Human Molecular Genetics, 2001, Vol. 10, No. 6 599-604
© 2001 Oxford University Press
PTEN coordinates G1 arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model
1Clinical Cancer Genetics and Human Cancer Genetics Programs, Comprehensive Cancer Center and the Division of Human Genetics, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA and 2CRC Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ, UK
Received 4 December 2000; Revised and Accepted 5 February 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene PTEN/MMAC1/TEP1 encodes a dual-specificity phosphatase that recognizes phosphatidylinositol-3,4,5-triphosphate and protein substrates. We have shown previously that over-expression of PTEN in a tetracycline-controlled inducible system blocks cell cycle progression and induces apoptosis in MCF-7 breast cancer cells. Here, we demonstrate that over-expression of wild-type PTEN leads to the suppression of cell growth through the blockade of cell cycle progression, an increase in the abundance of p27, a decrease in the protein levels of cyclin D1 and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-dead mutant, C124S, promotes cell growth and has the opposite effect on the abundance of p27, cyclin D1 levels and the phosphorylation of Akt. The G129E mutant, which does not have lipid phosphatase activity but retains protein phosphatase activity, behaves like C124S except that the former causes decreases in cyclin D1 levels similar to wild-type PTEN. Therefore, PTEN exerts its growth suppression through lipid phosphatase-dependent and independent activities and most likely, via the coordinate effect of both protein phosphatase and lipid phosphatase activities. Addition of either estrogen or insulin abrogates PTEN-mediated up-regulation of p27 and partially blocks PTEN-mediated growth suppression, whereas the combination of estrogen and insulin eliminates the alterations of p27 and cyclin D1 and completely blocks PTEN-mediated growth suppression. Our findings demonstrate that PTEN blocks cell cycle progression differentially through down-regulating the positive cell cycle regulator, cyclin D1, by its protein phosphatase activity, and up-regulating the negative cell cycle regulator, p27, by its lipid phosphatase activity.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene PTEN/MMAC1/TEP1 (1–3) has been implicated in a variety of human cancers. Germline mutations of PTEN are found in three inherited harmatoma tumour syndromes: (i) Cowden syndrome, which has a high risk of breast, thyroid and other cancers (4–7); (ii) Bannayan–Riley–Ruvalcaba syndrome (8,9); and (iii) a Proteus-like syndrome (10). Ectopic expression of PTEN suppresses tumourigenicity and cell growth (11,12) through G1 cell cycle arrest (13,14), apoptosis (14,15) or both (16–18).
PTEN contains the signature motif of protein tyrosine phosphatases and dual specificity protein phosphatases. It can dephosphorylate tyrosine-, serine- and threonine-phosphorylated peptides in vitro (19). A number of studies have clearly demonstrated that PTEN is a phosphatidylinositol-3 phosphatase and dephosphorylates PIP3, a product of phosphoinositol-3-kinase (PI3K), which is required for the phosphorylation and activation of PKB/Akt (15,20–23), a survival factor that protects various cell types against apoptosis (24). Consistent with the role of PTEN in the PI3K/Akt signalling pathway, several lines of evidence suggest that PTEN negatively regulates cell survival (14–17,20). PI3K also plays an important role in cell proliferation (25–29). Several proteins (30) including Akt, PAK and PKC, which phosphorylate and activate p70s6k, as well as MEK/MAPK (31) could potentially transduce mitogenic signals mediated by PI3K, but so far, Akt is the only one that has been found to be affected by PTEN. The role of Akt in the protection of cells from apoptosis is well documented but the role of Akt in PI3K-mediated cell cycle regulation is not clear. Cheney et al. (12) reported that PTEN blocks S phase entry by recruiting p27 into the cyclin E/CDK2 complex and inhibiting CDK2 kinase activity without affecting cyclin E or p27 expression (32). Li and Sun (33) demonstrated that stable clones from wild-type PTEN-transfected glioma cell lines resulted in the increase in the G1 population with a concomitant increase in p27 levels. Further, LY294002, the PI3K inhibitor, can mimic the effect of PTEN (33). The decrease in CDK2 kinase activity (32) or the increase in p27 was accompanied by the reduction of Akt phosphorylation, arguing that the inhibition of cell cycle progression by PTEN is solely mediated through its lipid phosphatase with subsequent blockade of the PI3K/Akt signalling pathway. However, direct evidence was lacking. In addition, it was also unclear whether the alteration of p27 levels or p27-associated CDK2 activity was the direct cause or a consequence of the inhibition of cell cycle progression.
