Combined targeting of AKT and mTOR synergistically inhibits proliferation of hepatocellular carcinoma cells

Abstract

Background: Due to the frequent dysregulation of the PI3K/AKT/mTOR signaling pathway, mTOR represents a suitable therapeutic target in hepatocellular carcinoma (HCC). However, emerging data from clinical trials of HCC patients indicate that mTOR inhibition by RAD001 (Everolimus) alone has only moderate antitumor efficacy which may be due to the feedback activation of AKT after mTOR inhibition. In this study, we analyzed the effects of dual inhibition of mTOR and AKT on the proliferation of HCC cell lines. In addition, we measured the feedback activation of each of the AKT isoforms after mTOR inhibition in HCC cell lines and their enzymatic activity in primary samples from HCC patients.

Methods: The activation status of specific AKT isoforms in human HCC samples and corresponding healthy liver tissue was analyzed using an AKT isoform specific in vitro kinase assay. AKT isoform activation after mTOR inhibition was analyzed in three HCC cell lines (Hep3B, HepG2 and Huh7), and the impact of AKT signaling on proliferation after mTOR inhibition was investigated using the novel AKT inhibitor MK-2206 and AKT isoform specific knockdown cells.

Results: AKT isoforms become differentially activated during feedback activation following RAD001 treatment. The combination of mTOR inhibition and AKT isoform knockdown showed only a weak synergistic effect on proliferation of HCC cell lines. However, the combinatorial treatment with RAD001 and the pan AKT inhibitor MK-2206 resulted in a strong synergism, both in vitro and in vivo. Moreover, by analyzing primary HCC tissue samples we were able to demonstrate that a hotspot mutation (H1047R) of PI3KCA, the gene encoding the catalytic subunit of PI3K, was associated with increased in vitro kinase activity of all AKT isoforms in comparison to healthy liver tissue of the patient.

Conclusion: Our results demonstrate that dual targeting of mTOR and AKT by use of RAD001 and the pan AKT inhibitor MK-2206 does effectively inhibit proliferation of HCC cell lines. These data suggest that combined treatment with RAD001 and MK-2206 may be a promising therapy approach in the treatment of hepatocellular carcinoma.

Figure 1

Feedback activation of AKT after mTOR inhibition is time- and dose dependent. (A) HCC cell lines Hep3B, HepG2 and Huh7 were treated for 24 h with increasing concentrations of RAD001 as indicated. Phosphorylation status of mTOR and AKT was analyzed by Western blot with phospho specific antibodies. (B) Dose dependent expression of pAKT (S473) and pAKT (T308) after 24 h treatment with RAD001 was quantified from three independent experiments as shown in A. Columns, mean percentage of three independent experiments; bars, SD, * p < 0.05. (C) Huh7 and (D) HepG2 cells were treated with 100 nM RAD001 up to 72 h, and cell lysates were prepared at the indicated time points. Where indicated, medium was removed after 48 h and replaced by fresh, 100 nM RAD001 containing medium. HSC70 served as loading control.

Figure 2

Differential and isoform specific activation of AKT1, AKT2 and AKT3. (A) Comparison of AKT isoform expression in HCC cell lines. Exponentially growing cells were seeded into 10 cm dishes and allowed to attach for 24 h. Cells were then lysed and analyzed by Western blot. One representative experiment out of three is shown. (B) HCC cells were treated over 24 h with the indicated concentration of RAD001. AKT isoform specific in vitro kinase assays were performed following quantitative AKT isoform immunoprecipitation, based on the same cell lysates as shown in Figure  1A and B. GSK3αbgr; fusion protein was used as an AKT substrate and phosphorylation at (S9/21) detected by Western blot. Columns, mean of three independent experiments; bars, SD. * p < 0,05; # p < 0,01.

Figure 3

Combining RAD001 with MK-2206 synergistically suppresses proliferation of HCC cell lines. (A) HCC cell lines were treated with the indicated concentration of MK-2206 over 24 h, and changes in mTOR- and AKT-signaling were analyzed by Western Blot. HSC70 served as loading control. (B) HCC cells were seeded into 96 well plates and incubated with increasing concentrations of either RAD001 (triangle), MK2206 (box), or a combination (inverted triangle) of both with a fixed ratio of 1:5. Controls were treated with DMSO only. Proliferation was analyzed after 72 h BrdU-incorporation. Asterisks indicate a significantly stronger inhibition of the drug combination compared to each compound alone, * p < 0.05. (C) HCC cell lines were treated with RAD001 (triangle) or a combination of RAD001 and MK-2206 (inverted triangle) with a constant concentration of 1.7 μM MK2206 for 72 h. Cells treated with DMSO only served as control. Proliferation was analyzed as in (B). Each data point represents mean of at least three independent experiments, normalized to controls; bars, SD. Asterisks indicate a significantly stronger inhibition of the drug combination compared to each compound alone, * p < 0.05. (D) Fractional effect plot for the effect of RAD001 and MK2206 as seen in (B). (E) Cell cycle analysis of HCC cell lines after 24 h treatment with 100 nM RAD001, 1.7 μM MK-2206, or the combination of both, compared to DMSO treated controls. Colums: mean of one representative experiment, performed in triplicates; bars: SD. The drug combination resulted in a significant increase of cells in G0/G1 phase compared to each drug alone and compared to controls in all three cell lines (p < 0.05).

Figure 4

Knockdown of AKT isoforms and mTOR inhibition synergistically inhibits HCC cell proliferation. (A) shRNA mediated knockdown of single AKT isoform in HCC cell lines, confirmed by Western blot. (B) Hep3B knockdown cells were treated with 100 nM RAD001, 1.7 μM MK-2206, the combination of both, or DMSO as control, over 24 h, and mTOR and AKT signaling pathway activity was analyzed by Western blot. HSC70 served as loading control. AKT isoform knockdown cells were treated with 100nM RAD001 over 24 h. (C) Cells transduced with non-target vector were also treated with 1.7 μM MK-2206 or a combination of 100 nM RAD001 and 1.7 μM MK-2206. Proliferation was analyzed by BrdU incorporation. Columns, mean of one experiment performed in triplicates, bars, SD. *, p < 0.01; **, p < 0.001.

Figure 5

RAD001 and MK-2206 synergistically suppress subcutaneous tumor growth in vivo. Huh7 cells were injected subcutaneously into SCID mice (n = 7 per group). After formation of palpable tumors, mice were treated with Placebo, RAD001, MK-2206 or the combination of both, respectively. (A) Treatment with RAD001 and MK-2206 significantly prolonged survival of tumor bearing mice compared to all other groups (p < 0.05). (B) Tumor volume was monitored over an 18 day period, presented as mean ± SEM. RAD + MK vs Placebo and RAD + MK vs MK-2206 alone was significant at day 15 (p < 0.05). Note that one mouse had to be withdrawn from the experiment in the RAD001 and MK-2206 treatment group at day 13 and 15 respectively, and two mice were withdrawn from the Placebo group at days 11 and 15. (C) AKT and mTOR signaling in tumor samples was analyzed by Western blot.

Figure 6

AKT isoforms are differentially activated in human HCC tissue samples. (A) Ten human HCC tissue samples obtained from specimen removed during surgery were analyzed by Western blot for expression of mTOR and AKT signaling proteins. HSC70 served as loading control. (B) Same samples, including two corresponding controls from surrounding normal liver tissue, were analyzed by AKT isoform specific in vitro kinase assay as described before. IgG served as loading control.