Molecular Cancer Research Targeting the PI3-Kinase Pathway in Cancer Bridging the Lab and the Clinic in Cancer Medicine
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Molecular Cancer Research 1:551-560 (2003)
© 2003 American Association for Cancer Research

Signaling and Regulation

The Role of Phosphatidylinositol 3'-Kinase and Its Downstream Signals in erbB-2-Mediated Transformation1

Kathleen M. Woods Ignatoski1, Donna L. Livant1, Sonja Markwart1, Navdeep K. Grewal1 and Stephen P. Ethier1

Department of Radiation Oncology and the Comprehensive Cancer Center, University of Michigan Health Systems, Ann Arbor, MI

Requests for reprints: Stephen P. Ethier, University of Michigan Medical School, 7312 CCGC, 1500 E. Medical Center Dr., Ann Arbor, MI 48109-0948. Phone: (734) 647-1008; Fax: (734) 647-9480. E-mail: spethier{at}umich.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
We previously demonstrated that erbB-2-overexpressing human mammary epithelial (HME) cells exhibit several transformed phenotypes including growth factor independence, anchorage-independent growth, motility, and invasiveness. Because phosphatidylinositol 3'-kinase (PI3K) is a major target of erbB-2 activation, we tested the contribution that PI3K and its downstream signaling pathways make to these phenotypes. Utilizing a constitutively active form of PI3K, p110CAAX, we show that PI3K can mediate most phenotypes observed in erbB-2-overexpressing cells. To identify pathways leading from PI3K to specific phenotypes, we expressed constitutively active AKT or PTEN in erbB-2-overexpressing cells or in HME cells. HME cells expressing constitutively active AKT were growth factor independent, anchorage independent and motile, but not invasive. PTEN expression blocked erbB-2-mediated invasion but none of the other phenotypes. Rottlerin blocked invasion induced by p110CAAX and erbB-2, suggesting that protein kinase C {delta} (PKC-{delta}) is the downstream effector of PI3K responsible for the invasive capacity of the cells. Consistent with these observations, phospho-AKT remained detectable in erbB-2 cells treated with LY294002 or expressing exogenous PTEN, but was abolished by treatment with the p38MAP kinase inhibitor SB202190. Thus, both PI3K-dependent and p38MAP kinase-dependent pathways lead to activation of AKT, and activation of PKC-{delta}, via PI3K, mediates invasion.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Amplification and overexpression of erbB-2 occurs in approximately 25% of human breast cancers (HBCs) (1, 2). Previously, we demonstrated that erbB-2 overexpression in HBC correlates with growth factor independence (3). To study the transforming function of erbB-2 in the absence of the other genetic alterations found in HBC, we used a bicistronic retroviral expression vector to overexpress erbB-2 in immortalized human mammary epithelial (HME) cells and demonstrated that erbB-2 overexpression can induce growth factor independence, anchorage independence, motility, and invasive capacity (4, 5). Because erbB-2 can mediate these transformed phenotypes, we set out to identify the signaling pathways activated by erbB-2 that mediate each of the transformed phenotypes.

erbB-2 is a member of the Type I class of receptor tyrosine kinases (6). Like the other members of the erbB family, which includes EGFR, erbB-3, and erbB-4, activation of erbB-2 occurs on dimerization, which results in intramolecular tyrosine phosphorylation of the cytoplasmic domain of the molecule. Furthermore, erbB-2 can form homodimers or heterodimers with other members of the erbB family (7). Indeed, erbB-2 has been shown to be the preferred binding partner for the other erbB family members (8), and erbB-2/EGFR and erbB-2/erbB-3 heterodimers activate diverse signaling pathways in cells (9). erbB-2/erbB-3 heterodimers may be particularly important, because this interaction results in the phosphorylation of the cytoplasmic domain of erbB-3, which by virtue of its six YXXM motifs, is a potent activator of phosphatidylinositol 3'-kinase (PI3K) (10). PI3K is composed of two subunits: the p85 regulatory subunit that binds activated growth factor receptors via its SH2 domains and the p110 catalytic subunit (11). There are at least three isoforms of each subunit, which may traffic to different intercellular compartments, and in that way, regulate different cellular characteristics (12). The p110 subunit is constitutively active and can phosphorylate several different phosphoinositides after being recruited to the cell surface by the regulatory subunit (13). Activation of PI3K results in the phosphorylation of the 3' position of phosphatidylinositol 4,5-bis-phosphate (PIP2) to yield phosphatidyl inositol 3,4,5-tris-phosphate (PIP3) (14). PIP3, in turn, activates several downstream signaling molecules either directly or indirectly, including AKT, mTor, and some isoforms of protein kinase C (PKC) (15–20). PTEN is a phosphatase that can dephosphorylate many phosphatidyl inositides. PIP3 is thought to be the major physiological substrate for PTEN in vivo and has therefore been implicated as an important negative regulator of PI3K signaling (21).

