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Cancer Research 68, 2895, April 15, 2008. doi: 10.1158/0008-5472.CAN-07-6268
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Soft Tissue Sarcoma Cells Are Highly Sensitive to AKT Blockade: A Role for p53-Independent Up-regulation of GADD45{alpha}

Quan-Sheng Zhu1, Wenhong Ren1, Borys Korchin1, Guy Lahat1, Adam Dicker4, Yiling Lu2, Gordon Mills2, Raphael E. Pollock1 and Dina Lev3

Departments of 1 Surgical Oncology, 2 Systems Biology, 3 Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas; and 4 Department of Radiation Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

Requests for reprints: Dina Lev, Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit 1104, Houston, TX 77030. Phone: 713-792-1637; Fax: 713-563-1185; E-mail: dlev{at}mdanderson.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The AKT signaling pathway is activated in soft tissue sarcoma (STS). However, AKT blockade has not yet been studied as a potential targeted therapeutic approach. Here, we examined the in vitro and in vivo effects of AKT inhibition in STS cells. Western blot analysis was used to evaluate the expression of AKT pathway components and the effect of AKT stimulation and inhibition on their phosphorylation. Cell culture assays were used to assess the effect of AKT blockade (using a phosphatidylinositol 3-kinase inhibitor and a specific AKT inhibitor) on STS cell growth, cell cycle, and apoptosis. Oligoarrays were used to determine gene expression changes in response to AKT inhibition. Reverse transcription–PCR was used for array validation. Specific small inhibitory RNA was used to knockdown GADD45{alpha}. Human STS xenografts in nude mice were used for in vivo studies, and immunohistochemistry was used to assess the effect of treatment on GADD45{alpha} expression, proliferation, and apoptosis. Multiple STS cell lines expressed activated AKT. AKT inhibition decreased STS downstream target phosphorylation and growth in vitro; G2 cell cycle arrest and apoptosis were also observed. AKT inhibition induced GADD45{alpha} mRNA and protein expression in all STS cells treated independent of p53 mutational status. GADD45{alpha} knockdown attenuated the G2 arrest induced by AKT inhibition. In vivo, AKT inhibition led to decreased STS xenograft growth. AKT plays a critical role in survival and proliferation of STS cells. Modulation of AKT kinase activity may provide a novel molecularly based strategy for STS-targeted therapies. [Cancer Res 2008;68(8):2895–903]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Use of standard chemotherapy (even in drug combinations) for soft tissue sarcoma (STS) therapy remains problematic due to toxicity, expense, and the markedly chemoresistant nature of these malignancies; the search for better systemic agents is therefore crucial (1). Molecularly targeted therapy has recently emerged as a new treatment paradigm that seeks to improve conventional systemic therapy by specifically and selectively targeting cancers while minimizing treatment-related morbidities. This approach has led to successful advances in several diseases, including gastrointestinal stromal tumor (2, 3), an STS subtype. To use targeted therapy for STS, an increased knowledge of potential targets and their roles in STS progression and metastasis is needed (4, 5).

One potential molecular target is AKT kinase and its signaling pathways. AKT, also called protein kinase B, is a serine-threonine kinase activated by phosphorylation of two critical residues: threonine 308 (T308) located in the activation loop and serine 473 (S473) at the COOH terminal portion of the protein (6, 7). AKT activation is mediated by phosphatidylinositol 3-kinase (PI3K), which in turn is activated by a multitude of cell surface receptors and other related molecules (8–10); the negative regulation of AKT activation is achieved via tumor suppressor genes, such as PTEN (11) and Src homology 2 domain-containing inositol 5-phosphatase1/2 (SHIP1/2; ref. 12). The role of AKT has been investigated in a variety of epithelial origin tumors; upon phosphorylation, AKT activates downstream pathways that promote protumorigenic, prometastatic processes (13). Activation of AKT has been found in brain (14), prostate (15), breast (16), lung (17), liver (18), gastric (19), colon (20), ovarian (21), and endometrial cancers (22), in association with cancer progression and chemoresistance. These findings have led to identification of several specific AKT inhibitors that are currently in clinical trials for a variety of epithelial malignancies.

Whereas not extensively explored, evidence points to potential involvement of the AKT pathway in STS development and progression. Recently, Hernando et al. reported increased expression of activated AKT in a large panel of human leiomyosarcoma, malignant fibrous histocytoma, and dedifferentiated liposarcoma (23); using a conditional PTEN knockout mouse model, they showed a critical role for the AKT pathway in smooth muscle transformation and leiomyosarcoma development. Tomita et al. identified a correlation between phosphorylated AKT (pAKT) expression in human STS specimens and subsequent tumor recurrence and patient survival (24). These findings suggest that determining the effect of AKT inhibition on STS in vitro and in vivo may facilitate inclusion of specific AKT targeted therapy in the anti-STS treatment armamentarium.

