Potential role of mTORC2 as a therapeutic target in clear cell carcinoma of the ovary

Abstract

The goal of this study was to examine the role of mTOR complex 2 (mTORC2) as a therapeutic target in ovarian clear cell carcinoma (CCC), which is regarded as an aggressive, chemoresistant histologic subtype. Using tissue microarrays of 98 primary ovarian cancers [52 CCCs and 46 serous adenocarcinomas (SAC)], activation of mTORC2 was assessed by immunohistochemistry. Then, the growth-inhibitory effect of mTORC2-targeting therapy, as well as the role of mTORC2 signaling as a mechanism for acquired resistance to the mTOR complex 1 (mTORC1) inhibitor RAD001 in ovarian CCC, were examined using two pairs of RAD001-sensitive parental (RMG2 and HAC2) and RAD001-resistant CCC cell lines (RMG2-RR and HAC2-RR). mTORC2 was more frequently activated in CCCs than in SACs (71.2% vs. 45.7%). Simultaneous inhibition of mTORC1 and mTORC2 by AZD8055 markedly inhibited the proliferation of both RAD001-sensitive and -resistant cells in vitro. Treatment with RAD001 induced mTORC2-mediated AKT activation in RAD001-sensitive CCC cells. Moreover, increased activation of mTORC2-AKT signaling was observed in RAD001-resistant CCC cells compared with the respective parental cells. Inhibition of mTORC2 during RAD001 treatment enhanced the antitumor effect of RAD001 and prevented CCC cells from acquiring resistance to RAD001. In conclusion, mTORC2 is frequently activated, and can be a promising therapeutic target, in ovarian CCCs. Moreover, mTORC2-targeted therapy may be efficacious in a first-line setting as well as for second-line treatment of recurrent disease developing after RAD001-treatment.

Figure 1. mTORC2 is frequently expressed and activated in ovarian clear cell carcinomas

Ovarian cancer tissue microarrays and normal ovarian tissues were stained with either anti-Rictor or anti-phospho-mTOR (Ser2481) antibodies. A, representative photographs of ovarian tissue microarray cores. Magnifications: x100, and x400 (inset). B, histogram indicating immunoreactivity profile. C, histogram indicating immunostaining profile by clinical stage. Proportion indicates proportion of medium/strong-staining tumors. *, p<0.05. D, mTORC2 activation status in four ovarian CCC cell lines. CCC cells were serum-starved overnight, after which the mTORC2 activity was determined by in vitro kinase assay. Briefly, the cell lysates were incubated with anti-Rictor antibody followed by incubation with protein A-agarose. Immunocomplexes were incubated in kinase reaction buffer, and phospho-AKT and Rictor were detected by Western blotting.

Figure 2. In vitro growth-inhibitory effect of AZD8055 on CCC cell lines

A, Chemical structures of RAD001 and AZD8055. B, Sensitivity of CCC cells to AZD8055 or RAD001. CCC cells were treated with the indicated concentrations of AZD8055 or RAD001 in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (**, significantly different between RAD001-treated and AZD8055-treated cells at given drug concentration; p<0.01). C, AZD8055, but not RAD001, induces apoptosis in ovarian CCC cells. RMG2 and HAC2 cells were treated with 10 nM AZD8055 or 10nM RAD001 for 24 h in the presence of 5% FBS. The cells were lysed, and DNA fragmentation was examined. Columns, mean; bars, SD. *, P < 0.05. D, The effect of AZD8055 or RAD001 on the phosphorylation of p70S6K, 4E-BP1, AKT, and PRAS40 in vitro. RMG2 and HAC2 cells were treated with 10 nM AZD8055 or 10 nM RAD001 for 6 h in the presence of 5% FBS. Cells were harvested, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with various antibodies.

