Regulation of AKT1 expression by beta-catenin/Tcf/Lef signaling in colorectal cancer cells

Susanne Dihlmann *, Matthias Kloor, Christine Fallsehr and Magnus von Knebel Doeberitz

Institute of Molecular Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 220/221, D-69120 Heidelberg, Germany

* To whom correspondence should be addressed. Tel: +49 6221 565 210; Fax: +49 6221 565 981; Email: susanne.dihlmann{at}med.uni-heidelberg.de


    Abstract
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The serine/threonine kinase AKT plays a critical role in controlling the balance between cell survival and apoptosis. Several reports implicated AKT in the molecular pathogenesis of different human malignancies and overexpression of AKT was recently demonstrated to be an early event in colorectal carcinogenesis. We report here the identification of nine putative Tcf/Lef-binding elements (TBEs) upstream to the ATG initiation site of the AKT1 gene. Four of these TBEs are located upstream of the transcriptional start, whereas five TBEs are situated in Exon 1 of the AKT1 gene. Accordingly, we hypothesized that AKT1 expression might be regulated by Wnt/ß-catenin signaling. To elucidate the regulation of AKT expression in colon cancer cells, we generated reporter constructs containing the luciferase gene under the control of different regions derived from the AKT1 promoter/enhancer. Transient expression of the constructs in colorectal cancer (CRC) cell lines resulted in significant activation of the reporter gene. Luciferase was stimulated 20- to 50-fold in SW480, SW948 and HCT116 CRC cells. In contrast, the AKT1 promoter/enhancer constructs showed only a weak response in 293 embryonic kidney cells. Coexpression of a constitutively active ß-catenin mutant in colon cancer cells further enhanced reporter gene activation from the AKT1 promoter/enhancer, whereas it was downregulated by introduction of either wild-type APC or dnTcf-4. In addition, immunohistochemical staining of tumor sections derived from CRC patients showed elevated expression levels of AKT1, correlating with enhanced cytoplasmic/nuclear expression of ß-catenin. In summary our data suggest that ß-catenin/Tcf contributes to the transcriptional regulation of the AKT1 gene.

Abbreviations: AP-1, activation protein 1; ARF, alternative reading frame; CBP, cAMP-responsive element binding protein; CRC, colorectal cancer; Lef, lymphocyte enhancer factor; MMP-7, matrix metalloproteinase-7; NF{kappa}B, nuclear factor kappa B; PML, promyelocytic leukemia; RT–PCR, reverse transcription–PCR; TBE, TCF-binding element; Tcf, T-cell factor


    Introduction
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The AKT family defines a family of closely related highly conserved cellular homologs of the viral oncoprotein v-akt (1). In humans, there are three members of the AKT gene family, designated AKT1, AKT2 and AKT3, which are located on different chromosomes. The AKT gene products, cytoplasmic serine/threonine (ser/thr)-specific protein kinases, are major downstream targets of numerous receptor tyrosine kinases signaling via phosphatidylinositol 3-kinase (2,3). Being a central player of multiple signaling pathways the AKT kinases control a range of diverse cellular functions, such as glucose metabolism, protein synthesis, cell cycle, cell survival and apoptosis (2). The degree of functional redundancy between AKT1, AKT2 and AKT3 is currently unclear, their different tissue-specific expression patterns, however, point to distinct functions (4).

Cumulative evidence suggests that AKT perturbations play an important role in tumorigenesis. Multiple reports describe an increased or constitutive activity of AKT isoforms in different cancers caused by gene amplification, mRNA overexpression, mutations leading to constitutive phosphorylation or inactivation of antagonists, such as PTEN (5). In colorectal cancer (CRC), AKT overexpression was recently described to be an early event during sporadic carcinogenesis (6). A strong immunoreactivity indicating high AKT expression was seen in 57% of adenomas and carcinomas derived from CRC patients and in AOM-induced rodent tumors, whereas normal colon mucosa exhibited no significant AKT expression. However, little is known about the mechanisms that cause AKT overexpression during colorectal carcinogenesis and about the consequences of AKT downregulation.

The most frequent initiating event in the pathogenesis of CRC is the inappropriate activation of the canonical Wnt-pathway (7). Several mutations in signaling components of this pathway result in stabilization of cytoplasmic ß-catenin and subsequent activation of specific target genes. In up to 80% of all sporadic adenocarcinomas and in the earliest stages of adenomas, this activation is caused by loss-of-function mutations in the APC gene. In addition, oncogenic mutations in one of the ser/thr-phosphorylation sites of ß-catenin lead to its stabilization and the transcription of target genes even in the absence of external Wnt-signals (8). Stabilized ß-catenin translocates to the nucleus where it binds to the transcription factors of the T-cell factor (Tcf)/lymphocyte enhancer factor (Lef) family. An increasing list of reports describes Wnt/ß-catenin-regulated target genes which promote carcinogenesis by driving proliferation, migration or tumor vascularization. (A list of known Wnt/ß-catenin-regulated target genes is available on http://www.stanford.edu/~rnusse/pathways/targets.html.)