We report that AKT activity blockade induces STS cell growth inhibition, G2 cell cycle arrest, and apoptosis both in vitro and in vivo using human STS xenograft murine models. Relevant to STS, which harbor a high rate of p53 mutations contributory to the STS chemoresistance phenotype (25), is the finding that antitumor effects induced by AKT inhibition were observable in both wtp53, as well as mutated p53 STS cell lines. In addition, we identified a p53-independent increase in GADD45{alpha}, which is at least partially responsible for AKT-induced STS growth inhibition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and reagents. Human SKLMS1 (leiomyosarcoma), HT1080 (fibrosarcoma), RD (rhabdomyosarcoma), A204 (unclassified sarcoma), SW872 (liposarcoma), SW684 (fibrosarcoma), and MES-SA and its multidrug resistant derived MES-SA/DX (uterine sarcoma) STS cell lines were obtained from American Type Culture Collection. Cells were cultured in DMEM (A204 in McCoy's 5A) supplemented with 10% FCS (Life Technologies, Inc.). p53 mutational status of these cells was previously determined by sequencing.5 The specific AKT kinase inhibitor A674563 (A563) was a kind gift from Abbott Laboratories; the PI3K inhibitor Ly294002 was purchased from Cayman Chemical. Doxorubicin (Ben Venue Laboratories) was obtained from the UTMDACC Pharmacy. Recombinant human epidermal growth factor (EGF; R&D Systems) was used for EGF receptor (EGFR) stimulation.

Commercially available antibodies were used to detect AKT, pAKT (S473), pGSK3 (S21/9), pMDM2 (S166), activated caspase-3, PTEN, SHIP2, EGFR, c-MET, HER2, and insulin-like growth factor-IR{alpha} (Cell Signaling); GADD45{alpha}, p53, p21/WAF1, MDM2, GSK3, and β-actin (Santa Cruz Biotechnology); and proliferating cell nuclear antigen (PCNA; Dako Cytomation). The Dead End Fluorometric TUNEL System (Promega) was used for TUNEL staining. Secondary antibodies included horseradish peroxidase (HRP)–conjugated (Universal kit HRP, Biocare Medical) and fluorescent secondary antibodies (antirabbit Alexa488 and antimouse Alexa 594, Jackson Immuno Research). Other reagents included CytoQ FC receptor block (Innovex Bioscience), Hoechst 33342 (Polysciences, Inc.), and propyl gallate (Acros Organics).

Western blot analysis. Western blot analysis was performed by standard methods. Briefly, 25 to 50 µg of proteins extracted from cultured cells were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were blocked and blotted with relevant antibodies. HRP-conjugated secondary antibodies were detected by enhanced chemiluminescence (Amersham Biosciences, Plc.). IRdye680-conjugated and IRdye800-conjugated secondary antibodies (Molecular Probes) were detected using Odyssey Imaging (LICOR Biosciences).

Measurement of cell proliferation. Cell growth assays were done using CellTiter96 cell prolifetation assay kit (Promega) as per manufacturer's instructions. STS cell lines were plated at concentrations of 1.5 x 103 to 4 x 103 cells per well (depending on cell doubling time) in 96-well plates. The next day, cells were treated with either 0.1% DMSO as control or different concentrations of LY294002 or A563 (for 24, 48, and 72 h). Absorbance was measured at a wavelength of 490 nm; absorbance values of treated cells are presented as a percentage of the absorbance of untreated cells. Drug concentrations required to inhibit cell growth by 50% (IC50) were determined by interpolation of dose-response curves.

Cell cycle analysis. STS cell monolayers were treated with relevant agents for varying time periods. Cells were harvested, washed, and fixed. Fixed cells were treated with 50 µg/mL RNase and stained with 50 µg/mL propidium iodide for 30 min. Cells were analyzed in a FACSCalibur, and data were analyzed with Cell Quest and Flowjo software or ModFitLT v3.1 software (Verity Software House).

Apoptosis assay. Apoptosis was measured using the Apoptosis Detection kit I (BD Biosciences). As a standard, 1 x 106/mL of cells per treatment condition were fixed and stained with 5 µL Annexin V–FITC (BD PharMingen) and 5 µL propidium iodide (Sigma). Flow cytometric analysis was performed for 1 x 104 cells and analyzed by FACScan (Becton Dickinson) using a single laser emitting excitation light at 488 nm. Data were analyzed by CellQuest software (Becton Dickson).

Caspase-3 apoptosis assay. DEVD-NucView 488 caspase-3 assay kit for live cells was purchased from Biotium, Inc. Apoptotic cells were detected per manufacturer's instructions. Briefly, STS cells grown on chamber slides were treated with A563 (1 µmol/L) or DMSO for 24 h. NucView substrate stock solution (5 µL from 0.2 mmol/L stock) was added to culture medium and incubated at room temperature for 30 min. Fluorescence was determined via a florescent microscope using FITC filters, and images were captured.

