To determine how AKT2 might contribute to tumor cell progression, a full-length, wild-type, human AKT2/protein kinase B (PKB)β cDNA was transfected into a panel of eight human breast and ovarian cancer cells. AKT2 transfectants demonstrated increased adhesion and invasion through collagen IV because of up-regulation of β1 integrins. In addition, AKT2 cells were more metastatic than control cells in vivo. Increased invasion by AKT2 was blocked by preincubation with an anti-β1 integrin function blocking antibody, exposure to wortmannin, and by expression of phosphatase and tensin homologue tumor suppressor (PTEN). Confocal microscopy performed on transfected human breast cancer cells showed that unlike AKT1, AKT2 protein predominantly localized adjacent to the collagen IV matrix during cellular attachment. Overexpression of AKT2, but not AKT1 or AKT3, was sufficient to duplicate the invasive effects of phosphoinositide 3-OH kinase (PI3-K) transfected in breast cancer cells. Furthermore, expression of kinase dead AKT2(181 amino acid methionine [M]), and not kinase dead AKT1(179M) or AKT3(177M), was capable of blocking invasion induced by either human epidermal growth factor receptor-2 (HER-2) overexpression or by activation of PI3-K. Taken together, these data indicate that AKT2 mediates PI3-K-dependent effects on adhesion, motility, invasion, and metastasis in vivo.

The PI3-K3 /AKT pathway can be activated in tumor cells by a number of mechanisms. Elevated levels of the regulatory subunit of PI3-K (p85; Ref. 1); mutations in the inter-src homology domain 2 region of the p85 regulatory subunit (2); and amplification of the PIK3CA gene, which encodes the p110α catalytic subunit of PI3-K (3), can all lead to constitutive activation of the PI3-K signaling pathway. Moreover, amplification and/or aberrant overexpression of a variety of growth factor receptor tyrosine kinases (e.g., HER-2, epidermal growth factor receptor; Refs. 4, 5); genetic loss or transcriptional down-regulation of the tumor suppressor gene, PTEN(6, 7); and mutations in ras are also known to activate the PI3-K/AKT pathway (8). Finally, AKT proteins themselves can be either amplified and overexpressed, or activated by a number of growth factors such as epidermal growth factor, HRG, IGF-1, IGF-II, basic fibroblast growth factor, platelet-derived growth factor, and insulin, indicating that there are multiple mechanisms by which the PI3-K/AKT pathway can be activated in cancer cells (9, 10, 11).

The kinases in the AKT family, which is composed of three members, AKT1/(PKBα), AKT2/(PKBβ), and AKT3/(PKBγ; Ref. 12), are independently activated by phosphorylation on serine residues at aa473 (AKT1), aa474 (AKT2), or aa472 (AKT3), as well as on threonine residues at aa308 (AKT1), aa309 (AKT2) or aa305 (AKT3). Both AKT1 and AKT2 appear to have similar substrates in vivo(13); however, several lines of evidence suggest that the physiological functions of AKT1 and AKT2 may not be entirely the same. First, subtle differences between AKT1 and AKT2 exist, such as in the specificity, level of activation, and location in various cell types (14, 15, 16, 17). Second, multimeric complexes formed by AKT proteins are restricted to individual isoforms, indicating the need to maintain specificity in interactions with other signaling proteins (12). In addition, although the AKT1 and AKT2 proteins require membrane localization for activity, only AKT2 appears to accumulate in the cytoplasm during mitosis (18) and in the nucleus during muscle cell differentiation (19). Microinjection of anti-AKT1 antibodies blocks cell cycle progression, whereas anti-AKT2 antibodies have no effect (19). Whereas AKT1 amplification has been detected in a single gastric carcinoma cell line, AKT2 amplification and overexpression is more frequently detected in a number of human tumors including breast and ovarian cancers (20, 21, 22, 23, 24). Finally, even in the presence of AKT1 and AKT3, AKT2-deficient mice show an inability to lower blood glucose (25), indicating that differences in substrate specificity may exist among the isoforms.

Much work has gone into understanding the role of AKT1 and its ability to promote cell survival. On the basis of the hypothesis that AKT function may not be redundant in cells, the present study was designed to determine how AKT2, specifically, contributes to tumor cell progression. To address this question, a panel of human breast and ovarian cancer cells lines stably expressing the AKT2 gene were developed. These studies indicate that an increased expression of AKT2 leads to increased cell invasiveness both in vitro and in vivo.

Cloning and Construction of AKT2, AKT3 Plasmids, and Other Constructs.

Full-length AKT2 cDNA was cloned from the OVCAR-3 cell line by, first, reverse transcribing then by PCR (RT-PCR) using the following primers: sense, 5′-TCC TGC ATG TCC TGC TGC CCT GAG-3′, and antisense, 5′-CAG CGG TGA CAG CGA GCG TGC-3′, designed and sequence-verified according to GenBank accession no. M95936. PCR primers, 5′-CTC AGA GGG GAG TCA TCA TGA GCG ATG TTA CC-3′ and 5′-AGA ATG AAA GAG ACT TAT TCT CGT CCA CTT GC-3′, were used to amplify the AKT3 cDNA from Marathon-Ready human brain cDNA (Clontech, Palo Alto, CA).

Dominant negative mutants of human AKT3 and AKT2 were made by changing the lysine residue at position 177 of AKT3 and the lysine residue at position 181 of AKT2 to a methionine residue. The C′70 AKT2 mutant, which lacks the 70 amino acids from the COOH terminus and has been previously reported to function as a dominant negative in transformation assays, was also generated by PCR (18). All of the AKT constructs [AKT2, AKT2(K181 amino acid methionine [M]), AKT3 and AKT3(K177M)] were tagged with a myc epitope and subcloned into pCDNA3.1 (Invitrogen).

myr-p110, wtp110, myr-AKT1, AKT1(K179M), and AKT1 were obtained from Upstate Biotechnology (Lake Placid, NY). Alternatively, full-length human AKT1 was obtained from Invitrogen (Carlsbad, CA) and was cloned from a SKBR3 cDNA library by RT-PCR for further confirmation of results. PTEN was cloned from a Marathon-Ready human brain cDNA (Clontech) by RT-PCR and epitope tagged with myc. The C124S catalytically inactive mutant of PTEN was used in some experiments.

Cell Lines and Transfections.

CaOV3, SK-OV-3, OV2008, MDA-MB-435, MDA-MB-231, T47D, MCF7, HBL-100, SKBR3, PC3, and PANC-1 were grown in Dulbecco’s modified Eagle’s High glucose medium (DMEM; Life Technologies, Inc., Rockville, MD) supplemented with 10% FCS (Irvine Scientific), 50 units/ml penicillin, and 50 μg/ml streptomycin (Irvine Scientific). HOSE cells, obtained from Dr. Beth Karlan (Cedars-Sinai Medical Center, Los Angeles, CA), was grown in 50% MCDB 105 (Sigma Chemicals) and Medium 199 (Life Technologies, Inc.) supplemented as described above. All of the AKT2 transfected clones were maintained in medium containing 500 μg of G418 (Life Technologies, Inc.). All of the cell lines were grown at 37°C in a humidified incubator containing 5% CO2.

Transfections were performed as described previously (27). To achieve the highest level of transfection efficiency, transient transfection assays were optimized using either LipofectAMINE (Life Technologies, Inc.) or LT-100 (Mirrus/Panvera, Madison, WI) according to the manufacturers’ instructions.

Localization of AKT2.

AKT2/YFP and AKT1/YFP were generated by subcloning full-length AKT1 and AKT2 in frame into pEYFP vector (Clontech). MDA-MB-435HER-2 cells were transiently transfected with LT-100 and 3 μg of AKT2/YFP, AKT1/YFP, and control YFP vector. Cells were replated onto collagen IV-coated coverslips and visualized ∼1–2 h postplating by Carl Zeiss LSM 310 Laser Scanning Confocal microscope.

Cell Attachment Assay.

A total of 2.5 × 107 cells were plated onto collagen I- and IV-, laminin-, and fibronectin-precoated plates (Becton Dickinson, Franklin Lakes, NJ). After 3–4 h, cells were removed and counted using a Coulter counter. Alternatively, ultra-low attachment 6-well plates (Corning Costar) were coated with fibronectin, laminin, collagen IV, or collagen I (Becton Dickinson) according to the manufacturer’s directions. These plates were selected because of their hydrophilic and neutrally charged surface, which prevents nonspecific cell attachment via hydrophobic and ionic interactions. In these experiments, 7500 cells were plated and visually quantitated 6–7 h postplating. In some experiments, cells were pretreated with 50 nm wortmannin for 30 min and plated in the presence of wortmannin or were transiently transfected with PTEN 72 h before plating.

Matrigel/Collagen IV Invasion Assay.

Matrigel, which is primarily composed of collagen IV and laminin, or human collagen IV, collagen I, laminin, or fibronectin were prepared according to the manufacturer’s directions (Becton Dickinson). Invasion assays were performed as described previously (28). The number of cells reaching the bottom chamber were quantitated by direct visual cell count after 48 h for the Matrigel invasion assay and after 12–15 h for the collagen IV invasion assay. Serum or 1 nm HRG (Lab Vision, Fremont, CA) was used as the chemoattractant. In some experiments, cells were pretreated with 50 nm wortmannin (Alexis Biochemicals, San Diego, CA) or LY294002 (Upstate Biotechnology) for 30 min and loaded onto the top chamber in the presence of the drug.

