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(The FASEB Journal. 2001;15:1953-1962.)
© 2001 FASEB

Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production

DOHOON KIM*,{dagger}, SUNHONG KIM*,{dagger}, HYONGJONG KOH*,{dagger}, SANG-OH YOON{dagger}, AN-SIK CHUNG{dagger}, KYOUNG SANG CHO*,{dagger} and JONGKYEONG CHUNG*,{dagger}

* National Creative Research Initiative Center for Cell Growth Regulation and
{dagger} Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305–701, Korea

1Correspondence: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373–1 Kusong-Dong, Yusong, Taejon 305–701, Korea. E-mail: jchung{at}mail.kaist.ac.kr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Akt/protein kinase B (PKB) serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. Here, we demonstrate for the first time a major role of Akt/PKB in the cell invasion properties of the highly metastatic cell line HT1080. Using confocal microscopic analyses of live samples, we found Akt/PKB to be localized in the leading edge membrane area of migrating HT1080 cells. This localization was dependent on phosphoinositide 3-kinase and required the lipid binding ability of the phosphoinositide binding pleckstrin homology domain of Akt/PKB. We examined the possible function of Akt/PKB in HT1080 invasion. Surprisingly, Akt/PKB potently promoted HT1080 invasion, by increasing cell motility and matrix metalloproteinase-9 (MMP-9) production, in a manner highly dependent on its kinase activity and membrane-translocating ability. The increase in MMP-9 production was mediated by activation of nuclear factor-{kappa}B transcriptional activity by Akt/PKB. However, Akt/PKB did not affect the cell-cell or cell-matrix adhesion properties of HT1080. Our findings thus establish Akt/PKB as a major factor in the invasive abilities of cancer cells.—Kim, D., Kim, S., Koh, H., Yoon, S.-O., Chung, A.-S., Cho, K. S., Chung, J. Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production.


Key Words: cell migration • PI3 kinase • NF-{kappa}B • MMP-9 • tumor invasion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE PROGRESSION OF a tumor in situ to an invasive tumor is a major prerequisite to cancer metastasis (1) and involves the acquisition of cell motility, novel cell adhesion properties, and extracellular protease production. For invasion, a cancer cell requires: 1) increased migration (motility), mediated by changes in the cytoskeleton; 2) a loss of cell-cell adhesion along with a gain of cell-matrix adhesion; and 3) increased expression and activation of extracellular proteases to degrade the extracellular matrix (ECM) and allow cell migration and invasion (1 2 3) .

Cell migration is an extremely complex process that requires the temporal and spatial coordination of multiple mechanisms such as actin polymerization leading to forward membrane extension, formation and release of focal adhesions acting as points of traction, and myosin motor activity leading to production of contractile force (4) . Although cell migration is crucial for normal physiological functions, embryonic development, inflammatory immune responses, wound repair, and angiogenesis, it also plays a critical role in cancer cell invasion and metastasis.

Different types of cell adhesion are mediated by a diverse group of cell surface proteins collectively classified as cell adhesion molecules. Cell-cell adhesions are formed particularly by the E-cadherin/catenin complex (5) , whereas adhesion of cells with extracellular matrix (ECM) proteins is mediated in a specific manner by integrins. In invasion and metastasis, single cells must separate from the solid tumor, which requires a loss of cell-cell adhesion (6) , and interact with the ECM, requiring a gain of cell-matrix adhesion properties.

The secretion of extracellular proteases plays an important role in immune functions, wound healing, and cancer cell invasion. Of these proteases, the matrix metalloproteinases (MMPs), a group of zinc-dependent ECM-degrading enzymes, are thought to play a critical role in tumor cell invasion (1) and have been shown to have increased expression correlated with the progression of various types of tumors (7 8 9) . The expression of MMP-2, MMP-7, and MMP-9 has been found to correlate with the metastatic potential of tumor cells (7 8 9) . Especially, MMP-9 (gelatinase B/92 kDa type IV collagenase) is expressed in a large variety of malignant cells and degrades collagen, a major component of the ECM and basement membrane (9 , 10) . It is interesting that MMP-9 has a nuclear factor-{kappa}B (NF-{kappa}B) binding site in its promoter region and is therefore expressed in an NF-{kappa}B-dependent manner (11) .

Akt/protein kinase B (Akt/PKB), first identified as the cellular homologue of the transforming oncogene v-Akt (12) , is a core component of the phosphoinositide 3-kinase (PI3K) signaling pathway. Akt/PKB is recruited to the plasma membrane and is activated by the binding of its pleckstrin homology (PH) domain to phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 3,4-bisphosphate (PIP2), the main products of PI3K (13 14 15) , in a manner that is also dependent on 3-phosphoinositide-dependent kinase 1 (PDK1) and a proposed PDK2. Activated Akt/PKB is a powerful promoter of cell survival, as it antagonizes apoptosis by phosphorylating and inactivating various components of the apoptotic machinery such as Bad (16) , caspase-9 (17) , and forkhead transcription factor family members (18) . It was recently shown that Akt/PKB activates the transcriptional activity of NF-{kappa}B, a family of transcription factors that when activated function to increase transcription of a wide range of genes, especially those involved in immune activation and cell survival (19 , 20) . In addition, Akt/PKB is involved in the regulation of cellular glucose metabolism through the inhibition of glycogen synthase kinase-3 (21) .