Microarray hybridization. Total RNA isolated from STS cells treated with relevant agents was used to synthesize cDNA as template for generating Biotin-16-UTP (nonradioactive) labeled cRNA target using TrueLabeling-AMP linear RNA amplification kit (SuperArray Bioscience Corporation). Labeled cRNA was purified using SuperArray ArrayGrade cRNA cleanup kit and quantified by spectrophotometry (1 absorbance unit, 40 µg/mL). Gene expression profiling was performed using OligoGEArray Human Cell Cycle OHS-020 (SuperArray Bioscience Corporation). This microarray is designed to profile the expression of 112 key genes in cell cycle regulation.6 Prehybridization (2 h) and hybridization (overnight) was performed in a hybridization oven (60°C) using 4 µg of labeled cRNA target. High stringency washing at 60°C (0.1 x SSC, 0.5% SDS) was followed by chemiluminescent detection. The array image was recorded using X-ray film and a flatbed desktop scanner to create grayscale (16 bits) files in TIFF format that were analyzed by GEArray Expression Analysis Suite online software.7

Reverse transcription-PCR. Reverse transcription–PCR (RT-PCR) was done as previously described (26). Briefly, total RNA was isolated from cultured STS cells using TRIzol reagent (Invitrogen Corp.) as per manufacturer instructions. Total RNA was reverse-transcribed using superscript II reverse transcriptase (Invitrogen), and 2 µL of the product were used as templates for multiplex PCR containing both target GADD45{alpha} and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers for normalization. PCR primers were designed using primer 3 software: GADD45{alpha}, 5'-GGAGAGCAGAAGACCGAAA-3' and 5'-TCACTGGAACCCATTGATC-3'; GAPDH, 5'-GAGCCACATCGCTCAGAC-3' and 5'-CTTCTCATGGTTCACACCC-3'. The PCR reaction solution contained 25 µL of Taq PCR Master Mix (Qiagen); 0.2 µmol/L of forward and reverse primers, respectively; 2 µL of cDNA and double-distilled water to final volume of 50 µL. PCR consisted of denaturation for 3 min at 94°C, 26 cycles of denaturation for 30 s at 94°C, annealing for 40 s at 56°C, and an extension for 50 s at 72°C. PCR cycles were terminated by an extension at 72°C for 7 min, and products were resolved on a 2% agarose gel.

Small inhibitory RNA knockdown of GADD45{alpha}. 5 x 105 RD cells were plated per well of six-well plate and incubated overnight at 37°C. The following morning, SmartPool GADD45{alpha} small inhibitory RNA (siRNA) or nontargeting siRNAs constructs (Dharmacon, Inc.) were transfected using Lipofectamine 2000 (Invitrogen) reagents according to manufacturer's instructions. Mock-transfected cells were treated with Lipofectamine 2000 only. Incubation time for transfection reagents was 24 h, at which time media was replaced with fresh regular media containing appropriate inhibitors (A563 or DMSO). The next day, cells were harvested for RT-PCR and cell cycle analysis by flow cytometry.

In vivo therapeutic animal model. All animal procedures and care were approved by the Institutional Animal Care and Usage Committee of UTMDACC. Animals received humane care as per the Animal Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals. Trypan blue staining–confirmed viable HT1080 STS cells (1 x 106/0.1 mL HBSS per mouse) were injected s.c. into the flank of 6-wk-old female nude/nude mice (National Cancer Institute/NIH; n = 20). Subcutaneous tumors were measured twice weekly by digital caliper; tumor volume was calculated as V = L x W 2 x {pi} / 6, where V is volume, L is length, and W is width. When average subcutaneous tumor volume reached ~100 mm3, mice were assigned into two treatment groups (n = 10): (a) control (vehicles only) and (b) A563 (20 mg/kg/bid, gavage). Mice were followed for tumor size and body weight and were sacrificed when control group tumors reached an average of 1.5 cm in largest dimension. Tumor was resected, weighed, and frozen or fixed in formalin and paraffin embedded for further immunohistochemical studies.

Immunohistochemical analysis. Immunohistochemistry was performed as previously described (27). Briefly, paraffin sections were dewaxed and rehydrated before antigen retrieval. Endogenous peroxidase activity was quenched with 0.6% hydrogen peroxide before blocking with horse serum. Staining with primary antibodies (described above) was done at concentrations based on manufacturer's recommendation. Biotinylated secondary antibodies were applied at 1:200 before ABC peroxidase system application (Vectastain ABComplex; Vector Laboratories, Inc.), 3,3'-diaminobenzidine color development (Sigma Chemical Co.), and Mayer's hematoxylin counterstaining. For immunofluorencence staining, fluorescence-conjugated secondary antibodies were used, followed by nuclear staining with Hoechst. In situ cell death detection kit (Roche Applied Science) was used as per manufacturer's instructions for TUNEL assay. Staining distribution and intensity were evaluated and scored by two independent reviewers (B.K. and Q.Z.). Photographs were obtained using a Leica DM4000B microscope (Leica Microsystems, Gmb) and a Leica HCxPL-S-APO 40x/0.75 numerical aperture objective lens. Images were captured using SPOT digital camera and were processed using SPOT advanced acquisition software (Diagnostic Instruments, Inc.).

