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Short Communications |
Kinase Is Frequently Elevated in Human Cancers and Its Constitutive Activation Is Required for Oncogenic Transformation in NIH3T3 Cells
From the Department of Pathology and Laboratory Medicine and the
Molecular Oncology Program,*
University of South Florida
College of Medicine and H. Lee Moffitt Cancer Center, Tampa, Florida;
the Cancer Research Department,
Berlex
Biosciences, Richmond, California; and the Kimmel Cancer
Center,
Thomas Jefferson University,
Philadelphia, Pennsylvania
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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signaling has been extensively studied throughout the last 6 years. It
has been shown that Akt is activated by a variety of stimuli in a
phosphoinositide-3-OH kinase (PI 3-kinase)-dependent
manner.6-9
Activation of Akt by growth factors depends on
the integrity of the PH domain, which binds to the PI 3-kinase product,
PI(3,4,5)P3, and phosphorylation of Thr-308 and
Ser-473 by PDK1 and PDK2/ILK, respectively. In addition, growth
factor-induced Akt activation is also mediated by Ras, Src, and
Gab1.10-12
In numerous cell types, it has been shown that
Akt induces survival and suppresses apoptosis induced by a variety of
stimuli, including growth factor withdrawal and loss of cell adhesion.
The mechanisms by which Akt promotes cell survival include
phosphorylation of the pro-apoptotic proteins BAD, caspase-9, Forkhead
transcription factors, and I
B kinase , resulting
in reduced binding of BAD to Bcl-XL, inhibition
of caspase-9 protease activity, Fas ligand gene transcription, and
activation of the nuclear factor-B cascade.13-17
Akt
has also been shown to inhibit the Raf-MEK-ERK pathway through
phosphorylation of Raf-1 in myotubes and overcome constitutively
activated MAPK-induced cell-cycle arrest in MCF7
cells.18,19 Although Akt/AKT1 is essential for cell survival and anti-apoptosis, alterations of Akt/AKT1 have not been consistently observed in any human malignancy. In fact, amplification of AKT1 has been reported in only a single gastric carcinoma.20 In this communication, we describe frequent activation of AKT1 in human carcinomas of prostate, breast, and ovary. We also demonstrate the biological significance of AKT1 activation in human cancer by showing that constitutively activated, but not wild-type, Akt is highly oncogenic in NIH3T3 cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All primary human cancer specimens were obtained from patients who underwent surgery at the H. Lee Moffitt Cancer Center and each sample contained at least 70% tumor cells as was confirmed by histological examination. The tissues were snap-frozen and stored at -70°C. Slides from each case were reviewed for grade and stage following the criteria of the American Joint Committee on Cancer, 1988 edition. NIH3T3 cells were cultured at 37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum. Transfection was performed with LipofectAMINE PLUS (Life Technologies, Inc., Rockville, MD). Stable clonal cell lines were established after G418 selection. Soft agar suspension and tumorigenesis assays were performed as previously described.21
Plasmids
Hemagglutin epitope (HA)-tagged wild-type, constitutively active (Myr-Akt and Akt-E40K), and dominant-negative (kinase-inactive mutant Myr-Akt-K179M) Akt were described previously.22
Immunoprecipitation and Western Blotting Analysis
The frozen tissue was lysed by a tissue tearor in a lysis buffer containing 20 mmol/L Tris-HCl (pH 7.5), 137 mmol/L NaCl, 15% (v/v) glycerol, 1% Nonidet P-40, 2 mmol/L phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin and leupeptin, 2 mmol/L benzamidine, 20 mmol/L NaF, 10 mmol/L NaPPi, 1 mmol/L sodium vanadate, and 25 mmol/L ß-glycerophosphate. An equal amount of protein was analyzed for protein expression and enzyme activity. For immunoprecipitation, lysates were precleared with protein A-protein G (2:1) agarose beads at 4°C for 20 minutes. After removal of the beads by centrifugation, lysates were incubated with the indicated antibody in the presence of protein A-protein G (2:1) agarose beads for 2 hours at 4°C. The beads were washed once with 50 mmol/L Tris-HCl (pH 7.5), 0.5 mol/L LiCl, 0.5% Triton X-10, twice with phosphate-buffered saline (PBS), and once with 10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl2, 10 mmol/L MnCl2, and 1 mmol/L dithiothreitol, all containing 20 mmol/L ß-glycerophosphate and 0.1 mmol/L sodium vanadate. Immunoprecipitates were subjected to in vitro kinase assays or Western blotting analysis. Protein expression was determined by probing Western blots of immunoprecipitates with phospho-Ser473 Akt (New England Biolabs, Beverly, MA) or anti-AKT1 (Santa Cruz Biotechnology, Santa Cruz, CA) antibody. Detection of antigen-bound antibody was performed with the ECL Western blotting analysis system (Amersham, Arlington Heights, IL).