We previously demonstrated that the invasive capacity of erbB-2-overexpressing cells is dependent on PI3K by using the PI3K inhibitor LY294002 and by transducing cells with an expression vector for the PTEN phosphatase (5). The present studies were aimed at determining which downstream effectors of PI3K are involved in driving growth factor-independent proliferation, anchorage-independent growth, motility, and invasive capacity, and to determine which of these phenotypes can be influenced by PTEN. The results indicate that transduction of a constitutively activated form of PI3K (p110CAAX) into immortalized HME cells results in the expression of most, but not all, of the altered phenotypes induced by erbB-2 overexpression. Furthermore, the data implicate AKT activation in growth factor-independent proliferation, anchorage-independent growth, and motility. The data also implicate PKC-{delta} in the invasive capacity that results from constitutive PI3K signaling. Finally, the results indicate that PTEN overexpression can block p110CAAX- and erbB-2-induced invasion, but that it does not completely block AKT activation or the phenotypes that result from the activation of this kinase.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Transformation of HME Cells by Expression of Constitutively Active PI3K
We have previously shown that erbB-2 signaling can mediate growth factor-independent proliferation, anchorage-independent growth, motility, and invasion when overexpressed in immortalized HME cells (4, 5). Because erbB-2 is a potent activator of PI3K, we performed experiments to test whether PI3K was sufficient to induce these transformed phenotypes. To carry out these experiments, a constitutively active form of the p110{alpha} subunit of PI3K (p110CAAX) was stably expressed in H16N2 cells, a normal, immortalized HME cell line, using a retroviral vector (Fig. 1A). Expression of p110CAAX in H16N2 cells was confirmed by Northern blot, and the cells were tested for the presence of several transformed phenotypes. Fig. 1 shows that H16N2 cells expressing p110CAAX could form colonies in soft agar (Fig. 1B). However, the expression of this isoform of p110 did not induce growth factor-independent proliferation in monolayer culture (not shown). Consistent with this observation was the finding that p110CAAX-induced soft agar growth also required the presence of exogenous insulin and epidermal growth factor (EGF). Thus, p110CAAX expression induced an anchorage-independent but growth factor-dependent phenotype. In separate experiments, we found that the PI3K inhibitor, LY294002, at 25 µM, could block erbB-2-mediated anchorage-independent growth, confirming the importance of PI3K activity in mediating this transformed phenotype (Fig. 1B).



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  		FIGURE 1. PI3K is sufficient to mediate several transformed phenotypes. A. p110CAAX expression. A Northern blot was probed for the exogenous p110 subunit of PI3K. The transcript size is about 5 kb due to the bicistronic nature of the plasmid—the plasmid size reflects the size of the p110 subunit plus the size of the Neo resistance gene. B. Anchorage-independent growth assay. Cells were grown for 3 weeks in soft agar and stained with the vital dye {rho}-iodonitrotetrazolium violet. Indicated cells received 25 µM LY294002 (LY) 3x/week at normal feedings. pNG and pTP are vector-only control cells. Assays were performed in duplicate at least twice. C. Phagocytic gold motility assay. Cells were plated on top of a layer of gold particles and allowed to attach and move for 24 h. Cleared areas were scored. Representative cells for each population are shown. The numbers in the bottom right corners are the means of area cleared by motile cells normalized to vector-only cells (pTP). The numbers are averages of the cleared area of 50–100 cells per cell type then normalized to vector-only cells. Assays were performed in duplicate twice. D. Phagocytic gold motility assay. Same as D except that the motility scores for all of the cell lines described in the manuscript are represented in graph form. The values are the normalized means of area cleared by motile cells. The numbers are averages of the cleared area of 50–100 cells per cell type then normalized to vector-only cells. Assays were performed twice in duplicate. E. SU-ECM invasion assays. erbB-2-overexpressing, p110CAAX-expressing, and p110CAAX cells expressing PTEN were subjected to SU-ECM invasion assays. Indicated cells received 25 µM LY294002 for 24 h before the experiment. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated twice.

 
Since we had previously shown that PI3K activity was required for erbB-2-mediated invasive capacity (5), experiments were performed to test whether p110CAAX could induce motility and invasiveness when expressed in H16N2 cells. Fig. 1, C and D, shows that expression of p110CAAX did induce a motile phenotype in HME cells. Using SU-ECM invasion assays, we also found that p110CAAX expression resulted in cells with invasive capacity. p110CAAX- and erbB-2-induced motility and invasive capacity (5) were blocked by the PI3K inhibitor LY294002 (Fig. 1, C–E).

