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. 2014 Feb 14;9(2):e88556.
doi: 10.1371/journal.pone.0088556. eCollection 2014.

Autophagy pathway is required for IL-6 induced neuroendocrine differentiation and chemoresistance of prostate cancer LNCaP cells

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Autophagy pathway is required for IL-6 induced neuroendocrine differentiation and chemoresistance of prostate cancer LNCaP cells

Pei-Ching Chang et al. PLoS One. .

Abstract

Prostate cancer (PCa) cells undergoing neuroendocrine differentiation (NED) are clinically relevant to the development of relapsed castration-resistant PCa. Increasing evidences show that autophagy involves in the development of neuroendocrine (NE) tumors, including PCa. To clarify the effect of autophagy on NED, androgen-sensitive PCa LNCaP cells were examined. Treatment of LNCaP cells with IL-6 resulted in an induction of autophagy. In the absence of androgen, IL-6 caused an even stronger activation of autophagy. Similar result was identified in NED induction. Inhibition of autophagy with chloroquine (CQ) markedly decreased NED. This observation was confirmed by beclin1 and Atg5 silencing experiments. Further supporting the role of autophagy in NED, we found that LC3 was up-regulated in PCa tissue that had relapsed after androgen-deprivation therapy when compared with their primary tumor counterpart. LC3 staining in relapsed PCa tissue showed punctate pattern similar to the staining of chromogranin A (CgA), a marker for NED cells. Moreover, autophagy inhibition induced the apoptosis of IL-6 induced NE differentiated PCa cells. Consistently, inhibition of autophagy by knockdown of beclin1 or Atg5 sensitized NE differentiated LNCaP cells to etoposide, a chemotherapy drug. To identify the mechanisms, phosphorylation of IL-6 downstream _targets was analyzed. An increase in phospho-AMPK and a decrease in phospho-mTOR were found, which implies that IL-6 regulates autophagy through the AMPK/mTOR pathway. Most important to this study is the discovery of REST, a neuronal gene-specific transcriptional repressor that is involved in autophagy activation. REST was down-regulated in IL-6 treatment. Knockdown experiments suggest that REST is critical to NED and autophagy activation by IL-6. Together, our studies imply that autophagy is involved in PCa progression and plays a cytoprotective role when NED is induced in PCa cells by IL-6 treatment. These results reveal the potential of _targeting autophagy as part of a combined therapeutic regime for NE tumors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The potentiating effect of androgen-deprivation on IL-6-induced autophagy in LNCaP cells.
(A) LNCaP-eGFP-LC3 cells were culture in 10% FBS, 2.5% FBS or 2.5% CDT supplemented RPMI 1640 in the absence (control) and presence of 100 ng/ml IL-6 for 48 hours. This was followed by fixation, nuclear counterstaining with DAPI (blue), and analysis by fluorescence microscopy (FITC, 63× magnification). (B) For each treatment, the percentage of cells with eGFP-LC3 punctate was calculated using the average from 15 microscopic fields; bars, SD.
Figure 2
Figure 2. IL-6 induces NED in LNCaP cells and this is concomitant with increased autophagy.
(A) LNCaP cells were treated with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 for 48 hours. The induced neurite elongation was assessed using brightfield microscopy images (40× magnification). (B) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars, SD. (C) LNCaP cells were treated as described in (A). Total cell lysates (TCLs) were prepared and then immunoblotted to detect tubulin III, androgen receptor (AR) and LC3. GAPDH was used as the loading control.
Figure 3
Figure 3. Chemical inhibition of autophagy flux suppresses IL-6-induced NED in LNCaP cells.
(A) LNCaP-eGFP-LC3 cells were treated with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6, in the absence and presence of 50 µM chloroquine (CQ) for 48 hours. This was followed by fixation, nuclear counterstaining with DAPI (blue), and analysis by fluorescence microscopy (FITC, 40× magnification). (B) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars, SD.
Figure 4
Figure 4. Knockdown of beclin1 suppresses IL-6 induced NED under androgen deprivation conditions.
(A) LNCaP-TR-shBeclin1 cells were treated with 1 µg/ml Dox for 48 hours. TCLs were prepared and immunoblotted to detect beclin1 using GAPDH as the loading control. (B) LNCaP-TR-shBeclin1 cells were treated for 48 hours with 1 µg/ml Dox in order to induce knockdown of beclin1 and they were then treated for another 48 hours with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 to induce cell NED. The inhibition of neurite elongation by beclin1 knockdown was assessed using brightfield microscopy images (40× magnification). (C) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars, SD. (D) TCLs were obtained from LNCaP-TR-shBeclin1 cells treated as described in (A) and these were then analyzed by immunoblotting using the indicated antibodies.
Figure 5