In Vivo Assays.

Nude (CD-1) mice (Charles River Laboratories, Wilmington, MA), five animals per group were injected with 1 × 106 cells i.p. and were observed once a week for tumor growth, ascites accumulation, body weight, and general health. At the end of 4–6 weeks, the animals were sacrificed and examined for tumor spread in the peritoneal cavity. Orthotopic injections (1 × 106) cells were performed by direct injection of cells through the nipple. SCID mice were used for the SKOV3 experiments. Animals were observed daily for signs of morbidity and were euthanized according to the guidelines established by the American Association for Laboratory Animal Care (AALAC).

Immunoblotting, Kinase Assay, Immunohistochemistry.

Immunoblotting was performed as described previously (27). Phospho-specific antibodies against AKT(S473, T308), phospho-GSK3-β (S9/21; Cell Signaling Technology, Beverly, MA) and anti-myc antibody (Invitrogen) were used according to the manufacturer’s direction. The phospho-AKT antibody was determined to also recognize phosphorylated AKT2. The AKT2-specific antibody (Upstate Biotechnology) did not cross-react with recombinant AKT1 or AKT3 in Western blot analysis. Integrin β1 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Integrin function-blocking antibody (29) and PDK1 antibody were from Upstate Biotechnology. PTEN antibody was from Cascade Bioscience (Winchester, MA).

A modified version of the in vitro AKT kinase assay (Cell Signaling Technology) was performed by first immunoprecipitating AKT2 using an AKT2-specific antibody.

AKT2 tumors were resected from animals, and immunohistochemistry using antibody to the myc epitope was used to validate the presence of an exogenous AKT2 gene product. Immunohistochemistry was performed according to the manufacturer’s direction (Invitrogen).

Stable Expression of AKT2 in Normal and Malignant Human Breast and Ovarian Cells.

An analysis of endogenous AKT2 expression in a variety of human breast and ovarian malignant cell lines was performed. All of the parental cell lines expressed various, but relatively similar, levels of AKT2 protein. (Fig. 1,A). Because AKT2 activity is known to be dependent on PI3-K activity, the cellular expression level of the PTEN phosphatase and basal AKT phosphorylation was also determined for these cells (Fig. 1,B). Five human malignant breast (MDA-MB-435, MDA-MB-231, MCF7, T47D, and SKBR3) and three human malignant ovarian (CaOV3, SKOV3, and OV2008) cell lines were transfected with a full-length myc-tagged AKT2 cDNA to evaluate the biological effects of AKT2 overexpression in transformed cells. In addition, the gene was transfected into two immortalized, nontransformed human cell lines, HOSE cells and HBL100 cell line. The relative expression levels of the various AKT2 clones were determined by Western blot analysis to detect the myc epitope-tagged AKT2 (Fig. 1,C). In addition to vector controls, MDA-MB-435 cells that stably expressed the upstream activating kinases of AKT2, PDK1, and PI3-K (p110) were used as positive controls in some experiments (Fig. 1, D and E). The levels of transfected AKT2 expression analyzed in this study were restricted to ∼2–4-fold higher than those found in parental cell lines and well within the range seen in natural (nonengineered) high AKT2 overexpressing human cells, OVCAR-3 (Fig. 1 F).

Exogenous expression of myc-tagged AKT2 in the transfected breast and ovarian cancer cells consistently resulted in a number of profound morphological changes that included increased cell size, multinucleation, accumulation of cytoplasmic vesicles, constitutive membrane ruffling, and lamellipodium formation (Fig. 1 G). Expression of a constitutively active mutant of PI3-K (myr-p110) in the MDA-MB-435 cells yielded a similar phenotype (data not shown), indicating that these effects are consistent with activation of this pathway.

AKT2 Overexpression Leads to Increased AKT2 Phosphorylation and Activity.

To verify activation of the AKT2 pathway, AKT2 phosphorylation was measured by Western blot analysis using a phospho-specific antibody that recognizes both ser473 of AKT1 and ser474 of AKT2. (Fig. 2,A). Like AKT1, AKT2 has been shown to phosphorylate GSK-3. Therefore, AKT2 catalytic activity was assessed by in vitro kinase assay using an AKT2-specific antibody and recombinant GSK-3β as the substrate (Fig. 2, A and B). These results indicate that increased expression of AKT2 leads to increased phosphorylation of S474 and activity of AKT2.

Overexpression of AKT2 Leads to Increased Adhesion and Invasion through Collagen IV.

Cellular invasion is thought to involve a three-step process that includes attachment, matrix degradation, and migration. Therefore, the attachment of CaOV3AKT2 and MDA-MB-435AKT2-expressing cells to plates precoated with a variety of extracellular matrices was evaluated (Table 1). Increased expression of AKT2 correlated with increased attachment to collagen I, collagen IV, and laminin. To confirm that this interaction was specific to the presence of the matrix, collagen IV was coated onto an ultra-low attachment dish (Corning Costar) to eliminate background plating caused by hydrophobic and ionic interactions. Predictably, only 2.7 and 0.3% MDA-MB-435control and MDA-MB-435AKT2 cells, respectively, attached to uncoated ultra-low attachment plates after 24 h. However, when collagen IV was coated onto these plates, an 11.3-fold increase in attachment was noted in the AKT2-overexpressing cell line as compared with controls (Fig. 3,A). Of interest, collagen IV is one of the major matrix components found in basement membrane throughout normal tissue and organ. Attachment studies with three other AKT2-transfected breast and ovarian cancer cell lines also occurred at an accelerated rate (data not shown), indicating that this was not a cell line-specific phenomenon. PI3-K signaling appeared to be required for this phenotypic effect because treatment with wortmannin or expression of the PTEN phosphatase gene blocked cellular attachment (Fig. 3 A).

In vitro invasion assays were subsequently performed to compare the ability of AKT2 cells to invade through fibronectin, laminin, collagen I, or collagen IV matrices. The data from these experiments demonstrate that both CaOV3AKT2- and MDA-MB-435AKT2-expressing cells invaded preferentially through collagen IV and to some extent through laminin (Fig. 3,B). Similar results were observed with stable transfectants of PDK1 and PI3-K (wtp110). In collagen IV invasion assays, MDA-MB-435AKT2-expressing cells had an average 5.4-fold increase (P = 0.025) in cell invasion when compared with controls. With the exception of two nontransformed cell lines HBL-100 and HOSE, similar results were obtained with all of the malignant cell lines transfected with wild-type AKT2 (i.e., MCF7, MDA-MB-435, MDA-MB-231, T47D, SKBR3, CaOV3, SKOV3, and OV2008), again indicating that increased invasion across multiple breast and ovarian cancer cell lines is generically associated with AKT2 overexpression (Table 2).

To assess whether increased invasion and attachment to collagen IV could be associated with changes in collagen-binding receptors, the level of β1 integrins, a component of both collagen- and laminin-binding receptors, was evaluated by Western blot analysis (30, 31). Expression of β1 integrin was found to be elevated in the AKT2 transfectants when compared with controls (Fig. 4,A). Whether this increase in β1 integrins occurs transcriptionally or posttranscriptionally is not known. Nevertheless, preincubation with an established function blocking monoclonal antibody to β1 resulted in a 70% decrease in AKT2-induced cell invasion, indicating that invasion through collagen IV is likely mediated through up-regulation of β1 integrins in these cells (Fig. 4 B).

Anoikis is a process by which epithelial cells undergo programmed cell death when they detach from their substratum, as during cell invasion. Therefore, AKT2 expressing cells were also evaluated for their ability to survive after invasion and under nutrient-deprived conditions. The number of cells that detached from the matrix and established themselves on the bottom plastic surface was determined 2 weeks or 4 weeks after invasion. The survival rate postinvasion was significantly higher in the AKT2-expressing cells at 2 weeks (10-fold increase) and at 4 weeks (250-fold increase) compared with control-expressing cells (Table 3).

In summary, AKT2 cells preferentially invade through collagen IV, which is consistent with the ability to attach to, and spread efficiently on, this matrix. Secondly, increased cell invasion is a generic consequence of AKT2 overexpression in human breast and ovarian cancer cells. Thirdly, increased survival of AKT2-expressing cells postinvasion is consistent with the ability of AKT proteins to suppress apoptosis caused by anoikis and nutrient deprivation.

The Invasive Effect of AKT2 in Malignant Cells Is Still Dependent on Upstream PI3-K Activity.

AKT2 proteins are activated by binding to PIP3 and by phosphorylation of S474 and T309. Therefore, it is not clear whether overexpression of wild-type AKT2 is sufficient to lead to its activation. Accordingly, experiments were performed to determine whether the biological and biochemical effects associated with the AKT2 expression that was observed in the present studies remain dependent on upstream PI3-K activity. To evaluate this, the effects of an inhibitor of PI3-K, wortmannin, on cell invasiveness as well as on AKT2 biochemical activity were studied. Cells cultures were propagated and then split to allow for simultaneous biological and biochemical analyses and either were plated onto Matrigel-coated Boyden chambers to determine cell invasiveness or were lysed to determine AKT2 phosphorylation. Cell invasiveness, phosphorylation of AKT2, and intracellular AKT2 activity were suppressed in the presence of wortmannin (Fig. 5,A). Similar results were also observed with another PI3-K inhibitor, LY294002 (76% inhibition; P = 0.0005). In contrast, pretreatment with the MEK inhibitor PD098059 (Fig. 5A) led to a slight decrease in AKT phosphorylation in one clone (C27) but had minimal effects on invasion, which indicated that this pathway may not play a significant role in AKT2-induced invasion.

