Rapamycin inhibits cell proliferation in type I and type II endometrial carcinomas: a search for biomarkers of sensitivity to treatment

Abstract

Objectives: Our goal was to evaluate the effect of rapamycin, an mTOR inhibitor, in type I and II human endometrial cancer tumor explants.

Methods: Short-term tissue culture with fresh endometrial cancer tumor explants was performed. Cell proliferation was assessed by MTS assay after treatment with rapamycin. Akt and PTEN status were documented by Western blotting. The effect of rapamycin on phosphorylated-S6 and 4E-BP-1 was also assessed by Western blotting. Real-time RT-PCR was used to quantify hTERT mRNA expression. Telomere length was determined by terminal restriction fragment Southern blotting.

Results: Thirteen fresh endometrial cancer tumor explants (nine Type I, four Type II) were placed in short-term culture and treated with rapamycin. Nine of the endometrial cancer tumors responded to rapamycin, with a median IC₅₀ of 11.4 nM. Sensitivity to rapamycin was independent of PTEN and Akt status. Tumors (13/13) had a reduction in phosphorylated-S6 and 10/13 had a reduction in phosphorylated 4E-BP-1. Rapamycin decreased hTERT mRNA expression in all of the endometrial cancer tumors. Telomere length did not correspond with responsiveness to this drug.

Conclusions: Rapamycin demonstrated activity in fresh endometrial tumor explants independent of PTEN and Akt status. Some tumors demonstrated a reduction in phosphorylated-S6 and 4E-BP-1 without a significant change in cellular proliferation, suggesting that additional pathways may modulate cellular proliferation. Thus, mTOR inhibitors may be a useful targeted therapy for both type I and type II endometrial cancers, but the search remains for a predictive biomarker of sensitivity to this therapy.

Fig. 1

Expression of the progesterone receptor (PR), estrogen receptor (ER), PTEN, phosphorylated-AKT (P-Akt) and β-actin in 13 human endometrial cancer tumor explants by Western blotting.

Fig. 2

Rapamycin inhibited phosphorylation of S6 in all of the human endometrial cancer tumor explants as determined by Western blotting [A]. In addition, rapamycin decreased phosphorylation of 4E-BP-1 in most of these tumor explants (77% or 10/13) [B]. Primary tumor cells were cultured for 24 h and then treated with 20 nM rapamycin (Ra) or control (C) for 24, 48 or 72 h. Little effect was seen on total S6 or total 4E-BP-1. This is a representative Western blot of 4 of the 13 endometrial cancer tumor explants examined.

Fig. 3

Rapamycin inhibits hTERT mRNA expression in human endometrial cancer tumor explants. Primary tumor cells were cultured for 48 h and then treated with 20 nM rapamycin or control for 48 h. hTERT mRNA levels were determined by real-time RT-PCR.

Fig. 4

Comparison of telomere length in human endometrial cancer tumor explants by terminal restriction fragment Southern blot analysis. Telomere length did not predict responsiveness to rapamycin among these samples.