is unknown whether cannabinoid receptors exert any influence on the molecular mechanisms associated to neuroendocrine differentiation and the possible effect of WIN 55,212-2 (WIN) cannabinoid, a non-selective agonist of CB1 and CB2. EXPERIMENTAL PROCEDURES: Neuroendocrine differentiation of prostate cancer LNCaP cells was induced by serum deprivation for 6 days. The levels of NE markers and signaling proteins were determined by Western blotting and qPCR. The levels of IL-6 secreted by LNCaP and NE cells were measured by ELISA and the levels of cannabinoid receptors were determined by qPCR. The effect of WIN55, 212-2 on prostate cancer cell viability was analyzed by the MTT cell viability assay. The involvement of signaling cascades was investigated by pharmacological inhibition and siRNA. The in vivo antitumor activity of WIN 55,212-2 was studied in athymic nude mice (nu/nu) which were injected subcutaneously in the right flank with 5 x 106 PC-3 cells, a mix of PC-3 and LNCaP cells, or a mix of PC-3 and NE cells in 0.1 ml of PBS + 0.5% BSA to induce prostate cell tumors. Tumor size was measured daily and calculated using the formula V(mm3 ) = 1/2(Length × Width2). When tumors reached a volume of 100 mm3 (approximately two weeks after transplantation), the mice were randomly divided into 2 groups (n =4) and treated daily s.c. with vehicle, 0.5 mg/kg WIN 55,212-2 (PC-3) or 1,5mg/Kg WIN 55,212-2 for 15 days. At the end of the study, the mice were sacrificed by placing them in a CO2 gas-filled chamber, and the excised tumors were recovered and homogenized in lysis buffer for protein quantification and Western blotting. All experiments were executed at least 3 times and performed by triplicate. The statistical significance of differences between the means was evaluated using the unpaired student’s t test. The level of significance was set at p ≤ 0.05. Data are presented as the mean ±.S. E of the number of experiments indicated. Calculations were performed using Instat (Graphpad Software). RESULTS AND CONCLUSIONS: The differentiated LNCaP cells exhibited neurite outgrowth and increased expression of the typical neuroendocrine (NE) markers neuron-specific enolase (NSE) and βIII-tubulin (βIII Tub). In accordance with previous reports showing a reduced expression and /or activity of AR (Wright et al. 2003), NE cells showed decreased AR and PSA expression besides to increased levels of NE markers. In addition, and according to previous reports (Cox et al. 1999), we corroborated that the neuroendocrine phenotype of LNCaP cells is reversible since the levels of the NE markers rise when SF medium is replaced with a complete medium and cells are maintained in culture for an additional 6 days. NE cells showed increased levels of the IL-6 secreted. Treatment with 3μM WIN diminished this secretion. The incubation of PC-3 cells with supernatant obtained from NE cells with IL-6 knocked down did not cause any effect on PC-3 cell viability. The presence of NE cells increased the mitogenic capacity of PC-3 cells, which is not mediated by the secretion of IL-6 from NE cells. The presence of NE cells stimulate the growth of tumor PC-3 cells. The PI3K/Akt/mTOR pathway in the neuroendocrine differentiation of LNCaP cells was hyper-activated as deduced by the significantly increased levels of phosphorylated Akt in Ser473, compared to control LNCaP cells. The treatment of cells either with the PI3K inhibitor LY294002, the Akt selective inhibitor Inhibitor IV (Akt Inh IV) or the mTOR inhibitor rapamycin, resulted in a decrease of the neuroendocrine markers NSE and βIII tubulin expression in NE cells. To analyze the role of AMPK in the NE differentiation of prostate LNCaP cells, cells were serum-deprived for 6 days and phosphorylation levels of AMPK in Thr172 and its well-stablished substrate acetyl-coenzyme A carboxylase (ACC) were monitored by Western blot. The results show that during neuroendocrine differentiation, there is a decrease in Thr172 phosphorylation of AMPK and in Ser79 of ACC, which indicates an inhibition of the AMPK pathway. To further analyze the function of AMPK on NED, LNCaP cells were treated with the AMPK pharmacological activator AICAR. This treatment rescued the decrease of ACC expression observed in NE cells, confirming that AMPK regulates its expression and notably reduces the expression of the NED markers NSE and βIII Tub. Our findings also demonstrate, for the first time, that AMPK is inhibited in NE differentiation of prostate cells. This work demonstrates that Akt activation is involved in the AMPK inactivation and this AMPK inhibition contributes to NE differentiation. To explore the status of autophagy in NE cells, the protein levels of LC3, p62, Atg1, Atg5 and LAMP2 were measured. The results show that autophagy is activated in neuroendocrine differentiated LNCaP cells. Long-term cell treatments with different pharmacological inhibitors of PI3K used to block autophagy (3-MA and LY294002) or Atg5 knockdown significantly blocked autophagy and prevented neuroendocrine differentiation of LNCaP cells, suggesting an essential role of autophagy activity in NE differentiation of LNCaP cells. Silencing of LAMP2 in NE cells significantly decreased the levels of the neuroendocrine marker βIII Tub but not those of NSE, resulting in an accumulation of the cargo protein p62 in both control and NE cells, indicating that knock down of LAMP2 blocks the fusion of autophagosomes and lysosomes. To investigate the effect of the pharmacological inhibition of the PI3K/Akt/mTOR pathway or pharmacological activation of AMPK on autophagy during NED, LNCaP cells were treated for 6 days with LY294002, Akt Inh IV, rapamycin or AICAR, respectively. All of these treatments produced the same blocking effect on autophagy. Taken together, these results demonstrate that pharmacological inhibition of PI3K activity abrogates autophagy flux and NE differentiation in LNCaP cells, suggesting an essential role of autophagy activity in NE differentiation of LNCaP cells. Our results also show that during NE differentiation, the expression of the cannabinoid receptors CB1 and CB2 dramatically decreases. Treatment with 3μM WIN 55,212-2 (a non-selective cannabinoid CB1 and CB2 receptor agonist) during 6 days inhibited neuroendocrine differentiation of LNCaP and PC-3 cells. In order to gain insight into the mechanism whereby the cannabinoid WIN prevents neuroendocrine differentiation of prostate LNCaP cells, we analyzed the PI3K/Akt/mTOR pathway. Addition of 3μM WIN during serum deprivation markedly reduced the increase in phosphorylated Akt in NE cells, implying a WIN-induced blocking of the PI3K/Akt pathway. When 3μM WIN was added to the serum-deprived medium, the inhibition of AMPK observed in NE cells, was prevented. Our results show an inhibition of this pathway by the cannabinoid WIN, indicating that WIN is probably acting at the beginning of the process and that this cannabinoid may be useful for the treatment of neuroendocrine cancers. In perspective, cannabinoids could represent a viable strategy to prevent or delay the progression of advanced prostate cancer with neuroendocrine differentiation features.