Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas

Abstract

The PI3K (phosphatidylinositol-3-kinase)/mTOR (mammalian target of rapamycin) pathway is frequently activated in endometrial cancer through various PI3K/AKT-activating genetic alterations. We examined the antitumor effect of NVP-BEZ235--a dual PI3K/mTOR inhibitor--and RAD001--an mTOR inhibitor--in 13 endometrial cancer cell lines, all of which possess one or more alterations in PTEN, PIK3CA, and K-Ras. We also combined these compounds with a MAPK pathway inhibitor (PD98059 or UO126) in cell lines with K-Ras alterations (mutations or amplification). PTEN mutant cell lines without K-Ras alterations (n = 9) were more sensitive to both RAD001 and NVP-BEZ235 than were cell lines with K-Ras alterations (n = 4). Dose-dependent growth suppression was more drastically induced by NVP-BEZ235 than by RAD001 in the sensitive cell lines. G1 arrest was induced by NVP-BEZ235 in a dose-dependent manner. We observed in vivo antitumor activity of both RAD001 and NVP-BEZ235 in nude mice. The presence of a MEK inhibitor, PD98059 or UO126, sensitized the K-Ras mutant cells to NVP-BEZ235. Robust growth suppression by NVP-BEZ235 suggests that a dual PI3K/mTOR inhibitor is a promising therapeutic for endometrial carcinomas. Our data suggest that mutational statuses of PTEN and K-Ras might be useful predictors of sensitivity to NVP-BEZ235 in certain endometrial carcinomas.

Figure 1. Copy number gain at the locus of

K-Ras (12p12.1) in the two group D cell lines. (A) SNP array ‘karyograms’ (250K) of HEC-50B cells. The graph shows the total copy number through chromosome 9p-X. The locus of K-Ras is amplified as indicated. (B) Array CGH of KLE cells. The graphs show total copy number throughout the entire genome (Left) and chromosome 12 (Right). The locus of K-Ras is amplified as indicated.

Figure 2. Inhibition of cell proliferation by NVP-BEZ235 and RAD001.

(A)–(D) WST-8 assay showing the sensitivity of endometrial cancer cells to NVP-BEZ235 and RAD001 at increasing concentrations of drug (nmol/L) for 72 h. The data was normalized to the value of control cells. (A) Four group A cell lines with double mutations of PIK3CA and PTEN, (B) Five group B cell lines with PTEN mutations, (C) Two group C cell lines with coexistent mutations of K-Ras and PIK3CA, and (D) Two group D cell lines with chromosomal copy number amplification at the locus of K-Ras. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D.

Figure 3. Inhibition of PI3K/mTOR signaling by NVP-BEZ235 and inhibition of mTOR signaling by RAD001 in endometrial cancer cell lines.

(A)–(B) Western blot of total lysates of HEC-88 (group A) and HEC-108 (group B) cells, treated with NVP-BEZ235 or RAD001 at concentrations ranging from 0 to 1,000 nmol/L with 10% fetal bovine serum (FBS). phospho-Akt (Ser473, Thr308), phospho-S6 (Ser235/236, Ser240/244), and phospho-4EBP1 (Thr 37/46) levels are shown with total Akt level for a loading control. (C) Western blot of total lysates of HEC-6 (group A) cells treated with NVP-BEZ235 or RAD001 at a dose of 100 nM for up to 48 hours with 10% FBS. Phosphorylation levels of the PI3K/mTOR signaling are shown with loading controls.

Figure 4. Flowcytometric analysis of cell cycle in cancer cells treated with either NVP-BEZ235 or RAD001.

(A–D) Cells (5×10⁵) were seeded in the presence of 10% serum and treated with NVP-BEZ235 or RAD001 for 48 h at a dose of 10 nM or 100 nM, respectively. A higher dose of NVP-BEZ235 (100 nM) significantly augmented the percentage of cells in the G0-G1phase of the cell cycle, compared with that of RAD001 (100 nM). (A)–(B); The data from two group A cells. (C)–(D); the data from two group B cells.

Figure 5. in vivo anti-tumor effect of NVP-BEZ235 and RAD001 in nude mice.

Subcutaneous xenograft tumors in athymic BALB/c mice were established in the injection of endometrial carcinoma cells. Mice were treated with a daily dose of 40 mg/kg (NVP-BEZ235) or with a daily dose of 2.5 mg/kg RAD001 or vehicle alone (control). Each treatment group contained 6 mice; (A) HEC-59 and (B) AN3CA. Tumor volumes were calculated by the formula {(major axis)*(minor axis)2/2}mm3 twice a week. Groups were compared at the end of treatment. Points, mean; bars, SD, *;p<0.05. (C) Appearance of subcutaneous tumors in HEC-59 xenografts. (D) Western blot of total lysates from the HEC-59 xenografts. p-Akt, p-FOXO1/3a, p-GSK3beta, p-S6 were assessed 1 and 24 h after the last drug administration. Total Akt and beta-actin were shown as loading controls.

Figure 6. Inhibition of cell proliferation and augmentation of G1 arrest by combination of a MEK inhibitor and NVP-BEZ235 (or RAD001) in cells with alterations in

K-Ras (mutation or amplification). (A)–(B) WST-8 assay was performed in HEC-1B (group C) and HEC-50B (group D) cell lines. All the experiments were repeated three times and each value is shown as the mean of three experiments +/− S.D. The combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 (or RAD001) significantly augmented anti-proliferative effect in these cell lines, compared with either inhibition alone (p<0.05 by Student's t-test). (C)–(D) Flowcytometric analysis of cell cycle in HEC-1B and HEC-50B cells. All experiments were repeated 3 times, and each value is shown as the mean of 3 experiments ± S.D. Combination of a MEK inhibitor (PD98059 or UO126) and NVP-BEZ235 significantly augmented the percentage of cells in the G0-G1phase of the cell cycle in these cells. *: p<0.05 by Student's t-test.

Figure 7. Inhibition of PI3K and MAPK signaling pathway by NVP-BEZ235, RAD001, and a MEK inhibitor (PD98059) in endometrial cancer cell lines with K-Ras alterations.

Western blot analysis of total lysates of HEC-1B and HHUA (group C) and HEC-50B and KLE (group D) cells treated with 1 of the PI3K (or MAPK) signal inhibitors or their combination with 10% FBS. Phospho-Akt, phospho-S6, and phospho-ERK levels are shown with total Akt, total S6, and total ERK levels.