Other recent studies have hinted at different roles for Akt/PKB besides cell survival and metabolism. Firtel’s group has demonstrated that the activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in the slime mold Dictyostelium (22) ; Bourne’s group demonstrated the polar localization of the PH domain of Akt/PKB during neutrophil chemotaxis (23) . In addition, Akt/PKB has been suggested to be involved in various aspects of angiogenesis (24) .

Inasmuch as cell motility and angiogenesis are both activities that are highly related to cancer cell invasion, we investigated whether Akt/PKB plays a role in controlling the invasive behavior of cancer cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids
Full-length Akt, Akt PH domain (amino acids 1–114), myristylated Akt, Akt with K179M mutation, Akt with R25C mutation, and Akt PH domain with R25C mutation were inserted into pEGFP-N1 (Clontech, Palo Alto, CA) and are referred to as GFP-Akt, GFP-AktPH, GFP-MyrAkt, GFP-AktK179M, GFP-AktR25C, and GFP-AktPHR25C, respectively. The amino acid mutations (K179M and R25C) were created by point mutations using the QuickChange Site Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Full-length Rac was cloned into pEGFP-N1 and referred to as GFP-Rac. Hemaglutinin (HA)-tagged full-length Akt and HA-tagged Akt with a K179M mutation cloned into pCMV5 were kind gifts from Dr. T. F. Franke and are referred to as pCMV5-Akt and pCMV5-AktK179M, respectively. HA-tagged myristylated Akt cloned into pECE was a kind gift from Dr. R. A. Roth and is referred to as pECE-MyrAkt. The p65 subunit of NF-{kappa}B was cloned into pJ3H and is referred to as pJ3H-p65. The MMP-9 luciferase reporter was constructed by inserting the MMP-9 promotor region (-670 to +3) in the pGL3 luciferase reporter vector (Promega, Madison, WI). The NF-{kappa}B reporter construct was purchased from Clontech.

Cell culture and transfection
The HT1080 human fibrosarcoma cell line was grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS and penicillin-streptomycin. Cells were transfected by using the LipoFectamine reagent (GibcoBRL, Grand Island, NY) according to the manufacturer’s instructions.

Confocal microscopy of live samples
Round cover slides (Fisher, Springfield, NJ) were coated with fibronectin or covered with a monolayer of a primary human fibroblast cell line to provide a suitable substrate for HT1080 migration. Meanwhile, cells were transfected with green fluorescent protein (GFP) fusion protein constructs and 24 h later were suspended with trypsin-EDTA and plated on the coated cover slides. Twenty-four hours later, the slides were loaded onto a cell perfusion chamber (Seoul Engineering Company, Seoul, Korea) set at 37°C. GFP-expressing cells were identified and visualized with a confocal microscope (Zeiss, Jena, Germany). Laser images were taken every 20 s, with an excitation track of 488 nm. The magnification was 400 x with a zoom factor of 1.4–2.0. The position of the field was constant unless otherwise indicated.

Wound healing and immunostaining
A standard immunostaining procedure was carried out to observe endogenous Akt. Briefly, cells were grown to near confluency on fibronectin-coated slides, and a wound was created with the blunt end of a yellow tip (Sarstedt, Numbrecht, Germany). Three hours later, cells were washed in ice-cold PBS and were fixed in 3.7% formaldehyde. After permeabilization with 0.2% Triton X-100, cells were blocked for 1 h in blocking solution containing goat normal serum and 2% bovine serum albumin (BSA). After cells were washed, they were incubated in 1:300 anti-Akt antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, washed, incubated in 1:500 fluorescein isothiocyanate-conjugated secondary antibody (Sigma, St. Louis, MO), and washed before preparation for slides.

Cell migration assays
A modified version of the standard transfilter migration assay (25) was performed. HT1080 cells were grown on 60-mm plates and were transfected with GFP blank vector for drug inhibitor experiments or were transfected with a GFP blank vector or GFP-Akt fusion constructs for plasmid transfection experiments. Transwell filters (diameter 6.5 mm, pore size 5 µm; Costar, Cambridge, MA) were coated on the lower side with 0.5 µg/µl type I collagen (Sigma) and were placed into the lower chamber containing medium supplemented with 1 µg/µl BSA (Sigma). Twenty-four hours after transfection, the number of GFP-expressing cells on each plate was counted by using a fluorescent microscope (Zeiss); this served as the premigration count. Approximately 5 x 104 cells from each plate were added to the upper compartment of the Transwell chamber and allowed to migrate for 16 h. For drug inhibitor experiments, the inhibitors were added to both the upper and lower compartments prior to migration. After 16 h, nonmigrated cells on the upper side of the membrane were removed with a cotton swab, migrated cells on the bottom surface of the membrane were fixed for 1 h in formaldehyde, and the membrane was prepared for slides. The number of GFP-expressing cells was counted for each membrane, and this number divided by the premigration count was used to obtain the index of migration. Thirty random fields were counted for each pre- and postmigration count, and cells displaying an apoptotic or necrotic morphology were excluded from the premigration counts. All assays were carried out in triplicate.