In a previous study, we have demonstrated that Wnt/ß-catenin regulated transcription is downregulated by aspirin and indomethacin through stabilization of ß-catenin phosphorylation (9). Expression profiling of a CRC cell line by oligonucleotide microarrays revealed downregulation of 41 genes in response to aspirin treatment including a set of genes reported to be Wnt/ß-catenin target genes (10) (http://ncbi.nlm.nih.gov/geo/). In addition, expression of the AKT gene was downregulated by aspirin, leading us to the hypothesis that AKT might be regulated by ß-catenin as well. In this paper, we report that transcription of AKT1 is indeed regulated by ß-catenin/Tcf–Lef in CRC cell lines and overexpression of the AKT1 kinase co-localizes with high levels of cytoplasmic and/or nuclear ß-catenin in sporadic colorectal tumor samples.


    MATERIALS AND METHODS
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Cell lines and tumor samples
Human CRC cell lines (SW480, SW948, HCT116, LS174T, HT-29, Colo320, CX-2 and LoVo) and the human embryonic kidney cell line 293 were provided by the tumor tissue core facility of the German Cancer Research Center (Tumorbank; DKFZ, Heidelberg, Germany). Cells were grown in RPMI 1640 (Life Technologies) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO2. Thirty-nine formalin-fixed, paraffin-embedded sporadic colorectal carcinoma samples were drawn from the archive of the Institute of Pathology, University Hospital Heidelberg.

Aspirin treatment of cell lines
Aspirin (acetylsalicylic acid) was purchased from Sigma, Germany. A 1 M stock solution was prepared in acetone and diluted in RPMI to a final concentration of 5 mM. Cells were starved in serum-free medium for 24 h for cell-cycle synchronization before addition of Tris-buffered 5 mM aspirin (pH 7–7.5) for 24 h as described previously (11). An equivalent volume of acetone was given to the cells that were used as controls.

RNA-extraction and real time RT–PCR
RNA was extracted from CRC cell lines using RNeasy Kit (Qiagen, Hilden, Germany). For cDNA synthesis, 2 µg of total RNA was included in a 20 µl reaction volume using hexamer primers and SuperScriptII reverse transcriptase (Invitrogen, Karlsruhe, Germany) following the manufacturer's instructions. For analysis of AKT1 transcripts by real-time PCR, 1 µl of 1:20 diluted cDNA was amplified by using the TaqMan Universal PCR Master Mix with the following primer pairs: Akt1Tq1: 5'-CATCACACCACCTGACCAAG-3' and Akt1Tq2: 5'-CGTGCCGAGTAGGAGAACTG-3'. Analysis was performed by an ABI PRISM® 7700 Sequence Detection System. Relative expression levels were determined by comparing {Delta}Ct-values of AKT1 amplificates in comparison with actinB amplificates of corresponding cDNA samples (Primer: ACTB76f: 5'-CCTAAAAGCCACCCCACTTCTC-3' and ACTB76r: 5'-ATGCTATCACCTCCCCTGTGTG-3') as described previously (12).

Expression plasmids
Expression plasmids encoding mutant ß-catenin ({Delta}45Cat), hTcf-4, dnTcf-4 and wild-type APC were kindly provided by B.Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA), pCS2+/Lef{Delta}N-VP16 was a kind gift from Andreas Hecht (University of Freiburg, Germany). The pRSV-lacZ vector was constructed at the DKFZ, Heidelberg, Germany.

Construction of AKT1 promoter/enhancer-luciferase reporter plasmids
For amplification of the AKT1 promoter/enhancer, genomic DNA was extracted from lymphocytes of a healthy individual and used as a template in 50 µl PCR (denaturation for 5 min at 95°C, followed by 40 cycles of 1 min at 95°C, 30 s at 60°C and 2 min 30 s at 72°C, and a final extension at 72°C). Two regions spanning 568 or 1401 bp, upstream of the translational start site were amplified by the following primers, Pro-568 for: 5'-CTTTCTGGGGATGCCTGT-3' or Pro-1401 for: 5'-CCGGGAGCTGTGTAGACT-3' and Pro-AKT1 rev: 5'-CTCTCCCTGTCCATGGTG-3'. The amplified fragments were introduced into pCR2.1 vector (‘TA cloning kit’ Invitrogen, Karlsruhe, Germany) and confirmed by sequencing. For construction of AKT1 promoter/enhancer-luciferase reporter plasmids, the corresponding regions (pro-568 and pro-1401) were excised form pCR2.1 and introduced into appropriate restriction sites of pGL3-Basic (Promega GmbH, Mannheim, Germany).