Although it is known that PTEN blocks PI3K signalling and that the PI3K pathway plays an important role in the regulation of both cell growth and cell survival, it is becoming evident that the function of PTEN in the regulation of cell growth goes beyond merely blocking the PI3K/Akt pathway. In addition to its well-documented lipid phosphatase activity, PTEN has protein phosphatase activity as well (19,34). It has been shown that the protein phosphatase activity of PTEN is implicated in cell migration and spreading, but whether it could also play a role in the regulation of cell growth has not yet been established. The N-terminal domain of PTEN has extensive homology to tensin (1), a cytoskeletal protein located at focal adhesions, downstream of which lie a panoply of pathways that regulate cellular integrity, cell-to-cell communication, cell–microenvironment interactions and cell migration. It is conceivable that PTEN may regulate cell growth through multiple signalling pathways, each of which could be regulated differently. Therefore, PTEN-mediated growth suppression may be dependent on the functional status of each particular signalling pathway with which PTEN interacts. To date, no factor which modulates PTEN function in this manner has been identified.
In this study, we show that the protein phosphatase activity of PTEN is necessary for its growth suppressive effect and demonstrate that PTEN inhibits cell cycle progression through the co-operation of its protein phosphatase, which leads to the down-regulation of cyclin D1, the positive regulator of CDK, and its lipid phosphatase activity, which leads to up-regulation of p27, the negative regulator of CDK.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODSREFERENCES |
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PTEN mutants, C124S and G129E, act as dominant negative lipid phosphatases
We previously reported that over-expression of wild-type PTEN in MCF-7 breast cancer cells by a tetracycline-controlled inducible system causes both G1 cell cycle arrest and apoptosis (16). PTEN possesses activity towards both phospholipid and protein. In order to understand whether the protein phosphatase activity could play a role in the regulation of cell growth and the cell cycle, we compared the effect of wild-type PTEN, G129E mutant PTEN and C124S mutant PTEN on cell growth and cell cycle progression. Germline mutations at the C124 position and the G129E mutation have been described in Cowden syndrome (7). To date, families with C124 mutations seem to have multi-organ involvement and a paucity of malignant breast disease, whereas families with G129E have malignant breast disease, based on small sample size (7,9). The C124S mutation results in a phosphatase-dead protein, with neither lipid nor protein phosphatase activity (22,34). The G129E mutation results only in loss of lipid phosphatase activity with retention of protein phosphatase activity (22). Three stable MCF-7 clones, MCF-7/PTEN-WT1 (wild-type), MCF-7/PTEN-CS3 (C124S) and MCF-7/PTEN-GE5 (G129E), which could be induced to express similar amounts of PTEN protein (Fig. 1, top), were chosen for the present investigation. When wild-type PTEN was induced in MCF-7 cells, the level of phosphorylated Akt was reduced (Fig. 1; WT Tet– versus Tet+) in accordance with the known relationship of functional PTEN, PI3K and Akt. In contrast, MCF-7 expressing C124S or G129E mutant PTEN resulted in increased phosphorylated Akt (Fig. 1; CS and GE). Thus, these observations suggest that both the C124S and G129E mutants are lipid phosphatase-dead. In addition, because MCF-7 cells have endogenous wild-type PTEN, this observation also suggests that both these mutants inactivate the lipid phosphatase activity of endogenous PTEN via a dominant negative mechanism.