The PTEN phosphatase has been shown to down-regulate the levels of PIP3 generated by PI3-K activity in cells (32, 33). Expression of endogenous PTEN in the MDA-MB-435 cell line is lower compared with other cell lines (Fig. 1,B). Therefore, transient transfection of PTEN was performed to reduce the formation of PIP3 in these cells. PTEN expression blocked both AKT2 phosphorylation and invasion (Fig. 5 B). The concurrent and significant decreases in both AKT2 phosphorylation and cell invasion after exposure to wortmannin and by PTEN demonstrate that these two phenomena appear to be dependent on the upstream PI3-K activity. In malignant cells in which PI3 kinase activity appears to be already elevated, expression of wild-type AKT2 was sufficient to induce invasion.

Overexpression of AKT2, but not AKT1 or AKT3, was Sufficient to Duplicate the Invasive Effects of PI3-K Transfected in Breast Cancer Cells.

To circumvent the possibility that increased cell invasion by AKT2 is secondary to genetic changes associated with stable expression and selection of AKT2-expressing clones, MDA-MB-435HER-2 cells were used to transiently express various constructs to determine their potential role in cell invasion. Because HRG has been shown to activate the PI3-K pathway and induce cell migration in HER-2-overexpressing cells (34), HRG was used as a chemoattractant in these studies. MDA-MB-435HER-2 cells, cotransfected with AKT2 or wild-type PI3-K, all displayed increased invasive potential over control cells at 12 h, whereas dominant negative AKT2 (a kinase-deficient version, K181MAKT2, and a COOH-terminal 70-amino-acid-truncation mutant, C′70AKT2) blocked invasion (Fig. 6,A). Among the three AKT isoforms, increased invasion appears to be restricted to AKT2 because a direct comparison of AKT1, -2, and -3, when expressed at equivalent levels, showed minimal or no effects with expression of AKT1 and AKT3 (Fig. 6,B). In addition, although dominant negative proteins are believed to function by sequestering upstream activators, only dominant negative AKT2 (K181MAKT2 or C70AKT2) blocked invasion, whereas dominant negative/kinase-dead versions of AKT1 (K179M) or AKT3 (K177M) did not (Fig. 6,B). Because AKTs are known to form large multimeric complexes, dominant negative proteins may be interfering with the level of activity in these complexes, which are specific to AKT1, AKT2, or AKT3. In four separate experiments, transient expression of both wild-type and myristylated AKT1 in the MDA-MB-435HER-2 cells resulted in no increase in invasion. In fact, expression of myr-AKT1 was associated with a mild inhibitory effect on this process and is consistent with previous results (35). Despite the absence of AKT1-stimulatory effects on cell invasion, stable expression of AKT1 in the MDA-MB-435 cells yielded a 2-fold increase in cell motility in response to IGF-1 (data not shown), which is also consistent with the observation that AKT proteins can up-regulate IGF-1 receptor (36). In three independent experiments, in which myristylated p110α (the catalytic subunit of PI3-K) was transiently transfected or stably expressed in cells, little or no increase in cell invasion was detected (Fig. 3 B). In contrast, expression of wild-type p110α did lead to increases in cell invasion similar to, or greater than, that seen with AKT2 transfection. The inconsistency between the myrp110 and wtp110 in invasion assays may be attributable to the inability of the cells expressing myrp110 to establish proper cell polarity in response to growth factor (HRG) stimulation (37).

AKT2 Predominantly Localizes Adjacent to the Collagen IV Matrix during Cellular Attachment.

Differences in the pattern of subcellular distribution between AKT1 and AKT2, are most likely caused by variations in their pleckstrin homology (PH) domain (aa107–147), and have been previously reported in adipocytes (14) and muscle cells (19). AKT1GFP fusion proteins in a canine kidney cell line (MDCK) were reported to localize to regions of cell-cell and cell-matrix interactions (38). To localize the AKT2 protein, MDA-MB-435HER-2 cells were transiently transfected with either an AKT2YFP fusion construct or an AKT1YFP fusion construct for comparison. These cells were selected because they exhibited elevated levels of PI3-K activity attributable to overexpression of the HER-2 oncogene (39). Ligand stimulation of MDA-MB-435HER-2 cells expressing either AKT1YFP or AKT2YFP showed immediate relocalization of both AKT1YFP and AKT2YFP to the plasma membrane, confirming that both fusion proteins were capable of responding equivalently to growth factor stimulation (data not shown). To determine the location of AKT1 and AKT2, transfected cells were plated onto collagen IV, and confocal scanning laser microscopy was performed ∼1–2 h postplating. Confocal images reveal that AKT2 expression is shifted toward the basal end of cells closest to the collagen IV matrix, whereas little or no expression of AKT2 is detected on the apical end of these cells (Fig. 7,A). In contrast, the distribution of AKT1 is found primarily in the middle section of the cell and in the nucleus rather than regions of cell-matrix interactions (Fig. 7 B). The existence of differences between the subcellular localization of AKT1 and AKT2 when plated on collagen IV raises the possibility that AKT2 may be more sensitive to detecting collagen IV in the external environment.

AKT2-expressing Cells Exhibit Increased Metastatic Potential in Vivo.

To assess both tumorigenic and metastatic potential, MDA-MB-435AKT2 were injected i.p. into nude mice in three independent experiments. Necropsies, performed 7–10 weeks postinoculation, revealed widespread dissemination of tumor cells in animals that received injections of the AKT2-expressing clones, compared with controls (Fig. 8, A and B). The AKT2 transfectants readily formed multiple adherent and nonadherent metastatic nodules throughout the peritoneum after inoculation (Fig. 8,B). Conversely, mice that received injections of the parental MDA-MB-435 cell line developed a single tumor mass, or at most two solitary tumor masses, accompanied by rare, loose tumor-cell clusters in the peritoneal cavity (Fig. 8,A). As much as 2 cc of nonadherent metastatic nodules were collected by centrifugation from the ascites fluid of AKT2-inoculated animals as compared with ∼0.2 cc (a 10-fold decrease) from the ascites of the vector control-injected animals. The most dramatic changes were observed in the histological evaluation of the animals. Microscopic analysis of tissues from animals that received injections of AKT2-expressing cells, after 4 weeks showed significant tumor infiltration in the pancreas, stomach, and duodenum as well as multiple masses throughout the peritoneum, diaphragm, and thorax (Fig. 8,B). In the mediastinal cavity, small tumor masses were found surrounding the esophagus and the associated blood and lymphatic vessels, and in the lung parenchyma (Fig. 8, C and D). Approximately 80% of the mediastinal lymph node was replaced by malignant cells (Fig. 8,E). Although there was no evidence of malignant cells in the bone marrow or brain, tumor cell infiltrates were detected in the nasal and paranasal sinuses. Conversely, examination of animals inoculated with control cells found one to two tumor masses in the peritoneal cavity but revealed no evidence of tumor metastases in the mediastinum, larynx, thyroid, bone marrow, or brain at 6 weeks postinjection. Lung parenchymal involvement was found in 13 (87%) of 15 animals inoculated with the AKT2 cells in three independent experiments but in only 2 (13%) of 15 animals given injections of control cells (Tables 4 and 5). Similar effects were observed with the CaOV3AKT2 transfectants, demonstrating that this phenomenon was not restricted to a single malignant cell line (data not shown). Immunohistochemical analysis of the tumor tissue from these animals revealed positive staining for myc-tagged AKT2 (Fig. 8,F). Although i.p. injections of MDA-MB-435, a human breast cancer cell line, are not usually performed to study metastasis, this assay was initially selected to assess not only the ability of cells to metastasize to the lung but also their adhesive ability to various tissues in vivo. To further extend these observations using traditional metastasis studies, an orthotopic implantation model, closely resembling the course of human breast metastases, i.e., involvement of the draining regional lymph nodes and lungs, was also performed (40). Orthotopic injection of MDA-MB-435AKT2 cells into the mammary fat pads of athymic mice yielded similar increases of metastasis to the lung. Necropsies performed 4 weeks postimplantation revealed the presence of a primary tumor at the site of injection. In addition, gross metastatic deposits were found in both the peritoneal cavity and pulmonary cavities as well as in the lung parenchyma (Table 5). Conversely, MDA-MB-435 control cells injected in the same manner resulted in larger primary tumors at the site of implantation in the mammary fat pad; however, microscopic examinations revealed small tumor cell infiltrates restricted to the lining of the peritoneal cavity as the only evidence of metastatic disease (Table 5). These data indicate that an aggressive metastatic phenotype in vivo may be related to biological processes, such as increased invasion and survival, that appear to be directly associated with the AKT2/PI3-K pathway.