Since p110CAAX activity appeared to be sufficient to induce most of the transformed phenotypes observed previously in erbB-2-overexpressing cells, we next examined the role of specific downstream signaling molecules in the PI3K pathway in each of the transformed phenotypes.

Role of AKT Activation in Transformed Phenotypes in HME Cells
AKT is known to be an important effector molecule in PI3K-activated signaling (5, 8). HME cells that overexpress erbB-2 have elevated levels of serine 473-phosphorylated AKT (Fig. 2A). To study the role of AKT in the transformed phenotypes described above, we transduced H16N2 cells with a retroviral vector containing a constitutively active form of AKT, Myr-AKT (Fig. 2B). The data in Fig. 2C show that expression of Myr-AKT in H16N2 cells induced anchorage-independent growth in H16N2 cells. Interestingly, whereas the presence of EGF in the culture medium was not necessary for the formation of colonies in soft agar, the presence of the growth factor resulted in the formation of larger colonies (Fig. 2C). Thus, like the results obtained with p110CAAX, expression of Myr-AKT resulted in anchorage-independent growth capacity, but did not induce complete growth factor independence in soft agar. Cells expressing p110CAAX did contain increased levels of serine-phosphorylated AKT (Fig. 2D). However, in contrast to the results with p110CAAX, Myr-AKT-expressing H16N2 cells were able to grow continuously in monolayer culture in the absence of EGF. Indeed, cells expressing constitutively active AKT are now routinely cultured without EGF, whereas vector control cells still require EGF.



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  		FIGURE 2. Myr-Akt mediates survival phenotypes. A. Activity of AKT in erbB-2 cells. Top panel is whole cell lysates probed for phospho-serine 473 of AKT (i.e., activated AKT; AKT-Pser473) and the bottom panel is total AKT in whole cell lysates. B. Expression of Myr-Akt (constitutively active AKT) shown by Western blot. Exogenous AKT is myc-tagged and therefore is a larger size protein than endogenous AKT. C. Anchorage-independent growth assay. Vector-only (pNG) and Myr-AKT cells were grown for 3 weeks in soft agar then stained with the vital dye {rho}-iodonitrotetrazolium violet. Vector-only cells grown in SFIHE (serum-free with insulin, hydrocortisone, and EGF) medium survive as single cells but do not make large colonies like those seen with Myr-AKT cells grown in SFIHE. Assays were performed in duplicate at least twice. D. Whole cell lysates were made from vector-only cells (pTP), erbB-2-overexpressing cells, or cells expressing p110CAAX. Proteins from the whole cell lysates were separated on gels, Western blotted, and probed for activated AKT-Pser 473 (top panel) or for total AKT (bottom panel). Blots were repeated twice. E. Phagocytic gold assay. The assay is the same as in Fig. 1D. Assays were performed in duplicate twice. F. SU-ECM invasion assay. erbB-2-overexpressing, p110CAAX-expressing, and Myr-AKT cells were subjected to SU-ECM invasion assays. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated at least twice.

 
Experiments were then performed to determine if expression of constitutively active AKT could induce a motile and invasive phenotype in H16N2 cells. Cells expressing Myr-AKT and cells transduced with control vector were tested for motility using the colloidal gold motility assay and for invasive capacity using the SU-ECM assay. The data in Fig. 2E show that constitutively active AKT did induce a motile phenotype in HME cells. However, Myr-AKT-transduced cells were not able to invade SU-ECM basement membranes (Fig. 2F). These results indicate that different effector arms of the PI3K pathway are involved in mediating different transformed phenotypes.

