Arsenic trioxide decreases AKT protein in a caspase-dependent manner

Arsenic trioxide (As₂O₃) is used clinically to treat acute promyelocytic leukemia but is less successful in other malignancies. To identify targets for potential combination therapies, we have begun to characterize signaling pathways leading to As₂O₃-induced cytotoxicity. Previously, we described the requirement for a reactive oxygen species–mediated, SEK1/c-Jun NH₂-terminal kinase (JNK) pathway to induce apoptosis. AKT inhibits several steps in this pathway; therefore, we postulated that As₂O₃ might decrease its activity. Indeed, As₂O₃ decreases not only AKT activity but also total AKT protein, and sensitivity to As₂O₃ correlates with the degree of AKT protein decrease. Decreased AKT expression further correlates with JNK activation and the release of AKT from the JNK-interacting protein 1 scaffold protein known to assemble the mitogen-activated protein kinase cascade. We found that As₂O₃ regulates AKT protein stability without significant effects on its transcription or translation. We show that As₂O₃ decreases AKT protein via caspase-mediated degradation, abrogated by caspase-6, caspase-8, caspase-9, and caspase-3 inhibitors but not proteosome inhibitors. Furthermore, As₂O₃ enhances the ability of a heat shock protein 90 inhibitor to decrease AKT expression and increase growth inhibition. This suggests that As₂O₃ may be useful in combination therapies that target AKT pathways or in tumors that have constitutively active AKT expression. [Mol Cancer Ther 2008;7(6):1680–7]

Arsenic trioxide (As₂O₃) is an effective treatment for patients with acute promyelocytic leukemia (APL) but has been less successful in other malignancies at tolerable doses. Several groups have begun preclinical and early-stage clinical efforts to develop rational combination therapies that increase the efficiency of As₂O₃. Some of these combinations show promise in non-APL acute myelogenous leukemia and multiple myeloma (1–3). To further identify therapeutic targets for such combination therapies, we have begun to characterize signaling pathways leading to As₂O₃-induced cytotoxicity.

As₂O₃ initiates many signaling cascades within a cell, but identifying those pathways essential for the induction of apoptosis is critical. As₂O₃ treatment results in production of reactive oxygen species (ROS) in leukemic cells (4, 5). Several compounds that enhance ROS production can sensitize cells to As₂O₃-induced apoptosis. We have shown that either l-buthionine sulfoximine (BSO; ref. 4), which depletes glutathione stores, or Trolox (6), a vitamin E analogue that selectively acts as a pro-oxidant in malignant cells (6, 7), can enhance As₂O₃-induced cytotoxicity. In addition, antioxidant compounds such as N-acetylcysteine inhibit As₂O₃-induced apoptosis (8). ROS production by As₂O₃ is linked to the induction of stress pathways, including mitogen-activated protein kinases (MAPK). As₂O₃ treatment results in c-Jun NH₂-terminal kinase (JNK) activation, which is required for maximal induction of apoptosis in several cell types (9). Genetic deletion of the upstream activator kinase SEK1/MAPK kinase 4 in mouse embryo fibroblasts inhibits both As₂O₃-induced JNK activity and apoptosis (9).

In addition to activated pathways leading to death, we hypothesized that survival signals would be inhibited by As₂O₃. AKT, a serine/threonine protein kinase, mediates the cell survival signals coming through phosphoinositide 3-kinase by phosphorylation and inactivation of several proapoptotic proteins, including BAD (10), caspase-9 (11, 12), and members of the forkhead family of transcription factors (13, 14), rendering them inactive. AKT also negatively regulates the MAPK pathways required for As₂O₃-induced apoptosis. AKT phosphorylates SEK1 on Ser⁷⁸ and inhibits its function (15). AKT also phosphorylates and inactivates ASK1, a MAPK kinase kinase upstream of SEK1 (15, 16). Oxidative stress initiates the dissociation of AKT from JNK-interacting protein 1 (JIP1), a scaffolding protein, which facilitates the activation of the ASK-SEK-JNK pathway (17). AKT binding to JIP1 negatively regulates this signal transduction pathway (17); thus, a decrease in AKT as a result of oxidative stress could enhance JNK signaling.