To determine the relative contribution of AKT2 to cell invasion and tumorigenicity, the highly invasive SKOV3 ovarian cancer cell line was transfected to stably express a dominant negative, kinase-dead version of AKT2 (K181MAKT2). This cell line was chosen because of its elevated AKT activity possibly attributable to amplification of p110α and HER-2 (Fig. 9,A). Stable expression of AKT2 in the SKOV3 ovarian cancer cell line increased invasion, whereas stable expression of kinase-deficient AKT2 decreased invasion (Fig. 9,B). To determine the effect of dominant negative AKT2 in vivo, SKOV3 vector control cells and SKOV3K181MAKT2 dominant negative cells were injected i.p. (orthotopic site for ovarian cancers) into SCID mice. A decrease in both tumorigenicity and metastatic spread was observed in animals injected with the dominant negative AKT2-expressing cell line (Fig. 9, C and D), indicating that AKT2 function is required for the tumorigenic growth of these cells.

The present study was directed at understanding the potential role, if any, of the PI3- kinase/AKT2 pathway in aggressive malignant cell behavior. Stable expression of AKT2 in eight breast and ovarian cancer cell lines increased their ability to invade through collagen IV, the major extracellular matrix component of basement membrane of most tissues and organs in humans. Several reports have confirmed that AKT plays a role in cell migration (36, 41, 42) and is thought to be important for maintaining morphological polarity during chemotaxis (37, 43). One study reported that AKT transfections promote invasion through up-regulation of the IGF-1 receptor (36). The present study indicates that another mechanism by which AKT2 may promote invasion is through increased expression of β1 integrins, a component of collagen IV-binding receptors. Increased β1 integrin expression has been shown to correlate with increased metastasis in some cancers (44, 45). AKT proteins can also regulate the synthesis of collagen IV (46), implying that an autocrine mechanism may be involved in AKT-induced cell invasion. Recent studies have also linked AKT activity with elevated levels of matrix metalloproteinases, demonstrating that AKT-expressing cells are also physically capable of degrading matrix (42, 47, 48). Whereas AKT1 is reported to participate in cell motility, only AKT2, in our studies using breast and ovarian cancer cells, was capable of duplicating the invasive effects of wild-type PI3-K. These data are similar to those of previous reports which have indicated that AKT1 does not mediate cell invasion in breast cancer cells (35, 49). The present study also indicates that AKT2 is an effector for PI3-K-dependent increases in cell invasion of human breast and ovarian cancer cells. On the basis of the observation that cell motility that is induced by many growth factors is dependent on active PI3-K signaling (34, 50, 51, 52), the role of AKT2 described in the present study may not be limited to breast and ovarian cancer cells. Stable expression of AKT2 in a pancreatic (Panc-1) and a prostate (PC3) cancer cell line also increases the invasive potential of these cells (Table 2).

Activated ras appears to influence metastatic ability by promoting cell proliferation while suppressing apoptosis at the secondary metastatic site (53). AKT2 proteins not only regulate cell migration and invasion but also the ability to suppress apoptosis postinvasion. However, AKT1 and AKT3 also contribute to tumor cell dissemination, survival, and proliferation. For example, the success of a newly established tumor to progress is also dependent on the ability of the tumor to recruit new vasculature, and AKT1 appears to regulate the production of vascular endothelial growth factor (54, 55). Furthermore, AKT3 overexpression correlates with hormone-unresponsive breast and prostate cancers, which are more aggressive and metastatic than their hormone-responsive counterparts (56). Consistent with these findings, an analysis of human breast cancer patients demonstrated that the presence of phosphorylated AKT in tumors correlates with an increase propensity to relapse with distant metastases (57). Overall, these findings suggest that activation of AKT1, AKT2, and AKT3 may be critical in coordinating events associated with late-stage tumor development and make the pathways in which they are involved a potential target for novel therapeutic interventions in human cancers.

Fig. 1.

Expression analysis of endogenous AKT2, PTEN, and transfected myc-tagged AKT2, PDK1, and p110α(PI3-K) in human breast and ovarian cancer cell lines. A, expression of endogenous levels of AKT2 in parent, nontransfected cells using an AKT2-specific antibody. For comparison, HOSE/AKT2C16, a normal cell line transfected with AKT2, which represents a 2–3-fold increase in expression, is shown as an indicator of the level of exogenous AKT2 expression used in the study. B, expression analysis to detect endogenous levels of PTEN in nontransfected cells using anti-PTEN antibody and to detect activation of AKT by phospho-specific antibody to Ser473 (which also recognizes Ser474 on AKT2). Only SKOV3 cell line has detectable levels of phosphorylated AKT. The AKT2-transfected cell line, HOSE/AKT2, shown as reference, also has elevated levels of phosphorylated AKT. A higher molecular-weight band in some lanes is caused by cross-reactivity to the PTEN antibody. C, Western blot analysis to detect myc-tagged AKT2 in the entire panel of AKT2 transfectants using anti-myc antibody. All of the lanes represent independent G418-resistant colonies. ∗, analyzed clones. D, expression of PDK1 in MDA-MB-435 cells transfected with full-length wild-type PDK1. E, phosphorylation of AKT in p110-transfected MDA-MB-435 cells. F, comparison of transfected AKT2 expression with natural amplified and overexpressing cell line, OVCAR-3. This was done to insure that the elevated levels in the AKT2-transfected cells were in a range similar to that seen in nature. Expression of AKT2 in clone 22 is significantly higher than is seen in the OVCAR-3 cell line and, therefore, was excluded in the analysis. G, photomicrograph of MDA-MB-435 cells stably expressing control vector (pcDNA3.1) or myc-tagged AKT2.

Fig. 1.

Expression analysis of endogenous AKT2, PTEN, and transfected myc-tagged AKT2, PDK1, and p110α(PI3-K) in human breast and ovarian cancer cell lines. A, expression of endogenous levels of AKT2 in parent, nontransfected cells using an AKT2-specific antibody. For comparison, HOSE/AKT2C16, a normal cell line transfected with AKT2, which represents a 2–3-fold increase in expression, is shown as an indicator of the level of exogenous AKT2 expression used in the study. B, expression analysis to detect endogenous levels of PTEN in nontransfected cells using anti-PTEN antibody and to detect activation of AKT by phospho-specific antibody to Ser473 (which also recognizes Ser474 on AKT2). Only SKOV3 cell line has detectable levels of phosphorylated AKT. The AKT2-transfected cell line, HOSE/AKT2, shown as reference, also has elevated levels of phosphorylated AKT. A higher molecular-weight band in some lanes is caused by cross-reactivity to the PTEN antibody. C, Western blot analysis to detect myc-tagged AKT2 in the entire panel of AKT2 transfectants using anti-myc antibody. All of the lanes represent independent G418-resistant colonies. ∗, analyzed clones. D, expression of PDK1 in MDA-MB-435 cells transfected with full-length wild-type PDK1. E, phosphorylation of AKT in p110-transfected MDA-MB-435 cells. F, comparison of transfected AKT2 expression with natural amplified and overexpressing cell line, OVCAR-3. This was done to insure that the elevated levels in the AKT2-transfected cells were in a range similar to that seen in nature. Expression of AKT2 in clone 22 is significantly higher than is seen in the OVCAR-3 cell line and, therefore, was excluded in the analysis. G, photomicrograph of MDA-MB-435 cells stably expressing control vector (pcDNA3.1) or myc-tagged AKT2.

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Fig. 2.

Increased phosphorylation and activation of AKT2 in the AKT2-transfected cell lines. In A, the level of AKT2 expression and phosphorylation in the CaOV3AKT2 transfectants was determined by Western blot analysis using an AKT2-specific antibody and a phospho-specific antibody to AKT that also recognizes Ser474 on AKT2. In vitro kinase assay using immunoprecipitated AKT2 and recombinant GSK3 as a substrate. Phosphorylated GSK3 was detected by Western blot analysis using a phospho-specific antibody to serine 9/21. Total GSK3 protein level is shown as control. In B, in vitro kinase assay performed with MDA-MB-435 AKT2 transfectants also show increased AKT2 activity.

Fig. 2.

Increased phosphorylation and activation of AKT2 in the AKT2-transfected cell lines. In A, the level of AKT2 expression and phosphorylation in the CaOV3AKT2 transfectants was determined by Western blot analysis using an AKT2-specific antibody and a phospho-specific antibody to AKT that also recognizes Ser474 on AKT2. In vitro kinase assay using immunoprecipitated AKT2 and recombinant GSK3 as a substrate. Phosphorylated GSK3 was detected by Western blot analysis using a phospho-specific antibody to serine 9/21. Total GSK3 protein level is shown as control. In B, in vitro kinase assay performed with MDA-MB-435 AKT2 transfectants also show increased AKT2 activity.

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Fig. 3.

Effect of AKT2 overexpression on cell attachment, motility, and invasion. In A, MDA-MB-435/vector and MDA-MB-435AKT2 cells, which were treated with 50 nm wortmannin for 30 min or transiently transfected with PTEN 72 h prior, were plated onto ultra-low attachment plates (Costar) coated with collagen IV. Increased attachment of AKT2-expressing cells to collagen IV was significantly greater because of the lack of nonspecific cell plating with these plates. Inhibition of PI3-K by both wortmannin and or by transient expression of the PTEN phosphatase blocked cell attachment. In B, MDA-MB-435 cells stably expressing AKT2, PDK1, myr-p110, and wt-p110 PI3 kinase were evaluated on their ability to invade through laminin, IV, collagen I, or fibronectin in Boyden chamber invasion assays. Both MDA-MB-435AKT2- and CaOV3AKT2-expressing cells preferentially invaded on collagen IV and to some extent on laminin.