Site directed mutagenesis
Mutation of TBE consensus sites (deletion of CTTTG; ‘{Delta}’) in the AKT1 promoter/enhancer reporter constructs was performed using ‘Gene Editor in vitro Site-Directed Mutagenesis System’ (Promega, Mannheim, Germany) according to the manufacturer's protocols with the following oligonucleotides (custom synthesized by Thermo Electron Corporation):

TBEdel1: 5'-GTCTTCCTCATGTTTGAATTT {Delta} CTTTCCTAGTCTGGGGAGCAGG-3';
TBEdel2: 5'-GACGGACTTGTCTGAACCTCT {Delta} TCTCCAGCGCCCAGCACTGGGC-3';
TBEdel5: 5'-AAGTACTTGGGGCATTTCCCT {Delta} GGTAAGGTGTGGTCTTCCCTG-3';
TBEdel6: 5'-GGGTGGGGGGACATCCAGAGGT {Delta} AGTCCAGCCCTCTGCCTCCAG-3'.
All constructs were confirmed by sequencing.

Transfection and reporter gene assays
Transient transfections were performed using SuperFect transfection reagent (Qiagen, Hilden, Germany) according to the manufacturer's instructions. In brief, 5 x 105 to 1 x 106 cells/well were grown in 6-well plates for 24 h and 1–4 µg of either pGL3-B-568- or pGL3-B-1401-luciferase reporter constructs was added to each well together with 1 µg of pRSV-lacZ for normalization. For coexpression of additional proteins, 1 µg of pCMV{Delta}45Cat, pCMV-APC, phTcf-4, pdnTcf-4 or p{Delta}Lef-VP16 was added as indicated in the figures. Empty vector (pBluescript II SK+, Stratagene, Heidelberg, Germany) was included to give a final volume of 6 µg. Cells were harvested after 24 h and lysates were prepared in Tris-PO4-luciferase buffer. Assays of luciferase and galactosidase activity were performed as described previously (13).

Immunoblotting
The monoclonal antibody directed against ß-catenin was purchased from Transduction Laboratories (BD Biosciences, Heidelberg, Germany), monoclonal anti-AKT1 (clone 2H10) and anti-Phospho-Akt (Ser473) were derived from Cell Signaling Technology (New England Biolabs, Frankfurt, Germany) and anti-actin (clone C4) was provided by ICN Biomedicals (GmbH, Eschwege, Germany). For immunoblotting of aspirin-treated cells complete cell lysates were prepared by adjusting 1 x 105 cells/µl in SDS-sample buffer (0.25 M Tris–HCl (pH 6.8); 20% glycerin; 4% SDS and 10% beta-mercaptoethanol). For immunoblotting of transfected cells equal amounts of each cell lysate in Tris-PO4-luciferase buffer derived from the transfection experiments were separated on a 10% SDS–polyacrylamide gel and blotted on nitrocellulose by semi-dry electroblotting. Equal loading was verified by staining with Ponceau S solution, before the blots were incubated with the indicated primary antibodies diluted in blocking buffer (Tris-buffered saline, 0.1% Tween-20 and 5% non-fat dry milk). Incubation with anti-AKT1 or anti-phospho-Akt (Ser473) was performed overnight at 4°C (dilution of 1:1000). Detection with anti-ß-catenin (dilution of 1:1000) and anti-actin (dilution of 1:2000) was done for 1 h at room temperature. After washing, rabbit-anti-mouse IgG peroxidase (Dako GmbH, Hamburg, Germany) was used as secondary conjugated antibody for visualization by enhanced chemiluminescence with ECL detection reagents (Amersham Biosciences, Freiburg, Germany) or SuperSignal West Femto-Substrate (Pierce, Rockford, IL), for detection of phosphorylation signals.

Immunohistochemistry
Serial formalin-fixed tissue sections of 39 sporadic colorectal carcinomas were stained using the Vectastain elite ABC detection system (Vector, Burlingame, CA, USA) according to the manufacturer's instructions. In brief, 2 µm sections were deparaffinized with xylene and passaged through decreasing concentrations of ethanol. Subsequently, antigen retrieval was performed by heating the slides in 10 mM citric acid in a microwave oven (two times 5 min, 560 W) at pH 6.0. Tissue sections were incubated with first antibody (anti-AKT1, clone 2H10; diluted 1:100 in PBS or anti-ß-catenin antibody diluted 1:200 in PBS) at 4°C overnight. After washing and incubation with secondary antibody (anti-mouse/anti-rabbit; Vector, Burlingame, CA, USA) for 30 min, staining reaction was done using DakoCytomation AEC Chromogen (Dako GmbH, Hamburg, Germany) as substrate.