Cell invasion assays
Cell invasion assays were performed as described for the cell migration assays, except that the Transwell filters were additionally coated on the upper side with 30 µg of Matrigel (Becton Dickinson, Bedford, MA).

Cell-matrix adhesion assays
A modified version of the standard static cell adhesion assay was performed. HT1080 cells were grown on 60-mm plates and were transfected with a GFP blank vector for drug inhibitor experiments or were transfected with a GFP blank vector or GFP-Akt fusion constructs for plasmid transfection experiments. Other 60-mm plates were coated overnight at 37°C with 10 µg/ml type I collagen (Sigma). Twenty-four hours after transfection, the number of GFP-expressing cells on each plate was counted using a fluorescent microscope; this served as the preadhesion count. For drug inhibitor experiments, cells were incubated with the inhibitor 16 h prior to the preadhesion count. Next, the cells on each plate were suspended with 1 mM EDTA/PBS and plated on the collagen-coated 60-mm plates. After various incubation periods, nonadherent cells were removed by agitation of the plate followed by washing once with DMEM. The number of adhered GFP-expressing cells was counted for each plate, and this number divided by the preadhesion count was used to obtain the percentage of adherence. Thirty random fields were counted for each pre- and postadhesion count, and cells displaying an apoptotic or necrotic morphology were excluded from the preadhesion counts.

Cell-cell adhesion assays
Cell-cell adhesion assays were performed as described for the cell-matrix adhesion assays, except the other 60-mm plates were covered with a 100% confluent monolayer of HT1080 cells instead of collagen.

Gelatin zymography
Production of MMPs by HT1080 cells was analyzed by gelatin zymography as described previously (26) . Briefly, cells were incubated in serum-free media for 2 days after transfection, or for 16 h after drug inhibitor treatment. The conditioned media were mixed with sample buffer and applied to a nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis column containing 0.1% gelatin. The lytic bands, indicating the presence of a protein with gelatinolytic activity, were visualized by Coomassie blue staining and were analyzed by using ImageQuant Software (Molecular Dynamics, Buckinghamshire, UK).

Luciferase assays
The NF-{kappa}B or MMP-9 reporter vector, internal Renilla luciferase control vector (pRL-TK), and other protein expression vectors were cotransfected as indicated in the figure legends. The total amount of transfected DNAs for all samples was 700 µg. All assays for firefly and Renilla luciferase activity were performed with one reaction tube sequentially, according to the manufacturer’s instructions (Promega). Luciferase activity was calculated as the firefly luciferase activity of the sample divided by the Renilla luciferase activity of the sample.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Akt/PKB is localized at the leading edge of migrating HT1080 cells
We investigated the localization of Akt/PKB in migrating metastatic cancer cells. The highly metastatic HT1080 cell line, which migrates in random directions over an appropriate two-dimensional substratum, were used as the model. Migrating HT1080 cells, transfected with a GFP-Akt fusion protein (GFP fused to the N terminus of Akt/PKB), were identified and observed using laser confocal microscopy of live samples. Remarkably, GFP-Akt was robustly and constantly localized at the leading edge, i.e., the membrane area at the front of the cell’s movement, in migrating HT1080 cells (Fig. 1A ). In contrast, in migrating HT1080 cells transfected with GFP protein, GFP was localized throughout the cytoplasm but not at the leading edge (Fig. 1B ).



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  		Figure 1. Akt/PKB was localized at the leading edge membrane of migrating HT1080 cells. A) In a cell perfusion chamber set at 37°C, migrating HT1080 cells expressing GFP-Akt were visualized via a laser confocal microscope and images were recorded every 20 s. B) Under the same conditions, migrating HT1080 cells expressing GFP were visualized and images were taken as in A. Images of the migrating cell at different recorded time points are shown. Arrowheads indicate the direction of the cell’s movement. The time for each image is relative to when the recording was initiated. Scale bar = 50 µm. The position of the field was constant throughout the recording. All experiments were repeated at least five times, and the images shown are representative.

Leading edge localization of Akt/PKB depends on PI3K activity
We examined the localization of the GFP-AktPH fusion protein (GFP fused to the PH domain of Akt/PKB) in migrating HT1080 cells. The PH domain of Akt/PKB binds PIP3 and PIP2, specific products of PI3K, and can thus be used as a marker to determine the localization of PI3K activity (22 , 23) . As shown in Fig. 2 (top and middle rows), AktPH displayed a pattern of leading edge localization identical to that of GFP-Akt, constantly remaining at the front of the cell’s movement even when the cell changed direction. However, on addition of the specific PI3K inhibitor LY294002 (25 µM), the leading edge localization of AktPH was instantly abolished, and AktPH became localized throughout the cytoplasm, although somewhat concentrated in the general nuclear area (Fig. 2 , bottom row). Also worth noting, addition of LY294002 caused the cell, which had been in constant motion, to stop immediately and completely. The abrogation of Akt leading edge localization and cell movement was also observed in GFP-Akt-transfected cells in response to 25 µM LY294002 (not shown). These results demonstrate that Akt/PKB is localized at the leading edge of migrating HT1080 cells in a PI3K-dependent manner and that the migration of the cell itself also depends on PI3K.