Fig. 3.

Effect of AKT2 overexpression on cell attachment, motility, and invasion. In A, MDA-MB-435/vector and MDA-MB-435AKT2 cells, which were treated with 50 nm wortmannin for 30 min or transiently transfected with PTEN 72 h prior, were plated onto ultra-low attachment plates (Costar) coated with collagen IV. Increased attachment of AKT2-expressing cells to collagen IV was significantly greater because of the lack of nonspecific cell plating with these plates. Inhibition of PI3-K by both wortmannin and or by transient expression of the PTEN phosphatase blocked cell attachment. In B, MDA-MB-435 cells stably expressing AKT2, PDK1, myr-p110, and wt-p110 PI3 kinase were evaluated on their ability to invade through laminin, IV, collagen I, or fibronectin in Boyden chamber invasion assays. Both MDA-MB-435AKT2- and CaOV3AKT2-expressing cells preferentially invaded on collagen IV and to some extent on laminin.

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Fig. 4.

Increased expression of collagen IV-binding receptors in MDA-MB-435AKT2 cells. A, Western blot analysis using anti-β1 integrin antibody. B, cells were pretreated with equivalent concentrations of control IgG or β1 function blocking monoclonal antibody. Invasion of AKT2-expressing cells through collagen IV is blocked to control levels with an antibody against β1 integrins.

Fig. 4.

Increased expression of collagen IV-binding receptors in MDA-MB-435AKT2 cells. A, Western blot analysis using anti-β1 integrin antibody. B, cells were pretreated with equivalent concentrations of control IgG or β1 function blocking monoclonal antibody. Invasion of AKT2-expressing cells through collagen IV is blocked to control levels with an antibody against β1 integrins.

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Fig. 5.

AKT2-induced invasion is dependent on PI3-K activity. In A, cells MDA-MB-435 control, MDA-MB-435 AKT2C27, and MDA-MB-435AKT2C28 were treated with DMSO control, 50 μm PD098059 (MEK inhibitor), or 30 nm wortmannin (PI3-K inhibitor) in serum-free medium for 90 min before invasion assay on Matrigel-coated Boyden chambers. Exposure to wortmannin blocked AKT2-induced cell invasion but exposure to the MEK inhibitor did not. Western blot analysis using phospho-specific antibody to Ser473 (first panel) or AKT2-specific antibody (second panel) indicated that a reduction in AKT2 phosphorylation and activity (third panel) correlated with a reduction in invasion. Fourth panel shows level of GSK3 protein used in the kinase assay. In B, transient expression of PTEN phosphatase into AKT2 expressing cells also blocked cell invasion and to some extent blocked AKT2 S473 phosphorylation (bottom panel).

Fig. 5.

AKT2-induced invasion is dependent on PI3-K activity. In A, cells MDA-MB-435 control, MDA-MB-435 AKT2C27, and MDA-MB-435AKT2C28 were treated with DMSO control, 50 μm PD098059 (MEK inhibitor), or 30 nm wortmannin (PI3-K inhibitor) in serum-free medium for 90 min before invasion assay on Matrigel-coated Boyden chambers. Exposure to wortmannin blocked AKT2-induced cell invasion but exposure to the MEK inhibitor did not. Western blot analysis using phospho-specific antibody to Ser473 (first panel) or AKT2-specific antibody (second panel) indicated that a reduction in AKT2 phosphorylation and activity (third panel) correlated with a reduction in invasion. Fourth panel shows level of GSK3 protein used in the kinase assay. In B, transient expression of PTEN phosphatase into AKT2 expressing cells also blocked cell invasion and to some extent blocked AKT2 S473 phosphorylation (bottom panel).

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Fig. 6.

Exogenous expression of AKT2 was sufficient to duplicate the invasive effects of PI3-K in breast cancer cells. MDA-MB-435HER-2 cells, which have elevated PI3-K activity because of overexpression of HER-2, was chosen to study the effects of various genes in inducing or inhibiting cell invasion. A, 2 μg of various myc epitope-tagged constructs, all of them driven by the cytomegalovirus (CMV) promoter, were transiently transfected 72 h before invasion assays. pCDNA3.1 was used as control vector for the transfection experiments. Approximately 40% of MDA-MB-435HER-2 were transfected as determined by control green fluorescent protein (GFP) plasmid. Cell invasion assays were performed with HRG (1 nm) as a chemoattractant. MDA-MB-435HER-2 cells, transfected with wt PI3-K(p110α) or AKT2, displayed increased invasive potential compared with control, whereas cells expressing dominant negative AKT2 (kinase-deficient AKT2K181M or C′70 term-truncated mutant of AKT2) displayed reduced invasive potential. No effect on invasion was detected with expression of myr-AKT1, AKT1, or dominant negative AKT1. B, MDA-MB-435HER-2 cells were transfected with wild-type AKT1, AKT2, AKT3, or kinase-dead AKT1(K179M), AKT2(K181M), AKT3 (K177M). Although a weak increase in invasion was detected with AKT3, only the expression of AKT2 resulted in a significant increase in cell invasion. As controls, expression of all AKT constructs were confirmed using a pan-AKT antibody that recognizes all three isoforms of AKT (transfected and endogenous) and by anti-myc antibody, which recognizes the epitope tag on all three of the transfected forms of AKT.

Fig. 6.

Exogenous expression of AKT2 was sufficient to duplicate the invasive effects of PI3-K in breast cancer cells. MDA-MB-435HER-2 cells, which have elevated PI3-K activity because of overexpression of HER-2, was chosen to study the effects of various genes in inducing or inhibiting cell invasion. A, 2 μg of various myc epitope-tagged constructs, all of them driven by the cytomegalovirus (CMV) promoter, were transiently transfected 72 h before invasion assays. pCDNA3.1 was used as control vector for the transfection experiments. Approximately 40% of MDA-MB-435HER-2 were transfected as determined by control green fluorescent protein (GFP) plasmid. Cell invasion assays were performed with HRG (1 nm) as a chemoattractant. MDA-MB-435HER-2 cells, transfected with wt PI3-K(p110α) or AKT2, displayed increased invasive potential compared with control, whereas cells expressing dominant negative AKT2 (kinase-deficient AKT2K181M or C′70 term-truncated mutant of AKT2) displayed reduced invasive potential. No effect on invasion was detected with expression of myr-AKT1, AKT1, or dominant negative AKT1. B, MDA-MB-435HER-2 cells were transfected with wild-type AKT1, AKT2, AKT3, or kinase-dead AKT1(K179M), AKT2(K181M), AKT3 (K177M). Although a weak increase in invasion was detected with AKT3, only the expression of AKT2 resulted in a significant increase in cell invasion. As controls, expression of all AKT constructs were confirmed using a pan-AKT antibody that recognizes all three isoforms of AKT (transfected and endogenous) and by anti-myc antibody, which recognizes the epitope tag on all three of the transfected forms of AKT.

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Fig. 7.

Cellular distribution of AKT2YFP and AKT1YFP in live cells using confocal microscopy. MDA-MB-435HER-2 cells were transiently transfected with either pEYFP fusion construct of AKT1 or AKT2. Cells were then plated onto collagen IV-coated coverslips. Confocal microscopy was performed within 1 h after plating. A and B, far left panel, a section through the basal end of the cell closer to the collagen IV matrix. Middle left panel, the middle section of the cell. Middle right panel, the apical end of the cell. Far right panel, the composite figure of the cell. In A, cellular distribution of AKT2YFP is found predominantly in the basal end of the cell adjacent to the collagen IV matrix. In B, cellular distribution of AKT1YFP is evenly distributed throughout the cell with most of the AKT1YFP being present in the middle section of the cell.

Fig. 7.

Cellular distribution of AKT2YFP and AKT1YFP in live cells using confocal microscopy. MDA-MB-435HER-2 cells were transiently transfected with either pEYFP fusion construct of AKT1 or AKT2. Cells were then plated onto collagen IV-coated coverslips. Confocal microscopy was performed within 1 h after plating. A and B, far left panel, a section through the basal end of the cell closer to the collagen IV matrix. Middle left panel, the middle section of the cell. Middle right panel, the apical end of the cell. Far right panel, the composite figure of the cell. In A, cellular distribution of AKT2YFP is found predominantly in the basal end of the cell adjacent to the collagen IV matrix. In B, cellular distribution of AKT1YFP is evenly distributed throughout the cell with most of the AKT1YFP being present in the middle section of the cell.

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Fig. 8.