    RESULTS
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Expression of AKT is reduced by aspirin
We have previously shown that transcription of Wnt/ß-catenin target genes is inhibited by aspirin and indomethacin in CRC cells (10,11). In the course of analyzing the molecular mechanisms of these non-steroidal anti-inflammatory drugs (NSAIDs) we observed that the protein level of AKT was similarly downregulated by aspirin in the absence of growth factors (Figure 1A). To investigate, whether this downregulation occurs at the transcriptional level, we examined expression of the three AKT isoforms, AKT1, 2 and 3 in response to aspirin by real-time PCR. The expression of AKT1 transcripts was significantly reduced in five of eight CRC cell lines on incubation with 5 mM aspirin for 24 h, whereas the level remained almost unchanged in 293 cells (Figure 1B). Expression of AKT2 and AKT3 was unaffected or clearly increased in response to aspirin in all cell lines tested (data not shown). Interestingly, the time course of AKT1 downregulation in colon cancer cells was the same as for Wnt/ß-catenin target genes (10; Figure 1A, and data not shown). Accordingly, we hypothesized that AKT1 expression might be regulated by Wnt/ß-catenin-signaling in colon cancer cells in a similar way.



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Fig. 1. Expression of AKT is reduced in response to aspirin treatment. (A) Equal cell numbers were treated with Tris-buffered 5 mM aspirin for the indicated times and lysed in Laemmli sample buffer. The lysates were separated by SDS–PAGE and probed by western blots with anti-AKT1 or anti-actin antibodies. (B) Relative AKT1 transcript levels in CRC cell lines following 24 h treatment with 5 mM aspirin versus untreated controls. Relative expression levels were determined by comparing {Delta}Ct-values of AKT1 amplificates with actinB amplificates of corresponding cDNA samples. Error bars indicate SD of two independent experiments from each cell line performed at least in duplicate.

 
Sequence analysis of the regions 5' to the initiating ATG of AKT1, 2 and 3 for TBE sites
To specify the functional interaction between Wnt/ß-catenin signaling and AKT1 gene expression, we scanned the AKT genes for regulating DNA-binding elements ~2.0 kb upstream to the initiating ATG. A search of the GenBank nucleotide database revealed the presence of nine putative ß-catenin/Tcf/Lef-binding sites (TBE) in the AKT1 gene [Accession no. NT_026437.11, (gi:29736559)] (Figure 2A), showing a high degree of homology to the core consensus sequence AGATCAAAGGG (14). Four of these TBEs are located upstream of the transcriptional start, whereas five TBEs are situated in Exon 1. In addition, one possible AP-1 binding site (ACTCAGT or TGAGTCA) and two potential nuclear factor kappa B (NF{kappa}B) binding elements were detected in the AKT1 promoter region (data not shown). Six potential TBEs were found in the promoter region of the AKT2 gene [Accession no. NT_011109, (gi:29800594)] and seven complete or partial TBEs were identified within 1340 nucleotides of the promoter region of the AKT3 gene [Accession no. NT_004836, (gi:51459207)]. Thus, although the probability of uncovering a TBE consensus sequence by chance is 1 in 2048 nt, these findings strongly suggest that the AKT genes might be targets of ß-catenin/Tcf signaling. Since we had initially identified AKT1 but not AKT2 and AKT3 expression to be affected by aspirin treatment we focused on regulation of AKT1 expression for further analysis. Whether AKT2 and AKT3 are targets of Wnt/ß-catenin signaling all the same will be subject to continuative studies.



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Fig. 2. (A) Location of Tcf-binding elements (TBEs) 2.0 kb upstream of the initiating ATG of the AKT1 gene [GeneBank accession no. NT_026437.11 (gi:29736559)]. The numbers indicate the position of each TBE starting x nucleotides upstream of the ATG initiation start site. for., forward orientation on reverse complement strand; rev., reverse orientation on reverse complement strand. (B) Schematic representation of the AKT1 promoter/enhancer reporter constructs. Open squares, TBE: potential Tcf/Lef-binding elements. (C) The AKT1 promoter/enhancer is endogenously activated in CRC cell lines. SW480, SW948 and HCT116 and 293 cell lines were transfected with the indicated amounts of two different reporter constructs. Shown are x-fold activations of the basal activity triggered by transfection of 1 µg of the empty vector pGL3Basic. Closed circles, activation of pGL3Basic; open circles, activation of pGL3-568; closed triangles, activation of pGL3-1401. Bars indicate ± SD of two independent experiments.

