Combination of RAD001 (everolimus) and docetaxel reduces prostate and breast cancer cell VEGF production and tumour vascularisation independently of sphingosine-kinase-1

Heba Alshaker,Yoshiaki Kawano,Torsten Böhler,Qi Wang,Tawfiq Arafat,Mathias Winkler,Dmitri Pchejetski,Robert Mills

doi:10.1038/s41598-017-03728-3

Abstract

Resistance to docetaxel is a key problem in current prostate and breast cancer management. We have recently discovered a new molecular mechanism of prostate cancer docetaxel chemoresistance mediated by the mammalian target of rapamycin (mTOR)/sphingosine-kinase-1 (SK1) pathway. Here we investigated the influence of this pathway on vascular endothelial growth factor (VEGF) production and tumour vascularisation in hormone resistant prostate and breast cancer models. Immunofluorescent staining of tumour sections from human oestrogen receptor (ER)-negative breast cancer patients showed a strong correlation between phosphorylated P70S6 kinase (mTOR downstream target), VEGF and SK1 protein expression. In hormone-insensitive prostate (PC3) and breast (MDA-MB-231 and BT-549) cancer cell lines the mTOR inhibitor RAD001 (everolimus) has significantly inhibited SK1 and VEGF expression, while low dose (5 nM) docetaxel had no significant effect. In these cell lines, SK1 overexpression slightly increased the basal levels of VEGF, but did not block the inhibitory effect of RAD001 on VEGF. In a human prostate xenograft model established in nude mice, RAD001 alone or in combination with docetaxel has suppressed tumour growth, VEGF expression and decreased tumour vasculature. Overall, our data demonstrate a new mechanism of an independent regulation of SK1 and VEGF by mTOR in hormone-insensitive prostate and breast cancers.

Highlights

Mammalian target of rapamycin is a protein kinase that is present in two distinct complexes: mammalian target of rapamycin (mTOR) complex 1 and mTOR complex 2[8]
To elucidate the mechanisms implicated in the effects of RAD001 in combination with docetaxel, we first analysed the effect of docetaxel on the expression of SK1 in MDA-MB-231
Similar to the previous findings in PC3 and DU145 prostate cancer cells[16], docetaxel did not decrease the levels of P70S6 Kinase (P70S6K) phosphorylation, SK1 mRNA or activity, but rather insignificantly increased them (Fig. 2)

Summary

Introduction

Mammalian target of rapamycin (mTOR) is a protein kinase that is present in two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)[8]. We revealed that RAD001 sensitizes PC3 and DU145 prostate cancer cells and tumours to docetaxel therapy through downregulation of mTOR/SK1 pathway[16]. We have investigated the effects of docetaxel/RAD001 combination therapy on VEGF regulation in hormone insensitive prostate and breast cancer cells. Our data from primary human breast tumours and human prostate cancer animal models have shown a correlated expression of SK1 and VEGF, which suggests a common regulatory mechanism. Contrary to chemoresistance that was shown in PC3 and DU145 cells[16], SK1 overexpression did not abrogate RAD001-induced reduction in VEGF in prostate (PC3) and breast (MDA-MB-231) cancer models, suggesting that the two pathways are regulated independently. Our results provide novel insights into the mechanisms of synergistic RAD001/docetaxel combination and highlight the potential for the use of this combined therapy in hormone-insensitive prostate and breast cancers

Methods

Results

Conclusion