Growth of MDA-MB-435/vector and MDA-MB-435AKT2 injected i.p. into nude mice. In A, growth of MDA-MB-435/vector control cell line remains localized; top panel, tumor growth in peritoneal cavity after 30 days, bottom panel, tumor growth in peritoneal cavity after 60 days. In B, growth of MDA-MB-435AKT2 cell line is dispersed as small tumor nodules (yellow arrow) throughout the peritoneal cavity after 30 days (top left panel) and 60 days (bottom left panel). Evidence of tumor cells invading the diaphragm (top right panel) or obstructing the intestines (bottom right panel) were frequently present. Lungs removed from MDA-MB-435AKT2-injected mice displayed tumor lesions on the surface and were often larger and fluid filled because of tumor emboli (middle inset). In D, H&E stain confirmed the presence of tumor cells in the blood vessels of lung tissue (black arrows) from mice that were given injections of AKT2-overexpressing cells compared with lung tissue from mice that were given injections of control cells (C). In E, also present in animals that received injections of AKT2-overexpressing cells are tumor cells that have completely filled 80% of the mediastinal lymph node (black arrow), indicating distant metastases. BV, blood vessels; LN, lymph node; Tumor, MDA-MB-435AKT2 cells. In F, H&E staining of tumor removed from mice that were given injections i.p. with AKT2-overexpressing cells (top panel), and immunohistochemistry performed with anti-myc antibody that recognizes myc epitope-tagged AKT2 confirmed the presence of transfected AKT2 in the tumor cells (bottom panel). Tumor, MDA-MB-435AKT2 cells.

Fig. 8.

Growth of MDA-MB-435/vector and MDA-MB-435AKT2 injected i.p. into nude mice. In A, growth of MDA-MB-435/vector control cell line remains localized; top panel, tumor growth in peritoneal cavity after 30 days, bottom panel, tumor growth in peritoneal cavity after 60 days. In B, growth of MDA-MB-435AKT2 cell line is dispersed as small tumor nodules (yellow arrow) throughout the peritoneal cavity after 30 days (top left panel) and 60 days (bottom left panel). Evidence of tumor cells invading the diaphragm (top right panel) or obstructing the intestines (bottom right panel) were frequently present. Lungs removed from MDA-MB-435AKT2-injected mice displayed tumor lesions on the surface and were often larger and fluid filled because of tumor emboli (middle inset). In D, H&E stain confirmed the presence of tumor cells in the blood vessels of lung tissue (black arrows) from mice that were given injections of AKT2-overexpressing cells compared with lung tissue from mice that were given injections of control cells (C). In E, also present in animals that received injections of AKT2-overexpressing cells are tumor cells that have completely filled 80% of the mediastinal lymph node (black arrow), indicating distant metastases. BV, blood vessels; LN, lymph node; Tumor, MDA-MB-435AKT2 cells. In F, H&E staining of tumor removed from mice that were given injections i.p. with AKT2-overexpressing cells (top panel), and immunohistochemistry performed with anti-myc antibody that recognizes myc epitope-tagged AKT2 confirmed the presence of transfected AKT2 in the tumor cells (bottom panel). Tumor, MDA-MB-435AKT2 cells.

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Fig. 9.

Effect of kinase-dead AKT2 in an invasive ovarian cancer cell line, SKOV3. In A, because of amplification of both HER-2 and PIK3CA (PI3-K) genes, phosphorylation of AKT is elevated in the SKOV3 cell line as compared with normal ovarian surface epithelial cells (HOSE). Stable expression levels of dominant negative AKT2(K181M) in clones 6, 7, and 14 in the SKOV3 cell line. In B, expression of dominant negative AKT2 in the SKOV3 cell line blocked invasion through collagen IV. Although a few cells invaded with clones 6 and 7, no surviving cells were detected after 48 h (bottom panel). In C, five animals per group (SCID mice) were given injections of 5 × 105 cells i.p. Necropsy of SKOV3control cell-injected mouse (left panel) and SKOV3(K181M)AKT2C14 cell-injected mouse (right panel) performed at 2.5 weeks show the presence of a large tumor mass in the control mouse and a smaller tumor mass in the dominant negative AKT2-injected mouse. White arrows, tumor mass. All of the SKOV3 control cell line-injected animals died approximately in 2.5 weeks with very large tumors in the peritoneal cavity. Mice that were given injections of SKOV3(K181M) AKT2 cells survived 5–6 weeks bearing only very small tumors as shown. In D, approximate tumor volume from tumors removed at 2.5 weeks from mouse injected with SKOV3 control cells and mouse injected with SKOV3AKT2K181M cells (cm scale).

Fig. 9.

Effect of kinase-dead AKT2 in an invasive ovarian cancer cell line, SKOV3. In A, because of amplification of both HER-2 and PIK3CA (PI3-K) genes, phosphorylation of AKT is elevated in the SKOV3 cell line as compared with normal ovarian surface epithelial cells (HOSE). Stable expression levels of dominant negative AKT2(K181M) in clones 6, 7, and 14 in the SKOV3 cell line. In B, expression of dominant negative AKT2 in the SKOV3 cell line blocked invasion through collagen IV. Although a few cells invaded with clones 6 and 7, no surviving cells were detected after 48 h (bottom panel). In C, five animals per group (SCID mice) were given injections of 5 × 105 cells i.p. Necropsy of SKOV3control cell-injected mouse (left panel) and SKOV3(K181M)AKT2C14 cell-injected mouse (right panel) performed at 2.5 weeks show the presence of a large tumor mass in the control mouse and a smaller tumor mass in the dominant negative AKT2-injected mouse. White arrows, tumor mass. All of the SKOV3 control cell line-injected animals died approximately in 2.5 weeks with very large tumors in the peritoneal cavity. Mice that were given injections of SKOV3(K181M) AKT2 cells survived 5–6 weeks bearing only very small tumors as shown. In D, approximate tumor volume from tumors removed at 2.5 weeks from mouse injected with SKOV3 control cells and mouse injected with SKOV3AKT2K181M cells (cm scale).

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

1

Supported by a grant from the UCLA/Revlon Women’s Cancer Research Program and by NIH Grant P01CA32737-14.

3

The abbreviations used are: PI3-K, phosphoinositide 3-OH kinase; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PDK1, PIP3-dependent kinase 1; PTEN, phosphatase and tensin homologue tumor suppressor; HER-2, human epidermal growth factor receptor-2; HRG, heregulin; MEK, MAPK kinase; EYFP, enhanced yellow fluorescent protein; MAPK, mitogen-activated protein kinase; IGF, insulin-like growth factor; PKB, protein kinase B; aa, amino acid; RT-PCR, reverse transcription-PCR; HOSE, human ovarian surface epithelial (cell); SCID, severe combined immunodeficient.

Table 1

Effect of AKT2 expression on cellular attachment to various matrices

Cells were added to plates precoated with various matrices (Becton Dickenson) and counted 3 h after plating. The number of cells counted is the average of three wells. The percentage of controls is listed within parentheses. The relative expression of AKT2 is with respect to nontransfected control cell line.

Cell lineRelative expression to controlsNo. of cells attached to matrix × 106 (% of controls)
Collagen ICollagen IVLamininFibronectin
435 control 3.34 (0%) 3.18 (0%) 1.48 (0%) 4.52 (0%) 
AKT2C27 4.44 (+33%) 4.13 (+30%) 2.00 (+35%) 4.77 (+5%) 
AKT2C28 4.28 (+28%) 4.21 (+32%) 2.55 (+72%) 2.19 (−50%) 
AKT2C23 7.45 (+123%) 5.65 (+78%) 5.20 (+250%) 4.87 (+8%) 
CaOV3 control 1.31 (0%) 1.34 (0%) 0.621 (0%) 0.951 (0%) 
AKT2C13 0.5 1.34 (+2%) 1.25 (−7%) 0.776 (+25%) 0.756 (−19%) 
AKT2C9 1.50 (+15%) 1.55 (+15%) 1.05 (+68%) 1.39 (+46%) 
AKT2C6 1.68 (+28%) 1.95 (+45%) 1.41 (+127%) 0.479 (−50%) 
Cell lineRelative expression to controlsNo. of cells attached to matrix × 106 (% of controls)
Collagen ICollagen IVLamininFibronectin
435 control 3.34 (0%) 3.18 (0%) 1.48 (0%) 4.52 (0%) 
AKT2C27 4.44 (+33%) 4.13 (+30%) 2.00 (+35%) 4.77 (+5%) 
AKT2C28 4.28 (+28%) 4.21 (+32%) 2.55 (+72%) 2.19 (−50%) 
AKT2C23 7.45 (+123%) 5.65 (+78%) 5.20 (+250%) 4.87 (+8%) 
CaOV3 control 1.31 (0%) 1.34 (0%) 0.621 (0%) 0.951 (0%) 
AKT2C13 0.5 1.34 (+2%) 1.25 (−7%) 0.776 (+25%) 0.756 (−19%) 
AKT2C9 1.50 (+15%) 1.55 (+15%) 1.05 (+68%) 1.39 (+46%) 
AKT2C6 1.68 (+28%) 1.95 (+45%) 1.41 (+127%) 0.479 (−50%) 
Table 2

AKT2 effects on cell invasion

AKT2-expressing clones were assayed on either Matrigel-coated or collagen IV-coated Boyden chambers. Statistical analysis was performed by taking the average of all of the AKT2-expressing clones invaded versus vector control group for three independent experiments. The number of clones analyzed for each cell line: five clones were analyzed for CaOV3 and MDA-MB-435; three clones for MDA-MB-231, T47D, SKOV3, SKBR3, and PC3; two clones for MCF7, PANC-1 and OV2008. For the normal cell lines, five clones were analyzed for HOSE, and two clones were analyzed for HBL-100.