Role of PTEN in Modulating Transformed Phenotypes in HME Cells
We have previously demonstrated that infection of erbB-2-overexpressing cells with a retroviral vector coding for the lipid phosphatase PTEN could block erbB-2-mediated invasion (4). On the basis of these observations, we hypothesized that studying PTEN's role in modulating transformed phenotypes would help to elucidate the downstream signaling events that are necessary for invasion, as well as other phenotypes. To this end, we expressed PTEN in erbB-2-overexpressing and p110CAAX-expressing cells to levels similar to endogenous PTEN expression levels (Fig. 3A) using a retroviral expression vector. The data in Fig. 1E show that, like in previous studies with erbB-2-expressing HME cells (5), PTEN blocked the invasive capacity of p110CAAX-expressing cells. By contrast, expression of PTEN did not block erbB-2- or p110CAAX-mediated anchorage-independent growth even when the cells were cultured in the absence of exogenous growth factors (Fig. 3B). PTEN expression at these levels also did not block erbB-2-mediated growth factor-independent proliferation in monolayer, but did transiently reduce the growth rate of the cells (Fig. 3C). The data in Fig. 3D show that erbB-2-mediated cell motility was also not blocked by exogenous PTEN expression. These results are in contrast to results obtained previously using the PI3K inhibitor LY294002, which was able to block all transformed phenotypes exhibited by erbB-2- and p110CAAX-expressing cells (Fig. 1, B, C, and E). The differential ability of PTEN and LY294002 to modulate the specific phenotypes induced by PI3K signaling suggests that in HME cells, PTEN only modulates the activity of specific isoforms of PI3K or is only operative in some cellular compartments.



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  		FIGURE 3. Exogenous PTEN expression blocked invasion only. A. Expression of exogenous PTEN by Western blot. Exogenous PTEN is FLAG-tagged so the exogenous band's mobility is retarded in the gel. B. Anchorage-independent growth assay. erbB-2-overexpressing cells expressing exogenous PTEN and p110CAAX-expressing cells expressing exogenous PTEN were grown for 3 weeks in soft agar then stained with the vital dye {rho}-iodonitrotetrazolium violet. Assays were performed in duplicate at least twice. C. Growth curve. erbB-2-overexpressing cells alone, with pTP control, or expressing exogenous PTEN were plated in a growth assay and fed with or without 25 µM LY294002 in normal feedings. Cell numbers were counted in triplicate on days 1, 3, 5, and 7. Assays were performed twice. D. Phagocytic gold assay. The assay is the same as in Fig. 1D. Assays were performed in duplicate twice. E. erbB-2-overexpressing cells were transfected with either the vector as a control or with PTEN WT. Then whole cell lysates were separated on gels, Western blotted, and probed for activated AKT-Pser 473 (top panel) or for total AKT (bottom panel). Blots were repeated three times.

 
Role of PI3K and p38 MAP Kinase in AKT Activation and Signaling
Because AKT activation can induce growth factor-independent proliferation, anchorage-independent growth, motility, but not invasion, and because PTEN expression blocked invasion, but not any other transformed phenotype, we hypothesized that AKT remained active in erbB-2-overexpressing cells that were transduced with exogenous PTEN. The data in Fig. 3E show that phospho-AKT was still detectable in erbB-2-overexpressing cells in the presence of exogenous PTEN. We also found that serine 473 phosphorylation levels of AKT in erbB-2-overexpressing cells were only partially reduced by the PI3K inhibitor LY294002 (Fig. 4A). Recently, Rani et al. (22) implicated p38MAP kinase in the activation of AKT. Therefore, we tested the ability of the p38MAP kinase inhibitor SB202190 to inhibit AKT activity in erbB-2-overexpressing cells. The data in Fig. 4B show that, whereas LY294002 resulted in a decrease in phospho-AKT levels, phosphorylated AKT was undetectable following exposure to SB202190. To determine if SB202190 had a direct effect on AKT kinase activity, AKT in vitro kinase assays were performed in the presence or absence of SB202190. Fig. 4C shows that SB202190 had no direct effects on AKT kinase activity because SB202190 could not block the kinase activity of isolated AKT. However, the data in Fig. 4D show that, whereas a 24-h incubation with SB202190 can totally block AKT phosphorylation, there is still residual AKT kinase activity under these conditions. These results suggest that the activation of AKT can also be influenced by p38MAP kinase in erbB-2-overexpressing cells. To be sure that the effects on AKT phosphorylation were indeed influenced by inhibiting p38MAPK and not by a non-specific effect of the SB202190 inhibitor, we incubated erbB-2-overexpressing cells with another p38MAPK inhibitor SB203580. Both inhibitors greatly reduced the amount of serine-phosphorylated AKT in erbB-2-overexpressing cells (Fig. 4E). However, the activity of p38MAP kinase was measured and no appreciable difference was seen in erbB-2-overexpressing cells compared to vector-only cells (data not shown). To demonstrate that p38MAPK-mediated AKT phosphorylation was due to erbB-2 signaling, we incubated the erbB-2-overexpressing cells with and without EGF. In the presence of EGF and SB202190, a condition in which AKT phosphorylation is greatly reduced (Fig. 4E), erbB-2-overexpressing cells still survived and proliferated slowly (Fig. 4F); whereas, in the absence of EGF, erbB-2-overexpressing cells incubated with SB202190 failed to grow and were not viable (Fig. 4F). Also, AKT-mediated transformed phenotypes were blocked when erbB-2-overexpressing cells were incubated with SB202190, including motility (Fig. 1D) and growth factor-independent growth (Fig. 4F). Taken together, these observations may account for the inability of PTEN or LY294002 to decrease phosphorylated serine 473-AKT levels in erbB-2-overexpressing cells and may also explain why PTEN does not abrogate altered phenotypes that are mediated by AKT signaling.



