RAD001 (Everolimus) inhibits growth of prostate cancer in the bone and the inhibitory effects are increased by combination with docetaxel and zoledronic acid

Abstract

Introduction: mTOR activity is increased in advanced prostate cancer (CaP) as a result of a high rate of PTEN mutations. RAD001 (Everolimus) is a new orally available mTOR inhibitor. The objective of our study was to evaluate the effects of RAD001 on the growth of CaP in the bone, both alone and in combination with docetaxel and zoledronic acid.

Methods: C4-2 CaP cells were injected into tibiae of mice and the animals were treated with RAD001, docetaxel, and zoledronic acid alone or in combination. Histomorphometrical analysis, serum PSA measurements, bone mineral density (BMD), and microCT were used to determine the effects of treatment on tumor and bone.

Results: All three agents alone decreased tumor volume, and RAD001 and docetaxel also decreased levels of serum PSA by 68% and 65%, respectively (both P < 0.01). Combinations of the agents were more effective in decreasing tumor volume than single agents. Three-drug treatment showed the greatest effect: 64% inhibition versus control (P < 0.01). Treatment with RAD001 interfered with the weight loss associated with growth of this tumor in the bone (non-RAD001 groups: 4.0% decrease in body weight, P = 0.0014; RAD001 groups: increase of 3.6% in body weight, P = 0.0037).

Conclusions: RAD001 inhibited growth of C4-2 cells in bone, an effect augmented by addition of docetaxel and zoledronic acid. Moreover RAD001 had a significant impact on maintenance of body weight. RAD001 may hold promise for its effects on both metastatic CaP and the important syndrome of tumor cachexia.

Fig. 1

Effects of RAD001 and TAX on serum PSA. RAD001 and TAX inhibited tumor growth in bone as demonstrated by decreases in serum PSA levels. Arrows mark start of treatment. A: RAD001: 5 mg/kg, P = 0.026; and 10 mg/kg, P = 0.029. B: TAX: 10 mg/kg, P = 0.0070; and 20 mg/kg, P = 0.0011. Results are plotted as mean ± SEM.

Fig. 2

Effects of RAD001, TAX, and ZOL alone and in combination on serum PSA. Treatment of C4-2 tumors in bone resulted in decreases in serum PSA levels. Treatment began at week 4 as indicated by arrow. A: Single-agent therapy. B: Combination therapy. C: Comparison of sacrifice serum PSA levels among groups. Results are plotted as mean ± SEM. Significant differences versus controls are denoted by: *P < 0.05, **P < 0.01.

Fig. 3

Effects of RAD001, TAX, and ZOL alone and in combination on tumor and bone. Bone histomorphometry was used to evaluate the effects of the treatment on tissue volume (TV) and tumor volume (TuV). Analyses were performed on longitudinal sections of tibiae (n = 5 per group). BMD and BHM analyses were used to evaluate the effects of the treatments on bone. Tissue volume (A), and tumor volume (B) were significantly decreased in all treatment groups versus the control group. The largest decreases in TV and TuV were observed with all three agents in combination. Comparison of BMD normalized to contralateral (non-tumored) tibiae for each treatment. C4-2 cells cause decreases in BMD. Treatment with low doses of ZOL alone or in combination preserved bone (C). Treatments with ZOL generally caused increases in BV. RAD001 treatment increased BV, but the combination of three agents did not (D). Results are plotted as mean ± SEM. Significant differences versus controls are denoted by: *P < 0.05, **P < 0.01.

Fig. 4

Micro-CT analyses. Analysis of the response of the bone to treatment was performed for trabecular bone (metaphysis) and cortical bone. A: Tissue volume at the metaphysis. B: Ratio of bone volume/tissue volume at the metaphysis. C: Cortical bone volume. D: Periosteal volume. Results are plotted as mean ± SEM. Significant differences versus untreated C4-2 xenografts are denoted by:*P < 0.05, **P < 0.01. Also shown for reference in these figures are the parameters of normal untreated tibiae. E: Representative examples of metaphyseal and cortical bone from groups treated with RAD001. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

RAD001 effects on bodyweight. Treatment with RAD001 inhibited weight loss caused by C4-2 growth in bone. For this analysis, animals were grouped based on whether or not they received RAD001. Total bodyweight was maintained in mice receiving RAD001, while animals with C4-2 tumors not treated with RAD001 experienced loss of body weight. There was a 10.7 ± 1.5% difference in final body weight between groups with and without RAD001 treatment (P < 0.001). Results are plotted as mean ± SEM.