 
The AKT1 promoter/enhancer is activated in CRC cells by ß-catenin/Tcf–Lef
Increased Wnt/ß-catenin signaling activity is a common feature of CRC cell lines. To examine whether the AKT1 promoter/enhancer is activated in CRC cell lines, we cloned two fragments of various lengths from a region spanning nucleotides –589 to –1991 5' to the translational start codon of the AKT1 gene upstream of the luciferase gene (Figure 2B). The resulting AKT1 promoter/enhancer constructs (pGL3B-568 and pGL3B-1401) were used for transfection of SW480 and SW948 cells, where Wnt/ß-catenin signaling is constitutively active due to the absence of functional APC. In addition, the constructs were analyzed in HCT116, harboring an oncogenic mutation in ß-catenin (15). In each of the three CRC cell lines, a dose-dependent induction of luciferase activity from pGL3B-1401, containing six of the putative TBE sites was observed (Figure 2C). pGL3B-568, lacking the TBE sites upstream of the transcriptional start site, was also activated in SW480 cells, albeit to a lesser extent than pGL3B-1401. In contrast, in 293 cells where endogenous Wnt/ß-catenin signaling is very low, activation of both constructs hardly exceeded background levels of the empty vector pGL3Basic. Thus, the AKT1 promoter/enhancer appears to be constitutively activated, particularly, in CRC cells.

Next we investigated the reporter constructs for responsiveness to ß-catenin and Tcf-4. According to the constitutively high endogenous ß-catenin levels, single coexpression of a degradation resistant ß-catenin mutant ({Delta}45-ß-catenin) had a weak but reproducible stimulatory effect on both pGL3B-568 and pGL3B-1401 reporter constructs in all CRC cell lines tested (Figure 3C and data not shown). Unexpectedly, overexpression of {Delta}45-ß-catenin was ineffective in 293 cells (Figure 3A), suggesting that it is insufficient for activating the AKT1 promoter, alone. We, therefore, tried to inhibit the endogenous AKT1 promoter activity in SW480 cells through a dominant negative Tcf-4-mutant [dnTcf-4, (16)]. dnTcf-4 lacks the ß-catenin binding domain, thereby binding to TBEs without activating transcription. As anticipated, ectopic expression of dnTcf-4 significantly suppressed activation of pGL3B-568 and pGL3B-1401 reporters in a dose-dependent manner to 55 and 45%, respectively, of the basal activity, in contrast to the empty vector, which was not significantly affected (Figure 3B).



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Fig. 3. (A) 293 embryonic kidney cells were cotransfected with the indicated reporter constructs and expression constructs for {Delta}45ß-catenin. Shown are x-fold activations related to the endogenous activation by the empty vector pGL3Basic. Bars indicate SE of five independent experiments. (B) Effects of a dominant negative mutant of Tcf-4 on the activity of the AKT1 promoter/enhancer. SW480 CRC cells were cotransfected with the indicated reporter constructs and expression constructs for dnTcf-4. Inhibition is shown as the percentage of endogenous activity of the 5'-UTR-constructs. (C and D) Effects of wild-type Tcf-4, wild-type APC or a dominant positive Lef-1-VP16 transcription factor on the activity of the AKT1 promoter/enhancer in SW948 CRC cells. Shown is x-fold activation/inhibition in response to coexpression of the indicated proteins related to the endogenous activity of the empty vector pGL3Basic. (E and F) Lysates derived from the cells in (C) and (D) and from cells transfected in parallel were subjected to immunoblotting with anti-AKT1, anti-P473-Akt, anti-ß-catenin or anti-actin antibodies. Note that both the relative levels of AKT1 and phospho-AKT1 correlate with stimulation or inhibition of its AKT1 promoter/enhancer. (B) and (C): bars indicate SE of two independent experiments, performed in duplicate. (D): bars indicate SD of two independent experiments.