Immunohistochemistry
Formalin-fixed paraffin-embedded sections were deparaffinized in xylene and rehydrated through graded alcohol to distilled water. The sections were subjected to antigen retrieval by boiling in a microwave for 30 minutes in 0.01 mol/L sodium citrate buffer (pH 6.0) and then exposed to 3% hydrogen peroxide diluted in water for 20 minutes at room temperature to block endogenous peroxidase activity. Sections were incubated in a blocking solution (PBS containing blocking serum) for 20 minutes followed by 20 minutes with an avidin/biotin blocking solution (Vector Laboratories, Burlingame, CA). The primary antibody to phospho-S473 Akt (Upstate Biotechnology, Lake Placid, NY) or PTEN (Upstate Biotechnology) was applied and incubated overnight at 4°C. After incubation, the slides were treated with biotinylated secondary antibody, washed, and treated with streptavidin and biotinylated horseradish peroxidase according to the manufacturer’s instruction (Vector Laboratories, Burlingame, CA). After washing, the signal was visualized by diaminobenzidine tetrahydrochloride. A negative control reaction with no primary antibody was always performed alongside the reaction-containing sample.
In Vitro Protein Kinase Assay
In vitro Akt kinase assays were performed as previously
described.11
Briefly, the reaction was performed in the
presence of 10 µCi of [
-32P]ATP (New
England Nuclear, Boston, MA) and 3 µmol/L of cold ATP in 30 µl of
buffer containing 20 mmol/L Hepes (pH 7.4), 10 mmol/L
MgCl2, 10 mmol/L MnCl2, and
1 mmol/L dithiothreitol. Histone H2B was used as
the exogenous substrate. After incubation at room temperature for 30
minutes, the reactions were stopped by adding protein-loading buffer,
and the products were separated in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis gels. Each experiment was
repeated three times. The relative amounts of incorporated
radioactivity were determined by autoradiography and quantitated with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
PI 3-Kinase Assay
Anti-p85 (Santa Cruz) antibody was used to immunoprecipitate p110
catalytic subunits of PI 3-kinase from the tumor lysate. The
immunoprecipitates were washed once with cold PBS, twice with 0.5 mol/L
LiCl, 0.1 mol/L Tris (pH 7.4), and finally with 10 mmol/L Tris (pH
7.5), 100 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid. The
presence of PI 3-kinase activity in immunoprecipitates was determined
by incubating the beads with reaction buffer containing 10 mmol/L HEPES
(pH 7.4), 10 mmol/L MgCl2, 50 µmol/L ATP, 20
µCi [-32P]ATP, and 10 µg
L--phosphatidylinositol-4,5-bis phosphate
(PI-4,5-P2; Biomol, Plymouth Meeting, PA) or
L--phosphatidylinositol-4-phosphate (Sigma Chemical Co.,
St. Louis, MO) for 20 minutes at 25°C. The reactions were stopped by
adding 100 µl of 1 mol/L HCl. Phospholipids were extracted with 200
µl of CHCl3CH3/MeOH.
Phosphorylated products were separated by thin-layer chromatography as
previously described.23
The conversion of
PI-4,5-P2 to PI-3,4,5-P3
and PI-4-P1 to PI-3,4-P2
was determined by autoradiography and quantitated by using a
PhosphorImager.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Despite the fact that the PI 3-kinase/Akt/AKT1 pathway is
essential for cell survival and anti-apoptosis, consistent alterations
of AKT1 in human primary tumors have not been well documented. We have
previously examined AKT1 and AKT2 alterations at the DNA and/or mRNA
levels in more than 100 cancer cell lines (including NCI 60 cancer cell
lines screen) and more than 300 primary tumors from various organs.
Amplification and/or overexpression of AKT2 were observed in 15 to 25%
of ovarian and pancreatic tumors examined,2,24,25
whereas
no AKT1 alteration at the DNA or mRNA level was detected (J. Q.