Statistical analysis. Cell culture assays were repeated at least thrice, and means ± SD was calculated. Cell lines were examined separately. For outcomes that were measured at a single time point, two sample t tests were used to assess the differences. Differences in xenograft growth in vivo were assessed using a two-tailed Student's t test. Significance was set at P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
STS cell lines express activated AKT. Previous reports have suggested a possible role for AKT activation in STS (24). To study the effect of AKT inhibition on STS cell growth, we initially evaluated the expression of pAKT in a panel of human STS cell lines of diverse histology (Fig. 1A ). AKT activation to varying levels was demonstrable in all STS cells examined by immunobloting for the pAKT S473 epitope. Moreover, increased AKT phosphorylation was observed when STS cells were under serum starvation conditions (Supplementary Fig. S1). Serum-independent pAKT expression is possibly an even better discriminator of STS cell AKT dependence.


Figure 1
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  		Figure 1. STS cells express high levels of activated and functional AKT. A, Western blot analysis demonstrating increased pAKT (S473) in a panel of STS cell lines. pAKT/total AKT ratio (calculated based on densitometry analysis) is recorded (bottom). B, Western blot analysis demonstrating the STS expression of several possible upstream AKT modifiers. C, EGF stimulation (80 ng/mL for 15 min) increases AKT phosphorylation in STS. D, AKT downstream targets (MDM2 and GSK3) are phosphorylated in STS cells.

 
There are multiple molecular mechanisms which could possibly result in the activation of AKT. Dysregulation in intrinsic components of the PI3K-AKT pathway, e.g., PTEN mutation resulting in the loss/dysfunction of the PI3K-negative regulator PTEN protein, has been identified in many cancers. Although not well described in STS, some evidence suggests that PTEN is dysregulated in STS, especially leiomyosarcomas (23). Screening human STS cells8 for PTEN expression (Fig. 1B), we observed that two cell lines (MES-SA, uterine sarcoma and its derivative MES-SA-Dx, and SW872, liposarcoma) do not exhibit PTEN protein. It is possible that the loss of PTEN in these cells is at least partially responsible for their pAKT expression. Similarly, we screened cells for the expression of another phosphatase that negatively regulates PI3K, SHIP2 (Fig. 1B). All STS cells were found to express SHIP2. As with STS cells expressing PTEN, we cannot exclude the possibility that loss of function, rather than loss of expression, of these phosphatases per se contributes to AKT activation.

The AKT pathway is a common point of convergence for a multitude of upstream activators, such as tyrosine kinase receptors, several of which have been previously shown to be dysregulated in STS (28, 29). For example, Western blot analysis (Fig. 1B) showed that all STS cells evaluated in the current study express at least one of the growth factor receptors examined and that most express multiple such receptors. Thus, it is possible that upstream modulators are at least partially responsible for the AKT phosphorylation observed in the STS cell lines. Moreover, stimulation of upstream receptors with an appropriate ligand (i.e., EGF/EGFR; Fig. 1C) can induce further AKT phosphorylation. These findings are of special importance in that the tumor microenvironment is rich in cytokines secreted by a variety of cells composing the tumor stroma, suggesting that AKT could possibly be even more activated in vivo.

To further evaluate whether AKT activation is of significance in STS cells, we examined the phosphorylation status of downstream AKT targets, such as MDM2 and GSK3. As shown in Fig. 1D, phosphorylation of MDM2 at Ser166 and phosphorylation of GSK3 at Ser21 ({alpha} subunit) and Ser9 (β subunit) were detectable at varying levels in all STS cell lines. These data suggest that AKT kinase is activated and functional in STS cells, mediating intracellular signaling and downstream target phosphorylation.

AKT blockade results in decreased STS cell downstream target phosphorylation and tumor cell growth inhibition. Next, we wanted to evaluate the effect of AKT inhibition on STS cells. Utilizing a commercially available inhibitor of the AKT activator PI3K (LY294002), we were able to show a dose-dependent reduction in AKT phosphorylation in all STS cell lines tested (Fig. 2A ). As suggested above, these data indicate that phosphorylation of AKT in STS is dependent upon PI3K activity. Treatment of the different STS cell lines with increasing doses (0.1–10 µmol/L) of LY294002 resulted in a dose-dependent decrease in cell proliferation, as was identified by MTS assay, with IC50s (at 48 h) ranging from 3.5 to 7.5 µmol/L (data not shown).