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  		FIGURE 4. AKT activation is at least partially dependent on p38MAPK. A. Vector-only (pTP) or erbB-2-overexpressing cells were incubated with or without 25 µM LY294002 for 24 h. Then whole cell lysates were separated on gels, Western blotted, and probed for activated AKT-Pser473 (top panel) or for total AKT (bottom panel). Note that all of the activation of AKT is not totally inhibited by LY294002. B. erbB-2-overexpressing cells were incubated for 24 h with either ethanol, 10 µM SB202190, 25 µM LY294002, or 10 µM SB202190 plus 25 µM LY294002. Whole cell lysates were separated on gels, Western blotted, and probed for AKT-Pser473 (top panel) or for total AKT (bottom panel). C. In vitro kinase assays for AKT activation were performed in the presence or absence of SB202190. AKT was immunoprecipitated from erbB-2-overexpressing cells and subjected to in vitro AKT kinase assays with or without 10 µM SB202190. Kinase assays were separated on SDS-PAGE and blotted to polyvinylidene difluoride (PVDF). Blots were probed with phospho-GSK (top panel; phosphorylated GSK indicates that the AKT in the immunoprecipitation was active) or AKT (bottom panel). Note that AKT kinase activity is not directly affected by the presence of SB202190. All experiments were done twice. D. In vitro kinase assays for AKT activation were performed after 24 h incubation of the cells with either 25 µM LY294002 (LY) or 10 µM SB202190 (SB). Kinase assays were separated on SDS-PAGE and blotted to PVDF. Blots were probed with phospho-GSK (top panel) or AKT (bottom panel). Kinase assays were repeated twice. Note that although all of the phosphorylation on Ser 473 is absent in the presence of SB202190, there is still some residual kinase activity. E. erbB-2-overexpressing cells grown with or without EGF were incubated for 24 h with either ethanol, 10 µM SB202190, or 10 µM SB203580. Whole cell lysates were separated on gels, blotted, and probed for AKT-Pser473 (top panel) or for total AKT (bottom panel). This was repeated twice. F. Growth curve. erbB-2-overexpressing cells were incubated with or without EGF ± SB202190, plated in a growth assay, and fed with or without 25 µM LY294002 in normal feedings. Cell numbers were counted in triplicate on days 1, 3, 5, and 7. Assays were performed twice.

 
Role of PKC-{delta} in PI3K-Induced Invasive Capacity
Since the AKT arm of the PI3K pathway is not involved in driving the invasive phenotype in HME cells, we investigated two other downstream mediators of PI3K for their role in invasion. mTor is an integral part of one of the effector arms of the PI3K pathway (16), and this enzyme can be blocked by Rapamycin. Fig. 5 shows that Rapamycin did not inhibit erbB-2-mediated invasion. Because some PKC isoforms are dependent on PI3K for activation, we investigated the role of PKC-{delta}, one of the isoforms dependent on PI3K, in mediating the invasive phenotype. The enzymatic activity of PKC-{delta} can be specifically blocked by using 5 µM of the inhibitor Rottlerin (23). The data in Fig. 5 show that, unlike Rapamycin, Rottlerin did block both erbB-2- and p110CAAX-mediated invasions (Fig. 5). Since Rottlerin can have non-specific effects on cell viability, we determined if Rottlerin had non-specific effects on the viability of cells by incubating erbB-2-overexpressing cells with ethanol or with 5 µM Rottlerin for 15 min, the time used for Rottlerin incubation for the invasion assays. By trypan blue exclusion, there was no effect of Rottlerin on cell viability, nor did Rottlerin influence the ability of the cells to reattach to the culture dish or decrease their subsequent ability to proliferate. Thus, we conclude that the effects we observed of Rottlerin on invasive capacity were not the result of non-specific toxicity (Table 1). However, Rottlerin has also been shown to inhibit MAPKAP-K2 and PRAK at concentrations around 5 µM (24). Therefore, we cannot rule out the possibility that these signaling molecules play a role in invasion. These results indicate that PI3K-mediated activation of PKC-{delta}, and possibly other PKC isoforms with pleckstrin homology domains, plays a role in the induction of the invasive phenotype in erbB-2-overexpressing cells and in p110CAAX-expressing HME cells.