Cheng and J.R. Testa, unpublished data). The essential role of AKT1
kinase in cell survival prompted us to examine if AKT1 kinase activity
is elevated in human cancer. We first examined whether anti-AKT1
antibody (D-17, Santa Cruz) specifically recognizes AKT1. HEK293 cells
were transiently transfected with HA-AKT1, HA-AKT2, or HA-AKT3, lysed,
and immunoprecipitated with monoclonal anti-HA antibody. The
immunoprecipitates were separated and detected with AKT1 antibody.
Figure 1A
showed that anti-AKT1 antibody
only reacted with HA-AKT1. We next immunoprecipitated AKT1 from lysates
prepared from frozen tumors of prostate, breast, and ovary with
anti-AKT1 antibody. The AKT1 immunoprecipitates were subjected to
in vitro kinase assays. Significantly increased AKT1 kinase
activity was observed in 16 of 30 prostate adenocarcinomas, 19 of 42
ductal breast cancers, and 11 of 23 ovarian serous adenocarcinomas. No
elevated AKT1 activity was detected in eight lobular breast carcinomas,
three endometrioid, and two borderline ovarian cancers examined,
implying that alteration of AKT1 kinase level primarily involves ductal
breast and serous ovarian carcinomas. Moreover, we observed that the
majority of AKT1 activated-tumors are high grade and stage III/lV
(Table 1)
, suggesting that activation of
AKT1 plays an important role in tumor progression rather than
initiation.
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).
To further demonstrate AKT1 activation and determine whether activated
AKT1 is derived from tumor cells or stromal tissues in the tumor
specimens with elevated AKT1 activity identified by in vitro
kinase assay, immunohistochemical staining of tumor paraffin sections
was performed with phospho-Ser473 Akt antibody. We first demonstrated
that the phospho-Ser473 Akt antibody is capable of recognizing
phosphorylated AKT1 by immunostaining paraffin sections prepared from
serum-starved and serum-starved/EGF-stimulated MCF7 cells (Figure 2
, panels 1 and 2). Phosphorylation
status of AKT1 in these cells was confirmed by Western blot analysis
with phospho-S473 Akt antibody (data not shown). The tumor paraffin
sections from the 16 prostate, 19 breast, and 11 ovarian tumor
specimens with elevated AKT1 kinase activity strongly immunoreacted
with phospho-Ser473 Akt antibody (Figure 2
, panels 3 to 5), whereas no
immunostaining was observed in normal tissues and the tumor samples
without increased AKT1 activity (Figure 2
, panel 6). Interestingly,
phosphorylated AKT1 was located in the tumor cell membrane and
cytoplasm but not the nucleus, which is in conflict with the previously
reported observation that activated Akt could translocate to the
nucleus in ectopically Akt-overexpressing cells.26,27
Our
data also showed that overexpression of GFP-tagged AKT1 translocates to
the nucleus in NIH3T3 cells after IGF-1 stimulation (data not shown),
indicating that activated Akt in the primary tumor cells could have a
different subcellular localization from the cells overexpressing
exogenous Akt.
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Because AKT1 kinase activity is regulated positively by PI
3-kinase and negatively by PTEN/MMAC1,28
we
examined PI 3-kinase activity and PTEN expression in the tumors
exhibiting AKT1 activation (Figure 3)
.
Elevated PI 3-kinase activity was observed in 7 of the 19 breast and 5
of the 11 ovarian carcinomas, but in none of the prostate tumors that
exhibit AKT1 activation. Immunohistochemical staining revealed no PTEN
expression in 10 of 16 prostate and 2 of 11 ovarian cancer specimens
with elevated AKT1 activity, whereas all breast carcinomas that showed
AKT1 activation expressed PTEN. Absence of PTEN is well correlated with
positive staining of phosphorylated AKT1 on the tumor tissue sections
(Figure 3
and data not shown). In addition, we performed single-strand
conformational polymorphism/sequencing analyses in AKT1-activated tumor
specimens that have neither PI 3-kinase nor PTEN alteration. No AKT1
mutation was detected, implying that there are other mechanisms leading
to AKT1 activation in the specimens without alterations of either PI
3-kinase or PTEN, which needs further investigation.