Here, we present data showing a correlation between sensitivity to As₂O₃-induced death and decreased AKT activity. We further show that As₂O₃ decreases total AKT protein levels without affecting transcription or translation of AKT. Instead, we found that As₂O₃ decreased AKT protein expression in a caspase-dependent manner. In addition, we show that As₂O₃ acts to enhance the growth inhibition seen with the heat shock protein 90 (hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), suggesting possible combination therapies to target AKT pathways.

As₂O₃, BSO, anti-β-actin antibody and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent were purchased from Sigma. LY294002, MG132, and caspase inhibitors were purchased from Calbiochem (EMD Biosciences). AKT antibody and the nonradioactive AKT kinase assay kit were purchased from Cell Signaling Technology.

NB4 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin/streptomycin. The arsenic-resistant NB4-MR-AsR2 and AsR3 cell lines (AsR2 and AsR3) were originally derived from the arsenic-sensitive NB4 cells and are cultured in the presence of 2 μmol/L As₂O₃ (4). AsR2 and AsR3 cells were washed thoroughly before all experiments. MDA-MB-468 cells were cultured in α-MEM containing 10% fetal bovine serum. MDA-MB-231 cells were cultured in DMEM/F-12 containing 10% fetal bovine serum. Breast cancer cells were plated at 10⁴ per well of a 24-well plate for 3-day growth curve analyses.

Quantitation of apoptotic cells was done as previously described (18). Cells were washed in 4°C PBS/5% fetal bovine serum/0.01 mol/L NaN₃, pelleted, and resuspended in 0.5 mL of hypotonic fluorochrome solution containing 50 μg/mL propidium iodide (Sigma), 0.1% sodium citrate, and 0.1% Triton X-100. Cells undergoing DNA fragmentation and apoptosis were shown to be those in which propidium iodide fluorescence was weaker than the typical G₀-G₁ cell cycle peak.

Cells were processed following the protocol from Cell Signaling Technology. Briefly, cells were lysed in 1× cell lysis buffer and 200 μL of protein extracts were incubated with AKT monoclonal antibody immobilized on agarose beads for 2 h at 4°C. Beads were then washed in 1× cell lysis buffer and in 1× kinase buffer. For the kinase assay, beads were resuspended in the 1× kinase buffer supplemented with 200 μmol/L ATP and glycogen synthase kinase 3α/β (GSK3α/β; Ser^21/9) fusion protein and incubated 30 min at 30°C. To stop the reaction, 2× SDS sample buffer was added to the samples, which were then boiled 5 min and loaded on a 12% SDS-PAGE gel. Samples were analyzed by immunoblotting with a phosphorylated GSK3α/β (Ser^21/9) antibody. Anti-AKT immunoblots were done as a control for the immunoprecipitation.

Assays for JNK activity were done as previously described (9). Briefly, following treatment, cells were washed and lysed in the presence of phosphatase and protease inhibitors. JNK1 was immunoprecipitated with an anti-JNK1 antibody (Santa Cruz Biotechnology, Inc.) and protein A-Sepharose beads by nutation at 4°C for 1 h. After washing, immune complexes were incubated with glutathione S-transferase-c-Jun (a gift from Sylvain Meloche, University of Montreal, Montreal, Quebec, Canada) and [γ-³²P]ATP for 30 min at 30°C. Glutathione S-transferase-c-Jun was visualized via SDS-PAGE and autoradiography. Immunoblotting was done before immunoprecipitation to ensure that treatment did not affect absolute JNK1 expression levels.

After treatment, cells were washed with cold PBS and lysed in radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 8), 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS] containing protease/phosphatase inhibitors. Protein concentration was measured with 5× Bradford reagent (Bio-Rad). Proteins were separated by SDS-PAGE gel and then transferred onto a nitrocellulose membrane. Membranes were blocked (1 h, room temperature) in TBS containing 0.1% Tween 20 (TBST) and 5% nonfat milk and incubated with anti-AKT (1:500), anti-JIP1, anti-β-actin, or anti-JNK1 (overnight, 4°C). The next day, membranes were washed in TBST and incubated with horseradish peroxidase–conjugated secondary antibody (1 h, room temperature). Membranes were then washed and peroxidase activity was visualized with the enhanced chemiluminescence method (GE Healthcare Biosciences-Amersham).

