RAD001 was provided by Novartis (Basel, Switzerland). MK-2206 was obtained from Selleck Chemicals (Houston, TX, USA). Stock solutions with a concentration of 10 mM were prepared and stored at −80°C. All RAD001 solutions were thawed and refrozen for a maximum of three times and then discarded. Antibodies against pan AKT, AKT1, AKT2, pAKT (S473), pAKT (T308), mTOR, pmTOR (S2448), pmTOR (S2481), Raptor, Rictor, pERK (T202/204), ERK, pS6 (S240/244), IRS-1 and pIRS-1 (S636/639) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against AKT2 and HSC-70 were purchased from Santa Cruz. AKT3 antibody was obtained from Millipore (Schwallbach, Germany). 7-AAD was obtained from BD Biosciences (Pharmingen, CA, USA).
The three hepatocellular carcinoma cell lines Hep3B, HepG2 and Huh-7 were a kind gift from Prof. Dr. H. Will at the Heinrich Pette Institute, Hamburg, Germany. All cell lines were maintained in DMEM, supplemented with 10% (v/v) FCS, and 1% (v/v) penicillin and streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. All cells were tested for mycoplasma contamination every 2–3 months.
Western Blot analysis
Western blot analysis was performed as described previously. Protein expression was quantified using an LAS-3000 Imager from Fuji (Raytest, Straubenhardt, Germany).
Lentiviral knockdown of AKT isoforms
pLKO.1-puro vector encoding either AKT1, AKT2, AKT3 or scrambled shRNA were purchased from Sigma-Aldrich (Taufkirchen, Germany). Generation of pseudotype lentiviruses and transduction were performed as previously described. Transduced cells were selected by addition of puromycin (Sigma-Aldrich, Taufkirchen, Germany) to culture medium (final concentration 1.5 μg/ml) for at least two weeks before experiments were carried out.
Proliferation and cell cycle assay
Proliferation was analyzed either by flow cytometry using the BrdU APC Flow Kit (BD, Pharmingen, CA, USA) or with the colorimetric BrdU ELISA Kit (Roche®, Basel, CH) as indicated in the figure legends. For FACS-based assays, cells were seeded into 10 cm dishes and allowed to attach overnight. Then, medium was replaced by medium containing RAD001, MK-2206, a combination of both, or DMSO as control. Final DMSO concentration in culture medium was 0.1% (v/v) in all experiments. For labeling BrdU (final concentration 10 μM) was added and cells were incubated for 12 to 16 h. For cell cycle analysis, cells were fixed in ice cold 70% ethanol for at least 6 h, washed and subsequently incubated with 5 μg 7-AAD and 5 μg RNAse A for one hour. Each experiment was performed in triplicates and has been repeated at least one time. Analysis was performed on BD Canto II flow cytometer (BD Pharmingen, CA, USA). Cell cycle analysis was performed using FlowJo 7.6.5 software.
For BrdU ELISA assays, cells were seeded into 96-well plates and allowed to attach overnight. Cells were then incubated for 72 h with different concentrations of MK-2206, RAD001, or a combination of both. Controls were treated with DMSO only. BrdU ELISA was performed as described by the manufacturer (Becton Dickenson, Heidelberg, Germany). Each experiment was repeated at least three times in quadruplicates.
Immunoprecipitation and AKT isoform specific in vitro kinase assay
Immunoprecipitation of AKT isoforms and subsequent in vitro kinase assay was performed as described before. Whole samples were analyzed by western blot technique probed with pGSK3α/ß (S9/21) and panAKT antibody. Subsequently,nitrocellulose membrane was incubated with secondary goat anti-mouse antibody (Santa Cruz Biotechnology, CA, USA) to detect mouse IgG levels for sample correction.
Primary human HCC samples
The human investigations were performed according to the Declaration of Helsinki after approval was obtained by the local ethics committee of the Medical Association Hamburg. From all patients, written informed consent was obtained prior to study related procedures. Tumor samples and samples of corresponding healthy liver tissue of HCC patients treated at the University Medical Center Hamburg Eppendorf, Department of Hepatobiliary and Transplant Surgery were stored at Indivumed (Hamburg, Germany) following the Indivumed Standard of Biobanking (http://www.indivumed.com). Genomic DNA isolation and sequence analysis was performed by Inostics (Hamburg, Germany). For protein analysis and kinase assays tissue samples were lysed by homogenisation of samples with Lysis Matrix-D (MP, USA) in NP-40 lysis buffer (containing: 50 mM HEPES pH 7.5, 150 mM NaCl, 1% NP-40, 2% aprotinin, 2 mM EDTA, 50 mM NaF, 10 mM NaPPi, 10% glycin, 1 mM vanadate and 1 mM PMSF) with the tissue lyser MX Pro (MP, USA).
Mutation analysis of PI3K, AKT and mTOR in HCC samples
Sequence analysis of genes from 10 HCC samples encoding the p85α adapter subunit of PI3K (exon 9–17 of PIK3RI), the p110α catalytic subunit of PI3Kα (exon 2 and 10–21 of PIK3CA), mTOR (exon 44–57 of FRAP1), AKT1 (exon 3–6), AKT2 (exon 2–5) or AKT3 (exon 1–3) were performed by amplification of the exons with primers located in the neighboring introns by PCR and sequenced using BigDye Terminator 3.1 according to the instructions by the manufacturer (Applied Biosystems). Primer sequences can be found in Additional file7: Table S1.
All experimental protocols were approved by local authorities (Ministry of Health and Consumer Protection, Hamburg, Germany, Permit Number G52/11). 2x10^6 Huh7 cells were injected subcutaneously into SCID mice (female, age 8 weeks, n = 7 per group, obtained from Charles River, Sulzfeld, Germany). Upon establishment of palpable tumours after three weeks, mice were randomly assigned to one of the four groups (Placebo, RAD001, MK-2206, or RAD001 and MK-2206), and treatment was started. Tumor growth was monitored by regular visual inspection and tumor dimensions were measured every 2–3 days. Tumor volume was calculated using the formula longest tumor diameter x (shortest tumor diameter)^2/2. RAD001, formulated as a microemulsion, was dosed at 1 mg/kg body weight and administered daily Monday through Friday. MK-2206 was formulated in a 30% (w/v) Captisol solution and administered Monday, Wednesday and Friday dosed at 100 mg/kg. A placebo microemulsion (provided by Novartis) and a 30% (w/v) Captisol solution served as placebo. The compounds were mixed immediately before administered by gavage in a total volume of 100 μl. Animals were treated until termination criteria (tumor ulceration, tumor size > 2 cm in largest diameter, loss of body weight exceeding 20%) were met, or for a maximum of 22 days when all mice in the placebo group had to be withdrawn. Xenograft primary tumours were harvested at necropsy and a portion of each tumor was snap frozen on liquid nitrogen for Western Blot analysis.
Student’s t-Test (unpaired, 2-tailed) was calculated based on the data of at least three independent experiments. Bonferroni correction for multiple testing was performed where applicable. Results were considered significant if p < 0.05. All error bars represent SD. Drug interactions were analyzed based on the median effect method of Chou and Talalay. CalcuSyn software (Biosoft, Cambridge, UK) was used to calculate the combination index (CI). CI values from 0.3 to 0.7 are considered to indicate synergism, CI values below 0.3 are considered to represent strong synergism, and values below 0.1 very strong synergism. IC50 values were calculated using CurveExpert Professional 1.3 software. Chi-squared test was used to test for significant differences in mouse survival.