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Protein phosphatase activity is involved in PTEN-mediated G1 arrest
If the growth suppression of PTEN is only mediated through its lipid phosphatase activity, which is known to counter the PI3K signalling pathway, one would expect that the G129E mutant might have a similar effect on cell growth and cell cycle distribution to that of the C124S mutant. As shown in Figure 2A, expression of wild-type PTEN led to growth suppression: the number of MCF-7/PTEN-WT cells after PTEN induction was 57.1% of that in the absence of PTEN over-expression. In contrast, expression of the C124S mutant promoted cell growth by 30%, whereas G129E had no effect when compared with the wild-type. The differences in cell number of the G129E mutant versus the wild-type and the C124S mutants reflect the relative contribution of the lipid and protein phosphatase activities, respectively. Therefore, it is clear from these observations that both protein and lipid phosphatase activities of PTEN are involved in growth suppression. This is in contrast with the conclusion drawn by Myers et al. (22), who stated that the lipid phosphatase rather than the protein phosphatase activity is important for its tumour suppressive activity, at least in a PTEN-null prostate cancer cell line. This discrepancy may be due to differences in PTEN signalling between a breast cancer cell line with endogenous PTEN and a PTEN-null prostate cancer cell lines or due to different expressional systems.
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Because PTEN is also known to effect G1 arrest, the ratio of cell number in the S phase to that in the G1 phase was compared amongst MCF-7 clones expressing wild-type and mutant PTEN. The S:G1 ratio mirrored the pattern of cell number among the three cell lines. As Figure 2B demonstrates, the S:G1 ratio of wild-type PTEN-expressing cells was decreased compared with native MCF-7 cells; the S:G1 ratio of the C124S mutant-expressing cells was increased; and the S:G1 ratio of the G129E cells was in between that of wild-type PTEN-expressing cells and C124S cells. No significant increase in the number of dead cells can be detected in all three lines at 48 h of induction (data not shown), thus confirming (16) that only G1 arrest is involved in growth suppression at this time point. In sum, because of the divergent responses in growth suppression and G1 arrest resulting from expression of the C124S phosphatase-dead mutant versus the G129E lipid phosphatase-dead mutant, it can be concluded that PTEN-mediated G1 arrest has a lipid phosphatase-dependent and independent component. The lipid phosphatase-independent component which mediates cell cycle arrest is most likely the protein phosphatase of PTEN.
PTEN down-regulates cyclin D1 and up-regulates p27
The G1:S cell cycle progression is controlled by several CDK complexes, such as CDK2-cyclin E, CDK4-cyclin D and CDK6-cyclin D, whose activities are dependent on the balance of cyclins and CDK inhibitors (CKIs), such as p27 and p21. To investigate whether PTEN-induced G1 cell cycle arrest is due to the down-regulation of cyclin expression or up-regulation of CKIs, we examined the protein levels of cyclin D1, E and H, all three of which are positive regulators of the G1:S cell cycle progression, as well as the protein levels of p27 and p21, which are negative regulators. As can be seen in Figure 3, the over-expression of wild-type PTEN led to an increase in p27 protein levels (Fig. 3, panel F) and a decrease in cyclin D1 levels (Fig. 3, panel C). Thus, PTEN likely inhibits cell cycle progression through coordinate effects on both cyclin D1 expression, a positive CDK regulator, and p27, a negative regulator of CDK.
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PTEN's protein and lipid phosphatase activities differentially regulate cyclin D1 and p27
To investigate the involvement of the protein and lipid phosphatase activities of PTEN in the regulation of either cyclin D or p27 expression, we compared the effects of wild-type PTEN, C124S and G129E mutants on cyclin D1 and p27 levels. Cyclin D1 protein levels were reduced in the cells over-expressing wild-type PTEN and the G129E mutant, both of which have intact protein phosphatase, but increased in cells over-expressing the phosphatase-dead mutant, C124S (Fig. 4, top). Taken together with the differential outcome on cell growth and cell cycle distribution between the C124S and G129E mutants (above), our data here demonstrate that a non-lipid phosphatase activity of PTEN negatively regulates cyclin D1 levels. Most likely, this non-lipid phosphatase activity which negatively regulates cyclin D1 levels and is necessary for PTEN's growth suppressive function is PTEN's protein phosphatase activity. This is in contrast to that reported by Paramio et al. (35) who showed that over-expression of PTEN in C33A cervical cancer cells inhibited cyclin D1 expression by blocking the PI3K/Akt pathway without affecting cell growth. This discrepancy may again be due to differences of PTEN signalling between breast and cervical cancer models or to different expressional systems. The protein levels of p27 were increased in wild-type PTEN-expressing cells and decreased in cells expressing C124S and G129E mutants (Fig. 4), both of which can act as dominant negative lipid phosphatases as evidenced by increased Akt phosphorylation (Figs 1 and 4). The inverse correlation between p27 and lipid phosphatase activity reflected by Akt phosphorylation clearly suggests that the lipid phosphatase activity of PTEN regulates p27 abundance. The effect of PTEN on p27 is consistent with that reported by Li et al. (33), but we provide direct evidence that the effect of PTEN on p27 is associated with its lipid phosphatase activity.
