Dual mTORC1/2 inhibition in a preclinical xenograft tumor model of endometrial cancer

Abstract

Objectives: Up to 70% of endometrioid endometrial cancers carry PTEN gene deletions that can upregulate mTOR activity. Investigational mTOR kinase inhibitors may provide a novel therapeutic approach for these tumors. Using a xenograft tumor model of endometrial cancer, we assessed the activity of mTOR and downstream effector proteins in the mTOR translational control pathway after treatment with a dual mTOR complex 1 and 2 (mTORC1/2) catalytic inhibitor (PP242) compared to that of an allosteric mTOR complex 1 (mTORC1) inhibitor (everolimus, RAD001).

Methods: Grade 3 endometrioid endometrial cancer cells (AN3CA) were xenografted into nude mice. Animals were treated with PP242, PP242 and carboplatin, carboplatin, RAD001, and RAD001 and carboplatin. Mean tumor volume was compared across groups by ANOVA. Immunoblot analysis was performed to assess mTORC1/2 activity using P-Akt, P-S6 and P-4E-BP1.

Results: The mean tumor volume of PP242+carboplatin was significantly lower than in all other treatment groups, P < 0.001 (89% smaller). The RAD001+carboplatin group was also smaller, but this did not reach statistical significance (P = 0.097). Immunoblot analysis of tumor lysates treated with PP242 demonstrated inhibition of activated P-Akt.

Conclusions: Catalytic mTORC1/2 inhibition demonstrates clear efficacy in tumor growth control that is enhanced by the addition of a DNA damage agent, carboplatin. Targeting mTORC1/2 leads to inhibition of Akt activation and strong downregulation of effectors of mTORC1, resulting in downregulation of protein synthesis. Based on this study, mTORC1/2 kinase inhibitors warrant further investigation as a potential treatment for endometrial cancer.

Keywords: Akt; Endometrial cancer; Preclinical study; Rapamycin; Translational regulation; eIF4E; mTOR pathway; therapeutics.

Fig. 1

Effect of PP242 catalytic and RAD001 allosteric rapalog inhibitors on mTOR downstream effector protein phosphorylation and protein synthesis in endometriod cancer cells in culture. (A) AN3CA cells in tissue culture were treated for 5 h or 10 h with 2.5 µM PP242 or 100 mM RAD001, considered maximal inhibitory doses based on IC₅₀ studies in a variety of cell types. Cells were lysed, equal amounts of soluble cell protein extracts resolved by SDS-PAGE and analyzed by protein immunoblotting with specific antibodies as shown. Phosphorylation immunoblotting of proteins was determined by immunoblotting membrane first with phospho-specific antibody then stripping the membranes followed by re-probing with non-phospho-specific antibodies. ECL was used for detection. Results shown are representative of three independent experiments. (B) Protein synthesis rates were determined 5 h after treatment of AN3CA cells with 2.5 µM PP242 or 100 mM RAD001, by [³⁵S]-methionine incorporation. Standard activity of label incorporation into nascent protein was determined by TCA precipitation and liquid scintillation counting. Results were normalized to control untreated cells. Standard errors of the mean (SEM) shown. *, P<0.05 by t-test.

Fig. 2

AN3CA endometrial tumor average response to treatment in a xenotransplant mouse model. Female BALB/c nu/nu mice were injected subcutaneously in the right flank with 2 × 10⁶ AN3CA cells, then mice randomized into treatment groups when tumors were 160 mm³. RAD001 (2.5 mg/kg) and PP242 (100 mg/kg) were administered by gavage on days 1–5 of each week, carboplatin (50 mg/kg, i.p.) was given on day 2 of the weekly cycle. Tumor sizes were determined by precision caliber twice weekly. Results represent the mean with SEM of two independent studies of 7–8 mice per treatment arm.

Fig. 3

Waterfall plot of individual AN3CA tumor responses to treatment with PP242, RAD001 without or with concurrent carboplatin. The data shown in Figure 3 were replotted to demonstrate individual final treatment responses per animal at the end of the 25 day treatment cycle. Each column represents one individual mouse corresponding to the different treatment groups.

Fig. 4

Effect of PP242 catalytic and RAD001 allosteric rapalog inhibitors on mTOR downstream effector protein phosphorylation in AN3CA endometriod tumors. Equal amounts of soluble protein extracts obtained from individual representative tumors were resolved by SDS-PAGE and analyzed by protein immunoblotting with specific antibodies as shown. Phosphorylation immunoblotting of proteins was determined by immunoblotting membrane first with phospho-specific antibody then stripping the membranes followed by re-probing with nonphospho- specific antibodies. ECL was used for detection. Extracts were prepared 2 h following the last drug treatment. Two tumors closest to the mean of tumor response in each treatment arm were chosen for analysis, identified as samples 1 and 2.