Figure 2
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  		Figure 2. AKT blockade inhibits AKT downstream target phosphorylation and induces STS growth inhibition. A, PI3K inhibitor LY294002 (4-h treatment) induced a dose-dependent reduction in AKT phosphorylation in RD cells (similar responses were observed in other STS cells). B, A563 does not affect AKT phosphorylation in STS cells (RD is shown), but directly inhibits its kinase activity. Western blot analysis demonstrating A563 (4-h treatment) dose-dependent inhibition of AKT downstream target phosphorylation (GSK3 and MDM2). C, IC50 values for different STS cell lines (48 h; MTS assays). Drug concentration required to inhibit cell growth by 50% (IC50) for each cell line was determined by interpolation from dose-response curve; columns, results of four independent experiments. D, A563 inhibits STS cell growth in a dose-dependent and time-dependent manner (MTS assays). Results were expressed as percentage of cell viability compared with control DMSO-treated cells; columns, results of four independent experiments.

 
Whereas PI3K blockade results in effective inhibition of AKT phosphorylation, it is not specific and possibly affects other signaling pathways (30, 31). Therefore, we next analyzed the efficacy of a specific AKT inhibitor, A563 (32). This compound is an ATP competitor that binds to the ATP site of the AKT kinase domain, inhibiting AKT-catalyzed phosphorylation activity. As depicted in Fig. 2B, A563 does not inhibit AKT phosphorylation per se, but blocks the phosphorylation of AKT downstream targets in a dose-dependent manner. Exposure of STS cells to increasing doses of A563 significantly decreased GSK3 and MDM2 phosphorylation (Fig. 2B). No effect on mitogen-activated protein kinase phosphorylation could be observed in STS cells after A563 treatment (data not shown).

Next, we determined the effect AKT activity blockade induced by A563 on STS cell proliferation. We examined the sensitivity of the different cell lines exposed to increasing concentrations of A563 for varying lengths of time (24, 48, and 72 hours). Our results indicate that all STS cell lines were sensitive to A563, with the IC50 values at 48 hours ranging from 0.22 ± 0.034 µmol/L (SW684) to 0.35 ± 0.06 µmol/L (SKLMS1; Fig. 2C). AKT inhibition induced growth inhibition in a dose-dependent and time-dependent manner (Fig. 2D). Cells expressing the highest level of pAKT (RD, MES-SA, and SW684; see densitometry in Fig. 1A) were found to be most sensitive to this inhibition. Additionally, because p53 mutations are the most common genetic alteration in STS and p53 mutated STS are more therapeutically resistant, it is important that no significant differences in response to A563 could be found when comparing STS cells bearing wtp53 (HT1080, A204, MES-SA) versus mutated p53 genes (SKLMS1, RD, SW684, SW872). This observation suggests the possible importance of further investigating the AKT inhibition-induced p53-independent pathway in STS.

AKT activity inhibition induces G2 cell cycle arrest and apoptosis in STS cells. Based on the observed effect of A563 on STS cell growth, we next evaluated the effect of A563 on STS cell cycle progression and apoptosis. Cell cycle analysis after treatment of STS cell lines with A563 using propidium iodide staining/fluorescence-activated cell sorting (FACS)–induced G2 cell cycle arrest in all STS cells tested (P < 0.05; Fig. 3A ). Additionally, an increase in apoptotic cells (cells in G0–sub-G1) could also be observed. When treating STS cells with A563, we observed clear morphologic changes, i.e., loss of elongated shape and cytoplasmic and nuclear condensation, confirming A563-induced apoptosis (Supplementary Fig. S2). To further quantitatively evaluate the effect of A563 on STS cell apoptosis, we used an Annexin V apoptosis detection assay (Fig. 3B). AKT inhibition induced significant early and late apoptosis in all STS cell lines tested (P < 0.01; Fig. 3B). Western blot analysis showed increase in caspase-3 cleavage (Fig. 3C), and using a NucView 488 caspase-3 assay kit, we detected apoptosis in living cells when treated with AKT kinase inhibitor, as shown in Fig. 3D. Similar to the cell growth assays, the effect of A563 on the cell cycle and apoptosis observed in the STS cells was independent of p53 mutational status, suggesting that these findings cannot solely be explained by wtp53 stabilization found to occur secondary to AKT inhibition (33). Taken together, these data indicate that AKT activity is highly correlated to proliferation and survival of STS cells. AKT blockade is effective in significantly inhibiting STS cell proliferation, as well as induction of G2 cell cycle arrest and apoptosis.


Figure 3
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  		Figure 3. AKT blockade induced G2 cell cycle arrest and apoptosis in STS cells. A, propidium iodide staining/FACS analysis shows A563 (1 µmol/L/24 h) induced G2 cell cycle arrest in STS cells. B, Annexin V staining demonstrating increase in early and late apoptosis in STS cell treated with A563 (1 µmol/L/24 h). C, A563 (24 h) induces a dose-dependent increase in cleaved caspase-3 in RD cells (Western blot analysis). D, caspase-3 apoptosis assay shows increased apoptosis in MES-SA cells treated with A563 (1 µmol/L/24 h). Nuclear DNA of apoptotic cells were stained green by the enzymatically released DNA dye. All figure panels are representative of three independent experiments.