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  		Figure 2. Leading edge localization of Akt/PKB depends on PI3K. Top row) Migrating HT1080 cells expressing GFP-AktPH were visualized and images were recorded as described earlier. Middle row) The position of the field was moved so that the cell would appear at the middle of the field, and images continued to be recorded every 20 s. Bottom row) The position of the field was moved so that the cell would appear at the middle of the field, 25 µM LY294002 was immediately added, and images continued to be recorded every 20 s. Images of the migrating cell at different recorded time points are shown. Arrowheads indicate the direction of the cell’s movement. The time for each image is relative to when the recording was initiated. Scale bar = 50 µm. The position of the field was constant throughout the recording except for the two field adjustments described. All experiments were repeated at least five times, and the images shown are representative.

Akt/PKB R25C mutants and Rac1 are not localized at the leading edge of migrating HT1080 cells
To further confirm the PI3K dependence of Akt/PKB leading edge localization, we created R25C mutants of GFP-Akt and GFP-AktPH, which cannot bind to PIP3 and PIP2, and examined their localization in migrating HT1080 cells. As expected, both GFP-AktR25C and GFP-AktPHR25C failed to display leading edge localization but did show a general cytoplasmic localization (Fig. 3A , B ). This result confirmed the PI3K dependence of Akt/PKB leading edge localization.



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  		Figure 3. AktR25C, AktPHR25C, and Rac do not display leading edge localization. Migrating HT1080 cells expressing GFP-AktR25C (A), GFP-AktPHR25C (B), or GFP-Rac (C) were visualized, and images were recorded as described before. Images of the migrating cell at different recorded time points are shown. Arrowheads indicate the direction of the cell’s movement. The time for each image is relative to when the recording was initiated. Scale bar = 50 µm. The position of the field was constant throughout the recording. All experiments were repeated at least five times, and the images shown are representative.

However, the small GTPase Rac1 is another important effector of PI3K and has been found to be a regulator of cytoskeletal structure during various cellular functions such as membrane ruffle formation and cell adhesion (27 , 28) . Therefore, we wondered whether GFP-Rac1 might display a localization pattern similar to that of Akt/PKB in migrating HT1080 cells. Rac1 did not display leading edge localization but was instead constantly localized throughout the entire membrane, likely because of its prenylation motif (Fig. 3C ). Inasmuch as Rac1 is both a PI3K effector and a membrane-localized protein, this result demonstrates the specificity of the leading edge localization of Akt/PKB, not only compared with other PI3K downstream components but also compared with other membrane-localized proteins. Interestingly, in a related study (29) , Hahn’s group demonstrated that although GFP-Rac is not preferentially localized to the leading edge of migrating cells, it is preferentially activated in the leading edge. This finding confirms that PI3K is preferentially active in the leading edge and is thus relevant to our study.

Endogenous Akt/PKB is localized at the leading edge of migrating HT1080 cells
We wanted to confirm whether endogenous Akt is indeed localized at the leading edge of migrating HT1080 cells. However, as the immunofluorescence procedure requires that cells be fixed, it is not possible to observe endogenous Akt/PKB localization in live migrating cells. Therefore, we used a wound-healing procedure to identify migrating cells and their direction of movement. An artificial wound was created with the blunt end of a yellow tip, and cells were fixed and immunostained. In cells migrating toward the acellular region, as evidenced by their sticking out beyond the wound front, endogenous Akt/PKB was found to be localized at the leading edge (Fig. 4A , B ). As expected, treatment with 50 µM LY294002 for 5 min abolished the leading edge localization of endogenous Akt (Fig. 4C , 4D ).



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  		Figure 4. Endogenous Akt is localized at the leading edge of migrating HT1080 cells during wound healing. A near-confluent HT1080 cell layer was given an artificial wound and 3 h later was immunostained for endogenous Akt as described earlier. A, B) Leading edge localization of endogenous Akt in cells migrating toward the acellular region. C, D) Abrogation of Akt leading edge localization by 50 µM LY294002. The acellular region is at the bottom side of each image for all parts of the figure. Scale bar = 50 µm. The images shown are representative.

Akt/PKB controls cell migration in a PI3K-dependent manner
Because Akt/PKB was shown to have highly specific localization during the migration of HT1080 cells, we next investigated whether Akt/PKB plays an active role in HT1080 migration. To this end, we used a modified version of the conventional transfilter migration assay, as described in Materials and Methods. As shown in Fig. 5A and C , overexpression of Akt/PKB nearly doubled the migration rate of HT1080 cells, whereas overexpression of myristylated Akt/PKB (MyrAkt), which is anchored to the plasma membrane and has a constitutively active kinase activity, increased the migration rate more than threefold. Kinase-dead Akt/PKB (AktK179M), which acts in a predominantly negative manner, significantly inhibited migration. The lipid-binding Akt/PKB mutant AktR25C failed to increase HT1080 migration. It is interesting that expression of AktPH, which is thought to compete with endogenous Akt/PKB for binding to the PI3K-generated phosphoinositides in the membrane, had an inhibitory effect on migration. These findings demonstrate that Akt/PKB promotes HT1080 migration in a manner that depends on both its membrane-translocating ability and its kinase activity.



