Akt inhibitor MK-2206 promotes anti-tumor activity and cell death by modulation of AIF and Ezrin in colorectal cancer

Abstract

Background: There is extensive evidence for the role of aberrant cell survival signaling mechanisms in cancer progression and metastasis. Akt is a major component of cell survival-signaling mechanisms in several types of cancer. It has been shown that activated Akt stabilizes XIAP by S87 phosphorylation leading to survivin/XIAP complex formation, caspase inhibition and cytoprotection of cancer cells. We have reported that TGFβ/PKA/PP2A-mediated tumor suppressor signaling regulates Akt phosphorylation in association with the dissociation of survivin/XIAP complexes leading to inhibition of stress-dependent induction of cell survival.

Methods: IGF1R-dependent colon cancer cells (GEO and CBS) were used for the study. Effects on cell proliferation and cell death were determined in the presence of MK-2206. Xenograft studies were performed to determine the effect of MK-2206 on tumor volume. The effect on various cell death markers such as XIAP, survivin, AIF, Ezrin, pEzrin was determined by western blot analysis. Graph pad 5.0 was used for statistical analysis. P < 0.05 was considered significant.

Results: We characterized the mechanisms by which a novel Akt kinase inhibitor MK-2206 induced cell death in IGF1R-dependent colorectal cancer (CRC) cells with upregulated PI3K/Akt signaling in response to IGF1R activation. MK-2206 treatment generated a significant reduction in tumor growth in vivo and promoted cell death through two mechanisms. This is the first report demonstrating that Akt inactivation by MK-2206 leads to induction of and mitochondria-to-nuclear localization of the Apoptosis Inducing Factor (AIF), which is involved in caspase-independent cell death. We also observed that exposure to MK-2206 dephosphorylated Ezrin at the T567 site leading to the disruption of Akt-pEzrin-XIAP cell survival signaling. Ezrin phosphorylation at this site has been associated with malignant progression in solid tumors.

Conclusion: The identification of these 2 novel mechanisms leading to induction of cell death indicates MK-2206 might be a potential clinical candidate for therapeutic targeting of the subset of IGF1R-dependent cancers in CRC.

Figure 1

MK-2206 inhibits Akt signaling in IGF1R-dependent CRC cells. A) & B) Loss of pAkt at Ser473 and T308 on treatment with increasing concentration of MK-2206 for 72 hours in GEO and CBS cells respectively. GAPDH is used as a loading control.

Figure 2

MK-2206 affects cell proliferation and cell death in vitro. A) MTT analysis shows reduction in cell proliferation on treatment with MK-2206. B) DNA fragmentation showing an increase in cell death with increasing concentration of MK-2206. C) Western blot analysis of various apoptotic members as pBad (Ser136), XIAP and Survivin. D) IP for 14-3-3 to determine the interaction with pBad (Ser136) and Bad showing a loss in the interaction on treatment with MK-2206. E) IP for Bad to determine the interaction with anti-apoptotic protein Bcl-x_L. (* = P < 0.01 and ** = P < 0.001).

Figure 3

MK-2206 inhibits the growth of colon tumor xenograft. A) Reduction in tumor size on treatment with MK-2206. B) Reduction in tumor volume in treated animals as compared to control animals. C) Reduction in the average tumor volume in animals treated with MK-2206 as compared to control animals. D) Reduction in tumor weight on treatment with Akt kinase inhibitor. (* = P < 0.01 and ** = P < 0.001).

Figure 4

MK-2206 inhibits Akt signaling in vivo: A) IHC images showing a reduction in pAkt at Ser473. B) Relative quantification was performed, followed by statistical analysis to determine the decrease in phosphorylation of Akt at Ser473 on treatment with MK-2206. C) Western blot analysis to confirm the loss of pAkt at Ser473 and Thr 308 in treated animals. (* = P < 0.01 and ** = P < 0.001).

Figure 5

Increased cell death and decreased cell proliferation on treatment with the allosteric Akt kinase inhibitor: TUNEL and Ki67 IHC was performed on control and treated samples A) Increased cell death on treatment with the inhibitor. B) Relative quantification was performed followed by statistical analysis to quantify the increase in death. There was a significant increase in cell death in treated animals. C) Shows a loss in cell proliferation on treatment with MK-2206. D) Bar graphs representing a highly significant loss in Ki67 staining in treated animals.

Figure 6

Increase in the expression and translocation of AIF on treatment with MK-2206 mediates cell death: A) western blot analysis showing an increase in the expression of AIF on treatment with MK-2206. Immunofluorescence was performed to study the translocation of AIF from mitochondria to the nucleus during cell death. B) Confocal images showing a reduced co-localization of mitotracker (red) and AIF (green) in treated as compared to control cells. C) Cellular fractionation to separate nucleus from cytosol was performed followed by western blot analysis for AIF. HDAC1 and GAPDH were used as compartmentalization control for nucleus and cytosol respectively. D) DNA fragmentation after treatment with AIF inhibitor results in reduction in cell death in presence and absence of MK-2206 thus confirming that MK-2206 causes AIF mediated cell death.

Figure 7

Loss of pEzrin on treatment with MK-2206 mediates cell death: A) Western blot analysis showing a reduction in the expression pEzrin (T567) on treatment with MK-2206 in vitro. B) Treatment with MK-2206 reduces the expression of pEzrin (T567) in vivo. Stable knockdown of Akt2 was performed in GEO cells. C) Western blot analysis showing a loss of pEzrin (T567) on knockdown of Akt2. No change in total Ezrin was observed on loss of Akt2. D) Western blot analysis showing loss of Akt1 does not affects the expression of pEzrin (T567). GAPDH is used as a loading control. E) Overall mechanism for induction of cell death by MK-2206. Akt kinase inhibitor MK-2206 mediates cell death by two different mechanisms. Loss of phosphorylation of Akt results in induction and translocation of AIF from the mitochondria to the nucleus, where it results in DNA fragmentation. On the other hand treatment with MK-2206 results in loss of pEzrin (T567), which results in loss of XIAP thus mediating cell death.