 
AKT inhibition up-regulated the expression of GADD45{alpha} independent of p53. To identify possible AKT downstream modulators responsible for the effect of AKT blockade on STS G2 cell cycle arrest in both wtp53 and mutp53 cells, we used the commercial Human Cell Cycle OligoGEArray to determine the expression profile of a panel of genes involved in cell cycle progression. Human RD cells (mutp53) and MES-SA cells (wtp53) were treated with A563 (1 µmol/L/12 hours), and total RNA from treated and untreated cells was used to identify changes in gene expression (Supplementary Fig. S3). Our results showed that GADD45{alpha}, a gene encoding for the growth arrest and DNA damage 45{alpha} protein, was the only gene up-regulated in both cell lines (5.69 ± 0.09–fold in MES-SA and 2.37 ± 0.62–fold in RD); therefore, we elected to study its possible role in AKT blockade-induced effects. Initially, we wanted to confirm the Oligoarray results in RD and MES-SA cell lines, as well as evaluate whether the GADD45{alpha} increase could also be seen in other human STS cells. Increase in GADD45{alpha} mRNA expression level after A563 treatment was identified in all STS cell lines tested (MES-SA, HT1080, A204-wtp53 and RD, SW684, SKLMS1-mutp53; Fig. 4A ). Furthermore, Western blot analysis showed that the changes observed in mRNA expression were translated to an increase in GADD45{alpha} protein levels in all STS cell lines treated with A563 (Fig. 4B). Our data also indicate that GADD45{alpha} induction can be observed after treatment with low doses of A563 (0.1 µmol/L) in both wtp53 and mutp53 cells (Fig. 4C), whereas p21 increase occurs only after exposure to a higher A563 dose and is dependent on p53 mutational status.


Figure 4
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  		Figure 4. AKT inhibition induces expression of GADD45{alpha}. A, increase in GADD45{alpha} mRNA in STS cells is shown (RT-PCR) after treatment with A563 (1 µmol/L/12 h). B, similarly, an increase in GADD45{alpha} protein is observed after A563 treatment (1 µmol/L/24 h; Western blot analysis). C, A563 (24 h) induces GADD45{alpha} expression in both wtp53 (MES-SA) and mutp53 (RD) STS cell lines, whereas A563-induced expression of p21 occurs only with a higher A563 dose and in cells harboring wtp53 (Western blot analysis). D, doxorubicin induces GADD45{alpha}, as well as p53 and p21 expression, only in wtp53 STS cells (A204 and MES-SA). A563-induced GADD45{alpha} is p53 status independent, whereas A563-induced p21 expression is observed only in wtp53 cells (Western blot analysis). All figure panels are representative of three independent experiments.

 
As its name implies, GADD45{alpha} has a role in cell cycle arrest and apoptosis in response to DNA damage in eukaryotic cells (34). The GADD45{alpha} gene was the first ever described p53 response gene; it was found to be transcriptionally up-regulated by p53 via a p53 consensus-binding site located in the third intron of the GADD45{alpha} gene (35). It has also been reported to be regulated in a p53-independent manner (34). To further evaluate whether the increase in GADD45{alpha} observed in STS cells after AKT inhibition is a general stress response or whether it is a specific response to AKT inhibition in these cells, we compared the effect of doxorubicin (the most commonly used chemotherapeutic agent for the treatment of STS) on GADD45{alpha} expression to that of A563 (Fig. 4D). Doxorubicin treatment resulted in an increase in GADD45{alpha} only in wtp53 STS cell lines (A204 and MES-SA). The chemotherapy induced stabilization and increase in p53 levels accompanied by increased p21 levels in these cells suggests that the wtp53 pathway is at least partially intact. Although not the focus of this manuscript, the lack of doxorubicin-mediated GADD45{alpha} induction in mutp53 cells (RD and SKLMS1) might possibly offer an additional explanation of our previous observation of increased doxorubicin resistance in STS harboring mutp53 (36, 37). In contrast, treatment with the AKT inhibitor increased the expression of GADD45{alpha} in both wtp53 and mutp53 cell lines, whereas p21 was only induced in wtp53 cells. These data suggest that AKT inhibition–induced up-regulation of GADD45{alpha} in STS cells is specific and independent of wtp53 protein function.