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  		FIGURE 5. erbB-2- and PI3K-mediated invasion is dependent on PKC-{delta}. erbB-2-overexpressing and p110CAAX-expressing cells were subjected to SU-ECM invasion assays. Indicated cells were incubated for 24 h with either 1 µM Rapamycin or for 15 min with 5 µM Rottlerin (a concentration specific for PKC-{delta}) and subjected to SU-ECM invasion assays. Invasion percentages are relative to the invasion capacity of untreated erbB-2 cells. Each assay was performed in duplicate and repeated at least twice.

 

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  		Table 1. Rottlerin Incubation Does Not Decrease Cell Viability

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
erbB-2 is amplified and overexpressed in approximately 25% of HBCs, and its overexpression correlates with poor prognosis (23, 25). However, the direct role that erbB-2 plays in cell transformation remains to be completely elucidated. To understand the mechanisms by which erbB-2 induces specific transformed phenotypes, we transduced erbB-2 into HME cells. We found that erbB-2 overexpression induces several transformed phenotypes (4, 5). Since PI3K is a major downstream effector of erbB-2 signaling, we set out to examine the role of PI3K and its downstream signaling pathways in each of these transformed phenotypes. We found that constitutive PI3K activity was sufficient to mediate anchorage-independent growth, motility, and invasion but not growth factor-independent proliferation.

AKT has long been known to play an important role in cell survival (26–28). Activated AKT can induce the transcription of anti-apoptotic molecules such as Bcl-2 and regulate proteins that participate in the induction of apoptosis (27–30). Our observations are consistent with a role for AKT in cellular survival. We found that constitutively active AKT confers both growth factor-independent and anchorage-independent growth capacity on HME cells. In a previous work, we demonstrated that normal HME cells require exogenous EGF for growth under serum-free conditions and that withdrawal of EGF from the culture medium results in progressive cell death (3). In addition, we have demonstrated that normal HME cells, when cultured under anchorage-independent conditions even in the presence of growth factors, undergo rapid cell death. Thus, the finding that constitutive AKT activation results in relaxed growth factor requirements in monolayer culture and the ability to survive when cultured in soft agar, are consistent with AKT's role in blocking programmed cell death.

We also found that Myr-AKT signaling does not play a role in the acquisition of the invasive capacity that results from activation of PI3K. Other studies in the literature have suggested that AKT is a downstream mediator of PI3K for invasion. In these studies, Boydon chambers coated with Matrigel were used to measure invasive capacity (31–33). It is possible that Boyden chamber assays yield data that more accurately reflect cell motility than actual invasion. Indeed, some breast cancer cells derived from highly invasive breast cancers, which are clearly invasive in other assays, and which form invasive tumors in nude mice, do not score as invasive in the Boyden chamber assay (34). In our laboratory, we use the SU-ECM assay developed by Livant et al. (35) to measure the invasive capacity of transformed cells. In this assay, cancer cells must cross a naturally occurring basement membrane, which has been shown to require the activation of matrix metalloproteinases (35 and unpublished data). We and others have found this assay to be a more accurate reflection of the invasive capacity of cancer cells. Our observations that AKT activation can mediate a motile phenotype are consistent with results obtained with Boyden chamber assays. However, our data do not point to a role for AKT in matrix metalloproteinase-mediated invasion of basement membranes.

The ability of p110CAAX to induce transformed phenotypes, including invasive capacity, the ability of exogenous PTEN to block invasion induced by either erbB-2 or p110CAAX, and the lack of involvement of AKT in invasive capacity, prompted us to examine other PI3K-activated pathways that could be involved in invasion. Because mTor has long been known to be activated by PI3K and because Rapamycin is a potent and specific inhibitor of this kinase, we used this drug to investigate the possible role of mTor in invasion. However, incubation of p110CAAX-expressing cells with concentrations of Rapamycin known to block mTor failed to substantially influence invasive capacity. These results prompted us to consider other possible effectors of PI3K activation. A number of molecules that contain pleckstrin homology domains can be modulated by PI3K signaling (36, 37). Although the PKC family of serine/threonine kinases has long been known to play important roles in growth regulation, it has recently been found that some members of this family, including PKC-{delta}, have pleckstrin homology domains and can be activated by PI3K (36, 38). Therefore, we chose to investigate this molecule for its role in PI3K-mediated invasion. The results presented here are consistent with this hypothesis, as Rottlerin completely blocked invasion induced by both erbB-2 and p110CAAX.

