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. 2016 Mar 15;114(6):659-68.
doi: 10.1038/bjc.2016.29. Epub 2016 Mar 8.

Androgen deprivation in LNCaP prostate tumour xenografts induces vascular changes and hypoxic stress, resulting in promotion of epithelial-to-mesenchymal transition

N M Byrne  1   2 , H Nesbitt  1 , L Ming  1 , S R McKeown  1 , J Worthington  3 , D J McKenna  1
Affiliations

Androgen deprivation in LNCaP prostate tumour xenografts induces vascular changes and hypoxic stress, resulting in promotion of epithelial-to-mesenchymal transition

N M Byrne et al. Br J Cancer. .

Abstract

Background: When single-agent androgen deprivation therapy (ADT) is administered for locally advanced prostate cancer, men usually relapse within 1-2 years with more malignant castrate-resistant disease. The reason for this is currently unknown. We now hypothesise that an initial treatment response that increases tumour hypoxia drives selection of more malignant tumours.

Methods: The LNCaP prostate tumour xenografts were analysed for physiological (oxygen and vasculature) and genetic (PCR array) changes during longitudinal treatment with ADT (bicalutamide, 6 or 2 mg kg⁻¹ daily for 28 days).

Results: Bicalutamide caused an immediate (within 24 h) dose-dependent fall in oxygenation in LNCaP-luc prostate tumours with a nadir of ≤ 0.1% oxygen within 3-7 days; this was attributed to a significant loss of tumour microvessels (window chamber study). The hypoxic nadir persisted for 10-14 days. During the next 7 days, tumours regrew, oxygenation improved and the vasculature recovered; this was inhibited by the VEGF inhibitor B20.4.1.1. Gene expression over 28 days showed marked fluctuations consistent with the physiological changes. Accompanying the angiogenic burst (day 21) was a particularly striking increase in expression of genes associated with epithelial-to-mesenchymal transition (EMT). In particular, insulin-like growth factor 1 (IGF-1) showed increases in mRNA and protein expression.

Conclusions: Hypoxic stress caused by ADT promotes EMT, providing a mechanism for the cause of malignant progression in prostate cancer.

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Figures

Figure 1
Figure 1
Bicalutamide effect on LNCaP-luc tumour growth, oxygenation and lung metastasis. The SCID mice (8 weeks old) bearing tumours of 100–200 mm3 were treated daily p.o. for 28 days with vehicle (0.1% DMSO in corn oil) or bicalutamide (2 or 6 mg kg−1). (A) Tumour growth. Results: mean±s.e; n⩾14 per group. (B) Oxygenation during treatment. All data points are significant (P⩽0.001) except days 0 and 28. (C) Quantification of metastasis in lungs of bicalutamide-treated mice compared with vehicle-treated mice. Student's t-test P-values: NS=not significant, **P<0.01.
Figure 2
Figure 2
Bicalutamide effect on vascularisation of LNCaP-luc tumour fragments in window chambers. The LNCaP-luc tumour fragments were allowed to vascularise for 7 days. At 15 min before imaging, the vasculature using a confocal microscope FITC-dextran was injected i.v. This was carried out before treatment (day 0) and on days 7, 14 and 21. Treatments: vehicle, bicalutamide 2 or 6 mg kg−1 (all p.o.), B20.4.1.1 (5 mg kg−1 i.p.) and bicalutamide 6 mg kg−1 with B20.4.1.1 administered on day 14. Images (× 10 magnification) are representative of ⩾3 mice per treatment. Scale bar=100 μM.
Figure 3
Figure 3
Stereological analysis of window chamber images. Histograms show changes in vessel parameters (A) MVD, (B) Branch points (C) length and (D) percentage coverage. Student's t-test P-values: *P<0.05, **P<0.01, ***P<0.001.
Figure 4
Figure 4
The PCR array analysis of the effect of bicalutamide on LNCaP gene expression. Mice bearing LNCaP-luc tumours (details in Figure 1) were treated daily for up to 28 days with vehicle or bicalutamide (2 mg kg−1). Mice were sacrificed, tumours removed and RNA extracted before treatment (day 0) and at the times indicated. Changes in gene expression were analysed using a PCR array and normalised to two reference genes. Fold changes were compared with day 0. (A) Dot plots showing longitudinal gene expression changes. (B) Changes in selected genes are detailed, bicalutamide-treated (shaded bars) compared with vehicle-treated controls. Results shown are mean±s.e. of ⩾5 pooled tumours analysed in duplicate.
Figure 5
Figure 5
Analysis of IGF-1 protein expression in LNCaP-luc tumours. Tumours were obtained as described in Figure 4, fixed, embedded and stained for IGF-1 immunoreactivity. (A) Representative images showing IGF-1 during vehicle or bicalutamide treatment (scale bar=25 μm) and (B) semi-quantification. Results are mean±s.e. of ⩾3 animals. Student's t-test P-values: NS=not significant, ***P<0.001.
Figure 6
Figure 6
Comparison of the effects of hypoxia and bicalutamide on LNCaP-luc cells. Gene expression of IGF-1, ITGA2 and TIMP1 was analysed in LNCaP-luc cells grown in vitro. (A) Cells were exposed to 4 or 24 h hypoxia (0.1%). (B) Cells were treated with bicalutamide for 24 or 168 h. (C) Gene expression in LNCaP cells isolated from tumours treated in vivo with vehicle (V1) or bicalutamide (B1). (D) Western blot of IGF-1 and TIMP1 in LNCaP-V1 and LNCaP-B1 cells. Results are mean±s.e. of 3 independent experiments. Student's t-test P-values: NS=not significant, *P<0.05, **P<0.01, ***P<0.001.
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