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We and others previously demonstrated that overexpression of
wild-type of Akt (WT-Akt) is unable to transform NIH3T3
cells.21,29
To determine whether activation of AKT1 in
human tumors has biological implication, we have introduced HA-tagged
constitutively active forms of Akt (Myr-Akt and Akt-E40K), wild-type,
and myristoylated kinase-inactive mutant (Myr-Akt-K179M) Akt into
NIH3T3 cells individually. After G418 selection, five stable clonal
cell lines from each transfection were obtained. Western blot and
in vitro kinase analyses revealed that all of the clonal
cell lines express Akt protein (Figure 4A)
. High levels of kinase activity were
detected in constitutively active Akt (Myr-Akt and
Akt-E40K)-transfected clonal cell lines. Figure 4A
shows Akt expression
and kinase activity in two clonal cell lines from each transfection.
Cells transfected with constitutively active forms of Akt, but not
Myr-Akt-K179M and WT-Akt were morphologically transformed, grew in
medium with low serum (0.1%), formed colonies in soft agar suspension,
and were highly tumorigenic in nude mice (Figure 4B)
. Tumors were
observed 1 to 3 weeks after the injection of constitutively activated
Akt-transfected cells in all mice, except clone 2 of Akt-E40K cells
(Figure 4
and Table 2
). Although vector
alone and WT-Akt-transfected NIH3T3 cells developed tumors in 1 of 25
and 2 of 25 mice, respectively, all of these tumors were detected after
38 days (Table 2)
. In addition, high levels of Myr-AKT1 protein and
kinase activity were observed in dissected tumors from Myr-Akt nude
mice but not in the tumors from vector or WT-Akt mice (Figure 4A)
.
These data suggest that kinase activity of Akt/AKT1 is essential for
oncogenic transformation in NIH3T3 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, has been demonstrated in
a number of different tumors.30-32
Constitutively
activated Stat3 and PI 3-kinase are able to induce malignant
transformation.33,34
Recent studies showed that among the
most critical tumor-cell survival pathways are those mediated by the
Akt/AKT1 kinase.6-9
In this report, we demonstrate
frequently elevated AKT1 kinase activity and phosphorylation of AKT1 in
human carcinomas of breast, prostate, and ovary. Moreover, we have also
shown that constitutively activated, but not wild-type, AKT1 is highly
tumorigenic in NIH3T3 cells. Furthermore, the majority of
AKT1-activated tumors are high grade and stage III/lV. These results
indicate that activation of AKT1 is a common occurrence in human
cancer, especially in more advanced tumors. We previously demonstrated that overexpression of wild-type AKT2, but not Akt/AKT1, in NIH3T3 cells resulted in malignant transformation.21 Ahmed and colleagues29 also showed that Akt is not tumorigenic when overexpressed in the nontumorigenic rat T-cell lymphoma cell line 5675. In contrast, v-akt-expressing 5675 cells were highly tumorigenic. Because v-akt arose by an in-frame fusion of the viral Gag and Akt, the oncogenic difference between v-akt and wild-type Akt/AKT1 may be because of myristoylation of the amino-terminus of v-akt.29,35 Several lines of evidence show that attachment of a membrane-targeting sequence (myristoylation/palmitoylation) to the amino-terminus of AKT1/Akt is sufficient to induce its maximal activation and that the PH domain of Akt is required for its membrane translocation and activation.9 We demonstrated, in this study, that overexpression of constitutively active forms of Akt (Myr-Akt and Akt-E40K) in NIH3T3 cell leads to oncogenic transformation, which supports the results obtained from chicken embryo fibroblasts and Rat1 cells.36,37 Taken collectively, these data suggest that the kinase activity of Akt contributes to the control of cell malignant transformation and that elevated AKT1 kinase activity plays an important role in development and/or progression of a subset of human cancers.
Previous studies have demonstrated that all of the tumor-associated PTEN mutants that have been biochemically characterized result in activation of AKT1.28 Recently, elevated PI 3-kinase activity has been observed in human ovarian cancer.32,38 As discussed above, Ras, Src, and Gab1 mediate growth factor signals to activate the PI 3-kinase/Akt pathway.10-12 Therefore, activation of AKT1 in human cancer could result from Ras mutation, overexpression/active mutation of growth factor receptor(s), AKT1 mutation, and Src activation. In the present report, we showed that the majority of cases with AKT1 activation had either PTEN down-regulation or PI 3-kinase activation, dependent on the tumor type. Activation of PI 3-kinase was frequently detected in breast and ovarian, but not prostate, carcinomas, whereas the absence of PTEN protein was observed in some prostate and ovarian carcinomas. However, missense mutations of PTEN, although uncommon, cannot be ruled out by immunohistochemistry, because they result in formation of full-length PTEN proteins that may be immunostained by anti-PTEN antibody. Nevertheless, no AKT1 mutation was found in the tumors examined, indicating that elevated AKT1 activity in human cancer results from alterations of upstream regulators of AKT1.