On the basis of these and other observations, we propose that in HBC cells, and in HME cells that overexpress erbB-2 or p110CAAX, different downstream effectors of the PI3K pathway are involved in mediating the different phenotypes exhibited by transformed cells. Thus, activation of AKT results in cells that can proliferate in the absence of EGF and which can survive in suspension culture. These findings are consistent with EGF's known role in activation of AKT in human epithelial cells. Furthermore, because normal human epithelial cells that are placed in suspension culture rapidly undergo programmed cell death, the ability of activated AKT to abrogate this phenotype is also in keeping with its known role as a survival factor.

One aspect of our observations that differs from other reports in the literature concerns our findings with exogenous PTEN. In our experimental system, overexpression of PTEN only reversed the invasive capacity of HME cells and had little or no influence on the other phenotypes that were induced by erbB-2 or p110CAAX. Thus, PTEN-transduced cells exhibited a transient decrease in growth rate in monolayer culture but were still motile and had the ability to grow in soft agar. Two factors have to be considered when interpreting these observations. First, breast cancer cells rarely develop mutations in PTEN (39, 40). Thus, PTEN's role in modulating cellular phenotypes in HME cells may differ from those played in other cell types. Second, because we used a retroviral vector to transduce PTEN into HME cells, the levels of exogenous PTEN expression were modest and probably substantially lower than in other studies in which plasmid vectors were used to transfect cells. With these considerations in mind, our data suggest that in breast cancer cells, phosphorylation of AKT can occur by pathways involving both PI3K and p38MAP kinase, which may act independently or together to activate AKT. p38MAPK appears to only activate AKT in the context of erbB-2 overexpression because SB202190 blocked AKT phosphorylation in erbB-2-overexpressing cells and not in EGFR- or FGFR-overexpressing cells (unpublished data). These results suggest that erbB-2 does not directly influence p38MAPK activity but that p38MAPK influences AKT phosphorylation specifically in erbB-2-overexpressing cells. The precise mechanism for p38MAPK's activation in the context of erbB-2 overexpression remains to be established. Thus, PI3K inhibition, either by LY294002 or by PTEN, does not abolish phosho-AKT levels, and PTEN does not block growth in monolayer or soft agar. By contrast, PTEN and LY294002 are able to completely block invasive capacity, suggesting that PI3K is the sole mediator of PKC-{delta} activation, and subsequent invasive capacity.

In summary, we have demonstrated that constitutive activation of the {alpha} isoform of the catalytic subunit of PI3K can induce many of the altered phenotypes exhibited by breast cancer cells, in general, and transformed cells that overexpress erbB-2. We have also shown that different downstream effectors of PI3K activity are responsible for mediating the many altered phenotypes exhibited by erbB-2-transformed HME cells, with AKT playing an important role in growth factor- and anchorage-independent survival and with PKC-{delta} playing an important role in the invasive capacity of transformed cells.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Cell culture
The base medium for H16N2-erbB2, H16N2-AKT, H16N2-erbB2/PTEN WT, H16N2-p110CAAX, and H16N2-pTP cells was Ham's F12 media supplemented with 0.1% BSA, 0.5 µg/ml fungizone, 5 µg/ml gentamicin, 5 mM ethanolamine, 10 mM HEPES, 5 µg/ml transferrin, 10 µM T3, 50 µM selenium, and 1 µg/ml hydrocortisone. H16N2-pTP and H16N2-p110CAAX cell medium was further supplemented with 10 ng/ml EGF. Cells infected with retroviral expression vectors were selected in 100 µg/ml geneticin (G418) for 2 weeks. All cell culture reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

For growth factor depletion assays, cells were plated at 3.5 x 104 in triplicate in six-well plates. Plating efficiencies were obtained on day 1 after plating, after which the growth factors were removed from the media. Cells in complete media and cells in growth factor-depleted media were counted on days 4, 7, and 11 after plating. Cell counts were performed as previously described on a Coulter model Z1 (Coulter Corporation, Miami, FL). Growth curves were performed in triplicate at least twice.

Retrovirus Construction
pTPerbB-2 retrovirus construction was previously described (4, 41). pTPPTEN(wt) retrovirus construction was also previously described (5). pNGMyrAKT retrovirus construction was done by digesting pCMV6AKT with SalI and EcoRI and ligating AKT into the XhoI-EcoRI-digested pNG3000. pNGp110CAAX retrovirus was made by digesting the p110CAAX plasmid (20) with BamHI and cloning p110CAAX into pBluescript pBC (Stratagene, La Jolla, CA). The orientation was tested by digesting the resulting plasmid with EcoRI. The p110CAAX was released with NotI and XhoI and inserted into pNG3000 into the NotI and EcoRI sites. The construct was sequenced to ensure that the ends were correct.