Cell lineTissueAverage fold increase invasion over controlsP
Malignant cells    
 CaOV3 Ovarian 13.6 0.007 
 MDA-MB-435 Breast 5.5 0.0001 
 MDA-MB-231 Breast 4.4 0.11 
 T47D Breast 2.9 0.07 
 SKOV3 Ovarian 2.3 0.07 
 SKBR3 Breast 2.3 0.0003 
 OV2008 Ovarian 1.7 0.19 
 MCF-7 Breast 1.4 0.38 
 PANC-1 Pancreas 2.8 0.007 
 PC3 Prostate 1.5 0.08 
Normal cells    
 HBL-100 Breast No invasion  
 HOSE Ovarian No invasion  
Cell lineTissueAverage fold increase invasion over controlsP
Malignant cells    
 CaOV3 Ovarian 13.6 0.007 
 MDA-MB-435 Breast 5.5 0.0001 
 MDA-MB-231 Breast 4.4 0.11 
 T47D Breast 2.9 0.07 
 SKOV3 Ovarian 2.3 0.07 
 SKBR3 Breast 2.3 0.0003 
 OV2008 Ovarian 1.7 0.19 
 MCF-7 Breast 1.4 0.38 
 PANC-1 Pancreas 2.8 0.007 
 PC3 Prostate 1.5 0.08 
Normal cells    
 HBL-100 Breast No invasion  
 HOSE Ovarian No invasion  
Table 3

Effect of AKT2 expression on long-term survival postinvasion

MDA-MB-435 control cells and MDA-MB-435AKT2C23 cells were plated onto Matrigel-coated invasion chambers. The number of cells invading and surviving were counted 2 weeks and 4 weeks postinvasion. P are given in parentheses.

Cell linesTimeNo. of cells surviving postinvasion
MDA-MB-435 control 2 wk 20 ± 5.1 
 4 wk 9 ± 5.7 
435AKT2C23 2 wk 199 ± 53 (P = 0.04) 
 4 wk 2261 ± 377.6 (P = 0.01) 
Cell linesTimeNo. of cells surviving postinvasion
MDA-MB-435 control 2 wk 20 ± 5.1 
 4 wk 9 ± 5.7 
435AKT2C23 2 wk 199 ± 53 (P = 0.04) 
 4 wk 2261 ± 377.6 (P = 0.01) 
Table 4

Effect of AKT2 overexpression on metastasis to the lung with MDA-MB-435 cells

In this assay, mice were given injections intraperitoneally. The animals were sacrificed 4–6 weeks after the injection, and lung tissues were removed, formalin fixed, and stained with H&E. Any evidence of tumor present on the surface of the lung or in any sections of the lung was scored as positive for lung metastasis. The number of colonies were not counted in this assay because tumor cells were often found as a single large mass or as occupying a large percentage of the lung tissue.

Cell lineIntraperitoneal injections (1 × 106 cells)
Relative AKT2 expression to controlsNo. of miceNo. of affected lungs
Control  15 
AKT2C28 10 
AKT2C23 
Cell lineIntraperitoneal injections (1 × 106 cells)
Relative AKT2 expression to controlsNo. of miceNo. of affected lungs
Control  15 
AKT2C28 10 
AKT2C23 
Table 5

Orthotopic injections into mammary fat pads (1 × 106 cells)

In this assay mice were given injections orthotopically into the mammary fat pads. The animals were sacrificed 4–6 weeks after the injection, and lung tissues were removed, formalin fixed, and stained with H&E. In this model, lung metastases were quantitated by the number of tumor cell colonies present in one 5-μm section of the lung.

Cell lineRelative AKT2 expression to controlsNo. of lungsNo. of tumor colonies (per 5-μm section)P
Control  0 ± 0.147  
AKT2C28 6.5 ± 1.52 0.0019 
Cell lineRelative AKT2 expression to controlsNo. of lungsNo. of tumor colonies (per 5-μm section)P
Control  0 ± 0.147  
AKT2C28 6.5 ± 1.52 0.0019 

We thank Drs. Gottfried Konecny, Giovanni Pauletti, and Malgorzata Beryt for critically reading the manuscript.