NB4 cells were lysed in buffer containing 50 mmol/L Tris (pH 8), 150 mmol/L NaCl, and 1% Triton X-100. After preclearing lysates, specific proteins were immunoprecipitated overnight at 4°C with anti-AKT antibody (sc-1618-R). Protein A-agarose beads were added for 4 h and then the beads were washed thrice in lysis buffer. Immunoprecipitates were run on SDS-PAGE gels and immunoblotted as described above.

Transient transfection of NB4 cells was done by electroporation using the Gene Pulser apparatus (Bio-Rad). For constitutively active AKT transfection, 1 × 10⁷ NB4 cells were transfected by electroporation with 10 μg of either empty vector pCMV (mock) or a constitutively active AKT (Dr. Adriane Stoica, Georgetown University, Washington, District of Columbia). Cells were treated with 2 μmol/L As₂O₃. After 48 h, cell proliferation was assessed by MTT assay.

NB4 cells were seeded in the presence or absence of 5 μmol/L As₂O₃ for 18 h. For the last 6 h, MG132, a proteasome inhibitor, was added to some cultures. Cells were then harvested and lysed with radioimmunoprecipitation assay buffer. Immunoblotting analysis was done using an anti-AKT polyclonal antibody (Cell Signaling Technology) and β-actin antibody (Sigma) as a loading control.

NB4 cells were treated with 5 μmol/L As₂O₃ in the presence or absence of Z-VAD (caspase inhibitor I), a pan-caspase inhibitor (Calbiochem), or with the Caspase Inhibitor Set II from Calbiochem: caspase-1 inhibitor VI (ZYVAD-fmk), caspase-2 inhibitor I (ZVDVAD-fmk), caspase-3 inhibitor II (Z-DEVD-fmk), caspase-5 inhibitor I (Z-WEHD-fmk), caspase-6 inhibitor I (Z-VEID-fmk), caspase-8 inhibitor (Z-IETD-fmk), caspase-9 inhibitor I (Z-LEHD-fmk), and caspase inhibitor III (Boc-D-fmk). After 6 or 24 h, cells were counted and then harvested to prepare whole-cell extracts. Immunoblotting analysis was done to evaluate AKT protein levels.

Caspase-3 activation was detected using the Caspase-Glo 3/7 Assay (Promega). NB4 and AsR2 cells were treated for 18 h. Subsequently, cells were incubated for 30 min at room temperature with an equal volume of Ac-DEVD-pNA substrate in the buffer provided. The amount of light was measured using a GloMax 20/20 luminometer (Promega) and normalized to cell number.

Based on our previous findings that an intact JNK signaling pathway is required for As₂O₃-induced apoptosis, we hypothesized that As₂O₃ may concomitantly decrease negative regulators of this pathway. We chose to assess AKT expression and activity in our model system of the arsenic-sensitive NB4 APL cell line after treatment for increasing amounts of time with 5 μmol/L As₂O₃. AKT activity was measured using a nonradioactive kinase assay with GSK3α/β (Ser^21/9) fusion protein as a substrate. Indeed, As₂O₃ decreases AKT activity within 18 h (Fig. 1A, top). In addition, we observed a time-dependent decrease in total AKT protein immunoprecipitated (Fig. 1A, bottom). To verify that this was not an artifact of the kinase assay, we then assessed total AKT levels by immunoblot and found a dose-dependent (Fig. 1B) and time-dependent (Fig. 1C) decrease in AKT protein levels, confirming our immunoprecipitation results that 5 μmol/L As₂O₃ decreases AKT by 6 to 8 h. In addition, longer exposures with low concentrations of As₂O₃ (0.5 μmol/L), well within the clinically achievable range, also decreased AKT protein expression (Fig. 1D).

The As₂O₃-induced decrease in AKT protein levels was not specific to APL cells. In both the IM9 B lymphoblastoid cell line and the HS-Sultan multiple myeloma cell line, As₂O₃ decreased AKT protein expression in a dose-dependent manner after 24 h, which correlated with the ability of As₂O₃ to activate JNK as assessed by in vitro kinase assay where JNK was immunoprecipitated from cell lysates and used to phosphorylate a glutathione S-transferase-c-Jun construct in vitro (Fig. 2A). Notably, higher doses of As₂O₃ were required to initiate the signaling events in these cell lines, which correlates with their decreased sensitivity to As₂O₃. A decrease in AKT levels correlated with sensitivity to As₂O₃ in breast cancer cell lines as well. AKT expression as well as AKT phosphorylation at Ser⁴⁷³ was decreased in MDA-MB-468 cells (Fig. 2B), which have a high level of constitutively active AKT. However, MDA-MB-231 cells, which are significantly less sensitive to As₂O₃-induced growth inhibition at day 3, did not have a decreased level of AKT after exposure to As₂O₃.