 
Since SW948 cells harbor relatively low levels of endogenous ß-catenin and proved to be a sensitive system to analyze modulations of the Wnt/ß-catenin pathway in previous investigations (9,11), we chose this cell line to study additional inhibiting and activating effects. Reconstitution of wild-type APC in SW948 cells resulted in a strong reduction of luciferase activity from both AKT1 promoter/enhancer constructs whereas single coexpression of wild-type Tcf-4 had no effect (Figure 3C). In contrast, co-expression of a dominant-positive Lef1{Delta}N-VP16 fusion protein lacking the ß-catenin binding domain and therefore constitutively activating transcription from Tcf/Lef-binding elements (17), strongly enhanced the activation of both AKT1 promoter/enhancer constructs (Figure 3D). Finally, expression of endogenous AKT1 corresponded well with the results obtained from the reporter assays. Immunoblots using extracts of the transfected cells confirmed that the amount of AKT1 increased on the ectopic expression of either {Delta}45-ß-catenin (Figure 3E) or Lef1{Delta}N-VP16 (Figure 3F) fusion protein, although it was significantly reduced in response to coexpression of APC (Figure 3E). The changes in protein levels were also reflected by the amount of phosphorylated AKT1, representing activated AKT1 kinase (Figure 3E, F). Taken together, these results strongly support the hypothesis that ß-catenin/Tcf/Lef protein complexes contribute to the regulation of AKT1 expression.

Specificity of the TBE sites in the AKT1 promoter/enhancer for regulation by ß-catenin/Tcf-4
Since the TBE motifs of the AKT1 promoter/enhancer are embedded in a long DNA region which might contain several other responsive elements, we next tested the specificity of the TBE sites for transactivation by ß-catenin/Tcf–Lef. To enhance the endogenous Wnt/ß-catenin signaling activity in SW948 cells we transiently cotransfected the cells together with {Delta}45-ß-catenin and Tcf-4 expression plasmids. Although we had observed only a weak effect by the ectopic expression of {Delta}45-ß-catenin alone (Figure 3C), coexpression of Tcf-4 together with {Delta}45-ß-catenin resulted in an almost 5-fold induction of the wild-type AKT1 promoter/enhancer (Figure 4B). Next, the four different TBEs alone or in combination were mutated and transiently transfected into SW948 cells (Figure 4A). In comparison to the wild-type construct, a loss of 30 to >70% of endogenous transactivation activity was observed with mutant AKT1 promoter/enhancer constructs (Figure 4B, halftone bars). Thus, the putative TBE sites exhibit promoter activation function in CRC cells. Interestingly, the mutated constructs were not completely resistant to transactivation by ß-catenin/Tcf-4 (Figure 4B, black bars). Removal of TBE1, 2, 5 and 6 in combination was most effective by reducing the activation of the AKT1 promoter/enhancer through ß-catenin/Tcf-4 to 40% of that obtained with the wild-type promoter/enhancer (1.9-fold versus 4.8-fold).



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Fig. 4. (A) Schematic representation of the mutated AKT1 promoter/enhancer reporter constructs. Open squares, TBE: potential Tcf/Lef-binding elements; cross, mutated TBE (5 nt were deleted by site directed mutagenesis). (B) Mutations in the Tcf/Lef binding sites reduce the activation of the AKT1 promoter/enhancer. SW948 CRC cells were cotransfected with the indicated reporter constructs and expression constructs for {Delta}45ß-catenin and Tcf-4. Bars indicate SD of two independent experiments.

 
AKT1 is upregulated in ß-catenin-activated human colorectal carcinomas
Overexpression of AKT protein was recently reported to be an early event during sporadic colon carcinogenesis (6). The precise nature of the AKT isoform and the reason for its upregulation however, was not clear. Coordinated expression of Wnt/ß-catenin target genes with increased cytoplasmic levels and/or with nuclear localization of ß-catenin has been shown in several studies (1823). We, therefore, analyzed serial sections from the archived blocks of 39 sporadic colorectal carcinomas for coexpression of ß-catenin and AKT1 by immunohistochemistry. In six of these tumor samples ß-catenin was exclusively located at the plasma membrane of tumor cells and normal cells. Nuclear and/or a strong cytoplasmic accumulation of ß-catenin was detected in the tumor cells of 33 samples (representative sample shown in Figure 5A and C). Of these 33 samples, 23 also displayed an intense AKT1 staining of the tumor cells. The level of AKT protein expression was associated with ß-catenin activation (Figure 5). Prominent overexpression of AKT1 was particularly observed in tumor areas where ß-catenin was strongly expressed in the cytoplasm or located in the nucleus of a high proportion of cells (representative sample in Figure 5A–D). In contrast, only a faint AKT staining was detected in tumors displaying exclusively membranous staining of ß-catenin (representative sample in Figure 5E and F).



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Fig. 5. Immunohistochemistry for ß-catenin (A, C and E) and AKT1 (B, D and F) expression using serial sections of human colorectal adenocarcinomas. A strong expression of AKT1 is visible in areas where ß-catenin is strongly expressed in the cytoplasm and/or nuclei of tumor cells (C and D), whereas a weak AKT1 expression correlates with membranous staining of ß-catenin (E and F). Specific staining is red, nuclear counterstaining is blue, arrows indicate nuclear staining of ß-catenin. Original magnification was 50x (A and B), 200x (C–F).