GADD45{alpha} knockdown attenuates G2 arrest induced by AKT inhibition. To further study whether AKT inhibition–induced GADD45{alpha} up-regulation is of functional significance in STS cells, we used smartpool siRNA directed against human GADD45{alpha} mRNA to knockdown its endogenous and inducible expression. RD cells were mock-transfected, transfected with nontargeting siRNA, or transfected with GADD45{alpha} smart pool siRNA for 24 hours, at which point cells were treated with 0.1%DMSO or A563 (1 µmol/L) for an additional 24 hours; cells were then harvested for total RNA isolation and for cell cycle analysis. As shown in Fig. 5A , RT-PCR showed that endogenous and AKT inhibition–induced GADD45{alpha} mRNA was knocked down effectively (>86% as measured by densitometry) after siRNA GADD45{alpha} transfection. Furthermore, when the cells were subjected to cell cycle analysis by FACS (Fig. 5B), GADD45{alpha} knockdown attenuated the AKT inhibition–induced G2 arrest. These data suggest that AKT inhibition–induced GADD45{alpha} is at least partially responsible for the observed G2 cell cycle arrest in response to A563 in STS cells.


Figure 5
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  		Figure 5. GADD45{alpha} knockdown attenuates G2 arrest induced by AKT inhibition. A, GADD45{alpha} siRNA transfection to RD cells decreased constitutive and A563-induced GADD45{alpha} expression compared with mock (Lipofectamine only) or nontargeting siRNA transfection (RT-PCR). B, AKT inhibition-induced G2 arrest was attenuated by GADD45{alpha} knockdown. RD cells transfected with nontargeting siRNA (top) exhibit increased G2 arrest after A563 treatment (1 µmol/L/24 h). No significant G2 arrest was observed in GADD45{alpha} siRNA transfected cells (bottom) after treatment with A563 (propidium iodide/FACS analysis). All figure panels are representative of three independent experiments.

 
AKT blockade decreases STS growth in vivo. To investigate if the effect of A563 on STS cells in culture can also be recapitulated in vivo, therapeutic animal studies were conducted using human fibrosarcoma (HT1080) subcutaneous xenografts. Treatment with A563 (20 mg/kg/bid, p.o.) was initiated when tumors reached 5 mm, tumor growth was compared with those of mice treated with vehicle only (5% dextrose). A563-treated mice exhibited slower tumor growth (Fig. 6A ) and a significant difference in tumor volume at the termination of the study (320.76 ± 86.8) compared with that in control group (667.92 ± 97.41; P < 0.01). No significant weight loss was observed in A563-treated mice compared with control groups. Death occurred in one of the A563-treated mice.


Figure 6
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  		Figure 6. AKT blockade decreases STS growth in vivo. A, A563 (per gavage, 20 mg/kg/bid) significantly inhibited HT1080 xenograft growth in nude mice (P < 0.01). B, H&E staining showed A563-induced necrosis in treated tumors (a, b). Immunohistochemical staining showed increased GADD45{alpha} (c, d) and decreased PCNA (e, f) expression in A563-treated tumors. TUNEL staining showed increased apoptotic cells in A563-treated tumors (g, h). All original images were captured at 400x magnification.

 
H&E staining of tumor specimens revealed pronounced tumor necrosis in A563-treated groups (Fig. 6B, a and b). Sections containing viable tumor cells were then selected for further immunohistochemical studies. A563-treated tumors were found to express increased levels of GADD45{alpha} (Fig. 6B, c and d) and decreased levels of PCNA (a nuclear marker for proliferation; Fig. 6B, e and f). Additionally, an increase in TUNEL assay staining levels (marker for apoptosis) in the A563-treated specimens was observed (Fig. 6B, g and h). Taken together, these data suggest that AKT blockade results in significant STS tumor growth inhibition, an observation of potential clinical utility.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Overall STS patient survival is ~50% at 5 years depending upon tumor size, histology, grade, location, and presence of regional or distant disease. Development of lung metastasis is particularly ominous, accounting for ~80% of sarcoma-specific deaths with the remainder attributable to aggressive local STS effects (38). The need to successfully control STS growth and dissemination via systemic approaches remains compelling but is hampered by the availability of few drugs available with meaningful STS efficacy; toxicity ratios and new and effective systemic agents are crucially needed. Several reports suggest a possible role for activated AKT in STS development and progression (39–41), rendering it a potentially attractive therapeutic target. It is therefore encouraging that our studies show that AKT signaling pathway blockade results in STS cell G2 arrest and apoptosis in vitro, as well as inhibition of STS growth in vivo. AKT blockade combined with conventional chemotherapy should be explored in STS; such combinations have shown effectiveness in other tumors (42, 43).

STS include >50 distinct histologic subtypes, which are grouped together due to their shared putative mesenchymal origin and unique clinical behaviors which distinguish them from the more common epithelial tumors. Due to their relative rarity (<1% of adult solid tumors), it is difficult to accrue sufficient numbers of individual STS histiotype patients needed to study the efficacy of new therapies. We have shown high expression of pAKT in STS cells of different histologic origin, perhaps due to an array of upstream modulators which could vary between different STS histiotypes. Functioning as a common convergence point of multiple dysregulated pathways operative in various STS, AKT per se may be more useful than its various upstream modulators as a target for STS therapy.