If the arsenic-induced decrease of AKT protein facilitates cell death, we hypothesized that cells resistant to As₂O₃-induced apoptosis would not decrease AKT expression in response to arsenic. To test this, we used arsenic-resistant NB4 subclones developed in our laboratory, which are grown under the selective pressure of 2 μmol/L As₂O₃. These cells have increased glutathione levels, which results in their resistance to As₂O₃ (4). When NB4 cells as well as the resistant clones AsR2 and AsR3 were treated with 5 μmol/L As₂O₃ for 24 h, AKT protein expression was decreased in the parental line but not in the resistant clones (Fig. 3A). Furthermore, when glutathione levels were decreased by a pretreatment with BSO for 18 h followed by 2 μmol/L As₂O₃ treatment for 24 h, the AKT protein decreased and apoptosis was induced (Fig. 3B; data not shown). AKT levels were not affected by treatment with BSO or As₂O₃ alone in these cell lines. We have previously shown that BSO, which decreases intracellular reduced glutathione levels, can sensitize these arsenic-resistant cells to As₂O₃-induced ROS production and apoptosis (4). Therefore, these data suggest that AKT is downstream of ROS generation by As₂O₃.

We then asked if pharmacologic inhibition of AKT in the arsenic-resistant cell lines could sensitize them to As₂O₃-induced death. AsR2 cells were treated with 2 μmol/L As₂O₃ over 2 days in the presence or absence of 2.5 μmol/L LY294002. LY294002 is an inhibitor of phosphoinositide 3-kinase, an activating kinase upstream of AKT. Interestingly, this inhibitor can overcome resistance to As₂O₃ in the AsR2 cells as assessed in growth curves, propidium iodide staining, and caspase-3 activation (Fig. 3C). To confirm that AKT down-regulation was necessary for As₂O₃-induced death, we asked whether overexpression of AKT in the NB4 cells results in resistance to As₂O₃-induced growth inhibition. Indeed, NB4 cells transiently transfected with a constitutively active AKT construct were completely resistant to a dose of As₂O₃ that substantially inhibited growth in untransfected or vector-transfected cells (Fig. 3D). Together, these data show that decreased AKT is an essential part of the mechanism of As₂O₃-induced apoptosis in APL cells, and resistance to As₂O₃ is associated with persistent AKT expression.

We then asked how AKT could inhibit As₂O₃-induced cell death. We have previously shown that JNK activation is critical for As₂O₃-induced apoptosis in NB4 cells. AKT is a negative regulator of this pathway, by binding to JIP1, the scaffold protein that integrates both positive and negative regulators of JNK signaling. When AKT is bound to JIP1, the MAPK cascade cannot assemble and JNK is not activated. AKT and JNK are coordinately and inversely regulated in our model system; therefore, we postulated that inhibition of AKT would increase JNK activation induced by As₂O₃. NB4 cells were serum starved for 24 h and subsequently treated for 24 h with 0.5 μmol/L As₂O₃ in the presence or absence of 2.5 μmol/L LY294002. As we have previously shown, 0.5 μmol/L As₂O₃ induced JNK activity at this time point (Fig. 4A). Addition of LY294002 enhanced the JNK activation alone and the combination of these drugs further enhanced JNK activation (Fig. 4A). These data further correlate decreased AKT activity with JNK activation. Next, we investigated whether As₂O₃-induced JNK activity was correlated with a decreased association of AKT with JIP1. Endogenous AKT and JIP1 were coimmunoprecipitated from NB4 cells treated for 24 h with control or 1 to 5 μmol/L of As₂O₃. AKT and JIP1 were strongly associated in untreated cells, but this association was significantly decreased even at the lowest dose of As₂O₃, which did not decrease AKT protein at this time point (Fig. 4B), indicating that As₂O₃-induced JNK activation correlates with decreased AKT-JIP1 binding. Interestingly, as AKT protein levels are decreasing with As₂O₃ treatment, JIP1 levels increase.