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Estrogen and insulin rescue PTEN-mediated growth suppression by circumventing PTEN-mediated p27 accumulation and cyclin D1 down-regulation
Because of the role of the sex steroid hormones in the genesis of breast carcinomas and their known role as a strong mitogen for breast and breast cancer cells, we sought to define whether estrogen, a strong growth stimulating factor in MCF-7 breast cancer cells, could modulate PTEN-mediated growth suppression in the context of cyclin D1 and p27 regulation. Similarly, because of the emerging role of the insulin/insulin-like growth factor pathways in carcinogenesis in general, and breast carcinogenesis specifically, we also sought to explore the effect of insulin on PTEN-mediated growth suppression in regard to cyclin D1 and p27. As Figure 5A illustrates, PTEN expression in estrogen-free media resulted in twice the growth inhibition compared to that in Dulbecco's modified Eagle's medium (DMEM) with 5% regular fetal bovine serum (FBS) (Fig. 5A, bar 1 versus bar 2). After adding purified estrogen to estrogen-free culture media, PTEN-mediated growth suppression was significantly reduced from 49.2 to 27.9% (Fig. 5A, bar 3), the latter of which is similar to that obtained with regular serum. Interestingly, insulin had an identical effect to that of estrogen (Fig 5A, bar 4); further, estrogen and insulin together completely abrogated PTEN-mediated growth suppression (Fig 5A, bar 5). To investigate whether estrogen and/or insulin inhibit/s PTEN-mediated growth suppression via modulation of cyclin D1 expression and/or p27 abundance, we compared the effect of PTEN on cyclin D1 and p27 levels in the presence and absence of estrogen and/or insulin. Either estrogen or insulin eliminated the effect of PTEN on p27 levels (Fig. 5B, panel 2) but not on cyclin D1 levels (Fig. 5B, panel 1), although either estrogen or insulin increased overall levels of cyclin D1. The effect of ectopic expression of PTEN on cyclin D1 was abrogated when estrogen and insulin were added together. Stimulation with insulin increased the overall levels of Akt phosphorylation, but did not alter the effect of PTEN on Akt phosphorylation (Fig. 5B, panel 3). Estrogen had no effect on the PTEN-associated decrease in either the basal level of Akt phosphorylation or the increased Akt phosphorylation in response to insulin stimulation.
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1.5 compared with lane 1 (PTEN off) where it is 0.75. The p27:tubulin ratio is There are two non-mutually exclusive interpretations for these observations with the two different mitogens. The first, and more conservative, is the implication of the involvement of two different pathways from PTEN down to p27 and from PTEN down to cyclin D. The second is that these observations may also suggest that the effect of PTEN on p27 and cyclin D1 are differentially regulated by each of its phosphatase activities.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODSREFERENCES |
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Since the isolation of PTEN, it was believed that only the lipid phosphatase activity of PTEN mediated growth suppression. Our observations, together with those of a recent report (36) suggest that both the protein and lipid phosphatase activities are involved in PTEN-mediated growth suppression, at least in the MCF-7 breast cancer model. The lipid phosphatase branch of PTEN up-regulates p27 by blocking PI3K signalling, whereas the protein phosphatase branch down-regulates cyclin D1. The bifurcation of PTEN's lipid and protein phosphatase signals ultimately converges at the CDKs for G1/S transition, and cell cycle progression is blocked. The growth suppressive effect of PTEN depends on cellular context, and the two phosphatase signalling pathways might be differentially modulated by mitogens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSION CONCLUSIONS MATERIALS AND METHODSREFERENCES |
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Cell culture
The MCF-7/Toff cell line (Clontech) and PTEN stable expressing clones were maintained in DMEM/10% FBS (Gibco BRL) with 100 U/ml penicillin G (Sigma), 100 µg/ml streptomycin sulfate (Sigma), 100 µg/ml geneticin and 1µg/ml tetracycline (Sigma).