 

    DISCUSSION
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Enhanced AKT kinase activity and subsequent downstream signal transduction is now accepted to play an important role in human malignancy. The mechanism for the regulation of AKT gene expression and its contribution to carcinogenesis, however, is largely unknown. Here we provide evidence for an interaction between Wnt/ß-catenin signaling and AKT gene expression in CRC. Our initial experiments on the mechanism of aspirin action revealed that, beside other effects on the protein level, aspirin downregulates AKT1 transcript expression in a subset of CRC cells. The reason why aspirin treatment differs between cell lines is still incompletely understood (24) and will be the subject of further studies. However, it is interesting to note that downregulation of AKT1 transcripts was confined to cell lines showing an increased Wnt/ß-catenin activity owing to the absence of wild-type APC (HT-29, Colo320, LoVo, SW480 and SW948) (15). Since we recently reported attenuation of Wnt/ß-catenin signaling to be one of the molecular mechanisms described for aspirin action (25), we assumed that AKT1 might be a transcriptional target of Wnt/ß-catenin signaling. Accordingly, we detected nine putative binding elements for Tcf/Lef transcription factors in the promoter/enhancer of the human AKT1 gene. Our studies in cultured cells show that the AKT1 promoter/enhancer is activated by ß-catenin or by the addition of a constitutive active Lef1{Delta}N-VP16 transcription factor, whereas it is suppressed on the reconstitution of wild-type APC or by the expression of dominant negative dnTcf-4. Furthermore, AKT1 overexpression co-localized with overexpression/nuclear localization of ß-catenin. We conclude from our data that human AKT1 is a direct target for ß-catenin/Tcf-4-mediated transactivation, although there might be additional regulating elements contributing to the regulation of AKT1 expression. Mutation of four of the TBEs reduced the endogenous activity and stimulation by TCF-4/ß-catenin, indicating that these sequences are critical for the responsiveness of the AKT1 promoter. The mutated constructs, however, were not completely resistant. This may be attributable to the remaining wild-type TCF-binding sites, TBE3 and TBE4 or to additional cryptic TCF-binding sites, which were not detected. Alternatively, ß-catenin could affect other promoter elements by an indirect mechanism. For instance, since ß-catenin was also described to stimulate the expression of c-jun and fra-1 transcription factors (26), this could result in the activation of the AP-1 binding site observed within the AKT1 promoter/enhancer. Such a synergistic stimulation of transcription by combinations of ß-catenin with c-jun and c-fos (which combine with AP-1) was already shown for other ß-catenin target genes, i.e. uPA, MMP-7, laminin-5{gamma}2 and osteopontin (18,22,27,28). Furthermore, PML and p300/CBP are involved in ß-catenin dependent transactivation of ARF and Siamois target genes (29). Whether similar cooperation of different transcription factors modulates AKT1 expression will require further study.

Cumulative evidence suggests that the extent of ß-catenin/Tcf-Lef responsiveness of promoters in target genes is determined not only by the sequence but also by the position of the individual TBEs. For example, the human matrix metalloproteinase-7 (MMP-7) promoter is transcriptionally activated by ß-catenin through TBEs located upstream of the transcriptional start site, whereas the mouse Mmp-7 promoter is repressed by ß-catenin through a TBE located downstream of its transcriptional start site (27,30,31). Accordingly, the finding that the AKT1 promoter/enhancer construct used in our study (pGL3B-1401) contains four TBEs upstream and two TBEs downstream of the transcriptional start site could be of functional relevance, for instance, for the interaction with other transcription factors. In this context, it is important to note that the AKT1 promoter/enhancer constructs displayed a different activation/repression response in 293 cells—different from the results obtained in CRC cells: the AKT1 promoter/enhancer construct containing TBE1–6 (pGL3B-1401) was resistant to endogenous transcription factors and to ectopic expression of ß-catenin, Lef1{Delta}N-VP16 and Tcf-4 (Figure 2C and Figure 3A and data not shown). In contrast, pGL3B-568, containing only the TBEs downstream of the transcriptional start site was endogenously activated 2-fold in 293 cells but repressed by ectopic expression of Tcf-4 (Figure 2C and data not shown). Thus, ß-catenin and Tcf-Lef transcription factors seem to differentially regulate target genes not only depending on the sequence and position of the TBEs, but also depending on the cellular context.