The effect of AKT inhibition on STS cells was reproducible using either Ly294002, a PI3K inhibitor, or A563, a potent direct inhibitor of AKT kinase activity. Whereas the effect of Ly294002 could be due to the inhibition of other PI3K downstream pathways, A563 is highly selective for the AKT pathway, suggesting its observed effects are directly related to AKT inhibition. However encouraging these results are in supporting our hypothesis, A563 cannot be used in the clinical arena. Others have shown that mice could not tolerate A563 for longer than 25 days, thereafter becoming moribund. Whereas a significant A563 effect on tumor growth was observed, tumors regrew rapidly upon A563 cessation (32). The dose-limiting toxicities of A563 and other pan-AKT inhibitors are mainly due to the physiologic role of AKT in insulin signaling and glucose metabolism (32, 44). Efforts to develop specific high affinity AKT inhibitors with less toxic side effects are currently under way. It may be a possible approach to use specific AKT1 inhibitors in lieu of pan-AKT inhibitors, such as A563. AKT2 inhibition is perhaps the major cause of A563 toxic effects and predominates in insulin signaling; whereas AKT null mice develop typical type II diabetes, AKT1 null mice do not (32).

Another approach to reducing severe metabolic side effects is via inhibiting individual AKT downstream substrates. There are caveats to such approaches: critical AKT downstream effectors may vary between different STS histiotypes or even within the same subtype, and inhibiting individual downstream components of the AKT pathway may miss key entities that are involved in the AKT-induced STS-promoting effects. However, encouraging preliminary results using mTOR inhibitors (a down stream target of AKT) in phase I/phase II clinical trials for advanced STS (45) suggests the feasibility of this approach and supports further study of potentially significant AKT downstream substrates in STS.

In the current study, we have identified GADD45{alpha} as a common AKT downstream target up-regulated after AKT inhibition in STS cells of different histologic background and p53 mutational status. The exact function of GADD45{alpha}, one of several growth arrest–inducible and DNA damage–inducible proteins, and its expression and role in STS is not well elucidated. It has been implicated in G2 cell cycle arrest and apoptosis, thereby serving as a tumor suppressor (46). GADD45{alpha} interacts with several important intracellular signaling molecules, such as proliferating cell nuclear antigen, cyclin B/CDC2 complex, p21/WAF1, histone, and aurora-A kinase, all of which participate in the regulation of DNA replication, DNA repair, and cell cycle progression (47). Our studies show that the GADD45{alpha} increase in response to AKT blockade at least partially mediates the anti-STS AKT inhibition–induced G2 cell cycle arrest. It is possible that loss of GADD45{alpha} expression and function in STS due to increased activated AKT may play a role in the dysregulated cell cycle progression of these tumors.

AKT-induced suppression of GADD45{alpha}, as well as AKT blockade-induced GADD45{alpha}, has not yet been extensively explored and may be due to several molecular mechanisms. AKT blockade is known to up-regulate and stabilize wtp53 expression through the inhibition of MDM2 phosphorylation (7); GADD45{alpha} is a known transcriptional downstream target of wtp53; however, this cannot solely explain the AKT inhibition–induced GADD45{alpha}, which also occurs in mutp53 cells. FOXO3 has also been identified as a GADD45{alpha} transcription activator (34, 48). AKT inhibition induces the translocation of cytoplasmic FOXO3 to the nucleus, thus increasing its transcriptional activity; previously. FOXO3 has been shown to increase GADD45{alpha} expression (34). In our studies, we have failed to show FOXO3 expression or subcellular localization in STS cells in response to AKT inhibition (data not shown); however, it is possible that other FOXO family members having similar functions might play a role in STS. Additionally, AKT inhibition–induced post transcriptional mechanisms resulting in GADD45{alpha} mRNA stability are also possible. For example, Zheng et al. (49) reported that nuclear factor-{kappa}B (NF-{kappa}B) inhibition resulted in posttranscriptional stabilization of GADD45{alpha} mRNA; AKT blockade inhibited the function of NF-{kappa}B (50). Current studies in our laboratory are investigating the STS-specific mechanisms resulting in AKT-induced GADD45{alpha} suppression and AKT inhibition–induced GADD45{alpha}.

In summary, we show that AKT inhibition results in significant antitumor activity against human STS in vitro and in vivo. Our results suggest the possibility of AKT blockade as a promising therapeutic intervention for the treatment of patients burdened by this disease.


   Acknowledgments
 
Grant support: Radiation Therapy Oncology Group seed grant.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Vincent L. Giranda (Abbot Laboratories) for kindly providing A674563 (A563).


   Footnotes
 
5 Information is available online in Cosmic database (www.samger.ac.uk). Back

6 For a comprehensive list of genes on the array see (http://geasuite.superarray.com). Back

7 For information see (http://geasuite.superarray.com). Back

8 All previously found to harbor wt PTEN (see www.sanger.ac.uk/genetics/CGP/CellLines). Back

Received 11/15/07. Revised 1/23/08. Accepted 2/11/08.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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