MK-2206 causes growth suppression and reduces neuroendocrine tumor marker production in medullary thyroid cancer through Akt inhibition

Abstract

Background: Development of targeted therapies for medullary thyroid cancer (MTC) has focused on inhibition of the rearranged during transfection (RET) proto-oncogene. Akt has been demonstrated to be a downstream target of RET via the key mediator phosphoinositide-3-kinase. MK-2206 is an orally administered allosteric Akt inhibitor that has exhibited minimal toxicity in phase I trials. We explored the antitumor effects of this compound in MTC.

Methods: Human MTC-TT cells were treated with MK-2206 (0-20 μM) for 8 days. Assays for cell viability were performed at multiple time points with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The mechanism of action, mechanism of growth inhibition, and production of neuroendocrine tumor markers were assessed with Western blot analysis.

Results: MK-2206 suppressed MTC cell proliferation in a dose-dependent manner (p ≤ 0.001). Levels of Akt phosphorylated at serine 473 declined with increasing doses of MK-2206, indicating successful Akt inhibition. The apoptotic proteins cleaved poly (ADP-ribose) polymerase and cleaved caspase-3 increased in a dose-dependent manner with MK-2206, while the apoptosis inhibitor survivin was markedly reduced. Importantly, the antitumor effects of MK-2206 were independent of RET inhibition, as the levels of RET protein were not blocked.

Conclusions: MK-2206 significantly suppresses MTC proliferation without RET inhibition. Given its high oral bioavailability and low toxicity profile, phase II studies with this drug alone or in combination with RET inhibitors are warranted.

Figure 1. Treatment with MK-2206 inhibits medullary thyroid cancer cell (MTC-TT) growth and neuroendocrine tumor marker production

MTT cellular viability assay demonstrates a dose-dependent reduction in MTC-TT cell growth over 8 days. Western blot analysis shows an associated decrease in expression of neuroendocrine tumor markers achaete-scute complex-like 1 (ASCL1) and chromogranin A (CgA). MTC-TT cells were plated into 24-well culture plates, incubated overnight, and then treated with MK-2206 (0–20µM) in standard cell media. Dimethyl sulfoxide (DMSO) was used as a treatment control. A) Absorbance values for MTT assays at each time point. Error bars represent standard error of the mean. B) Percentage reduction in growth at each time point. Error bars represent standard error of the percentage mean. One-way analysis of variance with Bonferroni correction demonstrated that all doses significantly reduced growth compared to DMSO control at all time points, p≤0.001. C) Protein lysates isolated after 4 days of treatment with MK-2206 (0–10 µM) show a significant dose-dependent decrease in production of ASCL1 and a marginal decrease in CgA. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 2. MK-2206 inhibits phosphorylation of Akt

Protein lysates of medullary thyroid cancer cells (MTC-TT) isolated after 4 days of treatment with MK-2206 (0–10 µM) demonstrate that increasing doses of MK-2206 reduce phosphorylation at the serine 473 residue of Akt (pAkt^S473), with marginal reduction at threonine 308 (pAkt^T308). Levels of total Akt protein remain stable with treatment. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 3. Growth suppression of medullary thyroid cancer cells (MTC-TT) by MK-2206 is mediated by apoptosis

Treatment of MTC-TT cells with MK-2206 (0–10 µM) for four days resulted in increased expression of apoptotic proteins cleaved poly-ADP ribose polymerase (PARP) and cleaved caspase-3, with corresponding decrease in apoptosis inhibitor survivin. Stable cyclin D1 levels suggest there is no associated change in cell cycle signaling. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 4. Treatment with MK-2206 causes no concomitant inhibition of RET (Rearranged during Transfection) activity in medullary thyroid cancer cells (MTC-TT)

Total RET protein levels are stable in MTC-TT lysates isolated after 4 days of treatment with MK-2206 (0–10 µM). Select phosphorylation residues (pRET^Y905 and pRET^Y1062) actually increase with treatment, indicating RET activity is not blocked by MK-2206 treatment. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 5. Schematic of potential combination treatment

The phosphoinositide-3-kinase (PI3K)/Akt pathway is highly active in many tumor cells, including medullary thyroid cancer. The pathway is activated by one of many receptor tyrosine kinases (RTK). Akt is activated downstream of PI3K, and then can activate or inhibit multiple targets that lead to cell proliferation and inhibition of cell cycle arrest and apoptosis. Activation of RET (Rearranged during Transfection) can also activated PI3K. Certain compounds, including LY294002, Sorafenib, Sunitinib, Vandetanib, and MK-2206 (in black circles) effectively inhibit proteins in these pathways, and thus combinations of compounds that target different components could be tested. PTEN, Phosphate and Tensin Homolog; PIP₂, phosphatidylinositol-3,4-diphosphate; PIP₃, phosphatidylinositol-3,4,5-triphosphate.