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Clinical Trial
. 2010 Oct 15;70(20):7992-8002.
doi: 10.1158/0008-5472.CAN-10-0585. Epub 2010 Aug 31.

Tubulin-targeting chemotherapy impairs androgen receptor activity in prostate cancer

Meng-Lei Zhu  1 , Craig M Horbinski, Mark Garzotto, David Z Qian, Tomasz M Beer, Natasha Kyprianou
Affiliations
Clinical Trial

Tubulin-targeting chemotherapy impairs androgen receptor activity in prostate cancer

Meng-Lei Zhu et al. Cancer Res. .

Abstract

Recent insights into the regulation of the androgen receptor (AR) activity led to novel therapeutic targeting of AR function in prostate cancer patients. Docetaxel is an approved chemotherapy for treatment of castration-resistant prostate cancer; however, the mechanism underlying the action of this tubulin-targeting drug is not fully understood. This study investigates the contribution of microtubules and the cytoskeleton to androgen-mediated signaling and the consequences of their inhibition on AR activity in human prostate cancer. Tissue microarrays from docetaxel-treated and untreated prostate cancer patients were comparatively analyzed for prostate-specific antigen (PSA) and AR immunoreactivity. The AR transcriptional activity was determined in prostate cancer cells in vitro, based on PSA mRNA expression and the androgen response element reporter activity. The interaction of AR with tubulin was examined by immunoprecipitation and immunofluorescence. Treatment of prostate cancer patients with docetaxel led to a significant translocation of AR. In untreated specimens, 50% prostate tumor cells exhibited nuclear accumulation of AR, compared with docetaxel-treated tumors that had significantly depleted nuclear AR (38%), paralleled by an increase in cytosolic AR. AR nuclear localization correlated with PSA expression. In vitro, exposure of prostate cancer cells to paclitaxel (1 μmol/L) or nocodazole (5 μg/mL) inhibited androgen-dependent AR nuclear translocation by targeting AR association with tubulin. Introduction of a truncated AR indicated the requirement of the NH(2)-terminal domain for AR-tubulin interaction. Our findings show that in addition to blocking cell division, docetaxel impairs AR signaling, evidence that enables new insights into the therapeutic efficacy of microtubule-targeting drugs in prostate cancer.

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Figures

Figure 1
Figure 1. Doclitaxel Suppresses PSA Expression in Human Prostate Tumors
Panel A reveals the PSA immunoreactivity pattern of prostate tissue array: from the left panel, untreated patients; right, Docetaxel-treated patients. Panel B shows representative images of individual prostate tumor TMAs from untreated control and docetaxel treated prostate cancer patients. Immunostaining for PSA was conducted as described in “Materials and Methods”. Panel C; Quantitative evaluation of PSA expression in tumor epithelial cells in prostate specimens from Docetaxel-treated and untreated prostate cancer patients was determine as described in “Materials and Methods”. * P< 0.01.
Figure 2
Figure 2. Microtubule Targeting Drugs Inhibit Ligand-dependent AR Transcriptional Activity
LNCaP cells were treated with DHT (1nM) in the presence or absence of Nocodazole (5ug/ml) or Paclitaxel (1μM). Panel A; PSA mRNA expression was determined by realtime PCR. Panel B; PSA protein levels were assessed by Western blotting and relative expression was quantitated (lower panel). Panel C; The AR transcriptional activity in response to microtubule-targeting drugs was determined using the ARE luciferase reporter vector in LNCaP cells.
Figure 3
Figure 3. Microtubule Targeting Inhibits Ligand-independent AR Transciptional Activity
Panel A; LNCaP cells were treated with a combination of DHT (0.1nM) and EGF (5nM) with or without Nocodazole (5μg/ml) or Paclitaxel (1μM). AR transcriptional activity was evaluated on the basis of PSA expression using realtime PCR. Panel B; LNCaP cells were treated with the following chemotherapeutic agents for 24-72hrs: TRAIL, Velcade, Doxasosin, Nocodazole (5μg/ml) or Paclitaxel (1μM) and cell death was determined using the MTT assay. Panel C; LNCaP cells were treated with DHT (1nM), in the presence or absence of Velcade or Doxasosin as shown. PSA mRNA expression was evaluated by Real-time PCR. * P<0.05
Figure 4
Figure 4. Docetaxel Suppresses AR Nuclear Translocation in Prostate Cancer
Panel A; AR protein expression levels in prostate cancer epithelial cells of Doclitaxel treated and untreated patients was evaluated by immunohistochemical staining. There was no significant change in AR levels in prostate epithelial cells between the two groups. Panel B reveals a representative image of the subcellular AR localization in human prostate tissue. AR presence was assessed in formalin-fixed, paraffin-embedded prostate cancer tissue microarrays via light microscopic examination, while blinded to treatment modality. The overall pattern of staining (specifically the presence or absence of AR localization in the nucleus, cytoplasm, or both) was determined for each tissue core. Left panel, tissue from untreated patients; right, tissue from Docetaxel-treated patients. Panel C indicates the percentage of nuclear and cytoplasmic AR in Docetaxel-treated and untreated tumors. For the prostate specimens from untreated patients 50% prostate cancer epithelial cells exhibited nuclear AR; In Docetaxel-treated patients, a reduction in nuclear translocation of AR, was paralleled by an increase in cytosolic AR Panel D; AR localization correlated with PSA levels in prostate epithelial cells.
Figure 5
Figure 5. Tubulin Interacts with AR
Panels A and B; Androgens induce AR nuclear translocation in LNCaP cells and pre-treatment of Paclitaxel and Nocodazole for 24hrs abrogated this AR nuclear translocation. Subcellular localization of AR was detected by fluoresencent staining (red) (40x magnification). Western blot analysis of the cellular compartments after subcellular fractionation also revealed that DHT-induced nuclear translocation of AR was blocked in response to either Paclitaxel or Nocodazole treatment (Panel C). GAPDH and PARP were used as loading controls. Panels D and E; LNCaP TβRII and CWR22 cells, respectively were treated with DHT (1nM), in the presence or absence of TGF-β (5ng/ml). Immunoprecipitation was performed by using the antibodies against either tubulin or AR to show the AR-tubulin association). Panel F; Truncated forms of AR transfected in PC-3 cells. Immunoprecipitation analysis of AR and tubulin interaction indicates that loss of ligand-binding and DNA-binding domain and hinge domain did not inhibit the AR-tubulin association (Panel G).
Figure 6
Figure 6. Androgens Inhibit Tubulin Expression in Prostate Cancer Cells
Panel A; Prostate cancer cells LNCaP, LNCaP TβRII and CWR22, were treated with DHT (1nM; for 72hrs) and tubulin expression was evaluated by Western blotting using GAPDH as internal normalizing control. Panel B; LNCaP TβRII and CWR22 cells were treated with DHT (1nM) with or without TGF-β (5ng/ml) for 72hrs. Tubulin expression was detected by immunofluorescence (red); nuclei were stained by DAPI (blue). Panel C illustrates the emerging mechanistic scenario: microtubules facilitate AR nuclear translocation and enhance downstream AR transcriptional activity in prostate cancer cells. Microtubule targeting chemotherapy blocks this pathway and suppresses AR signaling, via a negative feedback mechanism; AR signaling inhibits tubulin expression thus impairing the cytoskeleton structure and organization.
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