Modulation of Akt/mTOR signaling overcomes sunitinib resistance in renal and prostate cancer cells

Abstract

Tyrosine kinase inhibitors exhibit impressive activity against advanced renal cell carcinoma. However, recent clinical studies have shown an equivocal response to sunitinib in patients with castration-resistant prostate cancer. The tumor suppressor PTEN acts as a gatekeeper of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR cell-survival pathway. Our experiments showed that PTEN expression inversely correlates with sunitinib resistance in renal and prostate cancer cells. Restoration of PTEN expression markedly increases sensitivity of tumor cells to sunitinib both in vitro and in vivo. In addition, pharmacologic manipulation of PI3K/Akt/mTOR signaling with PI3K/mTOR inhibitor, GDC-0980, mTOR inhibitor, temsirolimus, or pan-Akt inhibitor, GSK690693, was able to overcome sunitinib resistance in cancer cells. Our findings underscore the importance of PTEN expression in relation to sunitinib resistance and imply a direct cytotoxic effect by sunitinib on tumor cells in addition to its antiangiogenic actions.

Figure 1

Differential induction of apoptosis by sunitinib in PTEN-positive and PTEN-negative renal and prostate cancer cell lines. (A) Cells were treated with sunitinib (25 μM) for 24 hours. The percentage of apoptotic cells was determined by the APO-BRDU DNA fragmentation assay followed by flow cytometry analysis as indicated in Materials and Methods. X-axis represents DNA content; Y-axis represents staining intensity. Representative data from one of five experiments are presented. (B) Western blot analysis of PTEN expression in renal and prostate cancer cell lines. Makhov et al.

Figure 2

Expression of PTEN in PTEN-negative renal 786-O and prostate PC-3 cancer cells sensitizes them to sunitinib-mediated apoptosis. (A) Western blot analysis of PTEN expression in 786-O and PC-3 cells stably transfected with either PTEN or control vectors. (B) PTEN and control vector transfected 786-O and PC-3 cells were treated with sunitinib (25 μM) for 24 hours. The percentage of apoptotic cells was determined by APO-BRDU assay followed by flow cytometry analysis. Data is representative of one of three experiments. (C) IL-6 and IL-8 levels in cell culture supernatants of 786-O and PC-3 cells treated with sunitinib for 12 hours. The assay was performed in triplicates. Results are expressed as the mean ± SEM. Makhov et al.

Figure 3

The effect of PTEN expression on tumor growth in vivo. PC-3-puro (squares) or PC-3-PTEN (circles) cells were inoculated s.c. in the flank region of 6 week old male C.B17/Icr-scid mice. Animals were treated with either sunitinib or control (vegetable oil) as detailed in Materials and Methods. (A) Dynamics of tumor growth in the experimental and control groups of animals. (B) Weights of xenograft tumors obtained from the experimental and control groups of animals. Data shown are mean of 5 mice in each group; bars, SEM. Makhov et al.

Figure 4

The effect of concomitant treatment with sunitinib and temsirolimus, GDC-0980 or GSK690693 on tumor growth in vitro and in vivo. (A) Chemical structures of sunitinib, temsirolimus, GDC-0980 and GSK690693. (B) Parental PTEN-negative 786-O and PC-3 cells were pre-treated with GDC-0980 (10 μM), GSK690693 (20μM) or temsirolimus (25 μM) for 1 hour followed by treatment with sunitinib (25 μM) for 24 hours. The percentage of apoptotic cells was determined by APO-BRDU assay. Representative data are from one of three experiments. (C) Parental PC-3 and 786-O cells were cultured with various combinations of sunitinib (25μM), temsirolimus (25μM), GDC-0980 (10 μM), and GSK690693 (20μM) for 12 hours. Expression and phosphorylation status of PI3K/Akt/mTOR signaling proteins was evaluated by western blotting using specific antibodies. (D) IL-6 and IL-8 levels in cell culture supernatants of parental 786-O and PC-3 cells treated with sunitinib (25μM) and temsirolimus (25μM) for 12 hours. The assay was performed in triplicate. Results are expressed as the mean ± SEM. (E) Subcutaneous PC-3 tumors were established in 6 week old male C.B17/Icr-scid mice. Treatment with sunitinib and/or temsirolimus and assessment of tumor growth were performed as described in Materials and Methods. Makhov et al.

Figure 5

Potential mechanisms of interaction between sunitinib and PI3K/Akt/mTOR inhibitors. Makhov et al.