Amplification and/or overexpression of AKT2, but not AKT1, have been detected in a subset of human ovarian, pancreatic, and breast cancers,2,24,25 suggesting that AKT2 may play a more important role in human malignancy. In the present study, frequent activation of AKT1 kinase in human cancers and malignant transformation resulting from expression of constitutively activated AKT1 provide the first evidence that AKT1 could have a role similar to that of AKT2 in human cancer. Comparison of the AKT and AKT2 protein and/or kinase levels in the same tumor will provide valuable information to better understanding of the importance of AKT1 and AKT2 in human malignancy. Expression of AKT1 and AKT2 protein and alterations of AKT2 at the kinase level in these series of specimens are currently under investigation.
Subcellular localization of activated AKT1 is controversial.9 Early studies on the subcellular localization of Akt/AKT1 revealed that, whereas c-akt is primarily cytosolic, v-akt is distributed equally between the cell membrane, the cytoplasm, and the nucleus in NIH3T3 cells.29 Recent studies showed that nuclear translocation of AKT1 and AKT2 in HEK293 and HeLa cells follows in short succession the insulin-induced translocation of AKT1 and AKT2 proteins to the cell membrane.26,27 However, all of these studies were performed in AKT1- or AKT2-transfected cells. In this report, we demonstrated that activated AKT1 in human primary tumors is distributed only in the plasma membrane and the cytosol, suggesting that activated endogenous AKT1 may not translocate to nucleus. In addition, we noted that immunoreaction to the phospho-Ser-473 antibody in tumor specimens is less strong, which could be because of either weak epitope of single phosphopeptide or epitope masking in paraffin section.
In summary, the data presented in this report showed that AKT1 kinase is frequently activated in human prostate, breast, and ovarian carcinomas. Elevated AKT1 kinase is an essential requirement for its oncogenic activity. These results provide the basis for understanding how the Akt pathway contributes to human oncogenesis. Further studies are required to determine the clinicopathological significance of AKT1 activation and to examine if overexpression of constitutively activated AKT1 develops prostate, breast, and ovarian tumors in transgenic mouse models using tissue-specific promoters.
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Acknowledgements |
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Footnotes |
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Supported by grants from the National Institutes of Health (CA77935) and the Department of Defense (OC990075).
M.S. and G.W. contributed equally to this work.
Accepted for publication May 4, 2001.
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References |
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) which contains the regulatory serine phosphorylation site. Biochem Biophys Res Comm 1999, 257:906-910[Medline]
B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 1999, 401:82-85[Medline]
B is a target of AKT in anti-apoptotic PDGF signalling. Nature 1999, 401:86-90[Medline]
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H. D. Skinner, J. Z. Zheng, J. Fang, F. Agani, and B.-H. Jiang Vascular Endothelial Growth Factor Transcriptional Activation Is Mediated by Hypoxia-inducible Factor 1{alpha}, HDM2, and p70S6K1 in Response to Phosphatidylinositol 3-Kinase/AKT Signaling J. Biol. Chem., October 29, 2004; 279(44): 45643 - 45651. [Abstract] [Full Text] [PDF]
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J. M. Stahl, A. Sharma, M. Cheung, M. Zimmerman, J. Q. Cheng, M. W. Bosenberg, M. Kester, L. Sandirasegarane, and G. P. Robertson Deregulated Akt3 Activity Promotes Development of Malignant Melanoma Cancer Res., October 1, 2004; 64(19): 7002 - 7010. [Abstract] [Full Text] [PDF]
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A. R. Uzgare and J. T. Isaacs Enhanced Redundancy in Akt and Mitogen-activated Protein Kinase-induced Survival of Malignant versus Normal Prostate Epithelial Cells Cancer Res., September 1, 2004; 64(17): 6190 - 6199. [Abstract] [Full Text] [PDF]
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