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  		Figure 5. Role of Akt/PKB and the PI3K pathway in HT1080 migration. A) Cells were transfected with 1 µg of GFP (GFP), GFP-Akt (Akt), GFP-MyrAkt (Myr), GFP-AktK179M (KD), GFP-AktR25C (RC), or GFP-AktPH (PH) and were assayed for migration through Transwell filters. Equal numbers (5 x 104) of cells were added to each well, with transfection rates of about 65%, 60%, 65%, 40%, 40%, and 65% for GFP, Akt, Myr, KD, RC, and PH, respectively. Of the control (GFP) cells added to the upper chamber, 16 ± 1.5% migrated to the bottom of the transfilter. B) Cells were transfected with 1 µg of GFP and treated with 50 µM LY294002 (LY), 50 µM PD98059 (PD), or 40 ng/ml rapamycin (RA) and assayed for migration through Transwell filters as described earlier; control cells (Cont) were untreated. C, D) Micrographs of the Transwell membranes used in (A) and (B), respectively; membranes were fixed with formaldehyde and viewed at 400 x magnification with an excitatory track of 488 nm. Values are expressed as "folds" of the control. Data are means ± SD of three separate experiments, each carried out in triplicate.

To determine whether Akt/PKB controls cell migration in a PI3K-dependent manner, we tested the effects of various specific inhibitors on HT1080 migration. As shown in Fig. 5B and D , LY294002, a specific PI3K inhibitor, inhibited migration by more than 60%. The specific mitogen-activated protein (MAP) kinase inhibitor PD98059 and rapamycin, a specific inhibitor of target of rapamycin (TOR), had no effect. This result confirms our observation (Fig. 2 , bottom row) that HT1080 migration is PI3K dependent.

Akt/PKB controls cell invasion in a PI3K-dependent manner
An increase in the migratory ability of a cell usually leads to an increase in invasive ability. Because Akt/PKB was found to modulate the migration of HT1080 cells, we examined whether Akt/PKB also affects their invasiveness, by analyzing their ability to invade a reconstituted ECM (Matrigel). As shown in Fig. 6A , the invasiveness of HT1080 cells was increased nearly threefold by Akt/PKB and more than fivefold by MyrAkt. Also, surprisingly, dominant-negative AktK179M completely inhibited HT1080 invasion, and AktR25C failed to promote invasion. AktPH had a significant inhibitory effect on invasion. Thus, the effects of Akt/PKB on invasion were similar to its effects on migration but were more dramatic. It is interesting that, as shown in Fig. 6B , both LY294002 and PD98059, but not rapamycin, significantly inhibited HT1080 invasion. Taken together, these results demonstrate that Akt/PKB strongly modulates cell invasion in a PI3K-dependent manner.



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  		Figure 6. Role of Akt/PKB and the PI3K pathway in HT1080 invasion. A) Cells were transfected with 1 µg of GFP (GFP), GFP-Akt (Akt), GFP-MyrAkt (Myr), GFP-AktK179M (KD), GFP-AktR25C (RC), or GFP-AktPH (PH) and were assayed for invasion through Matrigel (30 µg) on Transwell filters as described before. Cell numbers added and transfection rates were the same as those for the migration assays, and 11 ± 3% of the control (GFP) cells added to the upper chamber migrated to the bottom of the transfilter. B) Cells were transfected with 1 µg of GFP and were not treated (Cont) or were treated with 50 µM LY294002 (LY), 50 µM PD98059 (PD), or 40 ng/ml rapamycin (RA) and were assayed for invasion through Matrigel (30 µg) on Transwell filters as described before. Values are expressed as folds of the control. C) Cell death did not contribute to inhibition of cell invasion or migration by AktKD or LY294002. HT1080 cells were transfected with 1 µg of GFP-AktK179M or were treated with 50 µM LY294002, with or without 10 ng/ml TNF plus 1 µg/ml cycloheximide. Cell death was measured as the number of dead cells (round or ruptured morphology) vs. the total number of cells, or the number of transfected dead cells vs. the total number of transfected cells in the case of GFP-AktK179M transfection. GFP-AktK179M-transfected cells were incubated for 24 h after transfection and were washed before addition of TNF plus cycloheximide and counting to determine cell death. Values were calculated as cell death minus control untreated cell death. Data are means ± SD of three separate experiments, and each invasion assay was carried out in triplicate.