In our study, overexpression of the AKT protein kinase coincided with a strong cytoplasmic/nuclear staining for ß-catenin. However, there was no complete overlap, since part of the tumor samples displayed a weak AKT1 expression although ß-catenin was strongly overexpressed in these tumors. Given that the AKT1 promoter/enhancer contains additional binding elements, such as AP-1, NF{kappa}B and SP-1 binding sites, it is likely that AKT1 transcription may be further modulated by additional elements in the promoter via distinct complexes. This is in line with the expression pattern of AKT1 and its isoforms described in different tissues. In normal cells, basic levels of AKT1 and AKT2 are ubiquitously expressed (32). In addition, a recent study describes a differentiation state, distinct regulation of AKT isoforms in intestinal epithelial cells showing that the relative levels of AKT1 and AKT2 mRNA decrease during enterocyte differentiation (4). In contrast, in cancer cells constitutive overexpression of AKT1 and 2 seems to be a common event. Although AKT2 gene has been found to be amplified in ovarian, pancreatic and breast cancer (33,34), we describe here an additional mechanism resulting in the overexpression of AKT1 in CRC cells. It is well acknowledged that the expression of a kinase is not necessarily a reflection of its activity level. In our studies, the level of AKT1 phosphorylated at residue 473 runs in parallel with the total level of AKT1 in SW948 cells. This indicates that upregulation or downregulation of AKT1 on the transcriptional level may result in changes of AKT1 activity and consequently affect downstream effects. However, AKT1 kinase activity is further influenced by the presence and activity of growth factors, upstream kinases and antagonists, such as PTEN (3). Therefore, it is not surprising that we detected a strong immunohistochemical staining of both, total AKT1 and activated AKT1 (phosphorylated at residue 473) in some CRC samples, whereas in others high AKT1 levels did not correspond to the phosphorylation state (data not shown). At the same time, independent from regulation on the protein level, overexpression of AKT is likely to contribute to carcinogenesis by establishing the basis for enhanced AKT signaling activity.

Our finding that AKT1 expression is increased by ß-catenin/Tcf–Lef, may help to explain a link between aberrant Wnt/ß-catenin signaling and resistance to apoptosis, in the earliest stages of intestinal crypt cell dysregulation. Most sporadic colon cancers reduce apoptosis in consequence of truncation of the APC protein (35), but the mechanism downstream of APC inactivation through which apoptosis is dysregulated was not clear so far. In neurons and other tissues, AKT has been described to be an important mediator of cell survival and apoptosis by inhibiting pro-apoptotic downstream targets, such as BAD, Forkhead family members and caspase 9 (3,36). In addition, AKT1 was recently identified to be a major promoter of the suppression of apoptosis in human intestinal epithelial cells (4). Accordingly, increased AKT1 levels resulting from constitutively activated ß-catenin/Tcf–Lef complexes in intestinal cells showing high levels of nuclear ß-catenin might disrupt the balance between differentiation and apoptosis. Data from Fukumoto et al. (37) reported that AKT plays a role in Wnt-signaling by recruiting AKT into a complex with axin, glycogen synthase kinase-3b (GSK-3b) and dishevelled (Dsh) thereby increasing free ß-catenin levels. Given that these increased ß-catenin levels induce AKT transcription, this would result in a positive feedback loop of activated Wnt-signaling components and inhibition of apoptosis. Although we did not investigate cell viability in our experiments, a set of additional findings are consistent with the model of an interaction between the AKT-signaling and Wnt-signaling pathways. A recent immunohistochemical study described the detection of AKT upregulation to be an early event in sporadic colon carcinogenesis, since it was already obvious in 57% of premalignant adenomas. Furthermore, 57% of sporadic CRCs but only 22% of tumors that evolve via different genetic pathways such as HNPCC-associated carcinomas displayed increased AKT levels (6). Thus, the AKT-level runs parallel to the level of Wnt-signaling activity in colorectal tumors. Finally, according to our data, AKT1 expression decreased on reconstitution of wild-type APC in APC-deficient CRC cells. In summary, our data add AKT1 to the list of ß-catenin/Tcf–Lef target genes and propose a model by which aberrant Wnt/ß-catenin signaling is involved in early inhibition of apoptosis during colon carcinogenesis.


    Acknowledgments
 
We thank B.Vogelstein (Johns Hopkins Oncology Center, Baltimore, MD, USA) and A.Hecht (University of Freiburg, Germany) for kindly providing us with several cDNA expression plasmids. We also thank H.Youmans for technical assistance and C.Gurrola Diaz for help with generation of the AKT1 promoter/enhancer–reporter constructs. Part of this work was presented at the 95th Annual Meeting of the American Association for Cancer Research and was supported by an AFLAC-AACR Scholar-In-Training Award to S.D.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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Received January 13, 2005; revised April 7, 2005; accepted May 3, 2005.