Figure 3. Inhibition of mTORC1 by RAD001 induces mTORC2-mediated phosphorylation of AKT-PRAS40 signaling in CCC cells

A, RAD001 attenuates the phosphorylation of Rictor at Thr1135, which results in increased phosphorylation of AKT and PRAS40 in CCC cells. RMG2 and HAC2 cells were treated with 10 nM AZD8055 or 10 nM RAD001 for the indicated times in the presence of 5% FBS. B, Suppression of Rictor by shRNA inhibits RAD001-induced AKT phosphorylation. RMG2 and HAC2 cells plated in 6-well plates were transfected with 20nM control or Rictor shRNA. After 48 h, cells were treated with 10 nM AZD8055 or 10 nM RAD001 for 6 h in the presence of 5% FBS. Cells were harvested, lysed, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with various antibodies. C, Effect of S6K1 knockdown by siRNA on Rictor phosphorylation and mTORC2 activation. RMG2 and HAC2 cells plated in 6-well plates were transfected with 100 pmol control or S6K1 siRNA. After 48 h, cells were harvested, lysed, and equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with various antibodies. mTORC2 activity was determined by in vitro kinase assay as described in “Materials and Methods”. D, Model of mTORC1-inhibition mediated feedback activation of AKT. In a normal condition, S6K1 directly phosphorylates Rictor on Thr1135 which negatively regulates the ability of mTORC2 to phosphorylate AKT. mTORC1-inhibition by RAD001 triggers negative feedback mechanisms resulting in mTORC2-mediated phosphorylation of AKT-PRAS40.

Figure 4. mTORC2-mediated phosphorylation of AKT-PRAS40 signaling is responsible for resistance to RAD001

A, mTORC2 activation in RAD001-sensitive parental and RAD001-resistant CCC cells. RMG2, HAC2, RMG2-RR, and HAC2-RR cells were serum-starved overnight, after which mTORC2 activation was determined by in vitro kinase assay. B, Increased phosphorylation of AKT-PRAS40 signaling in RAD001-resistant cells compared to RAD001-sensitive cells. RMG2, HAC2, RMG2-RR, and HAC2-RR cells were serum starved overnight. Cells were harvested, lysed, and then equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with various antibodies. C, Suppression of Rictor by shRNA leads to the decreased phosphorylation of AKT and PRAS40 in RAD001-resistant CCC cells. RMG2-RR and HAC2-RR cells plated in 6-well plates were transfected with 20 nM control or Rictor shRNA and evaluated after 48 h. D, Treatment with AZD8055 attenuated the phosphorylation of AKT and PRAS40 in RAD001-resistant CCC cells. RMG2-RR and HAC2-RR cells were treated with 10 nM AZD8055 for 6 h. Cells were harvested, lysed, and then equivalent amounts (30 μg) of protein were subjected to SDS-PAGE and blotted with various antibodies.

Figure 5. Inhibition of mTORC2 prevents CCC cells from acquiring resistance to RAD001

A, Establishment of CCC cell lines stably transfected with control shRNA or Rictor shRNA. Note that knockdown of Rictor results in decreased AKT activity. B, Inhibition of mTORC2 activity prevents CCC cells from acquiring resistance to RAD001 in vitro. CCC cells stably transfected with control shRNA or Rictor shRNA were seeded into 96-well plates at a density of 3×10³/well. The cells were then treated with 100 nM RAD001 in the presence of 5% FBS. Cell growth was examined at day 3 and day 7 by cell counting as described in “Materials and Methods,” and the results are shown. Points, mean; bars, SD (*, P < 0.05). C, Inhibition of mTORC2-AKT activity during the course of RAD001 treatment prevents CCC cells from acquiring resistance to RAD001 in vivo. Athymic nude mice inoculated with CCC cells were administered placebo or 2.5 mg/kg RAD001 every 2 days for 4 weeks. Graphs are depicting weekly tumor volumes (mm³) for each treatment group. Points, mean; bars, SD (*, **, significantly different from the RAD001-treated or Rictor shRNA transfected tumors; P < 0.05). D, Effect of AZD8055 on proliferation of RAD001-resistant CCC cells in vitro. RAD001-resistant (RMG2-RR and HAC2-RR) cells were treated with the indicated concentrations of AZD8055 or RAD001 in the presence of 5% FBS for 72 h. Cell viability was assessed by MTS assay. Points, mean; bars, SD (**, p<0.01).