Although we took measures to ensure that negative effects from possible cell toxicity were minimized (as discussed later), as Akt/PKB and PI3K are mediators of antiapoptotic signaling we could not rule out that possible cell death from AktKD expression or LY294002 treatment may have contributed to their inhibitory effects on cell migration and invasion. Therefore, we assayed cell viability (Fig. 6C ). In the presence of tumor necrosis factor (TNF), the inhibition of Akt/PI3K by AktKD or LY294002 left cells unprotected from cell death, as evidenced by the high rate of cell death. However, under our noninsulted, normal experimental conditions, AktKD expression or LY294002 treatment per se did not result in significant cell death. Therefore, we were able to exclude the possibility that cell death contributed to the inhibition of invasion and migration by AktKD or LY294002.

Akt/PKB has no effect on cell-cell and cell-matrix adhesion
For a cancer cell to effectively invade the surrounding tissue, an increase in cell migration (motility), changes in adhesion properties, and increased extracellular protease expression are required (1 2 3) . Because the activating and inhibitory effects of Akt/PKB on cell invasion were significantly greater than its effect on cell migration (Fig. 6A vs. Fig. 5A ), we considered whether modulation of cell adhesion and extracellular protease expression properties by Akt/PKB also contributed to its overall effect on HT1080 invasion.

Cancer cells invading the host tissue break off their cell-cell contacts and make new contacts with the ECM, and therefore low cell-cell adhesion and high cell-matrix adhesion appear to be correlated with a highly invasive phenotype (3) . We performed modified versions of cell-cell adhesion and cell-collagen adhesion assays, as described in Materials and Methods. As shown in Fig. 7A and B , overexpression of Akt/PKB or its various mutants did not have a noticeable effect on either cell-cell or cell-collagen adhesion. We also failed to detect any significant changes in either the cell-matrix or cell-cell adhesion properties of HT1080 cells after treatment with LY294002 or other specific inhibitors (Fig. 7C , 7D ). Thus, we conclude that the cell-cell and cell-matrix adhesion properties of HT1080 cells are not regulated by Akt/PKB and therefore are not related to the promotion of cell invasion by Akt/PKB.



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  		Figure 7. Role of Akt/PKB and the PI3K pathway in HT1080 cell-cell and cell-matrix adhesion. A, B) Cells were transfected with 1 µg of GFP (GFP), GFP-Akt (Akt), GFP-MyrAkt (Myr), or GFP-AktK179M (KD) and were assayed for adhesion to type I collagen (A) or to other HT1080 cells (B) for various time periods. C, D) Cells were transfected with 1 µg of GFP and were not treated (Cont) or were treated with 50 µM LY294002 (LY), 50 µM PD98059 (PD), or 40 ng/ml rapamycin (RA); they were assayed for adhesion to type I collagen (C) or to other HT1080 cells (D). The data are means ± SD of three separate experiments.

Akt/PKB and PI3K are involved in the regulation of MMP-9 production
Changes in cancer cell adhesion or cell migration alone are not sufficient for efficient invasion of the surrounding tissue. The cancer cell must be also able to produce proteases in order to breach biological barriers such as basement membranes (30) . For the many known proteases, the expression of MMP-9 is regulated by NF-{kappa}B transcriptional activity (11) . NF-{kappa}B transcriptional activity, in turn, has been shown in recent studies to be activated by Akt/PKB (19 , 20) . Therefore, we suspected that MMP-9 plays a role in the promotion by Akt/PKB of cancer cell invasion.

The effect of Akt/PKB on the production of MMP-9 in HT1080 cells was determined by using a standard gelatin zymography procedure. As shown in Fig. 8A , Akt/PKB and MyrAkt affected MMP-9 production in a dose-dependent manner: MyrAkt increased MMP-9 production more than twofold, whereas dominant-negative AktK179M decreased MMP-9 production by more than 60%. Next, to determine whether the modulation of MMP-9 production depends on PI3K, we conducted zymography experiments with drug inhibitors. As shown in Fig. 8B , LY294002 dramatically decreased MMP-9 production. PD98059, which has been shown to decrease MMP-9 production in some cell types (31 , 32) , also decreased MMP-9 production but in a less dramatic fashion. In contrast, the TOR inhibitor rapamycin slightly activated MMP-9 production. Taken together, these results demonstrate that both Akt/PKB and PI3K are involved in the modulation of MMP-9 production.



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  		Figure 8. Role of Akt/PKB and the PI3K pathway in MMP-9 production in HT1080 cells. A) Cells were transfected with 1.4 µg of blank vector (lane 1, Cont), 700 ng or 1.4 µg of pCMV5-Akt (lanes 2 and 3, Akt), 700 ng or 1.4 µg of pECE-MyrAkt (lanes 4 and 5, Myr), or 1.4 or 2.1 µg of pCMV5-AktK179M (lanes 6 and 7, KD), and samples of the conditioned media were analyzed by gelatin zymography (middle panel). B) Cells were treated without (lane 1, Cont) or with 12.5, 25, or 50 µM LY294002 (lanes 2–4, LY), 25 or 50 µM PD98059 (lanes 5 and 6, PD), or 20 or 40 ng/ml rapamycin (lanes 7 and 8, RA), and samples of the conditioned media were analyzed by gelatin zymography (middle panel). The cell line utilized (which produces only MMP-2 and MMP-9) and the molecular weight of the band allowed us to determine that the band shown is MMP-9. The bands were quantified and presented as arbitrary densitometric units of gelatinolytic activity (upper panels). The ß-actin immunoblot of the cells (lower panels) demonstrates that equal numbers of cells were present during the conditioning of the media. The quantified data (upper panels) are means ± SD of three separate experiments; the gelatin zymography (middle panels) and ß-actin immunoblot (lower panels) data were from a representative experiment. The ß-actin immunoblots were for the same cells as those used for the gelatin zymography.