Figure 5. Knockdown of Atg5 suppresses IL-6 induced NED under androgen deprivation conditions.
(A) LNCaP-TR-shAtg5 cells were treated with 1 µg/ml Dox for 48 hours. TCLs were prepared and immunoblotted to detect Atg5 using GAPDH as the loading control. (B) LNCaP-TR-shAtg5 cells were treated for 48 hours with 1 µg/ml Dox to induce knockdown of Atg5 and then treated for another 48 hours with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 to induce cell NED. The inhibition of neurite elongation by Atg5 knockdown was assessed using brightfield microscopy images (40× magnification). (C) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars, SD. (D) TCLs obtained from LNCaP-TR-shAtg5 cells treated as described in (A) and these were then analyzed by immunoblotting using the indicated antibodies.
Figure 6
Figure 6. Up-regulation of the autophagy related genes LC3, the NE marker Chromogranin A (CgA), and the down-modulation of REST in relapsed PCa tissue compared to their primary tumor counterpart.
Representative IHC staining images of two pairs of tissue samples, namely primary and relapsed PCa specimens from the same individual, were stained using anti-LC3, anti-CgA, and anti-REST antibodies.
Figure 7
Figure 7. Inhibition of autophagy by CQ results in the cell death of IL-6-induced NE differentiated LNCaP cells.
(A) LNCaP cells were cultured in 2.5% CDT supplemented RPMI 1640 and then treated with 100 ng/ml IL-6 or vehicle control in the presence or absence of 50 µM CQ. Cell numbers were analyzed using a MTT cell viability assay at the indicated time points. Points, mean; bars, SD. (B) LNCaP cells were treated as described in (A) and then assayed for caspase-3/7 activity at the indicated time points. Each data point shown is the mean of three independent experiments; bars, SD.
Figure 8
Figure 8. Knockdown of beclin1 or Atg5 independently enhances the sensitivity of IL-6-induced NE differentiated LNCaP cells to etoposide.
LNCaP-TR-shBeclin1 (A) and LNCaP-TR-shAtg5 (B) cells were treated with Dox for 48 hours to induced either beclin1 or Atg5 knockdown, respectively, or left untreated. LNCaP control and LNCaP knockdown cells were pre-incubated with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6 for 48 hours followed by treatment with 20 µg/ml etoposide for 9 days before the MTT assay. Each data point shown is the mean of three independent experiments; bars, SD.
Figure 9
Figure 9. IL-6 treatment inhibits mTOR via the activation of AMPK pathway.
(A) LNCaP cells were treated for 48 hours with 2.5% CDT or 2.5% CDT plus 100 ng/ml IL-6. TCLs were prepared and immunoblotted using phospho-STAT3, phospho-Akt and phopho-ERK specific antibodies; immunoblotting to detect the non-phospho-counterparts of these proteins was used as the control. GAPDH was used as the loading control. (B) LNCaP cells were treated as described in (A) and immunoblotted using the antibodies as indicated and using GAPDH as the loading control.
Figure 10
Figure 10. Regulation of NED by REST in LNCaP cells.
(A) The level of REST protein declines during IL-6 treatment. LNCaP cells were treated with 100 ng/ml IL-6 for 48 and 96 hours. The expression level of REST was analyzed by immunoblotting using anti-REST antibody. GAPDH was used as the loading control. (B) LNCaP-TR-shREST cells were treated with or without Dox for 48 hours. TCLs were analyzed by immunoblotting using anti-REST antibody. (C) LNCaP-TR-shREST cells were treated with Dox for 6 days. The promotion of neurite outgrowth by REST knockdown was assessed using brightfield microscopy images (40× magnification). (D) The neurite elongation was quantified using the average from 3–5 microscopic fields; bars, SD. (E) LNCaP-TR-shREST cells were treated as described in (C). TCLs were prepared and analyzed by immunoblotting using the antibodies as indicated. (F) LNCaP-TR-REST cells were treated with 1 µg/ml Dox in the absence (control) or presence of 100 ng/ml IL-6 for 4 days. Inhibition of IL-6-induced neurite outgrowth by REST overexpression was assessed using brightfield microscopy images (40× magnification). (G) TCLs were obtained from LNCaP-TR-REST cells treated as described in (F); these were then analyzed by immunoblotting using the indicated antibodies. (H) RT-qPCR analysis of total RNA isolated from LNCaP-TR-shREST cells treated as described in (C). The relative mRNA levels of REST, Atg5, beclin1 and LC3 were normalized against GAPDH. Values from three independent data points are reported as mean±S.D.
Figure 11
Figure 11. The schematic model of IL-6 regulation of autophagy-dependent NED in PCa cells, LNCaP.
Based on our data, autophagy is essential for PCa cells NED and serves a cytoprotective role in NE differentiated PCa cells. IL-6 induces autophagy-dependent NED through activation AMPK/mTOR pathway and down-modulation of REST. Activation of AMPK inhibits mTOR, a key inhibitor of autophagy, which in-turn activates autophagy pathway. Down-modulation of REST level relieves gene silencer REST-mediated transcriptional repression as part of a relay mechanism found in IL-6 induced autophagy.

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