1
Gershtein E. S., Shatskaya V. A., Ermilova V. D., Kushlinsky N. E., Krasil’nikov M. A. Phosphatidylinositol 3-kinase expression in human breast cancer.
Clin. Chim. Acta
,
287
:
59
-67,  
1999
.
2
Philp A. J., Campbell I. G., Leet C., Vincan E., Rockman S. P., Whitehead R. H., Thomas R. J., Phillips W. A. The phosphatidylinositol 3′-kinase p85α gene is an oncogene in human ovarian and colon tumors.
Cancer Res.
,
61
:
7426
-7429,  
2001
.
3
Shayesteh L., Lu Y., Kuo W. L., Baldocchi R., Godfrey T., Collins C., Pinkel D., Powell B., Mills G. B., Gray J. W. PIK3CA is implicated as an oncogene in ovarian cancer[see comments].
Nat. Genet.
,
21
:
99
-102,  
1999
.
4
Slamon D. J., Clark G. M., Wong S. G., Levin W. J., Ullrich A., McGuire W. L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.
Science (Wash. DC)
,
235
:
177
-182,  
1987
.
5
Moscatello D. K., Holgado-Madruga M., Emlet D. R., Montgomery R. B., Wong A. J. Constitutive activation of phosphatidylinositol 3-kinase by a naturally occurring mutant epidermal growth factor receptor.
J. Biol. Chem.
,
273
:
200
-206,  
1998
.
6
Cantley L. C., Neel B. G. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.
Proc. Natl. Acad. Sci. USA
,
96
:
4240
-4245,  
1999
.
7
Zhou X. P., Gimm O., Hampel H., Niemann T., Walker M. J., Eng C. Epigenetic PTEN silencing in malignant melanomas without PTEN mutation.
Am. J. Pathol.
,
157
:
1123
-1128,  
2000
.
8
Rodriguez-Viciana P., Warne P. H., Dhand R., Vanhaesebroeck B., Gout I., Fry M. J., Waterfield M. D., Downward J. Phosphatidylinositol-3-OH kinase as a direct target of Ras[see comments].
Nature (Lond.)
,
370
:
527
-532,  
1994
.
9
Burgering B. M., Coffer P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction[see comments].
Nature (Lond.)
,
376
:
599
-602,  
1995
.
10
Liu A. X., Testa J. R., Hamilton T. C., Jove R., Nicosia S. V., Cheng J. Q. AKT2, a member of the protein kinase B family, is activated by growth factors, v-Ha-ras, and v-src through phosphatidylinositol 3-kinase in human ovarian epithelial cancer cells.
Cancer Res.
,
58
:
2973
-2977,  
1998
.
11
Liu W., Li J., Roth R. A. Heregulin regulation of Akt/protein kinase B in breast cancer cells.
Biochem. Biophys. Res. Commun.
,
261
:
897
-903,  
1999
.
12
Coffer P. J., Jin J., Woodgett J. R. Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation.
Biochem. J.
,
335
:
1
-13,  
1998
.
13
Mitsuuchi Y., Johnson S. W., Moonblatt S., Testa J. R. Translocation and activation of AKT2 in response to stimulation by insulin.
J. Cell. Biochem.
,
70
:
433
-441,  
1998
.
14
Calera M. R., Martinez C., Liu H., Jack A. K., Birnbaum M. J., Pilch P. F. Insulin increases the association of Akt-2 with Glut4-containing vesicles.
J. Biol. Chem.
,
273
:
7201
-7204,  
1998
.
15
Hill M. M., Clark S. F., Tucker D. F., Birnbaum M. J., James D. E., Macaulay S. L. A role for protein kinase Bβ/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes.
Mol. Cell. Biol.
,
19
:
7771
-7781,  
1999
.
16
Turinsky J., Damrau-Abney A. Akt kinases and 2-deoxyglucose uptake in rat skeletal muscles in vivo: study with insulin and exercise.
Am. J. Physiol.
,
276
:
R277
-R282,  
1999
.
17
Walker K. S., Deak M., Paterson A., Hudson K., Cohen P., Alessi D. R. Activation of protein kinase Bβ and γ isoforms by insulin in vivo and by 3-phosphoinositide-dependent protein kinase-1 in vitro: comparison with protein kinase Bα.
Biochem. J.
,
331
:
299
-308,  
1998
.
18
Cheng J. Q., Altomare D. A., Klein M. A., Lee W. C., Kruh G. D., Lissy N. A., Testa J. R. Transforming activity and mitosis-related expression of the AKT2 oncogene: evidence suggesting a link between cell cycle regulation and oncogenesis.
Oncogene
,
14
:
2793
-2801,  
1997
.
19
Vandromme M., Rochat A., Meier R., Carnac G., Besser D., Hemmings B. A., Fernandez A., Lamb N. J. Protein kinase Bβ/Akt2 plays a specific role in muscle differentiation.
J. Biol. Chem.
,
276
:
8173
-8179,  
2001
.
20
Bellacosa A., de Feo D., Godwin A. K., Bell D. W., Cheng J. Q., Altomare D. A., Wan M., Dubeau L., Scambia G., Masciullo V., et al Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas.
Int. J. Cancer
,
64
:
280
-285,  
1995
.
21
Miwa W., Yasuda J., Murakami Y., Yashima K., Sugano K., Sekine T., Kono A., Egawa S., Yamaguchi K., Hayashizaki Y., Sekiya T. Isolation of DNA sequences amplified at chromosome 19q13.1-q13.2 including the AKT2 locus in human pancreatic cancer.
Biochem. Biophys. Res. Commun.
,
225
:
968
-974,  
1996
.
22
Yuan Z. Q., Sun M., Feldman R. I., Wang G., Ma X., Jiang C., Coppola D., Nicosia S. V., Cheng J. Q. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.
Oncogene
,
19
:
2324
-2330,  
2000
.
23
Roy H. K., Olusola B. F., Clemens D. L., Karolski W. J., Ratashak A., Lynch H. T., Smyrk T. C. AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis.
Carcinogenesis (Lond.)
,
23
:
201
-205,  
2002
.
24
Ruggeri B. A., Huang L., Wood M., Cheng J. Q., Testa J. R. Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas.
Mol. Carcinog.
,
21
:
81
-86,  
1998
.
25
Cho H., Mu J., Kim J. K., Thorvaldsen J. L., Chu Q., Crenshaw E. B., III, Kaestner K. H., Bartolomei M. S., Shulman G. I., Birnbaum M. J. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKBβ).
Science (Wash. DC)
,
292
:
1728
-1731,  
2001
.
26
Cheng J. Q., Ruggeri B., Klein W. M., Sonoda G., Altomare D. A., Watson D. K., Testa J. R. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.
Proc. Natl. Acad. Sci. USA
,
93
:
3636
-3641,  
1996
.
27
Arboleda M. J., Eberwein D., Hibner B., Lyons J. F. Dominant negative mutants of mitogen-activated protein kinase pathway.
Methods Enzymol.
,
332
:
353
-367,  
2001
.
28
Konecny G., Untch M., Arboleda J., Wilson C., Kahlert S., Boettcher B., Felber M., Beryt M., Lude S., Hepp H., Slamon D., Pegram M. Her-2/neu and urokinase-type plasminogen activator and its inhibitor in breast cancer.
Clin. Cancer Res.
,
7
:
2448
-2457,  
2001
.
29
Casey R. C., Burleson K. M., Skubitz K. M., Pambuccian S. E., Oegema T. R., Jr., Ruff L. E., Skubitz A. P. β1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids.
Am. J. Pathol.
,
159
:
2071
-2080,  
2001
.
30
Elices M. J., Hemler M. E. The human integrin VLA-2 is a collagen receptor on some cells and a collagen/laminin receptor on others.
Proc. Natl. Acad. Sci. USA
,
86
:
9906
-9910,  
1989
.
31
Nykvist P., Tu H., Ivaska J., Kapyla J., Pihlajaniemi T., Heino J. Distinct recognition of collagen subtypes by α1β1 and α2β1 integrins. α1β1 mediates cell adhesion to type XIII collagen.
J. Biol. Chem.
,
275
:
8255
-8261,  
2000
.
32
Maehama T., Dixon J. E. The tumor suppressor. PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate.
J. Biol. Chem.
,
273
:
13375
-13378,  
1998
.
33
Stambolic V., Suzuki A., de la Pompa J. L., Brothers G. M., Mirtsos C., Sasaki T., Ruland J., Penninger J. M., Siderovski D. P., Mak T. W. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.
Cell
,
95
:
29
-39,  
1998
.
34
Adam L., Vadlamudi R., Kondapaka S. B., Chernoff J., Mendelsohn J., Kumar R. Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol-3 kinase.
J. Biol. Chem.
,
273
:
28238
-28246,  
1998
.
35
Shaw L. M., Rabinovitz I., Wang H. H., Toker A., Mercurio A. M. Activation of phosphoinositide 3-OH kinase by the α6β4 integrin promotes carcinoma invasion.
Cell
,
91
:
949
-960,  
1997
.
36
Tanno S., Mitsuuchi Y., Altomare D. A., Xiao G. H., Testa J. R. AKT activation up-regulates insulin-like growth factor I receptor expression and promotes invasiveness of human pancreatic cancer cells.
Cancer Res.
,
61
:
589
-593,  
2001
.
37
Chung C. Y., Funamoto S., Firtel R. A. Signaling pathways controlling cell polarity and chemotaxis.
Trends Biochem. Sci.
,
26
:
557
-566,  
2001
.
38
Watton S. J., Downward J. Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction.
Curr. Biol.
,
9
:
433
-436,  
1999
.
39
Peles E., Lamprecht R., Ben-Levy R., Tzahar E., Yarden Y. Regulated coupling of the Neu receptor to phosphatidylinositol 3′- kinase and its release by oncogenic activation.
J. Biol. Chem.
,
267
:
12266
-12274,  
1992
.
40
Leone A., Flatow U., VanHoutte K., Steeg P. S. Transfection of human nm23-H1 into the human MDA-MB-435 breast carcinoma cell line: effects on tumor metastatic potential, colonization and enzymatic activity.
Oncogene
,
8
:
2325
-2333,  
1993
.
41
Meili R., Ellsworth C., Lee S., Reddy T. B., Ma H., Firtel R. A. Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium.
EMBO J.
,
18
:
2092
-2105,  
1999
.
42
Park B. K., Zeng X., Glazer R. I. Akt1 induces extracellular matrix invasion and matrix metalloproteinase-2 activity in mouse mammary epithelial cells.
Cancer Res.
,
61
:
7647
-7653,  
2001
.
43
Servant G., Weiner O. D., Herzmark P., Balla T., Sedat J. W., Bourne H. R. Polarization of chemoattractant receptor signaling during neutrophil chemotaxis[see comments].
Science (Wash. DC)
,
287
:
1037
-1040,  
2000
.
44
Fujita S., Watanabe M., Kubota T., Teramoto T., Kitajima M. Alteration of expression in integrin β1-subunit correlates with invasion and metastasis in colorectal cancer.
Cancer Lett.
,
91
:
145
-149,  
1995
.
45
Morini M., Mottolese M., Ferrari N., Ghiorzo F., Buglioni S., Mortarini R., Noonan D. M., Natali P. G., Albini A. The α3β1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity.
Int. J. Cancer
,
87
:
336
-342,  
2000
.
46
Li X., Talts U., Talts J. F., Arman E., Ekblom P., Lonai P. Akt/PKB regulates laminin and collagen IV isotypes of the basement membrane.
Proc. Natl. Acad. Sci. USA
,
98
:
14416
-14421,  
2001
.
47
Kubiatowski T., Jang T., Lachyankar M. B., Salmonsen R., Nabi R. R., Quesenberry P. J., Litofsky N. S., Ross A. H., Recht L. D. Association of increased phosphatidylinositol 3-kinase signaling with increased invasiveness and gelatinase activity in malignant gliomas.
J. Neurosurg.
,
95
:
480
-488,  
2001
.
48
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.
FASEB J.
,
15
:
1953
-1962,  
2001
.
49
Keely P. J., Westwick J. K., Whitehead I. P., Der C. J., Parise L. V. Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K.
Nature (Lond.)
,
390
:
632
-636,  
1997
.
50
Bakin A. V., Tomlinson A. K., Bhowmick N. A., Moses H. L., Arteaga C. L. Phosphatidylinositol-3 kinase function is required for TGF-β-mediated epithelial to mesenchymal transition and cell migration.
J. Biol. Chem.
,
275
:
36803
-36810,  
2000
.
51
Bardelli A., Basile M. L., Audero E., Giordano S., Wennstrom S., Menard S., Comoglio P. M., Ponzetto C. Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis.
Oncogene
,
18
:
1139
-1146,  
1999
.
52
Nakanishi K., Fujimoto J., Ueki T., Kishimoto K., Hashimoto-Tamaoki T., Furuyama J., Itoh T., Sasaki Y., Okamoto E. Hepatocyte growth factor promotes migration of human hepatocellular carcinoma via phosphatidylinositol 3-kinase.
Clin. Exp. Metastasis
,
17
:
507
-514,  
1999
.
53
Varghese H. J., Davidson M. T., MacDonald I. C., Wilson S. M., Nadkarni K. V., Groom A. C., Chambers A. F. Activated ras regulates the proliferation/apoptosis balance and early survival of developing micrometastases.
Cancer Res.
,
62
:
887
-891,  
2002
.
54
Jiang B. H., Zheng J. Z., Aoki M., Vogt P. K. Phosphatidylinositol 3-kinase signaling mediates angiogenesis and expression of vascular endothelial growth factor in endothelial cells.
Proc. Natl. Acad. Sci. USA
,
97
:
1749
-1753,  
2000
.
55
Zhong H., Chiles K., Feldser D., Laughner E., Hanrahan C., Georgescu M. M., Simons J. W., Semenza G. L. Modulation of hypoxia-inducible factor 1α expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics.
Cancer Res.
,
60
:
1541
-1545,  
2000
.
56
Nakatani K., Thompson D. A., Barthel A., Sakaue H., Liu W., Weigel R. J., Roth R. A. Up-regulation of akt3 in estrogen receptor-deficient breast cancers and androgen-independent prostate cancer lines.
J. Biol. Chem.
,
274
:
21528
-21532,  
1999
.
57
Perez-Tenorio G., Stal O. Activation of AKT/PKB in breast cancer predicts a worse outcome among endocrine treated patients.
Br. J. Cancer
,
86
:
540
-545,  
2002
.