Akt/PKB induces MMP-9 via activation of NF-{kappa}B transcription activity
Because Akt/PKB has been shown to up-regulate NF-{kappa}B transcriptional activity in a variety of cells (19 , 20) , we suspected that the up-regulation of MMP-9 production in HT1080 cells by Akt/PKB might occur via increased NF-{kappa}B transcriptional activity. Therefore, we examined whether Akt/PKB increases NF-{kappa}B transcriptional activity in HT1080 cells, by using the luciferase assay system with an NF-{kappa}B reporter. As shown in Fig. 9A , cotransfection of Akt/PKB increased NF-{kappa}B reporter activity more than twofold compared with the control, whereas myristylated Akt/PKB increased reporter activity about 6.5-fold. However, cotransfection with AktK179M inhibited MMP-9 reporter activity more than twofold, whereas cotransfection with the p65 subunit of NF-{kappa}B, as a positive control, increased MMP-9 reporter activity more than sevenfold. Thus, we were able to determine that Akt/PKB modulates the transcriptional activity of NF-{kappa}B in HT1080 cells.



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  		Figure 9. Akt/PKB modulates MMP-9 transcription via NF-{kappa}B. A) HT1080 cells were transfected with the NF-{kappa}B reporter (10 ng) plus empty vector control (Cont, 500 ng), pCMV5-Akt (Akt, 300 ng), pCMV5-MyrAkt (Myr, 300 ng), pCMV5-AktK179M (KD, 500 ng), or pJ3H-p65 (p65, 500 ng). Thirty-six hours after transfection, lysates were assayed for luciferase activity. B) In the absence (black bars) or presence (white bars) of 50 µM PDTC, HT1080 cells were transfected with the MMP-9 reporter (200 ng) plus empty vector control (Cont, 500 ng), pCMV5-Akt (Akt, 300 ng), pCMV5-MyrAkt (Myr, 300 ng), pCMV5-AktK179M (KD, 500 ng), or pJ3H-p65 (p65, 500 ng). Thirty-six hours after transfection, lysates were assayed for luciferase activity. Values are expressed as folds of the control. Data are means ± SD of three separate experiments.

To determine whether the modulation of NF-{kappa}B transcriptional activity by Akt/PKB is responsible for its regulation of MMP-9 production, we performed luciferase assays with an MMP-9 reporter, either with or without the NF-{kappa}B inhibitor pyrrolidine dithiocarbamate (PDTC). In the absence of PDTC (Fig. 9B , black bars), Akt/PKB modulated MMP-9 reporter activity with the same pattern as that seen in Figs. 8A and 9A , confirming the modulation of MMP-9 production by Akt/PKB and further suggesting the involvement of NF-{kappa}B in the process. However, in the presence of the NF-{kappa}B inhibitor PDTC (50 µM), Akt/PKB, MyrAkt, AktK179M, and NF-{kappa}B p65 subunit had no effect on MMP-9 reporter activity (Fig. 9B , white bars), confirming that Akt/PKB modulates MMP-9 production by affecting the transcriptional activity of NF-{kappa}B.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In recent years, the PI3K pathway has been suggested to be highly involved in tumor cell invasion (33 , 34) . However, Akt/PKB, despite being perhaps the most widely studied component of the PI3K pathway for its involvement in cell survival, has not been investigated as a possible downstream mediator of PI3K for its involvement in cell invasion. Instead, research efforts have focused primarily on the Rho GTPase family member Rac, which has been implicated as a mediator in cytoskeletal regulation (27 , 28) , cell migration (4) , and cell adhesion properties (35) regulated by PI3K, leading to increased invasion (33 , 34) . Rac has also been shown to be involved in cell survival (36) .

In our study, we demonstrated that Akt/PKB plays a critical role in cell invasion as well. As with Rac1, Akt/PKB was found to modulate cell migration (Fig. 5A ), but unlike Rac1, Akt/PKB did not affect cell adhesion properties (Fig. 7A , 7B ) while modulating MMP-9 production (Fig. 8A ). Thus, we propose a new model for the promotion of cancer cell invasion by PI3K, in which Rac1 acts to modulate cell adhesion properties, Akt/PKB induces MMP-9 production, and both Rac1 and Akt/PKB promote cell migration.