Soft Agar Assays
Soft agar assays were performed as previously described (42). Briefly, dishes were coated with a 1:1 mix of the appropriate 2x medium for the cell line being studied and 1% Bactoagar. Cells were plated at 1 x 105 cells/well in a 1:1 mixture of appropriate 2x medium and 0.3% Bactoagar. Cells were fed 3x/week for 3 weeks, stained with 500 µg/ml {rho}-iodonitrotetrazolium violet (Sigma) overnight, photographed, and counted on an Accucount 1000 (Fisher Scientific, Pittsburgh, PA). Soft agar assays were done in duplicate and repeated at least twice.

Motility Assays
Phagocytic gold motility assays were performed as previously described (43). Briefly, coverslips were coated with 1% BSA, dipped in 100% ethanol, dried with a hair dryer, and placed in six-well dishes. Gold particles were prepared by combining 11 ml glass distilled H2O, 1.8 ml of a 14.5-mM AuCl4H solution (Sigma), and 6 ml of a 36.5-mM Na2CO3 solution and heating the mixture over a Bunsen burner. Immediately after the mixture reached the boiling point, 1.8 ml of a 0.1% formaldehyde solution were added. Coverslips were incubated at 4°C in the gold solution for up to 1 week before use. 2 x 104 cells were placed on coverslips, fed with the appropriate media, and incubated overnight. The first 50–100 cells seen in the microscope were photographed. Analysis of cleared areas was performed on a Macintosh power mac 850 using the public NIH Image 1.61 (developed at the U.S. NIH and available on the Internet at http://rsb.info.nih.gov/nih-image/). Each cell type was assayed in duplicate and each assay was repeated independently.

Invasion
Cells were suspended in 0.23% trypsin/EDTA (Life Technologies, Inc., Grand Island, NY) and placed on SU-basement membranes with or without FCS according to established methods (35) for 4 h at 37°C, the time required to observe maximal invasion percentages for normal and metastatic cells (35 and data not shown). The percentages of spread and adherent cells were evaluated in each assay to check viability before fixation in 2% formaldehyde and scored at 400x magnification using phase contrast optics. Viability ranged from 90% to 98% in all assays. Mean invasion percentages resulted from two independent determinations involving the scoring of all cells in contact with the invasion substrates. Each assay was performed twice. LY294002 (Sigma) stock solution was 25 mM in 100% ethanol. Rapamycin (Sigma) stock solution was 1 mM in 100% ethanol. Rottlerin (Calbiochem, San Diego, CA) stock solution was 50 mM. LY294002 and Rapamycin were added to fresh cell media to final concentrations of 25 and 1 µM, respectively, for 24 h before the start of the invasion assay. Rottlerin was added to a final concentration of 5 µM 15 min before the start of the invasion assay. Ten microliters of 100% ethanol were added to an identical plate as a control.

Protein Blots
Cells were incubated in 25 µM LY294002 (Sigma), 10 µM SB202190 (Calbiochem), or 10 µM SB203580 (Calbiochem). All 1000x stock solutions were made in 100% ethanol. Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% NP40, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, and 20 µg/ml leupeptin. Protein concentrations were equalized using the Lowry method. For whole cell lysates, Laemmli sample buffer was added and the samples were boiled. For immunoprecipitations, 1 µg of antibody was added to 1 mg of sample and incubated at 4°C for 1 h. Immune complexes were then bound to protein A/G beads for 1 h at 4°C. Immunoprecipitates were washed three times in lysis buffer. Laemmli sample buffer was added and the samples were boiled. Equal amounts of protein were separated by SDS-PAGE. The proteins were blotted to PVDF membranes and probed. AKT in vitro kinase assays were performed using a AKT kinase assay kit (Cell Signaling Technology, Beverly, MA). All AKT kinase assays were repeated at least three times. Ten micromolars SB202190 were added to the appropriate AKT kinase assay. AKT kinase assays with SB202190 were repeated twice.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We acknowledge Steven Kronenberg and Leslie Gransden for their assistance with the illustrations, and Dr. Christopher Chay and Dr. Sarah Parsons for critically reading this manuscript. We thank Dr. Thomas Franke for the gift of the Myr-AKT construct, Drs. Julian Downward and Anne Vojtek for the gift of the p110CAAX gene, Dr. Eric H. Radany for the gift of the pNG vector, and Dr. Jack Dixon for the gift of the PTEN gene.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 NIH grant number CA 70354-05. Back

Received June 6, 2002; revised March 13, 2003; accepted March 19, 2003.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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