Androgen deprivation results in time‐dependent hypoxia in LNCaP prostate tumours: Informed scheduling of the bioreductive drug AQ4N improves treatment response

Abstract

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide-treated and vehicle-only-treated tumours were re-established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 6 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.

Defining normoxia, physoxia and hypoxia in tumours—implications for treatment response

“…55 Studies in our laboratory showed that mice bearing LNCaP xenografts exposed to bicalutamide-induced hypoxia had increased metastasis to the lungs; this correlated with an increase in Bcl2 and reduction in Bax. 44 Gene amplification has also been reported in rodent tumour cells exposed ex vivo or in vivo to hypoxia; this was associated with an increase in metastases. 65,66 In both tissue culture and animal models, acute hypoxia/reoxygenation have been linked to the induction of DNA strand breaks and clearly, if these breaks are not repaired, they will result in further mutations.…”

“…43 More recently, we have shown that the median oxygen level of bicalutamide-treated LNCaP prostate xenografts remained below 0.1% oxygen for more than 10 days. 44 Overall, survival in this extreme stress will drive selection for malignant phenotypes that are governed by a Darwinian selection process. 45 …”

“…In LNCaP xenografts, we have found that the median oxygen level in vehicle-treated tumours is significantly reproducible (discussed below). 44 In humans, it has been shown that the median oxygen level in normal breast tissue was remarkably constant irrespective of the level of haemoglobin in the blood. This was in contrast to breast tumours, which showed both a markedly lower level of pO 2 than the normal tissue and also a fall, in this already low level, as the haemoglobin concentration decreased.…”

“…In addition, mice treated with bicalutamide had a marked increase in metastatic spread to the lungs, although this could be blocked successfully by treatment on Day 7 with a single dose of banoxantrone (AQ4N), a prodrug that specifically targets hypoxic cells. 44 The initial antivascular effect of the anti-androgen bicalutamide is not widely recognized, although previous studies, mostly using castration models, have provided much evidence that it is likely to occur (discussed in 44 ). Our studies showed that prostate tumour cells are very hypoxia tolerant.…”

“…However, tumours can adapt to this hypoxic insult and they recover with a more proangiogenic, and potentially malignant, phenotype. 44,83,84 This problem may be overcome in part, at least, by combination with other CCT. Hypoxia-activated prodrugs (HAPs) (discussed below) offer an additional approach, since they specifically target hypoxic cells.…”