We established that Akt/PKB is localized at the leading edge of migrating HT1080 cells, which provides an important clue for finding upstream and downstream components that interact with Akt during cancer cell migration. As the leading edge localization of Akt depended on PI3K and occurred in the absence of any chemotactic stimuli or migration-stimulating factors, we speculate that the binding of integrin with the substratum acts as the stimulatory signal for PI3K activation and subsequent Akt/PKB localization at the leading edge. In support of this hypothesis, integrins have been shown to be essential for cell migration and invasion (4 , 33) and have been shown to activate PI3K during this process (34) . In addition, although integrins are not preferentially concentrated at the leading edge of migrating cells, they are attached preferentially to the cytoskeleton at the leading edge (37) . The leading edge localization of Akt/PKB and its promotion of cell migration strongly imply that some modulation of the cytoskeleton occurs downstream of Akt/PKB, via a yet unidentified effector of Akt/PKB that is also localized in the leading edge. One highly possible downstream effector of Akt/PKB is filamin, which we have found binds with Akt/PKB in vivo (data not shown). Filamin, an actin-binding protein that plays a pivotal role in reorganizing the actin cytoskeleton, is required for cell migration (38) . Filamin was also shown to be localized at the leading edge of motile cells (39) . Another interesting candidate is p21-activated kinase (PAK), an important effector of Rac during its regulation of the cytoskeleton and cell migration (40 , 41) . Akt/PKB also stimulates PAK activity in a Rac-independent manner (42) , and thus it is possible that PAK is the effector that allows both Akt/PKB and Rac to promote cell migration. Further work is required to address the specific events that occur upstream and downstream of Akt/PKB in the promotion of cell migration.

We investigated cell migration and invasion by using modified versions of the transfilter migration and invasion assays. In the conventional transfilter migration and invasion assays (25) , equal numbers of cells are added to the upper chamber of the transfilter, and the cells that have migrated to the lower surface of the transfilter membrane are counted after a specific time period to determine the effect of a transfected gene or a drug treatment. However, in the case of DNA transfection, the proportion of untransfected or weakly expressing cells can dilute the effect of the gene observed in the migration assay. We therefore modified the original method. In our assay, cells were transfected with Akt/PKB and its mutants fused to GFP; GFP blank vector was the control. Immediately prior to suspension and application to the upper chamber, the number of GFP-expressing cells for each DNA transfection was counted. After migration or invasion, only the GFP-expressing cells were counted on each membrane. By counting only the GFP-expressing cells, we were able to eliminate the "noise" from untransfected or weakly expressing cells. In addition, as GFP-transfected cells were highly visible against a virtually null background, more accurate counting was possible, and cells displaying an apoptotic or necrotic morphology could be easily identified and excluded in the premigration count. Therefore, we used this type of modification in all cell migration and invasion assays (Figs. 5 and 6) and adhesion assays (Fig. 7) .

Studies of the regulation of MMP-9 production by using specific drug inhibitors have yielded various results according to cell type, some demonstrating the PI3K pathway (43 , 44) and others demonstrating the MAP kinase pathway (31 , 32) to be involved in the regulation of MMP-9. As shown in Fig. 8B , we found that both the PI3K and MAP kinase pathways are involved in the induction of MMP-9. Both pathways seem to contribute to MMP-9 production in a synergistic manner, as cotreatment with both LY294002 and PD98059 resulted in total inhibition of MMP-9 production (data not shown). As shown in Fig. 8A , we demonstrated the modulation of MMP-9 production by Akt/PKB, which explains for the first time the regulation of MMP-9 by PI3K. The inhibition of MMP-9 production by PD98059 explains its inhibitory effect on cell invasion (Fig. 6B ), although it affected neither cell migration (Fig. 5B ) nor adhesion (Fig. 7C and D ).

A limiting point of this study is that the observations refer to only one transformed tumor cell line and could be dependent on the particular genetic background of this cell line. However, we have conducted migration and invasion assays with a breast cancer cell line (MDA-MB-231) and observed similar results (data not shown). Also, in agreement with our findings, Tanno et al. (45) recently demonstrated that activated Akt promotes the invasion of pancreatic cancer cells. Thus, although it appears that Akt is involved in the invasive properties of many metastatic cell lines, further investigations with other metastatic cell lines are needed to determine whether the promotion of invasion by Akt is a general phenomenon.

In conclusion, we established a novel function of Akt/PKB separate from its functions in cell survival and while demonstrating a novel mechanism for the regulation of cancer cell invasion. This discovery provides a possible explanation of the multifaceted involvement of PI3K in cancer cell invasion, and especially MMP-9 production. Our findings have important clinical implications not only in cancer metastasis but also in angiogenesis, wound healing, and autoimmune disorders such as rheumatoid arthritis, in which cell migration and metalloproteinase production have important roles as well. Our findings may launch whole new avenues for research, in both clinical and cell biological fields, on the already well-studied protein Akt/PKB.


   ACKNOWLEDGMENTS
 
We are grateful to Drs. J. Blenis, T. F. Franke, and R. A. Roth for plasmids and reagents. We also thank the members of Dr. J. Chung’s laboratory. This work was supported by a Korea Research Foundation Grant (KRF-2000–015-DS0034).

Received for publication March 20, 2001. Revision received May 24, 2001.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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