Plasmid construction and transfection
The conserved glycine residue at codon 129 was mutated to glutamic acid by PCR-based site-directed mutagenesis and subsequently cloned into a tetracycline-controlled inducible expression vector, pUHD10-3, to generate pUHD10-3/PTEN.GE. The MCF-7/Toff cell line, which was stably transfected with the tetracycline-controlled transactivator expression plasmid pUHD15-1, was used to establish stable MCF-7/PTEN-GE cell lines as described previously (16).
Sub-confluent stock cells were washed twice with PBS, trypsinized by phenol red-free trypsin-EDTA (Gibco BRL) and diluted at a 1:3 ratio into MEBM [phenol red-free, (Clontech)], 5% charcoal/dextran-treated FBS (HyClone) and 1 µg/ml tetracycline for 3 days. Equal numbers of cells were plated into tetracycline-free culture medium to induce PTEN expression and into medium containing 1 µg/ml tetracycline as a control and cultured for 48 h, unless otherwise indicated. For testing the effect of estrogen and insulin on PTEN-mediated cell suppression, 10–7 M 17-ß-oestradiol (Sigma) or 10 µg/ml of insulin (Gibco BRL) was added to the culture medium immediately after induction of PTEN expression.
Cell growth and cell death assay
Cell growth was measured by methylene blue, as previously described (16). In brief, equal numbers of cells were plated into Tet+ and Tet– media in 12-well plates and cultured for 48 h. Absorption was measured at 595 nm. The ratio of the absorption in Tet– cultures to the absorption in Tet+ cultures at each time point was calculated and presented as a percentage of cell growth. Cells were prepared in p60 mm dishes. At the end of incubation, cells were trypsinized and washed into ice-cold PBS. Cells were then fixed by 40% ice-cold ethanol for 60 min. The fixed cells were washed with cold PBS and incubated at 37°C for 30 min in 0.5 ml PBS containing 10 mg/ml of propidium iodine (Sigma) and 5 mg/ml RNase A (New England Biolabs). DNA content was determined by FACS scan analysis (Becton Dickinson). Dead cells were determined by trypan blue staining as previously described (16).
Protein extraction and immunoblotting
Protein extraction and immunoblotting were performed precisely as detailed (16). The anti-PTEN monoclonal antibody 6H2.1, which was raised against the C-terminal of PTEN (37), and monoclonal anti-cyclin D1, anti-cyclin E antibodies, polyclonal anti-cyclin A, anti-cyclin B and anti-cyclin H antibodies (Santa Cruz Biotechnology) were used for western blot at 1:250 dilution. Polyclonal anti-Akt and anti-phospho-Akt-Ser 473 were purchased from New England BioLabs and used at 1:1000 dilution. Monoclonal anti-p21, anti-p27 antibodies (Signal Transduction Laboratory) and anti-
-tubulin (Sigma) antibodies were used at 1, 0.1 and 0.1 µg/ml, respectively.
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ACKNOWLEDGEMENTS |
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We thank Albert de la Chapelle and Gustavo Leone for critical review of this manuscript. This work was partially supported by the Susan G. Komen Breast Cancer Research Foundation (BCTR2000 462) to C.E. and the National Cancer Institute grant (P30CA16058) to The Ohio State University Comprehensive Cancer Center.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Ohio State University Human Cancer Genetics, 420 West 12th Avenue, Suite 690 TMRF, Columbus, OH 43210, USA. Tel: +1 614 292 2347; Fax: +1 614 688 3582/4245; Email: eng-1@medctr.osu.edu
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