We next investigated the mechanism by which As₂O₃ treatment decreases AKT expression. We found that As₂O₃ affects neither transcriptional activity of AKT1 as assessed by transient transfection and chromosomal immunoprecipitation assays nor the mRNA stability (Supplementary Fig. S1).¹

Therefore, we hypothesized that As₂O₃ induced degradation of AKT protein. AKT degradation has been shown to occur in a proteasome-dependent manner (19, 20). We treated NB4 cells with 5 μmol/L As₂O₃ for 18 h. Six hours before harvesting the cells, MG132, a potent reversible cell-permeable proteasome inhibitor, was added. Immunoblotting for total AKT protein levels showed that inhibiting the proteasome pathway could not rescue AKT protein from As₂O₃-induced degradation (Fig. 5A). AKT has also been shown to be a target of caspase-mediated degradation, particularly caspase-3, caspase-6, and caspase-7 (21–23). To test whether caspases mediate the As₂O₃-induced decrease in AKT protein levels, we treated NB4 cells for 6 or 24 h with Z-VAD, a pan-caspase inhibitor, alone or in combination with 5 μmol/L As₂O₃. Following Z-VAD treatment, AKT protein levels were protected from As₂O₃-induced decrease (Fig. 5B), suggesting that one or more caspases are involved in As₂O₃ effect on AKT protein. We then treated NB4 cells with a wide range of caspase inhibitors in combination with As₂O₃. Inhibitors of caspase-1, caspase-2, and caspase-5 had no effect on arsenic-induced AKT protein degradation, whereas caspase-3, caspase-6, caspase-8, and caspase-9 could each slightly inhibit As₂O₃ effect on total AKT protein levels but not as strongly as pan-caspase inhibitors (Z-VAD and Boc-D-fmk; Fig. 5C). Of the caspases implicated in this inhibitor experiment, caspase-3 is the furthest downstream and likely to integrate all the initiator caspase signals. To correlate caspase-3 activity with sensitivity to As₂O₃, we assessed caspase-3 activity in NB4 and AsR2 cells after 18 h of treatment with As₂O₃ alone or combined with Z-DEVD-fmk (Fig. 5D). As₂O₃ alone could increase caspase-3 activity in NB4 cells, which was decreased when As₂O₃ was used in combination with the caspase-3–specific inhibitor. However, in the arsenic-resistant AsR2 cell line, no increase in caspase-3 activity was detected in the presence or absence of As₂O₃ (Fig. 5D). Increased caspase-3 activity in the arsenic-sensitive NB4 cells correlates with the ability of As₂O₃ to decrease AKT protein levels.

17-AAG is a geldanamycin derivative currently undergoing clinical development, which binds to hsp90 and enhances degradation of hsp90 client proteins (24, 25). AKT is a hsp90 client protein, whose activity and, subsequently, expression are decreased after treatment with 17-AAG in vitro (26). Interestingly, As₂O₃ and 17-AAG act synergistically to induce apoptosis of HL-60 and Jurkat cells (26). We investigated whether As₂O₃ enhanced 17-AAG–induced cytotoxicity in NB4 cells. As₂O₃ (0.5 μmol/L), 17-AAG (0.125 or 0.25 μmol/L), and the combinations were used in 3-day growth curves to determine the effects of combination therapy. Indeed, the combination of As₂O₃ with 17-AAG was significantly more inhibitory than either drug alone (Fig. 6A). Higher concentrations of As₂O₃ (1 μmol/L) also enhanced the efficacy of 17-AAG even at 24 h (data not shown). Total AKT levels also were assessed at 24 h using immunoblotting. As₂O₃ and 17-AAG alone decreased total AKT levels, but the combination further decreased AKT expression (Fig. 6B and C). These data suggest that the enhanced cytotoxic effects of the combination treatment may be due to a decrease in prosurvival signals from AKT.

As₂O₃ is used effectively to treat patients with APL and shows promise as a treatment for multiple myeloma and myelodysplastic syndrome. Understanding the mechanism by which As₂O₃ induces apoptosis may identify potential targets for combination therapies and broaden the malignancies for which arsenic can provide effective treatment. Previous data from our laboratory and others show that generation of ROS and activation of the JNK signaling pathway mediate the arsenic-induced apoptotic signal in APL cells (4, 5, 8, 9). Based on these data, we hypothesized that As₂O₃ would inhibit negative regulators of this pathway.

