The VEGF pathway and the AKT/mTOR/p70S6K1 signalling pathway in human epithelial ovarian cancer

Abstract

Vascular endothelial growth factor (VEGF)-A inhibitors exhibit unseen high responses and toxicity in recurrent epithelial ovarian cancer suggesting an important role for the VEGF/VEGFR pathway. We studied the correlation of VEGF signalling and AKT/mTOR signalling. Using a tissue microarray of clinical samples (N=86), tumour cell immunohistochemical staining of AKT/mTOR downstream targets, pS6 and p4E-BP1, together with tumour cell staining of VEGF-A and pVEGFR2 were semi-quantified. A correlation was found between the marker for VEGFR2 activation (pVEGFR2) and a downstream target of AKT/mTOR signalling (pS6) (R=0.29; P=0.002). Additional gene expression analysis in an independent cDNA microarray dataset (N=24) showed a negative correlation (R=-0.73, P<0.0001) between the RPS6 and the VEGFR2 gene, which is consistent as the gene expression and phosphorylation of S6 is inversely regulated. An activated tumour cell VEGFR2/AKT/mTOR pathway was associated with increased incidence of ascites (chi(2), P=0.002) and reduced overall survival of cisplatin-taxane-based patients with serous histology (N=32, log-rank test, P=0.04). These data propose that VEGF-A signalling acts on tumour cells as a stimulator of the AKT/mTOR pathway. Although VEGF-A inhibitors are classified as anti-angiogenic drugs, these data suggest that the working mechanism has an important additional modality of targeting the tumour cells directly.

Figure 1

In endothelial cells, after activation, VEGFR2 is able to stimulate the AKT/mTOR signalling pathway through PI3K. More downstream 4E-BP1, P70S6K1 and S6 are phosphorylated. The mTORC2 complex (insensitive to rapamycin/FKBP12) has been shown to be an activator of AKT. The mTORC1 complex is sensitive to rapamycin (sirolimus) or derivates (everolimus, temsirolimus and deforolimus).

Figure 2

Schematic overview of patients included in the study.

Figure 3

(A–E) Serial slides with immunohistochemical staining for pVEGFR2(Tyr951) (A), pVEGFR2(Tyr996) (B), pS6 (C), p4E-BP1 (D), VEGF-A (E) of an ovarian cancer specimen. Note that there were locally places in which there was more prominent staining for pVEGFR2 with a concomitant expression of the ribosomal protein pS6.

Figure 4

A summary of immunohistochemical correlations.

Figure 5

The ²log relative gene expression correlations using an independent dataset of epithelial ovarian cancer samples. The RPS6 gene was significantly well correlated with the relative expression of VEGFR2. (vertical bars show 95% CI).

Figure 6

After 48 h RAD001 administration, prostate tissue showed a significant increase of normalised gene expression for RPS6 compared with 12 h (P<0.01—²log mean channel count increase of +0.28 95% CI (0.08–0.48)) and placebo-treated mice (P<0.001—²log mean channel count increase of +0.35 95% CI (0.17–0.53)), This effect was not observed for the expression of EIF4EBP1 (n.s.).

Figure 7

Kaplan–Meier survival curve (N=32 patients). Baseline expression of pS6 and pVEGFR2 were dichotomised into a high (+) and low (−) category using the median expression H-score value (log-rank test, P=0.041).