Several laboratories have investigated a role for AKT in As₂O₃-induced death, although the data are confusing. In one report, pretreatment with inhibitors of phosphoinositide 3-kinase had no effect on As₂O₃-induced death in NB4 cells but enhanced the death in MOLT-4 T-cell leukemia line (27). However, another report showed that a cotreatment with the same inhibitors enhances As₂O₃-induced apoptosis in NB4 cells but not the cytotoxicity associated with ionidamine, camptothecin, or cisplatin (28). The cytotoxic effect is correlated with a decrease in phosphorylated AKT but not total AKT (28). Our data show that cotreatment with phosphoinositide 3-kinase inhibitors can sensitize arsenic-resistant NB4 subclones (Fig. 3C) and confirm that overexpression of a constitutively active AKT construct rescues cells from As₂O₃-induced apoptosis (Fig. 3D). Of note, we also observed the As₂O₃-induced decrease of AKT protein in other cell lines, including several multiple myeloma lines and MDA-MB-468 breast cancer cells, which overexpress phosphorylated AKT (Fig. 2). In addition to confirming an association between dominant-negative AKT and As₂O₃-induced JNK activation and apoptosis, we focused on the observed decrease in AKT total protein and show that As₂O₃ does not decrease AKT transcription or translation (Supplementary Fig. S1) but causes decreased AKT protein expression that can be blocked by selective caspase inhibitors.

Several other apoptotic stimuli result in cleavage of AKT protein. Etoposide and Fas stimulation results in AKT cleavage in U937 and Jurkat cells (21). Tumor necrosis factor-α treatment of adipocytes causes AKT cleavage, which can be inhibited by caspase inhibitors but not proteosome inhibitors (29). Glucose deprivation–enhanced, tumor necrosis factor–related apoptosis-inducing ligand–induced cytotoxicity is associated with caspase-3–mediated AKT cleavage (30). Oxidative stress triggered by treatment with H₂O₂ also resulted in proteolysis of AKT (23). Cytokine withdrawal in 32D cells causes caspase-3–mediated cleavage at the Asp⁴⁶² residue (31), but mutation of this residue did not alter the ability of As₂O₃ to degrade AKT (data not shown). In addition, individual mutation of two other amino acids, previously shown to be important in AKT cleavage by caspases following death receptor activation (22), had no effect on As₂O₃-mediated AKT protein decrease (data not shown). Interestingly, hsp90 inhibitors target AKT for degradation via the ubiquitin-proteosome pathway (20).

We show that AKT and JNK are inversely activated in response to As₂O₃. In addition, As₂O₃ treatment decreases the association of AKT with JIP1, the scaffolding protein on which the MAPK cascade assembles. Despite several reports that AKT inhibits association of JNK with JIP1, it is unclear what triggers release of AKT from JIP1. Clearly, release occurs after As₂O₃ treatment at a time before total AKT protein is degraded (Fig. 4B). This suggests that release of AKT from the JIP1/JNK complex may be the initiating event that activates the JNK cascade leading to loss of AKT and induction of apoptosis.

Our data suggest that As₂O₃ could be used in combination with new targeted agents to increase the cytotoxicity and therapeutic spectrum. This includes agents that decrease AKT activity or expression, such as 17-AAG (Fig. 6). Alternatively, it may be used to enhance other promising drugs that unexpectedly increase AKT. CCI-779 (temsirolimus) is a rapamycin derivative that targets mammalian target of rapamycin, which lies downstream of AKT signaling. CCI-779 effectively inhibits growth of tumor cells in vitro and in vivo but also results in an increase in activated AKT via an ill-defined feedback mechanism (32, 33). Targeting the activated AKT by combining As₂O₃ with CCI-779 may result in enhanced tumor killing. Malignancies where the tumor suppressor PTEN is often silenced and AKT signaling is constitutively activated, such as glioblastoma multiforme, prostate cancer, endometrial cancer (34), and Herceptin-resistant breast cancer (35), are being targeted in clinical trials with mammalian target of rapamycin inhibitors. The combination of As₂O₃ and mammalian target of rapamycin inhibitors in these tumor settings is particularly intriguing and is currently under investigation in our laboratory.

All authors claim no conflict of interest.